Molecular Mechanisms of Pathogenesis of Acute and Chronic Liver Diseases

National Institute Of Allergy And Infectious Diseases

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Abstract

Over the past year, the Hepatic Pathogenesis Section has conducted an extensive program of basic and clinical research to study the pathogenesis of acute and chronic human liver disease, with a major focus on viral hepatitis and its long-term sequelae, cirrhosis and HCC, which contribute to a huge burden of disease worldwide. The mission of the section remains centered on translational research and the development of innovative strategies for prevention, diagnosis and treatment to reduce mortality from liver disease. 1. Pathogenesis of Acute Liver Failure and Acute Viral Hepatitis HBV is a major cause of ALF worldwide; about 1% of acute hepatitis B patients develop ALF. However, the molecular and immunological mechanisms dictating the different outcomes of acute HBV infection are poorly understood. A. Role of the humoral immunity in the pathogenesis of HBV ALF Building on our previous findings, which demonstrated a major role of the humoral immunity in HBV ALF, including an extensive intrahepatic production of IgM and IgG in germline configuration that exclusively target HBcAg with subnanomolar affinity, accompanied by complement deposition, we have conducted further investigations over the past year into the molecular basis of this response. In collaboration with Drs. Tsybovsky and Kwong from the VRC, we used cryogenic electron microscopy (cryo-EM) to compare the structures of the wild-type (ayw) and ALF-associated HBV core antigens, as well as their complexes with Fab fragments derived from ALF patients and chimpanzees with classic acute hepatitis B. The cryo-EM structures revealed distinct differences in binding characteristics between AHB- and ALF-derived Fabs. AHB-derived Fabs bound to either the tip or the floor of the HBcAg spikes via a canonical interaction that utilized complementarity determining regions (CDRs). In contrast, ALF-derived Fabs bound uniformly to the side of the HBcAg spikes, and some of them formed non-canonical contacts with HBcAg using the framework regions in addition to the CDRs. Notably, some ALF-derived Fabs utilized the deep insertion of their extended HCDR3 loop to form the majority of the antigen-contact interface. These structural features may explain how germline antibodies can exhibit unusually high binding affinity in the absence of somatic hypermutation. Our findings provide new insights into the immune mechanisms that may contribute to the pathogenesis of HBV-associated ALF. 2. Role of Hepatitis Viruses in the Pathogenesis of Chronic Liver Disease and HCC Over the past year, we have focused our research into the mechanisms by which hepatitis viruses contribute to chronic liver disease and HCC. HCC ranks sixth in incidence and second in mortality among cancers worldwide, with an overall 5-year survival rate lower than 20%. Despite significant progress in cancer survival overall, HCC has become the fastest-rising cause of cancer-related death in the US. Cirrhosis remains the major risk factor for the development of HCC, being present in 80-90% of the cases. Although hepatitis viruses account for over 70% of all HCC cases globally, the mechanisms whereby HBV, HCV and HDV promote hepatocarcinogenesis remain unknown. A key unresolved question is whether hepatitis viruses promote liver cancer indirectly through chronic inflammation, fibrosis, and liver regeneration, or directly, through the expression of oncogenic viral proteins, as seen with other tumor-associated viruses. Over the past year, we have focused particularly on the role of HDV, which is associated with a significantly higher risk of HCC compared to HBV alone. However, its role in oncogenesis has not yet been defined. A) Tumor microenvironment and intrahepatic HBV replication The increasing use of immune-based therapies in solid tumors underscores the importance of expanding our knowledge on the tumor microenvironment (TME) in HCC. The TME plays a critical role in intratumor heterogeneity and evolution, treatment failure, and, ultimately, disease outcome. In our earlier work, we identified two immune subtypes of HBV-related HCC—immune-high and immune-low (De Battista et al., 2024). This past year, we investigated the relationship between these immune phenotypes and intrahepatic HBV replication. (i) Intrahepatic hepatitis B surface antigen (HBsAg) expression differentiates immune-high and immune-low subtypes of HBV-associated HCC This year, we analyzed paired liver tissue samples (tumor and non-tumor) from 12 patients with HBV-HCC, equally divided between immune-high and immune-low phenotypes. All patients were under nucleos(t)ide analogue therapy at the time of surgery and had similar serum profiles: HBsAg-positive, HBeAg-negative, and anti-HBe-positive. HBV DNA levels were comparable across groups in both tumor and non-tumor tissue, as well as in serum. In contrast, HBsAg expression showed a striking difference. It was detected in all six immune-high tumors, with a diffuse cytoplasmic and membranous pattern, but only in one of six immune-low tumors. In non-tumor tissue, HBsAg was present in all but one immune-low patient. These findings provide the first evidence that HBsAg expression can distinguish immune-high from immune-low tumors and offer new insights into virus-host interactions in HBV-HCC. Moving forward, we plan to validate these findings in a larger cohort to assess whether intrahepatic HBsAg expression may serve as a biomarker for predicting response to immunotherapy. B. Role of HDV in hepatocarcinogenesis HDV causes the least common but most severe form of chronic viral hepatitis across all ages, leading to cirrhosis in up to 80% of cases, a major risk factor for the development of HCC. While cohort studies have shown that patients with chronic HDV infection have a significantly higher risk of developing HCC than those with HBV monoinfection, the molecular mechanisms of HDV -driven carcinogenesis remain unclear. HDV is a defective RNA virus that uses HBsAg as its envelope protein for virion formation, release, and transmission. However, HDV replication is independent from HBV and occurs exclusively in the nucleus. HDV encodes a single structural protein, the hepatitis delta antigen (HDAg), which exists in two isoforms with distinct functions: S-HDAg promotes viral replication whereas the L-HDAg inhibits replication and favors viral assembly. The obligatory symbiosis of HDV with HBV has made it difficult to dissect the mechanisms whereby HDV induces severe liver damage and HCC. Also, the limited availability of liver specimens from patients with chronic hepatitis D has been and continues to be a major obstacle in investigating HDV pathogenesis. Thus, whether HDV plays a direct role in hepatocarcinogenesis remains unknown. This past year, we performed a series of in vitro and in vivo studies that provide the first evidence that HDV exerts pro-oncogenic functions. We found that HDAg orchestrates a complex gene expression program, ultimately leading to enhanced cell proliferation and chromosomal instability. Importantly, the in vitro data were validated by in vivo findings in tumor tissue of patients with HDV HCC. Collectively, our data suggest a model whereby HDAg triggers a pro-proinflammatory and pro-oxidative environment that favors a mutagenic milieu in which DNA damage accumulates over the years, eventually leading to genome instability, which has emerged as a major culprit in cancer development and progression. (i) Spatial Compartmentalization of HBV and HDV in Coinfected Liver Tissue Mediated by HDV-Induced Suppression of HBsAg Given the obligatory dependence of HDV on HBsAg, we sought to understand how HDV-associated HCC develops in the context of HBV-HDV coinfection. Over the past year, we examined the spatial distribution of viral antigens and transcriptomic profiles in liver tissues from three patient cohorts: HDV-associated HCC (n=4), non-HCC HDV cirrhosis (n=7), and HBV-associated HCC (n=11). All HBV-HCC patients had received antiviral therapy prior to surgery or transplantation. We used RNA sequencing and in situ hybridization, combined with immunohistochemistry, to map the expression of viral markers (HDAg, HBsAg, HBcAg) and viral RNA. Our findings reveal minimal colocalization of HDAg and HBsAg within the same hepatocytes, indicating spatial compartmentalization of HBV and HDV. Further analysis suggests that HDV suppresses HBsAg expression via host gene modulation, likely contributing to the mutual exclusion of viral antigens. This study provides novel mechanistic insights into HDV pathogenesis and highlights the complex viral dynamics in coinfected liver tissue. Studies are ongoing to further characterize this compartmentalization and its implications for HCC development, especially in the context of HDV’s dependence on HBV for virion assembly and propagation. C. Characterization of broadly neutralizing antibodies against HCV for vaccine development Despite the availability of effective antiviral therapies, HCV remains a major global health challenge due to high rates of undiagnosed infections, limited access to treatment and ongoing transmission. Thus, a preventive vaccine is urgently needed to achieve long-term control and eventual elimination of HCV. However, the genetic heterogeneity of HCV poses a major challenge for the development of an HCV vaccine. Over the past year, we characterized a panel of neutralizing antibodies and their target epitopes on the HCV envelope proteins to support vaccine development. Using a phage display antibody library derived from a chronically infected patient, we identified a panel of VH1-69-derived antibody fragments (Fabs) that bind strongly to conserved regions of the E2 protein with dissociation constants in the low picomolar range. These antibodies showed broad neutralizing activity across HCV genotypes 1–6, with remarkably high potency. Structural analysis suggests that unlike other neutralizing antibodies, these Fabs target a unique site encompassing both the AR2A and AR3A antigenic regions of the E2 protein. These findings provide new insights into vaccine design strategies aimed at generating broadly neutralizing antibodies against HCV