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T cell immunity to hepatitis C virus: Lessons for a prophylactic vaccine

  • Robert Thimme
    Correspondence
    Corresponding author. Address: Department of Medicine II, Hugstetter Str. 55, 79106 Freiburg, Germany. Tel.: 0049 771 270 34030, fax: 0049 771 270 36100.
    Affiliations
    Department of Medicine II, Gastroenterology, Hepatology, Endocrinology and Infectious Diseases, Medical Center – University of Freiburg, Faculty of Medicine, Germany
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Open AccessPublished:September 28, 2020DOI:https://doi.org/10.1016/j.jhep.2020.09.022

      Summary

      There is consensus that HCV-specific T cells play a central role in the outcome (clearance vs. persistence) of acute infection and that they contribute to protection against the establishment of persistence after reinfection. However, these T cells often fail and the virus can persist, largely as a result of T cell exhaustion and the emergence of viral escape mutations. Importantly, HCV cure by direct-acting antivirals does not lead to a complete reversion of T cell exhaustion and thus HCV reinfections can occur. The current lack of detailed knowledge about the immunological determinants of viral clearance, persistence and protective immunity is a major roadblock to the development of a prophylactic T cell vaccine. This minireview highlights the basic concepts of successful T cell immunity, major mechanisms of T cell failure and how our understanding of these concepts can be translated into a prophylactic vaccine.

      Keywords

      Introduction

      HCV infection is a leading cause of death and morbidity from liver diseases. The WHO estimates that at least 71 million people are persistently infected and that no less than 400,000 people die each year from HCV infection.
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      Since the discovery of HCV more than 30 years ago, tremendous progress has been made in all aspects of HCV research, ranging from virology to immunopathology and finally to treatment. Indeed, the development and clinical implementation of direct-acting antivirals (DAAs) has revolutionised treatment with sustained virological response rates of almost 100%.
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      However, to achieve the ambitious WHO goal of global eradication by 2030, a prophylactic vaccine will be required.
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      Although several fundamental discoveries have been made over the last 3 decades in the field of adaptive immunity-mediated protection against HCV, unfortunately, they have not yet been successfully translated into a prophylactic vaccine.
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      This is partly due to the highly diverse nature and genetic variability of HCV, which hampers the efficacy of conventional vaccines mediated largely by B cell responses. Indeed, genetic variability is the main factor limiting the efficacy of both T cell immunity and neutralising antibodies, which are also known to play a strong role in mediating protective immunity.
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      HCV as a unique immunological model

      Several facets of HCV infection make it a unique human immunological model for the study of T cell immunity. For example, HCV infection leads to dichotomous outcomes, with only about 30% of patients acutely infected with HCV being able to clear the infection while the majority progress to chronic infection.
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      Innate and adaptive immune responses in HCV infections.
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      Immune responses to HCV and other hepatitis viruses.
      This is in clear contrast to other viruses that either always persist (HIV) or are cleared in most acutely infected adults (HBV). Thus, acute HCV infection provides a unique opportunity to study the immunological correlates that determine viral clearance vs. persistence. Notably, resolution of HCV infection can lead to long-lived protection against HCV persistence after reinfection with the virus, at least in some individuals.
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      • Cox A.L.
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      In contrast to HBV, recovery from HCV does not lead to sterilising immunity, but does at least lead to partially protective immunity. Indeed, immunity acquired by resolution of infection does not necessarily prevent reinfection but clearly reduces the risk of a persistent outcome when compared with primary infection.
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      • Urban G.
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      • et al.
      Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection.
      ,
      • Mehta S.H.
      • Cox A.
      • Hoover D.R.
      • Wang X.H.
      • Mao Q.
      • Ray S.
      • et al.
      Protection against persistence of hepatitis C.
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      Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection.
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      • Rice C.M.
      • et al.
      Previously infected and recovered chimpanzees exhibit rapid responses that control hepatitis C virus replication upon rechallenge.
      This is of relevance for vaccine development since it clearly sets the goal for an HCV vaccine, i.e. to interfere with development of viral persistence but not with infection.
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      • Barnes E.
      • Cox A.L.
      Approaches, progress, and challenges to hepatitis C vaccine development.
      ,
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      A prophylactic hepatitis C virus vaccine: a distant peak still worth climbing.
      ,
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      In patients progressing to chronic infection, adaptive immunity is often defective and not maintained.
      • Heim M.H.
      • Thimme R.
      Innate and adaptive immune responses in HCV infections.
      ,
      • Park S.H.
      • Rehermann B.
      Immune responses to HCV and other hepatitis viruses.
      Although a lot has been learnt about the possible mechanisms behind this T cell failure, there is still uncertainty around exactly how viral persistence develops despite the induction of adaptive immunity.
      Virus-specific CD4+ and CD8+ T cells play a central role in spontaneous elimination of acute HCV infection.
      Another fact that makes HCV a unique immunological model is that it can be cleared in almost all patients by short-term DAA therapy.
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      • Bukh J.
      • Houghton M.
      • Lemon S.M.
      • Lindenbach B.D.
      • et al.
      Critical challenges and emerging opportunities in hepatitis C virus research in an era of potent antiviral therapy: considerations for scientists and funding agencies.
      This is in contrast to other viral infections, such as HIV or HBV infection, where long-term therapy is typically required and viral clearance is not, or only rarely, achieved. Thus, in HCV, the impact of antiviral therapy-mediated antigen removal on recovery of impaired adaptive immunity can be studied and has indeed provided important insights into the fate of chronically stimulated cells (and the potential recovery of their functionality).
      • Rehermann B.
      • Thimme R.
      Insights from antiviral therapy into immune responses to hepatitis B and C virus infection.
      Herein, we will discuss current knowledge on T cell immunity in HCV infection, specifically the current understanding of correlates of protection, mechanisms of immune failure and possible reversion of impaired adaptive immunity, which must all be considered for successful vaccine design. Although broadly neutralising antibodies also play an important part in the adaptive immune response against HCV and present an attractive target for vaccine development, this review is focused on the role of HCV-specific T cell immunity.

      Evidence for role of adaptive immunity in outcome of HCV infection

      Adaptive immunity has clearly been linked to the outcome of infection in a series of studies performed in acutely HCV-infected humans and chimpanzees. These studies have been performed over the last 3 decades and thus also reflect immense methodical progress, ranging from pioneering work to identify relevant antigens (epitopes), comprehensive phenotypical and functional immune profiling, through to transcriptional and metabolic analyses.
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      T cell responses in hepatitis C virus infection: historical overview and goals for future research.
      By using these different approaches and heterogeneous cohorts, there is still an overall consensus that the emergence of multispecific T cell responses is temporarily linked to viral clearance.
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      Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response.
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      Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease.
      Indeed, although the onset of the HCV-specific T cell response is delayed for several weeks, in comparison to viraemia (which is detectable within 2 weeks of infection), its first appearance is linked to the sudden onset of viral control
      • Thimme R.
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      • Chang K.M.
      • Steiger C.
      • Ray S.C.
      • Chisari F.V.
      Determinants of viral clearance and persistence during acute hepatitis C virus infection.
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      • et al.
      Delayed induction, not impaired recruitment of specific CD8(+) T cells, causes the late onset of acute hepatitis C.
      (Fig. 1A). Subsequently, the HCV-specific T cell response is also associated with viral clearance that may occur within 6 months and after several weeks of an oscillating pattern of viraemia (Fig. 1A). The HCV-specific T cell response has also been associated with the onset of liver disease.
      • Thimme R.
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      • Steiger C.
      • Ray S.C.
      • Chisari F.V.
      Determinants of viral clearance and persistence during acute hepatitis C virus infection.
      This is further supported by co-culture experiments in vitro where HCV-specific CD8+ T cells inhibited HCV replication in human hepatoma cells by direct cytolytic and by non-cytolytic, cytokine-mediated effector functions.
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      All arms of the adaptive immune system, B cells, CD4+ and CD8+ T cells are involved in viral clearance.
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      Innate and adaptive immune responses in HCV infections.
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      Antibody responses in hepatitis C infection.
      Both CD4+ and CD8+ T cells target viral antigens from different viral regions, although non-structural proteins are the dominant targets.
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      HCV-specific T cell responses during and after chronic HCV infection.
      Indeed, immunodominant epitopes restricted by several HLA class I and class II alleles have been identified over the years. CD4+ T cell responses consist of Th1, Th17 and T follicular helper (Tfh) cells that have all been suggested to contribute to HCV clearance. Th17 cells may mediate their effect by secreting IL-21 for CD8+ stimulation.
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      Galectin-9 and IL-21 mediate cross-regulation between Th17 and Treg cells during acute hepatitis C.
      HCV-specific Tfh cells, however, contribute to antiviral immunity by inducing virus-specific antibodies.
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      Virus-specific CD4+ T cells have functional and phenotypic characteristics of follicular T-helper cells in patients with acute and chronic HCV infections.
      Memory virus-specific T cells can mediate protection of viral persistence after reinfection.
      Figure thumbnail gr1
      Fig. 1Course of HCV infection.
      (A) After infection, HCV can be cleared in association with a virus-specific CD4+ and CD8+ T cell response that is also temporally linked with ALT elevation. The T cell response is maintained after viral elimination. (B) Chronic persistent infection evolves despite induction of a somewhat weaker T cell response that is not maintained or only at low levels. ALT, alanine aminotransferase.

      Differences in adaptive immunity between acute resolving vs. persisting HCV infection

      The important role of adaptive immunity on the course of HCV infection is underpinned by differences observed with respect to outcome. Indeed, while a primary failure to mount a vigorous HCV-specific T cell response has been correlated with persistence in some patients, others have reported the presence of multispecific CD4+ and CD8+ T cell responses very early in infection and, notably, irrespective of the subsequent outcome of infection,
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      Hepatitis C virus-specific CD4+ T cell phenotype and function in different infection outcomes.
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      Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence.
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      • Thomas D.L.
      • Ray S.C.
      Comprehensive analyses of CD8+ T cell responses during longitudinal study of acute human hepatitis C.
      suggesting that different mechanisms may contribute to CD4+ T cell failure. Indeed, while a primary failure to induce T cell immunity seems to occur rarely, most patients are able to induce HCV-specific CD4+ T cell immunity. However, in persisting HCV infection, these responses decline more rapidly in their frequency and breadth than in patients who spontaneously clear the virus
      • Thimme R.
      • Oldach D.
      • Chang K.M.
      • Steiger C.
      • Ray S.C.
      • Chisari F.V.
      Determinants of viral clearance and persistence during acute hepatitis C virus infection.
      ,
      • Chen D.Y.
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      • Aneja J.
      • Matsubara L.
      • Robilotti B.
      • Hauck G.
      • et al.
      Hepatitis C virus-specific CD4+ T cell phenotype and function in different infection outcomes.
      ,
      • Schulze Zur Wiesch J.
      • Ciuffreda D.
      • Lewis-Ximenez L.
      • Kasprowicz V.
      • Nolan B.E.
      • Streeck H.
      • et al.
      Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence.
      ,
      • Lauer G.M.
      • Barnes E.
      • Lucas M.
      • Timm J.
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      • et al.
      High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection.
      • Gruener N.H.
      • Lechner F.
      • Jung M.C.
      • Diepolder H.
      • Gerlach T.
      • Lauer G.
      • et al.
      Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus.
      • Takaki A.
      • Wiese M.
      • Maertens G.
      • Depla E.
      • Seifert U.
      • Liebetrau A.
      • et al.
      Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C.
      (Fig. 1B). Thus, next to sufficient priming, maintenance of a virus-specific T cell response seems to be one of the key determinants associated with viral clearance, which appears to be especially relevant for CD4+ T cell responses. Phenotypic analysis revealed that virus-specific CD4+ and CD8+ T cells express activation markers and inhibitory receptors such as programmed cell death-1 (PD-1),
      • Urbani S.
      • Amadei B.
      • Tola D.
      • Massari M.
      • Schivazappa S.
      • Missale G.
      • et al.
      PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion.
      ,
      • Kasprowicz V.
      • Schulze Zur Wiesch J.
      • Kuntzen T.
      • Nolan B.E.
      • Longworth S.
      • Berical A.
      • et al.
      High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome.
      often irrespective of the outcome of infection, but that this expression declines with resolution of infection.
      • Chen D.Y.
      • Wolski D.
      • Aneja J.
      • Matsubara L.
      • Robilotti B.
      • Hauck G.
      • et al.
      Hepatitis C virus-specific CD4+ T cell phenotype and function in different infection outcomes.
      Downregulation of PD-1 increases expression of CD127 (the alpha-chain of the IL-7 receptor) which is required for T cell homeostatic proliferation; thus, these T cells share typical characteristics of memory T cells. Notably, higher CD127 expression on HCV-specific CD8+ T cells has been linked to the elimination of infection.
      • Shin E.C.
      • Park S.H.
      • Nascimbeni M.
      • Major M.
      • Caggiari L.
      • de Re V.
      • et al.
      The frequency of CD127(+) hepatitis C virus (HCV)-specific T cells but not the expression of exhaustion markers predicts the outcome of acute HCV infection.
      ,
      • Golden-Mason L.
      • Burton Jr., J.R.
      • Castelblanco N.
      • Klarquist J.
      • Benlloch S.
      • Wang C.
      • et al.
      Loss of IL-7 receptor alpha-chain (CD127) expression in acute HCV infection associated with viral persistence.
      Also, expression of the transcription factor T-bet that regulates the production of interferon-gamma and cytotoxic molecules in effector CD8+ T cells has been reported to be positively associated with outcome.
      • Kurktschiev P.D.
      • Raziorrouh B.
      • Schraut W.
      • Backmund M.
      • Wachtler M.
      • Wendtner C.M.
      • et al.
      Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction.
      In persisting infection, differentiation towards memory is blocked and the virus-specific CD8+ T cells that are present at low frequencies remain activated and express PD-1 and other inhibitory receptors in concert with a progressive loss of proliferation and cytokine production.
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      • et al.
      Coexpression of PD-1, 2B4, CD160 and KLRG1 on exhausted HCV-specific CD8+ T cells is linked to antigen recognition and T cell differentiation.
      Recent work has also shown differences in transcriptional T cell signatures in acute resolving vs. persisting infection. Indeed, metabolic, nucleosome and immune genes were found to be dysregulated in virus-specific CD8+ T cells early in acute persisting infection, which was linked to a lack of CD4+ T cell help.
      • Wolski D.
      • Foote P.K.
      • Chen D.Y.
      • Lewis-Ximenez L.L.
      • Fauvelle C.
      • Aneja J.
      • et al.
      Early transcriptional divergence marks virus-specific primary human CD8(+) T cells in chronic versus acute infection.
      Exhaustion-committed virus-specific CD8+ T cells were also shown to display a marked upregulation of transcription factors associated with impaired glycolytic and mitochondrial functions.
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      • et al.
      Targeting p53 and histone methyltransferases restores exhausted CD8+ T cells in HCV infection.
      Despite all these important insights, it is important to note that the exact immunological factors and their relative contribution to viral clearance vs. persistence are still not completely defined.

      Adaptive immunity in reinfection

      A hallmark of HCV infection is the absence of sterilising immunity upon re-exposure to the virus. However, several studies in humans
      • Osburn W.O.
      • Fisher B.E.
      • Dowd K.A.
      • Urban G.
      • Liu L.
      • Ray S.C.
      • et al.
      Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection.
      ,
      • Mehta S.H.
      • Cox A.
      • Hoover D.R.
      • Wang X.H.
      • Mao Q.
      • Ray S.
      • et al.
      Protection against persistence of hepatitis C.
      and chimpanzees
      • Bassett S.E.
      • Guerra B.
      • Brasky K.
      • Miskovsky E.
      • Houghton M.
      • Klimpel G.R.
      • et al.
      Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection.
      ,
      • Major M.E.
      • Mihalik K.
      • Puig M.
      • Rehermann B.
      • Nascimbeni M.
      • Rice C.M.
      • et al.
      Previously infected and recovered chimpanzees exhibit rapid responses that control hepatitis C virus replication upon rechallenge.
      have shown that the course of infection is significantly altered after reinfection, as indicated by a substantial reduction in the duration and magnitude of viraemia and a higher likelihood of resolving the reinfection compared to the primary infection (Fig. 2A). This is associated with a rapid emergence of memory virus-specific CD4+ and CD8+ T cell responses
      • Osburn W.O.
      • Fisher B.E.
      • Dowd K.A.
      • Urban G.
      • Liu L.
      • Ray S.C.
      • et al.
      Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection.
      ,
      • Mehta S.H.
      • Cox A.
      • Hoover D.R.
      • Wang X.H.
      • Mao Q.
      • Ray S.
      • et al.
      Protection against persistence of hepatitis C.
      ,
      • Bassett S.E.
      • Guerra B.
      • Brasky K.
      • Miskovsky E.
      • Houghton M.
      • Klimpel G.R.
      • et al.
      Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection.
      ,
      • Abdel-Hakeem M.S.
      • Bedard N.
      • Murphy D.
      • Bruneau J.
      • Shoukry N.H.
      Signatures of protective memory immune responses during hepatitis C virus reinfection.
      (Fig 2A). The central role of these T cell subsets in mediating protection has been proven in the chimpanzee model. Indeed, antibody-mediated depletion of CD4+ T cells before reinfection of immune chimpanzees resulted in persistent, low-level viraemia
      • Grakoui A.
      • Shoukry N.H.
      • Woollard D.J.
      • Han J.H.
      • Hanson H.L.
      • Ghrayeb J.
      • et al.
      HCV persistence and immune evasion in the absence of memory T cell help.
      (Fig. 2B). Incomplete control of HCV replication in the absence of adequate CD4+ T cell help was associated with the emergence of viral escape mutations within the MHC class I epitopes targeted by virus-specific CD8+ T cells. Meanwhile, depletion of CD8+ T cells led to prolonged viraemia that was first controlled when virus-specific CD8+ T cells reappeared
      • Shoukry N.H.
      • Grakoui A.
      • Houghton M.
      • Chien D.Y.
      • Ghrayeb J.
      • Reimann K.A.
      • et al.
      Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection.
      (Fig. 2C). Thus, combined, these results indicate that CD8+ T cells are the primary effector cells responsible for viral control while CD4+ T cells are critical for sustaining CD8+ T cell immunity and preventing the emergence of viral escape within CD8+ T cell epitopes. These results have sparked further interest in the concept of a T cell vaccine for HCV that does not prevent infection but induces a stronger immune response, thereby increasing the likelihood of viral clearance
      • Bailey J.R.
      • Barnes E.
      • Cox A.L.
      Approaches, progress, and challenges to hepatitis C vaccine development.
      (Fig 2D). However, it has to be noted that T cell-mediated protection can fail, as has been shown in re-exposed humans and chimpanzees, even when re-exposed with homologous virus.
      • Bukh J.
      • Thimme R.
      • Meunier J.C.
      • Faulk K.
      • Spangenberg H.C.
      • Chang K.M.
      • et al.
      Previously infected chimpanzees are not consistently protected against reinfection or persistent infection after reexposure to the identical hepatitis C virus strain.
      As for primary infection, the mechanisms by which HCV evades adaptive immunity in these instances are still not completely understood. Thus, it is a priority and prerequisite for the successful development of a T cell-based HCV vaccine to define the exact underlying mechanisms of T cell failure.
      T cell failure during chronic infection is primarily mediated by viral escape and T cell exhaustion.
      Figure thumbnail gr2
      Fig. 2Natural vs. vaccine-induced immunity during primary infection and reinfection.
      (A) Reinfection can be rapidly cleared in concert with a rapidly emerging memory response. (B) CD4+ T cell depletion before reinfection leads to chronic infection due to the emergence of viral escape mutations within CD8+ T cell epitopes. (C) After CD8+ T cell depletion, high HCV titres are maintained until CD8+ T cells reappear and eliminate the virus. (D) Concept of a prophylactic vaccine that induces a strong T cell response that is then rapidly expanding after infection, mirroring the T cell response after reinfection following natural primary infection.

      Failure of T cell immunity

      Failure of virus-specific CD8+ T cells – the main effector cells in HCV infection – has been attributed to 2 main mechanisms, T cell exhaustion and viral escape.
      T cell exhaustion is a consequence of chronic antigen stimulation in the context of inhibitory signalling and describes a state of profound loss of effector functions. Thus, T cell exhaustion is specific for a given antigen. Accordingly, unrelated antigen-specific CD8+ T cell responses in a given patient, such as influenza-specific CD8+ T cells, do not show markers of T cell exhaustion.
      • Alfei F.
      • Kanev K.
      • Hofmann M.
      • Wu M.
      • Ghoneim H.E.
      • Roelli P.
      • et al.
      TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.
      T cell exhaustion may thus contribute to viral persistence but may also limit immunopathology. T cell exhaustion is further characterised by high co-expression of inhibitory receptors, altered metabolism, impaired survival, a distinct transcriptional programme with an altered use of key transcription factors and a unique underlying epigenetic landscape.
      • Blank C.U.
      • Haining W.N.
      • Held W.
      • Hogan P.G.
      • Kallies A.
      • Lugli E.
      • et al.
      Defining ‘T cell exhaustion’.
      Indeed, these features have been identified in the majority of HCV-specific CD8+ T cells isolated from chronic infection.
      • Kasprowicz V.
      • Schulze Zur Wiesch J.
      • Kuntzen T.
      • Nolan B.E.
      • Longworth S.
      • Berical A.
      • et al.
      High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome.
      ,
      • Bengsch B.
      • Seigel B.
      • Ruhl M.
      • Timm J.
      • Kuntz M.
      • Blum H.E.
      • et al.
      Coexpression of PD-1, 2B4, CD160 and KLRG1 on exhausted HCV-specific CD8+ T cells is linked to antigen recognition and T cell differentiation.
      ,
      • Penna A.
      • Pilli M.
      • Zerbini A.
      • Orlandini A.
      • Mezzadri S.
      • Sacchelli L.
      • et al.
      Dysfunction and functional restoration of HCV-specific CD8 responses in chronic hepatitis C virus infection.
      • Radziewicz H.
      • Ibegbu C.C.
      • Fernandez M.L.
      • Workowski K.A.
      • Obideen K.
      • Wehbi M.
      • et al.
      Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression.
      • Nakamoto N.
      • Cho H.
      • Shaked A.
      • Olthoff K.
      • Valiga M.E.
      • Kaminski M.
      • et al.
      Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade.
      Recent work has shown that exhausted HCV-specific CD8+ T cells are not a homogeneous population but rather consist of distinct subsets. Indeed, the coexistence of a less differentiated TCF-1+CD127+PD-1+ memory-like and a PD-1hiEomeshiTOXhiCD127-terminally exhausted subset has been reported; these subsets also differ in their cytokine production and proliferative capacity
      • Wieland D.
      • Kemming J.
      • Schuch A.
      • Emmerich F.
      • Knolle P.
      • Neumann-Haefelin C.
      • et al.
      TCF1(+) hepatitis C virus-specific CD8(+) T cells are maintained after cessation of chronic antigen stimulation.
      (Fig. 3A). Most likely, TCF-1+CD127+PD-1+ memory-like cells serve as progenitors that sustain the immune response in chronic infection and give rise to the PD-1hiEomeshiTOXhiCD127-terminally exhausted subset after antigen encounter (Fig. 3A). Interestingly, the important biological role of the transcription factors TCF-1 (T cell factor 1) and TOX (thymocyte selection associated high mobility group box) was first reported in the lymphocytic choriomeningitis virus (LCMV) mouse model. Indeed, TCF-1 has been shown to sustain virus-specific CD8+ T cell responses to chronic viral infections,
      • Utzschneider D.T.
      • Charmoy M.
      • Chennupati V.
      • Pousse L.
      • Ferreira D.P.
      • Calderon-Copete S.
      • et al.
      T cell factor 1-expressing memory-like CD8(+) T cells sustain the immune response to chronic viral infections.
      while TOX has been shown to be a key inducer of exhaustion that translates persistent antigen stimulation into a specific transcriptional and epigenetic programme.
      • Khan O.
      • Giles J.R.
      • McDonald S.
      • Manne S.
      • Ngiow S.F.
      • Patel K.P.
      • et al.
      TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion.
      ,
      • Alfei F.
      • Kanev K.
      • Hofmann M.
      • Wu M.
      • Ghoneim H.E.
      • Roelli P.
      • et al.
      TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.
      Expression of TOX is thereby closely associated with the expression of inhibitory receptors such as PD-1. More specifically, overexpression of TOX leads to improved survival of a progenitor subset of exhausted CD8+ T cells that is also characterised by the expression of the transcription factor TCF-1.
      • Khan O.
      • Giles J.R.
      • McDonald S.
      • Manne S.
      • Ngiow S.F.
      • Patel K.P.
      • et al.
      TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion.
      ,
      • Alfei F.
      • Kanev K.
      • Hofmann M.
      • Wu M.
      • Ghoneim H.E.
      • Roelli P.
      • et al.
      TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.
      Thus, the translation from novel findings from the mouse to the human system underscores the important role of HCV as a highly relevant human immunological model. Exhaustion may be especially relevant at the site of infection, the liver, where antigen load is highest and where several immunosuppressive mechanisms that may further influence CD8+ T cell functionality are active.
      • Kroy D.C.
      • Ciuffreda D.
      • Cooperrider J.H.
      • Tomlinson M.
      • Hauck G.D.
      • Aneja J.
      • et al.
      Liver environment and HCV replication affect human T-cell phenotype and expression of inhibitory receptors.
      ,
      • Knolle P.A.
      • Thimme R.
      Hepatic immune regulation and its involvement in viral hepatitis infection.
      Figure thumbnail gr3
      Fig. 3CD8+ T cell characteristics in different phases.
      (A) HCV-specific CD8+ T cells are exhausted during chronic infection and exist as 2 subsets, TCF-1+CD127+PD-1+ memory-like and a PD-1hiEomeshiTOXhiCD127- exhausted T cell population. (B) After DAA-mediated cure, memory-like cells are maintained while terminally exhausted CD8+ T cells are lost. The memory-like cells do not resemble memory cells that are present after spontaneous resolution or vaccine induction as they maintain expression of PD-1 and impaired functionality. (C) Classical memory cells after spontaneous resolution are characterised by high TCF1 and CD127 expression and good functionality. (D) Vaccine-induced CD8+ T cells resemble CD8+ T cells present after spontaneous resolution. DAA, direct-acting antiviral; PD-1, programmed cell death 1; TOX, thymocyte selection associated high mobility group box.
      Viral escape mutations within the highly mutable HCV genome are a consequence of CD8+ T cell pressure and can lead to altered proteasome processing, reduced binding of peptide epitopes to MHC class I molecules or impaired recognition by the T cell receptor, all of which prevent recognition by CD8+ T cells.
      • Erickson A.L.
      • Kimura Y.
      • Igarashi S.
      • Eichelberger J.
      • Houghton M.
      • Sidney J.
      • et al.
      The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes.
      • Chang K.M.
      • Rehermann B.
      • McHutchison J.G.
      • Pasquinelli C.
      • Southwood S.
      • Sette A.
      • et al.
      Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus.
      • Timm J.
      • Lauer G.M.
      • Kavanagh D.G.
      • Sheridan I.
      • Kim A.Y.
      • Lucas M.
      • et al.
      CD8 epitope escape and reversion in acute HCV infection.
      Viral escape mutations typically occur during the first 6 months of infection
      • Cox A.L.
      • Mosbruger T.
      • Lauer G.M.
      • Pardoll D.
      • Thomas D.L.
      • Ray S.C.
      Comprehensive analyses of CD8+ T cell responses during longitudinal study of acute human hepatitis C.
      and affect about half of the CD8+ T cell-targeted epitopes.
      • Neumann-Haefelin C.
      • Timm J.
      • Spangenberg H.C.
      • Wischniowski N.
      • Nazarova N.
      • Kersting N.
      • et al.
      Virological and immunological determinants of intrahepatic virus-specific CD8+ T-cell failure in chronic hepatitis C virus infection.
      Indeed, the occurrence of viral escape mutations during long-term chronic infection is uncommon and probably reflects the absence of T cell-mediated selection pressure in that phase.
      • Cox A.L.
      • Mosbruger T.
      • Lauer G.M.
      • Pardoll D.
      • Thomas D.L.
      • Ray S.C.
      Comprehensive analyses of CD8+ T cell responses during longitudinal study of acute human hepatitis C.
      Viral escape also leaves an imprint on the phenotype of virus-specific CD8+ T cells.
      • Bengsch B.
      • Seigel B.
      • Ruhl M.
      • Timm J.
      • Kuntz M.
      • Blum H.E.
      • et al.
      Coexpression of PD-1, 2B4, CD160 and KLRG1 on exhausted HCV-specific CD8+ T cells is linked to antigen recognition and T cell differentiation.
      ,
      • Kasprowicz V.
      • Kang Y.H.
      • Lucas M.
      • Schulze zur Wiesch J.
      • Kuntzen T.
      • Fleming V.
      • et al.
      Hepatitis C virus (HCV) sequence variation induces an HCV-specific T-cell phenotype analogous to spontaneous resolution.
      Indeed, in the absence of ongoing antigen triggering, these cells do not display high levels of inhibitory receptors but rather express markers indicative of T cell memory, such as TCF-1 and CD127.
      • Wieland D.
      • Kemming J.
      • Schuch A.
      • Emmerich F.
      • Knolle P.
      • Neumann-Haefelin C.
      • et al.
      TCF1(+) hepatitis C virus-specific CD8(+) T cells are maintained after cessation of chronic antigen stimulation.
      Importantly, viral escape mutations can also have an impact on the replicative capacity of HCV, leading to compensatory mutations and/or rapid reversion when active immune selection pressure disappears.
      • Timm J.
      • Lauer G.M.
      • Kavanagh D.G.
      • Sheridan I.
      • Kim A.Y.
      • Lucas M.
      • et al.
      CD8 epitope escape and reversion in acute HCV infection.
      This fitness cost also contributes to the protective effect of specific HLA-types, e.g. HLA-B27, -B57 or -A3 types that are associated with a high level of spontaneous HCV clearance, which could be linked to dominant CD8+ T cell-targeted epitopes.
      • Neumann-Haefelin C.
      • McKiernan S.
      • Ward S.
      • Viazov S.
      • Spangenberg H.C.
      • Killinger T.
      • et al.
      Dominant influence of an HLA-B27 restricted CD8+ T cell response in mediating HCV clearance and evolution.
      • Kim A.Y.
      • Kuntzen T.
      • Timm J.
      • Nolan B.E.
      • Baca M.A.
      • Reyor L.L.
      • et al.
      Spontaneous control of HCV is associated with expression of HLA-B 57 and preservation of targeted epitopes.
      • Fitzmaurice K.
      • Petrovic D.
      • Ramamurthy N.
      • Simmons R.
      • Merani S.
      • Gaudieri S.
      • et al.
      Molecular footprints reveal the impact of the protective HLA-A∗03 allele in hepatitis C virus infection.
      Viral escape within these epitopes is not easily tolerated due to the fitness cost, thus hampering escape from CD8+ T cell immunity.
      • Kim A.Y.
      • Kuntzen T.
      • Timm J.
      • Nolan B.E.
      • Baca M.A.
      • Reyor L.L.
      • et al.
      Spontaneous control of HCV is associated with expression of HLA-B 57 and preservation of targeted epitopes.
      • Fitzmaurice K.
      • Petrovic D.
      • Ramamurthy N.
      • Simmons R.
      • Merani S.
      • Gaudieri S.
      • et al.
      Molecular footprints reveal the impact of the protective HLA-A∗03 allele in hepatitis C virus infection.
      • Dazert E.
      • Neumann-Haefelin C.
      • Bressanelli S.
      • Fitzmaurice K.
      • Kort J.
      • Timm J.
      • et al.
      Loss of viral fitness and cross-recognition by CD8+ T cells limit HCV escape from a protective HLA-B27-restricted human immune response.
      Clearly, such conserved viral regions that do not easily tolerate sequence variations are optimal targets for T cell-based vaccines. Recent work has shown that vaccines can induce T cell immunity to conserved regions of the virus.
      • von Delft A.
      • Donnison T.A.
      • Lourenco J.
      • Hutchings C.
      • Mullarkey C.E.
      • Brown A.
      • et al.
      The generation of a simian adenoviral vectored HCV vaccine encoding genetically conserved gene segments to target multiple HCV genotypes.
      Exhausted CD8+ T cells are comprised of a TCF-1+CD127+PD-1+ memory-like and a PD-1hiEomeshiTOXhiCD127-terminally exhausted subset.
      Both mechanisms of CD8+ T cell failure, T cell exhaustion and viral escape, are directly linked to the CD4+ T cell response. Indeed, during chronic infection, CD4+ T cells are only present at a very low frequency, display impaired proliferative and cytokine production, and are characterised by high expression of inhibitory receptors such as PD-1 and cytotoxic T-lymphocyte associated protein 4.
      • Chen D.Y.
      • Wolski D.
      • Aneja J.
      • Matsubara L.
      • Robilotti B.
      • Hauck G.
      • et al.
      Hepatitis C virus-specific CD4+ T cell phenotype and function in different infection outcomes.
      ,
      • Schulze Zur Wiesch J.
      • Ciuffreda D.
      • Lewis-Ximenez L.
      • Kasprowicz V.
      • Nolan B.E.
      • Streeck H.
      • et al.
      Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence.
      ,
      • Raziorrouh B.
      • Ulsenheimer A.
      • Schraut W.
      • Heeg M.
      • Kurktschiev P.
      • Zachoval R.
      • et al.
      Inhibitory molecules that regulate expansion and restoration of HCV-specific CD4+ T cells in patients with chronic infection.
      ,
      • Ackermann C.
      • Smits M.
      • Woost R.
      • Eberhard J.M.
      • Peine S.
      • Kummer S.
      • et al.
      HCV-specific CD4+ T cells of patients with acute and chronic HCV infection display high expression of TIGIT and other co-inhibitory molecules.
      Thus, lack of sufficient CD4+ T cell help is probably one of the main factors responsible for CD8+ T cell failure. It is currently unknown why CD4+ T cell responses are not sustained in persisting infection, however, most studies indicate that viral escape does not seem to play a role in this setting.
      • Fuller M.J.
      • Shoukry N.H.
      • Gushima T.
      • Bowen D.G.
      • Callendret B.
      • Campbell K.J.
      • et al.
      Selection-driven immune escape is not a significant factor in the failure of CD4 T cell responses in persistent hepatitis C virus infection.
      • Fleming V.M.
      • Harcourt G.
      • Barnes E.
      • Klenerman P.
      Virological footprint of CD4+ T-cell responses during chronic hepatitis C virus infection.
      • Lucas M.
      • Deshpande P.
      • James I.
      • Rauch A.
      • Pfafferott K.
      • Gaylard E.
      • et al.
      Evidence of CD4(+) T cell-mediated immune pressure on the Hepatitis C virus genome.
      Other mechanisms that contribute to CD8+ T cell failure such as suppressive regulatory T cells
      • Boettler T.
      • Spangenberg H.C.
      • Neumann-Haefelin C.
      • Panther E.
      • Urbani S.
      • Ferrari C.
      • et al.
      T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection.
      • Cabrera R.
      • Tu Z.
      • Xu Y.
      • Firpi R.J.
      • Rosen H.R.
      • Liu C.
      • et al.
      An immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection.
      • Sugimoto K.
      • Ikeda F.
      • Stadanlick J.
      • Nunes F.A.
      • Alter H.J.
      • Chang K.M.
      Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infection.
      are displayed in Box 1.
      Mechanisms of HCV-specific CD8+ T cell failure

      Restoration of adaptive immunity by DAAs

      A central question of high relevance, not only for vaccine design but for antigen-specific T cell immunity in general, is whether chronically stimulated CD8+ T cells remain exhausted once the relevant antigen is removed, or whether they regain their effector functions. By studying the fate of HCV-specific CD8+ T cells in patients undergoing DAA therapy, we recently showed that antigen withdrawal led to a loss of terminally exhausted cells, while memory-like progenitor CD8+ T cells (characterised by the expression of TCF-1+CD127+PD-1+) were still detectable, leading to a superior functionality of HCV-specific CD8+ T cells after HCV cure
      • Wieland D.
      • Kemming J.
      • Schuch A.
      • Emmerich F.
      • Knolle P.
      • Neumann-Haefelin C.
      • et al.
      TCF1(+) hepatitis C virus-specific CD8(+) T cells are maintained after cessation of chronic antigen stimulation.
      ,
      • Martin B.
      • Hennecke N.
      • Lohmann V.
      • Kayser A.
      • Neumann-Haefelin C.
      • Kukolj G.
      • et al.
      Restoration of HCV-specific CD8+ T cell function by interferon-free therapy.
      (Fig 3B). Notably, after antigen re-challenge in vivo in the context of a viral relapse, the TCF1+CD127+PD-1+ population expanded, which was accompanied by emergence of terminally exhausted EomeshiTCF1-CD127-PD-1hi HCV-specific CD8+ T cells, clearly indicating at least some kind of memory potential within the TCF1+CD127+PD-1 subset.
      • Wieland D.
      • Kemming J.
      • Schuch A.
      • Emmerich F.
      • Knolle P.
      • Neumann-Haefelin C.
      • et al.
      TCF1(+) hepatitis C virus-specific CD8(+) T cells are maintained after cessation of chronic antigen stimulation.
      However, these re-emerging cells failed to clear the virus. This is in agreement with a chimpanzee study showing persistence of HCV-specific CD8+ T cell immunity after DAA-mediated HCV elimination and also no protection from reinfection.
      • Callendret B.
      • Eccleston H.B.
      • Hall S.
      • Satterfield W.
      • Capone S.
      • Folgori A.
      • et al.
      T-cell immunity and hepatitis C virus reinfection after cure of chronic hepatitis C with an interferon-free antiviral regimen in a chimpanzee.
      Combined, these results indicate that HCV-specific CD8+ T cells that are maintained after DAA-mediated cure may have persistent defects that cannot simply be restored by viral elimination, as has also been reported by others.
      • Aregay A.
      • Owusu Sekyere S.
      • Deterding K.
      • Port K.
      • Dietz J.
      • Berkowski C.
      • et al.
      Elimination of hepatitis C virus has limited impact on the functional and mitochondrial impairment of HCV-specific CD8+ T cell responses.
      This is further supported by our recent finding that TOX, a critical transcription factor determining CD8+ T cell exhaustion, was highly detectable in HCV-specific CD8+ T cells during chronic infection and after DAA-mediated cure, but not after spontaneously resolved HCV infection
      • Alfei F.
      • Kanev K.
      • Hofmann M.
      • Wu M.
      • Ghoneim H.E.
      • Roelli P.
      • et al.
      TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.
      (Fig. 3A-C). These results are also in agreement with results obtained in the LCMV mouse model where TOX expression was irreversibly imprinted in chronically stimulated virus-specific CD8+ T cells, probably by TOX-dependent epigenetic programming that cannot simply be reversed by antigen elimination.
      • Khan O.
      • Giles J.R.
      • McDonald S.
      • Manne S.
      • Ngiow S.F.
      • Patel K.P.
      • et al.
      TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion.
      ,
      • Alfei F.
      • Kanev K.
      • Hofmann M.
      • Wu M.
      • Ghoneim H.E.
      • Roelli P.
      • et al.
      TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.
      However, and in contrast to late antiviral therapy, immune restoration is possible after early antiviral therapy against HCV.
      • Abdel-Hakeem M.S.
      • Bedard N.
      • Badr G.
      • Ostrowski M.
      • Sekaly R.P.
      • Bruneau J.
      • et al.
      Comparison of immune restoration in early versus late alpha interferon therapy against hepatitis C virus.
      Similarly, mouse studies have shown that early but not late antigen removal rescues CD8+ T cells from differentiation into exhaustion.
      • Utzschneider D.T.
      • Legat A.
      • Fuertes Marraco S.A.
      • Carrie L.
      • Luescher I.
      • Speiser D.E.
      • et al.
      T cells maintain an exhausted phenotype after antigen withdrawal and population reexpansion.
      Thus, combined, these results highlight the major challenge of overcoming T cell exhaustion late in chronically stimulated cells. Indeed, therapy with checkpoint blockade or the combination of antiviral therapy with vaccination failed to restore T cell immunity and antiviral activity in chronically HCV-infected patients
      • Kelly C.
      • Swadling L.
      • Capone S.
      • Brown A.
      • Richardson R.
      • Halliday J.
      • et al.
      Chronic hepatitis C viral infection subverts vaccine-induced T-cell immunity in humans.
      or chimpanzees.
      • Callendret B.
      • Eccleston H.B.
      • Satterfield W.
      • Capone S.
      • Folgori A.
      • Cortese R.
      • et al.
      Persistent hepatitis C viral replication despite priming of functional CD8+ T cells by combined therapy with a vaccine and a direct-acting antiviral.
      DAA therapy does not lead to complete restoration of exhausted T cell responses as evident by persistence of TOX+ CD8+ T cells.
      Of note, similar findings have been obtained for HCV-specific CD4+ T cells, which remained dysfunctional and at a very low frequency after DAA cure.
      • Hartnell F.
      • Esposito I.
      • Swadling L.
      • Brown A.
      • Phetsouphanh C.
      • de Lara C.
      • et al.
      Characterising HCV specific CD4+ T-cells following viral-vectored vaccination, directly acting anti-virals and spontaneous viral cure.
      Increased levels of regulatory T cells were also shown to persist,
      • Langhans B.
      • Nischalke H.D.
      • Kramer B.
      • Hausen A.
      • Dold L.
      • van Heteren P.
      • et al.
      Increased peripheral CD4(+) regulatory T cells persist after successful direct-acting antiviral treatment of chronic hepatitis C.
      indicating that alterations within the CD4+ T cell compartments remain largely unaffected after DAA cure. In contrast, a population of HCV-specific CD4+ T cells with a follicular T helper cell signature was maintained after therapy-induced elimination of persistent infection.
      • Smits M.
      • Zoldan K.
      • Ishaque N.
      • Gu Z.
      • Jechow K.
      • Wieland D.
      • et al.
      Follicular T helper cells shape the HCV-specific CD4+ T cell repertoire after virus elimination.

      Vaccine-induced T cells: success and failure

      Taken together, the studies discussed above teach us important lessons for the development of a T cell-based vaccine. Indeed, HCV-specific CD4+ and CD8+ T cells targeting different epitopes, especially in the non-structural proteins, are clearly linked with control of infection after HCV reinfection. Both T cell subsets are required, as helper and effector cells, respectively. These cells need to be induced rapidly and probably as importantly they need to be sustained. Thus, a prophylactic vaccine needs to efficiently induce both arms of the T cell response. However, observations that CD8+ T cells can fail to control infection because of the emergence of viral escape mutations and T cell exhaustion highlight the existing roadblocks that need to be overcome for a successful vaccine.
      Vigorous and functional HCV specific T cell responses can be induced by heterologous prime-boost vaccination strategies in healthy donors.
      The concept of a prophylactic T cell vaccine was first demonstrated in the chimpanzee model where vaccination with a recombinant replication-deficient adenovirus that expressed all non-structural proteins was able to protect vaccinated chimpanzees from acute hepatitis.
      • Folgori A.
      • Capone S.
      • Ruggeri L.
      • Meola A.
      • Sporeno E.
      • Ercole B.B.
      • et al.
      A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.
      Indeed, suppression of acute viraemia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8+ T cell responses that cross-reacted with vaccine and virus epitopes.
      • Folgori A.
      • Capone S.
      • Ruggeri L.
      • Meola A.
      • Sporeno E.
      • Ercole B.B.
      • et al.
      A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.
      Thus, these important findings have shown that it is possible to elicit effective immunity against heterologous HCV strains by just stimulating the cellular arm of the immune system. Subsequent analyses revealed that T cell responses in vaccinated animals appeared earlier, were more multifunctional and persisted at a higher frequency than in non-vaccinated animals.
      • Park S.H.
      • Shin E.C.
      • Capone S.
      • Caggiari L.
      • De Re V.
      • Nicosia A.
      • et al.
      Successful vaccination induces multifunctional memory T-cell precursors associated with early control of hepatitis C virus.

      Vaccine studies in humans

      The promising results obtained in chimpanzees have been translated to the human situation (Table 1). Indeed, two T cell vaccines designed to prevent HCV infection have been tested for immunogenicity in humans. A vaccine with the HCV core protein and ISCOMATRIX adjuvant was found to induce only weak responses in a subset of healthy volunteers and was thus not advanced into an at-risk population.
      • Drane D.
      • Maraskovsky E.
      • Gibson R.
      • Mitchell S.
      • Barnden M.
      • Moskwa A.
      • et al.
      Priming of CD4+ and CD8+ T cell responses using a HCV core ISCOMATRIX vaccine: a phase I study in healthy volunteers.
      In contrast, a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA)
      • Mally A.
      • Walker C.L.
      • Everitt J.I.
      • Dekant W.
      • Vamvakas S.
      Analysis of renal cell transformation following exposure to trichloroethene in vivo and its metabolite S-(dichlorovinyl)-L-cysteine in vitro.
      vector encoding the NS3, NS4, NS5A, and NS5B proteins that was tested in a phase I study conducted in healthy volunteers showed the induction of sustained memory and effector T cell populations.
      • Swadling L.
      • Capone S.
      • Antrobus R.D.
      • Brown A.
      • Richardson R.
      • Newell E.W.
      • et al.
      A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory.
      ,
      • Barnes E.
      • Folgori A.
      • Capone S.
      • Swadling L.
      • Aston S.
      • Kurioka A.
      • et al.
      Novel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man.
      These T cell memory responses further evolved over time with improvement of quality (proliferation and polyfunctionality) after heterologous MVA boost. Importantly, vaccine-induced CD4+ and CD8+ T cell subsets resemble memory T cells – in their phenotype and transcriptional profile (as indicated by high CD127 and low Eomes expression) – that are maintained after spontaneous resolution, thus indicating that they should rapidly expand in case of reinfection
      • Swadling L.
      • Capone S.
      • Antrobus R.D.
      • Brown A.
      • Richardson R.
      • Newell E.W.
      • et al.
      A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory.
      (Fig. 3C,D).
      Vaccine-induced T cells however failed to show a protective effect in a first human study.
      Table 1Prophylactic human T cell vaccine studies.
      Vaccine backboneAntigenStudyAuthors
      PrototypeCorePhase IDrane et al., Human Vaccine 2009
      Replication-deficient adenovirus vectorsNS3, NS4, NS5Phase ISwadling et al., Science Translational

      Medicine 2014
      Phase I/IICox et al., Open Forum Infectious Diseases 2019
      However, despite these promising findings, a randomised, multicentre, double-blind, placebo-controlled phase I and II clinical study, including 455 participants who injected drugs and received either the vaccine regimen or placebo, did not show a protective effect against the development of viral persistence.
      • Cox A.L.
      • Page K.
      • Melia M.
      • Veenhuis R.
      • Massaccesi G.
      • Osburn W.
      • et al.
      LB10. A randomized, double-blind, placebo-controlled efficacy trial of a vaccine to prevent chronic hepatitis C virus infection in an at-risk population.
      Importantly, T cell responses were induced, thus pointing to at least some vaccine-mediated immunological effects.
      • Cox A.L.
      • Page K.
      • Melia M.
      • Veenhuis R.
      • Massaccesi G.
      • Osburn W.
      • et al.
      LB10. A randomized, double-blind, placebo-controlled efficacy trial of a vaccine to prevent chronic hepatitis C virus infection in an at-risk population.
      Clearly, a detailed analysis will be needed to decipher the mechanisms of T cell failure in these vaccinees, e.g. to what degree viral escape and T cell exhaustion may have contributed to failure of vaccine-induced T cell immunity. Clearly, a major hurdle may be the complexity of different genotypes and T cell epitopes that are difficult to tackle with a vaccine covering a single genotype. Indeed, although immunisation with candidate HCV adenoviral vaccines can generate cross-reactive T cells at immunodominant epitopes, the degree of cross-reactivity varies between epitopes and may be HCV-subtype specific.
      • Kelly C.
      • Swadling L.
      • Brown A.
      • Capone S.
      • Folgori A.
      • Salio M.
      • et al.
      Cross-reactivity of hepatitis C virus specific vaccine-induced T cells at immunodominant epitopes.
      Although disappointing, the important findings of this vaccine trial are perhaps unsurprising, as they are in line with the overall ability of HCV to evade adaptive immunity, natural or vaccine-induced, and to establish persistent infection. They highlight the priority of defining the factors that determine these immunological events and we can expect to learn important lessons regarding vaccine-induced T cell failure from this study. In this context, it should also be noted that the vaccine did not include structural proteins such as core or E1/2 and was focused on non-structural proteins.

      Conclusion

      In the last decades, HCV taught us important lessons about the function, failure and fate of human virus-specific T cells in a highly relevant disease setting with implications for other chronic viral infections. However, there is still uncertainty about the exact correlates of protective immunity. Important questions remain such as: What are the mechanisms and the relative contribution of CD4+ and CD8+ T cell-mediated viral clearance? Why are these cells not maintained, or only maintained at a low frequency, in persisting infection? How can protective immunity be best induced and why is it not effective in all patients? What are the roles of B cells and neutralising antibodies? These central gaps in knowledge need to be addressed in the near future. Clearly, several of them, e.g. the T cell response at the site of infection, the liver, cannot be directly addressed in infected humans, thus, preclinical models are required. Chimpanzee HCV research is not possible anymore, however, the recent discovery of a highly related hepacivirus that infects rats and mice has created an important opportunity, with the potential to provide insights into subversion of immunity by hepaciviruses and to facilitate further development of vaccines.
      • Billerbeck E.
      • Wolfisberg R.
      • Fahnoe U.
      • Xiao J.W.
      • Quirk C.
      • Luna J.M.
      • et al.
      Mouse models of acute and chronic hepacivirus infection.
      ,
      • Ploss A.
      • Kapoor A.
      Animal models of hepatitis C virus infection.
      Also, further studies in acutely and chronically infected humans and individuals undergoing DAA therapy or vaccination are required to decipher the molecular, metabolic and transcriptional features of protective T cell immunity. In addition to the increasing knowledge on different CD4+ (e.g. follicular helper cell responses) and CD8+ (e.g. memory-like and exhausted) T cell subsets, rigorous studies on the nature of humoral immunity are needed for vaccine development. Looking back on what has been learnt about T cell immunology in HCV over the last 30 years, this author has little doubt that these important issues will be solved in the years to come with the help of new technologies, novel preclinical models and the emerging patient cohort treated with DAAs. The race is on!

      Abbreviations

      DAA, direct-acting antiviral; LCMV, lymphocytic choriomeningitis virus; MVA, modified vaccinia Ankara; PD-1, programmed cell death 1; TCF-1, T cell factor 1; Tfh, T follicular helper; TOX, thymocyte selection associated high mobility group box.

      Financial support

      The work was supported by Deutsche Forschungsgemeinschaft TRR 179, TP1.

      Conflict of interest

      The author declares no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgement

      I thank my long-term colleagues Drs. Bertram Bengsch, Tobias Boettler, Maike Hofmann and Christoph Neumann-Haefelin for helpful discussions and critically reading the manuscript and Catrin Tauber for excellent figure design. The work was supported by DFG TRR179, TP1.

      Supplementary data

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