CD8+ cell responses to hepatitis C virus (HCV) in the liver of persons with HCV-HIV coinfection versus HCV monoinfection

OBJECTIVE: Cellular immune responses are difficult to detect in the peripheral blood of persons with chronic hepatitis C virus (HCV) infection. We sought to determine whether T cell responses were present in the liver of patients with human immunodeficiency virus (HIV) and HCV coinfection. METHODS: T cells were expanded from liver-biopsy samples from 10 patients coinfected with HIV and HCV (median CD4(+) cell count, 456 cells/mm(3)) and 8 patients infected with HCV alone. CD8(+) cell responses were detected by use of a modified enzyme-linked immunospot (ELISpot) assay with recombinant vaccinia virus, and CD4(+) cell responses were detected by use of ELISpot with recombinant HCV proteins core, nonstructural (NS) 3, and NS5. RESULTS: Intrahepatic CD8(+) cell responses to HCV were detected in 7 of 10 patients coinfected with HCV and HIV (median frequency, 638 spot-forming cells [sfc]/1 x 10(6) cells) and were similar to those observed in patients singly infected with HCV (7/8; median, 647 sfc/1 x 10(6) cells). Intrahepatic HCV-specific CD4(+) cell responses were also comparable in both groups and correlated with the intrahepatic CD8(+) cell responses (r=0.59; P=.03). CONCLUSION: HCV-specific CD8(+) cell responses are present in the liver of persons with chronic HCV infection even when they are coinfected with HIV; these correlate with intrahepatic HCV-specific CD4(+) cell responses.     

Kinetics of intrahepatic hepatitis C virus (HCV)-specific CD4+ T cell responses in HCV and Schistosoma mansoni coinfection: relation to progression of liver fibrosis

The kinetics of intrahepatic hepatitis C virus (HCV)-specific CD4(+) T cell responses and their role in progression of fibrosis have not previously been characterized. Subjects with HCV/Schistosoma mansoni coinfection have a more rapid progression of HCV liver fibrosis than do those with HCV infection alone. The present prospective longitudinal study compared the liver histology, HCV-specific intrahepatic and peripheral CD4(+) T cell proliferative responses, and cytokines (enzyme-linked immunospot) in 48 subjects with unresolved acute HCV infection with or without S. mansoni coinfection, at 6-10 months after acute infection and at the end of follow-up (96+/-8.7 months), and the findings were correlated to the rate of progression of fibrosis per year. Coinfected subjects had significant worsening of fibrosis, compared with subjects with HCV infection alone. At baseline, subjects with HCV infection alone had stronger multispecific intrahepatic HCV-specific CD4(+) T helper 1 responses than did coinfected subjects, who had either no responses or weak, narrowly focused responses, and, over time, these T cell responses were maintained only in the liver. The rate of progression of fibrosis and virus load inversely correlated with intrahepatic HCV-specific CD4(+) T cell response. The present prospective analysis indicates that enhancement of progression of liver fibrosis is associated with failure to develop early, multispecific, HCV-specific CD4(+) Th1 responses, suggesting that novel therapeutic approaches inducing strong cellular immune responses might limit subsequent liver damage in individuals with chronic hepatitis C.     

Comparison of HCV-specific intrahepatic CD4+ T cells in HIV/HCV versus HCV

Persons with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) coinfection are at increased risk for progression to cirrhosis compared with persons with HCV alone, but the reasons for this are unclear. In chronic HCV, the mechanism of liver injury is presumed to be due to HCV-specific T cell destruction of hepatocytes, so it is paradoxical that immunosuppressed hosts have higher rates of fibrosis progression. We examined intrahepatic cellular immune responses to HCV antigens to determine whether there were qualitative or quantitative differences in subjects with and without HIV. Expanded, CD4-enriched, liver-infiltrating lymphocytes from 18 subjects with chronic HCV and 12 subjects with HIV/HCV were cultured in the presence of HCV core protein, nonstructural proteins NS3 and NS5, and recall antigens tetanus toxoid and Candida. Secretion of interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin (IL) 10 was determined using enzyme-linked immunosorbent spot assay. There were no significant differences in liver biopsy grade or stage for HIV/HCV versus HCV groups. There were no significant differences between groups in the secretion of IFN-gamma or TNF-alpha in response to HCV or recall antigens. However, there was a significant increase in IL-10 secretion in response to NS3 and NS5 in subjects with HCV compared with HIV and HCV coinfection. In conclusion, subjects with coinfection have an alteration of intrahepatic HCV-specific IL-10 cytokine response that may have implications for HCV-related disease progression.     

Features of the CD4+ T-cell response in liver and peripheral blood of hepatitis C virus-infected patients with persistently normal and abnormal alanine aminotransferase levels

BACKGROUND/AIMS: The liver is the primary site of hepatitis C virus (HCV) replication; intrahepatic T-cell responses may influence liver disease severity. METHODS: HCV-specific CD4(+) T-cell reactivity was investigated ex vivo in paired liver tissue and peripheral blood from 42 chronic HCV patients. RESULTS: The frequencies with which HCV-specific HLA class-II-restricted CD4(+) T-cell proliferation were observed were 29% in liver and 36% in peripheral blood. Among responses, non-structural-3 protein (NS3)-specific T-cell proliferation was dominant but non-exclusive and did rarely occur concurrently in liver infiltrate and peripheral blood suggesting liver compartmentalization of a CD4(+) T-cells population. Compared with 24 patients with abnormal ALT levels, 18 HCV carriers with persistently normal ALT levels had similar serum and liver viral loads but showed: (i) a low-activity grade and stage chronic hepatitis (P<0.001); (ii) less intrahepatic CD4(+) T-lymphocytes (P<0.01); (iii) less frequent intrahepatic (17 vs. 33%) and peripheral (17 vs. 38%) NS3-specific CD4(+) T-cell proliferation; (iv) less often in vitro T-helper (Th)1 (interferon-gamma) cytokine production (2 vs. 18%; P<0.001). CONCLUSIONS: Our data show a low frequency of intrahepatic HCV-specific HLA class-II-restricted CD4(+) Th1 responses in patients with chronic HCV. However, these Th1 responses are detected more often in those patients with overt clinical and histological disease.     

Stronger hepatitis C virus‐specific CD8+ T‐cell responses in HIV coinfection

Summary. Hepatitis C virus (HCV) is a widespread chronic infection that shares routes of transmission with human immunodeficiency virus (HIV). Thus, coinfection with these viruses is a relatively common and growing problem. In general, liver disease develops over years with HIV coinfection, when compared to decades in HCV monoinfection. The role of the immune system in the accelerated pathogenesis of liver disease in HIV/HCV coinfection is not clear. In this study, we compared the frequency, magnitude, breadth and specificity of peripheral blood CD4⁺ and CD8⁺ T‐cell responses between HCV‐monoinfected and HCV/HIV‐coinfected individuals and between HIV/HCV‐coinfected subgroups distinguished by anti‐HCV antibody and HCV RNA status. While HIV coinfection tended to reduce the frequency and breadth of anti‐HCV CD8⁺ T‐cell responses in general, responses that were present were substantially stronger than in monoinfection. In all groups, HCV‐specific CD4⁺ T‐cell responses were rare and weak, independent of either nadir or concurrent CD4⁺ T‐cell counts of HIV‐infected individuals. Subgroup analysis demonstrated restricted breadth of CD8⁺ HCV‐specific T‐cell responses and lower B‐cell counts in HIV/HCV‐coinfected individuals without anti‐HCV antibodies. The greatest difference between HIV/HCV‐coinfected and HCV‐monoinfected groups was substantially stronger HCV‐specific CD8⁺ T‐cell responses in the HIV‐coinfected group, which may relate to accelerated liver disease in this setting.     

An immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection

The CD4(+)CD25(+) regulatory T lymphocytes have been implicated in suppressing T cell immune responses. Our aim was to characterize the frequency, phenotype, function, and specificity of CD4(+)CD25(+) T cells in hepatitis C virus (HCV) infection. Peripheral CD4(+)CD25(+) cells from recovered (n = 15), chronic infected (n = 30), and normal control (n = 15) subjects were analyzed ex vivo for quantitation, phenotype, and effect on HCV-specific interferon gamma production and proliferation. CD4(+)CD25(+) specificity was determined by intracellular cytokine staining for interleukin 10 (IL-10). A higher proportion of CD4(+)CD25(+) were found in chronic infection (mean, 3.02%) when compared with recovered (1.64%, P = .001) and normal controls (2.27%, P = .02). CD4(+)CD25(+) cells display CD45RO(high), CD45RA(low), CD28(high), CD62L(high), and CD95(high) phenotype. HCV-specific interferon gamma activity was enhanced in peripheral blood mononuclear cells depleted of CD4(+)CD25(+) and suppressed in peripheral blood mononuclear cells enriched with CD4(+)CD25(+). Depletion of CD4(+)CD25(+) cells also enhanced HCV-specific CD4(+) and CD8(+) T cell proliferation. Cytokine analysis suggested CD4(+)CD25(+) cells secrete transforming growth factor beta (TGF-beta(1)) and IL-10. The inhibitory role for TGF-beta(1) was confirmed by anti-TGF-beta(1). Transwell studies showed CD4(+)CD25(+) mediated suppression to be dose dependent and requiring cell contact. CD4(+)CD25(+) cells showed HCV-specificity through IL-10 production, with a frequency ranging from 1.9% to 5.3%. A positive correlation was detected between CD4(+)CD25(+) T cell frequency and HCV RNA titer, whereas an inverse relation was found with liver inflammatory activity. In conclusion, CD4(+)CD25(+) T lymphocytes constitute a highly differentiated population and appear to play a role in viral persistence by suppressing HCV-specific T cell responses in a cell-cell contact manner.     

Liver-derived hepatitis C virus (HCV)-specific CD4(+) T cells recognize multiple HCV epitopes and produce interferon gamma

Virus-specific CD4(+) T-cell response at the site of inflammation is believed to play a decisive role for the course of viral disease. In hepatitis C virus (HCV) infection, the majority of studies focused on the peripheral blood T-cell response. In this study we analyzed intrahepatic virus-specific CD4(+) T-cell response and compared this with that in the peripheral blood. Liver and blood-derived T-cell lines were studied in 36 patients (18 with chronic hepatitis C and 18 with HCV-associated cirrhosis). Virus-specific interferon gamma (IFN-gamma) production at a single cell level to various HCV-proteins (core, nonstructural [NS] 3/4, NS5) were determined by enzyme-linked immunospot (ELIspot). Phenotyping was done by fluorescent-activated cell sorter analysis. In approximately half (16 of 36 [44%]) of intrahepatic T-cell lines a significant number of IFN-gamma spots were observed, whereas this was the case in only 19% (7 of 36 T-cell lines) in the blood. In relative terms, core and nonstructural proteins were recognized with the same frequency in both compartments, but HCV-specificity was significantly more often detected in liver tissue compared with the blood. Hepatitis activity index, viral load, and alanine transaminase levels did not correlate with the detection of HCV-specific CD4(+) T cells. All T-cell lines were dominated by CD4(+) T cells. In conclusion, HCV-specific CD4(+) T cells are multispecific, compartmentalize to the liver, and produce IFN-gamma. We speculate that our data would support the concept of compartmentalization of specific T cells at the site of inflammation and that a low frequency of specific T cells is associated with failure to clear the virus and a chronic course of disease.     

Specific cellular immune response and cytokine patterns in patients coinfected with hepatitis C virus and Schistosoma mansoni

Patients coinfected with hepatitis C virus (HCV) and Schistosoma mansoni show high incidence of viral persistence and accelerated fibrosis. To determine whether immunological mechanisms are responsible for this alteration in the natural history of HCV, the HCV-specific peripheral CD4(+) T cell responses and cytokines were analyzed in patients with chronic hepatitis C monoinfection, S. mansoni monoinfection, or HCV and S. mansoni coinfection. An HCV-specific CD4(+) proliferative response to at least 1 HCV antigen was detected in 73.3% of patients infected with HCV, compared with 8.6% of patients coinfected with HCV and S. mansoni. Stimulation with HCV antigens produced a type 1 cytokine profile in patients infected with HCV alone, compared with a type 2 predominance in patients coinfected with HCV and S. mansoni. In contrast, there was no difference in response to schistosomal antigens in patients infected with S. mansoni alone, compared with those coinfected with HCV and S. mansoni. These findings suggest that the inability to generate an HCV-specific CD4(+)/Th1 T cell response plays a role in the persistence and severity of HCV infection in patients with S. mansoni coinfection.     

Acute hepatitis C without and with schistosomiasis: correlation with hepatitis C-specific CD4(+) T-cell and cytokine response

BACKGROUND & AIMS: Immune responses during the first few months of acute hepatitis C virus (HCV) infection seem crucial for viral control, but the relationship of these responses to natural history is poorly characterized. METHODS: This prospective study investigated the HCV-specific CD4(+) and cytokine responses in patients with acute HCV hepatitis with or without Schistosoma mansoni coinfection, a parasitic infection with T helper (Th) 2 immune bias. HCV-specific CD4(+) proliferative responses and cytokine production in peripheral blood mononuclear cells were correlated with liver biopsy results at 6 months and at the end of follow-up. RESULTS: Whereas 5 of 15 patients with HCV alone recovered from acute HCV, all (17 of 17) patients with S. mansoni coinfection progressed to histologically proven chronic hepatitis. Coinfected patients had either absent or transient weak HCV-specific CD4(+) responses with Th0/Th2 cytokine production. The magnitude of the HCV-specific CD4(+) response at week 12 was inversely correlated with the fibrosis progression rate in chronically infected patients. CONCLUSIONS: Patients with acute hepatitis C and schistosomiasis coinfection cannot clear viremia and show rapid progression once chronic infection is established. This rapid progression is associated with a strong Th2 response in peripheral immune responses, suggesting that early development of vigorous Th1 responses not only facilitates clearance but delays disease progression.     

Racial difference in mortality among U.S. veterans with HCV/HIV coinfection

OBJECTIVES: This study was performed to examine the impact of viral coinfections and race on clinical and virological outcome of hepatitis C virus (HCV) infection. METHODS: Three groups of patients (265 HCV/HIV coinfected, 251 HCV monoinfected, 227 HIV monoinfected) were identified between 2000 and 2002 from the computerized patient record system at the Philadelphia VA Medical Center and analyzed for clinical and virological parameters. RESULTS: HCV/HIV coinfection was associated with higher frequency of liver function abnormalities (37% vs 21% vs 20%; p < 0.0003) and greater mortality (17% vs 6% vs 9% over 3 yr period, p = 0.0003, p = 0.027) compared to HCV or HIV monoinfection, respectively. However, HCV/HIV coinfection was not associated with worsened HIV-related parameters (CD4 count, HIV titer, and use of antiretroviral therapy) or increased HCV titers compared to HIV or HCV monoinfection in our population, respectively. Interestingly, mortality among HCV/HIV coinfected patients was significantly greater in white than in black patients (31% vs 15%, p = 0.011). This racial disparity in mortality was not apparent in the monoinfected groups and not explained by HBV coinfection or history of alcohol use disorder. CONCLUSIONS: We conclude that HCV/HIV coinfection is associated with worsened liver disease and higher mortality than HCV- or HIV-monoinfection without directly influencing CD4 count and HCV or HIV titers. Furthermore, we demonstrated a racial disparity in survival of HCV/HIV-coinfected patients that needs further investigation.     

Impact of HIV on host-virus interactions during early hepatitis C virus infection

BACKGROUND: Human immunodeficiency virus (HIV) may influence the outcome and natural history of hepatitis C virus (HCV) infection through an impact on acute HCV-specific T cell responses. METHODS: Fifty-five HIV-positive males with acute HCV infection were identified; monoinfected individuals (n = 8) were used for peripheral blood mononuclear cell comparison. In 14 coinfected and 8 HCV-monoinfected patients, HCV-specific T cell responses against a range of HCV antigens were assessed using interferon (IFN)-gamma enzyme-linked immunospot (ELISpot) and proliferation assays. E1/E2 region genetic diversity and the selection pressure on the virus were measured in 8 coinfected patients by use of cloned sequences over time. RESULTS: HCV persisted in 52 (95%) coinfected individuals. HCV/HIV coinfection significantly reduced IFN-gamma ELISpot responses versus those in HCV-monoinfected individuals, especially against nonstructural proteins (1/10 vs. 5/8; P = .008). In coinfected patients, increased HCV genetic diversity was observed between the first and subsequent time points, with no evidence for positive selection in the E1/E2 region sequenced. CONCLUSION: HIV coinfection is associated with increased rates of HCV persistence and a lack of critical CD4 T cell responses, with no evidence of immune selection pressure during early HCV infection. Loss of key cellular immune responses against HCV during acute disease may contribute to the failure of early host control of HCV in HCV/HIV-coinfected patients.     

Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infection

Hepatitis C virus (HCV) has a high propensity for persistence. To better define the immunologic determinants of HCV clearance and persistence, we examined the circulating HCV-specific T-cell frequency, repertoire, and cytokine phenotype ex vivo in 24 HCV seropositive subjects (12 chronic, 12 recovered), using 361 overlapping peptides in 36 antigenic pools that span the entire HCV core, NS3-NS5. Consistent with T-cell-mediated control of HCV, the overall HCV-specific type-1 T-cell response was significantly greater in average frequency (0.24% vs. 0.04% circulating lymphocytes, P =.001) and scope (14/36 vs. 4/36 pools, P =.002) among the recovered than the chronic subjects, and the T-cell response correlated inversely with HCV titer among the chronic subjects (R = -0.51, P =.049). Although highly antigenic regions were identified throughout the HCV genome, there was no apparent difference in the overall HCV-specific T-cell repertoire or type-1/type-2 cytokine profile relative to outcome. Notably, HCV persistence was associated with a reversible CD4-mediated suppression of HCV-specific CD8 T cells and with higher frequency of CD4(+)CD25(+) regulatory T cells (7.3% chronic vs. 2.5% recovered, P =.002) that could directly suppress HCV-specific type-1 CD8 T cells ex vivo. In conclusion, we found that HCV persistence is associated with a global quantitative and functional suppression of HCV-specific T cells but not differential antigenic hierarchy or cytokine phenotype relative to HCV clearance. The high frequency of CD4(+)CD25(+) regulatory T cells and their suppression of HCV-specific CD8 T cells ex vivo suggests a novel role for regulatory T cells in HCV persistence.     

Differential CD4(+) and CD8(+) T-cell responsiveness in hepatitis C virus infection

This study was performed to compare the vigor and phenotype of virus-specific CD4(+) and CD8(+) T-cell responses in patients with different virologic and clinical outcomes after hepatitis C virus (HCV) infection. The results show that a vigorous and multispecific CD4(+) proliferative T-cell response is maintained indefinitely after recovery from HCV infection whereas it is weak and focused in persistently infected patients. In contrast, the HCV-specific CD8(+) T-cell response was quantitatively low in both groups despite the use of sensitive direct ex vivo intracellular interferon gamma (IFN-gamma) staining. Furthermore, although HCV-specific cytolytic CD8(+) memory T cells were undetectable ex vivo, they were readily expanded from the peripheral blood of chronically HCV-infected patients but not from recovered subjects after in vitro stimulation, suggesting that ongoing viremia is required to maintain the HCV-specific memory CD8(+) T-cell response. HCV-specific CD8(+) T cells displayed a type 1 cytokine profile characterized by production of IFN-gamma despite persistent HCV viremia. The paradoxical observation that HCV-specific CD4(+) T cells survive and CD8(+) T cells are lost after viral clearance while the opposite occurs when HCV persists suggests the existence of differential requirements for the maintenance of CD4(+) and CD8(+) T-cell memory during HCV infection. Furthermore, the relative rarity of circulating CD8(+) effector T cells in chronically infected patients may explain the chronic insidious nature of the liver inflammation and also why they fail to eliminate the virus.     

Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees that recovered from hepatitis C

Hepatitis C virus (HCV) poses a global health problem because it readily establishes persistent infection and a vaccine is not available. CD4(+)CD25(+) T cells have been implicated in HCV persistence because their frequency is increased in the blood of HCV-infected patients and their in vitro depletion results in increased IFN-gamma production by HCV-specific T cells. Studying a well-characterized cohort of 16 chimpanzees, the sole animal model for HCV infection, we here demonstrate that the frequency of Foxp3(+)CD4(+)CD25(+) regulatory T cells (T(Regs)) and the extent of suppression was as high in spontaneously HCV-recovered chimpanzees as in persistently HCV-infected chimpanzees. Foxp3(+)CD4(+)CD25(+) T(Regs) suppressed IFN-gamma production, expansion, and activation-induced cell death of HCV-specific T cells after recovery from HCV infection and in persistent HCV infection. Thus, T(Reg) cells control HCV-specific T cells not only in persistent infection but also after recovery, where they may regulate memory T-cell responses by controlling their activation and preventing apoptosis. However, Foxp3(+)CD4(+)CD25(+) T(Reg) cells of both HCV-recovered and HCV-infected chimpanzees differed from Foxp3(+)CD4(+)CD25(+)T(Reg) cells of HCV-naive chimpanzees in increased IL-2 responsiveness and lower T-cell receptor excision circle content, implying a history of in vivo proliferation. This result suggests that HCV infection alters the population of Foxp3(+)CD4(+)CD25(+) T(Reg) cells.     

Intrahepatic CD8+ T-cell failure during chronic hepatitis C virus infection

The precise mechanisms responsible for the failure of intrahepatic hepatitis C virus (HCV)-specific CD8+ T cells to control the virus during persistent infection have not been fully defined. We therefore studied the CD8+ T-cell response in 27 HLA-A2-positive patients using four previously well-defined HLA-A2-restricted HCV epitopes. The corresponding HCV sequences were determined in several patients and compared with the intrahepatic HCV-specific CD8+ T-cell response. The results of the study indicate: (1) intrahepatic HCV-specific CD8+ T cells are present in the majority of patients with chronic HCV infection and overlap significantly with the response present in the peripheral blood. (2) A large fraction of intrahepatic HCV-specific CD8+ T cells are impaired in their ability to secrete interferon gamma (IFN-gamma). This dysfunction is specific for HCV-specific CD8+ T cells, since intrahepatic Flu-specific CD8+ T cells readily secrete this cytokine. (3) T-cell selection of epitope variants may have occurred in some patients. However, it is not an inevitable consequence of a functional virus-specific CD8+ T-cell response, since several patients with IFN-gamma-producing CD8+ T-cell responses harbored HCV sequences identical or cross-reactive with the prototype sequence. (4) The failure of intrahepatic virus-specific CD8+ T cells to sufficiently control the virus occurs despite the presence of virus-specific CD4+ T cells at the site of disease. In conclusion, different mechanisms contribute to the failure of intrahepatic CD8+ T cells to eliminate HCV infection, despite their persistence and accumulation in the liver.     

Vigorous hepatitis C virus-specific CD4+ and CD8+ T cell responses induced by protein immunization in the presence of Montanide ISA720 plus synthetic oligodeoxynucleotides containing immunostimulatory cytosine-guanine dinucleotide motifs

BACKGROUND: A prophylactic vaccine for hepatitis C virus (HCV) requires generation of strong humoral as well as CD4(+) and CD8(+) T cell responses. METHODS: The immunomodulatory effects of the combination of 2 adjuvants, synthetic oligodeoxynucleotides containing immunostimulatory cytosine-guanine dinucleotide motifs emulsified with Montanide ISA720 (M-ISA720/CpG), were investigated using the murine model. RESULTS: Administration of recombinant HCV (rHCV) nonstructural (NS) 3 and NS5B proteins plus M-ISA720/CpG (hereafter, "M-ISA720/CpG/rHCV protein") induced high anti-NS3 and anti-NS5B immunoglobulin (Ig) G titers, with the IgG2a isotype being predominant. NS3- and NS5B-specific interferon (IFN)- gamma - and interleukin-2-producing CD4(+) T cell responses, as assessed by enzyme-linked immunospot assay, were significantly more vigorous in mice immunized with M-ISA720/CpG/rHCV protein than in control mice immunized without adjuvant. NS3- and NS5B-specific IFN- gamma -producing CD8(+) T cell percentages, as measured by direct ex vivo intracellular cytokine staining assay, were, respectively, a mean+/-SD of 0.14% +/- 0.04% and 0.15% +/- 0.05% in mice immunized with M-ISA720/CpG/rHCV protein. Furthermore, boosting with recombinant NS3 expression plasmid DNA after priming with M-ISA720/CpG-adjuvanted rNS3 strikingly enhanced both CD4(+) and CD8(+) T cell responses. CONCLUSION: Immunization with M-ISA720/CpG/rHCV protein is capable of inducing potent humoral as well as HCV-specific T helper type 1-biased CD4(+) and CD8(+) T cell responses. A DNA boost after a protein prime--a reversal of the conventional approach--may provide an alternative path to the development of an effective HCV vaccine.     

Quantitative analysis of hepatitis C virus-specific CD8+ T cells in peripheral blood and liver using peptide-MHC tetramers

It is believed that the hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a role in the development of liver cell injury and in the clearance of the virus. To develop a direct binding assay for HCV-specific CTLs, we generated two peptide-MHC tetramers by using the recombinant HLA A2.1 molecule and A2-restricted T cell epitopes of the HCV NS3 protein. With these reagents we are able to detect specific CD8(+) cells in the blood of 15 of 20 HLA-A2(+), HCV-infected patients, at a frequency ranging from 0.01% to 1.2% of peripheral CD8(+) T cells. Phenotypic analysis of these specific cells indicated that there is a significant variation in the expression of the CD45 isoforms and CD27 in different patients. A 6-hour incubation of one patient's blood with NS3 peptides resulted in the activation of the epitope-specific CD8(+) cells, as indicated by their expression of CD69 and IFN-gamma. We also detected NS3-specific CD8(+) T cells in the intrahepatic lymphocyte population isolated from liver biopsies of two HCV-infected patients. The frequency of these specific CD8(+) cells in the liver was 1-2%, at least 30-fold higher than in the peripheral blood. All of the intrahepatic NS3-specific CD8(+) T cells were CD69(+), suggesting that they were activated CTLs. Direct quantitation and characterization of HCV-specific CTLs should extend our understanding of the immunopathogenesis and the mechanism of clearance or persistence of HCV.     

CD4+ T cell-dependent reduction in hepatitis C virus-specific humoral immune responses after HIV infection

BACKGROUND: Human immunodeficiency virus (HIV) infection adversely affects all stages of hepatitis C virus (HCV) infection, leading to increased rates of viral persistence, higher levels of HCV viremia, and accelerated progression of HCV-related liver disease. These disease interactions may result in part from impairment of B cell function, which is CD4(+) T cell dependent. METHODS: To determine the effect of HIV infection on B cell function, we compared HCV antibody levels and specificities in 29 HCV-infected persons before and after they acquired HIV and assessed the temporal correlation of these changes with overall CD4(+) T lymphocyte counts. RESULTS: The pre-HIV infection HCV antibody titer was a predictor of the subsequent titer for all antigens, and decreasing CD4(+) T cell numbers was strongly associated with a decrease in anti-HCV titers for several antigens. CD4(+) T cells counts of <500 cells/mm(3) were significantly associated with lower HCV antibody end-point titers. Higher HCV end-point titers were associated with fewer years from HIV infection and, for Core antigen, current drug use. CONCLUSIONS: HCV-specific antibody production is impaired by HIV infection, and loss of antibody production depends on CD4(+) T cell depletion. However, the decrease in titers is less significant in those who continue to actively inject drugs.     

The magnitude and breadth of hepatitis C virus-specific CD8+ T cells depend on absolute CD4+ T-cell count in individuals coinfected with HIV-1

CD8(+) T-cell responses are an essential antiviral host defense in persistent viral infections, and their sustained effectiveness is thought to be critically dependent on CD4(+) T-helper cells. To determine the relationship between HIV-1-induced CD4(+) T-cell depletion and hepatitis C virus (HCV)-specific CD8(+) T-cell responses during viral persistence, we studied 103 persons positive for HCV, 74 coinfected with HIV-1. CD8(+) T-cell responses to the entire HCV polyprotein were determined by using an interferon-gamma enzyme-linked immunospot (ELISpot) assay. Although HIV-1 infection by itself was not associated with a diminished HCV-specific response, HIV-1-associated CD4(+) depletion was associated with significantly lower HCV-specific CD8(+) T cells (R = 0.48, P < .0001). In contrast, declining CD4(+) counts over the same range were not associated with diminished Epstein-Barr virus (EBV)- (R = 0.19, P = .31) or HIV-1-specific (R = -0.13, P = .60) CD8(+) T-cell responses in persons infected with all viruses. These data indicate that frequencies of circulating HCV-specific CD8(+) T-cell responses are sensitive to absolute CD4(+) T-cell counts and provide a possible explanation for the accelerated HCV disease course in persons coinfected with HIV-1 and HCV.     

Characterization of HCV-specific Patr class II restricted CD4+ T cell responses in an acutely infected chimpanzee

Resolution of hepatitis C virus (HCV) infection is associated with strong and sustained virus-specific CD4+ T cell responses. In this study, we investigated the evolution of functional T cell responses during acute infection of a chimpanzee and the longevity of these lymphocytes in blood and liver after resolution of infection. Viremia increased through the first 3 weeks of infection and then remained stable until the onset of T cell responses at weeks 6 and 8 postinfection. CD4+ T cells targeting nonstructural HCV proteins were detected in proliferation assays by week 6 postinfection, but they failed to produce interferon gamma (IFN-gamma). HCV-specific CD4+ and CD8+ T cells with the ability to produce IFN-gamma appeared at week 8 when a rapid 10-fold reduction in plasma viremia was first observed. This cytokine response persisted through to week 24 when infection apparently resolved. T cell lines targeting 3 CD4+ T cell epitopes and 1 CD8+ T cell epitope were derived from liver and their Patr major histocompatibility complex (MHC) restriction elements were identified. In retrospective studies performed on cryopreserved peripheral blood mononuclear cells (PBMCs) collected at various timepoints after infection, the onset of an IFN-gamma response measured against the class II restricted epitopes correlated with viral clearance. In conclusion, the characterization of the HCV epitopes and MHC class II restriction elements described here will facilitate a detailed comparison of CD4+ T cell function in animals with resolved and persistent infections.