With several drugs in use that inhibit the hepatitis B virus polymerases (nucleos(t)ide analogues (or NUCs)), some argue that new direct-acting antiviral drugs, and new NUCs in particular, are not necessary. Here, we make the counter-argument that additional direct-acting antiviral drugs that can potently suppress hepatitis B virus replication, ideally via distinct mechanisms, are still needed and can provide additional therapeutic benefits.

Small-molecule HBV RNA destabilizing agents, such as the dihydroquinolizinones (DHQs), were first disclosed in a patent filing in 2015 and in peer reviewed literature in 2018. These compounds inhibit Poly-adenylating Polymerases 5 and 7 (PAPD5/7) and represent a novel antiviral strategy and their ability to degrade hepatitis B surface antigen (HBsAg) in cell culture and animal models generated considerable excitement and commercial interest. However, extrahepatic toxicity observed in preclinical and Phase I studies led to the discontinuation of several development programs. The subsequent emergence of liver-targeted PAPD5/7 inhibitors with improved safety profiles has rekindled interest in this therapeutic approach. Yet, with the apparent success of other investigational antivirals in reducing HBsAg levels, such as siRNAs, antisense oligonucleotides, and in at least one example, capsid assembly modulators (CAMs), questions remain as to whether RNA destabilizers still have a role in managing chronic hepatitis B (CHB). This review describes the current status of PAPD5/7 inhibitor development, evaluates the advantages and limitations of the approach, and considers potential strategies for integrating this class of molecules with other HBV therapies.

Chronic hepatitis B is associated with virus-specific and global T-cell dysfunction. We hypothesized that therapeutic reduction in serum HBV DNA, ALT, and HBsAg would restore HBV-specific T-cell function and modify T-cell regulatory phenotype, with associated posttreatment ALT flare. HBV-specific T-cell lymphoproliferative responses and global T-cell phenotype were prospectively examined at baseline, weeks 24, 48, 192, 216, and 240 in 34 adults with immune-active chronic hepatitis B treated with 192 weeks of tenofovir alone (n=21) or combined with pegylated interferon (PegIFN) in the first 24 weeks (n=13). HBV-specific T-cell IFNγ responses at weeks 0, 24, and 48 were examined by ELISpot assay ex vivo in 24 patients. Posttreatment flare was defined by serum ALT >5 times the upper limit of normal. Tenofovir therapy did not promote sustained induction of HBV-specific T-cell proliferative responses, regardless of PegIFN therapy or decreased serum HBsAg, HBV DNA, or ALT levels. Instead, HBV-specific T-cell IFNγ responses declined significantly by 48 weeks of therapy (p=0.008). Posttreatment ALT flare was associated with higher baseline %PD1+/CD8 (p=0.019), %PD1+/CD4 (p=0.039), and %CTLA4+/CD4 (p=0.003) T cells compared to non-flares, but without associated HBsAg loss or increased HBV-specific T-cell responsiveness. HBV-specific T-cell function was not restored after 192 weeks of tenofovir therapy and did not correlate with HBsAg levels before, during, or after therapy. Baseline global T-cell regulatory phenotype was a predictor for ALT flare post-therapy without associated HBsAg decline. These findings support the need for more novel immune-modulatory approaches to enhance HBV-specific T-cell responsiveness.

Introduction:Mutations in circulating nucleic acids can be used as biomarkers for the early detection and management of hepatocellular carcinoma (HCC). However, while circulating tumor DNA and microRNA have been extensively explored, circulating tumor mRNA and circulating mRNA mutants (ctmutRNA), which may provide advantages over other analytes, remain less well described. We previously reported the identification of 288 HCC selective ctmutRNA variants, called "candidates," from a small cohort of HCC patients using total RNAseq. The objective of the current study was to use targeted RNAseq to validate the specificity and sensitivity of these HCC selective variants in an independent cohort of patients with liver cirrhosis (LC). Methods:Several methods to isolate small extracellular vesicles and amplify mRNA from the circulation were compared. RNA was isolated, and the primers and probes selective for the 288 regions of interest were used with RNA from HCC (N = 50) and LC and no HCC (N = 35) patients. HCC tumor tissues (N = 11), a normal liver tissue and 3 cell lines were also studied. cDNA synthesis was followed by library construction using QIAseq RNA Fusion XP panel. QC analysis was carried out with an Agilent Bioanalyzer before sequencing on a NextSeq 550 instrument. A GATK HaplotypeCaller was used for variant calling and annotation carried out using snpEff. Results:Among the test panel of 288 ctmutRNA candidates in the original cohort, 75 were detected in the new cohort of plasma samples. Moreover, 388 other variants in proximity to the original lesions were also found in multiple HCC but not LC plasma samples. A subset of 36 HCC selective variants was able to identify all HCC patients. The most common tumor specific variants were Indels and SNPs. Novel mRNA fusion variants, corresponding to SENP7, HYI, SAR1A, RASA2, TUBA transcripts, etc., were identified in HCC and LC patients. Conclusion:Circulating RNA could be a robust analyte for noninvasive early detection of HCC and circulating RNA panels could be powerful tools in the entire spectrum of clinical management.

Despite the considerable clinical and economic burden imposed by hepatitis A virus (HAV) infection, both globally and in U.S., there are currently no available antiviral therapies for the treatment of type A hepatitis. Here we describe novel third-generation hepato-selective dihydroquinolizinones (HS-DHQs) with cellular uptake mediated by transport via hepatocyte-specific solute organic anion transporter family members 1B1 and 1B3 (OATP1B1-B3). The lead HS-DHQ compound, HS83128, demonstrates robust inhibition of the host cell TENT4A/B terminal nucleotidyltransferases required for efficient HAV RNA synthesis (IC50 6-25nM), and potent antiviral activity against HAV in cell culture (EC50 0.6 nM). Pharmacokinetic studies in CD-1 mice receiving comparable oral doses of HS83128 and a first-generation dihydroquinolizinone, RG7834, revealed a 5-fold increase in intrahepatic drug concentration and more than 10-fold improvement in liver versus nervous system tissue selectivity. Twice-daily oral administration of HS83128 rapidly arrested viral replication in HAV-infected Ifnar1-/- mice, reducing fecal virus shedding and cytokine markers of hepatic inflammation and reversing virus-induced liver injury. The hepato-selective nature of HS83128 may reduce the risk of neurologic and reproductive track toxicities observed with long-term administration of other dihydroquinolizinones, making it a candidate for the first antiviral therapy of hepatitis A.

Dihydroquinolizinones (DHQs) that inhibit cellular polyadenylating polymerases 5 and 7 (PAPD5 & 7), such as RG7834, have been shown to inhibit both hepatitis A (HAV) and hepatitis B virus (HBV) in vitro and in vivo. In this report, we describe RG7834-based proteolysis -targeting chimeras (PROTACs), such as compound 12b, (6S)-9-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-21-oxo-3,6,9,12,15,18-hexaoxa-22-azapentacosan-25-yl)oxy)-6-isopropyl-10-methoxy-2oxo-6,7-dihydro-2H-pyrido[2,1-a]isoquinoline-3-carboxylic acid. The PROTAC DHQs described here inhibited an HAV reporter virus in vitro with an IC50 of 277 nM. Although the PROTAC DHQs were also inhibitory to HBV, their activities were substantially less potent against HBV in vitro, being in the 10 to 20 mu M range, based on the reduction of HBsAg and HBV mRNA levels. Importantly, unlike RG7834, the incubation of cells in vitro with PROTAC DHQ 12b resulted in the degradation of PAPD5, as expected for a PROTAC compound, but curiously not PAPD7. PAPD5 polypeptide degradation was prevented when a proteasome inhibitor, epoxomicin, was used, indicating that proteasome mediated proteolysis was associated with the observed activities of 12b. Taken together, these data show that 12b is the first example of a PROTAC that suppresses both HAV and HBV that is based on a small molecule warhead. The possibility that it has mechanisms that differ from its parent compound, RG7834, and has clinical value, is discussed.

Death rates from primary liver cancer (hepatocellular carcinoma [HCC]) have continued to rise in the United States over the recent decades despite the availability of an increasing range of treatment modalities, including new systemic therapies. Prognosis is strongly associated with tumor stage at diagnosis; however, most cases of HCC are diagnosed beyond an early stage. This lack of early detection has contributed to low survival rates. Professional society guidelines recommend semiannual ultrasound-based HCC screening for at-risk populations, yet HCC surveillance continues to be underused in clinical practice. On April 28, 2022, the Hepatitis B Foundation convened a workshop to discuss the most pressing challenges and barriers to early HCC detection and the need to better leverage existing and emerging tools and technologies that could improve HCC screening and early detection. In this commentary, we summarize technical, patient-level, provider-level, and system-level challenges and opportunities to improve processes and outcomes across the HCC screening continuum. We highlight promising approaches to HCC risk stratification and screening, including new biomarkers, advanced imaging incorporating artificial intelligence, and algorithms for risk stratification. Workshop participants emphasized that action to improve early detection and reduce HCC mortality is urgently needed, noting concern that many of the challenges we face today are the same or similar to those faced a decade ago and that HCC mortality rates have not meaningfully improved. Increasing the uptake of HCC screening was identified as a short-term priority while developing and validating better screening tests and risk-appropriate surveillance strategies.

N-Alkyl-C1-dialkyl chains iminocyclitols with D or L-ribitol stereochemistry are synthesized with high diastereoselectivity after Grignard reagents addition to N-quaternary pyrrolines salts, and tested for antiviral activity in bovine viral diarrhea virus (BVDV), surrogate for hepatitis C virus (HCV). Dihedral angles are calculated from carbon chemical shift (δCn[ppm]) with 3-sphere method without building units. 3-Sphere, a hypersphere in 4D, under Hopf fibration and Lie algebra mathematics theories enable calculation of the dihedral angles from the NMR data (vicinal coupling constant 3JHnHn+1[Hz], chemical shift δCn[ppm]). Instead of 3D manifold equations on seven sets unit or six sets units are proposed equations between 4D – 2D, in function of the curvature. The relationship between the antiviral activity and the iminocyclitol structure reveals that monoalkyl chain, N-n-C1-dodecyl β-L-ribitol trifloroacetate salt 30 (IC50 1.5 uM) has higher antiviral activity in tangential space, relative to three alkyl chain, N-Methyl-C1-butil, nonyl-L-ribitol. HCl 26 (IC50 < 2 uM) with torus and Dupin cyclide coordinate, both with coordinates in 2D. Three alkyl chain isopropylidene protected pyrrolidine 25 has in 4D with all equations for calculation of the dihedral angles, and in protected pyrroline 19b double bond moves the coordinates in 2D.

Data description and detailed statistical information regarding model development. Supplementary Table 1. Patient information on samples from the University of Michigan. Supplementary Table 2. Patient information on samples from HALT-C. Supplementary Table 3. Patient information on samples from EDRN. Supplementary Table 4. Patient information on samples from Thomas Jefferson University. Supplementary Table 5. Patient information on samples from the University of Texas Southwestern Medical Center. Supplementary Table 6. Odds ratios for each predictor in univariate and multivariate logistic regressions. Supplementary Table 7. Indices of goodness-of-fit and apparent validation of candidate logistic regression models. Supplementary Table 8. Performance of leave one out cross validation (LOOCV). Supplementary Table 9. Cross Validation of bootstrap method. Supplementary Table 10. AUCs and IDIs from 3-fold Cross-Validation. Supplementary Table 11. Summary statistics of Doylestown model. Supplementary Table 12. Comparison of logistic regression, classification and regression tree (CART) and conditional inference tree (CTREE). Supplementary Figure 1. The distribution of AFP in each data set by cases and controls: Supplementary Figure 2. Quartiles for AFP in the individual patient sets. Supplementary Figure 3. Histogram of predictions and observe occurrence proportion of HCC for the top 4 models.

Supplementary Figure 1 from Novel Changes in Glycosylation of Serum Apo-J in Patients with Hepatocellular Carcinoma

PDF file, 354KB, N-linked glycosylation of total protein from 16 HCC tissue and adjacent liver tissue pairs.

Supplementary Table S1. Patient Characteristics for patients from the University of California at San Diego; Supplementary Table S2. Fitness of algorithms; Supplementary Table S3. Cross validations of potential models; Supplementary Table S4. Statistical inference of comparing models; Supplementary Figure S1. Scheme of study design;Supplementary Figure S2. Scatter plot; Supplementary Figure S3. AUROC for the individual components analyzed; Supplementary Figure S4. MALDI-TOF analysis of low molecular weight kininogen; Supplementary Figure S5. Glycopeptide analysis of tryptic glycopeptide 44-58; Supplementary Figure S6. Glycopeptide analysis of tryptic glycopeptide 197-208 showing identification of fucosylated glycopeptides; Supplementary Figure S7. Glycopeptide analysis of tryptic glycopeptide 289-300;

Background and rationale:Liver derived messenger ribonucleic acid (mRNA) transcripts were reported to be elevated in the circulation of hepatocellular carcinoma (HCC) patients. We now report the detection of high-risk mRNA variants exclusively in the circulation of HCC patients. Numerous genomic alleles such as single nucleotide polymorphisms (SNPs), nucleotide insertions and deletions (called Indels), splicing variants in many genes, have been associated with elevated risk of cancer. Our findings potentially offer a novel non-invasive platform for HCC surveillance and early detection. Approach:RNAseq analysis was carried out in the plasma of 14 individuals with a diagnosis of HCC, 8 with LC and no HCC, and 6 with no liver disease diagnosis. RNA from 6 matching tumors and 5 circulating extracellular vesicle (EV) samples from 14 of those with HCC was also analyzed. Specimens from two cholangiocarcinoma (CCA) patients were also included in our study. HCC specific SNPs and Indels referred as "variants" were identified using GATK HaplotypeCaller and annotated by SnpEff to filter out high risk variants. Results:The variant calling on all RNA samples enabled the detection of 5.2 million SNPs, 0.91 million insertions and 0.81 million deletions. RNAseq analyses in tumors, normal liver tissue, plasma, and plasma derived EVs led to the detection of 5480 high-risk tumor specific mRNA variants in the circulation of HCC patients. These variants are concurrently detected in tumors and plasma samples or tumors and EVs from HCC patients, but none of these were detected in normal liver, plasma of LC patients or normal healthy individuals. Our results demonstrate selective detection of concordant high-risk HCC-specific mRNA variants in free plasma, plasma derived EVs and tumors of HCC patients. The variants comprise of splicing, frameshift, fusion and single nucleotide alterations and correspond to cancer and tumor metabolism pathways. Detection of these high-risk variants in matching specimens from same subjects with an enrichment in circulating EVs is remarkable. Validation of these HCC selective ctmRNA variants in larger patient cohorts is likely to identify a predictive set of ctmRNA with high diagnostic performance and thus offer a novel non-invasive serology-based biomarker for HCC.

Globally, 296 million people are infected with hepatitis B virus (HBV), and approximately one million people die annually from HBV-related causes, including liver cancer. Although there is a preventative vaccine and antiviral therapies suppressing HBV replication, there is no cure. Intensive efforts are under way to develop curative HBV therapies. Currently, only a few biomarkers are available for monitoring or predicting HBV disease progression and treatment response. As new therapies become available, new biomarkers to monitor viral and host responses are urgently needed. In October 2020, the International Coalition to Eliminate Hepatitis B Virus (ICE-HBV) held a virtual and interactive workshop on HBV biomarkers endorsed by the International HBV Meeting. Various stakeholders from academia, clinical practice and the pharmaceutical industry, with complementary expertise, presented and participated in panel discussions. The clinical utility of both classic and emerging viral and immunological serum biomarkers with respect to the course of infection, disease progression, and response to current and emerging treatments was appraised. The latest advances were discussed, and knowledge gaps in understanding and interpretation of HBV biomarkers were identified. This Roadmap summarizes the strengths, weaknesses, opportunities and challenges of HBV biomarkers. Currently, there is no cure for hepatitis B virus (HBV) infection, which can lead to chronic liver disease and liver cancer, and only a few biomarkers are available. This Roadmap provides an overview of HBV serum biomarkers and their challenges.

HepatologyEarly View CORRESPONDENCE Letter to the editor: Importance of universal screening for chronic hepatitis B infection in adults in the United States Chari Cohen, Corresponding Author Chari Cohen Chari.Cohen@hepb.org Hepatitis B Foundation, Doylestown, Pennsylvania, USA Correspondence Chari Cohen, Hepatitis B Foundation, Doylestown, PA, USA. Email: Chari.Cohen@hepb.orgSearch for more papers by this authorKate Moraras, Kate Moraras Hepatitis B Foundation, Doylestown, Pennsylvania, USASearch for more papers by this authorMichalea Jackson, Michalea Jackson Hepatitis B Foundation, Doylestown, Pennsylvania, USASearch for more papers by this authorMaureen Kamischke, Maureen Kamischke Hepatitis B Foundation, Doylestown, Pennsylvania, USASearch for more papers by this authorRobert G. Gish, Robert G. Gish orcid.org/0000-0001-6306-3189 Hepatitis B Foundation, Doylestown, Pennsylvania, USASearch for more papers by this authorCarol L. Brosgart, Carol L. Brosgart Department of Medicine, Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California, USASearch for more papers by this authorMehlika Toy, Mehlika Toy Asian Liver Center, Department of Surgery, Stanford University School of Medicine, Palo Alto, California, USASearch for more papers by this authorDavid Hutton, David Hutton Department of Health Management and Policy, University of Michigan, Ann Arbor, Michigan, USASearch for more papers by this authorTimothy M. Block, Timothy M. Block Hepatitis B Foundation, Doylestown, Pennsylvania, USASearch for more papers by this authorSu Wang, Su Wang World Hepatitis Alliance, London, UK Saint Barnabas Medical Center, Livingston, New Jersey, USASearch for more papers by this authorSamuel So, Samuel So Asian Liver Center, Department of Surgery, Stanford University School of Medicine, Palo Alto, California, USASearch for more papers by this author Chari Cohen, Corresponding Author Chari Cohen Chari.Cohen@hepb.org Hepatitis B Foundation, Doylestown, Pennsylvania, USA Correspondence Chari Cohen, Hepatitis B Foundation, Doylestown, PA, USA. Email: Chari.Cohen@hepb.orgSearch for more papers by this authorKate Moraras, Kate Moraras Hepatitis B Foundation, Doylestown, Pennsylvania, USASearch for more papers by this authorMichalea Jackson, Michalea Jackson Hepatitis B Foundation, Doylestown, Pennsylvania, USASearch for more papers by this authorMaureen Kamischke, Maureen Kamischke Hepatitis B Foundation, Doylestown, Pennsylvania, USASearch for more papers by this authorRobert G. Gish, Robert G. Gish orcid.org/0000-0001-6306-3189 Hepatitis B Foundation, Doylestown, Pennsylvania, USASearch for more papers by this authorCarol L. Brosgart, Carol L. Brosgart Department of Medicine, Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California, USASearch for more papers by this authorMehlika Toy, Mehlika Toy Asian Liver Center, Department of Surgery, Stanford University School of Medicine, Palo Alto, California, USASearch for more papers by this authorDavid Hutton, David Hutton Department of Health Management and Policy, University of Michigan, Ann Arbor, Michigan, USASearch for more papers by this authorTimothy M. Block, Timothy M. Block Hepatitis B Foundation, Doylestown, Pennsylvania, USASearch for more papers by this authorSu Wang, Su Wang World Hepatitis Alliance, London, UK Saint Barnabas Medical Center, Livingston, New Jersey, USASearch for more papers by this authorSamuel So, Samuel So Asian Liver Center, Department of Surgery, Stanford University School of Medicine, Palo Alto, California, USASearch for more papers by this author First published: 24 December 2021 https://doi.org/10.1002/hep.32304Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Early ViewOnline Version of Record before inclusion in an issue RelatedInformation

Studies of human hepatitis B virus (HBV) immune pathogenesis are hampered by limited access to liver tissues and technologies for detailed analyses. Here, utilizing imaging mass cytometry (IMC) to simultaneously detect 30 immune, viral, and structural markers in liver biopsies from patients with hepatitis B e antigen+ (HBeAg+) chronic hepatitis B, we provide potentially novel comprehensive visualization, quantitation, and phenotypic characterizations of hepatic adaptive and innate immune subsets that correlated with hepatocellular injury, histological fibrosis, and age. We further show marked correlations between adaptive and innate immune cell frequencies and phenotype, highlighting complex immune interactions within the hepatic microenvironment with relevance to HBV pathogenesis.

Chronic hepatitis B (CHB) is characterized by high levels of hepatitis B virus (HBV) surface antigen (HBsAg) in blood circulation. A major goal of CHB interventions is reducing or eliminating this antigenemia; however, there are currently no approved methods that can do this. A novel family of compounds with a dihydroquinolizinone (DHQ) scaffold has been shown to reduce circulating levels of HBsAg in animals, representing a first for a small molecule. Reductions of HBsAg were a result of the compound's effect on HBsAg mRNA levels. However, commercial development by Roche of a DHQ lead compound, RG-7834, was stopped due to undisclosed toxicity issues. Herein we report our effort to convert the systemic RG7834 compound to a hepatoselective DHQ analog to limit its distribution to the bloodstream and thus to other body tissues.