Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

The emergence of SARS-CoV-2 variants that threaten the efficacy of existing vaccines and therapeutic antibodies underscores the urgent need for new antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells of COVID-19 patients. The three most potent antibodies targeted distinct regions of the RBD, and all three neutralized the SARS-CoV-2 variants B.1.1.7 and B.1.351. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the ACE2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five ultrapotent bispecific antibodies that inhibit authentic SARS-CoV-2 infection at concentrations of <1 ng/mL. Through a novel mode of action three bispecific antibodies cross-linked adjacent spike proteins using dual NTD/RBD specificities. One bispecific antibody was >100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, six of nine bispecific antibodies neutralized B.1.1.7, B.1.351 and the wild-type virus with comparable potency, despite partial or complete loss of activity of at least one parent monoclonal antibody against B.1.351. Furthermore, a bispecific antibody that neutralized B.1.351 protected against SARS-CoV-2 expressing the crucial E484K mutation in the hamster model. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

Adult Stem Cell-derived Complete Lung Organoid Models Emulate Lung Disease in COVID-19

SARS-CoV-2, the virus responsible for COVID-19, causes widespread damage in the lungs in the setting of an overzealous immune response whose origin remains unclear. We present a scalable, propagable, personalized, cost-effective adult stem cell-derived human lung organoid model that is complete with both proximal and distal airway epithelia. Monolayers derived from adult lung organoids (ALOs), primary airway cells, or hiPSC-derived alveolar type-II (AT2) pneumocytes were infected with SARS-CoV-2 to create in vitro lung models of COVID-19. Infected ALO-monolayers best recapitulated the transcriptomic signatures in diverse cohorts of COVID-19 patient-derived respiratory samples. The airway (proximal) cells were critical for sustained viral infection, whereas distal alveolar differentiation (AT2[->]AT1) was critical for mounting the overzealous host immune response in fatal disease; ALO monolayers with well-mixed proximodistal airway components recapitulated both. Findings validate a human lung model of COVID-19, which can be immediately utilized to investigate COVID-19 pathogenesis and vet new therapies and vaccines. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/344002v4_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1d1507aorg.highwire.dtl.DTLVardef@faa17forg.highwire.dtl.DTLVardef@80ceb1org.highwire.dtl.DTLVardef@81d61c_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIHuman lung organoids with mixed proximodistal epithelia are created C_LIO_LIProximal airway cells are critical for viral infectivity C_LIO_LIDistal alveolar cells are important for emulating host response C_LIO_LIBoth are required for the overzealous response in severe COVID-19 C_LI IN BRIEFAn integrated stem cell-based disease modeling and computational approach demonstrate how both proximal airway epithelium is critical for SARS-CoV-2 infectivity, but distal differentiation of alveolar pneumocytes is critical for simulating the overzealous host response in fatal COVID-19.

AI-guided discovery of the invariant host response to viral pandemics

We sought to define the host immune response, a.k.a, the "cytokine storm" that has been implicated in fatal COVID-19 using an AI-based approach. Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a seed gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. Surprisingly, this 166-gene signature was conserved in all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determines severity/fatality. Precise therapeutic goals were formulated and subsequently validated in high-dose SARS-CoV-2-challenged hamsters using neutralizing antibodies that abrogate SARS-CoV-2*ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine tracked with disease severity. Thus, the ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs. One Sentence SummaryThe host immune response in COVID-19. PANEL: RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSThe SARS-CoV-2 pandemic has inspired many groups to find innovative methodologies that can help us understand the host immune response to the virus; unchecked proportions of such immune response have been implicated in fatality. We searched GEO and ArrayExpress that provided many publicly available gene expression data that objectively measure the host immune response in diverse conditions. However, challenges remain in identifying a set of host response events that are common to every condition. There are no studies that provide a reproducible assessment of prognosticators of disease severity, the host response, and therapeutic goals. Consequently, therapeutic trials for COVID-19 have seen many more misses than hits. This work used multiple (> 45,000) gene expression datasets from GEO and ArrayExpress and analyzed them using an unbiased computational approach that relies upon fundamentals of gene expression patterns and mathematical precision when assessing them. Added value of this studyThis work identifies a signature that is surprisingly conserved in all viral pandemics, including Covid-19, inspiring the nomenclature ViP-signature. A subset of 20-genes classified disease severity in respiratory pandemics. The ViP signatures pinpointed the nature and source of the cytokine storm mounted by the host. They also helped formulate precise therapeutic goals and rationalized the repurposing of FDA-approved drugs. Implications of all the available evidenceThe ViP signatures provide a quantitative and qualitative framework for assessing the immune response in viral pandemics when creating pre-clinical models; they serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.

Oral drug repositioning candidates and synergistic remdesivir combinations for the prophylaxis and treatment of COVID-19

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. A high-throughput, high-content imaging assay of human HeLa cells expressing the SARS-CoV-2 receptor ACE2 was used to screen ReFRAME, a best-in-class drug repurposing library. From nearly 12,000 compounds, we identified 66 compounds capable of selectively inhibiting SARS-CoV-2 replication in human cells. Twenty-four of these drugs show additive activity in combination with the RNA-dependent RNA polymerase inhibitor remdesivir and may afford increased in vivo efficacy. We also identified synergistic interaction of the nucleoside analog riboprine and a folate antagonist 10-deazaaminopterin with remdesivir. Overall, seven clinically approved drugs (halofantrine, amiodarone, nelfinavir, simeprevir, manidipine, ozanimod, osimertinib) and 19 compounds in other stages of development may have the potential to be repurposed as SARS-CoV-2 oral therapeutics based on their potency, pharmacokinetic and human safety profiles.

Structural basis of a public antibody response to SARS-CoV-2

Molecular-level understanding of human neutralizing antibody responses to SARS-CoV-2 could accelerate vaccine design and facilitate drug discovery. We analyzed 294 SARS-CoV-2 antibodies and found that IGHV3-53 is the most frequently used IGHV gene for targeting the receptor binding domain (RBD) of the spike (S) protein. We determined crystal structures of two IGHV3-53 neutralizing antibodies +/- Fab CR3022 ranging from 2.33 to 3.11 [A] resolution. The germline-encoded residues of IGHV3-53 dominate binding to the ACE2 binding site epitope with no overlap with the CR3022 epitope. Moreover, IGHV3-53 is used in combination with a very short CDR H3 and different light chains. Overall, IGHV3-53 represents a versatile public VH in neutralizing SARS-CoV-2 antibodies, where their specific germline features and minimal affinity maturation provide important insights for vaccine design and assessing outcomes.

Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model

The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.