Immunology and Cell Biology (2006) 84, 239249

doi:10.1111/j.1440-1711.2006.01427.x

Review Article

Protective immunity against hepatitis C virus infection

L I S A N E L L I O T T,1,2 A N D R E W R L L OY D ,2 J O H N B Z I E G L E R 1 a n d R O S E M A RY A F F R E N C H 1,3
1

School of Womens and Childrens Health and 2Inflammatory Diseases Research Unit, School of Medical Sciences, The University of New South Wales, Sydney, New South Wales and 3Viral Immunology Group, Burnet Institute, Melbourne, Victoria, Australia
Summary There is increasing evidence that a small percentage of individuals exposed to the hepatitis C virus have the capacity to generate a strong cellular immune response against the virus and avoid persistent infection, and perhaps do so repeatedly after re-exposure. This article reviews the evidence that the responses identified in this unique group of individuals represent the protective immunity that will need to be elicited by hepatitis C virus vaccines. Key words: cellular immunity, hepatitis C virus, vaccination.

Introduction
Although protection from infection is the primary goal of preventive vaccination, in reality, protective vaccines generally seek to dramatically minimize the number of cells that become infected, rather than to absolutely prevent infection of any host cell. Most vaccines for prevention of viral infections induce strong antibody responses to neutralize viral particles before target cells can become infected, or allow rapid control of viral replication to minimize subsequent cell to cell transmission.17 These mechanisms are most effective when significant concentrations of high-affinity antibodies are generated.8,9 The use of vaccines that seek solely to elicit an antibody response currently appears inadequate to either prevent or control at least two of the major human viral pandemics, namely infections due to HIV and hepatitis C virus (HCV). Both of these viruses circulate as a complex quasispecies and are able to rapidly mutate to evade immunological control. There are currently a number of HIV and HCV vaccine candidates that seek to elicit cellular immunity, including both CD4 helper and CD8 CTL responses directed against multiple viral Ag. These candidates cannot absolutely prevent infection given that viral replication in cellular targets must be established before viral Ag can be presented to CTL, either through classical MHC class I restricted pathways or through cross-presentation. Accordingly, the goal of these vaccines is to prevent the establishment of persistent infection. The great majority of primary infections with HCV are asymptomatic.10,11 However, unlike other flaviviruses related to HCV, and unlike hepatitis B virus (HBV), HCV causes chronic infection in 5080% of adults who are exposed to it, resulting in progressive fibrotic liver injury that may ultimately lead to cirrhosis, liver failure and an increased risk of hepatocellular carcinoma.1216 Following primary infection,

viral persistence occurs in most cases despite the generation of HCV-specific antibodies and cellular immune responses, indicating that the immune response is functionally ineffective in the majority of exposed individuals. Studies in humans, and in chimpanzees, which provide the sole animal model for HCV disease, have shown that even after successful viral clearance, the immune system is often unable to prevent reinfection with either homologous or heterologous strains of HCV.1721 Therefore, in the case of HCV protective immunity may be defined as the ability of the immune system to prevent the establishment of chronic infection after exposure to HCV, whether that protection be with the first encounter or with re-exposure after previous infection episodes. Against this backdrop, recent data from studies in both humans and chimpanzees have challenged the dogma of a largely ineffective immune response and suggested that a small subset of individuals exposed to HCV, perhaps repeatedly, may develop cellular immunity against the virus, which confers protection against persistent infection. These findings may be analogous to those obtained in the context of HIV suggesting protection in unique subgroups such as the exposed, but uninfected, Kenyan sex workers.22,23 Understanding the characteristics of this immune response may provide key insights relevant to HCV vaccine development.

The primary immune response to HCV

Although the determinants of successful clearance of primary infection should not necessarily be taken as indicative of protective immunity, or of the best pathway to vaccine development, understanding the characteristics of such responses is an appropriate first step. As most episodes of primary HCV infection in humans are asymptomatic and therefore largely unavailable for intensive immunological and virological studies, important data have been obtained from studies in chimpanzees. Although this model has provided critical insights into HCV pathogenesis, an important caveat in extrapolating data to human infection is that there is a higher rate of clearance in chimpanzees.24 In addition, the limited availability and high cost of chimpanzees leads to

Correspondence: Rosemary A Ffrench, Viral Immunology Group, Burnet Institute, Commercial Road, Prahran, Vic. 3004, Australia. Email: ffrench@burnet.edu.au Received 28 September 2005; accepted 22 December 2005. 2006 The Authors Journal compilation 2006 Australasian Society for Immunology Inc.

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the publication of many studies that include sample sizes of only one or two chimpanzees.25,26

Humoral immune response

HCV-specific antibodies generally develop 28 weeks after exposure in humans and remain throughout the course of chronic infection. Antibody levels are generally low, with limited affinity maturation or isotype switching.27 In those who clear the virus, antibody levels gradually diminish in titre, with approximately 50% of individuals having no detectable antibodies by 20 years after primary infection.28,29 Although this humoral immunity may be part of an effective immune response in the minority of individuals who clear the primary infection, antibody generation is not an absolute requirement for viral clearance.3032 Studies have shown that individuals with hypogammaglobulinaemia clear the virus in proportions similar to the general population,3335 and recovery does not correlate to anti-HCV antibody titres or to the levels of antibodies directed to the envelope glycoproteins, E1 and E2,24,36,37 although it should be noted that these studies did not assess neutralizing antibodies. Both strain-specific and cross-reactive neutralizing antibodies against HCV have been identified in humans and chimpanzees.31,3842 High-titre neutralizing antibodies were able to protect chimpanzees from infection with homologous strains of HCV, but were ineffective against heterologous variants.31,40 The majority of chronically infected individuals develop cross-reactive neutralizing antibodies late in the course of primary infection, and this delay may be one of the mechanisms by which persistence occurs.38,41 Neutralizing antibodies were found in only two of seven acutely infected patients in a series of cases with primary infection, and their presence did not correlate to viral clearance,38 although the presence of neutralizing antibodies was associated with a decrease in viraemia.42 However, the presence of neutralizing antibodies in a commercial immune globulin preparation was associated with protection from HCV transmission, as infection only occurred in individuals using products that had been screened for HCV antibodies, suggesting that the exclusion had removed neutralizing antibodies.39 It is plausible that antibody development plays a role in the clearance of established viral variants, but fails to keep pace with emergent viral quasipecies.43 Persistence of an antibody response to HCV in chronic infection, like maintenance of strong CD8 responses, also depends on the presence of ongoing infection.44 It should be noted that assays used to detect neutralizing antibodies are based on pseudotyped virus and may only be able to reliably detect antibodies specific for that strain.

HCV genome have been able to detect responses in 60% of individuals with chronic infection.49 Responses in individuals with chronic infection are more narrowly directed and of a lower magnitude than those found in individuals who are able to clear the virus.49 Both CD4147,5052 and CD815359 responses appear to be required to control HCV infection. A strong, multispecific and sustained CD41 response has been associated with viral clearance,5052 with control of viraemia being inversely related to viral load.47 An increase in CD81 numbers has been associated with HCV-RNA levels5759 and effective immune control.5356 Chronic infection is associated with reduced or ineffective proliferation, antiviral cytokine production and lytic activity of CD41 and CD81 T cells.60,61 Viral clearance has been weakly associated with the class II MHC alleles DRB1 and DQB1 in a number of small ethnically restricted cohorts, suggesting that a superior ability to bind and present particular epitopes to CD41 cells may be important.6265 However, analysis of the specificity of CD81 CTL responses in 20 individuals who had cleared HCV showed that the dominant epitopes were highly varied, even among those sharing HLA alleles.49 This suggests that diverse responses directed against multiple epitopes are required to resolve primary infection. Consistent with this notion are data indicating that the emergence of CTL epitope variants early in the course of primary infection appears to contribute to HCV persistence.66

Protective immunity against HCV Reinfection

Early studies in the chimpanzee model showed that repeated exposure to heterologous and homologous strains of HCV can result in repeated episodes of acute hepatitis following each exposure, although reinfection was generally associated with reduced periods of viraemia.18,21 No difference between the humoral immune responses to heterologous and homologous challenges was identified, although cellular immune responses were not evaluated. It was concluded that natural infection does not prevent reinfection, producing early doubts about the feasibility of designing an effective HCV vaccine.18 More recent studies have shown that chimpanzees who had previously cleared HCV and were rechallenged did show an improved course of disease when compared with naive animals with primary infection. The re-exposed animals had reduced peak alanine aminotransferase levels, shorter periods of viraemia, lower peak HCV-RNA levels, as well as earlier production and higher levels of the antiviral cytokines IFN-g and TNF-a.19,20 In one study, rechallenged animals were viraemic for an average of 5 weeks, in comparison to the average of 14 weeks associated with primary infection.19 Rechallenged animals also had viral titres that were, on average, two logs lower than those recorded in the primary infection. HCVspecific cellular immune responses were detected in all animals within 2 weeks of rechallenge. Another study noted that rechallenged animals cleared the virus more rapidly despite the lack of antibodies to HCV envelope glycoproteins.20 Viral clearance rates following rechallenge in these animals have been correlated to the magnitude of the cellular immune responses generated after re-exposure. Animals with the most vigorous and sustained CD41 responses cleared viral RNA

Cellular immune response

Avigorous and broad cellular immune response early in primary infection has been associated with an increased likelihood of viral clearance,28,45,46 with responses found in 79100% of individuals who clear HCV, but in only 044% of those with chronic infection.29,47,48 However, these early assay systems predominantly used Ag based on the core and nonstructural (NS) viral protein NS3 sequences and were unable to detect responses directed against other HCV regions. In contrast, recent assays using overlapping peptides covering the entire

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rapidly, whereas animals with weak CD41 responses had persistent, low levels of circulating RNA.67 Depletion studies have shown that both CD41 and CD81 T cells are essential in preventing chronic infection after rechallenge.58,68 These findings indicate that this relative protection after re-exposure in the chimpanzee model is primarily mediated by a cellular immune response, although these studies did not assess neutralizing antibodies. Recent data in humans also suggest that individuals who have previously cleared HCV infection are less likely to develop chronic infection after re-exposure.32 A cohort of 164 injecting drug users (IDU) who had not previously been infected with HCV (i.e. who were anti-HCV antibody negative) and 98 IDU who had previously cleared HCV (i.e. who were anti-HCV positive and HCV-RNA negative) were followed for a 2 year period. New episodes of viraemia were detected in 21% of the IDU without prior infection and in 12% of IDU who had cleared an earlier HCV infection. Individuals with primary infection were 12 times more likely to develop chronic infection, and had average HCV-RNA levels two logs higher than those who became infected after having previously cleared HCV. It should be noted, however, that these findings were not confirmed in the recent analysis of reinfection episodes in a retrospective cohort study of Australian IDU.69 Nevertheless, these data suggest that the immune system is sometimes able to generate responses that are at least partially protective against establishment of chronic infection. As detailed analysis of the humoral and cellular immune responses against HCV was not undertaken in either of these studies, the characteristics of the immune responses in the setting of reinfection warrant further study. In particular, the patterns of response in those who efficiently clear the viral infection, and in those who do not, remain unclear.

infection.7479 As documented prior exposure has not always been evident in the subjects in these studies, such individuals may be referred to as being seronegative immune (Fig. 1). CD41 lymphocytes from 4 of 20 (20%) healthy, uninfected spouses of chronically infected individuals were shown to proliferate in response to recombinant HCV proteins, particularly NS3.74 The responses were maintained on repeated testing and all individuals remained antibody and RNA negative over 1 year of follow up, indicating that none were in the early stages of HCV infection. HCV-specific CD41 and CD81

Acute infection with clearance

9 10 11 12

Time (months)

Persistent infection

Exposure to HCV without apparent infection

Development of CD81 CTL responses against viral Ag is generally taken to indicate that established infection has previously occurred with processing and presentation of viral peptides through class I MHC. Accordingly, the identification of anti-HCV-specific CTL responses in the blood or liver of individuals who do not have evidence of current infection (i.e. anti-HCV antibodies, abnormal liver function tests or detectable HCV-RNA) implies that these individuals are likely to have been previously infected with HCV. The antibody-negative status may result either from seroreversion or from an initial failure to develop an antibody response in association with the primary infection. With regard to the former, long-term follow up of recipients of HCV-contaminated blood products has shown that of those who clear the primary infection, approximately 50% serorevert to become HCV antibody negative when tested by ELISA over the following decade.28,70 The serological responses to individual HCV Ag detected by immunoblot generally diminish gradually, with an indeterminate profile becoming evident over time, followed by ultimate disappearance of all serological reactivity. In a minority of cases, a failure of antibody synthesis may result from a coexisting immunodeficiency state, such as HIV infection.7173 Several cross-sectional studies of groups of seronegative individuals who are at risk of exposure to HCV have identified evidence of cellular immunity against the virus in the absence of

9 10 11 12

Time (months)

High-risk seronegative immune

Re-exposure

Time (months)

9 10 11 12

Figure 1 Three possible outcomes of hepatitis C virus (HCV) exposure. (A) Acute or self-limiting infection is associated with a strong cellular immune response, the development of HCV-specific antibodies (Ab) and viral clearance. (B) Some individuals have an ineffective cellular immune response and despite periods of viral control, they are ultimately unable to clear the virus and develop chronic disease. (C) A small percentage of individuals are exposed to the virus and develop an effective cellular immune response, resulting in viral clearance with transient viraemia and without antibody production. Periods of viraemia are shaded. Episodes of re-exposure to the virus are indicated by arrows in panel C. d, RNA; n, T-cell response; j, Ab.

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cellular immune responses were also identified in 2 of 10 laboratory personnel working in settings at risk for occupational exposure to HCV.75 It is possible that the peptide-induced CTL responses reported in these two subjects may have resulted from in vitro priming, as two rounds of prior stimulation with tetanus toxoid and IL-2 were used. However, the same two subjects (and none of the remaining eight individuals) also showed proliferative responses to several recombinant HCV proteins covering both structural and nonstructural regions. In another study, 7 of 29 (24%) seronegative individuals living with a chronically infected family member were found to have CTL responses directed against multiple HCV epitopes in a peptide-based [51Cr]-release assay using highly purified CD81 T cells.76 Limiting dilution analysis indicated that these precursor CTL were present in the circulation at very low or undetectable frequencies. However, HCV-specific effector CD81 T cells directed against both structural and nonstructural epitopes were found in four subjects (14%) from the same group when assessed by a sensitive enzyme-linked immunospot assay (ELISpot) with frequencies that were severalfold higher than those detected using the [51Cr]-release assay. Interestingly, the effector cells were confined to the CD45RO1CD28 subset, implying that these cells are continuously derived from a resting memory population and are insensitive to costimulation by APC and thus are possibly prone to apoptosis once they have carried out their functions.76 In combination, these results suggest that in some subjects subclinical HCV infection leads to establishment of immunological memory. The likely corollary of this finding is that maintenance of such T-cell responses requires repeated low-level stimulation induced by unapparent infections or prolonged retention of viral Ag in lymph nodes or elsewhere.76 Another study examined the sexual partners of 52 healthcare workers who developed HCV infection as a result of a needlestick injury. These individuals were prospectively followed for 48 weeks after their partners exposure.79 Viraemia with seroconversion was detected in 8 of 54 (15%) sexual partners, of whom 4 (50%) went on to develop chronic HCV infection. Of 44 partners without viraemia or antibody production, 14 (32%) had detectable HCV-specific cellular immune responses to recombinant proteins in a lymphocyte proliferation assay (LPA) and an IFN-g ELISpot assay. The responses were of a significantly lower magnitude and breadth than those detected in individuals who had cleared the virus, but of higher magnitude and more diverse than those individuals with chronic disease. An ELISpot assay using purified CD81 cells and HLA-A2 restricted peptides was used to assess CTL activity in A2-positive subjects. At week 48, positive responses were detected in 12 of 13 (92%) individuals with resolved infection, in 5 of 8 (63%) antibody and PCRnegative subjects, and in 4 of 13 (31%) chronically infected individuals. The HCV immunity associated with the sexual route of exposure in this report may indicate that inoculation via the mucosal route or in low dosage may preferentially promote effective immunity. This possibility is further supported by a study of babies born to HCV-infected mothers. Twenty of 28 (71%) HCV PCR-negative babies born to HCV PCR-positive mothers had proliferative responses to 38 core, E1 and NS3 peptides in a 5bromodeoxyuridine incorporation assay.80 Infants responded to a greater number of peptides, and had responses of a higher

magnitude, than their chronically infected mothers. In addition, HCV-specific cellular immune responses were also found in 13 of 127 (18%) seronegative individuals living with a chronically infected family member.81 IFN-g ELISpot responses based predominantly on CD41 T cells were detected using pools of overlapping peptides covering the core and NS3NS5b regions. Interestingly, the majority of those with responses were children of HCV-positive parents (12/13). It may be that the majority of exposed HCV PCR-negative children develop HCV-specific cellular immune responses early in life, with the frequency of these responses declining over the following decades. Studies of IDU who are likely to have been exposed to multiple HCV variants as a result of repeated sharing of contaminated injecting apparatus provide the strongest, but still circumstantial, evidence for the protective nature of these cellular immune responses. Considering the high prevalence and incidence of HCV infection among IDU82,83 and the fact that most IDU become infected within the first 23 years of initiation into injecting drug use,84 it is likely that seronegative individuals from such groups will provide key insights into potentially protective immune responses. For instance, in a cohort of 782 individuals who had been injecting drugs for more than 10 years, a small subgroup of 46 individuals (5.9%) were identified who remained seronegative, despite the fact that 12 of these 46 individuals (26%) reported injecting daily, with the majority reporting high-risk injecting practices such as injecting cocaine (n = 36; 78%) and using a shooting gallery (n = 11; 22%) in the last 6 months.85 Despite being HCV seronegative, this subgroup showed evidence of exposure to other bloodborne viruses, including hepatitis B in 29 of the 46 individuals (62%) and HIV in 11 (22%). When compared with the majority of this cohort who were HCV seropositive, the seronegative minority reported similar levels of high-risk behaviour, indicating that, at least on the basis of probabilities, some of the persistently seronegative individuals were likely to have been previously exposed to HCV. Interestingly, subsequent studies of eight individuals from this group found no evidence of HCV-specific cellular immune responses, using a recombinant vacciniaHCV CTL assay.86 In contrast, we have recently reported analysis of HCV-specific immune responses in a cohort of 38 high-risk, seronegative IDU who reported a median of 7 years of injecting drug use, with 20 episodes of injecting per month, and of whom approximately half reported sharing of injecting apparatus and half had serological markers of natural exposure to HBV.77 Effector CD81 Tcell responses were detected by HCVvaccinia-based, IFN-g ELISpot assays in 22 of 29 (76%) HCV seronegative subjects. Positive responses were associated with higher risk behaviour (P = 0.04). Despite negative anti-HCV ELISA results, 33 of 36 (92%) subjects showed faint bands against HCV NS proteins (NS3, NS4 and/or NS5) on recombinant immunoblot analysis. In 4 of 36 subjects (11%), these bands met the manufacturers criteria for a positive result, whereas results were indeterminate for 29 of 36 (81%) subjects. Given the behavioural profile of these subjects, the immune responses described are likely to have been primed and maintained by episodes of subclinical infection without classical seroconversion and may indicate a phenotype that is resistant to establishment of chronic HCV infection.

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Infection without seroconversion

Direct evidence for such a phenotype has recently been reported in both chimpanzees and humans. Transient viraemia and HCV-specific cellular immune responses were detected in two chimpanzees following sequential exposure to increasing infective HCV doses starting as low as one RNA (1) virion.87 Neither chimpanzee was anti-HCV positive after the initial infection with an estimated single virion; however, cellular immune responses were detected in PBMC both directly ex vivo and after expansion using a recombinant vacciniaHCV ELISpot assay. Comparable responses were also detected in intrahepatic lymphocytes after exposure to 1 or 10 RNA (1) virions. Increasing the dose to 100 RNA (1) virions induced both viraemia and seroconversion. This situation may be analogous to that studied in several experimental models, in which repeated or prolonged exposure to low levels of Ag has been shown to select for high-avidity T cells.8890 High-avidity T cells are associated with improved viral clearance rates in these models,91,92 which relates to the ability to initiate lysis of infected cells earlier in infection while Ag densities are low.93 In contrast, chimpanzees exposed to higher doses of virus may require both antibody and cellular immune responses for effective immune control. We have recently reported four cases of transient HCV viraemia with subsequent clearance, but without biochemical hepatitis or anti-HCV seroconversion, from a prospective cohort study of seronegative prison inmates. Subjects were screened monthly for incident infections and followed weekly from the onset of viraemia.78 After 26 months of viraemia, the subjects cleared HCV-RNA from the serum, but failed to develop anti-HCV antibodies over prolonged follow up. HCV-specific LPA and CTL responses were detected in both of the subjects who were assessed, with production of IFN-g in response to recombinant vacciniaHCV constructs also found in one. All subjects had risk factors for HCV transmission and despite repeatedly nonreactive HCV antibody ELISA results, all had weak, but consistent, serological reactivity against HCV NS proteins on immunoblot testing, including testing of stored samples collected well before the onset of viraemia. There have been previous reports of individuals who remained persistently viraemic without generating antibody responses, but these reports are limited by the use of first or second generation anti-HCV ELISA assays, which are likely to have reduced sensitivity.9497 Other reports of HIV coinfected individuals or subjects with hypogammaglobulinaemia have little relevance given the immunocompromised state.71,73 As these latter conditions were not present in the subjects in the prison study, these findings verify the occurrence of HCV infection in the absence of seroconversion in humans, and also support the evidence for the role of cellular immunity in facilitating clearance. In addition, given the high-risk profile of the inmates, the setting in which these cases occurred and the evidence for pre-existing immunity, these cases point to the likelihood that the episodes were of reinfection and to the existence of protective immunity that facilitated efficient viral clearance.

viduals who are exposed to HIV, but remain uninfected. Studies of such high-risk HIV seronegative subjects have identified HIV-specific cellular immune responses in uninfected babies born to infected mothers,98 health-care workers with occupational exposure99 and partners of HIV-positive individuals.100 The strongest evidence for protective immunity against HIV comes from sex workers in Nairobi who engaged in unprotected sex with clientele with a very high prevalence of HIV infection. These women were likely to have been exposed to numerous HIV variants, yet remained consistently uninfected.22,23 It has been hypothesized that these women may have received a low initial viral inoculum that efficiently primed a cell-mediated immune response in the absence of antibody synthesis.101 A diverse array of immune response characteristics have been implicated in this protective immunity, including a polarized Th1-cell response,102104 specific MHC class I and II alleles,105,106 CTL responses to envelope proteins and conserved peptide epitopes,100,107109 mucosal specific anti-HIV IgA production110,111 as well decreased expression of the chemokine co-receptors for viral entry, CXCR4 and CCR5.112,113 In addition, as several of these exposed, uninfected women were related, it is plausible that as yet unidentified genetic factors may have played a role.114 These immune responses may have been the result of HIV infection, but with extremely low levels of replication, as some individuals had HIV proviral DNA in resting CD41 cells at levels 104- to 106-fold lower than those in infected individuals well below the detection levels of conventional assays.115 Importantly, a number of these seronegative immune women have subsequently become infected with HIV following a period of reduction in frequency of, or an interruption to, sex work, suggesting that the protective mechanisms were maintained by repeated exposure to HIV.116 In many respects, HIV infection shares similar immunopathogenic mechanisms with HCV infection, as cellular immune responses are believed to be critical to the control of viral replication in both cases, but the viruses feature high levels of viral mutation allowing the development of escape mutants to subvert effective immune control. However, it should be noted that despite these similarities, HCV is not known to incorporate into the host genome. In addition, the viruses are generally transmitted via different routes, and hence do not generate identical patterns of immune response. HIV infection most commonly occurs via the genital mucosa and elicits CTL responses specific to mucosal virus.117 In contrast, HCV infection, which is almost exclusively transmitted from blood to blood, is associated with systemic CTL responses in the absence of mucosal CTL.118

Generation and maintenance of protective responses

It is well established in experimental systems that cross-reactivity in T-cell responses directed against two unrelated viruses can influence the generation of both the effector and memory T-cell populations. Lymphocytic choriomeningitis virus, Pichinde virus and vaccinia virus are recognized to have such cross-reactive epitopes, so that animals challenged with one virus have a rapid increase in cross-reactive clones when challenged with a second, unrelated virus.119 The initial antiviral responses appear to prime the immune system and increase clearance rates after subsequent infection with the unrelated, but crossreactive, virus.120 The phenomenon appears to be dependent

Analogy with HIV seronegative immune subjects

Cell mediated immune responses in viraemic, seronegative prisoners may well be comparable to those identified in rare indi-

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on selective reactivation of memory T cells with specificities to the earlier infection.121 These findings may be relevant to HCV, given that a cross-reactive epitope is shared between the influenza virus and the NS3 protein of HCV,122 and CD81 T cells specific for this epitope have been found in patients with acute HCV.123 In the dose-escalating reinfection study with two chimpanzees, one animal with pre-existing NS3-specific responses went on to clear the virus, whereas the other became chronically infected.87 Thus, it is plausible that individuals who have strong pre-existing responses to the crossreactive epitopes derived from influenza, or other Ag, are able to mount a more rapid and effective immune response when subsequently exposed to HCV. It is plausible that these individuals with protective immunity were able to generate an immune response directed against a critical region of the HCV genome, resulting in an increased ability to clear the virus. Studies of seronegative immune individuals have predominantly used a small set of selected Ag; hence, a comprehensive study of the specificity of these responses is needed (Table 1). However, given that no significant difference has been found between the specificity of cellular immune responses in individuals with self-limiting disease and that of individuals with chronic infection, it appears unlikely that responses directed against particular epitopes are the only mechanism responsible for protective immunity.124 Recent experimental data indicate that CD81 responses can be generated from uninfected cells, thus calling into question the dogma that the class I pathway is available only to replicating Ag. Dendritic cells are able to present Ag derived by phagocytosis of infected cells that have undergone apoptosis, thereby eliciting Ag-specific CD81 immune responses.125 This pathway may provide a mechanism by which individuals can be exposed to predominantly non-infectious HCV particles and generate HCV-specific responses without substantive viral replication being established. As an in vitro culture system to support HCV replication has only very recently been reported, very little is known about the propensity for HCV to produce defective virions. However, the key enzyme involved in replication of the virus, the RNAdependent RNA polymerase, has no proof-reading activity
Table 1 HCV-specific cellular immune responses in seronegative cohorts Risk factor for HCV exposure Spouses of chronically infected individuals74 Health-care workers75 Family members of chronically infected individuals76 Spouses of chronically infected individuals79 IDU77 IDU78 Spouses of chronically infected individuals81 Infants born to HCV PCR-positive mothers80 Assay and Ag used

and poor fidelity, with a calculated mutation rate of between 0.4 103 and 1.92 103 base substitutions per genome site per year.126 This is comparable to the mutation rate in HIV reverse transcriptase of between 103 and 104 substitutions per site.127 Thus, there is a high likelihood that defective virions are indeed produced during HCV replication in vivo. The role of ongoing Ag exposure in the maintenance of Agspecific cellular immune responses remains unresolved. It was initially argued that memory cells were only maintained by contact with trace amounts of specific Ag that remained after primary exposure.128 Several authors subsequently showed that both CD81 and CD41 memory T cells can survive in the absence of Ag.129131 However, there remains some dispute as to whether experimental evidence for Ag-independent T-cell memory provides a valid model of T-cell memory within a functioning immune system.132 It is therefore unclear whether the maintenance of HCV-specific T cells following viral clearance, or of those found in seronegative, immune individuals, requires repeated exposure to HCV Ag. The report that established HIV infection in seronegative immune sex workers may follow from a break in sex work suggests that regular Ag exposure is required to maintain HIV-specific cellular immune responses. It is plausible that a reservoir of infected cells with extremely low levels of HCV replication could remain protected from the immune system, such as within the liver, dendritic cells, PBMC or the brain.133138 Extrahepatic replication of HCV can occur in the lymph nodes, pancreas and, to a lesser extent, spleen, adrenal gland, bone marrow and thyroid tissue of patients coinfected with HIV; however, it remains unclear if this occurs in patients infected with HCV alone.139141 Importantly, recent studies using ultrasensitive PCR assays for HCV negative strand intermediates to screen patients who had previously cleared infection or had been successfully treated have shown that a significant proportion appear to harbour very low levels of residual virus in PBMC, but not in the liver.142,143 A more detailed understanding of the generation and maintenance of HCV-specific cellular immune responses in these seronegative immune individuals may provide key insights into the most appropriate directions for vaccine design. Future

No. subjects with HCV-specific cellular immune responses 4/20 2/11 7/29 14/44 22/29 two individuals 13/71 20/28

LPA: core, NS3-4, NS4 and NS5 proteins CTL and LPA: 10 core, E1 and NS3 peptides LPA: core, E1, E2, NS3, NS4 and NS5 proteins ELISpot and CTL: 20 core, E1, E2, NS3, NS4 and NS5 peptides LPA and ELISpot: core, NS3, NS4 and NS5 proteins ELISpot: 18 core, E1 and NS4 peptides ELISpot: coreNS2 and NS2-5 recombinant vaccinia virus constructs ELISpot and CTL: coreNS2 and NS2-5 recombinant vaccinia virus constructs LPA: core, NS3-4, NS4 and NS5 proteins ELISpot: 216 overlapping peptides covering core and NS3-NS5b BrdU incorporation: 38 core, E1 and NS3 peptides

BrdU, 5-bromodeoxyuridine; ELISpot, enzyme-linked immunospot assay; HCV, hepatitis C virus; IDU, injecting drug users; LPA, lymphocyte proliferation assay; NS, nonstructural.

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studies should focus on the contributions that high-avidity T cells and dominant protective epitopes play in the seronegative immune phenotype. In addition, the role of other host factors, including genetic polymorphisms and behavioural patterns in the setting of first exposure, as well as viral factors requires further study. This information may aid in the development of a vaccine that mimics the protective immune responses seen in these individuals without the need for repeated Ag exposure.

Conclusion
The combined evidence from these studies indicates that in some individuals, strong initial HCV-specific cellular immune responses are able to prevent the establishment of chronic HCV infection. In a small subset of cases, this immune response may confer the capacity to rapidly clear the virus after repeated exposures to multiple HCV genotypes without seroconversion. This protection against chronic infection is primarily mediated through HCV-specific cellular immune responses, indicating that this should be an important focus of vaccine design. Although successful clearance can occur without an antibody response, it remains unclear if the generation of antibodies, particularly neutralizing antibodies, would increase the effectiveness of vaccine candidates. Given the global burden of HCV disease, an understanding of how protective immune responses are generated is critical. Studies of subjects who fall into this unique group may provide key insights into the nature of these protective immune responses and guide vaccine development and immunotherapeutic options.

Acknowledgements
We would like to thank Professor Richard Strugnell and Associate Professor David Anderson for critical review of this article.

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