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Iron-Catalyzed Cross-Coupling Responses regarding Alkyl Grignards using Aryl Chlorobenzenesulfonates.
Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in severely ill patients and the pathophysiology of disease is thought to be immune-mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens, often promoting inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and non-human primates. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype. MC activation in humans was confirmed, through detection of the MC-specific protease, chymase, levels of which were significantly correlated with disease severity. These results support the association of MC activation with severe COVID-19, suggesting potential strategies for intervention.SARS-CoV-2 infection during pregnancy leads to an increased risk of adverse pregnancy outcomes. Although the placenta itself can be a target of virus infection, most neonates are virus free and are born healthy or recover quickly. Here, we investigated the impact of SARS-CoV-2 infection on the placenta from a cohort of women who were infected late during pregnancy and had tested nasal swab positive for SARS-CoV-2 by qRT-PCR at delivery. SARS-CoV-2 genomic and subgenomic RNA was detected in 23 out of 54 placentas. Two placentas with high virus content were obtained from mothers who presented with severe COVID-19 and whose pregnancies resulted in adverse outcomes for the fetuses, including intrauterine fetal demise and a preterm delivered baby still in newborn intensive care. Examination of the placental samples with high virus content showed efficient SARS-CoV-2 infection, using RNA in situ hybridization to detect genomic and replicating viral RNA, and immunohistochemistry to detect SARS-CoV-2 nucleocapsid protein. Infection was restricted to syncytiotrophoblast cells that envelope the fetal chorionic villi and are in direct contact with maternal blood. The infected placentas displayed massive infiltration of maternal immune cells including macrophages into intervillous spaces, potentially contributing to inflammation of the tissue. Ex vivo infection of placental cultures with SARS-CoV-2 or with SARS-CoV-2 spike (S) protein pseudotyped lentivirus targeted mostly syncytiotrophoblast and in rare events endothelial cells. Infection was reduced by using blocking antibodies against ACE2 and against Neuropilin 1, suggesting that SARS-CoV-2 may utilize alternative receptors for entry into placental cells.SARS-CoV-2 therapeutic antibody discovery efforts have met with notable success but have been associated with a generally inefficient process, requiring the production and characterization of exceptionally large numbers of candidates for the identification of a small set of leads. Here, we show that incorporating antibody-ligand blocking as part of LIBRA-seq, the high-throughput sequencing platform for antibody discovery, results in efficient identification of ultra-potent neutralizing antibodies against SARS-CoV-2. LIBRA-seq with ligand blocking is a general platform for functional antibody discovery targeting the disruption of antigen-ligand interactions.The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. SIS3 in vivo While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. link2 Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly-conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.Despite being FDA-approved for COVID-19, the clinical efficacy of remdesivir (Veklury ® ) remains contentious. We previously pointed out pharmacokinetic, pharmacodynamic and toxicology reasons for why its parent nucleoside GS-441524, is better suited for COVID-19 treatment. Here, we assess the oral bioavailability of GS-441524 in beagle dogs and show that plasma concentrations ∼24-fold higher than the EC 50 against SARS-CoV-2 are easily and safely sustained. These data support translation of GS-441524 as an oral agent for COVID-19.As the COVID-19 pandemic continues, the authorization of vaccines for emergency use has been crucial in slowing down the rate of infection and transmission of the SARS-CoV-2 virus that causes COVID-19. In order to investigate the longitudinal serological responses to SARS-CoV-2 natural infection and vaccination, a large-scale, multi-year serosurveillance program entitled SPARTA (SARS SeroPrevalence and Respiratory Tract Assessment) was initiated at 4 locations in the U.S. The serological assay presented here measuring IgG binding to the SARS-CoV-2 receptor binding domain (RBD) detected antibodies elicited by SARS-CoV-2 infection or vaccination with a 95.5% sensitivity and a 95.9% specificity. We used this assay to screen more than 3100 participants and selected 20 previously infected pre-immune and 32 immunologically nave participants to analyze their antibody binding to RBD and viral neutralization (VN) responses following vaccination with two doses of either the Pfizer-BioNTech BNT162b2 or the Moderna mRNA-1273 vaccine. Vaccination not only elicited a more robust immune reaction than natural infection, but the level of neutralizing and anti-RBD antibody binding after vaccination is also significantly higher in pre-immune participants compared to immunologically nave participants ( p less then 0.0033). Furthermore, the administration of the second vaccination did not further increase the neutralizing or binding antibody levels in pre-immune participants ( p =0.69). However, ~46% of the immunologically nave participants required both vaccinations to seroconvert.Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike S protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic, and through an ill-defined mechanism prevents in vitro infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ~5-10 fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both, a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2 virus. link3 The substantial ~5 fold or more decrease of half maximal effective concentrations (EC50 values) suggests a plausible treatment strategy based on the combined use of these inhibitors.Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies 1,2 . Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines 3,4 . In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naïve individuals 2,5-8 . The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern 4,9 . In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2-positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While ACE2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the variants of concern (VOC) B.1.1.7 and B.1.351 have similar cell-to-cell transmission capability.
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