a new coronavirus emerged in China and caused an acute respiratory disease now known as coronavirus disease 2019 (COVID-19) (1). The virus was identified to be a betacoronavirus related to severe acute respiratory syndrome coronavirus (SARS-CoV) and thus was named SARS-CoV-2 (2). In <2 decades, this virus is the third known coronavirus to cross the species barrier and cause severe respiratory infections in humans after SARS-CoV in 2003 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, yet with unprecedented spread compared with the earlier 2 viruses.Because of the rapid increase in number of cases and uncontrolled and vast spread worldwide, the World Health Organization has declared SARS-CoV-2 a pandemic. As of March 14, 2020, the virus had infected >130,000 persons in 122 countries, 3.7% of whom had died. (3). Rapid identification of the etiology and sharing of the genetic sequence of the virus, followed by international collaborative efforts initiated because of emergence of SARS-CoV-2, has led to rapid availability of real-time PCR diagnostic assays that support case ascertainment and tracking of the outbreak (4). Availability of these assays has helped in patient detection and efforts to contain the virus. However, validated serologic assays are still lacking and are urgently needed.Validated serologic assays are crucial for patient contact tracing, identifying the viral reservoir hosts, and epidemiologic studies. Epidemiologic studies are urgently needed to help uncover the burden of disease, in particular the rate of asymptomatic infections, and to get better estimates on illness and death. In ad-

The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV) in cell culture. This cross-neutralizing antibody targets a communal epitope on these viruses and may offer potential for prevention and treatment of COVID-19.

Coronaviruses cause respiratory tract infections in humans and outbreaks of deadly pneumonia worldwide. Infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host receptors and fuses the viral and cellular membranes. To understand the molecular basis of coronavirus attachment to oligosaccharide receptors, we determined cryo-EM structures of coronavirus OC43 S glycoprotein trimer in isolation and in complex with a 9-O-acetylated sialic acid. We show that the ligand binds with fast kinetics to a surface-exposed groove and that interactions at the identified site are essential for S-mediated viral entry into host cells, but free monosaccharide does not trigger fusogenic conformational changes. The receptor-interacting site is conserved in all coronavirus S glycoproteins that engage 9-O-acetyl-sialogycans, with an architecture similar to those of the ligand-binding pockets of coronavirus hemagglutinin esterases and influenza virus C/D hemagglutinin-esterase fusion glycoproteins. Our results demonstrate these viruses evolved similar strategies to engage sialoglycans at the surface of target cells.

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.

The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.

our coronaviruses mainly associated with common cold-like symptoms are endemic in humans, namely OC43, HKU1, NL63 and 229E, while three highly pathogenic zoonotic coronaviruses have emerged in the past two decades, leading to epidemics and a pandemic. Severe acute respiratory syndrome coronavirus (SARS-CoV) was discovered in Guangdong Province in China in 2002 and spread to five continents through air travel routes, infecting 8,098 people and causing 774 deaths. No cases were reported after 2004 1,2 . In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in the Arabian Peninsula, where it still circulates. It was exported to 27 countries, infecting a total of 2,494 individuals and claiming 858 lives as of January 2020 according to the World Health Organization 3 . A recent study further suggested that undetected zoonotic MERS-CoV transmissions are currently occurring in Africa 4 . A novel coronavirus, named SARS-CoV-2, was associated with an outbreak of severe pneumonia in Hubei Province, China, at the end of 2019 and has since infected over 121 million people and claimed more than 2.6 million lives worldwide during the ongoing COVID-19 pandemic 5,6 .SARS-CoV and SARS-CoV-2 probably originated in bats 5,7-10 , with masked palm civets and racoon dogs acting as intermediate amplifying and transmitting hosts for SARS-CoV [11][12][13] . Although MERS-CoV was also suggested to have originated in bats, repeated zoonotic transmissions occurred from dromedary camels 14,15 . The identification of numerous coronaviruses in bats, including viruses related to SARS-CoV-2, SARS-CoV and MERS-CoV, along with evidence of spillovers of SARS-CoV-like viruses to humans, strongly indicates that future coronavirus emergence events will continue to occur 5,[7][8][9][10][16][17][18][19][20] .The coronavirus spike (S) glycoprotein mediates entry into host cells and comprises two functional subunits mediating attachment to host receptors (S 1 subunit) and membrane fusion (S 2 subunit) [21][22][23][24][25][26][27] . As the S homotrimer is prominently exposed at the viral surface and is the main target of neutralizing antibodies (Abs), it is a focus of therapeutic and vaccine design efforts 28 . We previously showed that the SARS-CoV-2 receptor-binding domain (RBD, part of the S 1 subunit) is immunodominant, comprises multiple distinct antigenic sites, and is the target of 90% of the neutralizing activity present in COVID-19 convalescent plasma 29 . Accordingly, monoclonal Abs (mAbs) with potent neutralizing activity have been identified against the SARS-CoV-2, SARS-CoV and MERS-CoV RBDs and shown to protect against viral challenge in vivo [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] . The isolation of S309 from an individual recovered from SARS-CoV, which neutralizes SARS-CoV-2 and SARS-CoV through recognition of a conserved RBD epitope, demonstrated that potent neutralizing mAbs could inhibit β-coronaviruses belonging to different lineage B (sarbecovirus) clades 31 . An optimized version of S...

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect many animal species, including minks, cats, and dogs. To gain insights into SARS-CoV-2 infections in cats and dogs, we developed and validated a set of serologic assays, including ELISA and virus neutralization. Evaluation of samples from animals before they acquired coronavirus disease and samples from cats roaming SARS-CoV-2–positive mink farms confirmed the suitability of these assays for specific antibody detection. Furthermore, our findings exclude SARS-CoV-2 nucleocapsid protein as an antigen for serologic screening of cat and dog samples. We analyzed 500 serum samples from domestic cats and dogs in the Netherlands during April–May 2020. We showed 0.4% of cats and 0.2% of dogs were seropositive. Although seroprevalence in cats and dogs that had unknown SARS-CoV-2 exposure was low during the first coronavirus disease wave, our data stress the need for development of continuous serosurveillance for SARS-CoV-2 in these 2 animal species.

Globally accessible preventive and therapeutic molecules against SARS-CoV-2 are urgently needed. DARPin molecules are an emerging class of novel therapeutics based on naturally occurring repeat proteins (∼15 kDa in size) and can be rapidly produced in bacteria in large quantities. Here, we report the identification of 380 DARPin molecules specifically targeting the SARS-CoV-2 spike protein selected from a naïve library of 1012 DARPin molecules. Using extensive biophysical and biochemical characterization, (pseudo)virus neutralization assays and cryo-EM analysis, 11 mono-DARPin molecules targeting either the receptor binding domain (RBD), the S1 N-terminal-domain (NTD) or the S2 domain of the SARS-CoV-2 spike protein were chosen. Based on these 11 mono-DARPin molecules, 31 anti-SARS-CoV-2 multi-DARPin molecules were constructed which can broadly be grouped into 2 types; multi-paratopic RBD-neutralizing DARPin molecules and multi-mode DARPin molecules targeting simultaneously RBD, NTD and the S2 domain. Each of these multi-DARPin molecules acts by binding with 3 DARPin modules to the SARS-CoV-2 spike protein, leading to potent inhibition of SARS-CoV-2 infection down to 1 ng/ml (12 pM) and potentially providing protection against viral escape mutations. Additionally, 2 DARPin modules binding serum albumin, conferring an expected half-life of about 3 weeks in humans, were included in the multi-DARPin molecules. The protective efficacy of one multi-DARPin molecule was studied in a Golden Syrian hamster SARS-CoV-2 infection model, resulting in a significant reduction in viral load and pathogenesis. In conclusion, the multi-DARPin molecules reported here display very high antiviral potency, high-production yield, and a long systemic half-life, and thereby have the potential for single-dose use for prevention and treatment of COVID-19.