Hepatitis C Virus Vaccines in the Era of New Direct-acting Antivirals

Chao Shi; Alexander Ploss

Disclosures

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

In This Article

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 ( Table 2 ). 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.

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.

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