Immune control and failure in HCV infection--tipping the balance.

Epub ahead of print July 11, 2014 - doi:10.1189/jlb.4RI0214-126R

Review

Immune control and failure in HCV infection—tipping the balance Lynn B. Dustin,1 Siobhán B. Cashman,2 and Stephen M. Laidlaw2 University of Oxford, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, Oxford, United Kingdom RECEIVED FEBRUARY 28, 2014; REVISED JUNE 18, 2014; ACCEPTED JUNE 19, 2014. DOI: 10.1189/jlb.4RI0214-126R

ABSTRACT Despite the development of potent antiviral drugs, HCV remains a global health problem; global eradication is a long way off. In this review, we discuss the immune response to HCV infection and particularly, the interplay between viral strategies that delay the onset of antiviral responses and host strategies that limit or even eradicate infected cells but also contribute to pathogenesis. Although HCV can disable some cellular virus-sensing machinery, IFN-stimulated antiviral genes are induced in the infected liver. Whereas epitope evolution contributes to escape from T cell-mediated immunity, chronic high antigen load may also blunt the T cell response by activating exhaustion or tolerance mechanisms. The evasive maneuvers of HCV limit sterilizing humoral immunity through rapid evolution of decoy epitopes, epitope masking, stimulation of interfering antibodies, lipid shielding, and cell-to-cell spread. Whereas the majority of HCV infections progress to chronic hepatitis with persistent viremia, at least 20% of patients spontaneously clear the infection. Most of these are protected from reinfection, suggesting that protective immunity to HCV exists and that a prophylactic vaccine may be an achievable goal. It is therefore important that we understand the correlates of protective immunity and mechanisms of viral persistence. J. Leukoc. Biol. 96: 000 – 000; 2014.

Introduction An estimated 130 –185 million people worldwide are persistently infected with HCV [1–3]. HCV is transmitted primarily through percutaneous contact with infected blood, often

Abbreviations: DAA⫽directly acting antiviral drug, HBV⫽hepatitis B virus, HCV⫽hepatitis C virus, HVR⫽hypervariable, IFIT1⫽IFN-induced protein with tetratricopeptide repeats 1, IRES⫽internal ribosomal entry site, IRF3⫽IFNregulatory factor 3, ISG⫽IFN-stimulated gene, MAVS⫽mitochondrial antiviral signaling, MC⫽mixed cryoglobulinemia, MDA-5⫽melanoma differentiation antigen 5, nAb⫽antibodies with virus neutralization activity, PAMP⫽pathogen-associated molecular pattern, PD-1⫽programmed death-1, PKR⫽protein kinase R, poly U/UC⫽ poly uridine/uridine-cytidine, RIG-I⫽retinoic acid-induced gene-I, RIP1⫽receptor-interacting protein 1, SNP⫽single-nucleotide polymorphism, Tim-3⫽T cell Ig mucin 3, TBK1⫽TNFR-associated factor family member-associated NF-␬B activatorbinding kinase 1, TRAF⫽TNFR-associated factor, TRIF⫽Toll/IL-1R domain-containing adaptor-inducing IFN-␤, TRIM25⫽tripartite motif-containing protein 25

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through unsafe injection practices and illicit drug use; occupational, sexual, nosocomial, and vertical transmission also occur. Symptoms of acute HCV infection may be absent, mild, or nonspecific, and 50 – 80% of acute infections progress to chronicity. Many of those infected may not have symptoms for decades and thus, do not seek treatment until significant liver damage has occurred [4]. The long-term consequences of chronic HCV infection can be grave: patients may develop chronic liver disease leading to cirrhosis, hepatocellular carcinoma, and end-stage liver disease [4]. A liver transplant may be lifesaving but not curative, and reinfection of the liver graft is universal among those who harbor HCV at the time of transplant. HCV infection has surpassed HIV as a cause of death in the United States and increases the risk of early death from other causes [5, 6]. HCV/HIV coinfection is associated with accelerated liver disease, and chronic HCV infection may impact antiretroviral therapy for HIV [7]. HBV can exacerbate liver disease as a result of chronic HCV infection and vice versa [8]. Coinfection with HCV and parasites, such as Schistoma mansoni, may also lead to more rapid and severe liver disease than either pathogen alone [9]. The impacts of HCV infection on the immunopathogenesis of these and other human infections are still poorly understood and require additional study. While exciting new DAAs promise to revolutionize treatment for HCV infection [10], the virus is unlikely to disappear as a global health concern in the near future. Only a minority of patients infected with HCV has been diagnosed or treated to date [11, 12]. Barriers to treatment include lack of awareness of infection status, cost, uneven healthcare access, treatment contraindications, and concerns about side-effects. HCV treatment requires considerable investment of time by overburdened healthcare practitioners [13]. In industrialized countries, most incident HCV infections are in marginalized groups, such as i.v. drug users; such patients may not have regular health care, let alone access to specialists. Finally, DAAs come late in the game for the millions of patients who have

1. Correspondence: University of Oxford, Nuffield Dept. of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, Peter Medawar Bldg. for Pathogen Research, South Parks Rd., Oxford OX1 3SY, UK. E-mail: [email protected] 2. These authors provided equal contribution.

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been infected for decades. It is expected that the burden of HCV-related liver disease will continue to increase in the coming years. For all of these reasons, we cannot yet afford to declare victory. Perhaps the most likely route to conquering HCV will be through the development of an effective, widely available vaccine [14]. A major challenge to this goal is the extensive genetic diversity of HCV: there are seven major genotypes, whose nucleotide sequences vary from each other by 30% or more, and dozens of subtypes varying by at least 15% [15]. As discussed below, HCV evolves rapidly within an infected host. Vaccine development will benefit from a solid understanding of the immunological correlates of protection in those patients who clear infection without medical intervention. More than one-half of those who contract HCV develop chronic infection, and previous, spontaneous clearance does not afford universal protection from reinfection. The immune response to HCV can contribute to control of infection, and to the pathogenesis of chronic liver disease and extrahepatic diseases occurring in HCV patients.

HCV HCV is an enveloped RNA virus in the family Flaviviridae. Its genomic structure and replication mechanisms are detailed in recent reviews [16 –18]. The positive-sense ssRNA genome of HCV is ⬃9.6 kb and contains an IRES that hijacks the ribosome for cap-independent protein translation. The genome encodes a single polyprotein of ⬃3010 aa; the polyprotein is processed by host and viral proteases to yield three structural proteins (core (capsid) and the envelope glycoproteins (E1 and E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). It is believed that the structural proteins are incorporated into the viral particle with HCV genomic RNA, whereas nonstructural proteins are present in the infected cell. The RNA-dependent RNA polymerase of HCV lacks proofreading activity, and the virus exists within an infected host as a quasispecies or swarm of related viral sequences. This diversity permits rapid sequence drift and evolution under selection from host immune pressures and antiviral drugs. HCV replicates primarily in hepatocytes. Virus assembly is tightly linked to lipid and lipoprotein biosynthetic pathways, and circulating HCV behaves as a lipo-viral particle [19, 20] that usurps hepatocyte lipoprotein capture mechanisms for entry into susceptible cells [20]. Infected hepatocytes are clustered near each other, suggesting that virus spreads most efficiently in a cell-to-cell fashion [21].

INNATE IMMUNE RECOGNITION AND CONTROL OF HCV Innate antiviral mechanisms constitute the first line of defense against viral pathogens, while informing and directing the adaptive immune response [22]. The interplay between the host cell and virus at this early stage can set the stage for viral clearance or for chronic infection. As will be discussed in this section, HCV disables some innate virus-sensing and response 2 Journal of Leukocyte Biology


pathways [23, 24], yet HCV infection is clearly detected by innate mechanisms in the infected liver. HCV RNA is highly structured and replicates through a dsRNA intermediate, presenting the cell with a number of PAMPs whose recognition triggers IFN production and responses. ISGs are highly expressed in the infected liver. In the first weeks of infection, an exponential increase in HCV RNA slows abruptly with the onset of intrahepatic ISG expression; it is believed that this slowing represents ISG-dependent induction of an antiviral state that limits viral spread within the liver [25–29]. Viral replication persists, although perhaps at a reduced level, in the presence of ISG RNA and proteins. Which IFNs are produced and by which cells? Does the virus dampen IFN responses just enough to persist in the host, and does such dampening of responses explain the frequent failure of IFN-based therapies? PAMPs associated with HCV infection include the phosphorylated 5= end of the HCV RNA and the poly U/UC motif near its 3= end. Recognition of these PAMPs may be mediated by RIG-I, which with MDA-5 and Laboratory of Genetics and Physiology 2, forms a family of cytosolic RNA-binding proteins that contribute to viral recognition and antiviral responses (reviewed in ref. [30]). RIG-I has two amino-terminal caspase activation and recruitment domains, one RNA-binding helicase domain, and one carboxy-terminal regulatory domain. The carboxy-terminal domain binds RNA bearing a 5= triphosphate [31]; this binding may relax the structure of the molecule sufficiently to allow additional recognition of viral RNA bearing particular motifs, such as the poly U/UC sequence near the 3= end of the genome of HCV [32, 33]. RNA-bound RIG-I may activate TRIM25 E3 ubiquitin ligase, inducing K63-linked ubiquitination of RIG-I and activation of the MAVS protein [34]. MAVS is localized to intracellular membranes of mitochondria, mitochondrial-associated membranes near the endoplasmic reticulum, and peroxisomes; MAVS at different subcellular locations may have unique functions [23]. Activated MAVS forms prion-like aggregates that recruit TRAF2, -3, -5, and -6 [35]. These ubiquitin ligases catalyze the production of K63 polyubiquitin chains that then activate the downstream IRF3 and NF-␬B pathways required for IFN induction [35, 36] (Fig. 1A). The NS3-4A protease of HCV cleaves MAVS near its transmembrane domain and can thereby prevent MAVS-mediated stimulation of IFN induction in HCV-infected cells [37, 38]. MAVS cleavage has been observed in the HCV-infected liver [39, 40]. Importantly, it was reported recently that viral nucleocapsids containing 5= triphosphate viral RNA are recognized by RIG-I, independently of viral replication or genome translation [41]. Thus, RIG-I and MAVS could be activated before NS3-4A protein is expressed. The IRES of HCV may serve as an additional PAMP [42], but its recognition may serve to reduce antiviral responses. This RNA structure is recognized by PKR as early as 2 h postinfection, prompting a signaling cascade involving MAVS, TRAF3, and IRF3, but not RIG-I, to induce IFN-␤ and ISGs [43]. PKR phosphorylates and inactivates eukaryotic translation initiation factor-2␣. This may favor HCV replication by inhibiting host cell translation, including the translation of antiviral ISGs, but still allowing IRES-dependent translation of

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HCV RNA [42, 44]. By activating PKR, HCV may trigger ISG15 expression [43]. High ISG15 levels are associated with poor response to IFN-based HCV therapies [45, 46], and it is postulated that ISG15 promotes HCV replication by interfering with the ubiquitination reactions needed for RIG-I signaling [43] (Fig. 1A).

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Figure 1. Innate recognition of HCV RNA. These pathways are reviewed in detail in refs. [24, 36]. (A) RIG-I binds HCV RNA, resulting in a change in RIG-I conformation and the activation of E3 ubiquitin ligases (yellow shapes). Ubiquitinated RIG-I may activate MAVS, which forms prion-like aggregates that recruit additional ubiquitin ligases. Activated MAVS forms prion-like aggregates that recruit the E3 ubiquitin ligases TRAF2/5/6 and TRAF3. The ubiquitin ligases catalyze the production of K63 polyubiquitin chains and recruitment of enzymes that activate the downstream IRF3 and NF-␬B pathways. The NS3/4A protease of HCV can disable this pathway by cleaving MAVS near its transmembrane domain. ISG15, possibly induced by HCV RNA-stimulated PKR, may inhibit one or more ubiquitination steps. RNF135, ring finger protein 135; TRADD, TNFR type 1-associated death domain; NEMO, NF-␬B essential modifier; IKK, I␬B kinase. (B) TLR3 recognizes dsRNA within endosomal compartments and signals via the adaptor, TRIF, which recruits ubiquitin-conjugating enzymes, including TRAF3 and -6. These synthesize K63 polyubiquitin, which recruits enzymes that activate the IRF3 and NF-␬B transcription factors. The NS3-4A protease of HCV can disable TLR3 signaling by cleaving TRIF. PELI-1, protein pellino homolog 1; TAK, TGF ␤-activated kinase; TAB, TAKbinding protein.

TLR3 may contribute to establishment of an antiviral state through recognition of the dsRNA replication intermediate of HCV. After binding dsRNA within endosomal compartments, TLR3 activates IFN transcription via the adaptor TRIF. TRIF and K63 ubiquitin-modified TRAF3 activate TBK1, leading to IRF3 activation; TRIF also activates NF-␬B through TRAF6 and Volume 96, October 2014


RIP1 recruitment (reviewed in refs. [24, 36]; Fig. 1B). Given the localization of the RNA-binding motifs of TLR3 to the endosomal lumen— effectively, an extracellular compartment—it is unclear whether TLR3 recognizes hepatocyte infection in a cell-autonomous manner. Autophagy within an infected cell might provide a mechanism for such recognition. Alternatively, TLR3 recognition may depend on endocytic uptake of debris from neighboring infected hepatocytes [23]. Importantly, the NS3-4A protease of HCV may disable TLR3 signaling by cleaving TRIF [24, 47]. The relative impact of such cleavage on the detection of HCV infection by uninfected hepatocytes or patrolling immune cells is uncertain, as uninfected cells would not express NS3-4A. Despite its ability to interfere with MAVS and TRIF-mediated IFN induction, HCV stimulates innate antiviral pathways during acute and chronic infection. Early gene expression studies of acute HCV infection showed that an initial wave of ISG expression during acute infection slows viral replication but does not clear it [27–29]. Most studies do not report high expression of type I IFN (IFN-␣/␤) that could stimulate ISG expression [48]. More recent reports point instead to the type III or ␭ IFNs—IFNL1 (IL-29), IFNL2 (IL-28A), IFNL3 (IL-28B), and IFNL4 —which were unknown at the time of these earlier studies [27–29, 49, 50]. Patrolling dendritic cells may recognize HCV RNA in exosomes or debris from infected hepatocytes and then produce IFN-␣/␤ [51, 52] and IFN-␭ [53]. Importantly, hepatocytes themselves may express IFN-␣/␤ and IFN-␭. Studies of acute infection in primary human hepatocytes reveal IFN-␭ expression and ISG expression by HCV-infected cells, even in the presence of MAVS cleavage [54 –58]. Like IFN-␣/␤ and IFN-␥, IFN-␭ can inhibit HCV replication in hepatoma cells [59, 60] and primary liver cells [54]. HCV-infected primary liver cells produce IFN-␭ proteins at levels sufficient to limit HCV replication [54, 58]. IFN-␥ is produced later in infection by activated T and NK cells and is associated with viral clearance. Which ISGs are expressed, and how do they impact HCV replication? The ⬎300 ISGs and their antiviral functions are the subject of excellent recent reviews [24, 48, 61, 62]. IFN-␭ and the type I IFNs signal through distinct receptors and induce similar sets of ISGs, although with different kinetics [59, 63]. Among the genes induced in HCV-infected hepatocyte cultures are several ISGs with potential activity against HCV [24, 54, 57, 64]. These include oligoadenylate synthetases, whose product, 2=-5= oligoadenylate, may activate RNAse L to cleave HCV RNA; ISG20, an exonuclease; IFIT1 (ISG56), which may inhibit HCV IRES-mediated polyprotein translation; IRF1, IRF7, STAT1, RIG-I, and MDA-5, which may amplify IFN expression or response; and others, such as IFITM1, IFITM3, and radical S-adenosyl methionine domain-containing 2 (viperin), whose mechanisms of action against HCV are still incompletely understood [24]. Whereas specific, individual ISGs are able to reduce HCV replication in vitro, multiple ISGs may operate synergistically within infected cells. Why don’t IFNs and ISGs shut down HCV replication? This question may impact treatment as well, as chronically infected patients with high intrahepatic ISG expression are less likely to be cured by IFN-␣-based antiviral regimens than are those with 4 Journal of Leukocyte Biology


lower ISG expression before treatment [65, 66]. Several mechanisms may be at play. HCV-activated PKR is suggested to shut down translation of host cell ISG mRNAs without affecting HCV translation [44]. To determine whether such a mechanism operates in the infected liver, it will be necessary to define at the protein level which ISGs are expressed in infected cells and neighboring, uninfected cells. Stochastic variability in IFN responsiveness between cells may permit HCV to persist in a subpopulation of cells with reduced ISG expression [57, 67]. A number of studies have proposed that specific interactions between HCV proteins and ISGs may interfere with ISG function or with IFN signal transduction (reviewed in ref. [24]); many of these studies depended on overexpression of single HCV proteins in transfected cells and have not been confirmed in infected cells or in patients. Finally, HCV may benefit from reduced replication, as it would limit HCV antigen levels, reduce T cell recognition, prevent development of fulminant liver disease, and thereby, permit prolonged survival of host and virus. Studies documenting ISG expression in vivo during acute HCV infection [25–29] did not identify the cells responsible for IFN production or whether the infected cells themselves expressed ISGs. Importantly, ISG expression in the acutely infected liver does not predict whether the infection will clear or persist (this contrasts with chronic infection, where higher intrahepatic ISG expression correlates with poor response to IFN-␣-based antiviral therapies, as will be discussed below). In chronically infected liver cells, one study reported that ISG expression was largely undetectable in HCV RNA-positive hepatocytes [68]; however, other studies reported that ISG mRNA was readily detected in HCV-infected cells in vitro [57] and in vivo [21]. These observations suggest that viral recognition mechanisms are functional in the HCV-infected cell.

Novel IFNs, ISG expression, and IFN responsiveness Until recently, treatment for chronic HCV infection was based on pegylated IFN-␣, plus the nucleoside analog, ribavirin (reviewed in ref. [69]). This regimen effectively cleared HCV infection in approximately one-half of those treated, with significant differences in responsiveness among various patient populations and among the different viral genotypes. Whereas we would presume that IFN-␣ works, in part, through the induction of ISGs, gene-expression studies revealed that those patients who had high intrahepatic ISG expression before treatment were paradoxically less likely to benefit from IFN-␣-based antiviral therapy [65, 66]. Similarly, high blood levels of the IFN-responsive chemokine, CXCL10 (IFN-inducible protein 10), were found to correlate with poor responses to subsequent IFN-␣-based treatment (reviewed in ref. [70]). Several genome-wide association studies have identified SNPs near the IFNL3 (IL-28B) locus that are highly correlated with spontaneous and treatment-induced HCV clearance [71–74]. The mechanisms for this association are poorly understood, although it is believed that expression of IFNL3 or another gene in the region contributes to HCV control. Patients with clinically unfavorable IFNL3 genotypes had high intrahepatic ISG expression [66]. However, in vitro, HCV-infected liver cells (and uninfected adjacent cells) from donors with clinically un-

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T cells mediate protective and pathological roles in HCV infection. Self-limited, acute HCV infection is eliminated only following the appearance in the liver of T cells expressing IFN-␥ [25, 28, 29, 84, 91–93]. Successful immune responses to HCV target a diverse array of HCV epitopes, likely reducing viral escape options, and are sustained through HCV clearance and beyond. In contrast, early, broadly directed T cell responses wane in infections that progress to persistence (Fig. 2). CD4⫹ and CD8⫹ T cells are essential for resolution of infection, as demonstrated by studies in which chimpanzees that cleared HCV were reinfected after depletion of one subset or the other [94, 95]. What are the mechanisms by which T cells control HCV infection? IFN-␥ can inhibit HCV replication without killing infected cells and provides a mechanism by which limited num-

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NK cells may mediate virus control, tissue damage, and regulatory functions during HCV infection. NK cells are abundant in the normal liver [78] and although reduced in frequency, continue to make up a significant portion of the intrahepatic lymphoid compartment during HCV infection (reviewed in ref. [79]). NK cells integrate information from a diverse array of activating and inhibitory receptors [80] and must be “licensed” or educated to permit function and prevent autoreactivity [80, 81]. Evidence for an important role of NK cell function in the outcome of HCV infection includes the association of HCV spontaneous resolution [82, 83] and treatment-induced clearance [83] with genetic polymorphisms affecting NK cell-activating and inhibitory-triggering thresholds. NK cells are among the earliest immune responders to viral infection, but the roles of NK cells in control of HCV are incompletely understood [79]. These cells secrete cytokines, including IFN-␣, IFN-␥, and TNF-␣, which can inhibit viral replication, promote dendritic cell maturation, and induce production of chemokines that recruit lymphoid and inflammatory cells (reviewed in ref. [84]). NK cells can also mediate direct lysis of infected hepatocytes. NK cells may modulate adaptive immune responses by killing T cells and APCs [80]. Through early antiviral activities, NK cells may block T cell exhaustion driven by high antigen levels (reviewed in ref. [81]). NK cells are activated in patients with acute HCV infection, arguing against reports that HCV envelope proteins mediate direct inhibition of NK cell function [79, 85, 86]. Indeed, recent studies do not support the notion that the virus itself somehow inhibits NK cells [87]. Whereas there is a great deal of literature describing possible alterations in NK cell phenotype, subset distribution, and function during HCV infection, these reports are sometimes contradictory. Functional NK cell subsets include a CD56bright/ CD16negative subset that produces IFN-␥ and a CD56dim/ CD16positive subset that mediates cytotoxic activity. Patients with chronic HCV are reported to have altered ratios of cytotoxic and IFN-␥-producing subsets and increases in a functionally deficient subset of CD56negative/CD16positive NK cells [79, 88, 89]. Does this alteration represent dysfunction or a protective mechanism that limits NK-mediated liver damage? The

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hierarchies of receptors and ligands that may mediate NK cell interaction with infected hepatocytes are poorly characterized. It has not been demonstrated whether NK cells directly lyse infected hepatocytes in vivo or whether such lysis has a significant impact on viral loads. NK cells are affected by IFN-␣based antiviral treatment, and some reports demonstrate different NK responses to IFN-␣ in treatment responders versus nonresponders [90]. Whether these differences contribute to the efficacy of treatment is also not known.

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favorable IFNL3 genotypes expressed fewer ISGs and at lower levels compared with HCV-infected liver cells of the clinically favorable genotype [57]. Cells of the unfavorable IFNL3 genotypes were infected with HCV at a higher frequency [57]. A new, IFN-like gene, termed IFNL4, has been identified adjacent to IFNL3; polymorphisms tightly linked with the clinically favorable IFNL3 SNPs induce a loss-of-function mutation in IFNL4 [50]. The IFNL4 ⌬G allele, which permits IFNL4 protein expression, is associated with persistent HCV infection [75] and reduced cytolytic function of intrahepatic immune cells [76]. Recent reports suggest that IFNL4 is expressed in the HCV-infected liver [77]. More work is needed to understand whether and how IFNL4 expression could account for the association among IFNL3 genotypes, ISG expression, and IFN responsiveness.

viremia CD4 CD8


Figure 2. T cell responses in acute resolving and chronic HCV infection. (A) Patterns of viremia (HCV RNA) and liver cell death [alanine aminotransferase (ALT), which is released from damaged hepatocytes] during acute self-limiting HCV infection. (B) CD4⫹ and CD8⫹ T cells respond to multiple HCV epitopes until viremia is cleared and afterward. (C) Patterns of viremia and liver cell death in chronic infection. (D) CD4⫹ and CD8⫹ T cell responses wane as viremia persists. CD8⫹ T cells lose function as a result of exhaustion after loss of CD4⫹ T cell help. CD8⫹ T cells also select for variant virus sequences that escape immune detection.



bers of antigen-specific T cells may control infection; in contrast, cytolytic control of HCV replication is likely to depend on larger numbers of T lymphocytes [96]. A role for cytolytic activity is suggested by the correlation of T cell perforin expression and hepatocyte apoptosis, with resolution of infection in experimentally infected chimpanzees [97]. As will be discussed in this section, exhaustion of HCV-specific T cells is believed to contribute to persistent HCV infection. Cytolytic activity is lost early in the development of T cell exhaustion, whereas IFN-␥ production may survive [98]. If cytolysis of infected hepatocytes is indeed a requirement for resolution of infection, then the frequently observed “stunning” of CD8⫹ T cells, resulting in impaired proliferation, could contribute to the low rate of spontaneous clearance. Of note, adaptive immunity controls HCV infection to some extent, even in persistent infection; thus, HCV patients who have immunodeficiencies or HIV infection or undergo immunosuppressive treatment develop more severe liver disease [4]. Immunity to HCV reinfection is possible [99, 100]. Importantly, however, the presence of vigorous T cell responses after spontaneous clearance of HCV infection does not necessarily confer protection against subsequent challenge with homologous or heterologous HCV strains [101–103]. Chimpanzees experimentally inoculated with trace quantities of HCV showed induction of regulatory T cells and poor subsequent responses to HCV infection [104]. Immune responses to HCV come at a cost. HCV is not believed to be cytopathic directly [26, 105], although infected hepatocytes may have a reduced lifespan [26]. Liver damage in acute infection is attributed to T cell-mediated rather than virus-mediated damage [29, 105]. In chronic infection, much of this damage is misdirected: the bulk of inflammatory T cells in the liver does not appear to be HCV-specific [106 –108].

Failure of T cell immunity in chronic infection Adaptive immune responses arise only after weeks of infection, even in self-limiting infection (Fig. 2). Perhaps this delay is a result of inadequate innate-immune signaling due to viral strategies that reduce expression of IFNs and other cytokines. As HCV infection progresses to persistence, an initially broadly directed HCV-specific CD4⫹ and CD8⫹ T cell response weakens and narrows. It is possible that the waning of HCV-specific T cell function is, in part, an adaptive response to persistent high antigen load; reduced viral loads may permit T cells to recover their function [109]. Loss of CD4⫹ T cell help contributes to loss of CD8⫹ T cell function. The pathways leading to loss of CD4⫹ T cell help are poorly understood [110]. Whereas CD4⫹ T cell responses to HCV are difficult to identify in chronically infected patients [111, 112], they are readily observed early in the acute period— even in those who subsequently lose the responses and develop persisting infection [105, 112, 113]. Activated NK cells may facilitate viral persistence and CD4⫹ T cell failure through cytotoxic activity against activated CD4⫹ T cells [92, 114, 115]. This may benefit the host by minimizing inflammation and tissue destruction [114]. In contrast to CD8⫹ T cell responses, epitope escape appears to play a limited role in the failure of CD4⫹ T cell responses [116, 117]. Chronically evolving infections are asso6 Journal of Leukocyte Biology


ciated with reduced expression of the IL-7R␣ chain CD127 on HCV-specific CD4⫹ T cells [118]. Patients who develop persistent HCV infection have higher frequencies of Tim-3-expressing CD4⫹ T cells than those who clear infection, although this phenomenon is not limited to HCV-specific Th cells [119]. HCV-specific CD4⫹ T cells express higher levels of the inhibitory coreceptor PD-1; blockade of PD-1, TGF-␤, and IL-10 enhances T cell proliferation in vitro [120]. IL-2 supplementation is also reported to enhance the function of weakening CD4⫹ T cells [113]. In a cohort of injection drug users with acute HCV infection, increased regulatory T cell function and decreased Th17 function correlate with progression to chronicity [121]. HCV-specific CD8⫹ T cell responses fail as a result of T cell exhaustion and epitope escape. T cell exhaustion may represent an adaptive mechanism that limits immune-mediated pathologies [98]. In persistent HCV infection, the loss of effective CD4⫹ T cell help may doom HCV-specific CD8⫹ T cells to exhaustion. T cells expressing two or more inhibitory coreceptors (PD-1; 2B4; Tim-3; CTLA4; killer cell lectin-like receptor subfamily GF, member 1; or CD160) and reduced levels of the IL-7R␣ chain CD127 are readily observed in the blood and the liver as acute infection progresses to chronicity [122–125]. CD8⫹ T cells undergo exhaustion and death, at least in part, through loss of cytokine-mediated survival signals. Key cytokines involved in CD8⫹ T cell differentiation, function, and survival include the common-␥ chain receptor cytokine family members IL-2, IL-7, IL-15, and IL-21 [98, 126, 127]. Dysfunctional HCV-specific CD8⫹ T cells resemble cells undergoing cytokine withdrawal: they lose CD127 expression, gain PD-1 expression, and undergo caspase 9-mediated “death by neglect” as acute infection progresses toward persistence [128]. These cells may be rescued in vitro with IL-2, IL-7, and IL-15 supplementation [128]. Functional CD127⫹ CD8⫹ T cells are induced in chimpanzees successfully immunized with HCV [129]. Of note, IL-21 production by CD4⫹ T cells is associated with resolution of acute HCV infection [121]. The high replication rate of HCV (estimated at 1012 particles/day in an infected patient), coupled with its error-prone replication mechanism, generates a vast pool of viral sequence variants each day within an infected host [130]. T cell responses targeting an HCV epitope can and do select for variants lacking that epitope— clearing the way for viral persistence [131]. The impact of immune-mediated selection is seen in the selection of specific mutations in individuals expressing different HLA alleles [84, 106, 132–138]. Of interest, selection mediated by HLA-B alleles may be stronger than that mediated by HLA-A alleles [106]. Escape mutations may be more successful in individuals mounting a less-diverse T cell response [139, 140]. There seems to be little selective pressure for continued epitope escape in established chronic infection [141–143]. Viral sequence changes during infection represent multiple selective forces. HCV evolves within each new host to maximize its potential to replicate while avoiding immune-mediated clearance. Viable mutations are those that confer immunological escape yet retain replicative fitness [133, 144 –148]. Infection of a new host may impose a bottleneck on viral diversity

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as a result of a founder effect. In many cases, sequence changes restore consensus polypeptide sequences for a given HCV genotype [149]; this may represent a release of immunological pressure from the donor host. The host immune response selects for viral sequences that destroy immune recognition. However, viral evolution is constrained by the need to retain replicative fitness [146, 147, 150]. Some viral sequence changes that could mediate immunological escape are not tolerated, as they reduce viral-replicative fitness; T cell responses targeting these epitopes may be blunted by exhaustion and deletion [151, 152], as discussed in the previous paragraphs. Reductions in immune pressure may be associated with reversion of escape mutations [150]. Conversely, exhausted T cells may be released from their exhausted state by loss of the epitope they target: viral sequence mutations that eliminate recognition by an exhausted T cell clone are associated with functional recovery of those T cells [153].

EIA nAb Time (months//years)

Figure 3. Humoral immune responses in acute resolving and chronic HCV infection. (A) Rapid development of HCV nAb may contribute to spontaneous resolution of infection. Antibodies to HCV structural and nonstructural proteins can be detected by enzyme-linked immunoassay (EIA). Antibody levels may decline after infection is cleared. (B) Slower development of nAb responses may predispose to chronic infection. Antibody to structural and nonstructural proteins is detectable by EIA.

Caution: therapeutic reversal of T cell exhaustion? The discovery of exhausted, anergic, HCV-specific T cells in patients with chronic HCV infection raises the question of whether it is possible or desirable to reverse T cell exhaustion to permit immune control of the virus. Blockade of PD-1 during chronic lymphocytic choriomeningitis virus infection can restore immune-mediated control; however, PD-1 signaling also protects the body from immune-mediated tissue damage and death [154 –156]. Also important, most intrahepatic T cells express at least some inhibitory receptors, even in normal liver [125], and PD-1 is also expressed on HCV-specific memory CD8⫹ T cells after clearance of infection [157]. PD-1 blockade may not be sufficient to reverse T cell exhaustion [158] or restore immune control of HCV in an established persistent infection [159]. Blockade of Tim-3 [119, 121], CTLA-4 [160], and/or 2B4 [125] may also hold some immunotherapeutic promise.

ROLES OF B CELLS AND ANTIBODIES The roles of the humoral immune response in control and clearance of HCV infection are incompletely understood. Like T cell responses, antibody responses to acute HCV infection are delayed for weeks after the onset of viremia [161, 162] (Fig. 3). Clearance of acute HCV infection can occur in the absence of a detectable HCV-stimulated antibody response [105, 163–167]. Importantly, antibody responses wane after recovery in many patients [109, 168 –170]. Antibody responses target structural and nonstructural proteins. Antibodies targeting nonstructural proteins are useful for diagnosis and may contribute to opsonization, clearance of debris from infected cells, and inflammation, but it is unclear whether such antibodies can recognize virions or intact, infected cells. nAb, targeting E2 and to a lesser extent, E1, are readily detected in most persistently infected patients [166, 171, 172]. Acutely infected patients who will clear infection without treatment may rapidly develop broadly cross-reactive (effective against multiple HCV genotypes) nAb, whereas early nAb responses are weak and poorly cross-reactive in patients who

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progress to chronic infection [169, 170]. Rare, spontaneous clearance of chronic infection was also reported to occur after development of a broadly reactive nAb response [109]. A broadly cross-reactive nAb response may limit viral escape options, much as a broadly directed T cell response does, by targeting highly conserved epitopes. nAb may reduce viral loads sufficiently to release T cells from exhaustion caused by excessive antigenic stimulation [109]. nAb can provide passive protection against infection, and indeed, nAb activity in bulk human Ig preparations protected recipients from HCV infection before the advent of HCV serological screening [173–176]. Most serum nAb activity in persistently infected individuals rests in an IgG fraction [166].

Why doesn’t nAb mediate sterilizing immunity? HCV escapes from sterilizing humoral immunity by rapid sequence variation, stimulating the production of interfering antibodies, masking neutralization epitopes, and likely by concealing itself within lipoviral particles. Competition between nAb and interfering, non-neutralizing antibodies can disrupt nAb function. The CD81-binding loop region of E2 is targeted by neutralizing and non-neutralizing antibodies, with the latter limiting nAb access to key neutralization epitopes [177–180]. Neutralization epitopes may be masked by extensive glycosylation (see below) [181] and by virion association with lipoprotein particles [20]. Of note, ultrastructural studies of cell culture-derived HCV showed greater surface exposure of apolipoprotein E than of E2 [182]. Finally, HCV may spread between neighboring cells in a manner that does not expose it to circulating antibody [21, 68, 183–187]. The key to an effective nAb response may lie in the epitope targeted: many antibody responses are targeted toward the HVR sequence near the amino terminus of E2, which may act as a decoy [176, 188] and a shield, concealing conserved neutralization epitopes [189]. nAb targeting the HVR are often isolate-specific. HVR sequences are highly immunogenic but are under fewer functional constraints, so the virus can quickly escape HVR-binding nAb. Continuous generation of new quaVolume 96, October 2014


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In most patients, HCV has the ability to tip the scales in its favor through delay and distraction tactics (Fig. 4). The antiviral impact of the innate immune response is minimized by viral strategies that limit induction of IFNs and may interfere with the establishment of a virus-resistant state in the liver. Adaptive immunity may be delayed, in part, because of the success of HCV in limiting levels of IFN and inflammatory cytokine expression within the infected tissue. HCV escapes adaptive immune responses through rapid sequence changes that eliminate readily recognized epitopes. Additionally, the virus benefits from host mechanisms that protect the liver from destruction mediated by overzealous T cells. HCV avoids sterilizing humoral immunity through multiple camouflage strategies, concealing neutralization epitopes behind veneers of lipid and glycan, and promoting immune recognition of

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Whereas hepatocytes are the primary target of HCV infection, extrahepatic disease involving B lymphocyte dysfunction and malignancy is common in chronic HCV patients [201, 202]. MC is the most common extrahepatic manifestation, with some reports indicating the presence of detectable cryoglobulins in approximately one-half of unselected HCV patients (reviewed in refs. [203, 204]). In MC, the intravascular deposition of immune complexes typically containing IgM rheumatoid factor, polyclonal IgG, and viral RNA elicits an inflammatory reaction that can lead to vasculitis, nephropathy, and neuropathy [203, 204]; this apparently benign lymphoproliferative condition may progress to B cell non-Hodgkin lymphoma [205]. Of particular interest, successful antiviral therapy can result in complete regression of HCV-associated lymphoma [205]. B cells expressing rheumatoid factor-like IgM are clonally expanded in HCV patients with symptomatic MC, supporting the hypothesis that MC arises through antigen-driven stimulation of specific B cell clones [206, 207]. Notably, clonally expanded B cells in HCV-associated MC and lymphomas express a highly stereotyped antigen receptor encoded by rearranged VH1-69 and V␬3–20 variable region genes in many reports [206, 208 – 213]. Other mechanisms have been proposed for the development of HCV-associated MC and non-Hodgkin lymphoma but are inconsistent with current understanding of viral structure and replication (reviewed in ref. [162]). The model proposing that the E2 envelope protein of HCV induces polyclonal B cell activation by cross-linking CD81 on B cells [214] is inconsistent with clinical observations, in that B cell activation in MC is clonal rather than polyclonal [206, 208 –213]. Furthermore, it has not been demonstrated that intact circulating lipoviral particles [20] can mediate the activation shown with rE2. Whereas it has been suggested that HCV may infect B cells [215] and thereby cause mutations or functional changes, B

OUTLOOK

pt

Antibody responses, B cells, and extrahepatic disease

cells do not express the array of entry factors (including two tight-junction proteins, Claudin-1 and Occludin) needed for HCV infection of hepatocytes [20, 162, 216]. Cell culture-derived HCV of genotype 2a and HCV pseudoparticles of various genotypes do not enter primary B cells or B cell lines [216, 217]. Furthermore, levels of HCV RNA associated with patient lymphocytes are far below one copy/cell [218 –221], suggesting inefficient replication (if any) in human lymphocytes. The observation of similar clonally expanded B cells in multiple MC patients in different studies strongly suggests that a common antigenic stimulus plays a role in development of MC. Work is ongoing to probe the roles of viral and self-antigens in driving typical MC-related clonal expansion [222]; changes in cytokine levels [223] may also contribute.

Disru

sispecies allows HCV to evade humoral immunity by generating the raw material for selection of sequences conferring nAb escape [190 –192]. Whereas nAb responses select envelope sequence variation over time, envelope sequence changes are not observed in hypogammaglobulinemic patients [193–195]. Highly conserved and functionally constrained sequences involved in entry factor binding are bound by nAb with broad (multi-isolate) activity. These epitopes are protected by the HVR [176, 188, 189, 196] and as discussed in the next paragraph, by glycosylation. The ectodomains of the E1 and E2 envelope glycoproteins of HCV are heavily glycosylated, with glycans contributing almost one-half of the ectodomains’ apparent molecular weight. The immunodominant E2 contains nine N-linked glycosylation sites conserved across all HCV genotypes (two more are conserved in most isolates); these glycans limit nAb access to E1-E2 and thus protect them from neutralization [181]. Structural analysis of E2-nAb complexes showed that heavy glycosylation can mask neutralization epitopes [196]. Glycans play essential roles in assembly and folding of the E1-E2 heterodimer and in viral entry [181, 197–199]. Removal of the glycan shield increases the sensitivity of E2 to nAb activity [200].

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Figure 4. Tipping the balance? Many factors contribute to the success of HCV as a pathogen. These include the ability of HCV to delay innate and adaptive immune responses and its strategy of tactics of delay, distraction, disruptions, decoys, and disguises. The infected host can respond with innate mechanisms that limit virus replication and adaptive immune mechanisms that can mediate sterilizing immunity. The outcome of infection is strongly influenced by host genetic polymorphisms and host mechanisms that prevent excessive tissue damage.

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decoy epitopes. Perhaps the scales are now tipping. New drugs that sharply curtail viral replication and protein expression may enable infected cells to restore effective antiviral functions. Data collected from high-throughput virus sequencing in many infected patients may permit us to fine-tune the identification of “escape-proof” viral epitopes that should be included in future vaccine candidates. Recent work on E2 structure and nAb binding provides further insights for design of immunization strategies. Whereas the HCV crisis is far from over, there is room for optimism. Finally, a detailed knowledge of how this virus persists following immune recognition may prove useful in developing therapies for other known and emerging infections.

18. 19. 20. 21.

22. 23. 24. 25.

ACKNOWLEDGMENTS

26.

This work was funded by the U.S. National Institutes of Health (grants R01AI060561 and R01AI089957).

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KEY WORDS: immune evasion 䡠 innate immunity 䡠 interferons 䡠 T lymphocytes 䡠 B lymphocytes 䡠 NK cells

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