The 2020 Nobel Prize for Medicine or Physiology for the Discovery of Hepatitis C Virus: A Triumph of Curiosity and Persistence.

The 2020 Nobel Prize in Medicine or Physiology was awarded to Drs. Harvey Alter, Michael Houghton, and Charles Rice for their contributions to the discovery and characterization of the hepatitis C virus (HCV). Their achievements represent a remarkable triumph of biomedical science which allowed the development of curative therapy for HCV, that will save countless lives. This tribute provides a historical perspective of the laureates' seminal work leading to the discovery of the HCV and a synopsis of a forum hosted by the American Association for the Study of Liver Diseases to honor the laureates in which they offered their perspectives, advice for young investigators and what's left to accomplish in the field. Finally, others in the research community who have worked closely with one or more of the laureates, share some of their personal reflections and anecdotes.

The frequency of CD127(+) hepatitis C virus (HCV)-specific T cells but not the expression of exhaustion markers predicts the outcome of acute HCV infection.

T cells are exhausted and overexpress inhibitory molecules in chronic hepatitis C virus (HCV) infection. It is unclear whether this is the cause or consequence of HCV persistence. By studying serial blood and liver samples of chimpanzees during acute infection, we demonstrate that the early expression of the memory precursor marker CD127 on HCV-specific T cells, but not the expression of inhibitory molecules on those T cells or their ligands in the liver, predicts the outcome of acute infection.

Prospects for prophylactic and therapeutic vaccines against hepatitis C virus.

Natural cross-protective immunity is induced after spontaneous clearance of primary hepatitis C virus (HCV) infection. Although this suggests that effective prophylactic vaccines against HCV are possible, there are still several areas that require further study. Current data indicate that, at best, vaccine-induced immunity may not completely prevent HCV infection but rather prevent persistence of the virus. However, this may be an acceptable goal, because chronic persistence of the virus is the main cause of pathogenesis and the development of serious liver conditions. Therapeutic vaccine development is also highly challenging; however, strategies have been pursued in combination with current or new treatments in an effort to reduce the costs and adverse effects associated with antiviral therapy. This review summarizes the current state of HCV vaccines and the challenges faced for future development and clinical trial design.

Delayed induction, not impaired recruitment, of specific CD8⁺ T cells causes the late onset of acute hepatitis C.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is characterized by lack of immune-mediated liver injury despite a high level of HCV replication during the incubation phase, which lasts about 8 weeks. We investigated whether this results from delayed recruitment of HCV-specific T cells and whether it facilitates HCV persistence. METHODS: Six chimpanzees were infected with HCV; blood and liver samples were collected for 28 weeks and analyzed for immune cells and chemokines. RESULTS: Two chimpanzees developed self-limited infections, whereas the remaining 4 developed chronic infections. Levels of the chemokines CXCL10, CXCL11, CCL4, and CCL5 increased in blood and liver samples from all chimpanzees within 1 month of HCV infection. Chemokine induction correlated with intrahepatic type I interferon (IFN) responses in vivo and was blocked by neutralizing antibodies against IFN-ß in vitro. Despite the early-stage induction of chemokines, the intrahepatic lymphocytic infiltrate started to increase no earlier than 8 weeks after HCV infection, when HCV-specific, tetramer-positive CD8(+) T cells appeared in the circulation. The HCV-specific CD8(+) T cells expressed chemokine receptors when they were initially detected in blood samples, so they could be recruited to the liver as soon as they entered the circulation. CONCLUSIONS: Chemokines are induced during early stages of HCV infection, which requires a type I IFN-mediated response. The delayed onset of acute hepatitis does not result from delayed recruitment of HCV-specific T cells, but could instead be related to a primary delay in the induction of HCV-specific T cells. Divergent outcomes occur without evident differences in chemokine induction and T-cell recruitment.

Persistent growth of a human plasma-derived hepatitis C virus genotype 1b isolate in cell culture.

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNA(I) (VA RNA(I)), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-alpha/beta-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2'-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics.

Meta-analysis of hepatitis C virus vaccine efficacy in chimpanzees indicates an importance for structural proteins.

BACKGROUND & AIMS: Studies in patients and chimpanzees that spontaneously cleared hepatitis C virus (HCV) infections demonstrated that natural immunity to the virus is induced during primary infections and that this immunity can be cross protective. These discoveries led to optimism about prophylactic HCV vaccines, and several studies were performed in chimpanzees, although most included fewer than 6 animals. To draw meaningful conclusions about the efficacy of HCV vaccines in chimpanzees, we performed statistical analyses of data from previously published studies from different groups. METHODS: We performed a meta-analysis that compared parameters among naïve (n = 63), vaccinated (n = 53), and rechallenged (n = 36) animals, including peak RNA titer postchallenge, time points of peak RNA titer, duration of viremia, and proportion of persistent infections. RESULTS: Each vaccination study induced immune responses that were effective in rapidly controlling HCV replication. Levels of induced T-cell responses did not indicate vaccine success. There was no reduction in the rate of HCV persistence in vaccinated animals, compared with naïve animals, when nonstructural proteins were included in the vaccine. Vaccines that contained only structural proteins had clearance rates that were significantly higher than vaccines that contained nonstructural components (P = .015). CONCLUSIONS: The inclusion of nonstructural proteins in HCV vaccines might be detrimental to protective immune responses, and/or structural proteins might activate T-cell responses that mediate viral clearance.

Selection of hepatitis C virus resistant to ribavirin.

BACKGROUND: Given the side effects associated with intravenous injections of interferon, an interferon-free regimen for the treatment of HCV infections is highly desirable. Recently published clinical studies show that interferon-free combination therapies containing ribavirin are efficacious, suggesting that an interferon-free therapy could be adopted in the near future. Therefore, understanding HCV resistance to ribavirin could be of major importance. In an approach to understand the effect of ribavirin on HCV replication and HCV resistance, we have selected a ribavirin resistant mutant of HCV in vitro. METHODS: We serially passed the J6/JFH1 strain of HCV in Huh7D cells (a Huh7 cell derivative more permissive to HCV replication) in the presence of different concentrations of ribavirin. Virus replication was assessed by detection of HCV antigens by immunfluorscence of infected cells and titration of recovered virus present in the supernatant. cDNAs from virus RNA grown in 0 or 250 uM concentrations of ribavirin were synthesized by RT-PCR, and sequenced. RESULTS: A concentration of 125 uM of ribavirin did not have a dramatic effect on HCV replication, while 500 uM of ribavirin lead to viral extinction. Concentrations of 250 uM of ribavirin dramatically reduced virus replication which was sustained over six passages. At passage seven viral resurgence began and over two passages the level of virus reached that of the wild type virus grown without ribavirin. Virus recovered from these cultures were more resistant to 250 uM ribavirin than wild type virus, and showed no difference in replication relative to wild type virus when grown in the absence of ribavirin. The ribavirin resistant virus accumulated multiple synonymous and non-synonymous mutations that are presently being analyzed for their relationship to ribavirin resistance. CONCLUSIONS: It is possible to select a ribavirin resistant mutant of HCV that can replicate to levels similar to wild type virus grown without ribavirin. Analysis of the mutations responsible for the ribavirin resistance may aid in understanding the mechanism of action of ribavirin.

Challenge pools of hepatitis C virus genotypes 1-6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models.

Chimpanzees represent the only animal model for studies of the natural history of hepatitis C virus (HCV). To generate virus stocks of important HCV variants, we infected chimpanzees with HCV strains of genotypes 1-6 and determined the infectivity titer of acute-phase plasma pools in additional animals. The courses of first- and second-passage infections were similar, with early appearance of viremia, HCV RNA titers of >10(4.7) IU/mL, and development of acute hepatitis; the chronicity rate was 56%. The challenge pools had titers of 10(3)-10(5) chimpanzee infectious doses/mL. Human liver-chimeric mice developed high-titer infections after inoculation with the challenge viruses of genotypes 1-6. Inoculation studies with different doses of the genotype 1b pool suggested that a relatively high virus dose is required to consistently infect chimeric mice. The challenge pools represent a unique resource for studies of HCV molecular virology and for studies of pathogenesis, protective immunity, and vaccine efficacy in vivo.

Increased susceptibility of Huh7 cells to HCV replication does not require mutations in RIG-I.

BACKGROUND: The cytosolic retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor that senses HCV double-stranded RNA and triggers type I interferon pathways. The clone Huh7.5 of human hepatoma Huh7 cells contains a mutation in RIG-I that is believed to be responsible for the improved replication of HCV in these cells relative to the parental strain. We hypothesized that, in addition to RIG-I, other determinant(s) outside the RIG-I coding sequence are involved in limiting HCV replication in cell culture. To test our hypothesis, we analyzed Huh7 cell clones that support the efficient replication of HCV and analyzed the RIG-I gene. RESULTS: One clone, termed Huh7D, was more permissive for HCV replication and more efficient for HCV-neomycin and HCV-hygromycin based replicon colony formation than parental Huh7 cells. Nucleotide sequence analysis of the RIG-I mRNA coding region from Huh7D cells showed no mutations relative to Huh7 parental cells. CONCLUSIONS: We derived a new Huh7 cell line, Huh7D, which is more permissive for HCV replication than parental Huh7 cells. The higher permissiveness of Huh7D cells is not due to mutations in the RIG-I protein, indicating that cellular determinants other than the RIG-I amino-acid sequence are responsible for controlling HCV replication. In addition, we have selected Huh7 cells resistant to hygromycin via newly generated HCV-replicons carrying the hygromycin resistant gene. Further studies on Huh7D cells will allow the identification of cellular factors that increased the susceptibility to HCV infection, which could be targeted for anti-HCV therapies.

Virus-induced type I IFN stimulates generation of immunoproteasomes at the site of infection.

IFN-gamma is known as the initial and primary inducer of immunoproteasomes during viral infections. We now report that type I IFN induced the transcription and translation of immunoproteasome subunits, their incorporation into the proteasome complex, and the generation of an immunoproteasome-dependent CD8 T cell epitope in vitro and provide in vivo evidence that this mechanism occurs prior to IFN-gamma responses at the site of viral infection. Type I IFN-mediated generation of immunoproteasomes was initiated by either poly(I:C) or HCV RNA in human hepatoma cells and was inhibited by neutralization of type I IFN. In serial liver biopsies of chimpanzees with acute HCV infection, increases in immunoproteasome subunit mRNA preceded intrahepatic IFN-gamma responses by several weeks, instead coinciding with intrahepatic type I IFN responses. Thus, viral RNA-induced innate immune responses regulate the antigen-processing machinery, which occurs prior to the detection of IFN-gamma at the site of infection. This mechanism may contribute to the high effectiveness (95%) of type I IFN-based therapies if administered early during HCV infection.

History of the Discovery of Hepatitis A Virus.

Disease outbreaks resembling hepatitis A have been known since antiquity. However, it was not until World War II when two forms of viral hepatitis were clearly differentiated. After the discovery of Australia antigen and its association with hepatitis B, similar methodologies were used to find the hepatitis A virus. The virus was ultimately identified when investigators changed the focus of their search from serum to feces and applied appropriate technology.

Proteasome activator and antigen-processing aminopeptidases are regulated by virus-induced type I interferon in the hepatitis C virus-infected liver.

Many components of the class I antigen-processing pathway are thought to be regulated solely by interferon-gamma (IFN-gamma). Herein, we report type I IFN-mediated induction of proteasome activator (PA28) subunits alpha and beta, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and leucine aminopeptidase (LAP). This mechanism was initiated by either synthetic RNA (poly(I-C)) or by hepatitis C virus (HCV) RNA-mediated induction of type I IFN and abrogated by blocking of type I IFN. In serial liver biopsies of chimpanzees with acute HCV infection, increases in PA28 subunit and aminopeptidase mRNA levels correlated with intrahepatic type I IFN responses and preceded intrahepatic IFN-gamma responses by several weeks. Thus, viral RNA-induced type I IFN regulates the antigen-processing machinery early during viral infection and prior to IFN-gamma response. This mechanism may contribute to the high effectiveness of type I IFN-based therapies if administered early during acute HCV infection.

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV.

Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a novel coronavirus termed SARS-CoV. Due to the severity of this disease, the World Health Organization (WHO) recommends that manipulation of active viral cultures of SARS-CoV be performed in containment laboratories at biosafety level 3 (BSL3). The virus was inactivated by ultraviolet light (UV) at 254 nm, heat treatment of 65 degrees C or greater, alkaline (pH > 12) or acidic (pH < 3) conditions, formalin and glutaraldehyde treatments. We describe the kinetics of these efficient viral inactivation methods, which will allow research with SARS-CoV containing materials, that are rendered non-infectious, to be conducted at reduced safety levels.

Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay.

The availability of molecular protocols for the detection and quantitation of very low numbers of hepatitis C virus (HCV) particles in biological samples is an issue of interest in both clinical and analytical fields of HCV research. A sensitive and reproducible assay is described for HCV RNA quantitation using the TaqMan PCR fluorogenic real-time detection system to establish the levels of HCV RNA in chimpanzee plasma. Our TaqMan PCR protocol and synthetic full length HCV RNA template show that the threshold of sensitivity for our TaqMan PCR is two copies per reaction. As few as 10 genome copies per reaction could be quantitated maintaining a linear range. The accuracy of the TaqMan PCR test was comparable to commercial bDNA and Amplicor tests. The RNA standards of the laboratory were tested in parallel with a World Health Organization (WHO) International Standard for HCV RNA obtaining ratios of 2.7+/-0.7 RNA copies per HCV international unit (IU). Our method using RNA extracted from chimpanzee samples had an estimated sensitivity of 200 RNA copies/ml of plasma (approximately eight copies/reaction or 74 WHO IU/ml). Serial plasma samples from HCV-infected chimpanzees were analyzed using this methodology to evaluate its applicability, and RNA profiles were observed consistent with the evolution of the pathology in each animal. The present study therefore illustrates the high reproducibility, sensitivity and reliability of our TaqMan methodology, providing a useful method for HCV research to consistently detect and quantify viral RNA throughout a range of concentrations.

Engrafted human stem cell-derived hepatocytes establish an infectious HCV murine model.

The demonstrated ability to differentiate both human embryonic stem cells (hESCs) and patient-derived induced pluripotent stem cells (hiPSCs) into hepatocyte-like cells (HLCs) holds great promise for both regenerative medicine and liver disease research. Here, we determined that, despite an immature phenotype, differentiated HLCs are permissive to hepatitis C virus (HCV) infection and mount an interferon response to HCV infection in vitro. HLCs differentiated from hESCs and hiPSCs could be engrafted in the liver parenchyma of immune-deficient transgenic mice carrying the urokinase-type plasminogen activator gene driven by the major urinary protein promoter. The HLCs were maintained for more than 3 months in the livers of chimeric mice, in which they underwent further maturation and proliferation. These engrafted and expanded human HLCs were permissive to in vivo infection with HCV-positive sera and supported long-term infection of multiple HCV genotypes. Our study demonstrates efficient engraftment and in vivo HCV infection of human stem cell-derived hepatocytes and provides a model to study chronic HCV infection in patient-derived hepatocytes, action of antiviral therapies, and the biology of HCV infection.

Chimeric mouse model for the infection of hepatitis B and C viruses.

While the chimpanzee remains the only animal that closely models human hepatitis C virus (HCV) infection, transgenic and immunodeficient mice in which human liver can be engrafted serve as a partial solution to the need for a small animal model for HCV infection. The established system that was based on mice carrying a transgene for urokinase-type plasminogen activator (uPA) gene under the control of the human albumin promoter has proved to be useful for studies of virus infectivity and for testing antiviral drug agents. However, the current Alb-uPA transgenic model with a humanized liver has practical limitations due to the inability to maintain non-engrafted mice as dizygotes for the transgene, poor engraftment of hemizygotes, high neonatal and experimental death rates of dizygous mice and a very short time window for hepatocyte engraftment. To improve the model, we crossed transgenic mice carrying the uPA gene driven by the major urinary protein promoter onto a SCID/Beige background (MUP-uPA SCID/Bg). These transgenic mice are healthy relative to Alb-uPA mice and provide a long window from about age 4 to 12 months for engraftment with human hepatocytes and infection with hepatitis C or hepatitis B (HBV) viruses. We have demonstrated engraftment of human hepatocytes by immunohistochemistry staining for human albumin (30-80% engraftment) and observed a correlation between the number of human hepatocytes inoculated and the level of the concentration of human albumin in the serum. We have shown that these mice support the replication of both HBV and all six major HCV genotypes. Using HBV and HCV inocula that had been previously tittered in chimpanzees, we showed that the mice had approximately the same sensitivity for infection as chimpanzees. These mice should be useful for isolating non-cell culture adapted viruses as well as testing of antiviral drugs, antibody neutralization studies and examination of phenotypic changes in viral mutants.

Evidence for a functional RNA element in the hepatitis C virus core gene.

In the core protein-coding region of hepatitis C virus (HCV), evidence exists for both phylogenetically conserved RNA structures and a +1 alternative reading frame (ARF). To investigate its role in HCV infection, we introduced four stop codons into the ARF of a genotype 1a H77 molecular clone. The changes did not alter the core protein sequence, but were predicted to disrupt RNA secondary structures. An attenuated infection was established after inoculation of the mutant HCV RNA into an HCV naïve chimpanzee. The acute infection was atypical with low peak viremia, minimal alanine aminotransferase elevation, and early virus control by a diverse adaptive immune response. Sequencing circulating virus revealed progressive reversions at the third and then fourth stop codon. In cell culture, RNA replication of a genome with four stop codons was severely impaired. In contrast, the revertant genome exhibited only a 5-fold reduction in replication. Genomes harboring only the first two stop codons replicated to WT levels. Similarly, reversions at stop codons 3 and 4, which improved replication, were selected with recombinant, infectious HCV in cell culture. We conclude that ARF-encoded proteins initiating at the polyprotein AUG are not essential for HCV replication in cell culture or in vivo. Rather, our results provide evidence for a functionally important RNA element in the ARF region.

Hepatitis C virus epitope-specific neutralizing antibodies in Igs prepared from human plasma.

Neutralizing antibodies directed against hepatitis C virus (HCV) are present in Igs made from anti-HCV-positive plasma. However, these HCV-specific Igs are largely ineffective in vivo. The mechanism for the poor effectiveness is currently unknown. We hypothesize that the presence of nonneutralizing antibodies in HCV-specific Igs interferes with the function of neutralizing antibodies, resulting in the reduction or blockage of their effect. In the present study, we identified at least two epitopes at amino acid residues 412-419 (epitope I) and 434-446 (epitope II), located downstream of the hypervariable region I within the HCV E2 protein. We demonstrated that epitope I, but not epitope II, was implicated in HCV neutralization and that binding of a nonneutralizing antibody to epitope II completely disrupted virus neutralization mediated by antibody binding at epitope I. The dynamic interaction between nonneutralizing and neutralizing antibodies may thus play a key role in determining the outcomes of HCV infection. Further exploration of this interplay should lead to a better understanding of the mechanisms of neutralization and immune escape and may indicate pathways for the manufacture of an effective HCV-specific Ig product for immune prophylaxis of HCV infection.

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver.

We established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP+ cells expressing ALB were detected in close proximity to "beating" myocytes after 7 days of EB cultures. GFP+ cells increased in number, acquired hepatocyte-like morphology and hepatocyte-specific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP+ cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl(4)-induced liver injury. The transplanted GFP+ cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy.