SARS-CoV-2 infection triggers paracrine senescence and leads to a sustained senescence-associated inflammatory response.

Reports of post-acute COVID-19 syndrome, in which the inflammatory response persists even after SARS-CoV-2 has disappeared, are increasing1, but the underlying mechanisms of post-acute COVID-19 syndrome remain unknown. Here, we show that SARS-CoV-2-infected cells trigger senescence-like cell-cycle arrest2,3 in neighboring uninfected cells in a paracrine manner via virus-induced cytokine production. In cultured human cells or bronchial organoids, these SASR-CoV-2 infection-induced senescent cells express high levels of a series of inflammatory factors known as senescence-associated secretory phenotypes (SASPs)4 in a sustained manner, even after SARS-CoV-2 is no longer detectable. We also show that the expression of the senescence marker CDKN2A (refs. 5,6) and various SASP factor4 genes is increased in the pulmonary cells of patients with severe post-acute COVID-19 syndrome. Furthermore, we find that mice exposed to a mouse-adapted strain of SARS-CoV-2 exhibit prolonged signs of cellular senescence and SASP in the lung at 14 days after infection when the virus was undetectable, which could be substantially reduced by the administration of senolytic drugs7. The sustained infection-induced paracrine senescence described here may be involved in the long-term inflammation caused by SARS-CoV-2 infection.

Evaluation of Broad Anti-Coronavirus Activity of Autophagy-Related Compounds Using Human Airway Organoids.

To deal with the broad spectrum of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that threaten human health, it is essential to not only drugs develop that target viral proteins but also consider drugs that target host proteins/cellular processes to protect them from being hijacked for viral infection and replication. To this end, it has been reported that autophagy is deeply involved in coronavirus infection. In this study, we used airway organoids to screen a chemical library of autophagic modulators to identify compounds that could potentially be used to fight against infections by a broad range of coronaviruses. Among the 80 autophagy-related compounds tested, cycloheximide and thapsigargin reduced SARS-CoV-2 infection efficiency in a dose-dependent manner. Cycloheximide treatment reduced the infection efficiency of not only six SARS-CoV-2 variants but also human coronavirus (HCoV)-229E and HCoV-OC43. Cycloheximide treatment also reversed viral infection-induced innate immune responses. However, even low-dose (1 µM) cycloheximide treatment altered the expression profile of ribosomal RNAs; thus, side effects such as inhibition of protein synthesis in host cells must be considered. These results suggest that cycloheximide has broad-spectrum anti-coronavirus activity in vitro and warrants further investigation.

Elucidation of the liver pathophysiology of COVID-19 patients using liver-on-a-chips.

SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.

Design of a chimeric ACE-2/Fc-silent fusion protein with ultrahigh affinity and neutralizing capacity for SARS-CoV-2 variants.

Background: As SARS-CoV-2 continues to mutate into Variants of Concern (VOC), there is growing and urgent need to develop effective antivirals to combat COVID-19. Monoclonal antibodies developed earlier are no longer capable of effectively neutralizing currently active VOCs. This report describes the design of variant-agnostic chimeric molecules consisting of an Angiotensin-Converting Enzyme 2 (ACE-2) domain mutated to retain ultrahigh affinity binding to a wide variety of SARS-CoV-2 variants, coupled to an Fc-silent immunoglobulin domain that eliminates antibody-dependent enhancement and extends biological half-life. Methods: Molecular modeling, Surrogate Viral Neutralization tests (sVNTs) and infection studies of human airway organoid cultures were performed with synthetic chimeras, SARS-CoV-2 spike protein mimics and SARS-CoV-2 Omicron variants B.1.1.214, BA.1, BA.2 and BA.5. Results: ACE-2 mutations L27, V34 and E90 resulted in ultrahigh affinity binding of the LVE-ACE-2 domain to the widest variety of VOCs, with KDs of 93 pM and 73 pM for binding to the Alpha B1.1.7 and Omicron B.1.1.529 variants, and notably, 78fM, 133fM and 1.81pM affinities to the Omicron BA.2, BA2.75 and BQ.1.1 subvariants, respectively. sVNT assays revealed titers of ≥4.9 ng/ml, for neutralization of recombinant viral proteins corresponding to the Alpha, Delta and Omicron variants. The values above were obtained with LVE-ACE-2/mAB chimeras containing the FcRn-binding Y-T-E sequence which extends biological half-life 3-4-fold. Conclusions: The ACE-2-mutant/Fc silent fusion proteins described have ultrahigh affinity to a wide variety of SARS-CoV-2 variants including Omicron. It is proposed that these chimeric ACE-2/mABs will constitute variant-agnostic and cost-effective prophylactics against SARS-CoV-2, particularly when administered nasally.

Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

Exocyst complex component 2 is a potential host factor for SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an epidemic and spread rapidly all over the world. Because the analysis of host factors other than receptors and proteases has not been sufficiently performed, we attempted to identify and characterize host factors essential for SARS-CoV-2 infection using iPS cells and airway organoids (AO). Based on previous CRISPR screening and RNA-seq data, we found that exocyst complex component 2 (EXOC2) is one important host factor for SARS-CoV-2 infection. The intracellular SARS-CoV-2 nucleocapsid (N) expression level was decreased to 3.7% and the virus copy number in cell culture medium was decreased to 1.6% by EXOC2 knockdown. Consistently, immunostaining results showed that N protein-positive cells were significantly decreased by EXOC2 knockdown. We also found that EXOC2 knockdown downregulates SARS-CoV-2 infection by regulating IFNW1 expression. In conclusion, controlling the EXOC2 expression level may prevent SARS-CoV-2 infection and deserves further study.

SARS-CoV-2 disrupts respiratory vascular barriers by suppressing Claudin-5 expression.

In the initial process of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects respiratory epithelial cells and then transfers to other organs the blood vessels. It is believed that SARS-CoV-2 can pass the vascular wall by altering the endothelial barrier using an unknown mechanism. In this study, we investigated the effect of SARS-CoV-2 on the endothelial barrier using an airway-on-a-chip that mimics respiratory organs and found that SARS-CoV-2 produced from infected epithelial cells disrupts the barrier by decreasing Claudin-5 (CLDN5), a tight junction protein, and disrupting vascular endothelial cadherin-mediated adherens junctions. Consistently, the gene and protein expression levels of CLDN5 in the lungs of a patient with COVID-19 were decreased. CLDN5 overexpression or Fluvastatin treatment rescued the SARS-CoV-2-induced respiratory endothelial barrier disruption. We concluded that the down-regulation of CLDN5 expression is a pivotal mechanism for SARS-CoV-2-induced endothelial barrier disruption in respiratory organs and that inducing CLDN5 expression is a therapeutic strategy against COVID-19.

Cell response analysis in SARS-CoV-2 infected bronchial organoids.

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.

Biocompatible Films of Calcium Alginate Inactivate Enveloped Viruses Such as SARS-CoV-2.

The current pandemic is urgently demanding the development of alternative materials capable of inactivating the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus 2019 (COVID-19) disease. Calcium alginate is a crosslinked hydrophilic biopolymer with an immense range of biomedical applications due to its excellent chemical, physical, and biological properties. In this study, the cytotoxicity and antiviral activity of calcium alginate in the form of films were studied. The results showed that these films, prepared by solvent casting and subsequent crosslinking with calcium cations, are biocompatible in human keratinocytes and are capable of inactivating enveloped viruses such as bacteriophage phi 6 with a 1.43-log reduction (94.92% viral inactivation) and SARS-CoV-2 Delta variant with a 1.64-log reduction (96.94% viral inactivation) in virus titers. The antiviral activity of these calcium alginate films can be attributed to its compacted negative charges that may bind to viral envelopes inactivating membrane receptors.

Dual inhibition of TMPRSS2 and Cathepsin Bprevents SARS-CoV-2 infection in iPS cells.

It has been reported that many receptors and proteases are required for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Although angiotensin-converting enzyme 2 (ACE2) is the most important of these receptors, little is known about the contribution of other genes. In this study, we examined the roles of neuropilin-1, basigin, transmembrane serine proteases (TMPRSSs), and cathepsins (CTSs) in SARS-CoV-2 infection using the CRISPR interference system and ACE2-expressing human induced pluripotent stem (iPS) cells. Double knockdown of TMPRSS2 and cathepsin B (CTSB) reduced the viral load to 0.036% ± 0.021%. Consistently, the combination of the CTPB inhibitor CA-074 methyl ester and the TMPRSS2 inhibitor camostat reduced the viral load to 0.0078% ± 0.0057%. This result was confirmed using four SARS-CoV-2 variants (B.1.3, B.1.1.7, B.1.351, and B.1.1.248). The simultaneous use of these two drugs reduced viral load to less than 0.01% in both female and male iPS cells. These findings suggest that compounds targeting TMPRSS2 and CTSB exhibit highly efficient antiviral effects independent of gender and SARS-CoV-2 variant.