Overview
The immune response (IR) plays a pivotal role in the control of hepatitis C virus (HCV) infection. As a noncytopathic virus, HCV has developed successful mechanisms for evading the IR of the host. Successful evasion can lead to chronic infection, which culminates in cirrhosis and hepatocellular carcinoma in selected individuals. The IR to HCV infection is the first line of defense against viral replication. This newsletter will review the mechanisms by which HCV activates and evades the innate and adaptive antiviral defenses of the host.
Innate immune response
Study of the early innate IR in acute HCV is limited because the disease is often asymptomatic in humans. However, the chimpanzee (the only other animal susceptible to infection with HCV) provides a useful model for the study of the acute stages of infection. In the acute phase of HCV infection, the _expression of type 1 interferon (IFN) is one of the earliest responses of the innate IR. This is demonstrated in recent studies in chimpanzees, which showed a very early increase of IFN-response genes that preceded the _expression of T-cell surface markers by several weeks.1
Type 1 IFN, which consists of IFN-alfa and IFN-beta, provides the first line of defense against viral infections in general and has various effects on infected and immune cells. The mechanisms of antiviral action of type 1 IFN are not fully understood but include the following processes:
1. Downregulation of protein synthesis of infected cells by inducing cellular protein kinase PKR (which also reacts with HCV NS5A protein)
2. Increase of the major histocompatibility complex (MHC) _expression of antigen-presenting cells and target cells
3. Inhibition of viral replication by activation of Mx proteins or 2,5 oligoadenylate synthetase–induced ribonuclease (RNAse)
4. Upregulation of the activity of natural killer (NK) cells, dendritic cells (DC), and CD8+ cells
5. Induction of cell death by activation of apoptosis-inducing molecules such as the Fas ligand (FasL) and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)
The depression of type 1 IFN production by HCV appears to be an important mechanism that leads to hepatocellular injury. This mechanism was elegantly described by Zhang et al, who demonstrated that HCV primarily suppresses intracellular IFN-alfa production via the inhibitory effects of nonstructural proteins such as NS5A on the production of interferon regulatory factor (IRF)-7.2 HCV also evades the innate IR via depression of NK cell activity by binding the E2 protein on the viral envelope to the CD81 surface protein of NK cells.3 This process leads to the inhibition of NK cell activation, cytokine synthesis, and cytotoxicity, but spares T-cell function. The crosslinking of the E2 viral protein and the CD81 surface protein also activates T cells that bear the gamma-delta form of the T-cell receptor. The role of the CD81 surface protein as an HCV receptor has been substantiated by pseudotype technology, but some other CD81-bearing cells are also known to not allow HCV entry. The selective entry of HCV into CD81-bearing cells strongly suggests the presence of an unidentified co-receptor. Although these NK cells, once infected, are cytotoxic against hepatocytes and produce the cytokines TNF-alfa, IFN-gamma, and IL-8, they do not target the HCV infected cells (although the production of these cytokines can indirectly contribute to immunologically-mediated liver injury).3
Anti-HCV antibodies usually develop within 2-8 weeks of acute HCV infection. They are directed against epitopes from all HCV proteins but do not permit discrimination between acute and chronic infection. Although antibodies directed against the hypervariable region 1 (HVR-1) of the E2 protein are associated with potential neutralizing capacity and a self-limiting infection, their contribution to viral clearance remains controversial. This controversy is heightened as viral variability is increased in this region, raising the possibility of viral escape from humoral immunity with a positive correlation to the duration of clinical viremia.4 A more heterogeneous humoral immunity against HVR-1 is more likely to be associated with chronicity, although HCV clearance may also occur in the absence of a humoral response to enveloped proteins.5
Adaptive immune response
Cellular IRs are induced by dendritic cells (DC) that present antigens to CD4+ and CD8+ T cells in the lymph nodes, which, after priming, reenter the bloodstream and target the liver. The strength of the IR is governed by the stimulatory function of the DC, which, in turn, is determined by antigen processing, MHC _expression, and co-stimulation. However, the exact role of DCs remains limited, as direct DC analysis is difficult because of their very low frequency in peripheral blood. As a result, most research evaluating the cellular IR during the last 5 years has focused on virus-specific T cells, particularly since new techniques have enabled researchers to investigate cells directly ex vivo without prolonged in vitro stimulation.
Virus-specific CD8+ T cells are the main effector cells against HCV. They destroy infected cells through the release of granzymes and perforins and the secretion of inflammatory cytokines such as TNF-alfa and IFN-gamma. Virus-specific CD4+ T cells also assist in the regulation of the cellular IR via cytokine secretion, which, in turn, exerts direct antiviral effects and promotes the induction of virus-specific antibodies. Several groups have reported an excellent correlation between a multispecific, robust, and persistent HCV-specific CD4+ and CD8+ T-cell response and viral clearance during acute HCV infection.6 Controversial evidence suggests that the CD4+ response is maintained for several years following recovery from infection, while the CD8+ response may decrease over time.7 Contributors to persistent hepatocyte injury in HCV are likely to be oligospecific CD4+ and CD8+ T cells and nonspecifically reactive cytolytic cells. In contrast to the aforementioned multispecific and persistent HCV-specific CD4+ and CD8 T-cell response, HCV persistence is not only associated with a poor CD8+ and CD4+ T cell response, but this response is often directed to a limited range of nonstructural components of HCV.8
To determine whether diminished HCV-specific T-cell responses are the cause or the consequence of persistent infection, Cox et al studied CD8 responses during acute HCV in a series of at-risk intravenous drug users.9 In contrast to the results of earlier studies, the authors reported that the magnitude of the CD8+ response early in infection did not differ significantly between those subjects who cleared the infection (21%) and the majority of subjects, who became chronic carriers. A longitudinal analysis of these patients demonstrated a progressive loss in the strength and breadth of reactivity to HCV antigens in those who developed chronic HCV. This analysis strongly suggests that impaired CD8 responses are the consequence of functional T-cell exhaustion rather than the cause of persistent infection.
An important mechanism for the inhibition of HCV replication is believed to be mediated by noncytolytic means via IFN-gamma. In a study of 5 patients who were acutely infected with HCV via needlestick injury, Thimme et al reported that HCV eradication required a combination of CD38+ CD8+ T cells (which did not produce IFN-gamma) and CD38- CD8+ T cells (which were IFN-gamma producers and, presumably, acted noncytologically).10 These investigators demonstrated that the initial presence of HCV viremia and elevated alanine aminotransferase (ALT) levels were associated with activated CD38+ CD8+ T cells that did not secrete IFN-gamma, suggesting that these cells alone were insufficient to eradicate HCV. However, a switch in the functional activity of these CD8+ T cells caused by the loss of the activation marker CD38 was later observed. These T cells (CD38- CD8+) were now able to secrete IFN-gamma. This was associated with a rapid decrease in HCV RNA viral load in concert with a strong CD4+ specific response, suggesting that HCV-specific CD4+ T cells may contribute to CD8+ maturation. Therefore, HCV resolution appears to be associated with an early IFN-gamma response by CD8+ T cells, and any impairment of such cells, either quantitative or functional, leads to chronic infection.
Interestingly, once chronic HCV infection has been established, the IFN-gamma response from CD8+ T cells (type 1 cytokine-secreting T [Tc1] cell) is often weak and inefficient.11 Type 2 cytokine-secreting CD8+ T (Tc2) cells have been reported to intervene to combat the detrimental effects of Tc1 cells.12 Longitudinal studies show that Tc1 and Tc2 responses often fluctuate in relation to disease activity; Tc2 cells dominate when ALT levels are low and Tc1 cells are present when ALT levels rise.13,14 The pivotal role of intrahepatic HCV-specific CD8+ T cells was reinforced by the 2 important studies by Thimme et al and Xe et al, which demonstrated that sustained response to IFN-alfa treatment was associated with both detectable HCV-specific cytotoxic activity of liver-derived lymphocytes and a multispecific and vigorous T-cell response in the livers of chimpanzees who cleared acute HCV infection.
The precise mechanisms by which HCV evades the IR remain to be elucidated. Several viral proteins are known to inhibit IFN production directly and also through IFN-signaling of IFN-alfa and beta receptors. For example, NS5A reduces interferon-stimulated gene _expression; the downstream signaling of the receptors by the Janus kinase signal transducer and activator of transcription [Jak-STAT] is inhibited by HCV core protein; and IFN production as a result of dsRNA binding by TLR3 is antagonized by HCV protease 3/4A, which cleaves Toll-IL-1 receptor domain containing adaptor-inducing IFN-beta (TRIF) between amino acids 372 and 373. However, the manner in which HCV suppressed responses from intracellular dsRNA was unknown until Li et al recently reported that the NS3/4A protease of HCV eliminates the IFN response by targeting a newly-identified viral protein called mitochondrial antiviral signaling protein (MAVS; also called CARDIF, IPS-1, VISA). The finding that a single amino acid substitution in MAVS is protective against cleavage by NS3A/4 is a groundbreaking advance.15 Despite this landmark study, viral persistence in HCV remains unexplained. IR evasion strategies comprise several key components, including the suppressive effects of HCV on the IR, early high-level viremia and a late effector T-cell response, diminished CD4-T cell help, the suppressive effects of T regulatory cells, complex viral mutations, and a suboptimal neutralizing antibody response. Further work is required to assess the relative contribution of each evasion strategy. Understanding of these strategies will, hopefully, provide a framework for novel therapeutics.
References
1. Bigger CB, Brasky KM, Lanford RE. DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J Virol 2001;75:7059-66.
2. Zhang T, Lin RT, Li Y, et al. Hepatitis C virus inhibits intracellular interferon alpha _expression in human hepatic cell lines. Hepatology 2005;35:1225-36.
3. Crotta S, Stilla A, Wack A, et al. Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J Exp Med 2002;195:35-41.
4. Tseng CT, Miskovsky E, Houghton M, et al. Characterization of liver T-cell receptor gamma-delta T cells obtained from individuals chronically infected with hepatitis C virus (HCV): Evidence for these T cells playing a central role in the liver pathology associated with HCV infections. Hepatology 2001;33:1312-20.
5. Zibert A, Kraas W, Ross RS, et al. Immunodominant B-cell domains of hepatitis C virus envelope proteins E1 and E2 identified during early and late time points of infection. J Hepatol 1999;30:177-84.
6. Bassett SE, Thomas DL, Brasky KM, Lanford RE. Viral persistence, antibody to E1 and E2, and hypervariable region 1 sequence stability of HCV-inoculated chimpanzees. J Virol 1999;73:1118-26.
7. Gerlach JT, Diepolder HM, Jung MC, et al. Recurrence of hepatitis C virus after loss of CD4 (+) T-cell response in acute hepatitis C. Gastroenterology 1999;117:933-41.
8. Diepolder HM, Zachoval R, Hoffman RM, et al. Possible mechanism involving T-lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus infection. Lancet 1995;346:1006-7.
9. Cox AL, Mosbruger T, Lauer GM, et al. Comprehensive analyses of CD8+ T cell responses during longitudinal study of acute human hepatitis C. Hepatology 2005;42:104-12.
10. Thimme R, Oldach D, Chang K-M, et al. Determinants of viral clearance and persistence during acute hepatitis C infection. J Exp Med 2001;194:1395-406.
11. He XS, Rehermann B, Lopez-Labrador FX, et al. Quantitative analysis of hepatitis C virus-specific CD8(+) T cells in peripheral blood and liver using peptide-MHC tetramers. Proc Nat Acad Sci USA 1999;96:5692-7.
12. Nelson DR, Marousis CG, Ohno T. Intrahepatic hepatitis C virus-specific cytotoxic T lymphocyte activity and response to interferon alfa therapy in chronic hepatitis C. Hepatology 1998;28:225-30.
13. Cooper S, Erickson AL, Adams EJ, et al. Analysis of a successful immune response against hepatitis C virus. Immunity 1999;10:439-49.
14. Herrmann E, Neumann AU, Schmidt JM, et al. Hepatitis C virus kinetics. Antivir Ther 2000;5:85-90.
15. Li X-D, Sun L, Seth RB, et al. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci USA 2005;102:17717-22.
Author Spotlight
Sandeep Mukherjee, MBBCh, MPH, FRCPC
Assistant Professor, Department of Internal Medicine
Section of Gastroenterology and Hepatology
University of Nebraska Medical Center
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