Low Density Lipoprotein Receptor-Related Protein 1 (LRP1) is a host factor for RNA viruses including SARS-CoV-2 (preprint)

Viruses with an RNA genome are the main causes of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that rendered them resistant to the zoonotic Rift Valley fever virus (RVFV; family Phleboviridae, order Bunyavirales). This screen returned the Low Density Lipoprotein Receptor-Related protein 1 (LRP1, or CD91) as top hit, a 600 kDa plasma membrane protein known to be involved in a wide variety of cell activities. Inactivation of LRP1 expression in human cells reduced RVFV infection at the early stages of infection, including the particle attachment to the cell. In the highly LRP1-positive human HuH-7 cell line, LRP1 was required for the early infection stages also of Sandfly fever Sicilian virus (SFSV; family Phleboviridae, order Bunyavirales), vesicular stomatitis (VSV; family Rhabdoviridae, order Mononegavirales), Encephalomyocarditis virus (EMCV, family Picornaviridae), and the coronaviruses MERS-CoV, SARS-CoV-1, and SARS-CoV-2. While for RVFV, EMCV, and MERS-CoV the replication cycle could eventually catch up, LRP1 requirement for the late infection stage in HuH-7 cells was observed for SFSV, La Crosse virus (LACV; family Peribunyaviridae, order Bunyavirales), VSV, SARS-CoV-1, and SARS-CoV-2. For SARS-CoV-2, the absence of LRP1 stably reduced viral RNA levels in human lung Calu-3 cells, and both RNA levels and particle production in the hepatic HuH-7 cells. Thus, we identified LRP1 as a host factor that supports various infection cycle stages of a broad spectrum of RNA viruses.

Clinical grade ACE2 effectively inhibits SARS-CoV-2 Omicron infections (preprint)

The recent emergence of the SARS-CoV-2 variant Omicron has caused considerable concern due to reduced vaccine efficacy and escape from neutralizing antibody therapeutics. Omicron is spreading rapidly around the globe and is suspected to account for most new COVID-19 cases in several countries, though the severity of Omicron-mediated disease is still under debate. It is therefore paramount to identify therapeutic strategies that inhibit the Omicron SARS-CoV-2 variant. Here we report using 3D structural modelling that Spike of Omicron can still associate with human ACE2. Sera collected after the second mRNA-vaccination did not exhibit a protective effect against Omicron while strongly neutralizing infection of VeroE6 cells with the reference Wuhan strain, confirming recent data by other groups on limited vaccine and convalescent sera neutralization efficacy against Omicron. Importantly, clinical grade recombinant human soluble ACE2, a drug candidate currently in clinical development, potently neutralized Omicron infection of VeroE6 cells with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. These data show that SARS-CoV-2 variant Omicron can be readily inhibited by soluble ACE2, providing proof of principle of a viable and effective therapeutic approach against Omicron infections.

Development of a novel, pan-variant aerosol intervention for COVID-19 (preprint)

To develop a universal strategy to block SARS-CoV-2 cellular entry and infection represents a central aim for effective COVID-19 therapy. The growing impact of emerging variants of concern increases the urgency for development of effective interventions. Since ACE2 is the critical SARS-CoV-2 receptor and all tested variants bind to ACE2, some even at much increased affinity (see accompanying paper), we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here we show that intranasal administration of APN01 in a mouse model of SARS-CoV-2 infection dramatically reduced weight loss and prevented animal death. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers can now be initiated, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. This strategy could be used to develop a viable and rapidly actionable therapy to prevent and treat COVID-19, against all current and future SARS-CoV-2 variants. One Sentence SummaryPreclinical development and evaluation of aerosolized soluble recombinant human ACE2 (APN01) administered as a COVID-19 intervention is reported.

Structure-guided glyco-engineering of ACE2 for improved potency as soluble SARS-CoV-2 decoy receptor (preprint)

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.

IgA autoantibodies target pulmonary surfactant in patients with severe COVID-19 (preprint)

Complications affecting the lung are hallmarks of severe coronavirus disease 2019 (COVID-19). While there is evidence for autoimmunity in severe COVID-19, the exact mechanisms remain unknown. Here, we established a prospective observational cohort to study lung specific autoantibodies (auto-Abs). Incubation of plasma from severe COVID-19 patients with healthy human lung tissue revealed the presence of IgA antibodies binding to surfactant-producing pneumocytes. Enzyme-linked immunosorbent assays (ELISA) and protein pull-downs using porcine surfactant confirmed the presence of auto-Abs binding to surfactant proteins in severe COVID-19 patients. Mass spectrometry and ELISAs with recombinant proteins identified IgA auto-Abs that target human surfactant proteins B and C. In line with these findings, lungs of deceased COVID-19 patients showed reduced pulmonary surfactant. Our data suggest that IgA-driven autoimmunity against surfactant may result in disease progression of COVID-19.

Severe COVID-19 is associated with elevated serum IgA and antiphospholipid IgA-antibodies (preprint)

BackgroundWhile the pathogenesis of coronavirus disease 2019 (COVID-19) is becoming increasingly clear, there is little data on IgA response, the first line of bronchial immune defense. ObjectiveTo determine, whether COVID-19 is associated with a vigorous total IgA response and whether IgA autoantibodies are associated with complications of severe illness. Since thrombotic events are frequent in severe COVID-19 and resemble hypercoagulation of antiphospholipid syndrome (APS), our approach focused on antiphospholipid antibodies (aPL). Materials and methodsIn this retrospective cohort study we compared clinical data and aPL from 64 patients with COVID-19 from three independent centers (two in Switzerland, one in Liechtenstein). Samples were collected from April 9, 2020 to May 1, 2020. Total IgA and aPL were measured with FDA-approved commercially available clinical diagnostic kits. ResultsClinical records of the 64 patients with COVID-19 were reviewed and divided into a cohort with mild illness (mCOVID, n=26 [41%]), a discovery cohort with severe illness (sdCOVD, n=14 [22%]) and a confirmation cohort with severe illness (scCOVID, n=24 [38%]). Severe illness was significantly associated with increased total IgA (sdCOVID, P=0.01; scCOVID, P<0.001). Total IgG levels were similar in both cohorts. Among aPL, both cohorts with severe illness significantly correlated with elevated anti-Cardiolipin IgA (sdCOVID and scCOVID, P<0.001), anti-Cardiolipin IgM (sdCOVID, P=0.003; scCOVID, P<0.001), and anti-Beta2 Glycoprotein-1 IgA (sdCOVID and scCOVID, P<0.001). Systemic lupus erythematosus was excluded from all patients as a potential confounder of APS. ConclusionsHigher total IgA and IgA-aPL were consistently associated with severe illness. These novel data strongly suggest that a vigorous antiviral IgA-response triggered in the bronchial mucosa induces systemic autoimmunity.

Dysregulation in mTOR/HIF-1 signaling identified by proteo-transcriptomics of SARS-CoV-2 infected cells (preprint)

How Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remain largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected HuH7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a significant reduction in activated S6K1 and 4E-BP1 at 72 hours post infection. Unlike other human respiratory viruses, we found a significant inhibition of HIF-1 through the entire time course of the infection, suggesting a crosstalk between the SARS-CoV-2 and the mTOR/HIF-1 signaling. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe COVID-19 patients.