Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Initially, we show that many of the mutations observed in SARS-CoV-2 during mouse adaptation (at positions 417, 484, 501 of the spike protein) also arise in humans in variants of concern (VOC)2. Their appearance during mouse adaptation indicates that immune pressure is not required for their selection. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, we showed that elevated levels of an eicosanoid, prostaglandin D2 (PGD2) and of a phospholipase, PLA2G2D, contributed to poor outcomes in aged mice3,4. Using our virulent mouse-adapted SARS-CoV-2, we show that infection of middle-aged mice lacking expression of DP1, a PGD2 receptor, or PLA2G2D are protected from severe disease. Further, treatment with a DP1 antagonist, asapiprant, protected aged mice from a lethal infection. DP1 antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, and demonstrates that the PLA2G2D-PGD2/DP1 pathway is a useful target for therapeutic interventions. (Words: 254)

TMPRSS2 structure-phylogeny repositions Avoralstat for SARS-CoV-2 prophylaxis in mice

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target.1-3 We hypothesized drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo.4,5 Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested Kallikrein-related B1 inhibitor,6 inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle,5 Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.

K18-hACE2 Mice for Studies of COVID-19 Treatments and Pathogenesis Including Anosmia

The ongoing COVID-19 pandemic is associated with substantial morbidity and mortality. While much has been learned in the first months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation and many patients with this finding show no or only minor respiratory signs. Studies in animals experimentally infected with SARS-CoV-2, the cause of COVID-19, provide opportunities to study aspects of the disease not easily investigated in human patients. COVID-19 severity ranges from asymptomatic to lethal. Most experimental infections provide insights into mild disease. Here, using K18-hACE2 mice that we originally developed for SARS studies, we show that infection with SARS-CoV-2 causes severe disease in the lung, and in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Further, we show that infusion of convalescent plasma (CP) from a recovered COVID-19 patient provided protection against lethal disease. Mice developed anosmia at early times after infection. Notably, while treatment with CP prevented significant clinical disease, it did not prevent anosmia. Thus K18-hACE2 mice provide a useful model for studying the pathological underpinnings of both mild and lethal COVID-19 and for assessing therapeutic interventions.

Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract

BackgroundZoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors. Materials and methodsWe systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19. FindingsOur results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19. InterpretationThese results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPrevious studies of ACE2 mRNA transcript abundance in the human respiratory tract have suggested a possible association between ACE2 expression and age, sex, and the presence of comorbidities. However, these studies have provided conflicting results, as well as a lack of protein validation. Previous ACE2 protein studies have been limited by a paucity of lung tissue samples and reports that have produced contradictory results. Added value of this studyUsing a combination of single-cell RNA sequencing and immunohistochemistry, we describe ACE2 expression in the human respiratory tract. Staining protocols were optimized and validated to show consistent apical localization and avoid non-specific staining. We show ACE2 protein is found in subsets of airway cells and is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development for COVID-19, respectively. We show age, sex, and comorbidities do not increase ACE2 protein expression in the human respiratory tract. Implications of all the available evidenceACE2 protein abundance does not correlate with risk factors for severe clinical outcomes, but in some cases showed an inversed relationship. Features driving COVID-19 susceptibility and severity are complex, our data suggests factors other than ACE2 protein abundance as important determinants of clinical outcomes.