The recent outbreak of coronavirus disease caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241-and 149-fold more potent than the original EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examined, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.

The newly identified 2019 novel coronavirus (2019-nCoV) has caused more than 11,900 laboratory-confirmed human infections, including 259 deaths, posing a serious threat to human health. Currently, however, there is no specific antiviral treatment or vaccine. Considering the relatively high identity of receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. Here, we report for the first time that a SARS-CoV-specific human monoclonal antibody, CR3022, could bind potently with 2019-nCoV RBD (KD of 6.3 nM). The epitope of CR3022 does not overlap with the ACE2 binding site within 2019-nCoV RBD. These results suggest that CR3022 may have the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-nCoV infections. Interestingly, some of the most potent SARS-CoV-specific neutralizing antibodies (e.g. m396, CR3014) that target the ACE2 binding site of SARS-CoV failed to bind 2019-nCoV spike protein, implying that the difference in the RBD of SARS-CoV and 2019-nCoV has a critical impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD.

Background 24The COVID-19 pandemic caused by SARS-CoV-2 coronavirus threatens global public 25 health. Currently, neutralizing antibodies (NAbs) versus this virus are expected to 26 correlate with recovery and protection of this disease. However, the characteristics of 27 these antibodies have not been well studied in association with the clinical 28 manifestations in patients. 30 Methods 31Plasma collected from 175 COVID-19 recovered patients with mild symptoms were 32 screened using a safe and sensitive pseudotyped-lentiviral-vector-based neutralization 33 assay. Spike-binding antibody in plasma were determined by ELISA using RBD, S1, 34 and S2 proteins of SARS-CoV-2. The levels and the time course of SARS-CoV-2-35 specific NAbs and the spike-binding antibodies were monitored at the same time. 36 37 Findings 38 SARS-CoV-2 NAbs were unable to cross-reactive with SARS-CoV virus. SARS-CoV-39 2-specific NAbs were detected in patients from day 10-15 after the onset of the disease 40 and remained thereafter. The titers of NAb among these patients correlated with the 41 spike-binding antibodies targeting S1, RBD, and S2 regions. The titers of NAbs were 42 variable in different patients. Elderly and middle-age patients had significantly higher 43 plasma NAb titers (P<0.0001) and spike-binding antibodies (P=0.0003) than young 44 patients. Notably, among these patients, there were ten patients whose NAb titers were 45 under the detectable level of our assay (ID50: < 40); while in contrast, two patients, 46 showed very high titers of NAb, with ID50 :15989 and 21567 respectively. The NAb 47 titers were positive correlated with plasma CRP levels but negative correlated with the 48 lymphocyte counts of patients at the time of admission, indicating an association 49 between humoral response and cellular immune response.50 51 Interpretation 52The variations of SARS-CoV-2 specific NAbs in recovered COVID-19 patients may 53 raise the concern about the role of NAbs on disease progression. The correlation of 54 NAb titers with age, lymphocyte counts, and blood CRP levels suggested that the 55 interplay between virus and host immune response in coronavirus infections should be 56 further explored for the development of effective vaccine against SARS-CoV-2 virus. 57 Furthermore, titration of NAb is helpful prior to the use of convalescent plasma for 58 prevention or treatment. Sciences 64 65 66 67 All rights reserved. No reuse allowed without permission.

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Continuously emerging highly pathogenic human coronaviruses (HCoVs) remain a major threat to human health, as illustrated in past SARS-CoV and MERS-CoV outbreaks. The development of a drug with broad-spectrum HCoV inhibitory activity would address this urgent unmet medical need. Although previous studies have suggested that the HR1 of HCoV spike (S) protein is an important target site for inhibition against specific HCoVs, whether this conserved region could serve as a target for the development of broad-spectrum pan-CoV inhibitor remains controversial. Here, we found that peptide OC43-HR2P, derived from the HR2 domain of HCoV-OC43, exhibited broad fusion inhibitory activity against multiple HCoVs. EK1, the optimized form of OC43-HR2P, showed substantially improved pan-CoV fusion inhibitory activity and pharmaceutical properties. Crystal structures indicated that EK1 can form a stable six-helix bundle structure with both short α-HCoV and long β-HCoV HR1s, further supporting the role of HR1 region as a viable pan-CoV target site.