Developing advanced cell culture models to study COVID19-associated pulmonary fibrosis

Patients with severe COVID-19-associated pneumonia are at risk to develop pulmonary fibrosis. To study the underlying mechanisms, we aim to develop advanced cell culture models that reliably reflect COVID-19-related profibrotic microenvironment. To identify key cellular players, we performed pilot immunohistochemistry analysis on lung tissue from COVID-19 patients with fibrosis collected during autopsy. Results revealed diffuse alveolar damage with macrophage infiltration, and myofibroblast accumulation with enriched collagen deposition surrounding the damaged alveoli. To mimic SARS-CoV-2 infection in alveoli, we infected human primary type II alveolar epithelial cells (AEC2) and found enhanced signaling of profibrotic cytokine transforming growth factor beta (TGFbeta) in some donors. To recreate the early fibrotic niche, an alveolar-macrophage-fibroblast (AMF) tri-culture model was established. After infecting AEC2 with SARS-CoV-2 in this AMF model, gene expression analysis provided evidence for fibroblast-to-myofibroblast transition. Furthermore, we found that overexpression of SARS-CoV-2 papain-like protease (PLpro) can promote TGFbeta signaling in HEK293T and A549 cells. After infecting AEC2 with SARS-CoV-2 PLpro lentivirus in the AMF model, we found signs of epithelial-to-mesenchymal transition and fibroblast-to myofibroblast transition. In future studies, we will use a detailed analysis of COVID-19-associated lung fibrosis with other types of lung fibrosis, to further refine COVID-19-related fibrosis models, including lung-on-chip models.

RNA sequencing unravels unique host transcriptional responses to infection with SARS-CoV-2 in well differentiated primary human bronchial epithelial cells compared to other coronaviruses

As the causative agent of COVID-19, SARS-CoV-2 remains a global cause for concern. Compared to other highly pathogenic coronaviruses (SARS-CoV and MERS-CoV), SARS-CoV-2 exhibits stronger transmissibility but less lethality, indicating that SARS-CoV-2 displays unique characteristics, despite the partial genomic proximity. Thus, we aim to employ RNA sequencing to define transcriptional differences in epithelial responses following infection with SARS-CoV-2 compared to pathogenic SARS-CoV and MERS-CoV, and low pathogenic HCoV-229E. Primary human bronchial epithelial cells (PBEC) were differentiated for 6 weeks at the air-liquid interface (ALI) before parallel infection by the 4 different coronaviruses. After infection following apical application of coronaviruses at low dose, cells were harvested for bulk RNA sequencing. Results demonstrated that all tested coronaviruses efficiently infected ALI-PBEC. RNA sequencing analysis revealed that infection with SARS-CoV, MERS-CoV and HCoV-229E resulted in largely similar transcriptional responses by the epithelial cells. However, whereas infection with these viruses was accompanied by an increased expression of genes associated with JNK/AP-1 signalling, including FOS, FOSB and NR4A1, no such increase was observed following SARS-CoV-2 infection. Further, preliminary experiments indicated that an NR4A1 antagonist reduced viral replication in Calu-3 cells. In conclusion, these data suggest that SARS-CoV2-infected ALI-PBEC exhibit a unique transcriptional response compared to other coronaviruses, which might relate to the pathogenicity of the virus.

Antiviral Effects of Voclosporin on SARS-CoV-2 in Immunocompromised Kidney Patients

Background: Immunocompromised patients, including Kidney Transplant Recipients (KTRs) and lupus nephritis (LN) patients, are at increased risk for prolonged SARS-CoV-2 infection and developing COVID-19-related complications. Recently, we reported that voclosporin, a novel calcineurin inhibitor (CNI), demonstrated in vitro a more potent inhibitory effect on SARS-CoV-2 replication than other CNIs (tacrolimus and ciclosporin). Additionally the AURORA-2 study, where 216 LN patients were treated with voclosporin or placebo on top of standard of care, SARS-CoV-2 infection was detected in 12/100 placebo-treated patients (12%) compared to 7/116 voclosporin treated patients (6%) and more patients died due to COVID-19 in the placebo arm versus voclosporin arm (3% vs 0%). In this proof-of-concept study we assessed whether voclosporin demonstrated an added antiviral benefit in SARS-CoV-2 positive immunocompromised patients. Method(s): We performed a prospective, randomised, open-label, single-center, exploratory, proof of concept study in 20 KTRs with mild to moderate symptoms from a COVID-19 infection comparing time to viral clearance of SARS-CoV-2 between patients on standard immunosuppressive therapy with tacrolimus versus voclosporin (the VOCOVID study). Result(s): In the VOCOVID study no difference in time to viral clearance or time to clinical recovery was found between both treatment arms. Pharmacokinetic analysis demonstrated that adequate trough levels of voclosporin were reached from day 4-8 after randomization. Looking at specific timepoint in a post-hoc analysis, indeed a significantly better viral clearance of SARS-CoV-2 was observed in voclosporin treated patients at day 4-8. No safety concerns were raised. Conclusion(s): The present study provides evidence that voclosporin has a favorable benefit-risk profile for immunocompromised kidney disease patients who contract a SARS-CoV-2 infection while requiring the continuation of their immunosuppressants.

Efficacy assessment of newly-designed filtering facemasks during the sars-cov-2 pandemic

The SARS-CoV-2 pandemic resulted in shortages of production and test capacity of FFP2-respirators. Such facemasks are required to be worn by healthcare professionals when performing aerosol-generating procedures on COVID-19 patients. In response to the high demand and short supply, we designed three models of facemasks that are suitable for local production. As these facemasks should meet the requirements of an FFP2-certified facemask, the newly-designed facemasks were tested on the filtration efficiency of the filter material, inward leakage, and breathing resistance with custom-made experimental setups. In these tests, the facemasks were benchmarked against a commercial FFP2 facemask. The filtration efficiency of the facemask’s filter material was also tested with coronavirus-loaded aerosols under physiologically relevant conditions. This multidisciplinary effort resulted in the design and production of facemasks that meet the FFP2 requirements, and which can be produced at local production facilities. © The Author(s).