Innate and Adaptive Immune Responses in Chronic HCV infection

Abstract

Hepatitis C virus (HCV) remains a public health problem of global importance, even in the era of potent directly-acting antiviral drugs. In this chapter, I discuss immune response to acute and chronic HCV infection. The outcome of HCV infection is influenced by viral strategies that limit or delay the initiation of innate antiviral responses. This delay may enable HCV to establish widespread infection long before the host mounts effective T and B cell responses. HCV’s genetic agility, resulting from its high rate of replication and its error prone replication mechanism, enables it to evade immune recognition. Adaptive immune responses fail to keep up with changing viral epitopes. Neutralizing antibody epitopes may be hidden by decoy structures, glycans, and lipoproteins. T cell responses fail due to changing epitope sequences and due to exhaustion, a phenomenon that may have evolved to limit immune-mediated pathology. Despite these difficulties, innate and adaptive immune mechanisms do impact HCV replication. Immune-mediated clearance of infection is possible, occurring in 20–50% of people who contract the disease. New developments raise hopes for effective immunological interventions to prevent or treat HCV infection.

1. Introduction

Although estimates vary, it is believed that between 130 million and 200 million people worldwide are persistently infected with the hepatitis C virus, HCV (1–3). There is not yet an approved prophylactic vaccine. HCV is transmitted through percutaneous contact with infected blood. In most developed countries, blood screening has virtually eliminated the risk of infection through blood and blood products, but HCV transmission remains high in developing countries and also among people who inject drugs. Occupational, nosocomial, and vertical transmission are all observed, and sexual transmission of HCV may occur in some settings. Acute HCV infection may be asymptomatic or the symptoms may be nonspecific; thus, people may not know they are infected until many years later, when significant liver damage has occurred (4). Over 20–30 years, 15–30% of those chronically infected with HCV may develop long-term complications including cirrhosis; some of those can go on to develop hepatocellular carcinoma and/or end-stage liver disease (4, 5). HCV infection is now the leading indication for liver transplantation (6). Patients who harbor HCV at the time of transplantation experience recurrent infection of the grafted liver, frequently leading to accelerated fibrosis and cirrhosis (6).

Deaths from HCV now outstrip those from HIV infection in the developed world, and HCV infection increases mortality from other causes (7, 8). HCV complicates the outcome and treatment of other infectious diseases, and other infectious diseases complicate HCV pathogenesis and treatment. Thus, an estimated 20–30% of people with HIV infection worldwide are co-infected with HCV. HIV/HCV co-infection is associated with higher HCV viral loads, increased HCV chronicity, reduced response to anti-HCV therapy, and accelerated liver damage compared to HCV-mono-infection. Co-infected patients are also more likely to suffer kidney and neurocognitive disease than are HIV-mono-infected patients, and HCV co-infection can impact antiretroviral therapy for HIV (5, 9, 10). Hepatitis B virus (HBV) can exacerbate liver disease due to persistent HCV infection, while super-infection with HCV can exacerbate liver disease due to chronic HBV infection (11). Co-infection with HCV and liver-tropic parasites such as Schistoma mansoni may also lead to more rapid and severe liver disease than either pathogen alone (12). The immunopathogenic mechanisms of co-infection are still poorly understood and require additional study.

The landscape for HCV treatment is changing rapidly, and new directly-acting antiviral (DAA) drugs offer the hope that most patients who are treated can be cured (5, 13–16). At this time, however, most patients have not been either diagnosed or treated (17, 18). Among the numerous barriers to treatment are ignorance of infection status, uneven healthcare access, concern about side effects, and high drug prices (19). In addition, antiviral treatment will not immediately reverse liver disease in the millions of patients who have been infected for decades and in whom the burden of HCV-related liver disease will continue to increase dramatically in the coming years (20).

2. The goal of a vaccine

The availability of DAAs will not eliminate HCV as a global health problem. Ultimately, an effective, widely available vaccine will be needed to curb ongoing HCV transmission (21–23). While most HCV-infected patients progress to chronic hepatitis with persistent viremia, a significant minority (20–50%) of patients mount a successful immune response to HCV, resulting in spontaneous resolution of infection; recovery rates differ depending on factors such as age, race, sex, and genetics (5, 24–28). Thus, immune mediated control is possible. Can we stimulate a successful immune response, and thus protection from HCV persistence, with a vaccine? Several challenges have hindered vaccine development work to date. HCV presents extensive genetic diversity: there are seven major genotypes, whose nucleotide sequences differ from each other by 30% or more, and dozens of subtypes differing by at least 15% (29). Recent work has demonstrated that T cell immunity to HCV is likely to be genotype or even isolate-specific, even in subjects who spontaneously resolve infection (30). The inter-genotype and inter-subtype genetic diversity is compounded by HCV’s rapid evolution within each infected host. Also important, the correlates of protection from HCV infection are still incompletely understood. Discouragingly, those fortunate people who clear HCV infection without treatment have incomplete protection from re-infection (31–33).

3. Hepatitis C virus, antiviral therapy, and sites of replication

HCV is an enveloped RNA virus in the family Flaviviridae. Its genomic structure and replication mechanisms are detailed in several excellent recent reviews (16, 34–36). HCV has a single-stranded, positive sense RNA genome of approximately 9.6 kb (Figure 1). The RNA lacks 5´ cap and 3´ poly-A structures; translation occurs via and internal ribosomal entry site (IRES) that highjacks the ribosome to enable cap-independent protein translation. HCV RNA encodes a single polyprotein of approximately 3010 amino acids, which is processed co- and post-translationally by host and viral proteases to release ten mature proteins. These proteins are Core (capsid), the envelope glycoproteins E1 and E2, and seven non-structural proteins: p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The assembled virus is believed to incorporate HCV RNA plus core, E1, and E2 (37); p7 and NS2 are not required for HCV RNA replication but are required for virus assembly and egress; NS3-5B together make up the viral replicase while also contributing to assembly (36). RNA replication proceeds through a negative-sense RNA template. HCV’s NS5B RNA-dependent RNA polymerase lacks proofreading activity. As a result, the viral RNA changes rapidly and circulates within each infected host as a quasispecies--a swarm of related viral sequences. This availability of a diverse pool of viral sequences provides the raw material for rapid evolution under selection from host immune pressures and antiviral drugs.

Figure 1.

HCV RNA and polyprotein. Conserved RNA structures in the 5’ and 3’ untranslated regions (UTR) are illustrated. The genome has a single long reading frame encoding a viral polyprotein of approximately 3000 amino acids. The polyprotein is processed co- and post-translationally by host and viral proteases to release the three structural proteins, Core, E1, and E2 (labeled in blue) and the seven non-structural proteins (labeled in red).

Until recently, the gold standard for antiviral therapy involved prolonged treatment (24–48 weeks) with injected, PEGylated IFNα preparations plus the nucleoside analog, ribavirin. The treatment was difficult to tolerate and often ineffective. DAAs have dramatically changed the landscape for HCV patients, but the new drugs are expensive. DAAs target key steps in HCV replication and assembly. A detailed discussion of this topic is beyond the scope of this chapter, but the reader is referred to a number of excellent recent reviews (5, 15, 16). The first DAAs that came to market targeted the NS3-4A protease, blocking cleavage of the polyprotein and perhaps also of cellular targets. These first-generation DAAs, including Boceprevir and Telaprevir, were most effective when administered in combination with PEGylated IFNα and ribavirin; they were not equally effective in all HCV genotypes; and viral resistance to these drugs could be achieved with a small number of mutations (this is termed a low barrier to resistance). Subsequent generations of NS3-4A protease inhibitors are expected to have broader HCV genotype coverage and a higher genetic barrier to resistance. Development of drugs targeting other HCV proteins has raised hopes for the availability of an all-oral, IFN-free treatment regimen. Drugs that target NS5A (for example, Daclatasvir and Ledipasvir) have high potency, but some have a relatively low barrier to resistance. Drugs that target the NS5B polymerase include nucleoside analogs such as Sofosbuvir, which block polymerase activity in all HCV genotypes, and non-nucleoside drugs that may act by allosteric mechanisms. The non-nucleoside drugs target specific HCV genotypes. Combinations of these drugs act synergistically to inhibit HCV replication. All-oral drug combinations targeting 2 or 3 viral targets have shown extremely high potency in clinical trials. Additional drugs, targeting host factors required for HCV replication, are also in development. Host targets of these drugs include the microRNA miR-122, cyclophilin, and molecules required in HCV entry (16).

The primary site of HCV replication is the hepatocyte. Replication in blood cells and other tissues, if it occurs, is likely to be limited by several factors; these other cell types have not yielded robust and reproducible in vitro systems for HCV study. Importantly, HCV replication is dependent on the liver-specific miRNA, miR-122 (38–40). HCV particle assembly is tightly linked to host lipid and lipoprotein biosynthetic pathways (36, 37), and circulating HCV associates with host lipoproteins. Infectious HCV behaves as a lipo-viral particle (36, 41, 42) that takes advantage of hepatocyte lipoprotein capture mechanisms to gain entry into susceptible cells (42, 43). Many of the host factors involved in HCV entry are not expressed at significant levels in blood cells (44). Virus may be taken into many cell types without productive infection. Examination of biopsy material shows that infected hepatocytes are distributed in clusters of 4–50 cells (45), suggesting that HCV may spread efficiently from an infected “founder” cell to its closest neighbors (45–47).

4. Detecting and responding to HCV: Innate antiviral responses

Virus replication generates numerous pathogen-associated molecular patterns (PAMPs), and these in turn stimulate IFN and IFN-stimulated gene (ISG) expression. ISGs mediate numerous direct antiviral effects. IFNs, ISGs, inflammatory cytokines, and other signals derived from infected cells contribute to the initiation and regulation of adaptive immune responses. HCV, like many viral pathogens, has evolved strategies to blunt the innate antiviral response (48, 49). Thus, HCV RNA replication takes place in specialized, enclosed structures that may hide PAMPs from cellular detection (36). As discussed in this section, the viral NS3-4A protease specifically targets some proteins involved in virus detection. Other viral strategies to block IFN induction and signaling have also been proposed (reviewed in (50–52)). However, as discussed below, despite HCV’s ability to disable virus-sensing mechanisms within infected cells, antiviral pathways are initiated soon after virus infection and persist in the chronically-infected liver.

4.1. Recognition of viral RNA

HCV RNA has a phosphorylated 5´ end and a poly U/UC sequence near its 3´ end. These PAMPs together facilitate HCV RNA recognition by the cytosolic RNA binding protein, retinoic acid induced gene-I (RIG-I) (53–56). RNA-bound RIG-I triggers ubiquitination reactions and the formation of signaling complexes that ultimately activate the mitochondrial antiviral signaling protein (MAVS) (57). MAVS resides on intracellular membranes of mitochondria, mitochondrial-associated membranes near the endoplasmic reticulum, and peroxisomes. When activated, MAVS forms prion-like aggregates which recruit additional ubiquitin ligase activities (58). These ubiquitin ligases catalyze the production of K63 polyubiquitin chains, which facilitates protein-protein interactions that then activate the downstream interferon response factor-3 (IRF3) and NFκB pathways required for IFN induction (58, 59) (Figure 2A). Of interest, the various subcellular locations of MAVS are associated with distinct downstream activities (48, 60). Peroxisome-associated MAVS may be key to HCV recognition in hepatocytes because of its unique ability to stimulate IFNλ gene expression (60). HCV’s NS3-4A protease cleaves MAVS, releasing it from subcellular membranes, and can thereby prevent MAVS-mediated induction of IFN expression in HCV-infected cells (61, 62). MAVS cleavage has been observed in the HCV-infected liver (63, 64). However, RIG-I can recognize incoming viral RNA as it emerges from the nucleocapsid—before any viral replication or genome translation (65). This early recognition may explain, in part, why HCV-infected cells mount innate responses despite the presence of cleaved MAVS.

Figure 2.

Innate immune activation by HCV RNA. A) RIG-I binds to PAMPs in HCV RNA and changes its conformation, activating one or more E3 ubiquitin ligases (yellow shapes). Ubiquitinated RIG-I may activate MAVS, which then forms prion-like aggregates that recruit additional ubiquitin ligases. Recent evidence suggests that MAVS on peroxisomes is particularly important for induction of IFNλ. Subsequent ubiquitination steps stimulate recruitment of enzymes that activate the downstream IRF3 and NFκB pathways. HCV’s NS3/4A protease can disable this pathway by cleaving MAVS near its transmembrane domain. B) TLR3 recognizes dsRNA within endosomal compartments, signaling via the adaptor, TRIF. TRIF stimulates ubiquitin-conjugating enzymes, resulting in recruitment of enzymes that activate the IRF3 and NFκB transcription factors. HCV’s NS3-4A protease can disable TLR3 signaling by cleaving TRIF.

HCV RNA folds into complex structures featuring numerous stem-loops that can also act as PAMPs (Figure 1). These include the internal ribosomal entry site (IRES) near the 5´ end of the HCV genome. Innate recognition of this PAMP may paradoxically reduce antiviral responses (66). As early as 2 hours post infection, HCV IRES binding to protein kinase R (PKR) reportedly stimulates a signaling cascade involving MAVS, TRAF3, and IRF3, but not RIG-I, to induce IFN-β and ISG expression (67). PKR inhibits host cell protein translation by phosphorylating and inactivating the eukaryotic translation initiation factor, eIF2α. Such inhibition may reduce the translation of antiviral ISGs, without inhibiting the (eIF2α-independent) IRES-mediated translation of HCV RNA (66, 68). By activating PKR, HCV may also trigger ISG15 expression (67). Higher ISG15 levels are associated with poor response to IFN-based HCV therapies (69, 70), and ISG15 could promote HCV replication by interfering with the ubiquitination reactions needed for RIG-I signaling (67).

HCV RNA replication entails production of a double-stranded RNA (dsRNA) intermediate; dsRNA binds and activates the endosomal Toll-like receptor 3 (TLR3). TLR3 engagement stimulates IFN gene expression via the adaptor molecule, TIR-containing adaptor inducing IFNβ (TRIF). TRIF signals via ubiquitin ligase-dependent pathways to activate IRF3 and NFκB (reviewed in (49, 59, 71)) (Figure 2B). HCV’s NS3-4A protease may disable TLR3 signaling by cleaving TRIF, potentially disabling this pathway in infected cells (49, 72). Some questions remain about the role of TLR3, and of TRIF cleavage, in innate HCV recognition. TLR3’s RNA binding domains are localized in the endosomes rather than the cytosol; thus, they may not have access to HCV PAMPs within the infected cell. TLR3 may recognize HCV RNA taken up from neighboring infected cells or possibly in a cell-autonomous manner through autophagy. It is not clear whether or how NS3-4A-mediated TRIF cleavage affects the detection of HCV RNA by bystander or immune cells, since few if any of these cells would be infected—therefore, these cells are unlikely to express NS3-4A.

4.2 IFNs and ISGs

Despite the cleavage of MAVS and TRIF, it is clear that HCV stimulates innate antiviral pathways during acute and chronic infection. In acutely infected chimpanzees, hepatic ISG expression coincides with partial control of HCV replication (73–75). The sources and types of the IFN involved were not determined in these studies (73–76). Importantly, hepatocytes can express both type I (α/β) and type III (λ) IFNs, and can respond to these a well as to type II (γ) IFN.

Recent studies have shown that hepatocytes preferentially express type III IFNs in response to HCV infection, and that this expression is observed even in the presence of MAVS cleavage (77–81). HCV-infected primary liver cells produce IFNλ protein at levels sufficient to limit HCV replication (77, 81). Single nucleotide polymorphisms (SNPs) in and near the type III IFN genes are associated with the outcome of primary HCV infection (82, 83), IFNα-based antiviral therapy (84–88), and quantitative variation in ISG expression in vivo (89, 90) and in vitro (80). The type III IFN receptor, IFNLR, is preferentially expressed in epithelial tissues including the liver parenchyma (reviewed in (87)). Type III IFNs are encoded in a cluster on the long arm of human chromosome 19 (reviewed in (87, 88)). The genes are IFNL1, encoding the protein IFNλ1 (previously IL-29); IFNL2, encoding the protein IFNλ2 (previously IL-28A), IFNL3, encoding IFNλ3 (previously IL-28B), and the more recently discovered IFNL4, encoding IFNλ4.

How does polymorphism in this region influence HCV infection? Higher intrahepatic ISG expression at baseline is paradoxically associated with reduced likelihood of response to IFNα-based therapeutic regimens (91–93). The non-protective SNPs are associated with relatively higher ISG expression in patients with HCV infection (89), but not in healthy subjects (90). Because the important SNPs were close to the IFNL3 gene, initial studies focused on their effects on IFNL3 mRNA expression (85, 86, 89, 92, 94) or stability (95). Results were inconsistent (88). In 2013, researchers identified a novel gene, now termed IFNL4, in the IFNL3 region; this gene is transiently expressed in human hepatocytes following stimulation with a double-stranded RNA PAMP (96). The protective IFNL3/ IL28B SNPs are in linkage disequilibrium with polymorphisms in the IFNL4 gene. Specifically, a protective TT allele at rs368234815 disrupts the IFNL4 open reading frame, abolishing protein expression, while the non-protective IFNL4 ΔG allele at this site results in an intact open reading frame (96). The IFNL4 ΔG allele is believed to be the ancestral allele (88). The high frequency of the TT allele in some populations suggests that these populations are descended from survivors of some (presumably infectious) event that selected against expression of IFNλ4 (88). The non-protective IFNL4 ΔG allele is associated with development of persistent HCV infection (97). A different IFNL4 polymorphism, encoding a variant with diminished IFN activity, is also associated with improvements in spontaneous and treatment-induced clearance (98). IFNL4 is more distantly related to the other members of the IFNL gene family, but may have a similar protein structure (99) and has demonstrable antiviral activity (99). Despite the strong evidence that ability to express IFNλ4 contributes to development of persistent HCV infection, increased baseline ISG expression, and reduced efficacy of IFNα-based antiviral therapy, the mechanisms by which IFNλ4 may influence these disparate outcomes remain uncertain.

In vivo, IFNs produced by other cell types also undoubtedly contribute to the hepatic ISG response. Thus, dendritic cells (DCs) patrolling the liver may produce type I (100–102) and type III (103) IFNs following recognition of HCV RNA in exosomes or debris from infected hepatocytes. Recent work suggests that a very small subset of myeloid DCs expressing BDCA3/CD141 may play a key role in production of type III IFNs in response to HCV or debris from infected cells; type III IFN production by these cells is dependent on TLR3 signaling (104–106). Liver endothelial cells may also produce IFNs and amplify local IFN responses following detection of virus or debris (107). Natural killer cells and T cells also play key roles, as discussed in later sections of this chapter. IFNγ derived from activated T cells, in particular, is associated with viral clearance.

Which ISGs are expressed, and how do they impact HCV replication? There are more than 300 ISGs, mediating diverse antiviral functions as discussed in several excellent recent reviews (49, 76, 108–110). Although the type I and type III IFNs signal through distinct receptors, they induce similar sets of ISGs (albeit with somewhat different kinetics (87, 111, 112)). Among the ISGs expressed following HCV infection, several have potential activity against HCV (49, 77, 80, 113). These include oligoadenylate synthetases (OAS1 and OAS2), which synthesize 2’-5’ oligoadenylate that can, in turn, 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; RSAD2 (viperin) which may act on HCV through its interaction with lipid droplets (114); IFITM1 and IFITM3, which may act to inhibit viral entry and fusion. While specific individual ISGs are able to reduce HCV replication in vitro, multiple ISGs likely operate synergistically within infected cells.

4.3 Why doesn’t innate immunity clear HCV infection?

Importantly, the cycle of virus entry, virus detection, ISG induction, and innate antiviral response continues throughout chronic infection. HCV and ISG RNA can be found in the same cells in vitro (80) and during chronic infection in vivo (46) (but see (47)). The observation that ISG expression in the liver correlates with a sharp drop in viremia during acute HCV infection (73–75) strongly suggests that innate mechanisms control HCV infection in vivo. These observations raise the question of why endogenous IFNs and ISGs don’t cure HCV infection. Indeed, higher ISG expression before the start of therapy is associated with a poor outcome in IFN-based antiviral therapy (91–93). High intrahepatic ISG expression is now believed to correlate with polymorphisms in the IFNλ locus (89). Stochastic variability in IFN induction and/or responsiveness between cells may provide HCV with a continuous supply of susceptible cells with reduced ISG expression (80, 115, 116). Other mechanisms may also contribute. HCV-activated PKR could shut down translation of host cell ISG mRNAs without affecting HCV translation (68); to learn whether this 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. Researchers have proposed that HCV proteins, notably core, E2, and NS5A, may interfere with IFN signal transduction or block specific ISG functions (reviewed in (48–50)); of note, however, many of these studies have depended on overexpression of single HCV proteins in transfected cells, and have not been confirmed in infected cells or in patients. Finally, reduced HCV replication may be advantageous for a persisting virus because it limits antigen display and therefore T cell recognition, thus preventing development of fulminant liver disease and extending host survival.

5. Innate immune cells in chronic HCV infection

The liver environment is rich in cells that bridge innate and adaptive immunity. Here, I will touch briefly on natural killer (NK) cells, dendritic cells (DCs), and cells in the monocyte-macrophage lineage. For a recent review on these and other innate cells in the liver, the reader is referred to (117).

5.1 NK cells

NK cells are abundant in the liver in health and during HCV infection (118, 119). NK cell activation is mediated in part by integration of information from a diverse array of activating and inhibitory receptor-ligand pairs (120). Genetic polymorphisms in the numbers and types of these receptors, and their ligands, mandate that developing NK cells must be educated or “licensed” to function in each individual (120, 121). Of note, some genetic polymorphisms that affect NK cell activation thresholds are reported to correlate with spontaneous and treatment-induced clearance of HCV (122, 123).

NK cells are among the earliest immune responders to viral infection, but the roles of NK cells in control of HCV are still poorly understood (119). They may contribute to viral control through secretion of cytokines, including IFNα, IFNγ, and TNFα, that can drive ISG expression, inhibit viral replication, promote DC maturation, and promote release of chemokines that recruit lymphoid and inflammatory cells (reviewed in (50)). NK cells may also mediate direct lysis of infected hepatocytes, but it is not known whether this activity occurs in patients, how infected cells are recognized, or whether such lysis has a significant impact on viral loads. NK cells may modulate adaptive immune responses by killing antigen-presenting cells and activated CD4⁺ T cells (120); whether this mechanism operates during HCV infection is not yet known. Through early antiviral activities, NK cells may protect T cells from exhaustion due to high antigen levels (reviewed in (121)). NK cells are activated during acute HCV infection, arguing against reports that HCV envelope proteins inhibit NK cell function (119, 124, 125). In vitro studies also refute the hypothesis that HCV somehow inhibits NK cell function (126).

There is a great deal of literature describing possible alterations in NK cell phenotype, subset distribution, and function during HCV infection, but these reports are sometimes contradictory (119, 127, 128). Altered expression of specific activating and inhibitory receptors has been expressed in some studies, but the mechanisms of these alterations are not yet certain. Functional NK cell subsets include a CD56^bright/CD16^negative subset that produces IFNγ and a CD56^dim/CD16^positive subset that mediates cytotoxic activity. Altered ratios of cytotoxic to IFNγ-producing subsets have been reported in peripheral blood NK cells in patients with persistent HCV infection. Increased levels of a functionally deficient NK subset (CD56^negative/CD16^positive) are also reported. It is unclear whether altered NK cell subset distribution represents dysfunction, specific activation in the liver environment, or a protective mechanism that limits NK-mediated liver damage.

5.2. Dendritic cells (DCs)

DC subsets (129–131) influence the outcome of HCV infection through production of IFNs and other cytokines that directly affect viral replication and influence T cell activation, and through potent antigen presentation to T cells. Of the many DC subsets, three in particular have been studied for their role in HCV infection: plasmacytoid DCs (pDCs), which produce high levels of IFNs; myeloid or classical DCs (mDCs), which play essential roles in antigen presentation; and a minor population termed mDC2, identified by the expression of BDCA3/CD141, which may be especially potent IFNλ, producers (106). DCs may change their phenotypes and functions in response to different signals, thus there may be some fluidity among DC subsets (131).

Recognition of distinct PAMPs drives DCs to produce different cytokines, and these in turn promote different downstream immune responses (reviewed in (131)). pDCs preferentially recognize HCV via TLR7 (which binds single-stranded RNA), and produce higher levels of type I IFNs. The mDC2 subset appears to preferentially recognize HCV via TLR3 sensing of double-stranded RNA, and to produce particularly high levels of type III IFNs. The IFNs can act on hepatocytes and also on myriad other cell populations. DCs activated through engagement of the endosomal nucleic acid receptors TLR3, TLR7, or TLR9 produce IL-12; this cytokine supports TH1 differentiation and subsequent competence to produce IFNγ. IL-18 and IL-27 from DCs can synergize with IL-12 to promote TH1 development. Other PAMPs, including those binding TLR2, induce IL-10, which supports TH2 differentiation. There are reports that various HCV structural and non-structural proteins stimulate TLR2 on DCs, resulting in functional changes (reviewed in (117)). Some PAMPs, notably those derived from fungi, can promote release of IL-6 and IL-23, which support TH17 differentiation. Some PAMPs induce production of IL-10, retinoic acid, and TGF-β, supporting Treg differentiation. DCs can also release IL-15, which supports T cell survival. Type I IFN, IL-12, and IL-15 derived from DCs support NK cell activation, cytotoxicity, and survival (117).

HCV’s effects on DCs are controversial. Peripheral blood DCs are reduced in number in HCV patients (reviewed in (50, 117)), but this quantitative reduction is not accompanied by functional deficit (132–134). DCs may be reduced in the blood because of accumulation in the liver (135). Patients with HCV infection do not suffer from global immunological impairment, arguing against the notion that HCV exerts a broad inhibitory effect on DCs. Some groups report that DC function is impaired or cytokine production profiles altered in HCV patients (136–138). Cell culture-produced HCV was reported to inhibit IFNα production by DCs stimulated with TLR9 ligands (139); other studied functions were unaffected, and inhibition was independent of HCV infection or replicative capacity. Disruption of DC functions has been described in various vitro systems utilizing overexpression or high doses of individual HCV proteins (reviewed in (117)); the significance of these changes in vivo is not known. Arguing against an important role for HCV non-structural proteins within DCs, I would note that it is not clear that DCs support HCV infection. DCs do not express the full complement of entry factors required for infection of hepatocytes (44). The levels of HCV RNA associated with DCs in chronically infected patients are well below one RNA copy per cell (137, 140), suggesting that at most a minor population of patient DCs support any HCV replication. DC uptake of HCV for antigen presentation occurs independently of HCV entry factors (141). DCs may capture HCV (without productive infection) in part through the C-type lectin DC-SIGN (142, 143), perhaps permitting them to deliver HCV to susceptible cells.

An important role for DC activation and function is shown in prospective studies of people exposed to HCV through illicit drug use. Subjects who mounted a successful immune response, resulting in spontaneous resolution of infection, had reduced numbers of mature pDCs and increased evidence of mDC activation, including increased cytokine production in response to a diverse array of TLR ligands, increased expression of the T cell costimulator CD86, and reduced expression of inhibitory ligands such as PD-L1 and PD-L2 (144).

5.3 Monocytes, macrophages, and Kupffer cells

Monocytes, macrophages, and Kupffer cells (macrophages resident in the hepatic sinusoids) are abundant in the liver and are thought to play key roles in hepatic inflammation (145). These cell types express a variety of receptors that can detect pathogens and PAMPs at the cell surface and following internalization (145). Receptors at the cell surface include DC-SIGN, mannose receptor, and scavenger receptors. Human Kupffer cells are reported to produce inflammatory cytokines, notably IL-1β and IL-18, following interaction with HCV or HCV-infected cells (146–149). HCV replication is not required for this effect. The roles of IL-1β and IL-18 in chronic infection are not yet well understood, but they may contribute to intrahepatic and systemic inflammation, and increase production of IFNγ by NK cells (148). Kupffer cells, monocytes, and macrophages in the liver can modulate inflammation, immunity, and fibrosis through release of a host of other cytokines, including IL-10, IL-6, IL-12, TNFa and TGFβ, as well as reactive oxygen species (145). These cells may also present antigen to T cells; their expression of costimulatory ligands such as CD86, and co-inhibitory ligands including PD-L1, PD-L2, and galectin 9 may influence subsequent T cell activation and function (145).

6. T lymphocytes in HCV infection

T cells mediate both protective and pathological roles in HCV infection. Analysis of acute resolving infections suggests that T cells may control HCV infection both through IFNγ production and by killing infected cells. Spontaneous resolution of HCV infection follows the appearance in the liver of T cells expressing IFNγ (50, 74, 75, 150–153). In successful immune responses, these cells must target a broad array of HCV epitopes in order to reduce viral immune escape. These broadly-reactive T cell responses are sustained through spontaneous HCV clearance and for years afterward. In contrast, in infections that progress to chronicity, initial broadly directed T cell responses collapse (Figure 3). Both CD4⁺ and CD8⁺ T cell subsets are essential for resolution of infection, as demonstrated by studies in which chimpanzees that had previously cleared HCV were re-infected after depletion of one subset or the other (154, 155).

Figure 3.

T cell responses in acute resolving (A–B) and chronic (C–D) HCV infection. A) Patterns of viremia (HCV RNA) and liver cell death (alanine aminotransferase, ALT, which is released from damaged hepatocytes) in spontaneous resolution of HCV infection. B) CD4+ and CD8+ T cells respond to multiple HCV epitopes until viremia is cleared and afterwards. 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 due to exhaustion after loss of CD4+ T cell help. CD8+ T cells also select for variant virus sequences that escape immune detection.

IFNγ stimulates an antiviral program in hepatocytes, inhibiting HCV replication without killing infected cells; this mechanism allows limited numbers of antigen-specific T cells to mediate control, and offers the advantage of hepatocyte survival (156). However, the data support a key role for T cell-mediated killing of infected hepatocytes. T cell perforin expression, and hepatocyte apoptosis, correlated with spontaneous clearance of HCV in experimentally infected chimpanzees (157). Unlike IFNγ-dependent mechanisms, cytolytic control of HCV replication is likely to depend on larger numbers of T lymphocytes (156). As discussed below, persistent HCV infection is associated with exhaustion of HCV-specific T cells. Cytolytic activity is lost early in the development of T cell exhaustion, while IFNγ production may persist (158). If cytolysis of infected hepatocytes is required for resolution of infection, then loss of T cell proliferative capacity, associated with “stunned” HCV-specific T cells (159), could contribute to the low rate of spontaneous clearance.

Adaptive immunity exerts control over HCV infection even in persistent infection; thus, immunosuppressive therapy, immunodeficiencies, and HIV co-infection all exacerbate HCV-driven liver disease (4). Immunity to HCV re-infection is possible (31, 160). Unfortunately, however, even vigorous T cell responses after spontaneous clearance of HCV infection do not confer complete protection against subsequent challenge with homologous or heterologous HCV strains (161–163). Indeed, chimpanzees experimentally exposed to trace quantities of HCV showed induction of regulatory T cells and poor subsequent responses to HCV infection (164).

Immune responses to HCV are not always beneficial. While infected hepatocytes may have reduced lifespan (45, 165), HCV is not thought to be directly cytopathic. T cells, rather than virus, may be responsible for liver damage in acute infection (74, 166). In chronic infection, most of the inflammatory T cells infiltrating the liver are not even specific for HCV (167–169).

6.1. What happens to T cells in persistent HCV infection?

Immune responses to HCV are slow to develop even in infections that resolve without treatment (Figure 3). This delay may be due in part to HCV’s effects on innate immune signaling, as discussed in the previous section. As HCV infection progresses to persistence, the initial, broadly directed HCV-specific CD4⁺ and CD8⁺ T cell response weakens. The number of epitopes targeted is reduced due to failure of epitope-specific T cell populations and to viral evolution. The waning of HCV-specific T cell function may be an adaptive response that reduces T cell-mediated tissue damage in conditions of persistent, high antigen load; reduced viral loads may permit T cells to recover their function (170).

6.2. CD4+ T cell failure

HCV-specific CD4⁺ T cell help is lost as infection persists, and this loss contributes to the decline in function of HCV-specific CD8⁺ T cells. The pathways leading to loss of CD4⁺ T cell help are still not understood (171). HCV-specific CD4⁺ T cells are observable during acute infections, even those that progress to chronicity (166, 172, 173). However, HCV-specific CD4⁺ T cells are scarce in established chronic infection (172, 174). Studies in mice with chronic viral infection have shown that activated NK cells may facilitate viral persistence and CD4⁺ T cell failure by killing responding CD4⁺ T cells (152, 175, 176). Whether such a mechanism contributes to loss of HCV-specific CD4⁺ T cells in humans is not yet known. HCV-specific CD4⁺ T cells have lower expression of the IL-7 receptor alpha chain, CD127, during infections that progress to chronicity compared to infections that resolve without treatment (177). Given the importance of IL-7 in memory T cell survival and turnover (178), loss of this signal may contribute to the disappearance of CD4⁺ T cell responses to HCV. Patients with chronically-evolving HCV infection were reported to have increased expression of a negative regulator, Tim-3, on CD4⁺ T cells, but this observation was not limited to HCV-reactive T cells (179). HCV-specific CD4⁺ T cells express the inhibitory co-receptor programmed death-1 (PD-1) (180, 181) during acute and chronic infection. It has been reported that blockade of PD-1, TGFβ and IL-10 (181) or IL-2 supplementation (173) may partially restore functionality of HCV-specific CD4⁺ T cells. Regulatory T cells may play a role in HCV persistence; in a cohort of injection drug users with acute HCV infection, increased regulatory T cell function and decreased Th17 function correlated with progression to chronicity (182). Importantly, it is not clear whether these changes are causes or effects of CD4⁺ T cell failure in persisting HCV infection. Eliminating HCV-specific CD4⁺ T cells might benefit the host by minimizing inflammation and tissue destruction (175). Epitope escape appears to play a limited role in the failure of CD4⁺ T cell responses (183, 184); this contrasts with the prominent role of epitope escape in the failure of CD8⁺ T cell responses, discussed below.

6.3. CD8+ T cell failure

HCV-specific CD8⁺ T cell responses fail due to T cell exhaustion and epitope escape. CD8⁺ T cell exhaustion occurs through a multi-step process in which antigen-specific cells progressively lose effector functions and reduce their expression of cytokine receptors needed for memory CD8⁺ T cell homeostatic proliferation (158). Exhaustion may be protective in persistent infections because it reduces the risk that immune responses themselves will cause pathology (158). Exhausted, HCV antigen-specific CD8⁺ T cells can be observed in the blood and liver as acute infection progresses to chronicity (185–188). Features of these cells include expression of two or more inhibitory co-receptors (such as PD-1, 2B4, Tim-3, CTLA4, KLRGF1, or CD160), and reduced expression of the IL-7 receptor α chain, CD127. IL-7, one of the common-γ chain receptor cytokine family members, supports memory CD8⁺ T cell survival, function, homeostatic and antigen-dependent proliferation (158, 189, 190). Other members of this cytokine family, IL-7, IL-15, and IL-21, also play key roles in T cell survival and function. Down-regulation of CD127 makes CD8⁺ T cells less responsive to IL-7. Extensive CD127 down-regulation during the acute phase predicts HCV persistence in experimentally infected chimpanzees (191). Of note, however, transient loss of CD127 also identifies effector T cells (192). As acute HCV infection progresses toward persistence, HCV-specific CD8⁺ T cells lose CD127 expression, gain PD-1 expression, and undergo caspase 9-mediated “death by neglect” due to loss of cytokine survival signals (193). In contrast, protective HCV-specific CD8⁺ T cells elicited by an experimental HCV vaccine expressed CD127 (194). Exhausted CD8⁺ T cells may be rescued in vitro with IL-2, IL-7 and IL-15 supplementation (193). IL-21 production by CD4⁺ T cells is associated with resolution of acute HCV infection (182).

The association of exhausted HCV-specific T cells with chronic HCV infection raises the possibility of therapeutic efforts to reverse T cell exhaustion during chronic infection (171). Blockade of PD-1 during persistent LCMV infection in mice can restore immune-mediated control; however, PD-1 signaling also protects the body from immune-mediated pathology (195–197). Also important, intrahepatic T cells typically express at least some inhibitory receptors even in the absence of viral infection (188), and HCV-specific T cells continue to express PD-1 even after spontaneous clearance of infection (198). PD-1 blockade may not be sufficient to reverse T cell exhaustion (199) or restore immune control of HCV in an established persistent infection (200). Blockade of other inhibitory co-receptors, such as Tim-3 (179, 182), CTLA-4 (201), and/or 2B4 (188) might also hold some immunotherapeutic promise. The success of any therapeutic strategy aimed at blockade of inhibitory co-receptors would, of course, be contingent on the presence and survival of some critical number of antigen-specific T cells.

6.4. Immune escape and T cell responses

Another pathway to the failure of HCV-specific T cells is immunological escape. HCV’s error-prone replication mechanism produces amino acid changes in viral proteins at a dizzying rate (202). The resulting pool of novel viral RNA sequences provides the raw material for natural selection and rapid evolution under pressures that include immune recognition. HCV must evolve within each new host to maximize its potential to replicate and minimize immune-mediated clearance (203–209). As HCV infects a new host, viral sequence diversity may initially be limited by a founder effect. Those viral sequences capable of robust replication in the new environment have a selective advantage; sequence changes conferring efficient viral spread and replication sometimes restore consensus polypeptide sequences for a given HCV genotype (210). This may also represent a release of immunological pressure from the previous (donor) host. HCV may replicate for several weeks before the acutely infected host mounts an adaptive immune response. The onset of HCV-specific T cell responses is associated with signs of hepatocyte damage and a decline in viral load, suggesting that initial T cell responses mediate selection for viral sequence changes that abolish T cell recognition (211, 212), just as antibodies mediate selection for viral sequences that abolish antibody recognition (213, 214). The impact of T cell-mediated selection is seen in the selection of specific mutations in individuals expressing different HLA alleles (50, 167, 204, 215–220). For unknown reasons, selection mediated by HLA-B alleles may be stronger than that mediated by HLA-A alleles (167). Viral immune escape may be more successful in individuals mounting a less diverse T cell response (221, 222). Sequence changes are not unlimited, however, and viral immune escape is constrained by the need to retain replicative fitness (207, 208, 223). Some viral sequence changes that could mediate immunological escape are not tolerated because they reduce viral replicative fitness; T cell responses targeting these epitopes may be blunted by exhaustion and deletion (224, 225), as discussed in the previous paragraph. Once chronic infection is established, the selective pressure for continued T cell epitope escape is apparently lost (226–228). Reductions in immune pressure, for example during pregnancy, may be associated with reversion of escape mutations (223). Conversely, some 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 (229).

7. The humoral immune response

Antibody (Ab) responses are observed in chronic HCV infection, but their roles in control of HCV remain controversial (230, 231). As observed for T cell responses, Ab responses are delayed during acute HCV infection compared to other viral infections (232, 233) (Figure 4). Clearance of acute HCV infection can occur in the absence of a detectable HCV-stimulated Ab response (166, 234–239), and Abs to HCV can wane or vanish after spontaneous or treatment-induced resolution of infection (33, 170, 240, 241). Ab responses target both structural and non-structural proteins. However, all known virus-neutralizing Ab (nAb) target the envelope glycoproteins E1 and, especially, E2. It is not clear whether Abs against non-structural proteins can recognize intact infected cells or affect HCV replication in infected cells; these Abs might contribute to clearance and opsonization of debris from infected tissue.

Figure 4.

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

Lacking robust in vitro systems to study primary HCV isolates, researchers have turned to artificial in vitro systems to address questions of Ab-mediated neutralization of HCV infection (reviewed in (242)). Many recent studies have utilized lentiviral particles pseudotyped with HCV E1E2 heterodimers (HCVpp). HCVpp deliver reporter genes to hepatoma cell lines that express the HCV entry factors, SR-BI, CD81, Claudin-1 and Occludin (43, 242). Other studies have used chimeric cell-culture-derived HCV (HCVcc), substituting the core-NS2 regions (Figure 1) from HCV isolates of interest into the laboratory workhorse JFH-1 isolate (reviewed in (242)). Sera from patients with persistent HCV infection typically contain high-titer Abs able to neutralize infection by HCVpp bearing E1E2 from at least some genotypes (231, 237, 243, 244). Of interest, nAbs that block infection by HCVpp may be poorly effective against HCVcc (245).

Numerous studies support a role for nAbs in vivo. In chimpanzees, the closest animal model for human infection, early treatment with nAbs could prevent infection, and nAb infusion reduced viral load in persistent infection (246, 247). In immunodeficient mice with chimeric human livers, HCV infection can be prevented by nAb treatment before infection (248, 249), and persistent infection can be treated with nAb to induce an apparent cure (250). Similarly, HCV can infect the liver in mice expressing human CD81 and Occludin, and in these mice, too, broadly neutralizing monoclonal Abs can provide protection if administered before infection (251, 252) and even after establishment of infection (250). Importantly, the mouse models of HCV persistence used in these studies are, by necessity, immunodeficient, highlighting the important role played by nAb.

Humans, too, benefit from HCV-specific nAbs. Acutely infected patients destined to clear infection without treatment may develop an early, broadly crossreactive (effective against multiple HCV genotypes) nAb response, while early nAb responses are weaker and less broadly crossreactive in patients who progress to chronic infection (33, 170, 241, 253–255). A rare incidence of spontaneous clearance of chronic infection was reported to follow development of a broadly-reactive nAb response (170). Many broadly crossreactive nAbs target a restricted set of epitopes within E2, and are believed to block viral binding to entry factors (reviewed in (230, 231)). Broadly crossreactive nAbs may limit viral escape options, much as a broadly directed T cell response does, by targeting highly conserved epitopes. However, a discouraging report recently showed that nAb binding to highly conserved neutralizing epitopes is vulnerable to amino acid changes in E2 at sites outside of the nAb epitope (214). Some amino acid changes of this type may have a negative impact on viral infectivity (256). nAb may reduce viral loads sufficiently to release T cells from exhaustion caused by excessive antigenic stimulation (170). nAb can provide passive protection against infection and, indeed, nAb activity in bulk human immunoglobulin preparations protected recipients from HCV infection before the advent of HCV serological screening (249, 257–259). Most serum nAb activity in persistently infected individuals rests in an IgG fraction (237). The loss of CD4+ T cells in HIV co-infection reduces HCV-specific nAb breadth and titers (260).

7.1. Escape from nAb

HCV is thought to escape from sterilizing humoral immunity by rapid sequence variation, by stimulating the production of interfering antibodies, masking neutralization epitopes, and likely by concealing itself within lipoviral particles (reviewed in (230, 231)). Competition between nAb and interfering non-neutralizing Ab may disrupt nAb function: the CD81-binding loop region of E2 is targeted by both neutralizing and non-neutralizing Abs, with some reports suggesting that the non-neutralizing Abs may limit nAb access to key neutralization epitopes (261–264). This hypothesis is not universally accepted (231, 263–268). Neutralization epitopes may be masked by extensive glycosylation (see below) (269). HCV is assembled into lipoviral particles (42), and ultrastructural studies of HCVcc showed greater surface exposure of host apolipoprotein E than of viral E2 (270). While it is thought that lipoviral particle association may protect HCV from nAb, it is difficult to test this hypothesis quantitatively. Some HCV spread occurs between adjacent cells in a fashion that is resistant to E2-specific nAb (46, 47, 271–275). This type of spread may remain sensitive to smaller Ab-like reagents including an alpaca nanobody (276). Despite the multitude of potential escape mechanism, a recent report showed nAb-dependent cure of persistent HCV infection in immunodeficient mice bearing human liver grafts (250).

Structural and functional studies of E2 have revealed that key neutralization epitopes may be concealed behind a decoy structure—the so called first hypervariable (HVR1) sequence near the amino terminus of E2 (259, 277, 278). This sequence is highly immunogenic but elicits weakly cross-reactive, isolate-specific Abs. The HVR1 sequence is under few functional constraints, and indeed E2 lacking HVR1 is functional (278). HCV readily tolerates amino acid changes that abolish recognition by HVR1-specific Abs. Several reports link E2 sequence evolution to nAb escape during chronic infection (213, 254, 279, 280). Host nAb responses lag behind the shape-shifting E2 (213, 254). That nAbs fail to neutralise the dominant viral strain at a given time, yet successfully neutralise previously dominant viral strains in the same patient, demonstrates the continued evolution and escape of the virus under selective pressure from nAbs, with the humoral immune system always, sadly, one step behind (213). Whereas nAb responses select E1E2 sequence variation over time, envelope sequence changes are not observed in hypogammaglobulinemic patients (281–283). nAb with broad, multi-isolate reactivity bind to highly conserved and functionally constrained sequences involved in entry factor binding. These epitopes are protected by the HVR (259, 277, 278, 284) and, as discussed in the next paragraph, by glycosylation.

The ectodomains of HCV’s E1 and E2 envelope glycoproteins are heavily glycosylated: glycans contribute almost half of the ectodomains’ apparent molecular weight. E2, which is more immunogenic than E1, contains nine N-linked glycosylation sites conserved across all HCV genotypes (two additional sites are conserved in most isolates). Glycans play essential roles in assembly and folding of the E1E2 heterodimer, and are required for viral entry (269, 285–287). The glycans limit nAb access to E1E2, protecting virus from neutralization (269). Structural analysis of E2-nAb complexes showed that heavy glycosylation can mask neutralization epitopes (284). Removal of the glycan shield increases HCVpp sensitivity to nAb (288).

7.2 An immune response gone wrong? Mixed cryoglobulinemia

Hepatocytes are the primary target of HCV infection, but as discussed elsewhere in this book, B lymphocyte dysfunction and malignancy lead to extrahepatic disease in many chronic HCV patients (289, 290). Mixed cryoglobulinemia (MC) is a common extrahepatic manifestation (reviewed in (291, 292)). In MC, the intravascular deposition of immune complexes, containing IgM rheumatoid factor, polyclonal IgG, and viral RNA, elicits an inflammatory reaction that can lead to vasculitis, nephropathy, and neuropathy (291, 292); this apparently benign lymphoproliferative condition may progress to B cell non-Hodgkin lymphoma (293). Successful antiviral therapy may result in complete regression of HCV-associated lymphoma (293). 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 (294, 295). Notably, many reports indicate that clonally-expanded B cells in HCV-associated MC, as well as HCV-associated non-Hodgkin lymphomas, express a stereotypical antigen receptor encoded by rearranged VH1-69 (also called VH51p1) and Vκ3-20 (also known as kv325 and A27) variable region genes (294, 296–301).

Other mechanisms have been proposed for the development of HCV-associated MC and non-Hodgkin lymphoma, but these mechanisms are not supported by our current understanding of HCV tropism, structure, and replication (reviewed in (233)). Models proposing that HCV’s E2 envelope protein induces polyclonal B cell activation by crosslinking CD81 on B cells (302) are inconsistent with clinical observations, in that B cell activation in MC is clonally restricted rather than polyclonal (294, 296–301). Furthermore, it has not been demonstrated that intact circulating lipoviral particles (42) can mediate the activation reported in vitro with high concentrations of recombinant E2. While it has been suggested that HCV may infect B cells (303), and thereby cause mutations or functional changes, B cells do not express the array of entry factors (including two tight junction proteins, Claudin-1 and Occludin) needed for HCV infection of hepatocytes (42, 44, 233). Neither cell culture-derived HCV of genotype 2a, nor HCV pseudoparticles bearing envelope glycoproteins of various genotypes, can infect primary B cells or B cell lines (44, 304). Levels of HCV RNA associated with patient lymphocytes are far below one copy per cell (140, 305–307), suggesting that replication, if any, is inefficient in human lymphocytes. Observations of similar clonally expanded B cells in multiple MC patients, in different studies from different researchers, strongly suggest 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 (308); host genetic factors (309) and changes in cytokine levels (310) may also contribute.

8. Outlook

As we move into an era in which diagnosed HCV infections can be treated effectively (although not economically), a prophylactic vaccine remains a critical unmet need (21, 22, 311, 312). Therapeutic vaccines and immune enhancement strategies may also contribute to future treatment regimes. Understanding the mechanisms of immune control and failure will be essential to the development of effective vaccines. Significant progress has been made (22). Roles for both humoral and cell-mediated immunity must be considered. A prophylactic vaccine stimulating a robust, broadly-directed (30, 313), cross-reactive (multiple genotypes) (314), and polyfunctional (194, 313) T cell response is desirable if the goal is to prevent persistent HCV infection. This will be challenging because, even in subjects who have achieved spontaneous resolution of HCV infection, immunity may be largely strain or genotype-specific. Viral epitopes that can stimulate a universally protective anti-HCV immune response have not yet been identified. A prophylactic vaccine that stimulates production of broadly reactive nAb is appealing because of its potential to block even acute HCV infection. Emerging evidence indicates that broadly crossreactive nAb can treat established infection (250), underscoring the need for inclusion of the structural proteins, E1 and E2, in vaccine candidates.