Publications by authors named "Adolfo García-Sastre"

542 Publications

Introduction of Two Prolines and Removal of the Polybasic Cleavage Site Lead to Higher Efficacy of a Recombinant Spike-Based SARS-CoV-2 Vaccine in the Mouse Model.

mBio 2021 Mar 2;12(2). Epub 2021 Mar 2.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively unstable, a feature that might be enhanced by the presence of a polybasic cleavage site in SARS-CoV-2 spike. Exchange of K986 and V987 for prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus spike proteins. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin-converting enzyme 2 via adenovirus transduction. Variants tested include spike proteins with a deleted polybasic cleavage site, proline mutations, or a combination thereof, besides the wild-type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the K986P and V987P (PP) mutations completely protected from challenge in this mouse model. A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validate the choice of antigens that contain the PP mutations and suggest that deletion of the polybasic cleavage site may lead to a further-optimized design.
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http://dx.doi.org/10.1128/mBio.02648-20DOI Listing
March 2021

COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models.

Res Sq 2021 Feb 15. Epub 2021 Feb 15.

One year into the Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), effective treatments are still needed . Monoclonal antibodies, given alone or as part of a therapeutic cocktail, have shown promising results in patients, raising the hope that they could play an important role in preventing clinical deterioration in severely ill or in exposed, high risk individuals . Here, we evaluated the prophylactic and therapeutic effect of COVA1-18 , a neutralizing antibody isolated from a convalescent patient and highly potent against the B.1.1.7. isolate . In both prophylactic and therapeutic settings, SARS-CoV-2 remained undetectable in the lungs of COVA1-18 treated hACE2 mice. Therapeutic treatment also caused a dramatic reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 had a very strong antiviral activity in the upper respiratory compartments with an estimated reduction in viral infectivity of more than 95%, and prevented lymphopenia and extensive lung lesions. Modelling and experimental findings demonstrate that COVA1-18 has a strong antiviral activity in three different preclinical models and could be a valuable candidate for further clinical evaluation.
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http://dx.doi.org/10.21203/rs.3.rs-235272/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899470PMC
February 2021

Longitudinal Metabolomics of Human Plasma Reveals Robust Prognostic Markers of COVID-19 Disease Severity.

medRxiv 2021 Feb 8. Epub 2021 Feb 8.

There is an urgent need to identify which COVID-19 patients will develop life-threatening illness so that scarce medical resources can be optimally allocated and rapid treatment can be administered early in the disease course, when clinical management is most effective. To aid in the prognostic classification of disease severity, we performed untargeted metabolomics profiling of 341 patients with plasma samples collected at six longitudinal time points. Using the temporal metabolic profiles and machine learning, we then built a predictive model of disease severity. We determined that the levels of 25 metabolites measured at the time of hospital admission successfully predict future disease severity. Through analysis of longitudinal samples, we confirmed that these prognostic markers are directly related to disease progression and that their levels are restored to baseline upon disease recovery. Finally, we validated that these metabolites are also altered in a hamster model of COVID-19. Our results indicate that metabolic changes associated with COVID-19 severity can be effectively used to stratify patients and inform resource allocation during the pandemic.
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http://dx.doi.org/10.1101/2021.02.05.21251173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872388PMC
February 2021

Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression.

Sci Adv 2021 02 5;7(6). Epub 2021 Feb 5.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.
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http://dx.doi.org/10.1126/sciadv.abe7386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864571PMC
February 2021

The E484K mutation in the SARS-CoV-2 spike protein reduces but does not abolish neutralizing activity of human convalescent and post-vaccination sera.

medRxiv 2021 Jan 29. Epub 2021 Jan 29.

One year in the coronavirus disease 2019 (COVID-19) pandemic, the first vaccines are being rolled out under emergency use authorizations. It is of great concern that newly emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can escape antibody-mediated protection induced by previous infection or vaccination through mutations in the spike protein. The glutamate (E) to Lysine (K) substitution at position 484 (E484K) in the receptor binding domain (RBD) of the spike protein is present in the rapidly spreading variants of concern belonging to the B.1.351 and P.1 lineages. We performed in vitro microneutralization assays with both the USA-WA1/2020 virus and a recombinant (r)SARS-CoV-2 virus that is identical to USA-WA1/2020 except for the E484K mutation introduced in the spike RBD. We selected 34 sera from study participants based on their SARS-CoV-2 spike ELISA antibody titer (negative [N=4] versus weak [N=8], moderate [N=11] or strong positive [N=11]). In addition, we included sera from five individuals who received two doses of the Pfizer SARS-CoV-2 vaccine BNT162b2. Serum neutralization efficiency was lower against the E484K rSARS-CoV-2 (vaccination samples: 3.4 fold; convalescent low IgG: 2.4 fold, moderate IgG: 4.2 fold and high IgG: 2.6 fold) compared to USA-WA1/2020. For some of the convalescent donor sera with low or moderate IgG against the SARS-CoV-2 spike, the drop in neutralization efficiency resulted in neutralization ID50 values similar to negative control samples, with low or even absence of neutralization of the E484K rSARS-CoV-2. However, human sera with high neutralization titers against the USA-WA1/2020 strain were still able to neutralize the E484K rSARS-CoV-2. Therefore, it is important to aim for the highest titers possible induced by vaccination to enhance protection against newly emerging SARS-CoV-2 variants. Two vaccine doses may be needed for induction of high antibody titers against SARS-CoV-2. Postponing the second vaccination is suggested by some public health authorities in order to provide more individuals with a primer vaccination. Our data suggests that this may leave vaccinees less protected against newly emerging variants.
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http://dx.doi.org/10.1101/2021.01.26.21250543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852247PMC
January 2021

Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses.

Nat Microbiol 2021 Jan 28. Epub 2021 Jan 28.

Department of Cancer Biology and Global Center for Pathogens Research and Human Health, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.

The most frequent fetal birth defect associated with prenatal Zika virus (ZIKV) infection is brain calcification, which in turn may potentially affect neurological development in infants. Understanding the mechanism could inform the development of potential therapies against prenatal ZIKV brain calcification. In perivascular cells, bone morphogenetic protein (BMP) is an osteogenic factor that undergoes maturation to activate osteogenesis and calcification. Here, we show that ZIKV infection of cultivated primary human brain pericytes triggers BMP2 maturation, leading to osteogenic gene expression and calcification. We observed extensive calcification near ZIKV pericytes of fetal human brain specimens and in vertically transmitted ZIKV human signal transducer and activator of transcription 2-knockin mouse pup brains. ZIKV infection of primary pericytes stimulated BMP2 maturation, inducing osteogenic gene expression and calcification that were completely blocked by anti-BMP2/4 neutralizing antibody. Not only did ZIKV NS3 expression alone induce BMP2 maturation, osteogenic gene expression and calcification, but purified NS3 protease also effectively cleaved pro-BMP2 in vitro to generate biologically active mature BMP2. These findings highlight ZIKV-induced calcification where the NS3 protease subverts the BMP2-mediated osteogenic signalling pathway to trigger brain calcification.
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http://dx.doi.org/10.1038/s41564-020-00850-3DOI Listing
January 2021

The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and aged mice and is neutralized by convalescent and post-vaccination human sera.

medRxiv 2021 Jan 20. Epub 2021 Jan 20.

The current COVID-19 (coronavirus disease 19) pandemic, caused by SARS-CoV-2, disproportionally affects the elderly and people with comorbidities like obesity and associated type 2 diabetes mellitus. Small animal models are crucial for the successful development and validation of antiviral vaccines, therapies and to study the role that comorbidities have on the outcome of viral infections. The initially available SARS-CoV-2 isolates require adaptation in order to use the mouse angiotensin converting enzyme 2 (mACE-2) entry receptor and to productively infect the cells of the murine respiratory tract. We have "mouse-adapted" SARS-CoV-2 by serial passaging a clinical virus isolate in the lungs of mice. We then used low doses of this virus in mouse models for advanced age, diabetes and obesity. Similar to SARS-CoV-2 infection in humans, the outcome of infection with mouse-adapted SARS-CoV-2 resulted in enhanced morbidity in aged and diabetic obese mice. Mutations associated with mouse adaptation occurred in the S, M, N and ORF8 genes. Interestingly, one mutation in the receptor binding domain of the S protein results in the change of an asparagine to tyrosine residue at position 501 (N501Y). This mutation is also present in the newly emerging SARS-CoV-2 variant viruses reported in the U.K. (20B/501Y.V1, B1.1.7 lineage) that is epidemiologically associated with high human to human transmission. We show that human convalescent and post vaccination sera can neutralize the newly emerging N501Y virus variant with similar efficiency as that of the reference USA-WA1/2020 virus, suggesting that current SARS-CoV-2 vaccines will protect against the 20B/501Y.V1 strain.
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http://dx.doi.org/10.1101/2021.01.19.21249592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836140PMC
January 2021

Host-directed therapies against early-lineage SARS-CoV-2 retain efficacy against B.1.1.7 variant.

bioRxiv 2021 Jan 24. Epub 2021 Jan 24.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths worldwide and massive societal and economic burden. Recently, a new variant of SARS-CoV-2, known as B.1.1.7, was first detected in the United Kingdom and is spreading in several other countries, heightening public health concern and raising questions as to the resulting effectiveness of vaccines and therapeutic interventions. We and others previously identified host-directed therapies with antiviral efficacy against SARS-CoV-2 infection. Less prone to the development of therapy resistance, host-directed drugs represent promising therapeutic options to combat emerging viral variants as host genes possess a lower propensity to mutate compared to viral genes. Here, in the first study of the full-length B.1.1.7 variant virus, we find two host-directed drugs, plitidepsin (aplidin; inhibits translation elongation factor eEF1A) and ralimetinib (inhibits p38 MAP kinase cascade), as well as remdesivir, to possess similar antiviral activity against both the early-lineage SARS-CoV-2 and the B.1.1.7 variant, evaluated in both human gastrointestinal and lung epithelial cell lines. We find that plitidepsin is over an order of magnitude more potent than remdesivir against both viruses. These results highlight the importance of continued development of host-directed therapeutics to combat current and future coronavirus variant outbreaks.
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http://dx.doi.org/10.1101/2021.01.24.427991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836107PMC
January 2021

Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A.

Science 2021 02 25;371(6532):926-931. Epub 2021 Jan 25.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.
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http://dx.doi.org/10.1126/science.abf4058DOI Listing
February 2021

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Lipid Amphiphile Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine.

Angew Chem Int Ed Engl 2021 Jan 19. Epub 2021 Jan 19.

Mount Sinai School of Medicine: Icahn School of Medicine at Mount Sinai, Microbiology, UNITED STATES.

The search for vaccines that protect from severe morbidity and mortality as a result of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe the use of a novel amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. This amphiphile is water soluble and exhibits massive translocation to lymph nodes upon local administration, likely through binding to albumin, affording localized innate immune activation and a dramatic reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in the context of a licensed vaccine setting (i.e. the quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.
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http://dx.doi.org/10.1002/anie.202015362DOI Listing
January 2021

Chimeric Hemagglutinin-Based Live-Attenuated Vaccines Confer Durable Protective Immunity against Influenza A Viruses in a Preclinical Ferret Model.

Vaccines (Basel) 2021 Jan 11;9(1). Epub 2021 Jan 11.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Epidemic or pandemic influenza can annually cause significant morbidity and mortality in humans. We developed novel chimeric hemagglutinin (cHA)-based universal influenza virus vaccines, which contain a conserved HA stalk domain from a 2009 pandemic H1N1 (pH1N1) strain combined with globular head domains from avian influenza A viruses. Our previous reports demonstrated that prime-boost sequential immunizations induced robust antibody responses directed toward the conserved HA stalk domain in ferrets. Herein, we further followed vaccinated animals for one year to compare the efficacy and durability of these vaccines in the preclinical ferret model of influenza. Although all cHA-based immunization regimens induced durable HA stalk-specific and heterosubtypic antibody responses in ferrets, sequential immunization with live-attenuated influenza virus vaccines (LAIV-LAIV) conferred the best protection against upper respiratory tract infection by a pH1N1 influenza A virus. The findings from this study suggest that our sequential immunization strategy for a cHA-based universal influenza virus vaccine provides durable protective humoral and cellular immunity against influenza virus infection.
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http://dx.doi.org/10.3390/vaccines9010040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826668PMC
January 2021

MDA5 Governs the Innate Immune Response to SARS-CoV-2 in Lung Epithelial Cells.

Cell Rep 2021 01;34(2):108628

Immunity and Pathogenesis Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA. Electronic address:

Recent studies have profiled the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggest that cellular responses to viral challenge may affect disease severity. Yet the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remain to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. By screening 16 putative sensors involved in sensing of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Further analyses revealed that viral intermediates specifically activate the IFN response through MDA5-mediated sensing. Additionally, we find that IRF3, IRF5, and NF-κB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.
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http://dx.doi.org/10.1016/j.celrep.2020.108628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832566PMC
January 2021

A Newcastle Disease Virus (NDV) Expressing a Membrane-Anchored Spike as a Cost-Effective Inactivated SARS-CoV-2 Vaccine.

Vaccines (Basel) 2020 Dec 17;8(4). Epub 2020 Dec 17.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

A successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine must not only be safe and protective, but must also meet the demand on a global scale at a low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent coronavirus disease 2019 (COVID-19) vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine.
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http://dx.doi.org/10.3390/vaccines8040771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766959PMC
December 2020

A household case evidences shorter shedding of SARS-CoV-2 in naturally infected cats compared to their human owners.

Emerg Microbes Infect 2020 Dec 15:1-22. Epub 2020 Dec 15.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in domestic and wild cats. However, little is known about natural viral infections of domestic cats, although their importance for modelling disease spread, informing strategies for managing positive human-animal relationships and disease prevention. Here, we describe the SARS-CoV-2 infection in a household of two human adults and sibling cats (one male and two females) using real-time RT-PCR, an ELISA test, viral sequencing, and virus isolation. On May 5th, 2020, the cat-owners tested positive for SARS-CoV-2. Two days later, the male cat showed mild respiratory symptoms and tested positive. Four days after the male cat, the two female cats became positive, asymptomatically. Also, one human and one cat showed antibodies against SARS-CoV-2. All cats excreted detectable SARS-CoV-2 RNA for a shorter duration than humans and viral sequences analysis confirmed human-to-cat transmission. We could not determine if cat-to-cat transmission also occurred.
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http://dx.doi.org/10.1080/22221751.2020.1863132DOI Listing
December 2020

Mass Cytometry Defines Virus-Specific CD4 T Cells in Influenza Vaccination.

Immunohorizons 2020 Dec 11;4(12):774-788. Epub 2020 Dec 11.

Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, CA 94305;

The antiviral response to influenza virus is complex and multifaceted, involving many immune cell subsets. There is an urgent need to understand the role of CD4 T cells, which orchestrate an effective antiviral response, to improve vaccine design strategies. In this study, we analyzed PBMCs from human participants immunized with influenza vaccine, using high-dimensional single-cell proteomic immune profiling by mass cytometry. Data were analyzed using a novel clustering algorithm, denoised ragged pruning, to define possible influenza virus-specific clusters of CD4 T cells. Denoised ragged pruning identified six clusters of cells. Among these, one cluster (Cluster 3) was found to increase in abundance following stimulation with influenza virus peptide ex vivo. A separate cluster (Cluster 4) was found to expand in abundance between days 0 and 7 postvaccination, indicating that it is vaccine responsive. We examined the expression profiles of all six clusters to characterize their lineage, functionality, and possible role in the response to influenza vaccine. Clusters 3 and 4 consisted of effector memory cells, with high CD154 expression. Cluster 3 expressed cytokines like IL-2, IFN-γ, and TNF-α, whereas Cluster 4 expressed IL-17. Interestingly, some participants had low abundance of Clusters 3 and 4, whereas others had higher abundance of one of these clusters compared with the other. Taken together, we present an approach for identifying novel influenza virus-reactive CD4 T cell subsets, a method that could help advance understanding of the immune response to influenza, predict responsiveness to vaccines, and aid in better vaccine design.
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http://dx.doi.org/10.4049/immunohorizons.1900097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891553PMC
December 2020

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models.

bioRxiv 2020 Dec 1. Epub 2020 Dec 1.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, and analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.
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http://dx.doi.org/10.1101/2020.12.01.404483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724667PMC
December 2020

Pre-existing Hemagglutinin Stalk Antibodies Correlate with Protection of Lower Respiratory Symptoms in Flu-Infected Transplant Patients.

Cell Rep Med 2020 Nov 3;1(8):100130. Epub 2020 Nov 3.

Clinical Unit of Infectious Diseases, Microbiology and Preventive Medicine, Institute of Biomedicine of Seville, Virgen del Rocío University Hospital/CSIC/University of Seville, Seville, Spain.

Hemagglutination-inhibitory antibodies are usually highly strain specific with little effect on infection with drifted or shifted strains. The significance of broadly cross-reactive non-HAI anti-influenza antibodies against conserved domains of virus glycoproteins, such as the hemagglutinin (HA) stalk, is of great interest. We characterize a cohort of 40 H1N1pmd09 influenza-infected patients and identify lower respiratory symptoms (LRSs) as a predictor for development of pneumonia. A binomial logistic regression of log10 pre-existing antibody values shows that the probability of LRS occurrence decreased with increased anti-HA full-length and stalk antibody ELISA titers. However, a multilevel logistic regression model adjusted by other potential serocorrelates demonstrates that only antibodies directed against the stalk of HA correlate with protection from lower respiratory infection, limiting disease progression. Our predictive model indicates that a threshold of protective immunity based on broadly cross-reactive HA stalk antibodies could be feasible.
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http://dx.doi.org/10.1016/j.xcrm.2020.100130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691380PMC
November 2020

A chimeric hemagglutinin-based universal influenza virus vaccine approach induces broad and long-lasting immunity in a randomized, placebo-controlled phase I trial.

Nat Med 2021 01 7;27(1):106-114. Epub 2020 Dec 7.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Seasonal influenza viruses constantly change through antigenic drift and the emergence of pandemic influenza viruses through antigenic shift is unpredictable. Conventional influenza virus vaccines induce strain-specific neutralizing antibodies against the variable immunodominant globular head domain of the viral hemagglutinin protein. This necessitates frequent re-formulation of vaccines and handicaps pandemic preparedness. In this completed, observer-blind, randomized, placebo-controlled phase I trial (NCT03300050), safety and immunogenicity of chimeric hemagglutinin-based vaccines were tested in healthy, 18-39-year-old US adults. The study aimed to test the safety and ability of the vaccines to elicit broadly cross-reactive antibodies against the hemagglutinin stalk domain. Participants were enrolled into five groups to receive vaccinations with live-attenuated followed by AS03-adjuvanted inactivated vaccine (n = 20), live-attenuated followed by inactivated vaccine (n = 15), twice AS03-adjuvanted inactivated vaccine (n = 16) or placebo (n = 5, intranasal followed by intramuscular; n = 10, twice intramuscular) 3 months apart. Vaccination was found to be safe and induced a broad, strong, durable and functional immune response targeting the conserved, immunosubdominant stalk of the hemagglutinin. The results suggest that chimeric hemagglutinins have the potential to be developed as universal vaccines that protect broadly against influenza viruses.
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http://dx.doi.org/10.1038/s41591-020-1118-7DOI Listing
January 2021

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate.

EBioMedicine 2020 Dec 21;62:103132. Epub 2020 Nov 21.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States; Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States. Electronic address:

Background: Due to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus.

Methods: Here, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated.

Findings: We report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.

Interpretation: The results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2.

Funding: This work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants.
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http://dx.doi.org/10.1016/j.ebiom.2020.103132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679520PMC
December 2020

Physiology of cardiomyocyte injury in COVID-19.

medRxiv 2020 Nov 13. Epub 2020 Nov 13.

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System shows that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrate cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with SARS-CoV-2 in the presence of interleukins, with clinical findings, to investigate plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes, from healthy human subjects, with SARS-CoV-2 in the absence and presence of interleukins. We find that interleukin treatment and infection results in disorganization of myofibrils, extracellular release of troponin-I, and reduced and erratic beating. Although interleukins do not increase the extent, they increase the severity of viral infection of cardiomyocytes resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health system show that a significant portion of COVID-19 patients without prior history of heart disease, have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection can underlie the heart disease in COVID-19 patients.

One Sentence Summary: Cardiomyocytes derived from human induced pluripotent stem cells treated with interleukins and infected with SARS-CoV-2 in cultures, show increased release of troponin, disorganization of myofibrils, and changes in beating mirroring specific pathologies in some COVID-19 patients.
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http://dx.doi.org/10.1101/2020.11.10.20229294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668750PMC
November 2020

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies.

Vaccines (Basel) 2020 Nov 9;8(4). Epub 2020 Nov 9.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA.

The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals ( = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A "pooled serum" (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1.
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http://dx.doi.org/10.3390/vaccines8040666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712758PMC
November 2020

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.

Science 2020 12 5;370(6523):1473-1479. Epub 2020 Nov 5.

Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA, USA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.
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http://dx.doi.org/10.1126/science.abe3255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857409PMC
December 2020

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Modified Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine.

bioRxiv 2020 Oct 23. Epub 2020 Oct 23.

Department of Microbiology, Icahn School of Medicine at Mount Sinai New York, NY, USA.

The search for vaccines that protect from severe morbidity and mortality as a result of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Several vaccine candidates are currently being tested in the clinic. Inactivated virus and recombinant protein vaccines can be safe options but may require adjuvants to induce robust immune responses efficiently. In this work we describe the use of a novel amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile). This amphiphile is water soluble and exhibits massive translocation to lymph nodes upon local administration, likely through binding to albumin. IMDQ-PEG-CHOL is used to induce a protective immune response against SARS-CoV-2 after single vaccination with trimeric recombinant SARS-CoV-2 spike protein in the BALB/c mouse model. Inclusion of amphiphilic IMDQ-PEG-CHOL in the SARS-CoV-2 spike vaccine formulation resulted in enhanced immune cell recruitment and activation in the draining lymph node. IMDQ-PEG-CHOL has a better safety profile compared to native soluble IMDQ as the former induces a more localized immune response upon local injection, preventing systemic inflammation. Moreover, IMDQ-PEG-CHOL adjuvanted vaccine induced enhanced ELISA and in vitro microneutralization titers, and a more balanced IgG2a/IgG1 response. To correlate vaccine responses with control of virus replication in vivo, vaccinated mice were challenged with SARS-CoV-2 virus after being sensitized by intranasal adenovirus-mediated expression of the human angiotensin converting enzyme 2 (ACE2) gene. Animals vaccinated with trimeric recombinant spike protein vaccine without adjuvant had lung virus titers comparable to non-vaccinated control mice, whereas animals vaccinated with IMDQ-PEG-CHOL-adjuvanted vaccine controlled viral replication and infectious viruses could not be recovered from their lungs at day 4 post infection. In order to test whether IMDQ-PEG-CHOL could also be used to adjuvant vaccines currently licensed for use in humans, proof of concept was also provided by using the same IMDQ-PEG-CHOL to adjuvant human quadrivalent inactivated influenza virus split vaccine, which resulted in enhanced hemagglutination inhibition titers and a more balanced IgG2a/IgG1 antibody response. Enhanced influenza vaccine responses correlated with better virus control when mice were given a lethal influenza virus challenge. Our results underscore the potential use of IMDQ-PEG-CHOL as an adjuvant to achieve protection after single immunization with recombinant protein and inactivated virus vaccines against respiratory viruses, such as SARS-CoV-2 and influenza viruses.
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http://dx.doi.org/10.1101/2020.10.23.344085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587831PMC
October 2020

SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling.

Proc Natl Acad Sci U S A 2020 11 23;117(45):28344-28354. Epub 2020 Oct 23.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029;

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.
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http://dx.doi.org/10.1073/pnas.2016650117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668094PMC
November 2020

Polyreactive Broadly Neutralizing B cells Are Selected to Provide Defense against Pandemic Threat Influenza Viruses.

Immunity 2020 Dec 22;53(6):1230-1244.e5. Epub 2020 Oct 22.

Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, IL 60637, USA; Committee on Immunology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we found mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes were polyreactive. Polyreactive mAbs were preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augmented mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal that polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.
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http://dx.doi.org/10.1016/j.immuni.2020.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772752PMC
December 2020

Comparison of transgenic and adenovirus hACE2 mouse models for SARS-CoV-2 infection.

Emerg Microbes Infect 2020 Dec;9(1):2433-2445

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide pandemic with high morbidity and mortality. Development of animal models that recapitulate important aspects of coronavirus disease 2019 (COVID-19) is critical for the evaluation of vaccines and antivirals, and understanding disease pathogenesis. SARS-CoV-2 has been shown to use the same entry receptor as SARS-CoV-1, human angiotensin-converting enzyme 2 (hACE2) [1-3]. Due to amino acid differences between murine and hACE2, inbred mouse strains fail to support high titer viral replication of SARS-CoV-2 virus. Therefore, a number of transgenic and knock-in mouse models, as well as viral vector-mediated hACE2 delivery systems have been developed. Here we compared the K18-hACE2 transgenic model to adenovirus-mediated delivery of hACE2 to the mouse lung. We show that K18-hACE2 mice replicate virus to high titers in the nasal turbinates, lung and brain, with high lethality, and cytokine/chemokine production. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the nasal turbinates and lung, and no clinical signs of infection. The K18-hACE2 model provides a stringent model for testing vaccines and antivirals, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.
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http://dx.doi.org/10.1080/22221751.2020.1838955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655046PMC
December 2020

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.

Authors:
David E Gordon Joseph Hiatt Mehdi Bouhaddou Veronica V Rezelj Svenja Ulferts Hannes Braberg Alexander S Jureka Kirsten Obernier Jeffrey Z Guo Jyoti Batra Robyn M Kaake Andrew R Weckstein Tristan W Owens Meghna Gupta Sergei Pourmal Erron W Titus Merve Cakir Margaret Soucheray Michael McGregor Zeynep Cakir Gwendolyn Jang Matthew J O'Meara Tia A Tummino Ziyang Zhang Helene Foussard Ajda Rojc Yuan Zhou Dmitry Kuchenov Ruth Hüttenhain Jiewei Xu Manon Eckhardt Danielle L Swaney Jacqueline M Fabius Manisha Ummadi Beril Tutuncuoglu Ujjwal Rathore Maya Modak Paige Haas Kelsey M Haas Zun Zar Chi Naing Ernst H Pulido Ying Shi Inigo Barrio-Hernandez Danish Memon Eirini Petsalaki Alistair Dunham Miguel Correa Marrero David Burke Cassandra Koh Thomas Vallet Jesus A Silvas Caleigh M Azumaya Christian Billesbølle Axel F Brilot Melody G Campbell Amy Diallo Miles Sasha Dickinson Devan Diwanji Nadia Herrera Nick Hoppe Huong T Kratochvil Yanxin Liu Gregory E Merz Michelle Moritz Henry C Nguyen Carlos Nowotny Cristina Puchades Alexandrea N Rizo Ursula Schulze-Gahmen Amber M Smith Ming Sun Iris D Young Jianhua Zhao Daniel Asarnow Justin Biel Alisa Bowen Julian R Braxton Jen Chen Cynthia M Chio Un Seng Chio Ishan Deshpande Loan Doan Bryan Faust Sebastian Flores Mingliang Jin Kate Kim Victor L Lam Fei Li Junrui Li Yen-Li Li Yang Li Xi Liu Megan Lo Kyle E Lopez Arthur A Melo Frank R Moss Phuong Nguyen Joana Paulino Komal Ishwar Pawar Jessica K Peters Thomas H Pospiech Maliheh Safari Smriti Sangwan Kaitlin Schaefer Paul V Thomas Aye C Thwin Raphael Trenker Eric Tse Tsz Kin Martin Tsui Feng Wang Natalie Whitis Zanlin Yu Kaihua Zhang Yang Zhang Fengbo Zhou Daniel Saltzberg Anthony J Hodder Amber S Shun-Shion Daniel M Williams Kris M White Romel Rosales Thomas Kehrer Lisa Miorin Elena Moreno Arvind H Patel Suzannah Rihn Mir M Khalid Albert Vallejo-Gracia Parinaz Fozouni Camille R Simoneau Theodore L Roth David Wu Mohd Anisul Karim Maya Ghoussaini Ian Dunham Francesco Berardi Sebastian Weigang Maxime Chazal Jisoo Park James Logue Marisa McGrath Stuart Weston Robert Haupt C James Hastie Matthew Elliott Fiona Brown Kerry A Burness Elaine Reid Mark Dorward Clare Johnson Stuart G Wilkinson Anna Geyer Daniel M Giesel Carla Baillie Samantha Raggett Hannah Leech Rachel Toth Nicola Goodman Kathleen C Keough Abigail L Lind Reyna J Klesh Kafi R Hemphill Jared Carlson-Stevermer Jennifer Oki Kevin Holden Travis Maures Katherine S Pollard Andrej Sali David A Agard Yifan Cheng James S Fraser Adam Frost Natalia Jura Tanja Kortemme Aashish Manglik Daniel R Southworth Robert M Stroud Dario R Alessi Paul Davies Matthew B Frieman Trey Ideker Carmen Abate Nolwenn Jouvenet Georg Kochs Brian Shoichet Melanie Ott Massimo Palmarini Kevan M Shokat Adolfo García-Sastre Jeremy A Rassen Robert Grosse Oren S Rosenberg Kliment A Verba Christopher F Basler Marco Vignuzzi Andrew A Peden Pedro Beltrao Nevan J Krogan

Science 2020 12 15;370(6521). Epub 2020 Oct 15.

Quantitative Biosciences Institute (QBI) COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.
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http://dx.doi.org/10.1126/science.abe9403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808408PMC
December 2020

Scalable, effective, and rapid decontamination of SARS-CoV-2 contaminated N95 respirators using germicidal ultra-violet C (UVC) irradiation device.

medRxiv 2020 Oct 6. Epub 2020 Oct 6.

Importance: Particulate respirators such as N95 masks are an essential component of personal protective equipment (PPE) for front-line workers. This study describes a rapid and effective UVC irradiation system that would facilitate the safe re-use of N95 respirators and provides supporting information for deploying UVC for decontamination of SARS-CoV-2 during the COVID19 pandemic.

Objective: To assess the inactivation potential of the proposed UVC germicidal device as a function of time by using 3M 8211 - N95 particulate respirators inoculated with SARS-CoV-2.

Design: A germicidal UVC device to deliver tailored UVC dose was developed and snippets (2.5cm2) of the 3M-N95 respirator were inoculated with 106 plaque-forming units (PFU) of SARS-CoV-2 and were UV irradiated. Different exposure times were tested (0-164 seconds) by fixing the distance between the lamp (10 cm) and the mask while providing an exposure of at least 5.43 mWcm-2.

Setting: The current work is broadly applicable for healthcare-settings, particularly during a pandemic such as COVID-19.

Participants: Not applicable. Main Outcome(s) and Measure(s): Primary measure of outcome was titration of infectious virus recovered from virus-inoculated respirator pieces after UVC exposure. Other measures included the method validation of the irradiation protocol, using lentiviruses (biosafety level-2 agent) and establishment of the germicidal UVC exposure protocol.

Results: An average of 4.38x103 PFUml-1(SD 772.68) was recovered from untreated masks while 4.44x102 PFUml-1(SD 203.67), 4.00x102 PFUml-1(SD 115.47), 1.56x102 PFUml-1(SD 76.98) and 4.44x101 PFUml-1(SD 76.98) was recovered in exposures 2s,6s,18s and 54 seconds per side respectively. The germicidal device output and positioning was monitored and a minimum output of 5.43 mWcm-2 was maintained. Infectious SARS-CoV-2 was not detected by plaque assays (minimal level of detection is 67 PFUml-1) on N95 respirator snippets when irradiated for 120s per side or longer suggesting 3.5 log reduction in 240 seconds of irradiation.

Conclusions And Relevance: A scalable germicidal UVC device to deliver tailored UVC dose for rapid decontamination of SARS-CoV-2 was developed. UVC germicidal irradiation of N95 snippets inoculated with SARS-CoV-2 for 120s per side resulted in 100% (3.5 log in total) reduction of virus. These data support the reuse of N95 particle-filtrate apparatus upon irradiation with UVC and supports use of UVC-based decontamination of SARS-CoV-2 virus during the COVID19 pandemic.
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http://dx.doi.org/10.1101/2020.10.05.20206953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553180PMC
October 2020

SARS-CoV-2 infection, disease and transmission in domestic cats.

Emerg Microbes Infect 2020 Dec;9(1):2322-2332

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19) and responsible for the current pandemic. Recent SARS-CoV-2 susceptibility studies in cats show that the virus can replicate in these companion animals and transmit to other cats. Here, we present an in-depth study of SARS-CoV-2 infection, disease and transmission in domestic cats. Cats were challenged with SARS-CoV-2 via intranasal and oral routes. One day post challenge (DPC), two sentinel cats were introduced. Animals were monitored for clinical signs, clinicopathological abnormalities and viral shedding. examinations were performed at 4, 7 and 21 DPC. Viral RNA was not detected in blood but transiently in nasal, oropharyngeal and rectal swabs and bronchoalveolar lavage fluid as well as various tissues. Tracheobronchoadenitis of submucosal glands with the presence of viral RNA and antigen was observed in airways of the infected cats. Serology showed that both, principals and sentinels, developed antibodies to SARS-CoV-2. All animals were clinically asymptomatic during the course of the study and capable of transmitting SARS-CoV-2 to sentinels. The results of this study are critical for understanding the clinical course of SARS-CoV-2 in a naturally susceptible host species, and for risk assessment.
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http://dx.doi.org/10.1080/22221751.2020.1833687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594869PMC
December 2020