Dicoumarol is an effective post-exposure prophylactic for SARS-CoV-2 Omicron infection in human airway epithelium.

Repurposing existing drugs to inhibit SARS-CoV-2 infection in airway epithelial cells (AECs) is a quick way to find novel treatments for COVID-19. Computational screening has found dicoumarol (DCM), a natural anticoagulant, to be a potential SARS-CoV-2 inhibitor, but its inhibitory effects and possible working mechanisms remain unknown. Using air-liquid interface culture of primary human AECs, we demonstrated that DCM has potent antiviral activity against the infection of multiple Omicron variants (including BA.1, BQ.1 and XBB.1). Time-of-addition and drug withdrawal assays revealed that early treatment (continuously incubated after viral absorption) of DCM could markedly inhibit Omicron replication in AECs, but DCM did not affect the absorption, exocytosis and spread of viruses or directly eliminate viruses. Mechanistically, we performed single-cell sequencing analysis (a database of 77,969 cells from different airway locations from 10 healthy volunteers) and immunofluorescence staining, and showed that the expression of NAD(P)H quinone oxidoreductase 1 (NQO1), one of the known DCM targets, was predominantly localised in ciliated AECs. We further found that the NQO1 expression level was positively correlated with both the disease severity of COVID-19 patients and virus copy levels in cultured AECs. In addition, DCM treatment downregulated NQO1 expression and disrupted signalling pathways associated with SARS-CoV-2 disease outcomes (e.g., Endocytosis and COVID-19 signalling pathways) in cultured AECs. Collectively, we demonstrated that DCM is an effective post-exposure prophylactic for SARS-CoV-2 infection in the human AECs, and these findings could help physicians formulate novel treatment strategies for COVID-19.

Proteomic profiling of single extracellular vesicles reveals colocalization of SARS-CoV-2 with a CD81/integrin-rich EV subpopulation in sputum from COVID-19 severe patients.

Background: The global outbreak of COVID-19, and the limited availability of clinical treatments, forced researchers around the world to search for the pathogenesis and potential treatments. Understanding the pathogenesis of SARS-CoV-2 is crucial to respond better to the current coronavirus disease 2019 (COVID-19) pandemic. Methods: We collected sputum samples from 20 COVID-19 patients and healthy controls. Transmission electron microscopy was used to observe the morphology of SARS-CoV-2. Extracellular vesicles (EVs) were isolated from sputum and the supernatant of VeroE6 cells, and were characterized by transmission electron microscopy, nanoparticle tracking analysis and Western-Blotting. Furthermore, a proximity barcoding assay was used to investigate immune-related proteins in single EV, and the relationship between EVs and SARS-CoV-2. Result: Transmission electron microscopy images of SARS-COV-2 virus reveal EV-like vesicles around the virion, and western blot analysis of EVs extracted from the supernatant of SARS-COV-2-infected VeroE6 cells showed that they expressed SARS-COV-2 protein. These EVs have the infectivity of SARS-COV-2, and the addition can cause the infection and damage of normal VeroE6 cells. In addition, EVs derived from the sputum of patients infected with SARS-COV-2 expressed high levels of IL6 and TGF-ß, which correlated strongly with expression of the SARS-CoV-2 N protein. Among 40 EV subpopulations identified, 18 differed significantly between patients and controls. The EV subpopulation regulated by CD81 was the most likely to correlate with changes in the pulmonary microenvironment after SARS-CoV-2 infection. Single extracellular vesicles in the sputum of COVID-19 patients harbor infection-mediated alterations in host and virus-derived proteins. Conclusions: These results demonstrate that EVs derived from the sputum of patients participate in virus infection and immune responses. This study provides evidence of an association between EVs and SARS-CoV-2, providing insight into the possible pathogenesis of SARS-CoV-2 infection and the possibility of developing nanoparticle-based antiviral drugs.

SARS-CoV-2 induces cardiomyocyte apoptosis and inflammation but can be ameliorated by ACE inhibitor Captopril.

Although the clinical manifestation of COVID-19 is mainly respiratory symptoms, approximately 20% of patients suffer from cardiac complications. COVID-19 patients with cardiovascular disease have higher severity of myocardial injury and poor outcomes. The underlying mechanism of myocardial injury caused by SARS-CoV-2 infection remains unclear. Using a non-transgenic mouse model infected with Beta variant (B.1.351), we found that the viral RNA could be detected in lungs and hearts of infected mice. Pathological analysis showed thinner ventricular wall, disorganized and ruptured myocardial fiber, mild inflammatory infiltration, and mild epicardia or interstitial fibrosis in hearts of infected mice. We also found that SARS-CoV-2 could infect cardiomyocytes and produce infectious progeny viruses in human pluripotent stem cell-derived cardiomyocyte-like cells (hPSC-CMs). SARS-CoV-2 infection caused apoptosis, reduction of mitochondrial integrity and quantity, and cessation of beating in hPSC-CMs. In order to dissect the mechanism of myocardial injury caused by SARS-CoV-2 infection, we employed transcriptome sequencing of hPSC-CMs at different time points after viral infection. Transcriptome analysis showed robust induction of inflammatory cytokines and chemokines, up-regulation of MHC class I molecules, activation of apoptosis signaling and cell cycle arresting. These may cause aggravate inflammation, immune cell infiltration, and cell death. Furthermore, we found that Captopril (hypotensive drugs targeting ACE) treatment could alleviate SARS-CoV-2 induced inflammatory response and apoptosis in cardiomyocytes via inactivating TNF signaling pathways, suggesting Captopril may be beneficial for reducing COVID-19 associated cardiomyopathy. These findings preliminarily explain the molecular mechanism of pathological cardiac injury caused by SARS-CoV-2 infection, providing new perspectives for the discovery of antiviral therapeutics.

Repurposing CRISPR/Cas to Discover SARS-CoV-2 Detecting and Neutralizing Aptamers.

RNA aptamers provide useful biological probes and therapeutic agents. New methodologies to screen RNA aptamers will be valuable by complementing the traditional Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Meanwhile, repurposing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated systems (Cas) has expanded their utility far beyond their native nuclease function. Here, CRISmers, a CRISPR/Cas-based novel screening system for RNA aptamers based on binding to a chosen protein of interest in a cellular context, is presented. Using CRISmers, aptamers are identified specifically targeting the receptor binding domain (RBD) of the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two aptamer leads enable sensitive detection and potent neutralization of SARS-CoV-2 Delta and Omicron variants in vitro. Intranasal administration of one aptamer, further modified with 2'-fluoro pyrimidines (2'-F), 2'-O-methyl purines (2'-O), and conjugation with both cholesterol and polyethylene glycol of 40 kDa (PEG40K), achieves effective prophylactic and therapeutic antiviral activity against live Omicron BA.2 variants in vivo. The study concludes by demonstrating the robustness, consistency, and potential broad utility of CRISmers using two newly identified aptamers but switching CRISPR, selection marker, and host species.

Self-replicating RNA nanoparticle vaccine elicits protective immune responses against SARS-CoV-2

The creation of safe and effective vaccines that induce potent cellular and humoral immune responses against SARS-CoV-2 is urgently needed to end the global COVID-19 epidemic. Here we developed an alphavirus-derived self-replicating RNA (repRNA)-based vaccine platform encoding the receptor binding domain (RBD) of SARS-CoV-2 spike glycoprotein. The repRNA triggers prolonged antigen expression compared to conventional mRNA due to the replication machinery of repRNA. To improve the delivery and vaccine efficacy of repRNA, we developed a self-assembling liposome-protamine-RNA (LPR) nanoparticle with highly efficient encapsulation and transfection of repRNA. LPR-repRNA vaccines substantially activated type I interferon response and innate immune signaling pathways. Subcutaneous immunization of LPR-repRNA-RBD led to prolonged antigen expression, stimulation of innate immune cells and induction of germinal center response in draining lymph nodes. LPR-repRNA-RBD induced antigen-specific T-cell responses and skewed cellular immunity towards an effector memory CD8+ T cell response. Immunizations with LPR-repRNA-RBD triggered the production of anti-RBD IgG antibodies and induced neutralizing antibody response against SARS-CoV-2 pseudovirus. LPR-repRNA-RBD vaccines reduced SARS-CoV-2 infection and lung inflammation in mice. Altogether, these data suggest that LPR-repRNA platform can be a promising avenue for COVID-19 vaccine development. Graphical Zhang and colleagues develop a LPR nanovaccine platform encapsulating repRNA that encodes the RBD of SARS-CoV-2 spike glycoprotein (LPR-repRNA-RBD). Compared to conventional mRNA vaccines, LPR-repRNA vaccines enhance the magnitude and duration of antigen expression, activate innate immunity, induce antigen-specific cellular and humoral immune responses, and reduce SARS-CoV-2 infection and lung injury.

Inactivated vaccine-elicited potent antibodies can broadly neutralize SARS-CoV-2 circulating variants.

A full understanding of the inactivated COVID-19 vaccine-mediated antibody responses to SARS-CoV-2 circulating variants will inform vaccine effectiveness and vaccination development strategies. Here, we offer insights into the inactivated vaccine-induced antibody responses after prime-boost vaccination at both the polyclonal and monoclonal levels. We characterized the VDJ sequence of 118 monoclonal antibodies (mAbs) and found that 20 neutralizing mAbs showed varied potency and breadth against a range of variants including XBB.1.5, BQ.1.1, and BN.1. Bispecific antibodies (bsAbs) based on nonoverlapping mAbs exhibited enhanced neutralizing potency and breadth against the most antibody-evasive strains, such as XBB.1.5, BQ.1.1, and BN.1. The passive transfer of mAbs or their bsAb effectively protected female hACE2 transgenic mice from challenge with an infectious Delta or Omicron BA.2 variant. The neutralization mechanisms of these antibodies were determined by structural characterization. Overall, a broad spectrum of potent and distinct neutralizing antibodies can be induced in individuals immunized with the SARS-CoV-2 inactivated vaccine BBIBP-CorV, suggesting the application potential of inactivated vaccines and these antibodies for preventing infection by SARS-CoV-2 circulating variants.

Plasma cell-free DNA promise monitoring and tissue injury assessment of COVID-19.

Coronavirus 2019 (COVID-19) is a complex disease that affects billions of people worldwide. Currently, effective etiological treatment of COVID-19 is still lacking; COVID-19 also causes damages to various organs that affects therapeutics and mortality of the patients. Surveillance of the treatment responses and organ injury assessment of COVID-19 patients are of high clinical value. In this study, we investigated the characteristic fragmentation patterns and explored the potential in tissue injury assessment of plasma cell-free DNA in COVID-19 patients. Through recruitment of 37 COVID-19 patients, 32 controls and analysis of 208 blood samples upon diagnosis and during treatment, we report gross abnormalities in cfDNA of COVID-19 patients, including elevated GC content, altered molecule size and end motif patterns. More importantly, such cfDNA fragmentation characteristics reflect patient-specific physiological changes during treatment. Further analysis on cfDNA tissue-of-origin tracing reveals frequent tissue injuries in COVID-19 patients, which is supported by clinical diagnoses. Hence, our work demonstrates and extends the translational merit of cfDNA fragmentation pattern as valuable analyte for effective treatment monitoring, as well as tissue injury assessment in COVID-19.

Intranasal booster using an Omicron vaccine confers broad mucosal and systemic immunity against SARS-CoV-2 variants.

The highly contagious SARS-CoV-2 Omicron subvariants severely attenuated the effectiveness of currently licensed SARS-CoV-2 vaccines based on ancestral strains administered via intramuscular injection. In this study, we generated a recombinant, replication-incompetent human adenovirus type 5, Ad5-S-Omicron, that expresses Omicron BA.1 spike. Intranasal, but not intramuscular vaccination, elicited spike-specific respiratory mucosal IgA and residential T cell immune responses, in addition to systemic neutralizing antibodies and T cell immune responses against most Omicron subvariants. We tested intranasal Ad5-S-Omicron as a heterologous booster in mice that previously received intramuscular injection of inactivated ancestral vaccine. In addition to inducing serum broadly neutralizing antibodies, there was a significant induction of respiratory mucosal IgA and neutralizing activities against Omicron subvariants BA.1, BA.2, BA.5, BA.2.75, BF.7 as well as pre-Omicron strains Wildtype, Beta, and Delta. Serum and mucosal neutralizing activities against recently emerged XBB, BQ.1, and BQ.1.1 could also be detected but were much lower. Nasal lavage fluids from intranasal vaccination contained multimeric IgA that can bind to at least 10 spike proteins, including Omicron subvariants and pre-Omicron strains, and possessed broadly neutralizing activities. Intranasal vaccination using Ad5-S-Omicron or instillation of intranasal vaccinee's nasal lavage fluids in mouse nostrils protected mice against Omicron challenge. Taken together, intranasal Ad5-S-Omicron booster on the basis of ancestral vaccines can establish effective mucosal and systemic immunity against Omicron subvariants and multiple SARS-CoV-2 variants. This candidate vaccine warrants further development as a safe, effective, and user-friendly infection and transmission-blocking vaccine.

Phase-separated nucleocapsid protein of SARS-CoV-2 suppresses cGAS-DNA recognition by disrupting cGAS-G3BP1 complex.

Currently, the incidence and fatality rate of SARS-CoV-2 remain continually high worldwide. COVID-19 patients infected with SARS-CoV-2 exhibited decreased type I interferon (IFN-I) signal, along with limited activation of antiviral immune responses as well as enhanced viral infectivity. Dramatic progresses have been made in revealing the multiple strategies employed by SARS-CoV-2 in impairing canonical RNA sensing pathways. However, it remains to be determined about the SARS-CoV-2 antagonism of cGAS-mediated activation of IFN responses during infection. In the current study, we figure out that SARS-CoV-2 infection leads to the accumulation of released mitochondria DNA (mtDNA), which in turn triggers cGAS to activate IFN-I signaling. As countermeasures, SARS-CoV-2 nucleocapsid (N) protein restricts the DNA recognition capacity of cGAS to impair cGAS-induced IFN-I signaling. Mechanically, N protein disrupts the assembly of cGAS with its co-factor G3BP1 by undergoing DNA-induced liquid-liquid phase separation (LLPS), subsequently impairs the double-strand DNA (dsDNA) detection ability of cGAS. Taken together, our findings unravel a novel antagonistic strategy by which SARS-CoV-2 reduces DNA-triggered IFN-I pathway through interfering with cGAS-DNA phase separation.

HLA-Bw4 in association with KIR3DL1 favors natural killer cell-mediated protection against severe COVID-19.

Replicating SARS-CoV-2 has been shown to degrade HLA class I on target cells to evade the cytotoxic T-cell (CTL) response. HLA-I downregulation can be sensed by NK cells to unleash killer cell immunoglobulin-like receptor (KIR)-mediated self-inhibition by the cognate HLA-I ligands. Here, we investigated the impact of HLA and KIR genotypes and HLA-KIR combinations on COVID-19 outcome. We found that the peptide affinities of HLA alleles were not correlated with COVID-19 severity. The predicted poor binders for SARS-CoV-2 peptides belong to HLA-B subtypes that encode KIR ligands, including Bw4 and C1 (introduced by B*46:01), which have a small F pocket and cannot accommodate SARS-CoV-2 CTL epitopes. However, HLA-Bw4 weak binders were beneficial for COVID-19 outcome, and individuals lacking the HLA-Bw4 motif were at higher risk for serious illness from COVID-19. The presence of the HLA-Bw4 and KIR3DL1 combination had a 58.8% lower risk of developing severe COVID-19 (OR = 0.412, 95% CI = 0.187-0.904, p = 0.02). This suggests that HLA-Bw4 alleles that impair their ability to load SARS-CoV-2 peptides will become targets for NK-mediated destruction. Thus, we proposed that the synergistic responsiveness of CTLs and NK cells can efficiently control SARS-CoV-2 infection and replication, and NK-cell-mediated anti-SARS-CoV-2 immune responses being mostly involved in severe infection when the level of ORF8 is high enough to degrade HLA-I. The HLA-Bw4/KIR3DL1 genotype may be particularly important for East Asians undergoing COVID-19 who are enriched in HLA-Bw4-inhibitory KIR interactions and carry a high frequency of HLA-Bw4 alleles that bind poorly to coronavirus peptides.

Successful clearance of persistent SARS-CoV-2 asymptomatic infection following a single dose of Ad5-nCoV vaccine.

Persistent asymptomatic (PA) SARS-CoV-2 infections have been identified. The immune responses in these patients are unclear, and the development of effective treatments for these patients is needed. Here, we report a cohort of 23 PA cases carrying viral RNA for up to 191 days. PA cases displayed low levels of inflammatory and interferon response, weak antibody response, diminished circulating follicular helper T cells (cTfh), and inadequate specific CD4+ and CD8+ T-cell responses during infection, which is distinct from symptomatic infections and resembling impaired immune activation. Administration of a single dose of Ad5-nCoV vaccine to 10 of these PA cases elicited rapid and robust antibody responses as well as coordinated B-cell and cTfh responses, resulting in successful viral clearance. Vaccine-induced antibodies were able to neutralize various variants of concern and persisted for over 6 months, indicating long-term protection. Therefore, our study provides an insight into the immune status of PA infections and highlights vaccination as a potential treatment for prolonged SARS-CoV-2 infections.

Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mouse Models.

The outbreak of coronavirus disease 2019 (COVID-19) has resulted in a global pandemic due to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At the time of this manuscript's publication, remdesivir is the only COVID-19 treatment approved by the United States Food and Drug Administration. However, its effectiveness is still under question due to the results of the large Solidarity Trial conducted by the World Health Organization. Herein, we report that the parent nucleoside of remdesivir, GS-441524, potently inhibits the replication of SARS-CoV-2 in Vero E6 and other cell lines. Challenge studies in both an AAV-hACE2 mouse model of SARS-CoV-2 and in mice infected with murine hepatitis virus, a closely related coronavirus, showed that GS-441524 was highly efficacious in reducing the viral titers in CoV-infected organs without notable toxicity. Our results support that GS-441524 is a promising and inexpensive drug candidate for treating of COVID-19 and other CoV diseases.

Mouse models susceptible to HCoV-229E and HCoV-NL63 and cross protection from challenge with SARS-CoV-2.

Human coronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63) are endemic causes of upper respiratory infections such as the "common cold" but may occasionally cause severe lower respiratory tract disease in the elderly and immunocompromised patients. There are no approved antiviral drugs or vaccines for these common cold coronaviruses (CCCoV). The recent emergence of COVID-19 and the possible cross-reactive antibody and T cell responses between these CCCoV and SARS-CoV-2 emphasize the need to develop experimental animal models for CCCoV. Mice are an ideal experimental animal model for such studies, but are resistant to HCoV-229E and HCoV-NL63 infections. Here, we generated 229E and NL63 mouse models by exogenous delivery of their receptors, human hAPN and hACE2 using replication-deficient adenoviruses (Ad5-hAPN and Ad5-hACE2), respectively. Ad5-hAPN- and Ad5-hACE2-sensitized IFNAR-/- and STAT1-/- mice developed pneumonia characterized by inflammatory cell infiltration with virus clearance occurring 7 d post infection. Ad5-hAPN- and Ad5-hACE2-sensitized mice generated virus-specific T cells and neutralizing antibodies after 229E or NL63 infection, respectively. Remdesivir and a vaccine candidate targeting spike protein of 229E and NL63 accelerated viral clearance of virus in these mice. 229E- and NL63-infected mice were partially protected from SARS-CoV-2 infection, likely mediated by cross-reactive T cell responses. Ad5-hAPN- and Ad5-hACE2-transduced mice are useful for studying pathogenesis and immune responses induced by HCoV-229E and HCoV-NL63 infections and for validation of broadly protective vaccines, antibodies, and therapeutics against human respiratory coronaviruses including SARS-CoV-2.

Efficient Targeted Delivery of Bifunctional Circular Aptamer-ASO Chimera to Suppress the SARS-CoV-2 Proliferation and Inflammation.

Inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and excessive inflammation is the current task in the prevention and treatment of corona vireus disease 2019 (COVID-19). Here, a dual-function circular aptamer-ASO chimera (circSApt-NASO) is designed to suppress SARS-CoV-2 replication and inflammation. The chemically unmodified circSApt-NASO exhibits high serum stability by artificial cyclization. It is also demonstrated that the SApt binding to spike protein enables the chimera to be efficiently delivered into the host cells expressing ACE2 along with the infection of SARS-CoV-2. Among them, the SApt potently inhibits spike-induced inflammation. The NASO targeting to silence N genes not only display robust anti-N-induced inflammatory activity, but also achieve efficient inhibition of SARS-CoV-2 replication. Overall, benefiting from the high stability of the cyclization, antispike aptamer-dependent, and viral infection-mediate targeted delivery, the circSApt-NASO displays robust potential against authentic SARS-CoV-2 and Omicron, providing a promising specific anti-inflammatory and antiproliferative reagent for therapeutic COVID-19.

Biparatopic antibody BA7208/7125 effectively neutralizes SARS-CoV-2 variants including Omicron BA.1-BA.5.

SARS-CoV-2 Omicron subvariants have demonstrated extensive evasion from monoclonal antibodies (mAbs) developed for clinical use, which raises an urgent need to develop new broad-spectrum mAbs. Here, we report the isolation and analysis of two anti-RBD neutralizing antibodies BA7208 and BA7125 from mice engineered to produce human antibodies. While BA7125 showed broadly neutralizing activity against all variants except the Omicron sublineages, BA7208 was potently neutralizing against all tested SARS-CoV-2 variants (including Omicron BA.1-BA.5) except Mu. By combining BA7208 and BA7125 through the knobs-into-holes technology, we generated a biparatopic antibody BA7208/7125 that was able to neutralize all tested circulating SARS-CoV-2 variants. Cryo-electron microscopy structure of these broad-spectrum antibodies in complex with trimeric Delta and Omicron spike indicated that the contact residues are highly conserved and had minimal interactions with mutational residues in RBD of current variants. In addition, we showed that administration of BA7208/7125 via the intraperitoneal, intranasal, or aerosol inhalation route showed potent therapeutic efficacy against Omicron BA.1 and BA.2 in hACE2-transgenic and wild-type mice and, separately, effective prophylaxis. BA7208/7125 thus has the potential to be an effective candidate as an intervention against COVID-19.