Janus kinase-inhibition modulates hepatitis E virus infection.

Hepatitis E virus (HEV) usually causes a self-limiting disease, but especially immunocompromised individuals are at risk to develop a chronic and severe course of infection. Janus kinase (JAK) inhibitors (JAKi) are a novel drug class for the treatment of autoimmune inflammatory rheumatic disease (AIRD). As JAKs play a key role in innate immunity, viral infections and reactivations are frequently reported during JAKi treatment in AIRD patients. The aim of this study was to characterize the influence of JAKis on HEV replication. To this end, we evaluated liver enzymes of an AIRD patient under JAKi therapy with hepatitis E. Further, experiments with HEV (Kernow-C1 p6) were performed by infection of primary human hepatocytes (PHHs) followed by immunofluorescence staining of viral markers and transcriptomic analysis. Infection experiments in PHHs displayed an up to 50-fold increase of progeny virus production during JAKi treatment and transcriptomic analysis revealed induction of antiviral programs during infection. Upregulation of interferon-stimulated genes (ISG) was perturbed in the presence of JAKis, concomitant with elevated HEV RNA levels. The obtained results suggest that therapeutic JAK inhibition increases HEV replication by modulating the HEV-triggered immune response. Therefore, JAKi treatment and the occurrence of elevated liver enzymes requires a monitoring of potential HEV infections.

EGF receptor modulates HEV entry in human hepatocytes.

BACKGROUND AND AIMS: Being the most common cause of acute viral hepatitis with >20 million cases per year and 70,000 deaths annually, HEV presents a long-neglected and underinvestigated health burden. Although the entry process of viral particles is an attractive target for pharmacological intervention, druggable host factors to restrict HEV entry have not been identified so far. APPROACH AND RESULTS: Here we identify the EGF receptor (EGFR) as a novel host factor for HEV and reveal the significance of EGFR for the HEV entry process. By utilizing RNAi, chemical modulation with Food and Drug Administration-approved drugs, and ectopic expression of EGFR, we revealed that EGFR is critical for HEV infection without affecting HEV RNA replication or assembly of progeny virus. We further unveiled that EGFR itself and its ligand-binding domain, rather than its signaling function, is responsible for the proviral effect. Modulation of EGF expression in HepaRG cells and primary human hepatocytes affected HEV infection. CONCLUSIONS: Taken together, our study provides novel insights into the life cycle of HEV and identified EGFR as a possible target for future antiviral strategies against HEV.

In-depth analysis of T cell immunity and antibody responses in heterologous prime-boost-boost vaccine regimens against SARS-CoV-2 and Omicron variant.

With the emergence of novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs), vaccination studies that elucidate the efficiency and effectiveness of a vaccination campaign are critical to assess the durability and the protective immunity provided by vaccines. SARS-CoV-2 vaccines have been found to induce robust humoral and cell-mediated immunity in individuals vaccinated with homologous vaccination regimens. Recent studies also suggest improved immune response against SARS-CoV-2 when heterologous vaccination strategies are employed. Yet, few data exist on the extent to which heterologous prime-boost-boost vaccinations with two different vaccine platforms have an impact on the T cell-mediated immune responses with a special emphasis on the currently dominantly circulating Omicron strain. In this study, we collected serum and peripheral blood mononuclear cells (PBMCs) from 57 study participants of median 35-year old's working in the health care field, who have received different vaccination regimens. Neutralization assays revealed robust but decreased neutralization of Omicron VOC, including BA.1 and BA.4/5, compared to WT SARS-CoV-2 in all vaccine groups and increased WT SARS-CoV-2 binding and neutralizing antibodies titers in homologous mRNA prime-boost-boost study participants. By investigating cytokine production, we found that homologous and heterologous prime-boost-boost-vaccination induces a robust cytokine response of CD4+ and CD8+ T cells. Collectively, our results indicate robust humoral and T cell mediated immunity against Omicron in homologous and heterologous prime-boost-boost vaccinated study participants, which might serve as a guide for policy decisions.

Beyond the Usual Suspects: Hepatitis E Virus and Its Implications in Hepatocellular Carcinoma.

Hepatitis E virus infections are the leading cause of viral hepatitis in humans, contributing to an estimated 3.3 million symptomatic cases and almost 44,000 deaths annually. Recently, HEV infections have been found to result in chronic liver infection and cirrhosis in severely immunocompromised patients, suggesting the possibility of HEV-induced hepatocarcinogenesis. While HEV-associated formation of HCC has rarely been reported, the expansion of HEV's clinical spectrum and the increasing evidence of chronic HEV infections raise questions about the connection between HEV and HCC. The present review summarizes current clinical evidence of the relationship between HEV and HCC and discusses mechanisms of virus-induced HCC development with regard to HEV pathogenesis. We further elucidate why the development of HEV-induced hepatocellular carcinoma has so rarely been observed and provide an outlook on possible experimental set-ups to study the relationship between HEV and HCC formation.

Long-range allosteric regulation of the human 26S proteasome by 20S proteasome-targeting cancer drugs.

The proteasome holoenzyme is the major non-lysosomal protease; its proteolytic activity is essential for cellular homeostasis. Thus, it is an attractive target for the development of chemotherapeutics. While the structural basis of core particle (CP) inhibitors is largely understood, their structural impact on the proteasome holoenzyme remains entirely elusive. Here, we determined the structure of the 26S proteasome with and without the inhibitor Oprozomib. Drug binding modifies the energy landscape of conformational motion in the proteasome regulatory particle (RP). Structurally, the energy barrier created by Oprozomib triggers a long-range allosteric regulation, resulting in the stabilization of a non-productive state. Thereby, the chemical drug-binding signal is converted, propagated and amplified into structural changes over a distance of more than 150 Å from the proteolytic site to the ubiquitin receptor Rpn10. The direct visualization of changes in conformational dynamics upon drug binding allows new ways to screen and develop future allosteric proteasome inhibitors.

Automated Enrichment of Sulfanilamide in Milk Matrices by Utilization of Aptamer-Linked Magnetic Particles.

The present work demonstrates the first automated enrichment approach for antibiotics in milk using specific DNA aptamers. First, aptamers toward the antibiotic sulfanilamide were selected and characterized regarding their dissociation constants and specificity toward relevant antibiotics via fluorescence assay and LC-MS/MS detection. The performed enrichment was automated using the KingFisherDuo and compared to a manual approach. Verifying the functionality, trapping was realized in different milk matrices: (i) 0.3% fat milk, (ii) 1.5% fat milk, (iii) 3.5% fat milk, and (iv) 0.3% fat cocoa milk drink. Enrichment factors up to 8-fold could be achieved. Furthermore, it could be shown that novel implementation of a magnetic separator increases the reproducibility and reduces the hands-on time from approximately half a day to 30 min.

Aptamer-based trapping of phytosphingosine in urine samples.

Usually, small molecules like single metabolites used in clinical diagnostic can be quantified by instrumental approaches like LC-MS or bioanalytical techniques using antibodies or aptamers as selective receptors. The present work comprises the generation of aptamers with an affinity towards the medically relevant metabolite phytosphingosine via the previously reported just in time-Selection approach (Hünniger et al., 2014). The whole approach could be seen as a proof of concept to extend the existing just in time-Selection protocol for selection towards small molecules with dissociation constants in the low nanomolar range. Moreover it is conceivable that the shown methods could be quickly adapted to further scopes. Aptamers could be applied for clean-up or concentration processes prior to further analysis. As an example, we used the selected aptamers towards phytosphingosine bound to magnetic particles for affinity enrichment in both selection buffer and urine samples. As an outcome, enrichment factors of up to 9-fold (selection buffer)/4-fold (urine samples) were achieved by this approach.

The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design.

The proteasome is a validated target for anticancer therapy, and proteasome inhibition is employed in the clinic for the treatment of tumors and hematological malignancies. Here, we describe crystal structures of the native human 20S proteasome and its complexes with inhibitors, which either are drugs approved for cancer treatment or are in clinical trials. The structure of the native human 20S proteasome was determined at an unprecedented resolution of 1.8 angstroms. Additionally, six inhibitor-proteasome complex structures were elucidated at resolutions between 1.9 and 2.1 angstroms. Collectively, the high-resolution structures provide new insights into the catalytic mechanisms of inhibition and necessitate a revised description of the proteasome active site. Knowledge about inhibition mechanisms provides insights into peptide hydrolysis and can guide strategies for the development of next-generation proteasome-based cancer therapeutics.