A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees

Three percent of the world's population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8(+) T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses.     

Cellular immune responses associated with occult hepatitis C virus infection of the liver

Occult hepatitis C virus (HCV) infection is a type of recently identified chronic infection that is evidenced only by detection of HCV RNA in liver; patients consistently test negative for antibodies to HCV and HCV RNA in serum. Using ex vivo and in vitro measures of T-cell responses, we have identified functional virus-specific memory CD4(+) and CD8(+) T cells in the peripheral blood of patients with occult HCV infection. The features of the virus-specific T cells were consistent with immune surveillance functions, supporting previous exposure to HCV. In addition, the magnitudes of CD4(+) and CD8(+) T-cell responses were in parallel and correlated inversely with the extent of liver HCV infection. The detection of HCV-specific T cells in individuals in whom HCV RNA can persist in the liver despite the absence of viremia and antibodies indicates that HCV replication is prolonged in the face of virus-specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate that HCV-specific cellular immune responses are markers not only of previous exposure to and recovery from HCV but also of ongoing occult HCV infection.     

Human immunodeficiency virus type 1-hepatitis C virus coinfection: intraindividual comparison of cellular immune responses against two persistent viruses

Both human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) lead to chronic infection in a high percentage of persons, and an expanding epidemic of HIV-1-HCV coinfection has recently been identified. These individuals provide an opportunity for simultaneous assessment of immune responses to two viral infections associated with chronic plasma viremia. In this study we analyzed the breadth and magnitude of the CD8(+)- and CD4(+)-T-lymphocyte responses in 22 individuals infected with both HIV-1 and HCV. A CD8(+)-T-lymphocyte response against HIV-1 was readily detected in all subjects over a broad range of viral loads. In marked contrast, HCV-specific CD8(+)-T-lymphocyte responses were rarely detected, despite viral loads in plasma that were on average 1,000-fold higher. The few HCV-specific responses that were observed were relatively weak and limited in breadth. CD4-proliferative responses against HIV-1 were detected in about half of the coinfected subjects tested, but no proliferative response against any HCV protein was found in these coinfected persons. These data demonstrate a major discordance in immune responses to two persistent RNA viruses. In addition, they show a consistent and profound impairment in cellular immune responses to HCV compared to HIV-1 in HIV-1-HCV-coinfected persons.     

Full-breadth analysis of CD8+ T-cell responses in acute hepatitis C virus infection and early therapy

Multispecific CD8(+) T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8(+) T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8(+) T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8(+) T-cell responses, as well as CD4(+) T-cell responses. Rapid recrudescence also occurred despite broad CD8(+) T-cell responses. Importantly, in vivo suppression of CD3(+) T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8(+) T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.     

Failure of innate and adaptive immune responses in controlling hepatitis C virus infection

Effective innate and adaptive immune responses are essential for the control of hepatitis C virus (HCV) infection. Indeed, elimination of HCV during acute infection correlates with an early induction of innate and a delayed induction of adaptive immune responses. However, in the majority of acutely HCV-infected individuals, these responses are insufficient to clear the virus and persistence develops. In recent years, different mechanisms responsible for the failure of innate and adaptive immune responses have been identified. These include the proteolytic cleavage of molecules playing key roles in the induction of the interferon response, manipulation of interferon-induced effector proteins, interference with CD8+ T-cell function or immune escape in T- and B-cell epitopes. In this review, we discuss the possible roles of innate and adaptive immune responses in HCV clearance and the different evasion strategies used by the virus to escape these immune responses.     

Enhanced and sustained CD8+ T cell responses with an adenoviral vector-based hepatitis C virus vaccine encoding NS3 linked to the MHC class II chaperone protein invariant chain

Potent and broad cellular immune responses against the nonstructural (NS) proteins of hepatitis C virus (HCV) are associated with spontaneous viral clearance. In this study, we have improved the immunogenicity of an adenovirus (Ad)-based HCV vaccine by fusing NS3 from HCV (Strain J4; Genotype 1b) to the MHC class II chaperone protein invariant chain (Ii). We found that, after a single vaccination of C57BL/6 or BALB/c mice with Ad-IiNS3, the HCV NS3-specific CD8(+) T cell responses were significantly enhanced, accelerated, and prolonged compared with the vaccine encoding NS3 alone. The AdIiNS3 vaccination induced polyfunctional CD8(+) T cells characterized by coproduction of IFN-γ, TNF-α and IL-2, and this cell phenotype is associated with good viral control. The memory CD8(+) T cells also expressed high levels of CD27 and CD127, which are markers of long-term survival and maintenance of T cell memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8(+) T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice demonstrated that this protection was mediated primarily through IFN-γ production. On the basis of these promising results, we suggest that this vaccination technology should be evaluated further in the chimpanzee HCV challenge model.     

Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C

As acute hepatitis C virus (HCV) infection is clinically inapparent in most cases, the immunologic correlates of recovery are not well defined. The cellular immune response is thought to contribute to the elimination of HCV-infected cells and a strong HCV-specific T-helper-cell (Th) response is associated with recovery from acute hepatitis C (ref. 2). However, diagnosis of resolved hepatitis C is based at present on the detection of HCV-specific antibodies and the absence of detectable HCV RNA, and detailed comparison of the humoral and cellular immune response has been hampered by the fact that patient cohorts as well as HCV strains are usually heterogeneous and that clinical data from acute-phase and long-term follow-up after infection generally are not available. We studied a cohort of women accidentally exposed to the same HCV strain of known sequence and found that circulating HCV-specific antibodies were undetectable in many patients 18-20 years after recovery, whereas HCV-specific helper and cytotoxic T-cell responses with an interferon (IFN)-gamma-producing (Tc1) phenotype persisted. The data indicate these HCV-specific CD4 + and CD8+ T cells are biomarkers for a prior HCV exposure and recovery. Because of undetectable antibodies against HCV, the incidence of self-limited HCV infections and recovery may be underestimated in the general population.     

Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection

The factors leading to spontaneous clearance of hepatitis C virus (HCV) or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance is key to the development of more effective therapeutic and prophylactic strategies. Here, using a sensitive neutralization assay based on infectious HCV pseudoparticles (HCVpp), we have studied the kinetics of humoral responses in a cohort of acute-phase patients infected during a single nosocomial outbreak in a hemodialysis center. The 17 patients were monitored for the spontaneous outcome of HCV infection for 6 months before a treatment decision was made. Blood samples were taken frequently (15 +/- 4 per patient). Phylogenetic analysis of the predominant virus(es) revealed infection by only one of two genotype 1b strains. While all patients seroconverted, their sera induced two opposing effects in HCVpp infection assays: inhibition and facilitation. Furthermore, the ability of sera to facilitate or inhibit infection correlated with the presence of either infecting HCV strain and divided the patients into two groups. In group 1, the progressive emergence of a relatively strong neutralizing response correlated with a fluctuating decrease in high initial viremia, leading to control of viral replication. Patients in group 2 failed to reduce viremia within the acute phase, and no neutralizing responses were detected despite seroconversion. Strikingly, sera of group 2, as well as naive sera, facilitated infection by HCVpp displaying HCV glycoproteins from different genotypes and strains, including those retrieved from patients. These results provide new insights into the mechanisms of viral persistence and immune control of viremia.     

Characterization of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine

Current therapies for the treatment of hepatitis C virus (HCV) infection are only effective in a restricted number of patients. Cellular immune responses, particularly those mediated by CD8(+) CTLs, are thought to play a role in the control of infection and the response to antiviral therapies. Because the Core protein is the most conserved HCV protein among genotypes, we evaluated the ability of a Core prototype vaccine to prime cellular immune responses in rhesus macaques. Since there are serious concerns about using a genetic vaccine encoding for Core, this vaccine was a nonclassical ISCOM formulation in which the Core protein was adsorbed onto (not entrapped within) the ISCOMATRIX, resulting in approximately 1-microm particulates (as opposed to 40 nm for classical ISCOM formulations). We report that this Core-ISCOM prototype vaccine primed strong CD4(+) and CD8(+) T cell responses. Using intracellular staining for cytokines, we show that in immunized animals 0.30-0.71 and 0.32-2.21% of the circulating CD8(+) and CD4(+) T cells, respectively, were specific for naturally processed HCV Core peptides. Furthermore, this vaccine elicited a Th0-type response and induced a high titer of Abs against Core and long-lived cellular immune responses. Finally, we provide evidence that Core-ISCOM could serve as an adjuvant for the HCV envelope protein E1E2. Thus, these data provide evidence that Core-ISCOM is effective at inducing cellular and humoral immune responses in nonhuman primates.     

The clearance of hepatitis C virus infection in chimpanzees may not necessarily correlate with the appearance of acquired immunity

Clearance of hepatitis C virus (HCV) infection in humans and chimpanzees is thought to be associated with the induction of strong T-cell responses. We studied four chimpanzees infected with HCV derived from an infectious full-length HCV genotype 1b cDNA. Two of the chimpanzees cleared the infection to undetectable levels for more than 12 months of follow-up; the other two became persistently infected. Detailed analyses of HCV-specific immune responses were performed during the courses of infection in these chimpanzees. Only weak and transient T helper responses were detected during the acute phase in all four chimpanzees. A comparison of the frequency of gamma interferon (IFN-gamma)-producing CD4(+) and CD8(+) T cells in peripheral blood by ELISpot assay did not reveal any correlation between viral clearance and T-cell responses. In addition, analyses of IFN-gamma, IFN-alpha, and interleukin-4 mRNA levels in liver biopsies, presumably indicative of intrahepatic T-cell responses, revealed no distinct pattern in these chimpanzees with respect to infection outcome. The present study suggests that the outcome of HCV infection in chimpanzees is not necessarily attributable to HCV sequence variation and that chimpanzees may recover from HCV infection by mechanisms other than the induction of readily detectable HCV-specific T-cell responses.     

Kinetic analysis by real-time PCR of hepatitis C virus (HCV)-specific T cells in peripheral blood and liver after challenge with HCV

Intrahepatic virus-specific CD8(+) T cells are thought to be important for the control of hepatitis C virus (HCV) infection, yet the precise kinetics for the expansion of epitope-specific T cells over the course of infection are difficult to determine with currently available methods. We used a real-time PCR assay to measure the frequency of clonotypic HCV-specific CD8(+) T cells in peripheral blood and snap-frozen liver biopsy specimens of two chimpanzees (Pan troglodytes) with previously resolved HCV infection who were rechallenged with HCV. In response to rechallenge, the magnitude of each clonotypic response was 10-fold higher in the liver than in the blood, and the peak clonotype frequency was concurrent with the peak viral load. The higher frequency of HCV-specific clonotypes in the liver than in peripheral blood was maintained for at least 3 months after the clearance of viremia. After antibody-mediated CD8(+) T-cell depletion and another viral challenge, the rebound of these clonotypes was seen prior to an appreciable reconstitution of CD8(+) T-cell values and, again, at higher frequencies in the liver than in peripheral blood. These data demonstrate the importance of intrahepatic virus-specific CD8(+) T cells for the clearance of infection and the rapid kinetics of expansion after virus challenge.     

Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates

We have previously reported the production of hepatitis C virus-like particles (HCV-LP) using a recombinant baculovirus containing the cDNA of the HCV structural proteins (core, E1, and E2). These particles resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice. Here we present evidence on the immunogenicity of HCV-LP and the effects of novel adjuvant systems in a nonhuman primate model, the baboon. Three groups of four baboons were immunized with HCV-LP, HCV-LP and adjuvant AS01B (monophosphoryl lipid A and QS21), or HCV-LP and the combination of AS01B and CpG oligodeoxynucleotides 10105. After four immunizations over an 8-month period, all animals developed HCV-specific humoral and cellular immune responses including antibodies to HCV structural proteins and gamma interferon(+) (IFN-gamma(+))CD4(+) and IFN-gamma(+)CD8(+) T-cell responses. The immunogenicity of HCV-LP was only marginally enhanced by the use of adjuvants. The overall HCV-specific immune responses were broad and long lasting. Our results suggest that HCV-LP is a potent immunogen to induce HCV-specific humoral and cellular immune responses in primates and may be a promising approach to develop novel preventive and therapeutic modalities.     

Retention of CD4+ CD25+ FoxP3+ regulatory T cells in the liver after therapy-induced hepatitis C virus eradication in humans

Following infection with the hepatitis C virus (HCV), in most cases immunity fails to eradicate the virus, resulting in slowly progressing immunopathology in the HCV-infected liver. We are the first to examine intrahepatic T cells and CD4(+) CD25(+) FoxP3(+) regulatory T cells (Treg) in patients chronically infected with HCV (chronic HCV patients) during and after antiviral therapy by collecting multiple aspiration biopsy samples from the liver at different time points. We found that intrahepatic Treg frequencies were increased upon alpha interferon and ribavirin administration in about 50% of chronic HCV patients, suggesting stronger regulation of intrahepatic immunity by Treg during antiviral therapy. After cessation of antiviral therapy, the frequency of intrahepatic Treg remained above baseline in the large majority of livers of individuals who successfully cleared the virus. The phenotype of those Treg that were retained in the liver months after therapy-induced clearance of HCV RNA indicated a reduced contribution of effector memory cells. Our findings, gathered by multiple samplings of the liver, indicate that successful antiviral therapy of chronic HCV patients does not lead to normalization of the local immune response to a resting state comparable to that for healthy livers. The continuous presence of high numbers of Treg, with a phenotype reflecting a relatively weak suppressive activity, suggests ongoing residual regulation of immunopathology. These findings provide important insight into the dynamics of the immune response to HCV, as well as the effect of therapy on intrahepatic immunity.     

Adaptive immune responses to hepatitis C virus: from viral immunobiology to a vaccine

Hepatitis C virus (HCV) causes chronic infection in approximately two-thirds of cases, leading to chronic hepatitis, liver cirrhosis, liver disease, liver failure, and hepatocellular carcinoma in a substantial proportion of the 170 million HCV-infected individuals worldwide. It is generally accepted that the cellular immune response plays the most important role in determining the outcome of HCV infection. First, vigorous, multispecific and sustained CD4+ and CD8+ T-cell responses are associated with viral clearance. Second, depletion studies in chimpanzees, the only other host of HCV besides humans, have shown that both CD4+ and CD8+ T-cells are required for virus elimination. Third, the host's human leukocyte antigen alleles, which restrict the repertoire of CD4+ and CD8+ T-cell responses, influence the outcome of infection. Of note, protective immunity has been demonstrated in population-based studies, as well as in experimentally infected chimpanzees. Thus, a detailed understanding of the mechanisms contributing to the failure of the antiviral immune response should allow successful development of prophylactic and therapeutic vaccination strategies.     

Discordant outcomes following failure of antiretroviral therapy are associated with substantial differences in human immunodeficiency virus-specific cellular immunity

Many individuals chronically infected with human immunodeficiency virus type 1 (HIV-1) experience a recrudescence of plasma virus during continuous combination antiretroviral therapy (ART) due either to the emergence of drug-resistant viruses or to poor compliance. In most cases, virologic failure on ART is associated with a coincident decline in CD4(+) T lymphocyte levels. However, a proportion of discordant individuals retain a stable or even increasing CD4(+) T lymphocyte count despite virological failure. In order to address the nature of these different outcomes, we evaluated virologic and immunologic variables in a prospective, single-blinded, nonrandomized cohort of 53 subjects with chronic HIV-1 infection who had been treated with continuous ART and monitored intensively over a period of 19 months. In all individuals with detectable viremia on ART, multiple drug resistance mutations with similar impacts on viral growth kinetics were detected in the pol gene of circulating plasma virus. Further, C2V3 env gene analysis demonstrated sequences indicative of CCR5 coreceptor usage in the majority of those with detectable plasma viremia. In contrast to this homogeneous virologic pattern, comprehensive screening with a range of antigens derived from HIV-1 revealed substantial immunologic differences. Discordant subjects with stable CD4(+) T lymphocyte counts in the presence of recrudescent virus demonstrated potent virus-specific CD4(+) and CD8(+) T lymphocyte responses. In contrast, subjects with virologic failure associated with declining CD4(+) T lymphocyte counts had substantially weaker HIV-specific CD4(+) T lymphocyte responses and exhibited a trend towards weaker HIV-specific CD8(+) T lymphocyte responses. Importantly the CD4(+) response was sustained over periods as long as 11 months, confirming the stability of the phenomenon. These correlative data lead to the testable hypothesis that the consequences of viral recrudescence during continuous ART are modulated by the HIV-specific cellular immune response.     

Hepatitis C virus infection among First Nation and non-First Nation people in Manitoba, Canada: a public health laboratory study

Demographic information and laboratory test results on 136 169 clinical serum specimens submitted to the public health laboratory in Manitoba, Canada, for hepatitis C virus (HCV) testing between January 1995 and December 2003 were analyzed. The difference in the clearance rates of HCV infection, without therapeutic intervention, and the HCV genotypes infecting First Nation and non-First Nation people were studied. The rates of co-infection of HCV-positive individuals with other hepatitis viruses were also compared between the two study groups. The results of the analyses of the data indicated that there was a 4.4-fold increase in the number of specimens tested and a 4.9-fold decrease in HCV antibody (anti-HCV) positive cases during the study period. The proportion of specimens submitted for testing from First Nation individuals was lower than their proportion in the Manitoba population. Our study also indicated that there was a significantly higher proportion of First Nation patients who had self-limiting infection (patients cleared the infection and became HCV RNA negative without anti-HCV treatment) in comparison to non-First Nation patients. The proportion of First Nation females who had self-limiting infection was significantly higher than non-First Nation females. HCV genotype 1 infection represented more than 60% of HCV infection in Manitoba. The rate of individuals positive for the hepatitis A virus antibody in the HCV-positive population was higher among First Nation than non-First Nation individuals. On the other hand, there were more HCV-infected First Nation patients than non-First Nation patients who were not immune to the hepatitis B virus. The data indicate that fewer First Nation patients seek anti-HCV therapy in comparison to non-First Nation. In conclusion, the differences in the rates of HCV self-limiting infection between First Nation and non-First Nation individuals in Manitoba may reflect the genetic differences between the two cohorts, which may consequently affect the immune response to the HCV infection.     

Distinct CD4+ CD8+ double-positive T cells in the blood and liver of patients during chronic hepatitis B and C

CD4(+) and CD8(+) T cells, the main effectors of adaptive cellular immune responses, differentiate from immature, non-functional CD4(+)CD8(+) double-positive T (DPT) cells in the thymus. Increased proportions of circulating DPT lymphocytes have been observed during acute viral infections; in chronic viral diseases, the role and repartition of extra-thymic DPT cells remain largely uncharacterized. We performed a phenotypic analysis of DPT cells in blood and liver from patients chronically infected by hepatitis C (HCV) or B (HBV) viruses. The highest percentages of DPT cells, predominantly CD4(high)CD8(low), were observed in patients infected by HCV, while HBV-infected patients mostly displayed CD4(low)CD8(high) and CD4(high)CD8(high) DPT cells. All proportions of DPT cells were higher in liver than in blood with, for each subpopulation referred to above, a correlation between their frequencies in these two compartments. In HCV patients, intra-hepatic DPT cells displayed more heterogeneous activation, differentiation and memory phenotypes than in the blood; most of them expressed CD1a, a marker of T cell development in the thymus. Ex vivo, the inoculation of liver slices with HCV produced in cell culture was accompanied by a disappearance of CD8(high) cells, suggesting a direct effect of the virus on the phenotype of DPT cells in the liver. Our results suggest that, in half of the patients, chronic HCV infection promotes the production of DPT cells, perhaps by their re-induction in the thymus and selection in the liver.     

CD8+ T-cell responses to different HIV proteins have discordant associations with viral load

Selection of T-cell vaccine antigens for chronic persistent viral infections has been largely empirical. To define the relationship, at the population level, between the specificity of the cellular immune response and viral control for a relevant human pathogen, we performed a comprehensive analysis of the 160 dominant CD8(+) T-cell responses in 578 untreated HIV-infected individuals from KwaZulu-Natal, South Africa. Of the HIV proteins targeted, only Gag-specific responses were associated with lowering viremia. Env-specific and Accessory/Regulatory protein-specific responses were associated with higher viremia. Increasing breadth of Gag-specific responses was associated with decreasing viremia and increasing Env breadth with increasing viremia. Association of the specific CD8(+) T-cell response with low viremia was independent of HLA type and unrelated to epitope sequence conservation. These population-based data, suggesting the existence of both effective immune responses and responses lacking demonstrable biological impact in chronic HIV infection, are of relevance to HIV vaccine design and evaluation.     

Transmission of clonal hepatitis C virus genomes reveals the dominant but transitory role of CD8⁺ T cells in early viral evolution

The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8(+) T cells is a dominant force driving early amino acid replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8(+) T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute-phase nonsynonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome. Moreover, the ratio of nonsynonymous to synonymous mutations, a measure of positive selection pressure, was increased 50-fold in class I epitopes compared with the rest of the HCV genome. Finally, some mutation of the clonal H77C genome toward a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the majority of these amino acid substitutions occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8(+) T cells. This influence of immune pressure on viral evolution appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.