Search

A multi-component microarray, applying a novel analysis algorithm, was developed for quantitative evaluation of the SARS-CoV-2 vaccines' immunogenicity. The array enables simultaneous quantitation of IgG, IgM, and IgA, specific to the SARS-CoV-2 spike, receptor binding domain, and nucleocapsid proteins. The developed methodology is based on calculating an apparent immunoglobulin signal from the linear range of the fluorescent read-outs generated by scanning the microarray slides at different exposure times. A dedicated algorithm, employing a rigorous set of embedded conditions, then generates a normalized signal for each of the unique assays. Qualification of the multi-component array performance (evaluating linearity, extended dynamic-range, specificity, precision, and accuracy) was carried out with an in-house COVID-19, qRT-PCR positive serum, as well as pre-pandemic commercial negative sera. Results were compared to the WHO international standard for anti-SARS-CoV-2 immunoglobulins. Specific IgG, IgM, and IgA signals obtained by this array enabled successful discrimination between SARS-CoV-2 q-RT-PCR positive (seroconverted SARS-CoV-2 patients) and negative (naïve) samples. This array is currently used for evaluation of the humoral response to BriLife, the VSV-based Israeli vaccine during phase I/II clinical trials.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis in 2019. Currently, the main method for identification of SARS-CoV-2 is a reverse transcription (RT)-PCR assay designed to detect viral RNA in oropharyngeal (OP) or nasopharyngeal (NP) samples. While the PCR assay is considered highly specific and sensitive, this method cannot determine the infectivity of the sample, which may assist in evaluation of virus transmissibility from patients and breaking transmission chains. Thus, cell-culture-based approaches such as cytopathic effect (CPE) assays are routinely employed for the identification of infectious viruses in NP/OP samples. Despite their high sensitivity, CPE assays take several days and require additional diagnostic tests in order to verify the identity of the pathogen. We have therefore developed a rapid immunofluorescence assay (IFA) for the specific detection of SARS-CoV-2 in NP/OP samples following cell culture infection. Initially, IFA was carried out on Vero E6 cultures infected with SARS-CoV-2 at defined concentrations, and infection was monitored at different time points. This test was able to yield positive signals in cultures infected with 10 pfu/ml at 12 hours postinfection (PI). Increasing the incubation time to 24 hours reduced the detectable infective dose to 1 pfu/ml. These IFA signals occur before the development of CPE. When compared to the CPE test, IFA has the advantages of specificity, rapid detection, and sensitivity, as demonstrated in this work.

Graphical abstract Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC–MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC–MS analysis. A sensitive and specific LC–MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03831-5.

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 108 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03214-w.

Breast tumors are characterized by an extensive desmoplastic stroma, abundantly populated by fibroblasts. Cancer-associated fibroblasts (CAF) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. CAF also orchestrate tumor-promoting inflammation in multiple tumor types, including breast cancer. However, the mechanisms through which normal tissue fibroblasts are reprogrammed to tumor-promoting CAFs are mainly obscure. Here, we show that mammary fibroblasts can be educated by breast cancer cells to become activated to a proinflammatory state that supports malignant progression. Proteomic analysis of breast cancer cell–secreted factors identified the secreted proinflammatory mediator osteopontin, which has been implicated in inflammation, tumor progression, and metastasis. Osteopontin was highly secreted by mouse and human breast cancer cells, and tumor cell–secreted osteopontin activated a CAF phenotypes in normal mammary fibroblasts in vitro and in vivo. Osteopontin was sufficient to induce fibroblast reprogramming and neutralizing antibodies against osteopontin-blocked fibroblast activation induced by tumor cells. The ability of secreted osteopontin to activate mammary fibroblasts relied upon its known receptors CD44 and αVβ3 integrin. Strikingly, osteopontin silencing in tumor cells in vivo attenuated stromal activation and inhibited tumor growth. Our findings establish a critical functional role for paracrine signaling by tumor-derived osteopontin in reprograming normal fibroblasts into tumor-promoting CAFs. Cancer Res; 75(6); 963–73. ©2015 AACR.

Figure S3: Knockdown of OPN does not affect tumor cell growth in vitro.

Table 2: Summary of proteomics analysis: highest intensity extracellular proteins in tumor cell CM.

Supplementary Table 1: Conditioned medium proteome. Intensity values (Log2 values) are indicated for each proteins with and without label-free quantification (LFQ) normalization. PEP- posterior error probability. 'Extracellular' column indicates the proteins annotated as such according Gene Ontology Cellular Compartment (GOCC).

Figure S2: CD44 expression is up regulated in CAFs and in activated NMFs.

Figure S1: OPN does not affect NMFs proliferation.

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, represses translation, and induces mRNA degradation, while the host elicits an 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-mediated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5′UTR of SARS-CoV-2 drives cap-independent translation and promotes expression of NSP1 in an eIF4E-independent and Torin1-resistant manner. Upon expression, NSP1 further enhances cap-independent translation. However, the sub-genomic 5′UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin1. We conclude that the combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5′UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.

Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24 h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.

We have previously published research on the anti-viral properties of an alkaloid mixture extracted from Nuphar lutea, the major components of the partially purified mixture found by NMR analysis. These are mostly dimeric sesquiterpene thioalkaloids called thiobinupharidines and thiobinuphlutidines against the negative strand RNA measles virus (MV). We have previously reported that this extract inhibits the MV as well as its ability to downregulate several MV proteins in persistently MV-infected cells, especially the P (phospho)-protein. Based on our observation that the Nuphar extract is effective in vitro against the MV, and the immediate need that the coronavirus disease 2019 (COVID-19) pandemic created, we tested here the ability of 6,6′-dihydroxythiobinupharidine DTBN, an active small molecule, isolated from the Nuphar lutea extract, on COVID-19. As shown here, DTBN effectively inhibits SARS-CoV-2 production in Vero E6 cells at non-cytotoxic concentrations. The short-term daily administration of DTBN to infected mice delayed the occurrence of severe clinical outcomes, lowered virus levels in the lungs and improved survival with minimal changes in lung histology. The viral load on lungs was significantly reduced in the treated mice. DTBN is a pleiotropic small molecule with multiple targets. Its anti-inflammatory properties affect a variety of pathogens including SARS-CoV-2 as shown here. Its activity appears to target both pathogen specific (as suggested by docking analysis) as well as cellular proteins, such as NF-κB, PKCs, cathepsins and topoisomerase 2, that we have previously identified in our work. Thus, this combined double action of virus inhibition and anti-inflammatory activity may enhance the overall effectivity of DTBN. The promising results from this proof-of-concept in vitro and in vivo preclinical study should encourage future studies to optimize the use of DTBN and/or its molecular derivatives against this and other related viruses.

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-2’s 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 spike’s S1 subunit. The assay’s 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 CtAbstract FigureGraphic abstractSchematic representation of sample collection and analysis. The figure was created using BioRender.com

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis. Rapid and reliable clinical diagnosis is essential for effectively controlling transmission. The gold standard assay for SARS-CoV-2 identification is the highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR); however, this assay depends on specialized reagents and may suffer from false results. Thus, additional assays based on different approaches could be beneficial. Here, we present a novel method for SARS-CoV-2 identification based on mass spectrometry. The approach we implemented combines a multistep procedure for the rational down-selection of a set of reliable markers out of all optional in silico derived tryptic peptides in viral proteins, followed by monitoring of peptides derived from tryptic digests of purified proteins, cell-cultured SARS-CoV-2, and nasopharyngeal (NP) swab matrix spiked with the virus. The marker selection was based on specificity to SARS-CoV-2 and on analytical parameters including sensitivity, linearity, and reproducibility. The final assay is based on six unique and specific peptide markers for SARS-CoV-2 identification. The simple and rapid (2.5 h) protocol we developed consists of virus heat inactivation and denaturation, tryptic digestion, and identification of the selected markers by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The developed assay enabled the identification of 104 PFU/mL SARS-CoV-2 spiked into buffer. Finally, the assay was successfully applied to 16 clinical samples diagnosed by RT-qPCR, achieving 94% concordance with the current gold standard assay. To conclude, the novel MS-based assay described here is specific, rapid, simple, and is believed to provide a complementary assay to the RT-qPCR method.

The COVID-19 pandemic caused by SARS-CoV-2 imposes an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we show the development of a replication competent recombinant VSV-∆G-spike vaccine, in which the glycoprotein of VSV is replaced by the spike protein of SARS-CoV-2. In-vitro characterization of this vaccine indicates the 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 is implemented. We show that a single-dose vaccination results in a rapid and potent induction of SARS-CoV-2 neutralizing antibodies. Importantly, vaccination protects hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss, and alleviation of the extensive tissue damage and viral loads in lungs and nasal turbinates. Taken together, we suggest the recombinant VSV-∆G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2., Here, the authors generate a replication-competent VSV based vaccine expressing SARS-CoV-2 spike protein and show protection in the hamster model with one dose. Analysis of the antibody response in mice shows induction of neutralizing antibodies and suggests a desirable Th1-biased response to the vaccine.

Bacillus anthracis overcomes host immune responses by producing capsule and secreting toxins. Production of these virulence factors in response to entering the host environment was shown to be regulated by atxA, the major virulence regulator, known to be activated by HCO3- and CO2. While toxin production is regulated directly by atxA, capsule production is independently mediated by two regulators; acpA and acpB. In addition, it was demonstrated that acpA has at least two promotors, one of them shared with atxA. We used a genetic approach to study capsule and toxin production under different conditions. Unlike previous works utilizing NBY-HCO3- medium under CO2 enriched conditions, we used a sDMEM-based medium. Thus, toxin and capsule production can be induced in ambient or CO2 enriched atmosphere. Using this system, we could differentiate between induction by 10% NRS, 10% CO2 or 0.75% HCO3-. In response to high CO2, capsule production is induced by acpA in an atxA-independent manner, with little to no toxin (protective antigen PA) production. atxA is activated in response to serum independently of CO2, inducing toxin and capsule production in an acpA or acpB dependent manner. HCO3- was also found to activate atxA, but in non-physiological concentrations. Our findings may help explain the first stages of inhalational infection, in which spores germinating in dendritic cells require protection (by encapsulation) without affecting cell migration to the draining lymph-node by toxin secretion.

The first case of SARS-CoV-2 was discovered in Israel in late February 2020. Three major outbreaks followed, resulting in over 800,000 cases and over 6,000 deaths by April 2021. Our aim was characterization of a serological snapshot of Israeli patients and healthy adults in the early months of the COVID-19 pandemic. Sera from 55 symptomatic COVID-19 patients and 146 healthy subjects (early-pandemic, reverse transcription-quantitative PCR [qRT-PCR]-negative), collected in Israel between March and April 2020, were screened for SARS-CoV-2-specific IgG, IgM, and IgA antibodies, using a 6-plex antigen microarray presenting the whole inactivated virus and five viral antigens: a stabilized version of the spike ectodomain (S2P), spike subunit 1 (S1), receptor-binding-domain (RBD), N-terminal-domain (NTD), and nucleocapsid (NC). COVID-19 patients, 4 to 40 days post symptom onset, presented specific IgG to all of the viral antigens (6/6) in 54 of the 55 samples (98% sensitivity). Specific IgM and IgA antibodies for all six antigens were detected in only 10% (5/55) and 4% (2/55) of the patients, respectively, suggesting that specific IgG is a superior serological marker for COVID-19. None of the qRT-PCR-negative sera reacted with all six viral antigens (100% specificity), and 48% (70/146) were negative throughout the panel. Our findings confirm a low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population prior to the COVID-19 outbreak. We further suggest that the presence of low-level cross-reacting antibodies in naive individuals calls for a combined, multiantigen analysis for accurate discrimination between naive and exposed individuals. IMPORTANCE A 6-plex protein array presenting the whole inactivated virus and five nucleocapsid and spike-derived SARS-CoV-2 antigens was used to generate a serological snapshot of SARS-CoV-2 seroprevalence and seroconversion in Israel in the early months of the pandemic. Our findings confirm a very low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population. We further propose that the presence of low-level nonspecific antibodies in naive individuals calls for a combined, multiantigen analysis for accurate discrimination between naive and exposed individuals enabling accurate determination of seroconversion. The developed assay is currently applied to evaluate immune responses to the Israeli vaccine during human phase I/II trials.

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 5’UTR 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 5’UTRs 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 5’UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.

rVSV-ΔG-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 108 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-ΔG-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-ΔG-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.

The poly- δ- d-glutamic acid capsule of Bacillus anthracis plays a major role in this bacterium pathogenicity. Capsule synthesis relies on a 5 gene operon; capB, C, A, D and E that are regulated by acpA and acpB, that respond to the major virulence regulator - atxA. We took a genetic approach to examine the involvement of acpA and acpB in capsule production in vitro and on B. anthracis virulence in vivo. To complement the effect of the mutations on capsule accumulation in vitro, we applied our toxin independent systemic infection method to study their effects in vivo. We found that though the roles of acpA and axpB are redundant in vitro, deleting acpA had a significant effect on pathogenicity, mainly on the time to death. As expected, deletion of both acpA and acpB resulted in loss of capsule accumulation in vitro and full attenuation in vivo, indicating that capsule production depends exclusively on acpA/B regulation. To identify additional effects of acpA and acpB on pathogenicity via non-capsule related virulence pathways, we bypassed acpA/B regulation by inserting the pagA promotor upstream to the cap operon, diverting regulation directly to atxA. This resulted in restoration of capsule accumulation in vitro and virulence (in intravenous or subcutaneous inoculation) in vivo. To test for additional pXO2-based genes involved in capsule production, we cloned the pagAprom-capA-E into the chromosome of VollumΔpXO2, which restored capsule accumulation. These results indicate that of the pXO2 genes, only capA-E and acpA are required for capsule production.

Objectives Environmental surfaces have been suggested as likely contributors in the transmission of COVID-19. This study assessed the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contaminating surfaces and objects in two hospital isolation units and a quarantine hotel. Methods SARS-CoV-2 virus stability and infectivity on non-porous surfaces was tested under controlled laboratory conditions. Surface and air sampling were conducted at two COVID-19 isolation units and in a quarantine hotel. Viral RNA was detected by RT-PCR and infectivity was assessed by VERO E6 CPE test. Results In laboratory-controlled conditions, SARS-CoV-2 gradually lost its infectivity completely by day 4 at ambient temperature, and the decay rate of viral viability on surfaces directly correlated with increase in temperature. Viral RNA was detected in 29/55 surface samples (52.7%) and 16/42 surface samples (38%) from the surroundings of symptomatic COVID-19 patients in isolation units of two hospitals and in a quarantine hotel for asymptomatic and very mild COVID-19 patients. None of the surface and air samples from the three sites (0/97) were found to contain infectious titres of SARS-Cov-2 on tissue culture assay. Conclusions Despite prolonged viability of SARS-CoV-2 under laboratory-controlled conditions, uncultivable viral contamination of inanimate surfaces might suggest low feasibility for indirect fomite transmission.

Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary morbidities rendered SARS-CoV-2-refractive CD-1 mice susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low doses of the acute lung injury stimulants bleomycin or ricin caused severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates greater than 50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart, and serum of low-dose ricin-pretreated mice compared with non-pretreated mice. Furthermore, lung extracts prepared 2-3 days after viral infection contained subgenomic mRNA and virus particles capable of replication only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against the SARS-CoV-2 receptor binding domain (RBD). Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of coronavirus disease 2019 (COVID-19) pathologies in patients with preexisting morbidities.

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.

The COVID-19 pandemic was a stark reminder that a barren global antiviral pipeline has grave humanitarian consequences. Pandemics could be prevented in principle by accessible, easily deployable broad-spectrum oral antivirals. Here we report the results of theCOVID Moonshot, a fully open-science, crowd sourced, structure-enabled drug discovery campaign targeting the SARS-CoV-2 main protease. We discovered a novel chemical series that is differentiated from current Mpro inhibitors in that it maintains a new non-covalent, non-peptidic scaffold with nanomolar potency. Our approach leveraged crowdsourcing, high-throughput structural biology, machine learning, and exascale molecular simulations and high-throughput chemistry. In the process, we generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. In a first for a structure-based drug discovery campaign, all compound designs (>18,000 designs), crystallographic data (>840 ligand-bound X-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2,400 compounds) for this campaign were shared rapidly and openly, creating a rich open and IP-free knowledgebase for future anti-coronavirus drug discovery.

Severe manifestations of COVID-19 are mostly restricted to people with comorbidities. Here we report that induced mild pulmonary morbidities render SARS-CoV-2-refractive CD-1 mice to be susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low-doses of the acute-lung-injury stimulants bleomycin or ricin caused a severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates of >50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart and serum of low-dose-ricin pretreated, as compared to non-pretreated mice. Notably, the deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against SARS-CoV-2 RBD. Thus, viral cell entry in the sensitized mice seems to involve viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. In summary, we present a novel mice-based animal model for the study of comorbidity-dependent severe COVID-19.

Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24 h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.

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-ΔG-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-ΔG-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-ΔG-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-ΔG-spike as a safe, efficacious and protective vaccine against SARS-CoV-2 infection.

SummaryThe 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.

As Bacillus anthracis spores pose a proven bio-terror risk, the treatment focus has shifted from exposed populations to anthrax patients and the need for effective antibiotic treatment protocols increases. The CDC recommends carbapenems and Linezolid (oxazolidinone), for the treatment of anthrax, particularly for the late, meningeal stages of the disease. Previously we demonstrated that treatment with Meropenem or Linezolid, either as a single treatment or in combination with Ciprofloxacin, fails to protect rabbits from anthrax-meningitis. In addition, we showed that the failure of Meropenem was due to slow BBB penetration rather than low antibacterial activity. Herein, we tested the effect of increasing the dose of the antibiotic on treatment efficacy. We found that for full protection (88% cure rate) the dose should be increased four-fold from 40 mg/kg to 150 mg/kg. In addition, B. anthracis is a genetically stable bacterium and naturally occurring multidrug resistant B. anthracis strains have not been reported. In this manuscript, we report the efficacy of classical β-lactams as a single treatment or in combination with β-lactamase inhibitors in treating anthrax meningitis. We demonstrate that Ampicillin based treatment of anthrax meningitis is largely efficient (66%). The high efficacy (88-100%) of Augmentin (Amoxicillin and Clavulonic acid) and Unasyn (Ampicillin and Sulbactam) makes them a favorable choice due to reports of β-lactam resistant B. anthracis strains. Tazocin (Piperacillin and Tazobactam) proved inefficient compared to the highly efficient Augmentin and Unasyn.

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human induced pluripotent stem cell-derived kidney organoids with SARS-CoV-2. Single cell RNA-sequencing indicated injury and dedifferentiation of infected cells with activation of pro-fibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in Long-COVID., Graphical Abstract, Jansen, Reimer, Nagai et al report that SARS-CoV-2 infects kidney cells and is associated with kidney fibrosis in patients. Using single cell transcriptomics of infected kidney organoids, they show that SARS-CoV-2 causes kidney injury and stimulates pro-fibrotic signaling. Viral infection in organoids was inhibited by a recently developed protease blocker.

Cancer-Associated Fibroblasts (CAFs) were shown to orchestrate tumour-promoting inflammation in multiple malignancies, including breast cancer. However, the molecular pathways that govern the inflammatory role of CAFs are poorly characterised. In this study we found that fibroblasts sense damage-associated molecular patterns (DAMPs), and in response activate the NLRP3 inflammasome pathway, resulting in instigation of pro-inflammatory signalling and secretion of IL-1β. This upregulation was evident in CAFs in mouse and in human breast carcinomas. Moreover, CAF-derived inflammasome signalling facilitated tumour growth and metastasis, which was attenuated when NLRP3 or IL-1β were specifically ablated. Functionally, CAF-derived inflammasome promoted tumour progression and metastasis by modulating the tumour microenvironment towards an immune suppressive milieu and by upregulating the expression of adhesion molecules on endothelial cells. Our findings elucidate a mechanism by which CAFs promote breast cancer progression and metastasis, by linking the physiological tissue damage response of fibroblasts with tumour-promoting inflammation., Cancer associated fibroblasts (CAFs) are known to promote pro-tumorigenic inflammation. Here, the authors show that CAFs sense tissue damage and activate NLRP3 inflammasome and pro-inflammatory IL-1β secretion, and CAF-derived inflammasome signalling promotes breast tumour growth and metastasis.

In nature, Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis usually infecting grazing animals. Taking advantage of their stability and ability to survive harsh conditions for decades, this deadly bacterium was stockpiled during the twentieth century as a bio-weapon by the great nations. The 1972 convention that prohibited the development, production and stockpiling of bio-weapons reduced these nation-level productions but increased the probability that knowhow, and in some cases weapon grade spores, will become available for use by terror groups, thus creating a new threat—bio-terror. In modern history there were two documented bio-terror events as well as one accidental discharge from an army facility and other industrial exposures that resulted in human exposure to B. anthracis spores. These incidents demonstrate the power of B. anthracis spores as a bio-terror agent and the challenges that are associated with such release/use. In this chapter, we will use the published data regarding these events together with experimental data obtained from animal experiments, to discuss the challenges associated with the use of B. anthracis spores as a bio-terror agent and the ways to counteract them. We will go through the different challenges of patient diagnosis and treatment, discuss the challenges of monitoring the environment and decontamination. In addition we will describe the available forensic tools and discuss the challenges of identifying spore production prior to dissemination.

Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro , may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets.

Nonencapsulated (∆pXO2) Bacillus anthracis strains are commonly used as vaccines and for anthrax research, mainly in the mouse model. Previously, we demonstrated that the infection of rabbits, intranasally or subcutaneously, with the spores of a fully virulent strain results in the systemic dissemination of the bacteria, meningitis, and death, whereas ∆pXO2 strains are fully attenuated in this animal model. We used the intravenous inoculation of rabbits to study the pathogenicity of the ∆pXO2 strain infection. Bacteremia, brain bacterial burden, and pathology were used as criteria to compare the Vollum∆pXO2 disease to the wild type Vollum infection. To test the role of adhesion in the virulence of Vollum∆pXO2, we deleted the major adhesion protein BslA and tested the virulence and immunogenicity of this mutant. We found that 50% of the rabbits succumb to Vollum∆pXO2 strain following i.v. infection, a death that was accompanied with significant neurological symptoms. Pathology revealed severe brain infection coupled with an atypical massive bacterial growth into the parenchyma. Contrary to the Vollum strain, deletion of the bslA gene fully attenuated the ∆pXO2 strain. Though the Vollum∆pXO2 cannot serve as a model for B. anthracis pathogenicity in rabbits, deletion of the bslA gene prevents central nervous system (CNS) infections, possibly leading to the generation of a safer vaccine.