A vaccine protecting against chronic infection with hepatitis C virus (HCV) is urgently needed to address the ongoing public health crisis caused by this insidious pathogen—a major cause of liver cirrhosis and cancer. Efforts toward development of vaccines were initiated after the discovery of HCV in 1989, but to date, no vaccine has been licensed. An increased understanding of HCV protective immunity and HCV envelope glycoprotein structure and function is paving the way toward rational vaccine design and evaluation.
HCV is mainly transmitted through blood, but sexual and perinatal transmissions do occur. Worldwide, most transmissions are caused by unsafe medical procedures, but ∼20% occur in people who inject drugs (PWID), an important risk group in high-income countries; another risk group in high-income countries is men who have sex with men (1). The World Health Organization (WHO) estimates that 1.5 million new infections occur annually, and incidence is increasing in many countries, including the US, where transmission is fueled by the opioid crisis. About 75% of infections progress to chronic hepatitis C, estimated to affect 58 million individuals worldwide. Chronic hepatitis C leads to liver cirrhosis in ∼20% of cases, which also comes with an ∼5% annual risk for the development of hepatocellular carcinoma. Annually, HCV-induced liver diseases cause ∼290,000 deaths worldwide.
Efficient, curative, and direct-acting antivirals have so far had limited effect on global HCV prevalence. Without population screening programs, ∼80% of chronic infections are undiagnosed because specific symptoms often only appear when severe and potentially irreversible liver damage has developed. Further, reinfections are common in risk groups. Thus, it currently appears unrealistic to achieve the WHO target of eliminating viral hepatitis as a major public health threat by 2030. In the future, achieving this target globally will likely require a vaccine, and the availability of a vaccine is predicted to result in aggregated cost savings of several billion US dollars compared with testing and treatment alone (2).
HCV belongs to the Flaviviridae family and is an enveloped positive-sense, single-stranded RNA virus classified into eight major genotypes with ∼30% sequence difference. This extensive genetic heterogeneity challenges vaccine development. The virus particle contains the structural proteins Core, which encapsidates the RNA genome, and E1 and E2, which are highly heterogeneous envelope glycoproteins that form heterodimers interacting with host cell receptors during viral entry into hepatocytes. E1 and E2 are the main targets of neutralizing antibodies (nAbs) and thus of vaccine approaches that target B cells. The nonstructural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) mediate viral processing inside the host cell and are thus primarily targeted by vaccine approaches that aim to elicit T cell–mediated responses.
In preclinical studies, a range of HCV vaccine candidates has been explored—based on viral vectors, recombinant inactivated viruses, virus-like particles (VLPs), nanoparticles, recombinant proteins, peptides, DNA, and mRNA—showing varying immunogenicity (1). Among these, only one candidate has proceeded to a clinical protection study. In 2021, a phase 1/2 trial (NCT01436357) in PWID provided evidence that a viral vector vaccine encoding NS3-NS5B, based on chimpanzee adenovirus 3 for priming and modified vaccinia Ankara virus for boosting, induced HCV-specific T cells. However, this vaccine candidate did not protect against chronic HCV infection (1, 3). The most advanced B cell–targeting HCV vaccine candidate, an MF59-adjuvanted E1-E2 heterodimer vaccine, was protective in chimpanzees and had the capacity to induce broadly neutralizing antibodies (bnAbs) in a phase 1 trial reported in 2010 (NCT00500747). However, immunogenicity was low, with <50% of vaccinees mounting nAbs (1). Rational design of next-generation vaccines is now guided by improved understanding of HCV protective immunity, viral immune escape mechanisms, and the structure of the HCV envelope glycoproteins and neutralizing epitopes.
HCV protective immunity is feasible: ∼25% of acute infections are cleared, and clearance is more likely after reinfection (1, 4). Early studies on protective immunity focused on evaluating T cells. Later, studies of nAbs were enabled by the development of suitable recombinant in vitro systems: Panels of HCV E1-E2 containing pseudoparticles, which allow studies of HCV cell entry, and of cell culture–adapted HCV, which allows studies of the complete viral life cycle, now represent HCV genetic and antigenic diversity (5–8). Important insights were initially gained from chimpanzee challenge studies, which are no longer feasible; studies now rely on humans at risk of infection. Together, such studies have clarified that both humoral and cellular immune responses appear to be involved in the natural clearance of HCV infection; however, they may not always contribute equally (1, 5). Thus, in any individual, nAbs or T cells might be the dominant drivers of viral clearance.
Clearance has been associated with sustained CD4+ and CD8+ T cell responses, with low expression of inhibitory receptors, such as programmed cell death protein 1 (PD1), on T cells as well as with induction of high-titer nAbs (1, 5). Both nAbs with narrow specificity and bnAbs, which target epitopes that are conserved across HCV strains, have been associated with natural clearance in humans (1, 5, 8). Such antibody responses are also induced during chronic HCV infection, which suggests that antibody titers and timing, in relation to the onset of infection and the emergence of viral immune escape variants within the infected individual, likely determine the prospects of antibody-driven clearance (1, 5). The demonstration of persisting memory B and T cells after natural infection also holds promise for vaccine-induced protection (9).
HCV genetic heterogeneity and mutational immune escape call for vaccines that target conserved epitopes. Although the vaccine candidate eliciting T cells was not protective in the phase 1/2 trial (3), similar viral vector vaccines are currently being optimized to increase the magnitude and breadth of T cell responses by using conserved epitopes (1, 10). Alternatively, T cell epitopes could be presented using mRNA, recombinant protein, and peptide vaccine platforms (1). The protective potential of nAbs was demonstrated in passive transfer experiments in chimpanzees and human liver chimeric mice (5, 11). Therefore, emerging vaccine candidates that target B cells to induce potent bnAbs should be evaluated in humans.
Conserved neutralizing epitopes on the envelope glycoproteins that are targeted by bnAbs include continuous epitopes in antigenic site 412 (AS412) and AS434 and noncontinuous conformation-sensitive epitopes in antigenic region 3 (AR3). These epitopes contribute to the E2 “neutralizing face” and the binding site of the key HCV receptor, CD81, on hepatocytes. Antibodies targeting this neutralizing face likely interfere with receptor binding and hepatocyte entry (5). Heavy-chain variable regions of such bnAbs are often encoded by the immunoglobulin heavy variable 1-69 (IGHV1-69) gene, making it relevant to explore germ line–targeting vaccine approaches using antigens designed to engage and guide affinity maturation of germline B cells encoding bnAb precursors—a strategy explored for HIV vaccines (5). Although several studies have refined the HCV E2 and AR3 structure (5), the structure of E1-E2 and the conserved noncontinuous conformation-sensitive epitope AR4, which is at the base of E2 but requires E1 for stable presentation, was only recently resolved (12). AR4-targeting antibodies might stabilize E1-E2 in a prefusion state, thereby inhibiting viral fusion with host cells (12). The bnAbs induced by natural HCV infection in humans often target the neutralizing face or AR4, although AS412-targeting antibodies are less frequent, and responses targeting multiple epitopes tend to have increased neutralization breadth (13).
How can a vaccine antigen be designed to elicit potent bnAbs? Emerging studies on the determinants of HCV immunogenicity in humans identified highly immunogenic HCV strains and revealed favorable viral features that can inform antigen engineering (8, 13). During repeated infections in humans, bnAb responses were promoted by antigenically related E1-E2 that were characterized by high exposure of conserved epitopes targeted by bnAbs (8). However, structural flexibility of the envelope glycoproteins and limited accessibility of conserved neutralizing epitopes constitute HCV immune escape mechanisms and hamper induction of bnAbs during natural infection and vaccination (5). Epitope stabilization and exposure could be achieved by structure-based antigen design or by genetic modification of determinants of epitope exposure identified in recombinant cell culture–adapted HCV, such as certain E2 residues, E2 glycosylation motifs, and E2 hypervariable region 1 (HVR1) (5, 14). Regions that induce nonfavorable antibody responses might be deleted from vaccine antigens. For example, HVR1 is immunodominant but leads to induction of non-nAbs or nAbs with narrow specificity that are rapidly rendered inefficient by mutational viral escape (1, 5). E1-E2 could be presented by recombinant inactivated HCV. VLP, nanoparticle, mRNA, and protein vaccine platforms could be used to present E1-E2 or E2 alone, which is often more technically feasible. Alternatively, relevant epitopes could be presented on scaffolds. Adjuvants to enhance immunogenicity are also being explored.
A protective vaccine may need to engage both arms of the adaptive immune system. Vaccination of mice with a viral vector encoding conserved T cell epitopes and E2 without its three highly variable regions induced T cells and nAbs (10). An mRNA vaccine platform could similarly deliver T and B cell antigens, and inactivated virus and VLP vaccines can elicit not only nAbs but also T cells, modulated by an adjuvant. Alternatively, combination or prime-boost regimens could be used to activate T and B cells (1).
There is a critical need to facilitate the transition of promising preclinical vaccine candidates from animal immunogenicity studies to clinical efficacy trials. Currently, there is no robust immunocompetent HCV permissive animal model for vaccine efficacy evaluation. Instead, certain aspects of HCV vaccine–induced protection can be studied in transgenic immunocompromised and humanized mice. The protective potential of vaccine-induced nAbs can be evaluated in passive transfer studies in human liver chimeric mice (11). In addition, the overall capacity of certain vaccine approaches to induce protective immunity can be studied in surrogate animal models using HCV-related viruses (11). Currently, assessment of the protective potential of preclinical vaccine candidates needs to largely rely on knowledge-based evaluation of potency, breadth, and epitope specificity of vaccine-induced immune responses in vitro compared with protective immune responses. Therefore, there is a push to establish protocols for a controlled human infection model, where healthy volunteers are intentionally infected with HCV, to facilitate fast and cost-effective clinical efficacy testing of promising vaccine candidates (15). Additional clinical efficacy trials could then assess the protective capacity of selected candidates in individuals at risk of infection, reflecting real-world settings with regard to exposure to heterogeneous HCV strains and variable immune status of important vaccine target groups (see the figure).
Overcoming HCV immune escape mechanisms requires rational vaccine design based on the molecular understanding of viral features and host protective immunity. In the absence of robust immunocompetent animal challenge models, efforts should be directed at aligning assays for evidence-based characterization and prioritization of vaccine candidates and at facilitating accelerated clinical vaccine testing. Experiences gained on the cumbersome path toward an HCV vaccine might inform vaccine development for other complex viral targets.
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