Cap-independent translation and a precisely localized RNA sequence enable SARS-CoV-2 to control host translation and escape anti-viral response (preprint)

Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represseses translation and induces mRNA degradation, while the host elicits anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-meidated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5UTR of SARS-CoV-2 exhibits an IRES-like activity and promotes expression of NSP1 in an eIF4E-independent and Torin-1 resistant manner. Upon expression, NSP1 enhances cap-independent translation. However, the sub-genomic 5UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin-1. The combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.

Nonclinical Safety and Immunogenicity of an rVSV-ΔG-SARS-CoV-2-S vaccine in mice, hamsters, rabbits and pigs (preprint)

rVSV-{Delta}G-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. Nonclinical safety, immunogenicity and efficacy studies were conducted in 4 animal species, using multiple dose levels (up to 10e8 PFU/animal) and various dosing regimens. There were no treatment related mortalities in any study, or any noticeable clinical signs. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings gave cause for safety concern in any of the studies. There was no viral shedding in urine, nor viral RNA detected in whole blood or serum samples 7 days post vaccination. The rVSV-{Delta}G-SARS-CoV-2-S vaccine immune response gave rise to neutralizing antibodies, cellular immune response, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph node. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive IFNAR KO mice. Vaccine virus replication and distribution in K18 hACE2 transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The rVSV-{Delta}G-SARS-CoV-2-S vaccine was well tolerated locally and systemically and elicited an effective immunogenic response up to the highest dose tested, supporting further clinical development.

Spike vs nucleocapsid SARS-CoV-2 antigen detection: application in nasopharyngeal swab specimens (preprint)

Public health experts emphasize the need for quick, point-of-care SARS-CoV-2 detection as an effective strategy for controlling virus spread. To this end, many "antigen" detection devices were developed and commercialized. These devices are mostly based on detecting SARS-CoV-2s nucleocapsid protein. Recently, alerts issued by both the FDA and the CDC raised concerns regarding the devices tendency to exhibit false positive results. In this work we developed a novel alternative spike-based antigen assay, comprised of four high-affinity, specific monoclonal antibodies, directed against different epitopes on the spikes S1 subunit. The assays performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an in-house nucleocapsid-based assay, composed of antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR positive and negative nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike-assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct<25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct<25) while less specific (87% specificity). Despite being out-performed by qRT-PCR, we suggest that there is room for such tests in the clinical setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/21253148v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@2cdc04org.highwire.dtl.DTLVardef@12090daorg.highwire.dtl.DTLVardef@10603dforg.highwire.dtl.DTLVardef@1e84cfa_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphic abstractSchematic representation of sample collection and analysis. The figure was created using BioRender.com

COVID Moonshot: Open Science Discovery of SARS-CoV-2 Main Protease Inhibitors by Combining Crowdsourcing, High-Throughput Experiments, Computational Simulations, and Machine Learning (preprint)

Herein we provide a living summary of the data generated during the COVID Moonshot project focused on the development of SARS-CoV-2 main protease (Mpro) inhibitors. Our approach uniquely combines crowdsourced medicinal chemistry insights with high throughput crystallography, exascale computational chemistry infrastructure for simulations, and machine learning in triaging designs and predicting synthetic routes. This manuscript describes our methodologies leading to both covalent and non-covalent inhibitors displaying protease IC50 values under 150 nM and viral inhibition under 5 uM in multiple different viral replication assays. Furthermore, we provide over 200 crystal structures of fragment-like and lead-like molecules in complex with the main protease. Over 1000 synthesized and ordered compounds are also reported with the corresponding activity in Mpro enzymatic assays using two different experimental setups. The data referenced in this document will be continually updated to reflect the current experimental progress of the COVID Moonshot project, and serves as a citable reference for ensuing publications. All of the generated data is open to other researchers who may find it of use.

A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies (preprint)

Antibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized in vitro selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel antibody generation.

Induced pulmonary comorbidities render CD-1 mice sensitive to SARS-CoV-2 (preprint)

Severe manifestations of COVID-19 are mostly restricted to persons with comorbidities, and they form a significantly high proportion of those which develop life-endangering lung injury. Nevertheless, COVID-19 animal models established to date are not based on preexistence of comorbidities. Here we report that mild pulmonary injury induced by administration of acute-lung-injury stimulants, renders outbred CD-1 mice to be sensitive to SARS-CoV-2. Following intranasal pretreatment of mice with low doses of ricin or bleomycin, SARS-CoV-2 infection caused a severe disease manifested by sustained body loss and mortality rates of >50%. Low-dose-ricin pretreated mice displayed markedly higher levels of viral RNA than mice not pretreated with ricin, not only in the nasal turbinate, trachea and lungs but also in the serum and heart. The deleterious effects of SARS-CoV-2 infection in ricin-pretreated mice were effectively alleviated by passive transfer of polyclonal and monoclonal antibodies generated against SARS-CoV-2 or SARS-CoV-2 RBD. Notably, viral cell entry in the sensitized mice model seems to involve viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. In summary, we present a novel animal model in mice that express native murine ACE2 yet are susceptible to genetically unaltered SARS-CoV-2, for the study of comorbidity-dependent COVID-19 pathology and treatment.

A genome-wide CRISPR/Cas9 knock-out screen identifies the DEAD box RNA helicase DDX42 as a broad antiviral inhibitor (preprint)

Genome-wide CRISPR/Cas9 knock-out genetic screens are powerful approaches to unravel new regulators of viral infections. With the aim of identifying new cellular inhibitors of HIV-1, we have developed a strategy in which we took advantage of the ability of type 1 interferon (IFN) to potently inhibit HIV-1 infection, in order to create a cellular environment hostile to viral replication. This approach led to the identification of the DEAD-box RNA helicase DDX42 as an intrinsic inhibitor of HIV-1. Depletion of endogenous DDX42 using siRNA or CRISPR/Cas9 knock-out increased HIV-1 infection, both in model cell lines and in physiological targets of HIV-1, primary CD4+ T cells and monocyte-derived macrophages (MDMs), and irrespectively of the IFN treatment. Similarly, the overexpression of a dominant-negative mutant of DDX42 positively impacted HIV-1 infection, whereas wild-type DDX42 overexpression potently inhibited HIV-1 infection. The positive impact of endogenous DDX42 depletion on HIV-1 infection was directly correlated to an increase in viral DNA accumulation. Interestingly, proximity ligation assays showed that DDX42, which can be mainly found in the nucleus but is also present in the cytoplasm, was in the close vicinity of HIV-1 Capsid during infection of primary monocyte-derived macrophages. Moreover, we show that DDX42 is also able to substantially decrease infection with other retroviruses and retrotransposition of long interspersed elements-1 (LINE-1). Finally, we reveal that DDX42 potently inhibits other pathogenic viruses, including Chikungunya virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Aglycone polyether ionophores as broad-spectrum agents inhibit multiple enveloped viruses including SARS-CoV-2 in vitro and successfully cure JEV infected mice (preprint)

Infections with zoonotic viruses, such as flaviviruses, influenza virus, and the SARS-CoV-2 pandemic coronavirus constitute an increasing global risk. Hence, an urgent need exists for the development of broad-spectrum antivirals to prevent such outbreaks. Here, we show that the maduramycin and CP-80,219 aglycone polyether ionophores exhibit effective broad-spectrum antiviral activity, against various viruses, including Japanese encephalitis virus (JEV), Dengue virus (DENV), Zika virus (ZIKV), and Chikungunya virus (CHIKV), while also exhibiting promising activity against PR8 influenza virus and SARS-CoV-2. Moreover, liposome-encapsulated maduramycin and CP-80,219 provide full protection for mice from infection with JEV in vivo. Mechanistic studies suggest that aglycone polyether ionophores primarily inhibit the viral replication step without blocking endosome acidification to promote the fusion between viral and cellular membranes. The successful application of liposomes containing aglycone polyether ionophores in JEV-infected mice offers hope to the development of broad-spectrum antiviral drugs like penicillin back to 1940s.

Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir (preprint)

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3'-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3'-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3'-exonuclease that recognizes mismatches. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis (preprint)

This study describes the development of a novel assay for SARS-CoV-2 identification using LC-MS/MS analysis. A multi-step procedure for the rational down-selection of a set of markers has leaded to the discovery of six SARS-CoV-2 specific and sensitive markers, enabling the reliable identification of the virus. A rapid and simple assay was developed, successfully applied to clinical nasopharyngeal samples. The assay may potentially serve as a complementary approach for SARS-CoV-2 identification.

A single dose of recombinant VSV-{triangleup}G-spike vaccine provides protection against SARS-CoV-2 challenge (preprint)

The COVID-19 pandemic caused by SARS-CoV-2 that emerged in December 2019 in China resulted in over 7.8 million infections and over 430,000 deaths worldwide, imposing an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we generated a replication competent recombinant VSV-{Delta}G-spike vaccine, in which the glycoprotein of VSV was replaced by the spike protein of the SARS-CoV-2. In vitro characterization of the recombinant VSV-{Delta}G-spike indicated expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in vivo model for COVID-19 was implemented. We show that vaccination of hamsters with recombinant VSV-{Delta}G-spike results in rapid and potent induction of neutralizing antibodies against SARS-CoV-2. Importantly, single-dose vaccination was able to protect hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss of the immunized hamsters compared to unvaccinated hamsters. Furthermore, whereas lungs of infected hamsters displayed extensive tissue damage and high viral titers, immunized hamsters lungs showed only minor lung pathology, and no viral load. Taken together, we suggest recombinant VSV-{Delta}G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2 infection.

LAMP-Seq: Population-Scale COVID-19 Diagnostics Using a Compressed Barcode Space (preprint)

The ongoing SARS-CoV-2 pandemic has already caused devastating losses. Exponential spread can be slowed by social distancing and population-wide isolation measures, but those place a tremendous burden on society, and, once lifted, exponential spread can re-emerge. Regular population-scale testing, combined with contact tracing and case isolation, should help break the cycle of transmission, but current detection strategies are not capable of such large-scale processing. Here we present a protocol for LAMP-Seq, a barcoded Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method that is highly scalable. Individual samples are stabilized, inactivated, and amplified in three isothermal heat steps, generating barcoded amplicons that can be pooled and analyzed en masse by sequencing. Using unique barcode combinations per sample from a compressed barcode space enables extensive pooling, potentially further reducing cost and simplifying logistics. We validated LAMP-Seq on 28 clinical samples, empirically optimized the protocol and barcode design, and performed initial safety evaluation. Relying on world-wide infrastructure for next-generation sequencing, and in the context of population-wide sample collection, LAMP-Seq could be scaled to analyze millions of samples per day.