Immunity to hepatitis C virus: stunned but not defeated

Microbes and Infection 4 (2002) 57–65 www.elsevier.com/locate/micinf

Review

Immunity to hepatitis C virus: stunned but not defeated Paul Klenerman *, Michaela Lucas, Ellie Barnes, Gillian Harcourt Nuffıeld Department of Medicine, University of Oxford, Peter Medawar Building, South Parks Road, Oxford, OX1 3SY, UK

Abstract Hepatitis C virus (HCV) readily causes a persistent infection, although some individuals spontaneously control infection. ‘Successful’ immune responses appear to be multi-specific and sustained-including a major role for CD4+ T cells. Some antiviral CD8+ T cells show reduced capacity to secrete antiviral cytokines either temporarily (‘stunning’) or in the long term (‘stunting’). The co-ordination of multiple immune effector functions may be required to gain control of HCV. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: HCV; CD8+ T lymphocyte; Tetramer; CD4+ T lymphocyte; NKT cell

1. Introduction HCV is a positive-stranded RNA virus with a genetic structure similar to that of flaviviruses. It causes a disease of worldwide importance since it is estimated that over 170 million people are infected. Unlike hepatitis B virus, the infection is persistent in the majority of cases, and a significant proportion of these will go on to develop progressive fibrosis, cirrhosis, liver failure or hepatocellular carcinoma. Although treatment is available, in the form of combination therapy with interferon-alpha and ribavirin, this leads to control or eradication of the virus in less than half of those treated with common viral genotypes. However, spontaneous clearance after acute infection occurs in about one in five cases; thus natural resistance to the virus is common, unlike, for example HIV. In light of the above, from the immunological point of view there are a number of interesting issues. 1) How do patients clear HCV spontaneously, i.e. what is a protective immune response? 2) Why do induced immune responses fail to clear the virus, i.e. how does it escape? 3) Which aspects of the immune response are critical to enhance/induce in a vaccine? 4) How are immune effector cells involved in the response to treatment? 5) In those with persistent infection, what are the roles of various immune responses in determining disease progression/pathology?

* Corresponding author. Tel.: +44-1865-281885; fax: +44-1865-281236. E-mail address: [email protected] (P. Klenerman). © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 1 2 8 6 - 4 5 7 9 ( 0 1 ) 0 1 5 1 0 - 6

Since the discovery of HCV over a decade ago, some of these issues have been addressed, and the resulting data form the basis of this review. However, various problems mean that answering the question ‘what constitutes a protective immune response against HCV?’ is not possible—yet. These problems include: 1) lack of patients presenting acutely with infection; 2) variability of viral strains; 3) variable natural history and slow progression; 4) lack of cell culture models; 5) lack of assays for neutralisation; 6) compartmentalisation of immune responses in the liver; 7) weak cellular immune responses in persistent carriers; 8) highly ‘individualised’ responses—lack of consistency between individuals. For this review we introduce a conceptual framework for understanding the differences between persistent high-level virus infection versus clearance, then discuss various arms of the immune response in turn. At the end of each section the main points are summarised. Finally we return to the ‘holistic’ aspects to discuss a simple model of successful and unsuccessful anti-HCV responses.

2. Basis of antiviral immunity Antiviral immune responses comprise an enormous array of cellular types, subtypes, effector functions, and signalling/recruitment molecules; however, a simple framework is laid out in Fig. 1. A useful model to explore this framework is lymphocytic choriomeningitis virus (LCMV) in the mouse [1]. LCMV is an RNA virus, which like HCV

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Fig. 1. Players in the immune response against HCV and their roles. APC, antigen-presenting cell; NKT, natural killer T cell; NK, natural killer cell; IFN-alpha, interferon-alpha.

can set up infections which can either persist or be controlled to very low levels. Furthermore, since some strain/dose/mouse combinations are hepatotropic, acute hepatitis can occur [2]. As a model it is of limited value in understanding the pathology of chronic liver inflammation, but it is very relevant in understanding ‘correlates of protection’. LCMV infection is controlled initially by innate immune mechanisms, such as interferons. Mice lacking normal interferon-alpha and -beta pathways are highly susceptible to infection—despite having at the outset normal ‘adaptive’ immunological repertoires [3]. Thereafter, the dose, route and, importantly, the kinetics of infection play an important role in defining outcome. Delivery of viral antigen in the right context (primarily on dendritic cells within lymphoid tissue) is a key step. Induced CD8+ T lymphocytes, which recognise a limited set of viral peptides play the major role in initial control of LCMV—and the ability to lyse infected cells is generally important here. However, in the liver, suppression of replication within hepatocytes (but not Kuppfer cells) can occur without lytic activity. This is similar to work in a murine transgenic model of HBV, where the important mediators appear to be interferon-gamma and tumour necrosis factoralpha (TNF-alpha) [4]. Nevertheless, while the CD8+ lymphocytes are present within liver tissue, and antigen is expressed on hepatocytes, immune-mediated pathology can occur, whether lysis is absolutely required for suppression of virus or not. Activated T lymphocytes can cause death of infected cells (or potentially innocent ‘bystander’ cells) through release of lytic granules or by Fas/FasL interactions. The rate of generation of responses versus the rate of replication of the virus is key in determining the degree of liver pathology.

Early, vigorous responses lead to relatively little hepatitis because few infected cells are involved, whereas the same effector response, delayed by only a few days, can then lead on to fulminant hepatitis simply due to the increase in the number of targets killed [5]. Viral escape through mutation can occur during acute infection, but only under relatively special circumstances, i.e. if the CD8+ T lymphocyte responses are artificially focused on a single viral epitope—and if the viral load is also very high. In these situations the mutable RNA genome is able to throw up mutations at a rate which leads to selection of ‘escape mutants’—genomes where the particular peptide epitope recognised is mutated to prevent T cell activation [6]. Outside the acute setting (i.e. beyond the first couple of weeks of infection), CD4 and B cell compartments play a very important role in sustaining control of the virus. In mice where these responses are deficient, leading to lack of CD4 help for CD8 cells, or loss of the capacity to make IgG neutralising antibodies, viral recrudescence is inevitable [7]. Finally, in any situation where the viral load is very high, especially if the virus is widely distributed in diverse organs (including the liver) T cells (both CD8 and CD4, but more easily the former) may go through a process of ‘exhaustion’. This is related to antigen-induced cell death and deletion, although the ‘exhaustogenic mechanism’ and the ‘exhaustogenic site(s)’ are not clear [8]. This provides a framework for understanding HCV. Here the relevant issues are ‘what do the specificity, dynamics and function of the various arms of the immune response look like, and how do they correlate with outcome?’ The lessons from the LCMV model relevant for HCV may be summarised as follows: 1) dynamics of early innate antiviral responses are crucial; 2) dynamics of induced

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antiviral responses are also crucial; 3) broad vigorous responses in T cell compartment lead to initial control (not elimination); 4) broad vigorous responses in multiple compartments lead to long-term control (not elimination); 5) lytic function is important in many tissues, but not strictly in hepatocytes; 6) lytic function can cause immunopathology in hepatocytes; 7) immune escape by mutation occurs under conditions of high viral load and focused immune responses; 8) immune escape through exhaustion occurs under special conditions of high viral load and particular strains.

3. CD8+ T lymphocyte responses to HCV 3.1. A protective role? From first principles, from the LCMV model, and from lessons from other human infections such as HIV, CD8+ T lymphocytes ought to play a significant role in control of HCV. CD8+ T lymphocytes are able to recognise virally infected cells in the liver (or potentially other infected cell types)—in theory before they release new virions—and also, through secretion of antiviral cytokines, protect neighbouring cells. In HIV, there is a good deal of data linking CD8+ T lymphocyte responses with acute and long-term control of virus [9]. The situation in HCV is, however, less clear. In acute disease, CD8+ T cell responses are relatively easy to detect by a variety of methods, in both man and in primate models [10–15]. A ‘broad’ response, i.e. against multiple peptide epitopes, has been seen in situations where virus is controlled. Some of these responses are very vigorous, i.e. high numbers of virus-specific CD8+ T lymphocytes as detected by MHC class I peptide tetramers, or by functional analyses such as interferon-gamma release (ELISpot). The cells are also highly activated, as measured by surface expression of molecules such as CD38 and MHC class II. Indeed the proportion of activated cells in the

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HCV-specific compartment correlates nicely with the degree of liver inflammation as judged by alanine aminotransferase (ALT)—at least during acute disease [11]. The difficulty with attributing a protective role to CD8+ T lymphocyte responses alone is that activated responses are also seen in those who fail to clear the virus. Similar epitopes may be recognised, and at least one of these, NS3 1073-81, restricted by HLA-A2, is highly conserved ([16] and unpublished results). The important difference that seems to be emerging from various studies is that in those where virus persists, these CD8+ T cell responses are not sustained [10,11,17,18]. This is of some interest, since in other situations where viral antigen is continuously present, such as HIV, this serves to drive equivalent responses over long periods of time. However, by comparison, the responses in chronic HIV infection are relatively strong (as measured in the blood—it is possible that in the liver the enrichment may change the picture slightly) [9]. Initial papers, where in vitro restimulations were used to amplify these HCV-specific populations, led to an impression that numerous cells were devoted to HCV control [19]. However, in more recent publications, particularly those in which cytokine release is measured ex vivo, the numbers of antigen responsive cells appears to be very low [17,18]. Despite these low-level responses, it has been possible to address issues of phenotype and function. The phenotype of the cells (independent of whether virus is present or not) is as follows. They are low in CD62L and high in CD45RO (indicating a memory phenotype), low in activation markers CD69, CD38 and class II (indicating a ‘resting’ phenotype), and high in CD27 and CD28 but low in perforin (see Fig. 2) [10,20]. The meaning of the latter is not clear in terms of effector function, but represents potentially an ‘immature’ phenotype [21]. Lack of perforin in memory populations is not restricted to HCV, but is also seen in HIV and Epstein Barr virus (EBV). Interestingly, in contrast to acute disease, the level of activation of antigen-specific cells does not

Fig. 2. Example of CD8+ T lymphocyte immune response against HCV. A response against NS3 1073-81 peptide determined by use of class I peptide tetramers is shown. The FACS plot illustrates a stable response from a patient in whom virus is no longer present. The CD8+ lymphocyte population is illustrated. The cells have been stained with a class I peptide tetramer shown on the y axis, and an antibody against intracellular perforin, shown on the x axis. All tetramer-positive cells are perforin low (upper left-hand population).

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correlate with ALT, i.e. liver inflammation [11]. This may be due to compartmentalisation of the activated cells within the liver.

some situations, CD8+ responses show a reduced capacity to secrete antiviral cytokines.

3.2. Intact antiviral function?

4. CD4+ T lymphocyte responses

One point of interest is that the ability of HCV-specific cells to release antiviral cytokines appears in some cases to be lower than that of other CD8+ responses. This phenomenon, first observed in acute HCV infection, was described initially as ‘stunning’—a transient loss of function associated perhaps with overexposure to viral antigen (akin to ‘exhaustion’) [10]. However, more recent studies suggest that similar phenotypes may be sustained in the long term and even remain after antigen load is lowered [20]. The phenotype may represent some failure of the inducing environment (‘stunting’ as opposed to ‘stunning’. It is not clear whether this is organ specific (i.e. to do with particular cell subtypes presenting antigen in the liver) or virus specific (various genes in HCV such as core, could potentially have immunomodulatory functions). 3.3. Other problems in the CD8 arena? One of the issues facing those of us trying to understand immune responses to HCV is the issue of specificity. Early studies did much to define responses which could be detected in patients infected with HCV—often using predicted epitopes and then using culture techniques to identify responder populations in vitro [22,23]. Many of these responses are HLA-A2 restricted, which is of value given the prevalence of this allele in human populations. This work, has, however, led to a particular focus on these individual peptides—without clear evidence that they are representative of the response as a whole. In one closely studied patient, the HLA-A2-restricted responses, although very large, represented only two out eight total responses [11]. In other cases, where a similar approach has been used, multiple new epitopes have been observed [24]. Much more work needs to be done to try and understand what really constitutes the important responses within any one individual—how many and how much, which restriction element and how variable the peptide? This work may give us a better understanding of the true role of the overall CD8+ response—rather than just snapshots of it—over the next few years. To summarise, the CD8+ T lymphocyte responses to HCV include the following. 1) Acutely, these may be vigorous: up to 8% of CD8 T lymphocytes directed against a single epitope. 2) Multiple peptide epitopes may be recognised: up to eight. 3) The acutely induced cells are highly activated—but only transiently. 4) Expansions are seen in those who fail to clear virus as well as in those who control virus; the exact differences in terms of numbers, specificity and function are not yet clear. 5) In those who fail to clear virus, CD8+ responses become hard to detect. 6) In

4.1. A protective role? While the information on CD8 responses gives us tantalising glimpses of a protective role, the information on CD4 responses, although limited, appears to be more consistent. Like CD8+ T lymphocytes, broad and sustained responses appear to correlate with control as opposed to carriage [25–28]. This comes from studies of acute disease, but is backed up also by cross-sectional studies. Interestingly, it is also backed up by an association between particular class II genotypes—HLA DRB1*1101 in linkage disequilibrium with HLA-DQB1*0301—with control of acute infection [29]. Exactly how the class II allele may lead to protection is not clear, and this allele is not protective in all populations, although other class II associations have been made. The protective class II molecule may present particular peptides which are either highly conserved or somehow well expressed/processed/presented, for example in nonstructural proteins [30]. A recent study has picked up several of these by computer prediction, and they turned out to be well recognised [31]. Using another approach, we identified the group of patients with the appropriate haplotype to have larger numbers of CD4+ T cell responses, particularly responses to multiple epitopes [28]. Once again, the effect of liver compartmentalisation must be taken into account—it has recently been shown that an enrichment of CD4+ responses is seen in the liver, by a factor of about two-fold overall [32]. Interestingly, similar compartmentalisation was seen early after liver transplantation, although the presence of such infiltrates did not in this case correlate with outcome [33]. 4.2. Intact antiviral function? Again, outside acute disease, if virus persists, CD4 responses appear to wane—in some studies reappearance of virus correlates with disappearance of specific CD4 cells [25]. As for CD8+ responses, some of this may depend on how the responses are measured. It seems likely that some clones stop secreting interferon-gamma during acute disease and instead secrete regulatory cytokines such as IL-10. Thus ‘loss’ of responses may instead represent a switch in phenotype, a phenomenon which may in some cases result from viral mutation within CD4 epitopes [34]. In the current absence of class II tetramers for HCV, we can only speculate that the stunned or stunted CD4 cells may also exist. Reactivation of such cells by therapy (see below) indicates that they are not ‘exhausted’ or deleted, but rather in a non-functional state in the presence of high viral loads.

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4.3. Other problems in the CD4 arena Again, as for CD8+ lymphocytes, remarkably few class II restricted epitopes have been mapped. Early reports indicated that a conserved epitope in NS3 may be associated specifically with viral control, although other workers identified core as a potential target [26,35]. What is likely is that broad responses to conserved epitopes with appropriate function and dynamics are crucial, rather than a very large response to one particular peptide. Assuming that such responses are protective, perhaps in concert with CD8 responses, it is still not clear what their role is. They may act as direct antiviral effectors, assuming the target cell, or perhaps one nearby, can present peptide via class II. (This cell obviously need not be infected, e.g. a Kuppfer cell, merely picks up local or circulating antigen.) Alternatively they may act to support CD8+ responses as has been demonstrated in murine models [36]. Very likely they act to help B cell responses—in the generation or maintenance of antibodies which can block virus entry or in some way interfere with infectivity. The function of these cells at a detailed level requires further detailed analysis; perhaps in appropriate model settings, where the liver compartment can be adequately assessed. To summarise the CD4+ T lymphocyte responses to HCV: 1) multiple proteins may be targeted; 2) multiple responses are associated with clearance; 3) clearance and multiple responses show class II associations; 4) in the presence of virus, CD4+ responses become hard to detect; 5) compartmentalisation in the liver may occur; 6) the functional role of these responses has not been fully defined, although they appear to be important in control of viraemia; 7) major expansions may be seen after interferon therapy, even if they have been undetectable previously.

5. B cell responses As stressed above, even in the LCMV model, where CD8+ T cells play a central role, nothing can be achieved in the long term without B cell activity [7]. In LCMV, the demonstration of neutralisation in vitro is relatively straightforward, and this can be understood largely as blocking viral entry, even though the structures involved have not been finely resolved. The situation in HCV is much more complex, and so few data exist on which to judge the role of antiviral antibodies. Envelope protein E2 is a likely target and this is known to bind to a cellular receptor CD81 [37]. Antibodies that block the interaction between HCV E2 and CD81 have been used in an indirect assay for ‘neutralisation of binding’. Indirect evidence for a role for neutralising antibodies in acute disease comes from studies where variation in the hypervariable region of E2 was associated with viral escape and poor outcome [38]. In other words, escape (through mutation) from early neutralising antibodies may play a critical role in long-term outcome. This needs

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to be tied in with a simultaneous (dynamic) analysis of cellular immune responses in order to understand the relative roles of the immune effector arms [39]. It is also not clear what the form of the envelope proteins is on the surface of the virion, and what their exposure to potential neutralising antibodies is, in view of the fact that they are likely to be complexed with host lipoproteins.

6. Antigen presentation As hinted above, the fact that T cell function may be impaired even very early on in infection suggests that the inducing environment, i.e. the antigen-presenting cell, may be abnormal. There is some evidence that dendritic cells (DCs) can be infected by HCV, and also that expressed HCV genes may inhibit DC function—particularly a reduction in IL-12 production [40]. Even if the DCs are not themselves infected, the presence of viral antigens in circulating fluids may potentially influence function (e.g. by binding CD81). However, it is important to remember that although specific responses to HCV may be impaired, if there are problems in induction of responses to other antigens, they are quite subtle, and are not obvious clinically. Responses to EBV, for example, appear to be normal at the CD8+ level [10,20]. However, much more work needs to be done to establish the effects of HCV at the level of the DC. Also, the effects of interferon-alpha therapy on the DC in treated patients warrants attention.

7. Other subsets: innate immunity Since much of the host–virus war is played out in the battleground of the liver, it is worth considering that numerous other subsets are present in this organ [41]. There are large numbers of NK cells, gamma-delta T cells and also an unusual subset known as ‘NKT’ cells. These latter cells possess a restricted alpha–beta TCR (V alpha 24–V beta 11) combined with numerous surface markers common to NK cells (the equivalent subset in mice are NK1.1-positive T cells). Interestingly they can be triggered to produce large amounts of either pro-inflammatory or regulatory cytokines. Their role in HCV is not yet known although in a murine HBV model their activity can be antiviral. They have been observed in HCV-infected livers using CD1d-lipid tetramers (the cells recognise alpha-galactosyl ceramide presented by the non-polymorphic CD1d molecule) [42].

8. The immune response and antiviral therapy Therapy with interferon-alpha, more recently in combination with ribavirin, is the mainstay of HCV treatment. This therapy is only partially effective: although most patients become HCV RNA negative (by PCR) on treat

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Fig. 3. Additional effect of ribavirin on antiviral immune responses. V, viral load. For explanation of the experimental set-up see text. Upper left, infection of wild-type mice with high dose ‘exhaustogenic’ virus. Lower left, minimal effect of antiviral drug on the same mice. Upper right, heavy chain transgenic mice infected with the same dose of virus also show high viral loads—due in this case to escape from antibody through mutation. Lower right, additive effect of drug and antibody, with prevention of escape.

ment, many relapse over the 6 months after therapy has stopped. Clearly, this suggests that the immune responses that evolve during therapy play a role in controlling the virus immediately thereafter. Both interferon-alpha and also ribavirin can potentially affect immune induction, although interferon is likely to be the most potent biologically. However, as in acute disease, which immune responses are induced and how they might act is not understood. Some work has identified interferon-gamma-secreting CD4 responses, which emerge shortly after treatment is commenced, as an important correlate of response to treatment [43]. Others have suggested that both CD4+ and CD8+ T cell responses may show a switch to a Th1 profile by ribavirin therapy [44]. The role of neutralising antibodies arising during treatment and influencing outcome has not really been completely addressed, for the reasons stated above. However, an interesting observation from the LCMV model might be relevant. Mice transgenic for the heavy chain of an antibody directed against the envelope glycoprotein—and with neutralising capacity—show a very high level of antibody responses after viral infection. If high viral loads are present, escape occurs rapidly. In a normal mouse, such viral loads lead to exhaustion of T cell responses. Interestingly, ribavirin has anti-LCMV activity, although as for HCV, this is relatively weak on its own, and does not prevent exhaustion in the wild-type mouse. However, in the transgenic situation, it appears that such activity is sufficient to prevent escape by mutation away from the selective force of neutralising antibody [45]. Thus drugs are acting here as a fourth arm of the immune response: increasing the ‘breadth’ of antiviral activity and preventing viral escape (see Fig. 3).

In a recent cross-sectional study we observed that treatment was associated with an increase in responsiveness at the CD4+ T cell level—in both those who were successfully cleared of virus and in those who failed [28]. Interestingly in this cohort, there was a specific increase in responses to epitopes in HCV core, a finding which has been backed up by data from a more recent prospective study (E. Barnes et al., manuscript in preparation). Obviously, simply inducing responses, even those which may be sustained, is not sufficient. It is possible that some of these induced responses may be re-awakenings of dormant responses—even some against infections with other genotypes—a modern version of the phenomenon of ‘original sin’ (G. Harcourt et al., manuscript in preparation). However, as with acute infection, certain epitopes may be more ‘protective’ than others. Again, there is a weak effect of HLA class II genes to back up this idea. Since clearance of virus on therapy is associated with enhancement of cellular (and possibly humoral responses, although we do not know this yet), but is not of itself always sufficient in the long term, a potential approach against HCV might be as follows. Firstly achieve viral control with interferon-alpha, and then vaccinate at this point, with a boost at the end of treatment. It is possible that doing this, rather than relying on the host’s own efforts alone might enhance the long-term control of virus by broadening CD4+ T cell responses or inducing CD8+ T cell or B cell responses. An alternative approach might be to follow the HIV model. This arises from the contradiction that while removing high viral loads enhances immune responsiveness (and in the HCV case interferon-alpha may augment this), it also removes antigen. Viral antigen is required to promote cellular immune responses, perhaps particularly CD8 responses. Thus while on therapy, intermittent re-exposure to the autologous virus (by removing the drug cover) may lead to stepwise enhancement of immune responses and, ultimately, control even when drugs are removed [46]. Unfortunately, while this works well in those acutely infected with HIV, we do not know yet how it works in those with established infection (the usual case in HCV) and where the ‘palette’ of cellular responses still available to boost may be more limited. It does have the advantage of boosting with autologous sequences, which is a major problem in HCV across its multiple genotypes.

9. An “holistic” approach: implications for a vaccine By breaking the immune response down to its component parts we gain some ideas about the roles of various mediators, but the LCMV model teaches us that we cannot understand antiviral immunity in this way. A recent experiment confirms this view. In the absence of CD8+ T lymphocytes (let us imagine they have been exhausted or the virus has escaped), the CD4+ responses and B cell responses are markedly enhanced—due to excess antigen.

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Control over viral replication can be gained, but if CD8+ responses do not return, this is only temporary—virus exhausts CD4 T cell responses and escapes B cell responses [47,48]. Once this state is achieved, the lack of CD4 responses means it is no longer possible to generate new antiviral responses in the T or B cell compartment. To use a chess analogy, the pluripotential CD4 responses are acting as the queen, protected initially by numerous effector CD8 cells (which are more like pawns), and B cells (bishops and rooks, providing sweeping coverage of the board to keep the opposition pinned down). Once the queen is lost, however, the position becomes very difficult. This analogy may be relevant for HCV. Early innate responses, and then induced CD8 and CD4 T cell responses, probably play a key role but are subject to the dual threats of exhaustion and escape. By bringing the viral load down effectively and early, they protect themselves from these threats, but may not be sufficient to control virus in the long term without emerging antiviral antibody cover. If any one arm fails initially, the viral load increases and with it the capacity to escape and exhaust [39]. Sustained high viral loads lead to CD4 dysfunction (the fact that they are recoverable with therapy suggests they are not entirely deleted), and facilitate escape from antibodies. Much of this depends on the kinetics of the early responses in relation to viral kinetics. Underpinning all this, variation in host genes controlling, for example, interferon responses, probably plays a vital early role. In terms of a vaccine, the implication from this model is that breadth is the key: breadth of responses in the T cell compartment and breadth in terms of inducing humoral and cellular immunity. There are numerous obstacles to overcome here—notably the issue of variability. However, various genes such as core are relatively conserved and at least would provide a solid ‘cellular’ basis to underpin other responses. Much more work is needed, though, to really define what is ‘protective’ about protective responses—at the level of peptides.

10. Conclusions HCV, although small in terms of its genome, is nevertheless a complex virus. Unculturable, often unstainable, and frequently unstable, defining in clear terms the successes and failures of the immune system against it is still a difficult task. Nevertheless, progress has been made in defining the numbers, phenotype and function of some aspects of the CD4+ and CD8+ T lymphocyte responses. More detail is required, both in terms of peptides, but also function. The role of non-classical cells needs attention and the effects of the hepatic environment are not fully understood. Considering the enormous amount of time and effort that have gone into defining these factors in HIV, it is not surprising that many questions still remain unanswered for

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HCV, but those involved should learn from progress in the HIV field. The lesson from this is that painstaking mapping of responses—coupled with the new technologies available to immunologists—can ultimately make sense of some of the phenomena observed in patients, and point us in the direction of successful vaccines

Acknowledgements This work was sponsored by the Wellcome trust and also by the EU (QLK2-CT-1999-00356). We are grateful to Annie Lorton, Jane Collier and Simon Hellier for their help with clinical studies, Rodney Phillips for support in the lab, and Philip Goulder for his helpful comments and suggestions.

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