Inhibition of SARS-CoV-2-mediated thromboinflammation by CLEC2.Fc.

Thromboinflammation is the major cause of morbidity and mortality in COVID-19 patients, and post-mortem examination demonstrates the presence of platelet-rich thrombi and microangiopathy in visceral organs. Moreover, persistent microclots were detected in both acute COVID-19 and long COVID plasma samples. However, the molecular mechanism of SARS-CoV-2-induced thromboinflammation is still unclear. We found that the spleen tyrosine kinase (Syk)-coupled C-type lectin member 2 (CLEC2), which was highly expressed in platelets and alveolar macrophages, interacted with the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (SARS-CoV-2 RBD) directly. Unlike the thread-like NETs, SARS-CoV-2-induced aggregated NET formation in the presence of wild-type (WT), but not CLEC2-deficient platelets. Furthermore, SARS-CoV-2 spike pseudotyped lentivirus was able to induce NET formation via CLEC2, indicating SARS-CoV-2 RBD engaged CLEC2 to activate platelets to enhance NET formation. Administration of CLEC2.Fc inhibited SARS-CoV-2-induced NET formation and thromboinflammation in AAV-ACE2-infected mice. Thus, CLEC2 is a novel pattern recognition receptor for SARS-CoV-2, and CLEC2.Fc and may become a promising therapeutic agent to inhibit SARS-CoV-2-induced thromboinflammation and reduced the risk of post-acute sequelae of COVID-19 (PASC) in the future.

Seroprevalence of COVID-19 in Taiwan revealed by testing anti-SARS-CoV-2 serological antibodies on 14,765 hospital patients.

BACKGROUND: Coronavirus Disease 2019 (COVID-19) has become a worldwide pandemic and affected more than 227 countries or territories, resulting in more than 25 million cases with over 0•85 million deaths, as of September 2, 2020. Taiwan has been successful in countering the COVID-19 outbreak, however, the potential risk for asymptomatic infections and the prevalence rates remain unknown. We aimed to estimate the seroprevalence of COVID-19 in Taiwan via serologically testing hospital patients with neither symptoms indicative of nor positive nucleic acid test for SARS-CoV-2 infection. METHODS: Residual specimens from laboratory blood tests for outpatient and emergency department patients visiting a medical centre in Taipei, Taiwan, within one week in May and another in July, 2020, were collected. We used Elecsys Anti-SARS-CoV-2 Assay to screen and further validated cases with high cutoff index by a confirmatory ELISA assay. We also analysed antibody responses against SARS-CoV-2 along disease progression in four nucleic acid test confirmed COVID-19 patients. FINDINGS: Blood samples from a total of 14,765 patients were tested. The unweighted seroprevalence of anti-SARS-CoV-2 antibodies was 0•07% [95% CI, 0•04%-0•13%]; after weighting with the population demographics of Taiwan, the estimated overall seroprevalence was 0•05% [95% CI, 0•02%-0•10%]. Furthermore, based on data of the four COVID-19 cases, the seroconversion dates for IgM were as early as 9 days and that for IgG 11 days after symptoms onset. INTERPRETATION: We screened the anti-SARS-CoV-2 antibodies in a small-scale population-based study and observed an approximately 0•05% seroprevalence of COVID-19, indicating that the current containment protocols emphasising mask wearing, hand washing, social distancing and mandatory quarantine for all incomers are effective in Taiwan. FUNDING: Taipei Veterans General Hospital, Taipei, Taiwan.

CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation.

BACKGROUND: Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We incubated SARS-CoV-2 with neutrophils in the presence or absence of platelets to observe NET formation. We further isolated extracellular vesicles from COVID-19 patients' sera (COVID-19-EVs) to examine their ability to induce NET formation. RESULTS: We demonstrated that antagonistic mAbs against anti-CLEC5A mAb and anti-TLR2 mAb can inhibit COVID-19-EVs-induced NET formation, and generated clec5a-/-/tlr2-/- mice to confirm the critical roles of CLEC5A and TLR2 in SARS-CoV-2-induced lung inflammation in vivo. We found that virus-free extracellular COVID-19 EVs induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A) and TLR2. Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation was attenuated dramatically in clec5a-/-/tlr2-/- mice. CONCLUSIONS: This study demonstrates that SARS-CoV-2-activated platelets produce EVs to enhance thromboinflammation via CLEC5A and TLR2, and highlight the importance of CLEC5A and TLR2 as therapeutic targets to reduce the risk of ARDS in COVID-19 patients.

Rapid generation of mouse model for emerging infectious disease with the case of severe COVID-19.

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.

C-type lectins and extracellular vesicles in virus-induced NETosis.

Dysregulated formation of neutrophil extracellular traps (NETs) is observed in acute viral infections. Moreover, NETs contribute to the pathogenesis of acute viral infections, including those caused by the dengue virus (DV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Furthermore, excessive NET formation (NETosis) is associated with disease severity in patients suffering from SARS-CoV-2-induced multiple organ injuries. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and other members of C-type lectin family (L-SIGN, LSECtin, CLEC10A) have been reported to interact with viral glycans to facilitate virus spreading and exacerbates inflammatory reactions. Moreover, spleen tyrosine kinase (Syk)-coupled C-type lectin member 5A (CLEC5A) has been shown as the pattern recognition receptor for members of flaviviruses, and is responsible for DV-induced cytokine storm and Japanese encephalomyelitis virus (JEV)-induced neuronal inflammation. Moreover, DV activates platelets via CLEC2 to release extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs). The DV-activated EXOs (DV-EXOs) and MVs (DV-MVs) stimulate CLEC5A and Toll-like receptor 2 (TLR2), respectively, to enhance NET formation and inflammatory reactions. Thus, EVs from virus-activated platelets (PLT-EVs) are potent endogenous danger signals, and blockade of C-type lectins is a promising strategy to attenuate virus-induced NETosis and intravascular coagulopathy.

Genetic variants that influence SARS-CoV-2 receptor TMPRSS2 expression among population cohorts from multiple continents.

The World Health Organization recently announced that pandemic status has been achieved for coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Exponential increases in patient numbers have been reported around the world, along with proportional increases in the number of COVID-19-related deaths. The SARS-CoV-2 infection rate in a population is expected to be influenced by social practices, availability of vaccines or prophylactics, and the prevalence of susceptibility genes in the population. Previous work revealed that cellular uptake of SARS-CoV-2 requires Angiotensin Converting Enzyme 2 (ACE-2) and a cellular protease. The spike (S) protein on SARS-CoV-2 binds ACE-2, which functions as an entry receptor. Following receptor binding, transmembrane protease serine 2 (encoded by TMPRSS2) primes the S protein to allow cellular uptake. Therefore, individual expression of TMPRSS2 may be a crucial determinant of SARS-CoV-2 infection susceptibility. Here, we utilized multiple large genome databases, including the GTEx portal, SNP nexus, and Ensembl genome project, to identify gene expression profiles for TMPRSS2 and its important expression quantitative trait loci. Our results show that four variants (rs464397, rs469390, rs2070788 and rs383510) affect expression of TMPRSS2 in lung tissue. The allele frequency of each variant was then assessed in regional populations, including African, American, European, and three Asian cohorts (China, Japan and Taiwan). Interestingly, our data shows that TMPRSS2-upregulating variants are at higher frequencies in European and American populations than in the Asian populations, which implies that these populations might be relatively susceptible to SARS-CoV-2 infection.

Immunologic aspects of characteristics, diagnosis, and treatment of coronavirus disease 2019 (COVID-19).

On March 11, 2020, the World Health Organization declared the worldwide spread of the infectious disease COVID-19, caused by a new strain of coronavirus, SARS-CoV-2, as a pandemic. Like in all other infectious diseases, the host immune system plays a key role in our defense against SARS-CoV-2 infection. However, viruses are able to evade the immune attack and proliferate and, in susceptible individuals, cause severe inflammatory response known as cytokine storm, particularly in the lungs. The advancement in our understanding of the mechanisms underlying the host immune responses promises to facilitate the development of approaches for prevention or treatment of diseases. Components of immune system, such as antibodies, can also be used to develop sensitive and specific diagnostic methods as well as novel therapeutic agents. In this review, we summarize our knowledge about how the host mounts immune responses to infection by SARS-CoV-2. We also describe the diagnostic methods being used for COVID-19 identification and summarize the current status of various therapeutic strategies, including vaccination, being considered for treatment of the disease.