Journal of Hepatology Update: Hepatitis C

Basic

A prophylactic hepatitis C virus vaccine: A distant peak still worth climbing Thomas F. Baumert1,2,3,4,⇑, Catherine Fauvelle2,3, Diana Y. Chen1, Georg M. Lauer1,⇑ 1

Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical School, USA; 2Inserm Unité 1110, France; 3Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg, France; 4Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France

Summary Hepatitis C virus (HCV) infects an estimated more than 150 million people and is a leading cause of liver disease worldwide. The development of direct-acting antivirals (DAAs) will markedly improve the outcome of antiviral treatment with cure of the majority of treated patients. However, several hurdles remain before HCV infection can be considered a menace of the past: High treatment costs will most likely result in absent or limited access in middle and low resource countries and will lead to selective use even in wealthier countries. The limited efficacy of current HCV screening programs leads to a majority of cases being undiagnosed or diagnosed at a late stage and DAAs will not cure virus-induced end-stage liver disease such as hepatocellular carcinoma. Certain patient subgroups may not respond or not be eligible for DAA-based treatment strategies. Finally, reinfection remains possible, making control of HCV infection in people with ongoing infection risk difficult. The unmet medical needs justify continued efforts to develop an effective vaccine, protecting from chronic HCV infection as a mean to impact the epidemic on a global scale. Recent progress in the understanding of virus– host interactions provides new perspectives for vaccine development, but many critical questions remain unanswered. In this review, we focus on what is known about the immune correlates of HCV control, highlight key mechanisms of viral evasion that pose challenges for vaccine development and suggest areas of further investigation that could enable a rational approach to vaccine design. Within this context we also discuss insights from

Keywords: Hepatitis C; Vaccine; T cells; Antibodies. Received 2 June 2014; received in revised form 4 August 2014; accepted 5 September 2014 ⇑ Corresponding authors. Addresses: Inserm U1110, Université de Strasbourg, 3 Rue Koeberlé, F-67000 Strasbourg, France. Tel.: +33 368853703 (T.F. Baumert). Massachusetts General Hospital, Warren 1019A, 55 Fruit Street, Boston, MA 02114, USA. Tel.: +1 617 724 7515 (G.M. Lauer). E-mail addresses: [email protected] (T.F. Baumert), glauer@mgh. harvard.edu (G.M. Lauer). Abbreviations: DAA, direct-acting antivirals; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HHV, human herpes viruses; CMV, cytomegalovirus; EBV, Epstein-Barr virus; nAbs, neutralizing antibodies; TRL, triglyceride-rich lipoproteins; LDL, low-density lipoproteins; LVP, lipoviral particles; VLP, viruslike particles.

recent HCV vaccination studies and what they suggest about the best way to go forward. Ó 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction The arrival of powerful new direct-acting antivirals (DAA) will dramatically change the care of patients chronically infected with hepatitis C virus (HCV) [1]. Individuals who are aware of the infection, have access to state of the art medical care and have prescription coverage for these drugs can expect to be cured from the infection in almost all cases – yet the question remains how many of the more than 150 million people in the world, infected with HCV, will fall into this group; so far the following key challenges remain [1]: First, high costs will limit access to drugs even in high resource countries where it remains to be seen to what degree the new treatments will reach key patient populations, especially those with the highest risk profiles [2]. In countries with less medical and financial resources, some of which have the highest incidence of HCV infection, the hurdles will even be higher [3]. Even with dramatically reduced prices for the new DAA therapies that have recently been announced by the pharmaceutical industries for countries such as India and Egypt [3], it is far from clear into how many successfully treated individuals this will translate. Second, in the absence of effective screening programs, HCV infection is often diagnosed at a late stage or rarely diagnosed at all (in low and middle income countries) and DAAs will not cure already established liver disease such as hepatocellular carcinoma. Third, DAAs may have limitations in certain clinical scenarios such as genotype 3 infection, advanced or decompensated liver disease, transplantation, treatment experienced patients. Furthermore, their use in children and pregnant women remains to be determined [1,4]. Fourth, reinfection remains possible even after successful curative therapy, especially in patient populations with continued exposure to HCV. Importantly, in this context, the United States is experiencing a revitalized epidemic of intravenous

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JOURNAL OF HEPATOLOGY

Key Points •

A Resolving infection Neutralizing Ab Total Ab Strength of Ab CD8 T cells CD4 T cells ALT Viral load

0

An effective prophylactic HCV vaccine remains a critically important weapon to halt the global HCV epidemic

•

It is unclear whether sterilizing immunity will be feasible, but protection from chronic infection would be as effective in preventing liver disease

•

Both T cells and neutralizing antibodies are important for HCV control

•

HCV viral diversity is the greatest challenge to an effective vaccine

•

Testing HCV vaccines in clinical trials and learning from failure is key for advancing the field

Infection

24 36 Weeks after infection

48

Neutralizing Ab Total Ab

Infection

There is reason to be optimistic about the possibility of an effective HCV vaccine. In contrast to most other chronic viral infections in humans, such as human immunodeficiency virus (HIV), human herpes virus (HHV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV), HCV infection is spontaneously resolved in a significant number of infected individuals [10] (Fig. 1). In addition, the 20–30% of patients with spontaneous control of HCV are also much more likely to clear HCV after a second infection [11]. Furthermore, spontaneous clearance has been described even in patients after long-term chronic infection [12]. These observations suggest that with the right immune response, chronic hepatitis C is preventable. Given that spontaneous resolution of HCV does not reliably prevent infection upon reexposure to the virus [11], the feasibility of a vaccine conferring sterilizing immunity is questionable. While complete prevention of infection would be preferable, a vaccine that reliable terminates infection (Fig. 2) would have comparable impact, since acute infection with HCV is usually asymptomatic with low morbidity and almost zero mortality [10]. On the other hand, serious hurdles to an effective HCV vaccine are evident and pose a major challenge. Foremost is the extreme diversity of the virus that dwarfs even that of HIV. With less than 75% conservation at the amino acid level between each of the 7 major genotypes [13], a pan-genotypic vaccine would be a major accomplishment. In addition, despite intense basic and clinical immunological research in HCV and HIV infection, the key elements of a successful immune response remain ill-defined.

12

B Persistent infection

0

How likely is an effective HCV vaccine?

Basic

heroin use, resulting in increasing number of new infections with HCV [5]. Interrupting transmission networks through aggressive treatment programs in this patient group may be possible [6], but likely will be less efficient than a prophylactic vaccine. Given these challenges and despite the justified enthusiasm about the new therapies available now and in the near future, it is plausible that it will take an efficient HCV vaccine to really curb or even end this epidemic on a global scale [7]. Although detailed studies, modelling the impact of a vaccine on HCV eradication, are limited, previous data estimate that a prophylactic HCV vaccine with 80% efficacy would have a substantial impact on the incidence of chronic HCV infection and would be highly economically attractive [8,9].

12

24 36 Weeks after infection

48

Fig. 1. Dichotomous natural outcome of HCV infection. After exposure to HCV, patients display high levels of viraemia within days. Adaptive immune responses (T cells and antibodies) develop after 6–10 weeks, in almost all patients, at which time viraemia is at least partially controlled in most subjects and elevated liver enzymes may appear. Maintenance of HCV-specific CD4 and CD8 T cell responses and early development of neutralizing antibodies are associated with spontaneous eradication of HCV, usually within 24 weeks after infection (A). In contrast, subjects developing chronic HCV infection quickly lose CD4 responses, followed by a decline of the CD8 response and a delayed neutralizing antibody response (B).

Therefore, it is not exactly clear what kind of immune response needs to be elicited by a vaccine in order to be effective. In the end, these questions will most likely be answered by combining additional translational research in acute and chronic HCV infection with clinical trials of vaccines in humans. These attempts at vaccination might not be successful right away, but carefully planned human trials and the analysis of the clinical, virological and immunological features of vaccination and the dissection of the response to HCV exposure or infection will contribute essential information to improve vaccine strategies.

T cell responses and control of HCV infection Evidence from chimpanzee and human studies support critical roles for CD4 and CD8 T lymphocyte responses in the control of primary and secondary HCV infection [14–28]. The strongest

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Journal of Hepatology Update: Hepatitis C

A Sterilizing immunity Neutralizing Ab Strength of Ab CD8 T cells

Basic

CD4 T cells ALT Viral load

i.e. the duration until T cell responses appear, have an impact on disease outcome is unclear and will be hard to ascertain in human infection, given the challenges in identifying the specific date of exposure. But if the timing of the response is important, a vaccination that enables a much earlier T cell response to infection would provide a critical advantage to achieve viral control. Other key determinants for the effectiveness of the T cell response could be the breadth and magnitude of the response, targeting of certain epitopes or viral regions, T cell functionality and T cell regulation. In the next paragraphs we will lay out what is currently known about T cell responses in spontaneously resolving vs. chronic HCV infection and what still needs to be defined in the future. CD4 T cells

Vaccination

HCV exposure

B Prevention of chronicity Neutralizing Ab

Vaccination

HCV exposure

Fig. 2. Two pathways to a prophylactic HCV vaccine. The ideal HCV vaccine would induce sterilizing immunity, i.e. would completely prevent any HCV infection. Such a vaccine would most likely be based on strongly cross-reactive neutralizing antibodies to HCV (A). Since so far sterilizing immunity has appeared elusive, many vaccine approaches have focused on preventing chronic infection, and thus liver disease (B). This is based on the observation of natural immunity against HCV and is expected to be primarily mediated by HCV-specific CD4 and CD8 T cells, though recent data suggest that induction of neutralizing antibodies could further enhance efficacy.

evidence comes from T cell depletion experiments in the chimpanzee model, where a lack of either CD4 or CD8 T cells during exposure and early infection was associated with persistent viraemia [18,26]. The importance of CD4 and CD8 cells for the outcome of HCV is further supported by human population studies that identified strong associations between disease resolution and certain human leukocyte antigen (HLA) class I and II alleles in various ethnic populations [29–34]. Since in almost all cases spontaneous control of HCV is only achieved in the acute phase of infection (first 24 weeks, Fig. 1), the main focus has been to study acute phase hepatitis to determine the relationship between HCV-specific T cell responses and viral clearance. Interestingly, HCV-specific CD4 T helper- and CD8 cytotoxic T lymphocytes (CTL) responses develop quite late, being first detectable in the blood 6–10 weeks after infection in both self-limited and chronically evolving hepatitis C [22–24,35,36], despite high levels of HCV viraemia already detectable within a week of exposure [15,37]. Whether the kinetics of the response,

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The lack of HCV-specific CD4 T cell responses that proliferate after antigenic stimulation in vitro is the hallmark of persistent HCV infection [38–40]. In contrast, self-limited infection is characterized by a broad range of HCV antigens inducing CD4 proliferation in vitro and these assays remain positive for years and even decades after the infection is cleared [39,40]. This is indicative of a severe defect in HCV-specific CD4 T cells already at the earliest stage of the host immune response. Apart from this specific defect, no conclusive differences have been observed so far between HCV-specific CD4 T cells from different infection outcomes in the initial phase of acute hepatitis. It was previously assumed that the breadth and magnitude of the CD4 response was directly linked to HCV clearance, since only individuals who had spontaneously resolved HCV had detectable Th-1 responses, targeting a broad range of epitopes [39,41]. However, a recent study from our laboratory has shown that most individuals progressing to chronic infection develop CD4-responses against HCV antigens with similar breadth and magnitude as those with self-limited infection during the earliest phase of the T cell response [42]. Only with the establishment of persistent viraemia are additional defects observed beyond the lack of CD4 T cell proliferation, the most important being the physical deletion of HCV specific CD4 T cells from the blood. Thus, a key feature of a successful immune response against HCV is the maintenance of functional virus specific CD4 T cell responses in the face of ongoing viral replication. Whether such responses should preferably target certain epitopes or viral regions is not known, as previous studies were unable to identify immunogenic regions associated with distinct outcomes [42]. Another unanswered question is whether the phenotype of the CD4 T cell response, elicited by a vaccine, is relevant. Only recently have we begun to fully appreciate the heterogeneity of the CD4 T cell response, with Th1, Th2, Th17, Th22, T follicular helper and T regulatory cells being described [43], and how these different CD4 T cell populations orchestrate the immune response in very distinct ways. In this context it needs to be remembered that most studies on the CD4 T cell response in the aforementioned studies in HCV infection were performed by functional tests skewed to detect Th1 responses, potentially missing the contribution of other Th subsets to disease resolution or HCV persistence. Extending our viewpoint, Kared et al. recently reported that the expansion of Th17- HCV-specific CD4 T cells, characterized as CD161hiCCR6+CD26+ and producing IL-17A and IL-21, was associated with disease resolution [44]. In contrast, reduced frequencies of IL-21 producing HCV-specific CD4 T cells

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combined with exhaustion and reduced proliferation of HCV-specific CD8 T cells were observed in viral persistence. The reduction of IL-21 producing virus-specific CD4 cells in chronically evolving infection appeared to be through Gal-9 expressing regulatory T cells that activate TIM-3 on HCV-specific CD4 T cells [44]. Defects were compensated in culture with supplementation of IL-21 that promoted cell proliferation, as well as cell survival and the inhibition of exhaustion of HCV-specific CD8 T cells. Another interesting observation was contributed by Rowan et al. who have suggested a possible role for Th-17 cells in the eradication of HCV [45], although the production of IL-17 could be suppressed by IL-10 and transforming growth factor beta (TGF-b), excreted by monocytes in response to NS4 protein [46]. Besides these studies, our understanding of the role of different CD4 T cell populations in HCV, as well as in other acute and chronic viral infections, remains rather limited and warrants more detailed research. Based on our current knowledge, the aim for a vaccine is to induce a broadly directed CD4 response against HCV that responds quickly and lastingly in the face of a viral challenge (Fig. 2). Whether it is necessary to fine-tune that response, for example in terms of the functionality or phenotype of the induced CD4 T cells, is currently an open question. CD8 T cells HCV-specific CD8 T cells have higher frequencies compared to CD4 T cell responses and direct visualization and phenotyping of CD8 T cells using HLA multimers is technically much easier; thus, many more studies have been performed on the early CD8 response than on the CD4 response in HCV infection. Despite the plethora of studies on CD8 T cells, it is still not entirely clear what kind of CD8 T cell response is required to achieve full control of HCV. Again, as with CD4 responses, vigorous and broadly directed CD8 responses were observed in self-limited disease [17,28], but if individuals were studied within the initial 6 months of HCV infection, similarly strong CD8 responses could be seen in patients with a chronic course of infection [22]. What we understand better, compared to the CD4 responses, are the different mechanisms that render the HCV-specific CD8 T cell response debilitated and unable to control the virus throughout the progression to persistent viraemia.

Generally, at least two pathways are involved in viral evasion from the CD8 response in chronic hepatitis C (Table 1). One is the emergence of HCV variants that carry amino acid substitutions in or around targeted CD8 epitopes that are associated with a lack of recognition by epitope-specific CD8 T cells. CD8 T cell-mediated viral escape occurs already during acute infection [47] and once persistent viraemia is fully established, up to 50% or more of all targeted CD8 epitopes can be mutated, depending on the cohort [48–50]. These mutations typically come at a cost for the virus, since often the escape variants show less replicative fitness than the original infecting virus [51,52]. Notably, carriers of the HLA class I alleles B⁄27 and B⁄57 have a higher likelihood of clearing HCV [53,54] and this has been associated with CD8 responses against conserved epitopes within the non-structural protein 5B, with escape mutations within these epitopes resulting in a profound viral fitness cost [54–56]. In some cases, the virus evolved even further, with additional mutations that compensate for the initial loss of viral fitness [52]. However, this multi-step process requires time, especially when viral replication is somewhat diminished, which might explain why targeting certain epitopes confers a higher likelihood of viral control. Besides viral fitness, other factors such as T cell receptor (TCR) breadth, diversity, functional avidity, and lack of CD4 T cell helper cells may also play a role in determining the emergence of viral escape mutations. Whether any of these factors are actually decisive in allowing the virus to escape and thus, are primarily the cause for viral persistence, or whether viral escape mutations are just a consequence of other factors allowing viral persistence with continuous high HCV replication rates is unknown. But with regards to vaccine development, it is important to keep in mind that certain qualities of the vaccine-induced CD8 response might influence the likelihood of viral escape. The other major mechanism, impeding effectiveness of the HCV-specific CD8 T cell response in chronic infection, is T cell exhaustion, which is characterized by an increasing failure of T cells to exert their effector functions and at its final step by physical deletion of virus-specific T cell populations [57,58]. Growing evidence suggests that intrinsic regulatory pathways (such as regulation through T cell inhibitory receptors) as well as extrinsic regulatory pathways (such as immunoregulatory cytokines or regulatory T cells) contribute to T cell exhaustion. In terms of intrinsic regulatory pathways, it has been shown that

Table 1. Mechanisms of escape from T cell responses.

Escape route Mechanism Viral escape HCV genetic variability (based on high transcription error rates due to lack of proof-reading function of the viral polymerase) leads to emergence of viral mutations variants that are not recognized by the existing T cell response. Frequently observed in response to CD8 T cells; escape from CD4 responses is less studied but seems rare T cell Activation of various T cell inhibitory pathways (e.g., PD-1, CTLA4, TIM-3) exhaustion leads to viral exhaustion. Co-expression/activation of multiple receptors (intrinsic) leads to more severe exhaustion. Better examined for CD8 T cells, but also operative on CD4 T cells T cell exhaustion (extrinsic)

T cell regulatory cytokines (e.g., IL-10) and regulatory cells (e.g. FoxP3+ CD4 Tregs) inhibit T cell function. Complexity and relevance of these interactions not fully understood

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[Ref.] Timm J. et al., J Exp Med 2004 Cox A. L. et al., J Exp Med 2005 Kuntzen T. et al., J Virol 2007 Erickson A.L. et al., Immunity 2001 Fuller M.J. et al., Hepatology 2010 Urbani S. et al., J Virol 2006 Kasprowicz V. et al., J Virol 2008 Bengsch B. et al., PLoS Pathog 2010 McMahan R.H. et al., J Clin Invest 2010 Kroy D. et al., Gastroenterology 2014 Accapezzato D. et al., J Clin Invest 2004 Sugimoto K. et al., Hepatology 2003 Rushbrook S.M. et al., J Virol 2005 Kared H. et al., PLoS Pathog 2013

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Basic

JOURNAL OF HEPATOLOGY

Basic

Journal of Hepatology Update: Hepatitis C virus-specific CD8 T cells in chronic infection may simultaneous express different inhibitory receptors such as PD-1, CD160, CTLA-4, TIM-3, 2B4, and KLRG1 [59]. The number of receptors simultaneously expressed by a CD8 T cell seems to determine the severity of T cell dysfunction [59] and the extent to which the dysfunction is reversible. However, the relationship between expression of inhibitory receptors and T cell exhaustion is not straightforward, since during the early immune response T cells typically express high levels of T cell inhibitory receptors, especially PD-1 [60], and this is observed even in subjects who spontaneously control HCV [61], or after immunization with the highly efficacious yellow fever vaccine [62]. Continued stimulation with antigen drives higher levels of inhibitory receptor expression and exhaustion in chronic infection [63]; but the sweet spot for the perfect equilibrium between the physiological role of these pathways, in preventing an overshooting immune response that could result in immune–mediated disease, and their potential contribution to T cell failure, has yet to be defined. Similar questions remain for extrinsic regulatory pathways, for example IL-10 producing T cells or CD4+Foxp3+ regulatory T cells, that have been suggested to expand in blood and liver during chronic infection and are thought to contribute to HCV-specific CD8 T cell exhaustion [41,64–68]. Overall, for the vaccine to be effective against HCV persistence, there is little doubt that robust and broad T cell responses are essential in an immune response to HCV (Fig. 2). However, more work is needed to better define functional and phenotypic properties of T cell responses that are associated with disease resolution, so that we could harness this information to ensure a vaccine-induced response that is effective, long-lasting and resistant to HCV-mediated immune evasion.

in patients spontaneously clearing HCV infection [73]. Interestingly, in a well-characterized patient with established chronic HCV infection who later eliminated HCV spontaneously, clearance also correlated with the appearance of nAbs [12]. Further support for the contribution of nAbs in the control of HCV comes from studies in frequently exposed people who inject drugs intravenously. Osburn et al. observed an increased rate of viral clearance after HCV re-exposure compared to primary infection (83% clearance after re-exposure vs. 25% during primary infection) [74]. Importantly, whereas cross-reactive nAbs are rarely present in the sera of patients who develop chronic HCV infection, they are detected in 60% of the subjects who cleared the secondary infection [74]. The potential capacity of nAbs to prevent HCV infection is also supported by observations in liver transplantation with liver graft infection. A retrospective study revealed that treatment of HCV-infected patients by hepatitis B immunoglobulins (HBIG), hypothesized to incidentally contain anti-HCV antibodies, during liver transplantation (LT) decreased HCV recurrence [75]. Interestingly, HCV-negative patients undergoing LT, treated by the same anti-HCV contaminated HBIG and exposed to HCV, presented a lower frequency of HCV acquired infection [75]. These observations indicate that nAbs may protect against HCV infection, especially in the context of LT where viral evasion from nAbs has been described as a key feature of liver graft infection [76,77]. Overall there is compelling clinical evidence that nAbs, targeting HCV, are contributing to both viral clearance during early infection and protection against de novo HCV infection. These observations support the concept that the development of an effective HCV vaccine should include the induction of broadly cross-neutralizing antibodies (Fig. 2). Mechanisms of viral evasion from host neutralizing antibodies

Impact of neutralizing antibodies for prevention and control of HCV infection Neutralizing antibody (nAb) responses are critical components of the host defence during viral infections and are recognized as a key element in the protective immune response against infection elicited by many prophylactic vaccines [69,70]. While their potential role in preventing chronic infections with hypervariable viruses such as HIV or HCV has been less clear, recent studies in chimpanzees and clinical cohorts as well as mechanistic studies in HCV cell culture and small animal models have revitalized interest in the role of nAbs for protection against chronic HCV infection. Neutralizing antibodies and control of HCV infection in clinical cohorts Several studies have investigated the role of antibodies in the control of HCV infection. Whereas viral clearance can occur in the absence of nAbs in humans and chimpanzees [71], several studies have demonstrated that nAbs may have an important role in controlling infection. Pestka et al. studied the role of nAbs during acute HCV infection [72]. The follow-up over 17 years in a cohort of women accidentally infected by the same HCV strain reveals a strong correlation between viral clearance and the rapid induction of nAbs during the acute phase of HCV infection [72]. These conclusions were recently supported by Osburn et al. who observed more rapidly developing broad humoral responses

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The efficacy of HCV nAb and also the design of an efficient vaccine are greatly impacted by the considerable capacity of HCV to alter its sequence and thus to escape humoral and cellular immune responses. Notably, nAbs are mainly blocking viral entry by targeting the HCV envelope glycoproteins (GPs) E1 and E2 and their interaction with host entry factors. However, multiple mechanisms enable the virus to persist in the presence of neutralizing antibodies and evade virus-neutralization (Table 2). HCV circulates in patients as a pool of variants, genetically distinct and continuously evolving. Even during established chronic infection, the virus continues to respond to immune pressure through the selection of mutants that are not neutralized by circulating nAbs [78]. A similar observation has been made in acute liver graft infection, where predominantly such viral escape variants infect the liver graft [76]. HCV can escape neutralization through single point mutations such as modifications of glycosylation sites [79,80]. The virus can also disseminate using cell-to-cell transmission, thus avoiding the extracellular compartment with its presence of nAbs. [81,82]. In addition HCV utilizes host-specific factors to escape nAbs. Fofana et al. also demonstrated that mutations in the GP E2, from a variant selected during LT, conferred viral escape to humoral responses by altering the use of the T cell receptor CD81 [77]. Escape through altered use of the scavenger receptor B1 has also been described [83]. HCV circulates in the blood in association with triglyceride-rich lipoproteins (TRL) and lowdensity lipoproteins (LDL) forming hybrid lipoviral particles

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JOURNAL OF HEPATOLOGY Table 2. Mechanisms of evasion from antibody responses.

[Ref.] Dowd K.A. et al., Gastroenterology 2009

Gal-Tanamy M. et al., Proc Natl Acad Sci 2008

Von Hahn T. et al., Gastroenterology 2007 Liu L. et al., Hepatology 2012 Ray S.C. et al., J Exp Med 2005 Helle F. et al., J Virol 2007 Helle F. et al., J Virol 2010

Glycosylation of structural proteins Cell-to-cell transmission

Numerous glycans present at the surface of HCV GPs reduce access of nAbs to their epitopes HCV can disseminate directly using cell-to-cell transmission thus avoiding extracellular exposure to antibodies

Timpe J.M. et al., Hepatology 2008 Brimacombe C.L. et al., J Virol 2011 Xiao F. et al., PLoS Pathogens 2014

Association with lipoproteins Induction of interfering antibodies Mutations altering entry factor use

HCV association with lipoproteins reduces viral sensitivity to nAbs by obscuring epitope to which antibodies bind

André P. et al., J Virol 2002 Grove J. et al., J Virol 2008

Basic

Escape route Mechanism Viral diversity HCV genetic variability allows the virus its adaptation to the host environment and evasion from humoral responses

Efficient neutralization of HCV by nAbs can be disturbed by the presence of Zhang P. et al., Proc Natl Acad Sci 2009 interfering antibodies produced incidentally by the host Mutations in the GPs confer viral escape to humoral responses by altering the use of the host cell receptors

Fofana I. et al., Gastroenterology 2012

GP, envelope glycoproteins; nAbs, neutralizing antibodies.

(LVP) [84]. By obscuring epitopes to which nAbs bind, these associations may reduce the sensitivity of particles to antibody mediated neutralization and facilitate HCV persistence [83,84]. Finally, the immune system itself may incidentally enable HCV persistence by the presence of antibodies capable to interfere and block activity of nAbs [85,86]. Despite these challenges, broadly cross-neutralizing monoclonal antibodies, directed against the E2 envelope glycoprotein that can efficiently block HCV infection of various genotypes, have been isolated from HCV-chronically infected patients or immunized animals [87–89]. The characterization of these antibodies also led to a better understanding of the GPs structure, which is essential for defining epitopes that promise to be effective immunogens. Just recently Kong et al. have successfully characterized the structure of the E2 core by obtaining a crystal of the E2 core in association with the fragment antigen-binding region (FAb) of a high avidity binding human monoclonal antibody [90]. Besides revealing a globular structure of E2 that has also been affirmed by others [91], this study identified an essential and conserved epitope on E2 that may be used for vaccine design [90]. Undoubtedly, the effectiveness of HCV to evade host immune responses remains a major challenge for the development of a protective vaccine. However, recent progress in the better understanding of both HCV structure and immunology have renewed the hope that a sterilizing, antibody-based vaccine for HCV might be a possibility.

Prophylactic and therapeutic vaccine approaches in chimpanzees and humans Within the last two decades, several prophylactic and therapeutic vaccine candidates based on diverse strategies (induction of antibody vs. T cell responses), using different delivery approaches

(recombinant proteins, peptides, DNA, virus-like particles, viral vectors, yeast) and targeting different regions of the HCV polyprotein have been developed in preclinical animal models including rodents and chimpanzees. Several candidates were also evaluated in clinical trials in healthy volunteers or in HCV infected patients (Table 3). In this overview we will focus on the more advanced vaccine candidates, for which data in either chimpanzees or humans are publicly available. Prophylactic vaccine candidates A prophylactic vaccine remains the most effective approach to decisively impact an epidemic or even eradicate a pathogen globally. In the case of HCV infection, such a vaccine would ideally confer sterilizing immunity, but the reliable prevention of chronic infection would be an acceptable alternative, since almost all the sequelae of HCV infection result later during persistent infection. Prevention of HCV chronicity is the aim of the prophylactic T cell vaccine approach that is currently the furthest in clinical development and that has shown by far the strongest immunogenicity with regard to eliciting T cell responses. The original idea was to use the excellent immunogenicity of adenoviruses, and through selection of adenovirus serotypes that rarely infect humans (human adenovirus 6, Ad6; chimpanzees adenovirus 3, ChAd3) avoid interference with pre-existing nAbs. Folgori et al. tested the efficacy of two immunizations by MRKAd6, coding for HCV NS proteins, followed by boost injections of MRKAd24NSmut and later a plasmid DNA encoding NS proteins in chimpanzees [92]. The vaccination induced vigorous and broadly directed T cell responses, with a further significant enhancement of CD4 and CD8 T cell responses after MRKAd24NSmut and DNA boosts. After HCV challenge, all animals became infected but HCV replication was contained (100 times lower

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Journal of Hepatology Update: Hepatitis C Table 3. Examples for vaccine approaches hat have been investigated in preclinical studies in chimpanzees or clinical trials in humans.

Approach Peptides/

Prophylactic/ therapeutic Prophylactic

Immunogen-adjuvant E1E2

Stage of development Chimpanzees

Current status [Ref.]

Phase I (30 volunteers) Phase I (60 volunteers) Phase I (50 volunteers) Phase I/II (128 volunteers/60 patients)

Published

Phase II (50 patients) Phase II (71 patients) Phase II (28 patients) Chimpanzees

Published

Firbas C. et al., Vaccine 2006; Klade C.S. et al., Gastroenterology 2008; Schlaphoff V. et al., Vaccine 2007 Klade C.S. et al., Vaccine 2012

Completed

NCT00601770

Published

El-Awady M.K. et al., Vaccine 2013

Published

Chimpanzees Phase II (15 patients) Phase II

Published Published Recruiting

Elmowalid G.A. et al., Proc Natl Acad Sci USA 2007 Forns X. et al., Hepatology 2000 Alvarez-Lajonchere L. et al., J Viral Hepat 2009 NCT01335711

Chimpanzees

Published

Folgori A. et al., Nat Med 2006

Phase I (40 volunteers) Phase I/II (68 + 276 IDU) Phase I (19 volunteers /14 patients) Phase I (15 patients)

Published Recruiting

Barnes E. et al., Sci Transl Med 2012 NCT01436357

Recruiting

NCT01296451

Published

Habersetzer F. et al., Gastroenterology 2011 DiBisceglie A.M. et al., Gastroenterology 2014

Published

proteins

Basic

Core-iscomatrix™ E1E2-MF59C.1 E1E2 Therapeutic

IC41poly-L-arginine

IC41imiquimod IC41

HCV-LP

Prophylactic

DNA vector

Prophylactic Therapeutic

Viral vector

Prophylactic

Therapeutic

E1E2 (Cenv3) Core E1E2 E2 Core E1E2 (CIGB-230) NS3/4A (ChronVac-C) Ad6NSmut DNA-NSmut Ad6NSmut ChAd3NSmut AdCh3NSmut MVA-NSmut AdCh3NSmut MVA-NSmut TG4040

Published Recruiting Published

Choo Q.L. et al., Proc Natl Acad Sci USA 1994; Meunier J.C. et al., J Infect Dis 2011 Drane D. et al., Hum Vaccines 2009 Frey S.E. et al., Vaccine 2010; Stamataki Z. et al., J Infect Dis 2011 NCT01718834

TG4040 + SOC

Phase II (153 patients)

Published

Ad6NSmut ChAd3NSmut Ad6NSmut MVA-Nsmut + SOC

Phase I (32 patients) Phase I (9 patients)

Completed

NCT01094873

Completed

NCT01701336

SOC, standard of care; HCV-LP, HCV-like particles.

viral loads) and 4/5 immunized chimpanzees cleared the virus with significantly shorter duration of viraemia compared to unvaccinated animals. No increase in liver enzymes was observed in any of the immunized animals [92]. Based on these results, a phase I clinical trial was initiated with 40 healthy individuals, evaluating the safety and efficacy of immunization using a single or double prime of Ad6NSmut vector, followed by a boost with the ChAd3NSmut vector, or by using the inverse vaccination sequence [93]. Both vectors induced specific and broad crossreactive T cell responses with cytokine secretion in the vaccinees. Importantly, a functional and proliferating long-term T cell population was still detected after one year [93]. Two additional clinical trials (phase I and phase I/II) are currently recruiting S40

participants (NCT01296451, NCT01436357) with a further modified approach, employing ChAd3NSmut followed by a modified vaccinia Ankara (MVA) virus, the latter being selected for its efficacy in boosting T cell responses [94]. Clinical trial NCT01436357 is the first trial to enrol at risk subjects (injecting drug users) and will therefore have the capacity to evaluate vaccine efficacy in humans. A very distinct approach, employing recombinant proteins and targeting the HCV envelope, was based on the observation that recombinant E1E2 proteins induced cross-nAbs in chimpanzees and seemed to at least partially protect them from HCV infection [95,96]. A phase I clinical trial in 60 healthy volunteers immunized with different doses of HCV E1E2 proteins (genotype

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1a) combined with MF59C.1 adjuvant [97] demonstrated good safety of the vaccine and the induction of envelope-specific CD4 T cell and humoral responses [97]. The neutralization capacity of Abs elicited by the vaccine was tested on HCV pseudoparticles. Evidence for antibodies with neutralizing activities was detected in a significant number but not in all subjects [97]. An in depth analysis of sera from patients of the same cohort showed that all sera neutralized against in vitro infection with heterologous 1a, 1b, and 2a strains [98]. HCV core protein adjuvanted with the ISCOMATRIX™ (that is known for its capacity to increase CD4 and CD8 T cell responses) has also been tested in a phase I clinical trial on 30 healthy individuals [99]. Specific humoral responses (Abs titres >10) were detected in all except one subject (23/24) whereas CD8 T cell responses were detected only in 2/8 subjects who received the highest dose [99]. The use of recombinant HCV proteins in vaccine design in the previously discussed vaccine approaches present some disadvantages especially concerning the correct folding of the proteins and the processes of production [100–102]. To overcome these limitations, Elmowalid et al. developed virus-like particles (VLPs), expressing HCV structural proteins, as a potential vaccine candidate [101]. In this study, performed on chimpanzees, all animals immunized with HCV-like particles (HCV-LPs) developed specific CD4 and CD8 T cell responses with cytokine production. Upon HCV challenge, all four animals became infected but after ten weeks three of them cleared the virus and just one continued to have intermittent viraemia with low HCV RNA titres. After vaccination an increase in the magnitude of peripheral and intrahepatic T cell and proliferative responses against the HCV structural proteins was observed [101]. A clinical trial in humans has not yet been reported using this approach. Other concepts based on subviral/virus-like or native particles are currently being pursued using recombinant hepatitis B virus (HBV) subviral particles expressing HCV envelope epitopes [102], recombinant cell culture-derived HCV (HCVcc) [103] or retroviral pseudotypes expressing HCV envelope glycoproteins [100]. Therapeutic vaccine candidates The recent licensing of DAAs is rapidly changing the care of chronically HCV infected patients, including difficult-to-treat populations, such as patients with liver cirrhosis or HIV coinfection, and it is expected that the vast majority of patients on treatment will achieve sustained virologic response (SVR) [1]. Thus, the impact of therapeutic vaccines in the arsenal of antiviral treatment is most likely limited and most pharmaceutical companies have halted the clinical development of therapeutic vaccines. For this reason, but also because of the rather limited efficacy in published studies, we will only briefly summarize some well-characterized concepts for immunotherapeutic approaches as examples. Despite the so far disappointing results, important lessons might be learned from immunotherapeutic trials in chronic hepatitis C, especially for new approaches to attempt immunotherapeutic control of chronic HBV infection. A strong indication of the difficulties in using therapeutic vaccines as monotherapy for chronic HCV infection comes from a trial using the vaccine approach based on chimpanzee adenoviruses, expressing all HCV NS proteins, already described as a prophylactic vaccine above. While vaccination in healthy volunteers induced extremely broad and vigorous T cell responses [93], this

approach resulted in no significant changes in HCV viraemia when administered in chronically infected patients and only caused a boost of some T cell responses [104]. Importantly, sequencing of the patients’ viruses revealed that only the T cell responses that did not target the autologous circulating virus were preferentially boosted after vaccination [104]. This supports the general immunogenicity of the vaccine, but also highlights the challenge of invigorating exhausted T cell responses that continue to recognize the circulating virus, without the addition of antiviral therapy. Two concepts which have been studied in detail in clinical trials are T cell vaccines using peptides and recombinant poxviruses. IC41 is a peptide vaccine, containing five synthetic peptides from core, NS3 and NS4 proteins that correspond to conserved HLA-A2 restricted CD8 and promiscuous CD4 epitopes, together with poly-L arginine as an adjuvant. Phase I and phase II clinical trials demonstrated that the approach is safe and well-tolerated, though the induced T cell responses were relatively modest, [105–107], especially when compared to responses, induced by the viral vectors described above. No significant changes in HCV RNA were observed when patients with chronic HCV infection were vaccinated [106]. IC41 was also supplemented with imiquimod (a TLR7 agonist) and while there was some reduction of viraemia after 16 weeks, there was no significant correlation between HCV levels and T cell immune responses [108]. Another phase II clinical trial, investigating HCV virological responses following biweekly immunization with IC41, is currently ongoing (NCT00601770). One of the most intensively studied concepts in patients is the use of recombinant poxviruses, expressing HCV proteins containing immunodominant epitopes. TG4040 is a recombinant poxvirus vaccine that expresses the hepatitis C virus (HCV) proteins NS3, NS4, and NS5B. In an open-label, dose-escalating study in patients with mild chronic hepatitis C (CHC), the viral-vectorbased vaccine TG4040 had a good safety profile, but induced weak HCV-specific cellular immune responses and had no sustained effect on the viral load [109]. Several important lessons should be drawn from these studies: The first is that new therapeutic vaccines need to be tested in healthy individuals first and not just in patients with chronic HCV infection. Otherwise one cannot discriminate whether a vaccine is just not sufficiently immunogenic or whether the severe exhaustion of T cells in chronic infection prevented a measurable impact on the T cell response by an otherwise immunogenic agent. Second, when increased immune responses are observed after vaccination in chronic infection, it needs to be tested whether these responses target the circulating virus in the individual patient. Boosting T cells that are not exhausted, due to not seeing the antigen, is easier to achieve but will have no impact on viral replication, since they have no target in the infecting viral strain. Finally, apart from the selection of the best vaccine vector, the question of the right antigen remains open and relevant. The use of a large part of the HCV genome will probably be the best to include in a HCV vaccine, given the typically broad T cell response that is associated with spontaneous resolution of infection and that most likely limits the ability of the virus to evade the immune response. However, antigens need to be carefully chosen, allowing to optimize and broaden the immune response while avoiding potentially detrimental effects, such as peptide competition or the dominance of responses with limited efficacy.

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JOURNAL OF HEPATOLOGY

Journal of Hepatology Update: Hepatitis C

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Conclusions Despite the recent dramatic progress in antiviral therapies for chronic HCV infection, a prophylactic HCV vaccine remains an extremely desirable asset in order to control the HCV epidemic in all populations and in all regions of the world. Recent advances in vaccine design have finally led to vaccination approaches that reliable induce robust CD4 and CD8 T cell responses, similar to what is observed in acute HCV infection. Through ongoing clinical trials in persons at high risk for HCV infection, we should soon find out whether the pre-existence of broadly directed and vigorous T cell responses is sufficient to reliably prevent chronic HCV infection, and, if protection can be achieved, whether it extends significantly beyond the HCV genotype from which the vaccine was derived. The recent structural characterization of the interaction of cross-neutralizing antibodies with the viral envelope glycoprotein E2 as well as understanding of the molecular mechanisms of viral evasion from neutralizing antibodies, has stimulated new concepts for the rational design of B cell vaccines. Thus, in the meantime it seems prudent to further investigate how neutralizing antibodies can contribute to HCV clearance or even establish sterilizing immunity, how immunity can be broadened to cover as many HCV strains as possible, and what kind of T cell response profile is most strongly associated with efficient control of HCV. Finally, since the chimpanzee model is no longer available in the US and Europe, novel and innovative approaches to assess the efficacy and safety of future vaccine candidates are urgently needed.

Financial support G.M. Lauer is supported by the National Institutes of Health (NIH) Grant U19 AI082630, NIH Grant U19 AI066345 and NIH Grant R01 AI105035. T.F. Baumert is supported by the French Cancer Foundation (ARC IHU201301187), French Research Agency (LABEX ANR-10-LAB-28) and the European Commission (ERC2008-AdG-233130-HEPCENT, EU FP7 HepaMab; INTERREG-IVRhin Supérieur-FEDER-Hepato-Regio-Net2012).

Conflict of interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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