Bridging the gap: A new tool to down select HCV vaccine candidates

Currently, the elimination of HCV relies on diagnostics to identify active infection, drugs to treat infected people, and harm reduction approaches to minimize the risk of infection. However, the incidence of new HCV infections continues to outpace the number of curative treatments in many countries; only 11 countries are on track to achieve the World Health Organization’s 2030 elimination targets. The missing public health tool to reduce incidences of hepatitis C is a vaccine that prevents HCV infection. The ideal HCV vaccine should induce both cellular and humoral immunity, and such immunity must be sufficiently broad to be effective against at least a majority of circulating genotypes. Neutralizing antibodies targeting multiple distinct epitopes within the E1E2 glycoprotein region and CD4+ and CD8+ T cells targeting multiple regions of the virus should be generated by future vaccines to minimize the risk of immune escape and progression to chronic infection. A successful vaccine will substantially increase the number of countries able to achieve elimination and reduce the cost of elimination.1 Multiple obstacles have slowed the development of a hepatitis C vaccine, including its high genomic diversity, which exceeds that of HIV, difficulty in directly culturing the virus isolated from infected people in cell lines limiting the available strains for characterization, the uncertainty of the exact correlates of immune protection including the relative importance of cellular responses versus neutralizing antibodies in protection, and the lack of immune-competent small animal challenge models to test the efficacy of vaccine candidates. To overcome these obstacles, novel technologies and methodologies have been developed and have led to the discovery of multiple promising candidate vaccines in preclinical studies over the past 5 years, with many showing an ability to elicit broadly neutralizing antibodies in small animals as assessed in in vitro assays. The chimpanzee is the only immune-competent animal model that can recapitulate HCV infection in humans and has been invaluable for our understanding of the virus. However, the chimpanzee is no longer available for biomedical research. The discovery that the fulminant hepatitis C isolate JFH1 was able to replicate in Huh 7.5 cells provided the first system in which to study the complete viral life cycle in vitro as well as evaluate neutralizing antibodies to HCV in infected people and vaccine-derived immune serum using virions that more closely resemble those produced in natural infection. To overcome the inability to readily grow intact isolates from other genotypes and subtypes of HCV, an alternative approach was adopted. Intergenotypic recombinant viruses were generated in which the Core-NS2 region of representative isolates of each major genotype of HCV was chimerized onto the backbone of the JFH1 nonstructural region. In most cases, adaptive mutations were required to confer chimeric viruses with the ability to replicate in Huh 7.5 liver cell lines.2 These cell culture–derived chimeric viruses allowed the assessment of neutralizing antibodies present in immune serum to cross-neutralize different genotypes and subtypes of HCV.2 However, the introduction of these viruses into primary liver cells requires further adaptive mutations for efficient replication, and so there is a lack of viral genetic unity between in vitro and in vivo systems. More recently, Bankwitz et al3 have developed a panel of 13 chimeric HCV viruses representing 5 genotypes and 9 distinct subtypes without adaptive mutations and could be grouped into 6 distinct neutralization clusters. While useful for short-term experiments, this virus panel is not genetically stable in the long term, acquiring adaptive mutations for sustained growth, even in cell culture. The immune-deficient chimeric urokinase-type plasminogen activator (uPA)-severe combined immunodeficiency (SCID) mouse (uPA-SCID) can be reconstituted with primary human liver cells and supports the replication of HCV in vivo4. Passive transfer of immunoglobulins into these reconstituted mice before challenge can determine the capacity of immune serum to achieve protection.5,6 In these studies, the viral inoculum used was immune serum from a chronically infected patient, patient H. Using a J6/JFH1 chimeric virus, Lindenbach et al showed that cell culture–derived virus could also infect human liver reconstituted uPA-SCID mice.7 However, an extension of the utility of the human liver-uPA-SCID model beyond supporting infection with cell culture–derived genotype 2a viruses JFH1 and J6/JFH1 has not to date been achieved but is critical to validate the breadth of in vitro neutralization potency of vaccine-induced antisera in an in vivo model that recapitulates bona vide HCV replication in human liver cells. An article by Collignon et al8 has established a system where the neutralization activity of monoclonal antibodies or immune serum to hepatitis C can be evaluated using the same viruses in vitro and in vivo. By growing JFH1-based Core-NS2 genotype 1–6 cell culture-adapted recombinant viruses in human liver uPA-SCID mice, they identified mouse adaptive mutations within the E1E2 region and reverse engineered these into the parental genome. These adaptive mutations were genetically stable in the case of genotype 1–5 variants, while some strains acquired additional adaptive mutations when cultured in Huh 7.5 cells. Importantly, the viruses showed robust replication with an ability to passage these viruses. Reverse-engineered mouse-adapted variant viruses or genomic RNA of genotypes 1a, 1b, 2b, 2c, 3a, 4a, 5a, and 6a were then used to infect human liver-uPA-SCID mice where they achieved high titer infection of 107–108 genome equivalents/mL and sustained infection beyond 4 weeks. Because these viruses contain mutations in the E1E2 region, well-defined broadly neutralizing monoclonal antibodies were used to assess changes in their sensitivity to neutralization compared to the parental viruses, with some increases and decreases in sensitivity to neutralization noted. To confirm that mouse-adapted virus variants can be used to assess neutralization in vivo in the human liver-uPA-SCID mouse, proof-of-concept experiments using well-characterized potent broadly neutralizing monoclonal antibody AR4A were performed. Mice received an infusion of AR4A and were then challenged with selected mouse-adapted virus variants. No virus was detected in the passively immunized mice, while control animals were able to maintain virus replication for at least 8 weeks, demonstrating that AR4A neutralizes these recombinant viruses with defined adaptive mutations both in vitro and in vivo. The development of this system by Collignon et al could be a valuable tool for HCV vaccine development by further de-risking and prioritizing promising vaccines in preclinical development. Researchers will be able to evaluate immune sera in cell culture–based neutralization assays and then verify the neutralizing activity of immune serum, using the same virus panel, in human-liver-uPA-SCID challenge experiments that more closely resemble the virus replication cycle and the viral-lipoprotein content observed in natural infection. The ability to test immune serum in this system could be essential for down selection and prioritization of the most promising vaccine candidates and will bridge a gap between preclinical and clinical testing. The remaining limitations of using this panel of JFH1-based Core-NS2 genotype 1–6 adapted recombinant viruses in human liver uPA-SCID mouse model are the lack of a functional immune system and the inability to evaluate whether cellular immune responses generated by vaccine candidates contribute to protection. While this panel of chimeric viruses includes isolates of all major genotypes of HCV, only 3 of the 6 neutralization clusters identified by Bankwitz et al3 are presented, and so there remains a gap in the full assessment of the breadth of neutralization. Finally, the range in neutralization sensitivity of this panel of viruses needs to be considered in the evaluation of the neutralizing activity of immune serum. Here, a panel of antigenically and genetically diverse hepatitis C glycoproteins that can be pseudotyped into retroviral particles (HCVpp) may prove valuable. This panel of 15 glycoproteins, mainly derived from genotype 1a glycoproteins,9 exhibit various sensitivities to neutralization from tier 1 (easiest to neutralize) to tier 4 (most difficult to neutralize). While HCVpp is a reliable model to test for entry-blocking antibodies, there are subtle differences in the entry process between HCVpp and HCV virions derived from human hepatocytes due to many factors, including different arrangements and content of glycoproteins E1E2 on their surface, different lipoprotein content, and receptor dependencies. Recently, there has been significant optimism in the field of HCV vaccine development as many promising candidates have been described as having the ability to generate humoral and/or cellular immune responses. Furthermore, mRNA-based vaccines offer a new platform to induce a protective immune response against HCV that may be simpler and cheaper to develop. In addition, the development of a human challenge model for hepatitis C is progressing that will accelerate the clinical development of HCV vaccines by allowing the assessment of the most promising candidates in humans with intact immune systems to assess the level and type of protection afforded.10 Preclinical screening of promising candidates in the system of Collignon and colleagues could be an effective means by which to select the most promising candidates for human challenge studies. This is an exciting time for HCV vaccine development. Combined with highly effective drugs, the successful addition of an effective vaccine would put the elimination of HCV-induced viral hepatitis within reach.

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