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.

Delivery of spike-RBD by bacterial type three secretion system for SARS-CoV-2 vaccine development.

COVID-19 pandemic continues to spread throughout the world with an urgent demand for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a bacterial vector COVID-19 vaccine (aPA-RBD) that carries the gene for the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Live-attenuated strains of Pseudomonas aeruginosa (aPA) were constructed which express the recombinant RBD and effectively deliver RBD protein into various antigen presenting cells through bacterial type 3 secretion system (T3SS) in vitro. In mice, two-dose of intranasal aPA-RBD vaccinations elicited the development of RBD-specific serum IgG and IgM. Importantly, the sera from the immunized mice were able to neutralize host cell infections by SARS-CoV-2 pseudovirus as well as the authentic virus variants potently. T-cell responses of immunized mice were assessed by enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) assays. aPA-RBD vaccinations can elicit RBD-specific CD4+and CD8+T cell responses. T3SS-based RBD intracellular delivery heightens the efficiency of antigen presentation and enables the aPA-RBD vaccine to elicit CD8+T cell response. Thus, aPA vector has the potential as an inexpensive, readily manufactured, and respiratory tract vaccination route vaccine platform for other pathogens.

Neutralization Effect of Sera against Delta and Omicron in Patients Recovering from COVID-19 and Inactivated Vaccine Recipients.

This study aims to analyze the serum neutralization capacity against Delta and Omicron variants in three clusters of individuals, including those who had recovered from COVID-19 and those who had received two and three doses of inactivated vaccine. Pseudovirus neutralization tests were performed on serum samples. The neutralizing titers between different groups were compared using the Wilcoxon's signed-rank test. Among the two-dose vaccinees, the neutralization titers of the Omicron variant were reduced by approximately 3.1-fold compared to the wild-type virus (p < 0.05). Meanwhile, among the three-dose vaccinees, the neutralization titers for Delta and Omicron variants were 3.5-fold (p < 0.05) and 5.0-fold (p < 0.05) lower, respectively, as compared to the wild-type virus. In addition, among the recovering patients, the neutralization titers for Delta and Omicron variants were 3.9-fold (p < 0.05) and 29.1-fold (p < 0.05) lower, respectively, as compared to the wild-type virus. Overall, only 12.0% (11/92) of participants showed neutralizing titers against Omicron above the detection limit. The ability to neutralize wild-type pseudovirus was significantly boosted in three-dose vaccinees as compared to two-dose vaccinees. Sera from recovered patients showed greater neutralizing titers for the wild-type and Delta pseudoviruses than the two- and three-dose inactivated vaccine groups. The present study revealed a loss of neutralizing activity against the Omicron variant in almost all samples. Moreover, the immunization effect obtained through natural infection is more robust than that from the active immunization method of vaccination.

Peptide-based inhibitors hold great promise as the broad-spectrum agents against coronavirus.

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome (MERS), and the recent SARS-CoV-2 are lethal coronaviruses (CoVs) that have caused dreadful epidemic or pandemic in a large region or globally. Infections of human respiratory systems and other important organs by these pathogenic viruses often results in high rates of morbidity and mortality. Efficient anti-viral drugs are needed. Herein, we firstly take SARS-CoV-2 as an example to present the molecular mechanism of CoV infection cycle, including the receptor binding, viral entry, intracellular replication, virion assembly, and release. Then according to their mode of action, we provide a summary of anti-viral peptides that have been reported in peer-reviewed publications. Even though CoVs can rapidly evolve to gain resistance to the conventional small molecule drugs, peptide-based inhibitors targeting various steps of CoV lifecycle remain a promising approach. Peptides can be continuously modified to improve their antiviral efficacy and spectrum along with the emergence of new viral variants.

Peptide-based inhibitors hold great promise as the broad-spectrum agents against coronavirus

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome (MERS), and the recent SARS-CoV-2 are lethal coronaviruses (CoVs) that have caused dreadful epidemic or pandemic in a large region or globally. Infections of human respiratory systems and other important organs by these pathogenic viruses often results in high rates of morbidity and mortality. Efficient anti-viral drugs are needed. Herein, we firstly take SARS-CoV-2 as an example to present the molecular mechanism of CoV infection cycle, including the receptor binding, viral entry, intracellular replication, virion assembly, and release. Then according to their mode of action, we provide a summary of anti-viral peptides that have been reported in peer-reviewed publications. Even though CoVs can rapidly evolve to gain resistance to the conventional small molecule drugs, peptide-based inhibitors targeting various steps of CoV lifecycle remain a promising approach. Peptides can be continuously modified to improve their antiviral efficacy and spectrum along with the emergence of new viral variants.

A nanomaterial targeting the spike protein captures SARS-CoV-2 variants and promotes viral elimination.

The global emergency caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic can only be solved with effective and widespread preventive and therapeutic strategies, and both are still insufficient. Here, we describe an ultrathin two-dimensional CuInP2S6 (CIPS) nanosheet as a new agent against SARS-CoV-2 infection. CIPS exhibits an extremely high and selective binding capacity (dissociation constant (KD) < 1 pM) for the receptor binding domain of the spike protein of wild-type SARS-CoV-2 and its variants of concern, including Delta and Omicron, inhibiting virus entry and infection in angiotensin converting enzyme 2 (ACE2)-bearing cells, human airway epithelial organoids and human ACE2-transgenic mice. On association with CIPS, the virus is quickly phagocytosed and eliminated by macrophages, suggesting that CIPS could be successfully used to capture and facilitate virus elimination by the host. Thus, we propose CIPS as a promising nanodrug for future safe and effective anti-SARS-CoV-2 therapy, and as a decontamination agent and surface-coating material to reduce SARS-CoV-2 infectivity.

Caspase-1 and Gasdermin D Afford the Optimal Targets with Distinct Switching Strategies in NLRP1b Inflammasome-Induced Cell Death.

Inflammasomes are essential complexes of innate immune system, which form the first line of host defense against pathogens. Mounting evidence accumulates that inflammasome signaling is highly correlated with coronavirus disease 2019 (COVID-19). However, there remains a significant gap in our understanding of the regulatory mechanism of inflammasome signaling. Combining mathematical modeling with experimental analysis of NLRP1b inflammasome signaling, we found that only the expression levels of caspase-1 and GSDMD have the potential to individually switch cell death modes. Reduction of caspase-1 or GSDMD switches cell death from pyroptosis to apoptosis. Caspase-1 and GSDMD present different thresholds and exert distinct pathway choices in switching death modes. Pyroptosis switches to apoptosis with an extremely low threshold level of caspase-1, but with a high threshold of GSDMD. Caspase-1-impaired cells employ ASC-caspase-8-dependent pathway for apoptosis, while GSDMD-impaired cells primarily utilize caspase-1-dependent pathway. Additionally, caspase-1 and GSDMD can severally ignite the cooccurrence of pyroptosis and apoptosis. Landscape topography unravels that the cooccurrence is dramatically different in caspase-1- and GSDMD-impaired cells. Besides pyroptosis state and apoptosis state, a potential new "coexisting" state in single cells is proposed when GSDMD acts as the driving force of the landscape. The "seesaw model" is therefore proposed, which can well describe the death states that are controlled by caspase-1 or GSDMD in single cells. Our study sheds new light on NLRP1b inflammasome signaling and uncovers the switching mechanisms among various death modes, providing potential clues to guide the development of more rational control strategies for diseases.

A Retinol Derivative Inhibits SARS-CoV-2 Infection by Interrupting Spike-Mediated Cellular Entry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the global pandemic and life-threatening coronavirus disease 2019 (COVID-19). Although vaccines and therapeutic antibodies are available, their efficacy is continuously undermined by rapidly emerging SARS-CoV-2 variants. Here, we found that all-trans retinoic acid (ATRA), a vitamin A (retinol) derivative, showed potent antiviral activity against all SARS-CoV-2 variants in both human cell lines and human organoids of the lower respiratory tract. Mechanistically, ATRA directly binds in a deep hydrophobic pocket of the receptor binding domain (RBD) located on the top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the "down" RBDs and locks most of the S trimers in an RBD "all-down" and ACE2-inaccessible inhibitory conformation. In summary, our results reveal the pharmacological biotargets and structural mechanism of ATRA and other retinoids in SARS-CoV-2 infection and suggest that ATRA and its derivatives could be potential hit compounds against a broad spectrum of coronaviruses. IMPORTANCE Retinoids, a group of compounds including vitamin A and its active metabolite all-trans retinoic acid (ATRA), regulate serial physiological activity in multiple organ systems, such as cell growth, differentiation, and apoptosis. The ATRA analogues reported to date include more than 4,000 natural and synthetic molecules that are structurally and/or functionally related to ATRA. Here, we found that ATRA showed potent antiviral activity against all SARS-CoV-2 variants by directly binding in a deep hydrophobic pocket of the receptor binding domain (RBD) located on top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the "down" RBDs and locks most of the S trimers in an RBD "all-down" and ACE2-inaccessible inhibitory conformation, suggesting the pharmacological feasibility of using ATRA or its derivatives as a remedy for and prevention of COVID-19 disease.

Microplastics interact with SARS-CoV-2 and facilitate host cell infection

Microplastics (MP) pollution is a global issue that raises concerns about potential toxicity for environmental and human health. The notion that SARS-CoV-2 is more stable when adsorbed on plastic surfaces urged us to examine whether the virus can attach to MP, which may facilitate infection upon inhalation or ingestion. Here, we describe that MP can bind SARS-CoV-2 pseudovirus on their surface and enhance infection of human cells in vitro. This enhanced in vitro infectivity was confirmed with authentic SARS-CoV-2, in parallel with increased expression of inflammation-related caspase-3, IL-8 and TNF-alpha genes. These results suggest that the presence of MP in the environment or in our respiratory or gastrointestinal tracts has the potential to interact with SARS-CoV-2, and potentially increase viral infectivity and spreading.

A glucose-like metabolite deficient in diabetes inhibits cellular entry of SARS-CoV-2.

The severity and mortality of COVID-19 are associated with pre-existing medical comorbidities such as diabetes mellitus. However, the underlying causes for increased susceptibility to viral infection in patients with diabetes is not fully understood. Here we identify several small-molecule metabolites from human blood with effective antiviral activity against SARS-CoV-2, one of which, 1,5-anhydro-D-glucitol (1,5-AG), is associated with diabetes mellitus. The serum 1,5-AG level is significantly lower in patients with diabetes. In vitro, the level of SARS-CoV-2 replication is higher in the presence of serum from patients with diabetes than from healthy individuals and this is counteracted by supplementation of 1,5-AG to the serum from patients. Diabetic (db/db) mice undergo SARS-CoV-2 infection accompanied by much higher viral loads and more severe respiratory tissue damage when compared to wild-type mice. Sustained supplementation of 1,5-AG in diabetic mice reduces SARS-CoV-2 loads and disease severity to similar levels in nondiabetic mice. Mechanistically, 1,5-AG directly binds the S2 subunit of the SARS-CoV-2 spike protein, thereby interrupting spike-mediated virus-host membrane fusion. Our results reveal a mechanism that contributes to COVID-19 pathogenesis in the diabetic population and suggest that 1,5-AG supplementation may be beneficial to diabetic patients against severe COVID-19.

A single-cell transcriptomic landscape of the lungs of patients with COVID-19.

The lung is the primary organ targeted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making respiratory failure a leading coronavirus disease 2019 (COVID-19)-related mortality. However, our cellular and molecular understanding of how SARS-CoV-2 infection drives lung pathology is limited. Here we constructed multi-omics and single-nucleus transcriptomic atlases of the lungs of patients with COVID-19, which integrate histological, transcriptomic and proteomic analyses. Our work reveals the molecular basis of pathological hallmarks associated with SARS-CoV-2 infection in different lung and infiltrating immune cell populations. We report molecular fingerprints of hyperinflammation, alveolar epithelial cell exhaustion, vascular changes and fibrosis, and identify parenchymal lung senescence as a molecular state of COVID-19 pathology. Moreover, our data suggest that FOXO3A suppression is a potential mechanism underlying the fibroblast-to-myofibroblast transition associated with COVID-19 pulmonary fibrosis. Our work depicts a comprehensive cellular and molecular atlas of the lungs of patients with COVID-19 and provides insights into SARS-CoV-2-related pulmonary injury, facilitating the identification of biomarkers and development of symptomatic treatments.

Generation and Effect Testing of a SARS-CoV-2 RBD-Targeted Polyclonal Therapeutic Antibody Based on a 2-D Airway Organoid Screening System.

Coronavirus disease 2019 (COVID-19) is an acute respiratory infectious disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The US FDA has approved several therapeutics and vaccines worldwide through the emergency use authorization in response to the rapid spread of COVID-19. Nevertheless, the efficacies of these treatments are being challenged by viral escape mutations. There is an urgent need to develop effective treatments protecting against SARS-CoV-2 infection and to establish a stable effect-screening model to test potential drugs. Polyclonal antibodies (pAbs) have an intrinsic advantage in such developments because they can target rapidly mutating viral strains as a result of the complexity of their binding epitopes. In this study, we generated anti-receptor-binding domain (anti-RBD) pAbs from rabbit serum and tested their safety and efficacy in response to SARS-CoV-2 infection both in vivo and ex vivo. Primary human bronchial epithelial two-dimensional (2-D) organoids were cultured and differentiated to a mature morphology and subsequently employed for SARS-CoV-2 infection and drug screening. The pAbs protected the airway organoids from viral infection and tissue damage. Potential side effects were tested in mouse models for both inhalation and vein injection. The pAbs displayed effective viral neutralization effects without significant side effects. Thus, the use of animal immune serum-derived pAbs might be a potential therapy for protection against SARS-CoV-2 infection, with the strategy developed to produce these pAbs providing new insight into the treatment of respiratory tract infections, especially for infections with viruses undergoing rapid mutation.

SARS-CoV-2 promotes RIPK1 activation to facilitate viral propagation.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the ongoing global pandemic that poses substantial challenges to public health worldwide. A subset of COVID-19 patients experience systemic inflammatory response, known as cytokine storm, which may lead to death. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important mediator of inflammation and cell death. Here, we examined the interaction of RIPK1-mediated innate immunity with SARS-CoV-2 infection. We found evidence of RIPK1 activation in human COVID-19 lung pathological samples, and cultured human lung organoids and ACE2 transgenic mice infected by SARS-CoV-2. Inhibition of RIPK1 using multiple small-molecule inhibitors reduced the viral load of SARS-CoV-2 in human lung organoids. Furthermore, therapeutic dosing of the RIPK1 inhibitor Nec-1s reduced mortality and lung viral load, and blocked the CNS manifestation of SARS-CoV-2 in ACE2 transgenic mice. Mechanistically, we found that the RNA-dependent RNA polymerase of SARS-CoV-2, NSP12, a highly conserved central component of coronaviral replication and transcription machinery, promoted the activation of RIPK1. Furthermore, NSP12 323L variant, encoded by the SARS-CoV-2 C14408T variant first detected in Lombardy, Italy, that carries a Pro323Leu amino acid substitution in NSP12, showed increased ability to activate RIPK1. Inhibition of RIPK1 downregulated the transcriptional induction of proinflammatory cytokines and host factors including ACE2 and EGFR that promote viral entry into cells. Our results suggest that SARS-CoV-2 may have an unexpected and unusual ability to hijack the RIPK1-mediated host defense response to promote its own propagation and that inhibition of RIPK1 may provide a therapeutic option for the treatment of COVID-19.

Evidence of SARS-CoV-2 Transmission Through the Ocular Route.

PURPOSE: Currently, the coronavirus disease 2019 (COVID-19) pandemic is raging around the world. However, the transmission of its pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is not fully clear. It is still controversial whether the ocular transmission of SARS-CoV-2 exists. This review aimed to summarize the evidence of SARS-CoV-2 ocular transmission. METHODS: Online articles were searched till October 23, 2020 in Pubmed, Embase, and websites of World Health Organization, Centers for Disease Control and Prevention COVID-19, American Academy of Ophthalmology, and American Society of Cataract and Refractive Surgery under the search strategy of (((("COVID-19"[Mesh]) OR ("SARS-CoV-2"[Mesh])) OR (2019 novel coronavirus)) OR (2019-nCoV)) AND (((("Conjunctivitis"[Mesh]) OR (Ocular Surface)) OR ("Eye"[Mesh])) OR ("Ophthalmology"[Mesh])). The language was not restricted. After screening, 1445 records were excluded and 168 references original articles were finally included. RESULTS: Cells of ocular surface express both the receptor of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), offering molecular bases for the ocular susceptibility to SARS-CoV-2. Accumulated COVID-19 patients presented conjunctivitis as the initial or the only symptom. Whether COVID-19 patients had ocular symptoms or not, SARS-CoV-2 was detectable on the ocular surface, and the isolated virus was infectious, proving that the ocular surface can not only be a reservoir but also a source of contagion. SARS-CoV-2 may reach the ocular surface by hand-eye contact and aerosols. Once SARS-CoV-2 reaches the ocular surface, it may transfer to other systems through the nasolacrimal system or hematogenous metastasis. CONCLUSION: The ocular surface can serve as a reservoir and source of contagion for SARS-CoV-2. SARS-CoV-2 can be transmitted to the ocular surface through hand-eye contact and aerosols, and then transfer to other systems through nasolacrimal route and hematogenous metastasis. The possibility of ocular transmission of SARS-CoV-2 cannot be ignored.

Single-cell transcriptomic atlas of primate cardiopulmonary aging.

Aging is a major risk factor for many diseases, especially in highly prevalent cardiopulmonary comorbidities and infectious diseases including Coronavirus Disease 2019 (COVID-19). Resolving cellular and molecular mechanisms associated with aging in higher mammals is therefore urgently needed. Here, we created young and old non-human primate single-nucleus/cell transcriptomic atlases of lung, heart and artery, the top tissues targeted by SARS-CoV-2. Analysis of cell type-specific aging-associated transcriptional changes revealed increased systemic inflammation and compromised virus defense as a hallmark of cardiopulmonary aging. With age, expression of the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) was increased in the pulmonary alveolar epithelial barrier, cardiomyocytes, and vascular endothelial cells. We found that interleukin 7 (IL7) accumulated in aged cardiopulmonary tissues and induced ACE2 expression in human vascular endothelial cells in an NF-κB-dependent manner. Furthermore, treatment with vitamin C blocked IL7-induced ACE2 expression. Altogether, our findings depict the first transcriptomic atlas of the aged primate cardiopulmonary system and provide vital insights into age-linked susceptibility to SARS-CoV-2, suggesting that geroprotective strategies may reduce COVID-19 severity in the elderly.

A human circulating immune cell landscape in aging and COVID-19.

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.

Imaging and clinical features of patients with 2019 novel coronavirus SARS-CoV-2.

BACKGROUND: The pneumonia caused by the 2019 novel coronavirus (SARS-CoV-2, also called 2019-nCoV) recently break out in Wuhan, China, and was named as COVID-19. With the spread of the disease, similar cases have also been confirmed in other regions of China. We aimed to report the imaging and clinical characteristics of these patients infected with SARS-CoV-2 in Guangzhou, China. METHODS: All patients with laboratory-identified SARS-CoV-2 infection by real-time polymerase chain reaction (PCR) were collected between January 23, 2020, and February 4, 2020, in a designated hospital (Guangzhou Eighth People's Hospital). This analysis included 90 patients (39 men and 51 women; median age, 50 years (age range, 18-86 years). All the included SARS-CoV-2-infected patients underwent non-contrast enhanced chest computed tomography (CT). We analyzed the clinical characteristics of the patients, as well as the distribution characteristics, pattern, morphology, and accompanying manifestations of lung lesions. In addition, after 1-6 days (mean 3.5 days), follow-up chest CT images were evaluated to assess radiological evolution. FINDINGS: The majority of infected patients had a history of exposure in Wuhan or to infected patients and mostly presented with fever and cough. More than half of the patients presented bilateral, multifocal lung lesions, with peripheral distribution, and 53 (59%) patients had more than two lobes involved. Of all included patients, COVID-19 pneumonia presented with ground glass opacities in 65 (72%), consolidation in 12 (13%), crazy paving pattern in 11 (12%), interlobular thickening in 33 (37%), adjacent pleura thickening in 50 (56%), and linear opacities combined in 55 (61%). Pleural effusion, pericardial effusion, and lymphadenopathy were uncommon findings. In addition, baseline chest CT did not show any abnormalities in 21 patients (23%), but 3 patients presented bilateral ground glass opacities on the second CT after 3-4 days. CONCLUSION: SARS-CoV-2 infection can be confirmed based on the patient's history, clinical manifestations, imaging characteristics, and laboratory tests. Chest CT examination plays an important role in the initial diagnosis of the novel coronavirus pneumonia. Multiple patchy ground glass opacities in bilateral multiple lobular with periphery distribution are typical chest CT imaging features of the COVID-19 pneumonia.