Abstract Background Acute hepatitis B virus (HBV) infection encompasses a broad clinical spectrum, from self-limited hepatitis to acute liver injury (ALI), acute liver failure (ALF), subacute liver failure, and ALF on chronic HBV reactivation. This study investigates how host and viral factors interact to shape the clinical spectrum of HBV-associated ALF. Methods We analyzed 231 serial serum samples from 49 well-characterized patients including 36 with HBV-associated ALF and 13 with acute hepatitis B. Among ALF patients, 7 had ALI, 7 acute and 11 subacute liver failure, and 11 ALF on chronic HBV. Viral factors, including HBV replication, genotype, sequence diversity and viral evolution by next-generation sequencing (NGS) were assessed alongside host immune responses and cytokine profiles by proteomics. Results Demographics were similar across groups. Mortality was highest in subacute liver failure and ALF on chronic HBV (82%) and intermediate in acute liver failure (43%). The three forms of ALF differed by clinical, virologic, and immunologic features. Acute liver failure was characterized by markedly elevated alanine aminotransferase and high IgM anti-HBc titers, whereas ALF on CHB showed higher serum HBV DNA, HBV RNA and HBcrAg levels. HBV genotype A predominated (63%), followed by D (15%), C (9%), and others (2–6%). NGS of the basal core promoter and precore/core regions revealed highly homogeneous viral populations in acute and subacute liver failure throughout follow-up, while ALF on CHB showed marked viral heterogeneity. Genetic diversity was genotype-dependent, unrelated to disease severity, and significantly lower in genotype A than D. Acute liver failure was associated with broad type 1 cytokine response (i.e. IP-10, MIP-1α, MIP-1β, TNF-α, and IFN-γ), restricted type 2 cytokine response (i.e. IL-4 and sCD137), along with proinflammatory (i.e. MCP-1, Il-6, and IL-8) and homeostatic (i.e. IL-7) cytokines, whereas subacute liver failure and ALF on chronic HBV exhibited low and restricted, but distinct cytokine profiles. Elevated GM-CSF and PDGF-BB at admission predicted transplant-free survival. Conclusions Distinct clinical, serologic, virologic, and cytokine profiles correlate with disease severity, highlighting differing pathogenic mechanisms across HBV-associated ALF forms. Early biomarkers, including GM-CSF and PDGF-BB, may help predict recovery and guide individualized management to improve survival.

Synopsis of the 20 top-scored pathways associated with WLT-unique genes identified in the three tumors.

Clusters of the 37 genes involved in the cell cycle and differentially expressed in malignant hepatocytes derived from HDV-HCC.

Synopsis of the 20 top-scored pathways associated with genes identified in malignant hepatocytes derived from HDV-, HBV- and HCV-HCC.

Genes associated with the twenty top-scored pathways of malignant hepatocytes of HDV-HCC.

INTRODUCTION: Hepatitis E virus (HEV) infection rarely causes icteric hepatitis, yet 10%–40% of adult Americans have serological evidence of previous infection. The aim of this study was to investigate the incidence, presentation, and outcome of acute and previous HEV infection in a large cohort of patients with suspected drug-induced liver injury (DILI). METHODS: Serum samples from 2012 patients enrolled in the DILI Network were tested for anti-HEV immunoglobulin G (IgG). Those with detectable anti-HEV IgG underwent testing for anti-HEV IgM; those with detectable anti-HEV immunoglobulin m (IgM) were tested for HEV RNA. RESULTS: Anti-HEV IgG was detected in 407 (20%) patients and associated with increasing subject age and earlier year of enrollment. The median age of seropositive subjects was more than a decade higher than seronegative subjects (59.8 vs 48.7 years). The overall prevalence of anti-HEV declined from 22% (2004–2011) to 18% (2012–2019), suggestive of a cohort effect. The frequency of acute hepatitis E (median ALT = 1231 IU/L) also decreased from 3% (2004–2008) to 1.2% (2009–2013) to 0.6% (2014–2019). These results suggest that acute HEV infection is usually subclinical and was much more frequent in this cohort before 2004. DISCUSSION: Acute HEV infection accounts for less than 1% of suspected American DILI cases and is more frequent in older men. Previous HEV infection is also most commonly seen in older individuals. Clinicians should consider testing for unsuspected acute HEV infection in older adult patients with acute hepatocellular DILI and jaundice.

INTRODUCTION:HCC is the third leading cause of cancer-related death worldwide, with chronic viral hepatitis accounting for more than 70% of the cases. Therapeutic options are limited and ineffective. The increasing use of immune-based therapies in solid tumors highlights the need to expand our knowledge on the immunologic microenvironment of HCC.METHODS:Access to liver samples from 20 well-characterized patients with HCC associated with HCV (n = 9) or HBV (n = 11) gave us the opportunity to study the immunologic landscape in these tumors. For each patient, RNA-sequencing was performed on the tumor and surrounding nontumorous tissue.RESULTS:We found that both HCV- and HBV-HCC are associated with a predominance of downregulated genes (74% and 67%, respectively). Analysis of the immune landscape using a curated gene list showed 216 of 2481 (9%) immune genes in HCV-HCC and 164 of 2560 (6%) in HBV-HCC. However, only 8 immune genes (4%) were upregulated in HCV-HCC and 27 (16.5%) in HBV-HCC. HCV-HCC was characterized by an enrichment of downregulated genes related to T-cell activation and oxidative stress. The dramatic downregulation of immune genes related to T-cell activation in HCV-HCC prompted us to perform an extensive immunohistochemistry analysis on paraffin-embedded liver specimen. Interestingly, we found a significant reduction of immune-cell infiltration (CD3, CD8 and CD20 positive cells) within the tumor. Moreover, we observed that HCV-HCC is characterized by an enrichment of M2-like CD68-positive cells. These data are consistent with the dramatic downregulation of immune-cell infiltration seen in HCV-HCC. Conversely, HBV-HCC was characterized by upregulation of genes related to monocyte/macrophage activation and cell cycle control, and downregulation of genes involved in various cell metabolisms.CONCLUSION:This study demonstrates a distinctive molecular signature and immune landscape in HCC of different viral etiology, which could provide new insights into pathogenesis and lead to the development of novel immune-based therapies.

Older age at the time of infection with hepatitis viruses is associated with an increased risk of liver fibrosis progression. We hypothesized that the pace of fibrosis progression may reflect changes in gene expression within the aging liver. We compared gene expression in liver specimens from 54 adult donors without evidence of fibrosis, including 36 over 40 y old and 18 between 18 and 40 y old. Chitinase 3-like 1 (CHI3L1), which encodes chitinase-like protein YKL-40/CHI3L1, was identified as the gene with the greatest agedependent increase in expression in liver tissue. We investigated the cellular source of CHI3L1 in the liver and its function using liver tissue specimens and in vitro models. CHI3L1 expression was significantly higher in livers of patients with cirrhosis of diverse etiologies compared with controls represented by patients who underwent liver resection for hemangioma. The highest intrahepatic CHI3L1 expression was observed in cirrhosis due to hepatitis D virus, followed by hepatitis C virus, hepatitis B virus, and alcohol-induced cirrhosis. In situ hybridization of CHI3L1 messenger RNA (mRNA) identified hepatocytes as the major producers of CHI3L1 in normal liver and in cirrhotic tissue, wherein hepatocytes adjacent to fibrous septa showed higher CHI3L1 expression than did those in more distal areas. In vitro studies showed that recombinant CHI3L1 promotes proliferation and activation of primary human hepatic stellate cells (HSCs), the major drivers of liver fibrosis. These findings collectively demonstrate that CHI3L1 promotes liver fibrogenesis through a direct effect on HSCs and support a role for CHI3L1 in the increased susceptibility of aging livers to fibrosis progression.

INTRODUCTION: The aim of this study was to determine the role of hepatitis E virus (HEV) infection in a large cohort of prospectively enrolled patients with severe acute liver injury (ALI). METHODS: Serum samples from 594 consecutive adults enrolled between 2008 and 2018 in the US Acute Liver Failure Study Group ALI registry were tested for anti-HEV IgM and anti-HEV IgG levels. Those with detectable anti-HEV IgM underwent further testing for HEV RNA using real-time polymerase chain reaction. RESULTS: The median age of patients was 38 years; 41% were men and 72% Caucasian. Etiologies of ALI included acetaminophen hepatotoxicity (50%), autoimmune hepatitis (8.9%), hepatitis B virus (8.9%), and idiosyncratic drug-induced liver injury (7.9%). Overall, 62 patients (10.4%) were negative for anti-HEV IgM but positive for IgG, whereas only 3 men (0.5%) were positive for both anti-HEV IgM and IgG. These 3 cases were initially diagnosed as having indeterminate, HEV, and hepatitis B virus-related ALI. One of these patients had detectable HEV RNA genotype 3, and another anti-HEV IgM+ patient had detectable HEV antigens by immunohistochemistry on liver biopsy. On multivariate modeling, older (odds ratio: 1.99) and non-Caucasian subjects (odds ratio: 2.92) were significantly more likely to have detectable anti-HEV IgG (P < 0.0001). DISCUSSION: Acute HEV infection is an infrequent cause of ALI in hospitalized North American adults. The anti-HEV IgG+ patients were significantly older and more likely to be non-Caucasian. These data are consistent with other population-based studies that indicate exposure to HEV in the general US population is declining over time and might reflect a cohort effect.

Hepatitis B virus (HBV) is a major cause of acute liver failure (ALF) worldwide. While liver damage in classic acute hepatitis B is believed to be T-cell mediated, the pathogenesis of HBV-associated ALF remains largely unknown. Access to liver specimens from well-characterized patients with HBV-associated ALF provided us with the opportunity to perform next-generation sequencing (NGS) of the entire VH repertoires of IgM and IgG from the livers of four ALF patients, a control liver donor and a patient with chronic HBV infection. We found that ALF is not associated with expansion of specific B-cell lineages. However, NGS showed that the intrahepatic VH repertoires from ALF patients were characterized by the abundant presence of antibodies in germline configuration in contrast to their marginal prevalence in controls. Moreover, NGS identified a large number of VH genes in germline configuration with identical VDJ sequences in the IgM and IgG repertoires in all four ALF patients, indicating that isotype switch from IgM to IgG had occurred without somatic hypermutation. The results of this study indicate that the presence of intrahepatic antibodies in unmutated germline configuration is a broad phenomenon in the global antibody repertoire generated from total RNA derived from whole-liver tissue that is strongly associated with ALF, suggesting a major role of T cell-independent humoral immunity in the pathogenesis of ALF.

Transmission to chimpanzees of a precore hepatitis B virus (HBV) mutant implicated in acute liver failure (ALF) in humans did not cause ALF nor the classic form of acute hepatitis B (AHB) seen upon infection with the wild-type HBV strain, but rather a severe AHB with distinct disease features. Here, we investigated the viral and host immunity factors responsible for the unusual severity of AHB associated with the precore HBV mutant in chimpanzees. Archived serial serum and liver specimens from two chimpanzees inoculated with a precore HBV mutant implicated in ALF and two chimpanzees inoculated with wild-type HBV were studied. We used phage-display library and next-generation sequencing (NGS) technologies to characterize the liver antibody response. The results obtained in severe AHB were compared with those in classic AHB and HBV-associated ALF in humans. Severe AHB was characterized by: (i) the highest alanine aminotransferase (ALT) peaks ever seen in HBV transmission studies with a significantly shorter incubation period, compared to classic AHB; (ii) earlier HBsAg clearance and anti-HBs seroconversion with transient or undetectable hepatitis B e antigen (HBeAg); (iii) limited inflammatory reaction relative to hepatocellular damage at the ALT peak with B-cell infiltration, albeit less extensive than in ALF; (iv) detection of intrahepatic germline antibodies against hepatitis B core antigen (HBcAg) by phage-display libraries in the earliest disease phase, as seen in ALF; (v) lack of intrahepatic IgM anti-HBcAg Fab, as seen in classic AHB, but at variance with ALF; and (vi) higher proportion of antibodies in germline configuration detected by NGS in the intrahepatic antibody repertoire compared to classic AHB, but lower than in ALF. This study identifies distinct outcome-specific features associated with severe AHB caused by a precore HBV mutant in chimpanzees, which bear closer resemblance to HBV ALF than to classic AHB. Our data suggest that precore HBV mutants carry an inherently higher pathogenicity that, in addition to specific host factors, may play a critical role in determining the severity of acute HBV disease.

Studies performed in chimpanzees at the National Institutes of Health (NIH) demonstrated a significant difference in ALT levels during acute hepatitis of different viral etiologies, with a hierarchy in the extent of liver damage according to the infecting virus: the highest level was in HDV superinfection, followed by infection with a precore HBV mutant, HBV/HDV coinfection, and, lastly, wild-type HBV infection. Our study demonstrates that both the virus and host are important in disease pathogenesis and offers new insights into their roles. We found that distinct cytokine profiles were associated with disease severity and clinical outcome. In particular, resolution of classic acute hepatitis B (AHB) correlated with a predominant Th1 response, whereas HBV/HDV coinfection showed a predominant proinflammatory response. Severe AHB and HDV superinfection showed a restricted cytokine profile and no evidence of Th1 response. The lack of cytokines associated with adaptive T-cell responses toward the precore HBV mutant and HDV superinfection argues in favor of a direct cytopathic effect of these viruses. ABSTRACT Historical studies conducted in chimpanzees gave us the opportunity to investigate the basis for the different severities of liver damage and disease outcome associated with infection with wild-type hepatitis B virus (HBV) versus a precore HBV mutant, HBV/hepatitis D virus (HDV) coinfection, and HDV superinfection. Weekly samples from 9 chimpanzees were studied for immune responses by measuring plasma levels of 29 cytokines in parallel with alanine aminotransferase (ALT) levels and viral kinetics. Comparison of classic acute hepatitis B (AHB) with severe or progressive AHB and HBV/HDV coinfection or superinfection identified distinct cytokine profiles. Classic AHB (mean ALT peak, 362 IU/liter) correlated with an early and significant induction of interferon alpha-2 (IFN-α2), IFN-γ, interleukin-12 p70 (IL-12 p70), and IL-17A. In contrast, these cytokines were virtually undetectable in severe AHB (mean ALT peak, 1,335 IU/liter), characterized by significant elevations of IL-10, tumor necrosis factor alpha (TNF-α), and MIP-1β. In progressive AHB (mean ALT peak, 166 IU/liter), there was a delayed and lower-magnitude induction of cytokines. The ALT peak was also delayed (mean, 23.5 weeks) compared to those of classic (13.5 weeks) and severe AHB (7.5 weeks). HBV/HDV coinfection correlated with significantly lower levels of IFN-α2, IFN-γ, and IL-17A, associated with the presence of multiple proinflammatory cytokines, including IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-15. Conversely, HDV superinfection induced the highest ALT peak (1,910 IU/liter) and was associated with a general suppression of cytokines. Our data demonstrate that the most severe liver damage, caused by an HBV precore mutant and HDV, correlated with restricted cytokine expression and lack of Th1 response, raising the question of whether these viruses are directly cytopathic. IMPORTANCE Studies performed in chimpanzees at the National Institutes of Health (NIH) demonstrated a significant difference in ALT levels during acute hepatitis of different viral etiologies, with a hierarchy in the extent of liver damage according to the infecting virus: the highest level was in HDV superinfection, followed by infection with a precore HBV mutant, HBV/HDV coinfection, and, lastly, wild-type HBV infection. Our study demonstrates that both the virus and host are important in disease pathogenesis and offers new insights into their roles. We found that distinct cytokine profiles were associated with disease severity and clinical outcome. In particular, resolution of classic acute hepatitis B (AHB) correlated with a predominant Th1 response, whereas HBV/HDV coinfection showed a predominant proinflammatory response. Severe AHB and HDV superinfection showed a restricted cytokine profile and no evidence of Th1 response. The lack of cytokines associated with adaptive T-cell responses toward the precore HBV mutant and HDV superinfection argues in favor of a direct cytopathic effect of these viruses.