BACKGROUND & AIMS:The function and quantity of CD8+ T-cell responses is superior in acute vs. chronic HBV infection. However, whether different HBV-derived epitopes are targeted in these distinct courses of HBV infection remains unclear. METHODS:We screened peripheral blood mononuclear cells from 56 patients with chronic HBV infection genotype D and 32 patients with acute/resolved HBV infection to identify responses to overlapping peptides (18mers) covering the full HBV genotype D proteome. We then performed experimental fine-mapping of the minimal optimal epitopes. RESULTS:Patients with acute HBV infection showed a broad HBV-specific CD8+ T-cell epitope repertoire. After spontaneous resolution, the strength of HBV-specific CD8 T-cell responses decreased, whereas the broad epitope landscape was preserved. In chronic HBV infection, the specificity of HBV-specific CD8+ T-cell responses to HBV antigens was shifted, with a lack of functional HBsAg-specific CD8+ T-cell responses (p = 0.0007), as recently described. Interestingly, patients with chronic HBV infection showed significantly more HLA-B-restricted HBV-specific CD8+ T-cell responses than HLA-A-restricted responses (33 vs. 19), whereas this distribution was balanced in patients with acute/resolved HBV infection (16 vs. 27; p = 0.0364). Most (79.6%) of the detected responses showed conserved autologous viral sequences. This observation was confirmed in a broader sequence dataset with no evidence for HLA-B-driven CD8+ T-cell selection pressure. CONCLUSIONS:In contrast to acute/resolved infection, conserved HLA-B-restricted epitopes are dominant in chronic HBV infection, making them interesting candidates for immunotherapeutic approaches towards functional cure of chronic HBV infection. IMPACT AND IMPLICATIONS:To date, there is no treatment to achieve functional cure of chronic HBV infection. Immunotherapy boosting dysfunctional HBV-specific CD8+ T cells is an interesting approach, but relies on selection of optimal target epitopes. Using an unbiased approach, we found that the HBV-specific CD8+ T-cell epitope repertoire in chronic HBV infection is not well covered by previously described epitopes. We characterized 28 novel, primarily HLA-B-restricted epitopes that are dominantly targeted in chronic HBV infection. These might be used as a "toolbox" for immunological studies and immunotherapeutic approaches.

BACKGROUND AND AIMS:Bulevirtide reduces viremia in chronic hepatitis D virus (HDV) infection, but long-term treatment is required in most patients to prevent relapse. Sustained treatment responses may be fostered by therapy-induced amelioration of HDV-specific CD8+ T-cell responses that are exhausted due to high viremia and antigen loads during chronic HDV infection. We therefore studied the effect of bulevirtide monotherapy on HDV-specific CD8+ T-cell repertoire, phenotype, and functionality. METHODS:28 HDV-infected cirrhotic patients starting bulevirtide treatment were followed for 40-120 weeks on treatment. HLA-class-I typing and HDV sequencing was performed. HDV-specific CD8+ T cells were analyzed longitudinally using optimal epitopes and overlapping peptides spanning the L-HDAg. Ex vivo high-dimensional flow cytometry analysis of peptide/HLA class I tetramer+ CD8+ T cells was performed longitudinally in selected patients. RESULTS:In 42% of patients, an HDV-specific CD8+ T-cell response was detectable at baseline, but did not substantially increase during treatment. Importantly, a large majority of HDV-specific CD8+ T-cell responses targeted viral epitopes with sequence variations consistent with viral escape mutations. Only one HLA-B*35-restricted HDV-specific CD8+ T-cell epitope was conserved and continuously targeted throughout therapy. HDV-specific CD8+ T cells targeting this conserved epitope displayed a predominant terminally exhausted phenotype at baseline that shifted longitudinally to a memory-like phenotype with suppression of HDV load. CONCLUSION:Albeit based on small patient numbers, antiviral treatment with bulevirtide could ameliorate exhausted HDV-specific CD8+ T cells targeting conserved HDV epitopes. However, as the majority of HDV-specific CD8+ T-cell responses target escaped viral epitopes, this does not translate to a substantial improvement in overall HDV-specific CD8+ T-cell immunity. Our findings may explain in part why long-term bulevirtide treatment is required to prevent viral relapse.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a common comorbidity of psoriasis, a chronic non-communicable inflammatory skin disease. This research project aimed at investigating the effect of herbal hepatoprotective drugs and silymarin on chronic inflammation in cellular models of MASLD and psoriasis. Three-dimensional (3D) liver spheroids were obtained from HepG2/LX-2 cells, and 3D multicellular liver spheroids were generated from human primary hepatocytes, Kupffer cells and liver endothelial cells. The spheroids were transformed to steatotic and inflamed MASLD-like spheroids by treating them with free fatty acids, fructose and LPS. Then the MASLD-like spheroids were treated with the herbal drugs Hepar SL™ and Iberogast™, the flavonolignan silymarin, the monoclonal anti-IL-17A antibody ixekizumab, and the acetyl-CoA carboxylase inhibitor firsocostat. Subsequently, the degree of steatosis and release of pro-inflammatory cytokines was assessed. In vitro generated psoriasis-like keratinocytes were treated with the same compounds or were exposed to conditioned media (CM) from MASLD-like spheroids. The herbal compounds demonstrated various anti-inflammatory effects in both MASLD-like spheroids and psoriasis-like keratinocytes. Silymarin also displayed an anti-steatotic effect in MASLD-like spheroids. In psoriasis-like keratinocytes, some herbal compounds reduced the psoriatic phenotype and inflammation markers, as did CM from MASLD-like spheroids treated with herbal compounds. This indicates that hepatoprotective herbal drugs, particularly silymarin, can modulate steatosis and inflammatory markers in in‑vitro models of MASLD and psoriasis. The clinical relevance of these findings remains to be established in dedicated pharmacokinetic and clinical studies.

Virus-specific CD4+ T cells are essential for coordinating adaptive immunity during infection, but their differentiation and maintenance in chronic infection remain unclear. Using human hepatitis C virus (HCV) infection as a model, we assessed the determinants of virus-specific CD4+ T cell immunity in acute, spontaneously cleared, chronic, and therapeutically cured infections. During acute infection, multiple subsets of progenitor CD4+ T cells emerged, including subsets that are also found in chronic infection. In chronic infection, stem-like Bcl-2+ CD4+ T cells and T-bet+ effector CD4+ T cells existed in a progenitor/progeny relationship. Following therapy-mediated HCV cure, these cells retained their chronic signature but formed a stable memory pool that persisted for years and was distinct from HCV-specific CD4+ T cell memory after spontaneous clearance. Collectively, our findings highlight differences in CD4+ T cell fates that depend on infection outcomes and reveal common principles of CD4+ and exhausted CD8+ T cell maintenance during and after chronic infection.

The hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. Although most infections are self-limiting, zoonotic genotypes can persist in immunocompromised individuals. Transmitted via the fecal-oral route, HEV has been suggested to directly infect the intestinal epithelium, a tissue with high regenerative capacity. Here, we demonstrate that HEV predominantly infects proliferative transit-amplifying and intestinal stem cells within the crypts of human pluripotent stem cell-derived intestinal organoids (hIOs). Supporting this, we detected HEV RNA in the intestinal crypts of an HEV-infected patient. We further found that HEV infection spreads through cell division and is maintained in hIOs for more than 40 days, contrasting with acute hepatitis A virus, whose infections are rapidly cleared from hIOs. Given the self-renewal ability and metabolic constraints of proliferative intestinal progenitor cells, our findings suggest that intestinal crypts could serve as reservoirs for chronic HEV infection and highlight the intestinal crypt as a primary target for viral infection in the gastrointestinal tract.

Natural killer (NK) cell responses are modulated by type-I interferons (IFNs) in viral infection. Chronic hepatitis C virus (HCV) infection, marked by robust IFN signatures, shows NK cells with reduced cytokine release but heightened cytotoxicity. Comparable alterations occur in chronic hepatitis B virus (HBV) infection even without a pronounced IFN milieu, implying additional regulatory layers. We analyzed NK cells from healthy donors and patients with chronic HBV or HCV and found conserved expression patterns of interferon-stimulated genes (ISGs) such as IFITM3, IRF1, IFIT2, and ISG20 that correlated with NK cell differentiation state. These genes are governed by fate-determining transcription factors, including ETS1, FLI1, and Eomes, and appear to be constitutively expressed rather than driven by persistent IFN exposure. Network analysis suggested that NK cell ISGs participate not only in antiviral defense but also in processes such as transport and metabolism, underscoring their role in shaping NK responses during health and chronic viral infection.

Hepatitis E virus (HEV) is a major cause of acute hepatitis and mainly transmitted faecal-orally. HEV particles present in faeces are naked (nHEV), whereas those found in the blood are quasi-enveloped (eHEV) with a cell-derived lipid membrane. Despite its global health impact, the cellular life cycle of HEV remains poorly understood, particularly regarding the mechanisms of viral entry into host cells. To address this knowledge gap, we develop a high content RNA-FISH-based imaging assay that allows for the investigation of the entry pathways of both naked and quasi-enveloped HEV particles. Surprisingly, we find that integrin α3, previously implicated in nHEV cell entry, is not expressed in the cell types that are most permissive for HEV infection. Instead, we identify integrin β1 (ITGB1) pairing with different α-integrins as the key player mediating nHEV cell entry. Our results indicate that the interaction of nHEV with ITGB1 facilitates entry through Rab11-positive recycling endosomes. In contrast, eHEV particles do not interact with ITGB1 and enter cells using a classical endocytic route via Rab5a-positive early endosomes. The entry of both types of HEV particles requires endosomal acidification and proteolytic cleavage by lysosomal cathepsins, which ultimately results in delivery of the HEV genome to the cytoplasm.

INTRODUCTION:Complex mixed presentations of severe mental disorders (SMD) with treatment resistance pose major challenges in clinical practice. The role of novel neuronal antibodies in cerebrospinal fluid (CSF) is largely unexamined in this context. METHODS:A well-studied paradigmatic case of a 36-year-old female patient is reported. RESULTS:She presented with attention-deficit/hyperactivity disorder-like symptoms (including frequent sensory overload), severe pain (pre-diagnosed as fibromyalgia and somatoform pain disorder), and motor tics. In addition, she developed secondary depressive symptoms. Various psychopharmacological treatment attempts were unsuccessful or not tolerated. The diagnostic routine work-up with a wide range of blood tests, electroencephalography (EEG), routine magnetic resonance imaging (MRI), cerebrospinal fluid (CSF) analyses, and [18F]fluorodeoxyglucose positron emission tomography revealed no clear pathological findings. Tissue-based assays using CSF material found strong immunoglobulin G antibody staining specifically directed against a cell population in the thalamus. Neurotransmitter measurements detected low GABA, glutamate and serotonin concentrations as well as high dopamine levels in the CSF. Different MRI-based analyses indicated no neurostructural alterations in the thalamus; however, left mesiotemporal volume loss was identified. The independent component analysis of the EEG showed left temporal theta waves, partly resembling spike-wave-complexes. Immunotherapy using high-dose steroids resulted in a partial improvement with subjectively reduced stimulus overload, intermediate disappearance of pain, and fewer tics. The improvement could not be objectified psychometrically/neuropsychologically. The mesiotemporal volume loss was no longer present. There were no relevant changes in further research MRI measurements of the thalamus including arterial spin labeling, diffusion tensor imaging, and diffusion microstructure imaging from pre to post-immunotherapy. DISCUSSION:Novel antibodies against strategic brain structures, such as the thalamus, might be associated with some complex SMD. Further immunopsychiatric research in this direction holds promise for a better understanding of similar patients.

Intrahepatic immune responses are often insufficient to control hepatitis virus infections. A recent study by Venzin and colleagues demonstrates a detailed mechanism by which an intrahepatic tricellular network and the cytokine IL-27 can augment virus-specific immunity.

Background A rise in paediatric cases of acute hepatitis of unknown origin (AHUO) was observed in 2022, some requiring liver transplantation. A link to adeno-associated virus 2 infection and CD4 + T-cell mediated disease was reported in cohorts in the UK and USA but does not explain all cases. Objective To determine the intrahepatic immune cell interactions in the inflamed liver and a possible contribution of SARS-CoV-2 infection. Design Patients with acute non-A non-E hepatitis (10/12 AHUO, 2/12 subacute) during February 2022–December 2022 undergoing liver biopsy were recruited in a European patient cohort. Hepatological, virological, histopathological and highly multiplexed spatial and single-cell analyses of liver biopsies were performed. Results Patients were negative for adenoviral and SARS-CoV-2 PCR. Three patients had a positive adenoviral serology and 10/12 patients had a history or serological evidence of SARS-CoV-2 infection. Imaging mass cytometry identified significant intrahepatic immune infiltration with an enrichment of CD8 + T-cells. The highest CD8 infiltration and concomitant peripheral immune activation were observed in patients with the most severe hepatitis. CD8 + T-cell infiltration was connected to histomorphological interface hepatitis and bridging necrosis. Cellular neighbourhood analysis indicated disease-associated microanatomic interactions between CX3CR1 + endothelial and myeloid cell populations, interacting with effector CD8 + T-cells suggesting a pathogenic cellular triad. Of note, we detected intrahepatic SARS-CoV-2 antigens in ACE2-expressing cells in the areas with significant pathology in 11/12 samples using several different detection methods. 10/12 patients were treated with corticosteroid therapy and no liver transplantation was required. Conclusions We identified a possible manifestation of an immune-mediated postacute sequel to COVID-19 associated with a characteristic immune infiltrate in children with AHUO. COVID-19 testing should be considered in paediatric AHUO.