April 14, 2013

Expert Review of Gastroenterology and Hepatology

Chao Shi, Alexander Ploss

Expert Rev Gastroenterol Hepatol. 2013;7(2):171-185.

Abstract and Introduction
Abstract

Hepatitis C virus (HCV) infection is a major global health problem as it has a high propensity for establishing chronicity. Chronic HCV carriers are at risk of developing severe liver disease including fibrosis, cirrhosis and liver cancer. While treatment has considerably improved over the years, therapy is still only partially effective, and is plagued by side effects, which contribute to treatment failure and is expensive to manage. The drug development pipeline contains several compounds that hold promise to achieve the goal of a short and more tolerable therapy, and are also likely to improve treatment response rates. It remains to be seen, however, how potent antiviral drug cocktails will affect the hepatitis C burden worldwide. In resource-poor environments, considerable costs, inadequate infrastructure for medical supervision and distribution may diminish the impact of future therapies. Consequently, development of novel therapeutic and prophylactic strategies is imperative to contain HCV infection globally.

Prospects in HCV Treatment

An estimated 170 million people, or 3% of the world population, are chronically infected with hepatitis C virus (HCV) (Figure 1). Persistent HCV infection leads to liver cirrhosis and can culminate eventually in hepatocellular carcinomas. Since the discovery of HCV as a causative agent for non-A non-B hepatitis in 1989, constant efforts have been made to improve the outcome of hepatitis C patients. Before 1990, HCV was an incurable disease and monotherapy with IFN-α resulted in a sustained virologic response (SVR) in only 10% of the treated patients.[1] Combination therapies of pegylated interferon (peg-IFN) with ribavirin (RBV) were later applied and became the standard-of-care for HCV. This combination treatment improved SVR rates, but fell short of curing HCV infection in more than 50% of patients with HCV genotype 1 and had an even worse outcome or was contraindicated in patients with comorbidities such as HIV infection, cirrhosis, transplant recipients or in African–Americans,[2,3] thus creating a need for more effective therapies. The introduction of direct-acting antivirals (DAAs), which are inhibitors of virally encoded protein functions, to the market represents a milestone in HCV therapy. Incivek® (generic name: telaprevir; Vertex, MA, USA) and Victrelis™ (generic name: boceprevir; Merck, NJ, USA), two drugs that interfere with the virally encoded NS3/4A protease, were approved by the US FDA in 2011. Addition of telaprevir or boceprevir to the peg-IFN/RBV regimen increases SVR rates in certain clinical trial cohorts to 60–70%.[4–7] In the meantime, many candidates of HCV DAAs, including the next generation of protease inhibitors, NS5A inhibitors and polymerase inhibitors, are at the late stage of development. Recent clinical trials have demonstrated that combinations of orally administered DAAs with different mechanisms of action can cure chronic HCV infection with 90% rate,[8–10] although the optimal results remain to be confirmed in larger patient cohorts. The availability of these new, presumably more potent DAAs is expected to revolutionize the standard-of-care of HCV infection, with a promise to cure HCV with an all-oral, IFN-free cocktail regimen. In addition, drugs targeting host factors that are essential for HCV replication, such as cyclophilin A and miR122, are also in the pipeline. A drastic expansion of the ammunition for treating HCV infection is expected in the next few years.

779243-fig1

Figure 1.

Relative hepatitis C virus prevalence and distribution of common genotypes. The numbers in the figure indicate the most prevalent HCV genotype(s) in the respective regions. Data taken from WHO (2006) and [20,152].

Because HCV, unlike HIV and hepatitis B virus (HBV), does not integrate into the host genome, successful treatment with antiviral therapies is able to eradicate the virus from individuals. A 90% cure rate of new antiviral drugs suggests that the number of existing patients will shrink in the USA and other developed counties, where effective treatment can be applied. Moreover, as a consequence of implementation of rigorous blood supply screening for HCV since 1991, the number of new HCV infections in the USA fell from a peak of 180,000/year in the mid-1980s to 16,000/year in 2009 ([11] and CDC data). Currently, the most common cause of ongoing HCV transmission is the sharing of contaminated needles or syringes by injection drug users (IDUs; in the USA) and unsafe medical practices (globally). Some studies have projected the prevalence in the USA to decline from 3.2 million in 2005 to 2.5 million in the 2020s without considering the utilization of more effective antiviral regimens.[12,13] Using a similar but simplified approach, the authors predict that with the application of new DAAs, which can potentially improve the rate of SVR from 50 to 90%, the infected population in the USA will decline below 2 million in 2020s (Figure 2 & ). Furthermore, since the new regimens can be applied to patients who were previously ineligible for the standard-of-care treatment due to their insensitivity or intolerance to interferons, the percentage of patients receiving treatment is expected to increase. Currently, only 10–27% of people diagnosed with HCV infection are offered treatment.[14] If we assume the treatment rate increases to 50% with the application of new DAAs, the projection of the US prevalence will be below half a million in 2020s (Figure 2). However, this optimistic outlook comes with caveats.

Table 1. Variables and assumptions used in the projection.

Variable Base value Explanation Ref.
Prevalence of chronic infection 3,000,000 in 2012 2.7–3.9 million CDC data and [153]
Incidences of new infection 15,000/year Estimated 16,000 for 2009 by CDC, assuming remains stable
Spontaneous clearance rate 18%
[16,154]
Diagnosis rate of infected 20–70% of existing20% of newly infected Increases with ages as symptoms exacerbate; assume 70% in 2022
Treatment rate of diagnosed 10% with peg-IFN/RBV50% with DAAs Increased rate with DAAs due to improved tolerance and efficacy
SVR 50% with peg-IFN/RBV90% with DAAs
[8–10]
Mortality of infected Average: 21,000/year Mortality increases with ages, but decreases with application of DAAs

As new infections in the USA occurs mainly among injection drug user, the transmission rate within this population is assumed stable, given that many prevention programs have been in place, and that low penetration of treatment in this population will keep the number of the infected at a sustained level. DAA: Direct-acting antiviral; Peg-IFN: Pegylated interferon; RBV: Ribavirin; SVR: Sustained virologic response.

779243-fig2

Figure 2.

A projection of hepatitis C virus burdens in the USA for the next decade. Assuming 50% sustained virologic response rate for peg-IFN/ribavirin regimen and 90% for DAAs combo regimen. DAA: Direct-acting antiviral; HCV: Hepatitis C virus; peg-IFN: Pegylated interferon; RBV: Ribavirin.

Unmet Demands

First, access to the new drugs is limited due to their high costs. Depending on the duration of treatment, a regimen of telaprevir or boceprevir costs US$30,000–50,000.[15] According to the current paradigms, peg-IFN/RBV, which costs $35,000 per course, still needs to be added to the treatment with either DAAs. Meanwhile, additional expenses for managing the side effects, among which are anemia, rash and depression, must be considered. Because HCV infection is prevalent among marginalized groups with lower incomes, who usually lack adequate coverage by medical insurance, the penetration of new treatment will probably be low. Moreover, since most new incidences of HCV infection in the USA are acquired through sharing of contaminated needles or syringes by IDUs, a low penetrance of treatment in this population will keep the number of new infections at a sustained level. Even if a certain rate of treatment can be achieved in the high-risk population of IDUs, frequent re-exposure and reinfection can still pose a problem, but may be ameliorated if an at least partially protective vaccine were available. Therefore, hepatitis C may become more of a social problem than a medical one. This critical issue will be difficult to tackle without sufficient political support.

Second, although HCV drug development is moving forward rapidly, the efforts to identify chronic HCV carriers lag behind. As 80% of people do not exhibit any symptoms following initial infection, most people with HCV are unaware of their infection status.[16] This is especially a problem for the high-risk population, which do not have access to routine medical screening. The low rate of diagnosis not only leads to an underestimation of overall HCV burden, but also limits the utilization of effective treatments.

Third, HCV infection will remain a global epidemic in the foreseeable future. Even if it will be possible to treat all the infected individuals in developed countries with new DAA regimens, HCV infection is likely to persist in the vast majority who live in the developing world, where the medical infrastructure cannot support and afford the treatment. Those developing countries also face the additional challenge of a high transmission rate owing to inadequate screening of blood products and an increasing number of IDUs.[17] Given the constant human migration, it is impossible for the developed countries to insulate themselves from the epidemic elsewhere. Furthermore, as most new drugs were designed to treat HCV genotype 1, because it is difficult to cure with peg-IFN/RBV, and it is predominant in developed counties, the effectiveness of those drugs for controlling infection with other genotypes remains to be tested. This is particularly problematic as nongenotype 1 viruses are widely distributed around the globe (Figure 1).

HCV Vaccines

In general, vaccination has been considered the most effective approach for combatting infectious diseases. A lesson learned from fighting HIV is that emphasis on prevention as well as treatment is important for controlling the pandemic.[18] In addition to reducing the transmission with more efficient blood screening and prevention-focused programs for IDUs, a prophylactic vaccine for HCV will fill the gaps of treatment discussed earlier, and provide a long-term solution for the disease. The target population for the vaccine will be the high-risk groups in developed countries, including healthcare workers, IDUs and men who have sex with men, and the entire population of developing countries. Optimally, a putative HCV vaccine would be effective against all HCV genotypes, would have minimal side effects, administered in a single shot and could be produced and distributed at minimal cost. However, after more than two decades of research, no HCV vaccine is currently available.

The development of a vaccine for HCV is hampered by several challenges. The first is the tremendous genetic and antigenic diversity of the virus. Currently there are seven genotypes being recognized, with sequence dissimilarity of 30–35% across the genome.[19,20] Most genotypes contain genetic diversity within the group, and can be further categorized into viral quasispecies.[21] As a positive sense, single-stranded RNA virus, HCV replicates its genome rapidly through the highly error-prone polymerase. This feature, together with the remarkable capability of the virus to produce progeny during chronic infection, enables HCV to generate a vast reservoir of genetic diversity, which provides the basis for selecting a variant with higher replicative fitness and capability of immune evasion. This also suggests that HCV is more likely to escape the immune protection elicited by a vaccine designed for a limited number of epitopes. The new antiviral drugs are facing the same challenge, as HCV variants will emerge with mutated binding sites for inhibitors (reviewed in[22]). To avoid resistance, a combination of drugs acting with different mechanisms needs to be used. Similarly, a successful vaccine is likely to contain multiple conserved epitopes that minimize the risk of viral escape. Thus, many of the current vaccination approaches in clinical development, discussed in the following sections, include numerous viral proteins encompassing larger numbers of epitopes to alleviate this problem. Additional complexity stems from the putatively different sensitivities of usually linear T-cell epitopes and frequently conformational antibody epitopes to mutational sequence diversity. While amino acid mutations in critical residues can readily abrogate T-cell antigen recognition, sequence variations that do not alter shape or charge of a viral antigen may not necessarily affect antibody recognition. The second challenge for vaccine development is incomplete understanding in the immunology of HCV infection. This is largely due to a lag in the development of experimental systems. Researchers were not able to grow HCV and test the ability of antibodies to neutralize HCV in vitro until recently.[23–26] Because of a narrow species tropism of HCV, chimpanzees are the only in vivo experimental model with competent immune system available for HCV vaccine research, but those studies are limited due to ethical concerns, restricted availability and prohibitively high costs. As a result, much of our knowledge in HCV immunology relies on the studies of a very small number of animals. Clinical studies on infected patients have been informative and probably less biased by sample size, but are hampered by limited access to the relevant tissue compartment, that is, the liver and usually limited information on the dose, timing and antigenic composition of the transmitted viral genome. The lack of accessible animal models is also a hurdle for testing the efficacy of vaccine candidates.

Current HCV Vaccine Research
Immune Responses to HCV & Correlates of Protection

Despite the challenges in studying HCV, considerable progress has been in made in characterizing anti-HCV immune responses. It has been estimated that approximately 20% of individuals are able to clear the infection spontaneously following acute HCV infection, whereas the rest progresses to chronicity.[27] Longitudinal studies of the two cohorts of patients during and after acute infection have defined immunologic correlates that are associated with viral clearance.

A strong T-cell response, characterized by the production of effector cytokines including IFN-γ, and broad epitope specifically correlate with the resolution of acute infection.[28–34] After clearance of the acute infection, memory T cells are maintained, but whether they can provide protection against reinfection is incompletely understood.[35–39] While usually not resulting in sterilizing immunity, that is, prevention of acute infection after re-exposure especially to antigenically more divergent HCV strains, adaptive immunity protects against progression to chronic infection following repeated HCV exposure.[40,41] As chronic infection persists, the number of epitopes recognized decreases and T-cell responses are often lost.[42,43] Since HCV-associated morbidity and mortality are caused by chronic infection, a vaccine, even if it only prevents viral persistence, would greatly ameliorate the problem. Although neutralizing antibodies are present during the chronic phase of infection, these antibodies are not able to clear the virus.[44,45] Several mechanisms of viral escape from antibody-mediated neutralization have been postulated and tested (reviewed in [46]). Recently, several human monoclonal antibodies against HCV envelope protein E1 or E2, which show crossneutralizing capability, were identified.[47–50] These antibodies were able to prevent infection of heterologous HCV in the HCV pseudoparticle and HCV cell culture model system, suggesting passive prophylaxis with exogenous neutralizing antibodies or eliciting high-affinity antibodies with similar specificity representing a viable strategy to prevent or more efficiently control HCV infections.

Although resolution of the infection is dependent on adaptive immunity, innate responses are also observed early after HCV infection. Type I interferons and interferon response pathways are induced in the liver at early stages of infection regardless of the clinical outcomes.[29,51,52] The fact that various strategies have evolved in the HCV life cycle to interfere with the IFN response[53] indicates that the innate response exerts a significant pressure on HCV. Moreover, recent genome-wide association studies have identified single-nucleotide polymorphisms in the IL-28B gene locus that correlate with spontaneous clearance of an acute HCV infection and predict to a certain extent how likely patients with a given combination of IL28B alleles are to respond to peg-IFN/RBV therapy.[54–57] Genetic analysis has also revealed the important role of natural killer (NK) cells, which produce IFN-γ and are abundant in the liver. Genetic polymorphisms that affect the threshold of NK-cell activation influence the clinical outcomes of HCV infection.[58] Recent genetic data suggest that taking into account the combination of polymorphisms within the loci of IL28B, HLA-C and its ligands, the killer immunoglobulin-like receptors has greater predictive value for clearance of HCV infection.[59] These results not only highlight the importance of innate immunity during HCV infection, but also suggest the efficacy of a certain vaccine may depend on the genetic features of recipients.

Approaches of Vaccination

Along with the efforts in improving our understanding of the basic immunology of HCV infection, various approaches have been taken to develop vaccines. Specific approaches in the development of both prophylactic and especially therapeutic vaccines against HCV infection have been recently reviewed in great detail elsewhere.[60] In this article, the authors limit the discussion on general principals pertaining to the different vaccination approaches and highlight candidate vaccination approaches that are in active clinical development.

Prophylactic Vaccination

Prophylactic vaccinations aim at preventing infection often through the induction of a pathogen-specific humoral immune response. However, the role of neutralizing antibodies in protection against HCV infection remains controversial.[61] Although only few founder viruses appear to initiate the HCV infection during transmission,[62] antigenically diverse viral variants are readily produced once HCV starts to replicate. The antigenic diversity poses further challenges to the prophylactic vaccination approach. Early attempts focused on inducing the production of neutralizing antibodies against envelope proteins of HCV, E1 and E2. This was inspired by the success of HBV vaccines, which induce antibodies against HBV surface antigens, thereby preventing viral entry and infection. Induction of HCV envelope-specific antibodies in naive chimpanzees by vaccination with recombinant E1 and E2 or DNA yielded protection from virus challenge.[63,64] Similarly, immunization of healthy human volunteers with HCV envelope glycoproteins elicits antibodies that crossneutralize heterologous virus strains in vitro.[65,66,201] A major challenge remains in the identification of suitable immunogens that elicit broadly neutralizing antibody responses. The major antigen determinants within the viral envelope are in the hypervariable-region 1 of the E2 glycoprotein, which, as the name implies, is not necessarily suitable to confer broad protection against antigenically diverse viruses. It has been speculated that more broadly shared epitopes will become accessible when the HVR1 region is deleted from the viral envelope. However, the idea of engineering the immunogenicity of HCV by exposing better-conserved epitopes remains to be tested. Furthermore, analysis of the structural details of (conformational) epitopes recognized by antibodies with broad neutralizing activity may provide a starting point for the design of immunogens capable of eliciting antibodies with similar activity.[67,68]

Prophylactic vaccination approaches are not limited to those geared towards inducing neutralizing antibodies. Clinical trials are ongoing to assess the efficacy, safety and immunogenicity based on the sequential use of adeno- and/or modified vaccinia Ankara (MVA) vectors expressing HCV nonstructural proteins NS3-NS5B.[202,203] Conceivably, combining the approaches that prime both humoral and cellular immunity would protect more efficiently against HCV challenge, although the concept remains to be tested in suitable animal models and/or clinical trials.

Therapeutic Vaccination

The main rationale of therapeutic vaccination is to bolster new and/or restore ineffective previously primed antiviral adaptive immune responses to neutralize circulating virus and eliminate infected cells. Optimally, therapeutic vaccination, conceivably in combination with standard-of-care treatment, would eventually result in complete control of the previously established viral infection or at least significantly mitigates liver disease progression. Treatment of chronic HCV infection has considerably improved in recent years and numerous directly acting antiviral and host-targeting antiviral drug candidates have shown remarkable efficacy in clinical trials. These advances may ultimately reduce the need for therapeutic vaccines. However, as outlined earlier, the new treatment, while being expensive, is not effective in all patient populations, and is plagued with considerable side effects. Consequently, more cost-effective alternatives are required to improve treatment options, particularly in resource-poor environments.

Therapeutic vaccine trials have demonstrated that HCV-specific immune responses can be primed in chronically infected individuals, resulting in transient reductions in HCV RNA titers in subsets of patients.[69–71] However, to date, no therapeutic vaccine candidates have achieved sustained SVRs. Considering that immune exhaustion is frequently associated with chronic HCV infection, the fact that partially functional T-cell responses can be primed is still remarkable. These observations also argue that a better understanding of mechanisms of immune exhaustion is needed to pair therapeutic vaccinations with specific immunomodulatory regimens to bolster antiviral immunity. A plethora of approaches has been undertaken towards developing a therapeutic vaccine against HCV infection (reviewed in detail in [60,72]). These can be broadly divided into peptide- or protein-based vaccines, DNA vaccines, viral vector vaccines – including recombinant adenovirus, MVA, alphavirus or paramyxovirus vectors – recombinant yeast-based vaccines and vaccination approaches based on dendritic cells (DCs). Of those, some have advanced into early clinical development assessing their safety and immunogenicity (reviewed in detail in[72]), but only few are currently being actively pursued ( ). For example, it was previously demonstrated that HCV antigen expression from DNA can result in robust induction of HCV-specific humoral and T-cell immunity, depending on the antigen combination. Currently, administration with a plasmid expressing NS3/4a of HCV genotype 1 and subsequent in vivo electroporation is being tested in combination with peg-IFN and RBV in chronically infected HCV patients. In contrast to plasmid DNA vaccines, viral vectors are highly immunogenic and also allow for the expression of an antigen combination of choice. From a regulatory perspective of safety, insufficient or incomplete attenuation of replication of viral vector is a major concern. Replication incompetent adenoviral and MVA vectors have been extensively tested in this respect. To induce anti-HCV immunity, adenoviruses alone and/or with MVA expressing HCV nonstructural proteins in combination with standard-of-care therapy are currently being evaluated for their potential to restore dysfunctional T-cell response and to broaden HCV-specific T-cells' immunity. Saccharomyces cerevisiae, being nonpathogenic in humans but highly immunogenic, can be engineered to express heterologous proteins and thus present a desirable vaccine platform. The impact of vaccination with inactivated S. cerevisiae engineered to express a fusion protein of HCV core and NS3 in combination with peg-IFN and RBV is being investigated.

Table 2. Approaches for therapeutic vaccination in clinical development.

Strategy Approach Examples in clinical development ClinicalTrials.gov identifier Sponsor Outcome
Peptides Viral peptides coupled with adjuvants to induce humoral and cellular immunity
No active clinical trials
NA
DNA vaccines Expression of HCV protein(s) from a DNA plasmid In vivo electroporation of DNA plasmid expressing HCV NS3/4a NCT01335711 ChronTech Pharma AB Unpublished
Viral vector vaccines Use of viral vectors for delivering HCV RNA; produces a broader range of HCV epitopes and highly immunogenic AdV vector expressing HCV NS3-5B NCT01094873 Okairos Unpublished
MVA and AdV vectors expressing HCV NS3-5BMVA vector expressing NCT01296451 Okairos Unpublished
HCV NS3, NS4 and NS5B (+peg-IFN/RBV) NCT01055821 Transgene
Recombinant yeast-based vaccines Expression of HCV protein(s) in yeast to induce innate and adaptive immunity Inactivated Saccharomyces cerevisiae expressing NS3-core fusion protein (+peg-IFN/RBV) NCT00606086 GlobeImmune Unpublished
Vaccines based on DCs Infusion of ex vivo stimulated DCs loaded with HCV antigens
No active clinical trials
NA

AdV: Adenovirus; DC: Dendritic cell; HCV: Hepatitis C virus; MVA: Modified vaccinia Ankara; NA: Not applicable; NS: Nonstructural; Peg-IFN: Pegylated interferon; RBV: Ribavirin.

Clinical trial IDs obtained from [205].

Alternative Paths

Improved Design & Selection of Immunogens. Immunogen selection is a formidable challenge when thinking about a HCV vaccine due to the extreme diversity of the virus. Most of the currently licensed effective vaccines have been developed by inoculating attenuated or inactivated pathogens, or isolated antigenic components of a given pathogen. However, live-attenuated, or even inactivated, virus is not an easy approach for HCV. Safety issue is of course a concern, but more importantly, a robust replication system that allows large-scale production of HCV particles, and further purification for vaccine usage is not yet available. Selection of individual proteins and even combinations thereof does not cover adequately the genetic and antigenic complexity of the different HCV genotypes and existing quasispecies in a given patient. While some regions within the HCV open reading frame are more conserved across genotypes, they do not necessarily contain the most potent epitopes. Advances in computational and structural biology offer putative solutions to overcome this hurdle.

Reverse vaccinology starts with genomic sequences of the pathogen and uses bioinformatics tools to predict potentially antigenic protein products of the sequences. The protein candidate can then be tested with experimental systems.[73] Recent success in applying reverse vaccinology to develop vaccines against meningococus B has demonstrated the effectiveness and efficiency of this approach.[74] The selection of antigenic regions, especially putative antibody epitopes, can be further refined and verified using 3D structures of a given pathogen derived protein superimposed with the linear and/or conformational epitopes of known potent neutralizing antibodies pointing towards the 'Achilles heel' of a (viral) pathogen. Crystal structures of the HIV envelope have been solved and numerous very potent antibodies have been identified;[75–82] but this recent gain in knowledge has not yet translated into effective structure-based vaccine design for HIV.[83,84] In order to use structure-based approaches for designing vaccine candidates for HCV, some critical gaps need to be closed. Although, the HCV E2 proteins can now be purified under presumably native conditions[85] and based on biochemical data a model of E2 has been put forward,[86] high-resolution crystal structures for the viral envelope remain elusive. Considerable progress has been made in the identification of broadly crossreactive monoclonal antibodies.[47,49,50,87–92] The assumption that a reconstructed antigen designed to fit a neutralizing antibody will be an efficient immunogen to elicit protective antibodies in vivo remains to be proven. Nevertheless, as a supplemental approach, structure-based antigen design may help improve the efficacy of vaccine candidates identified by empirical immunogenicity trials.

To design polyvalent vaccine antigens for T-cell-based vaccines, a computational approach was developed for HIV[93,94] and has also been considered for HCV.[95] Such artificial antigens are comprised of sets of 'mosaic' proteins, which are computationally generated recombinants assembled from fragments of natural sequences using a genetic algorithm. Mosaic proteins are similar to natural proteins, but are optimized to maximize the coverage of common potential T-cell epitopes found in a population of natural sequences, and to minimize the inclusion of rare epitopes to avoid vaccine-specific responses. Sets of mosaic proteins provide coverage of the most common 9-mers in the circulating population, and enable the delivery of these variants in the form of intact proteins that could be processed naturally and delivered readily in a vaccine. Adenovirally expressed mosaic HIV-1 vaccines have been shown to expand the breadth and depth of cellular immune responses in rhesus monkeys;[96] however, it has yet to be proven that mosaic HIV vaccines are more immunogenic than conventional antigen combinations in clinical trials; theoretically, HCV-mosaic vaccines hold promise as more potent immunogens to elicit T-cell responses with pan-genotypic specificity.

For any of the aforementioned approaches to define potentially more potent immunogens, new or refined platforms are needed to elicit immunity in vivo. Expression of HCV protein antigen in various viral vectors, including replication incompetent MVA or adenoviral vectors, induces protective immune responses against diverse pathogens and cancer in various animal models, and can induce robust and sustained cellular immunity in humans. However, for the most commonly used serotype 5 adenoviruses, most humans have neutralizing antibodies, which can diminish the immunogenicity of such vaccines. Recently, more than 1000 adenoviruses from chimpanzees have been isolated and sequenced, which can induce potent cellular immunity across multiple species.[97] In clinical trials, these simian adenovirus vectors appear to be safe and highly immunogenic. Although there is so far no side-by-side comparison of vaccine efficacy to demonstrate its superiority to other nonhuman ones, these simian adenoviruses provide a viable alternative to their human counterparts as vectors for vaccine delivery.[98,99] Harnessing the inherent immunogenicity of activated DCs, the central orchestrator of adaptive immunity in vivo, may serve as one alternative to complement and boost viral vaccine vectors (reviewed in [100]). To avoid the need of isolation and expansion of autologous DCs in vitro, which would limit the utility of the approach for widespread use, direct targeting of antigens to DCs in vivo is currently being explored. It was previously demonstrated that antigens fused antibodies binding to cell-type-specific uptake receptors on the surface of DCs can induce antigen-specific immune responses in vivo (reviewed in [101]), and thus may be applied to induce in particular T-cell immunity to HCV with novel, tailored HCV antigens.

Passive Prophylaxis. Although neutralizing antibodies induced by natural infection or vaccines are not sufficient to prevent HCV infection,[102] strong and broadly reactive antibodies to HCV from exogenous sources can be used in postexposure prophylaxis or prevention of recurrent HCV infection after liver transplantation. Indeed, it has been shown that immunoglobulins prepared from chronic HCV patients can prevent hepatitis C in liver recipients, if antibodies are administered to patients without prior exposure to the virus.[103] However, those immunoglobulins have so far failed to prevent reinfection in HCV patients who have undergone liver transplantation.[104] Recent development of human monoclonal antibodies has enabled in vitro production of anti-HCV antibodies with defined specificity at a large scale. The observation that these monoclonal antibodies are able to neutralize diverse HCV quasispecies in human liver-chimeric mice[50] has raised the hope that potent antibodies at high dosages will be effective for postexposure prophylaxis in humans. To achieve a high, sustained dose of antibodies, vector-mediated gene delivery approaches are being explored. In the case of HIV, overexpression of broadly neutralizing antibodies using an adeno-associated virus vector was able to fully protect humanized mice from HIV infection.[105] Similar studies are sought to test whether this approach can produce effective prophylaxis against HCV.

Immunotherapeutic Approaches. As an immunotherapeutic approach, immune cells with antiviral activities are transferred to enhance the endogenous immune response in the recipient. In a study, HCV-infected patients who have undergone liver transplantation were infused with lymphocytes extracted from the liver allografts.[106] These lymphocytes include abundant NK and NK T cells, and were treated in vitro with IL-2/anti-CD3 mAbs before infusion. This treatment markedly lowered the HCV RNA titers in patients and completely prevented HCV infection in human liver-chimeric mice.[106] Furthermore, it has been shown that CD56+ cells obtained from the peripheral blood mononuclear cells show anti-HCV activity.[107] However, unlike T and B cells, NK cells lack an antigen-recognition receptor for distinguishing healthy and infected cells. Instead, their responses depend on the signals from inhibitory and activating receptors. A putative solution may be to guide NK cells to HCV-infected target cells using bispecific antibodies binding to an invariant domain of an activating receptor and viral antigens/antigen–MHC complexes on the target cell.

The efficacy of HCV vaccines may also be enhanced by immunomodulatory treatments. As overly activated T-cell response can cause excessive tissue damage, many regulatory mechanisms are in place to keep T-cell activity in check.[108] For instance, T cells express the inhibitory receptor programmed death-1 (PD-1)[109] and T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3).[110] Ligation of these regulatory receptors by their ligands induces negative signals to the responding cells and leads to reduction of cytokine production and cell proliferation.[111,112] Moreover, Foxp3+ Treg, which is a specialized CD4+ T cell, can suppress the function and proliferation of effector T cells.[113] These regulatory mechanisms together limit T-cell responses during chronic infection, and also affect the efficacy of a vaccine. It has been shown that blockade of PD-1 or Tim-3 can enhance the proliferation and cytotoxicity of HCV-specific cytotoxic T lymphocytes.[114] With this rationale, the effect of inhibitory receptor blockade or Treg depletion on the efficacy of a HCV vaccine needs to be examined.

Recent Progress in the Model Systems for Vaccine Studies

The development of an infectious cell culture system for HCV in 2005[23,115,116] marked a major milestone for hepatitis C research. It has not only enabled in-depth studies of the HCV life cycle but has also aided HCV vaccine research. The cell culture system for HCV has not only been applied to study the efficiency of neutralizing antibodies,[46,117,118] but also to understand the mechanisms of immune evasion of HCV.[47,119] However, the infectious cell culture system relies primarily on a single HCV molecular clone derived from a Japanese genotype 2a infected patient with fulminant hepatitis (JFH1), replicate efficiently both in tissue culture and in animal models. Subsequently, intergenotypic chimeras consisting of the core through NS2 regions of representative genomes of all seven HCV genotypes have been constructed.[120–123] These tools have become indispensable for in vitro and in vivo assays of HCV entry and neutralization. However, a broad spectrum of infectious clones of all HCV genotypes capable of replicating in cell culture is still missing. Despite these advances, a huge gap still exists between in vitro experimental systems and clinical studies. Although the efficacy of preventive vaccines can be assessed in individuals with high risks of exposure, such as IDUs and healthcare workers, the low incidence rates of infection in developed countries requires a large number of participants to be recruited in order to detect statistically significant differences. Consequently, it may be more feasible to conduct clinical trials in developing countries with high prevalence and incidence rates of new infections. Testing of preventative and especially therapeutic vaccines in human trials without adequate preclinical safety assessment in animal models can be problematic, since vaccine induced and/or exacerbated responses may lead to more severe hepatitis and in worst-case scenario to acute liver failure. Furthermore, vaccine dosing, formulations and vaccination schedules often need to be empirically tested to define regimens that result not only in maximal immunogenicity but also efficacy. However, animal models that can be infected with HCV and thus are suitable for testing of vaccine candidates are sparse ().

Table 3. Models for in vivo vaccine testing.

Models for in vivo tests Humans (clinical trials) Chimpanzees Mouse-adapted HCV Genetically humanized mice Xenotransplantation models
HCV life cycle in vivo
Entry Yes Yes ? (in vitro: yes) Yes Yes
Replication Yes Yes ? ? Yes
Assembly Yes Yes ? ? Yes
Immunity
Innate Yes Yes Impairment needed? Yes Yes
Adaptive – cellular Yes Yes Impairment needed? Yes Limited
Adaptive – humoral Yes Yes Impairment needed? Yes Impaired
Pathogenesis Hepatitis, fibrosis, cirrhosis, HCC Milder than in humans Not yet tested Not yet tested Evidence for fibrosis
Validated for vaccine testing Yes Yes No Limited No
Practical considerations
Costs High High Low Low Medium
Throughput Low Low High High Medium
Heterogeneity High High Low Low High

It is not known whether the respective parts of the HCV life cycle are supported.

It is unclear whether innate and/or adaptive responses need to be suppressed.

HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus.

Chimpanzees have been the central animal model to study HCV infection. Besides humans, they are the only species that is naturally susceptible to HCV infection. Chimpanzees partially recapitulate the natural course of infection in humans. While acute hepatitis is somewhat milder in chimpanzees than in humans, experimentally infected animals also frequently progress to chronicity.[74,124,125] Subclinical hepatitis, after many years of chronicity, however, is also not uncommon in humans without risk factors for rapid progression including, older age or alcohol intake. The chimpanzee model has been of critical importance for defining the nature of protective immunity following reinfection. Experiments in chimpanzees demonstrated that clearance of a primary infection with HCV does not provide sterilizing immunity against challenges with homologous or heterologous viruses.[83,84] However, despite their utility, the use of chimpanzees in biomedical research is intensely debated due to ethical concerns. In fact, strong public opposition against the use of large apes has led to the ban of chimpanzee research in many countries. Undoubtedly, an NIH moratorium on 'nonessential chimpanzee research'[204] is likely to constrain HCV vaccine development and HCV research in general in the future. Furthermore, experiments in chimpanzees are associated with very high cost, and usually only few animals, which are genetically heterogeneous, remain available. Consequently, the interpretation of data derived from experiments on a small number of chimpanzees is often statistically difficult.

Accessible animal models, in particularly immunocompetent small rodents and primates, are urgently needed. Significant progress has been recently made in attempts to adapt HCV to infect nonpermissive species and to engineer the host environment to provide an environment that is more conducive to viral infection. While many new tools are still in the early phases of development, they hold promise to dramatically improve our ability to preclinically assess vaccine candidates in the near future.

Adapting HCV to Infect Nonpermissive Species

The full life cycle of HCV can be divided into three critical steps: cell entry, replication and assembly/egress/release of viral particles. Host factors are involved in each step. For entering its target cell, the hepatocyte, HCV requires numerous cellular factors, including glycosaminoglycans,[126,127] low-density lipoprotein receptor, scavenger receptor class B type I (SCARB1),[128–131] CD81,[132] two tight junction proteins, claudin-1[133] and occludin (OCLN).[134,135] More recently, the cholesterol absorption receptor Niemann-Pick C1-like 1[136] and two receptor tyrosine kinases, EGF receptor and EphrinA2[137] have been implicated in the viral uptake pathway into human cells (reviewed in [138]). The difference in the ability of HCV to engage these host factors helps define the species tropism of HCV. In murine cells, HCV appears to less efficiently engage CD81 and OCLN, which precludes viral entry.[134] However, given the error-prone replication of HCV genome, it is conceivable to 'train' HCV to utilize murine orthologs of HCV entry factors. Critical proof-of-concept for this approach was recently provided in a study, in which a strain of HCV was selected to use murine CD81.[139] This virus acquired mutations in the E1 and E2 envelope proteins, which facilitated viral entry into murine cells in the absence of human entry factors. It remains to be tested whether the 'murine-tropic' HCV can actually infect mice in vivo. A mouse model would certainly be very attractive for HCV vaccine research; as multiple inbred and outbred lines are available, mice can be generated in large numbers at fairly low costs and numerous tools to analyze vaccine and virally induced immune responses are available. However, since the evolutionary divergence of mouse and man 65 million years ago, these two species have inhabited different ecological niches and have been challenged with minimally overlapping groups of pathogens. Therefore, the human and mouse immune systems, evolving to meet these challenges, have accumulated many differences,[140] making genes related to immunity, together with genes involved in reproduction and olfaction, the most divergent between the two species.[141] These differences are likely to affect the quality of an antiviral immune response and consequently may lessen the utility of the system for testing vaccines and therapeutics targeting human HCV.

Consequently, adaptation of HCV to other, more closely related species to humans needs to be pursued to ameliorate some of the caveats. Small nonhuman primates, such as Rhesus monkeys are more similar to humans but they are also resistant to HCV infection[142] possibly due to the inability of HCV to counteract antiviral innate immune responses.[143] Nonetheless, adaptation of HCV to small nonhuman primates offers several considerable advantages. Given the greater similarity to humans, it may potentially be easier to overcome putative incompatibilities between virally encoded proteins and host factors. Furthermore, rhesus monkeys have been extensively used in biomedical research, and a plethora of tools is available. Studies conducted in monkeys may translate more readily into clinical results. Macaques also offer a better platform for pharmacokinetic studies for HCV vaccines and drugs. In addition, the fact that a large fraction of HCV carriers is coinfected with HIV must be considered when assessing vaccine candidates. A macaque model for HIV/HCV coinfection is of interest and significant clinical relevance, especially when a simian-tropic HIV-1 is already available.[144,145]

Engineering the Hosts to Accommodate HCV

Another direction researchers have taken to create an animal model is to engineer the host environment to support HCV infection. Host adaptation can be achieved by actually transplanting human tissues to humanize relevant tissue compartments or by genetic adaptation of the host species. It has been demonstrated that engraftment of human hepatocytes into suitable xenorecipients can render mice susceptible to HCV infection.[146] In these xenotransplantation models, liver injury is induced in the recipients before transplantation to provide a growth stimulus to the engrafted human hepatocytes. Currently, the urokinase plasminogen activator transgenic mouse and fumary lacetoacetate hydrolase deficient mouse, both being susceptible to endogenous liver injury, were used in combination with severe immunodeficiency for liver xenotransplantation models.[147–149] Human liver-chimeric mice can be successfully infected with HCV.[146,147,150] However, a major drawback of these human liver-chimeric mice is the lack of a functional immune system. Although the efficacy of neutralizing antibodies and antiviral drugs can be assessed in those mice, it is difficult to study the immune responses induced by vaccines. Recently, an alternative xenorecipient strain has been constructed, in which FK506 binding protein fused to active caspase 8 is transgenically expressed.[125] Upon injection of the FK506 dimerizer, AP20187 apoptosis is induced in mouse hepatocytes. Remarkably, mice injected with a mixture of autologous human hepatoblasts, nonparenchymal cells and hematopoietic stem cells resulted in measurable human hepatic and hematopoietic chimerism. Dually engrafted mice not only supported HCV viremia at very low levels but also mounted antigen-specific viral immune response.[125] Although the functionality of the human immune system still requires substantial improvement, these and similar xenotransplant models may become a suitable platform to analyze vaccine induced, human anti-HCV immunity in a small animal model.

Alternatively, mice have been genetically modified to be more permissive for HCV infection. Based on the previous discovery that human CD81 and OCLN constitute the minimal set of entry factors required for viral uptake into rodent cells,[134] mice were engineered to express adenovirally delivered human entry factors.[151] These genetically humanized mice support HCV entry. Importantly, this mouse is fully immunocompetent, and it is thus suitable for immunization and challenge studies. However, in order for this model to gain additional utility, for example, to study virus-induced immunity or to evaluate therapeutic vaccine candidates, the recapitulation of the entire life cycle in an inbred, genetically humanized mouse would be critical. Nevertheless, the introduction of a small animal model with a competent immune system, would be an enabling tool in HCV vaccine research.

Expert Commentary

The field of HCV research has witnessed exciting breakthroughs in recent years. Two specific antiviral inhibitors have reached the market, and many more are at the late stage of development. Combination therapies with the new antivirals are projected to cure HCV infection efficiently in most patients who can afford the treatment. However, the socially disadvantaged, who face the highest risk of HCV infection in developed countries, along with the vast majority in the developing world, are less likely to gain immediate access to the current medical innovations. Thus, a vaccine administered to high-risk populations in both developed and, in particular, developing countries represents a cost-effective alternative in the era of new antivirals. Immunological control of HCV is possible, but the genetic and antigenic diversity of the virus poses a major challenge to vaccine development, yet substantial progress has been made. Various vaccination approaches are being exploited, having yielded several candidates for human trials. Vaccine research is prominently advanced by new methodologies adopted for in vitro and in vivo studies on HCV infection. The introduction of small animal models will enable a better understanding in the immunology of HCV infection and more rigorous preclinical tests on vaccine candidates. If we are experiencing a harvest season for DAAs, the coming one is for HCV vaccines.

Five-year View

Based on data from recently completed and ongoing clinical trials, it is foreseeable that potent combinations of directly acting and/or host-targeting antivirals that can effectively cure chronic HCV infection in most patient population will reach the market. However, predicted high costs and potential side effects requiring medical supervision may lessen the impact of pharmacological intervention in resource-poor environments. By contrast to the diverse portfolio of compounds, which currently fill the drug development pipelines, only few vaccination approaches are being evaluated in clinical trials, making it unlikely that a pan-genotypic vaccine will become accessible within the next half decade. Advances in the ability to grow HCV in vitro has provided critical insights in HCV biology but has also led to the construction of new tools to monitor the efficacy of vaccination approaches. The ban of the use of chimpanzees in many countries or the severely diminished resources allocated to chimpanzee research in others, such as the USA, will undoubtedly slow down the development of prophylactic and therapeutic vaccines. Considerable effort has been made in the development of alternative animal models yet falling short of a fully immunocompetent animal model that is readily susceptible to HCV infection and mimics closely HCV pathogenesis. Continued animal engineering may yield such an in vivo system, which is urgently needed to preclinically evaluate vaccine candidates.

Sidebar
Key Issues
  • Adequate resources need to be devoted to obtain more accurate data on the prevalence and rate of spreading of hepatitis C virus (HCV).

  • The tremendous activity in anti-HCV drug development needs to be extended to HCV vaccine development.

  • Continued research activities to provide further insights into the mechanisms of HCV clearance and persistence.

  • Additional virological tools including a broad array of infectious clones covering all HCV genotypes are needed to effectively test vaccine-induced immunity in vitroand in vivo.

  • Integration of novel computational and structure-based design of vaccine candidates to improve epitope selection. These attempts probably require 3D structures of the viral particle and high-resolution crystal structure of the viral envelope. Furthermore, alternative vaccine delivery vehicles are needed to efficiently induce immunity against HCV.

  • In the absence of chimpanzees, fully immunocompetent small animal models that recapitulate the entire HCV life cycle are urgently needed for testing vaccine candidates, particularly the ones for prophylactic purpose, prior to entering clinical trials.

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Acknowledgements
The authors thank Naglaa Shoukry and Leonia Bozzacco for critical reading of the manuscript.

Expert Rev Gastroenterol Hepatol. 2013;7(2):171-185. © 2013 Expert Reviews Ltd.

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