Sofía Pérez-Del-Pulgar

HBV recurrence after liver transplantation (LT) can occur despite removal of the infected liver and antiviral prophylaxis. Timing and mechanisms of HBV and HDV recurrence remain unclear. We evaluated post-transplant viral kinetics to address this gap. Twelve HBV patients undergoing LT, including five with HDV-coinfection, were prospectively enrolled. Serial serum and liver samples were rigorously collected before, during, and post-LT to characterize viral kinetics. HBV-DNA, HDV-RNA, HBsAg, and HDAg were detected in all explanted livers, whereas cccDNA was found only in HBV-monoinfected patients. Serum HBV-DNA, detectable in five recipients before LT, became undetectable within hours from LT. In HBV-monoinfected patients, baseline HBsAg levels were low (< 3logIU/mL) and cleared rapidly after LT, with faster disappearance in those receiving HBIG (5 days vs. 6 months). Among HDV-recipients, all treated with HBIG, HBsAg clearance was rapid when baseline HBsAg levels were 10IU/mL (12 h) and delayed at higher levels (6 months). HDV-RNA kinetics paralleled HBsAg decline. Post-transplant liver biopsies (reperfusion, 3- and 12-months) tested negative for viral markers, except for isolated intrahepatic HDV detection at 3-months in one patient. No intrahepatic HBV reinfection was observed post-LT. Occasional intrahepatic HDV detection supports the need for improved prophylactic strategies in HDV-recipients.

Background: Withdrawal of prolonged nucleos(t)ide analogue (NA) treatment results in hepatitis B surface antigen (HBsAg) loss in some subjects with chronic hepatitis B (CHB), potentially revealing immune correlates of functional cure. Objective: We investigated whether baseline or longitudinal changes in humoral immunity correlated with outcome of discontinuing prolonged NA treatment. Design: Global memory B cells (MBC) and T follicular helper cells (Tfh) were analysed by flow cytometry. HBs (small surface)/HBc (core)-MBC were quantified by ex-vivo bait staining and function assessed by cultured ELISpots (enzyme-linked immunosorbent spots). Immune parameters assessed at end-of-treatment (EOT), 12 and 48 weeks after treatment withdrawal (and at 4–8 years in a subset) were correlated with intrahepatic and longitudinal serum viral markers and alanine transaminase (ALT). Results: Individuals on prolonged NA had comparable frequencies of HBc-MBC and HBs-MBC, although the latter were PD-1 ^hi and functionally defective. Following treatment withdrawal, increases in class-switched HBc-MBC were frequently temporally linked with hepatic flares. Subjects achieving HBsAg loss had an increase in activated global MBC detectable at EOT that become more marked by week 48, accompanied by significant increases in plasmablasts. HBs-MBC in those with HBsAg loss showed significant reductions in PD-1, trends to increased activation (CD71) and function and a more robust correlation with Tfh, compared with HBsAg persistence. MBC changes were maintained 4–8 years after HBsAg seroconversion. Conclusion: Differences in global and HBs-specific B cell immunity associate with HBsAg loss, whereas HBc-MBC temporally associates with flares, following withdrawal of prolonged NA treatment. Our results underscore the need to further explore the potential of B cell targets for monitoring and enhancing HBV functional cure in larger cohorts.

Background and Aim: PD‐1‐expressing T cells within the HBV‐infected liver constitute a target of novel immunotherapeutics. Our aim was to investigate the impact of viral suppression on PD‐1 expression on intrahepatic versus circulating lymphocyte populations from chronic hepatitis B (CHB) patients. Methods: Twenty‐two CHB patients, nine of them on nucleos(t)ide analogues (NUCs), had paired blood, liver fine needle aspirations (FNAs) and biopsies. A subset had a follow‐up FNA after treatment initiation ( n = 4) or discontinuation ( n = 4). Intrahepatic (iHBV‐DNA and cccDNA) and serum (HBV‐DNA, HBsAg, HBcrAg and cirB‐RNA) viral markers were quantified. Flow cytometry was used for immunophenotyping PBMCs and intrahepatic lymphocytes. An independent liver FNA scRNAseq dataset was used to consolidate our results. Results: PD‐1 expression on tissue‐resident memory CD8 T cells (T _RM ) correlated with both iHBV‐DNA and cccDNA, as well as surrogate markers of cccDNA transcriptional activity (cirB‐RNA and HBcrAg) in CHB patients with mild hepatitis. These associations were not reflected in circulating T cells. PD‐1 expression intensity on CD8 T _RM was lower in NUC‐treated than in naive patients, changes that were again not detectable in the circulation. Longitudinal analysis showed that viral load rebound induced by NUC discontinuation had the potential to drive re‐expression of high levels of PD‐1 on CD8 T _RM . Conversely, therapy initiation and subsequent viral suppression reversed these changes. scRNAseq results further extended the profiling of these PD‐1 + CD8 T _RM , showing a phenotype consistent with bystander activation in response to subclinical liver damage. Conclusions: Intrahepatic viral markers correlate with PD‐1 expression on global liver‐resident T cells of CHB patients with mild hepatitis, with a reduction after prolonged NUC therapy and re‐expression following treatment withdrawal.

Background & Aims: Predicting clinical and virological outcomes in HBeAg‐negative (HBeAg‐neg) chronic infection often requires long‐term monitoring. Our study explored whether a single measurement of quantitative HBsAg (qHBsAg), HBV core‐related antigen (HBcrAg), and circulating HBV RNA (cirB‐RNA) can define the natural course of untreated HBeAg‐neg chronic infection patients.Methods: To this aim, we included 128 naïve HBeAg‐neg chronic infection patients, stratified according to qHBsAg levels in: (1) 10–1000 IU/mL, (2) 1000–10 000 IU/mL, and (3) > 10 000 IU/mL.Results: HBcrAg and cirB‐RNA were detected in 27% and 19% of patients with qHBsAg > 1000 IU/mL but rarely detected in patients with qHBsAg < 1000 IU/mL. After a median follow‐up of 5.1 years, 9.4% of patients lost HBsAg, and 8.5% experienced an increase in HBV DNA > 2000 IU/mL. qHBsAg < 1000 IU/mL was the only factor independently associated with functional cure.Conclusions: In untreated HBeAg‐neg chronic infection patients, single‐point cirB‐RNA and HBcrAg do not offer additional predictive value over qHBsAg < 1000 IU/mL for spontaneous HBsAg loss.

The study of hepatitis C virus (HCV) replication in cell culture is mainly based on cloned viral isolates requiring adaptation for efficient replication in Huh7 hepatoma cells. The analysis of wild-type (WT) isolates was enabled by the expression of SEC14L2 and by inhibitors targeting deleterious host factors. Here, we aimed to optimize cell culture models to allow infection with HCV from patient sera. We used Huh7-Lunet cells ectopically expressing SEC14L2, CD81, and a GFP reporter with nuclear translocation upon cleavage by the HCV protease to study HCV replication, combined with a drug-based regimen for stimulation of non-modified wild-type isolates. RT-qPCR-based quantification of HCV infections using patient sera suffered from a high background in the daclatasvir-treated controls. We therefore established an automated image analysis pipeline based on imaging of whole wells and iterative training of a machine learning tool, using nuclear GFP localization as a readout for HCV infection. Upon visual validation of hits assigned by the automated image analysis, the method revealed no background in daclatasvir-treated samples. Thereby, infection events were found for 15 of 34 high titer HCV genotype (gt) 1b sera, revealing a significant correlation between serum titer and successful infection. We further show that transfection of viral RNA extracted from sera can be used in this model as well, albeit with so far limited efficiency. Overall, we generated a robust serum infection assay for gt1b isolates using semi-automated image analysis, which was superior to conventional RT-qPCR-based quantification of viral genomes.

The study of hepatitis C virus (HCV) replication in cell culture is mainly based on cloned viral isolates requiring adaptation for efficient replication in Huh7 hepatoma cells. The analysis of wild type (WT) isolates has been enabled by expression of SEC14L2 and by inhibitors targeting deleterious host factors. Here, we aimed at optimizing cell culture models to allow infection with HCV from patient sera. We used Huh7-Lunet cells ectopically expressing SEC14L2, CD81 and a GFP reporter with nuclear translocation upon cleavage by the HCV protease to study HCV replication, combined with a drug-based regimen for stimulation of non-modified wildtype isolates. RT-qPCR based quantification of HCV infections using patient sera suffered from high background in the daclatasvir treated controls. We therefore established an automated image analysis pipeline based on imaging of whole wells and iterative training of a machine-learning tool, using nuclear GFP localization as a readout for HCV infection. Upon visual validation of hits assigned by the automated image analysis the method revealed no background in daclatasvir treated samples. Thereby, infection events were found for 15 of 34 high titer HCV genotype (gt) 1b sera, revealing a significant correlation of serum titer and successful infection. We further show that transfection of viral RNA extracted from sera can be used in this model as well, albeit with so far limited efficiency. Overall, we generated a robust serum infection assay for gt1b isolates using semi-automated image analysis, which was superior to conventional RT-qPCR based quantification of viral genomes.

Background: The risk of hepatocarcinoma in HCV cirrhotic patient responders after treatment with DAAs decrease, but HCC still occurs. A correlation between specific miRNAs and the development of hepatocarcinoma have been highlighted. Aim: To investigate miRNA expression in HCV-infected cirrhotic patients treated with DAAs, regarding whether or not they developed HCC at follow-up. Methods: A total of 73 outpatients with HCV-related cirrhosis treated with DAAs were enrolled, 28 of which had HCC. Samples were collected at the start and at the end of treatment. In the screening phase, 172 miRNAs were analyzed at baseline. Differentially expressed miRNAs were validated in the entire cohort. Results: In the validation phase, at baseline and in patients treated for 12 weeks, miR-28-5p was confirmed to be more highly expressed in the HCC group compared to the non-HCC group. In all of the patients treated for 12 weeks, at end of the treatment we found a significant downregulation in miR-132-3p, miR-133b-3p, miR-221-3p and miR-324-3p. In the HCC group, miR-28-5p was significantly downregulated after DAA therapy as well as in HCC patients treated for 24 weeks. Conclusion: In the HCC group, miR28-5p was differently expressed both at baseline and at the end of therapy with DAAs. This difference in expression should suggest its involvement in HCC development.

Supplementary Data from Hepatocellular Carcinoma in Mongolia Delineates Unique Molecular Traits and a Mutational Signature Associated with Environmental Agents

Supplementary Figure from Hepatocellular Carcinoma in Mongolia Delineates Unique Molecular Traits and a Mutational Signature Associated with Environmental Agents