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COVID-19 pandemic has severely impacted the public health and social economy worldwide. A safe, effective, and affordable vaccine against SARS-CoV-2 infections/diseases is urgently needed. We have been developing a recombinant vaccine based on a prefusion-stabilized spike trimer of SARS-CoV-2 and formulated with aluminium hydroxide and CpG 7909. The spike protein was expressed in Chinese hamster ovary (CHO) cells, purified, and prepared as a stable formulation with the dual adjuvant. Immunogenicity studies showed that candidate vaccines elicited robust neutralizing antibody responses and substantial CD4+ T cell responses in both mice and non-human primates. And vaccine-induced neutralizing antibodies persisted at high level for at least 6 months. Challenge studies demonstrated that candidate vaccine reduced the viral loads and inflammation in the lungs of SARS-CoV-2 infected golden Syrian hamsters significantly. In addition, the vaccine-induced antibodies showed cross-neutralization activity against B.1.1.7 and B.1.351 variants. These data suggest candidate vaccine is efficacious in preventing SARS-CoV-2 infections and associated pneumonia, thereby justifying ongoing phase I/II clinical studies in China (NCT04982068 and NCT04990544).

To curb the pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), multiple platforms have been employed toward a safe and highly effective vaccine. Here, we develop a novel cell-based vaccine candidate, namely K562-S, by utilizing human cell K562 as a cellular carrier to display Spike (S) protein of SARS-CoV-2 on the membrane. Analogous to the traditional inactivated vaccine, K562-S cells can be propagated to a large scale by culturing and completely lose their viability after exposure to X-ray irradiation or formalin. We in turn demonstrated high immunogenicity of formalin-inactivated K562-S vaccine in both mouse and non-human primates and its protective efficacy in mice. In mice, immunization with inactivated K562-S vaccines can elicit potent neutralizing antibody (nAb) responses persisting longer than 5 months. We consequently showed in a hACE2 mouse model of SARS-CoV-2 infection that a two-shot vaccination with adjuvanted K562-S rendered greater than 3 log reduction in viral lung load and concomitant ameliorated lung pathology. Of importance, the administration of the same regimen in non-human primates was able to induce a neutralizing antibody titer averaging three-fold higher relative to human convalescent serum. These results together support the promise of K562-based, S-protein-expressing vaccines as a novel vaccination approach against SARS-CoV-2. Importantly, with a powerful capacity to carry external genes for cell-based vectors, this platform could rapidly generate two- and multiple-valent vaccines by incorporating SARS-CoV-2 mutants, SARS-CoV, or MERS-CoV.

The ongoing SARS-CoV-2 pandemic poses a severe global threat to public health, as do influenza viruses and other coronaviruses. Here, we present chimpanzee adenovirus 68 (AdC68)-based vaccines designed to universally target coronaviruses and influenza. Our design is centered on an immunogen generated by fusing the SARS-CoV-2 receptor-binding domain (RBD) to the conserved stalk of H7N9 hemagglutinin (HA). Remarkably, the constructed vaccine effectively induced both SARS-CoV-2-targeting antibodies and anti-influenza antibodies in mice, consequently affording protection from lethal SARS-CoV-2 and H7N9 challenges as well as effective H3N2 control. We propose our AdC68-vectored coronavirus-influenza vaccine as a universal approach toward curbing respiratory virus-causing pandemics.

Significance SARS-CoV-2 primarily targets the lung and enters the body through ACE2 receptors. However, the expression of ACE2 is extremely low in the airways. In this study, we utilized APEX2 proximity-labeling techniques and identified nonmuscle myosin heavy chain IIA (MYH9) as an ACE2 coreceptor to promote SARS-CoV-2 infection of human lung cells, as well as infection of pan-coronavirus. We demonstrated that MYH9 colocalized with SARS-CoV-2 S primarily at the membrane, which interacts with the S2 subunit and the S1-NTD subunit through its C-terminal domain, and enhances SARS-CoV-2 entry in an ACE2-dependent manner. Our results define MYH9 as a key host factor that facilitates SARS-CoV-2 and pan-coronavirus infection, which may serve as a potential target for future clinical intervention strategies., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), binds to host receptor angiotensin-converting enzyme 2 (ACE2) through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. However, the expression of ACE2 is extremely low in a variety of human tissues, especially in the airways. Thus, other coreceptors and/or cofactors on the surface of host cells may contribute to SARS-CoV-2 infection. Here, we identified nonmuscle myosin heavy chain IIA (MYH9) as an important host factor for SARS-CoV-2 infection of human pulmonary cells by using APEX2 proximity-labeling techniques. Genetic ablation of MYH9 significantly reduced SARS-CoV-2 pseudovirus infection in wild type (WT) A549 and Calu-3 cells, and overexpression of MYH9 enhanced the pseudovirus infection in WT A549 and H1299 cells. MYH9 was colocalized with the SARS-CoV-2 S and directly interacted with SARS-CoV-2 S through the S2 subunit and S1-NTD (N-terminal domain) by its C-terminal domain (designated as PRA). Further experiments suggested that endosomal or myosin inhibitors effectively block the viral entry of SARS-CoV-2 into PRA-A549 cells, while transmembrane protease serine 2 (TMPRSS2) and cathepsin B and L (CatB/L) inhibitors do not, indicating that MYH9 promotes SARS-CoV-2 endocytosis and bypasses TMPRSS2 and CatB/L pathway. Finally, we demonstrated that loss of MYH9 reduces authentic SARS-CoV-2 infection in Calu-3, ACE2-A549, and ACE2-H1299 cells. Together, our results suggest that MYH9 is a candidate host factor for SARS-CoV-2, which mediates the virus entering host cells by endocytosis in an ACE2-dependent manner, and may serve as a potential target for future clinical intervention strategies.

Frequent outbreaks of coronaviruses underscore the need for antivirals and vaccines that can counter a broad range of coronavirus types. We isolated a human antibody named 76E1 from a COVID-19 convalescent patient, and report that it has broad-range neutralizing activity against multiple α- and β-coronaviruses, including the SARS-CoV-2 variants. 76E1 also binds its epitope in peptides from γ- and δ-coronaviruses. 76E1 cross-protects against SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and therapeutic murine animal models. Structural and functional studies revealed that 76E1 targets a unique epitope within the spike protein that comprises the highly conserved S2' site and the fusion peptide. The epitope that 76E1 binds is partially buried in the structure of the SARS-CoV-2 spike trimer in the prefusion state, but is exposed when the spike protein binds to ACE2. This observation suggests that 76E1 binds to the epitope at an intermediate state of the spike trimer during the transition from the prefusion to the postfusion state, thereby blocking membrane fusion and viral entry. We hope that the identification of this crucial epitope, which can be recognized by 76E1, will guide epitope-based design of next-generation pan-coronavirus vaccines and antivirals.

Background The receptor-binding domain (RBD) variants of SARS-CoV-2 could impair antibody-mediated neutralization of the virus by host immunity; thus, prospective surveillance of antibody escape mutants and understanding the evolution of RBD are urgently needed. Methods Using the single B cell cloning technology, we isolated and characterized 93 RBD-specific antibodies from the memory B cells of four COVID-19 convalescent individuals in the early stage of the pandemic. Then, global RBD alanine scanning with a panel of 19 selected neutralizing antibodies (NAbs), including several broadly reactive NAbs, was performed. Furthermore, we assessed the impact of single natural mutation or co-mutations of concern at key positions of RBD on the neutralization escape and ACE2 binding function by recombinant proteins and pseudoviruses. Results Thirty-three amino acid positions within four independent antigenic sites (1 to 4) of RBD were identified as valuable indicators of antigenic changes in the RBD. The comprehensive escape mutation map not only confirms the widely circulating strains carrying important immune escape RBD mutations such as K417N, E484K, and L452R, but also facilitates the discovery of new immune escape-enabling mutations such as F486L, N450K, F490S, and R346S. Of note, these escape mutations could not affect the ACE2 binding affinity of RBD, among which L452R even enhanced binding. Furthermore, we showed that RBD co-mutations K417N, E484K, and N501Y present in B.1.351 appear more resistant to NAbs and human convalescent plasma from the early stage of the pandemic, possibly due to an additive effect. Conversely, double mutations E484Q and L452R present in B.1.617.1 variant show partial antibody evasion with no evidence for an additive effect. Conclusions Our study provides a global view of the determinants for neutralizing antibody recognition, antigenic conservation, and RBD conformation. The in-depth escape maps may have value for prospective surveillance of SARS-CoV-2 immune escape variants. Special attention should be paid to the accumulation of co-mutations at distinct major antigenic sites. Finally, the new broadly reactive NAbs described here represent new potential opportunities for the prevention and treatment of COVID-19.

Background Epidemic outbreaks caused by SARS-CoV-2 are worsening around the world, and there are no target drugs to treat COVID-19. IFN-κ inhibits the replication of SARS-CoV-2; and TFF2 is a small secreted polypeptide that promotes the repair of mucosal injury and reduces the inflammatory responses. We used the synergistic effect of both proteins to treat COVID-19. Methods We conducted an open-label, randomized, clinical trial involving patients with moderate COVID-19. Patients were assigned in a 1:1 ratio to receive either aerosol inhalation treatment with IFN-κ and TFF2 every 24 h for six consecutive dosages in addition to standard care (experimental group) or standard care alone (control group). The primary endpoint was the time until a viral RNA negative conversion for SARS-CoV-2 in all clinical samples. The secondary clinical endpoint was the time of CT imaging improvement. Data analysis was performed per protocol. This study was registered with chictr.org.cn, ChiCTR2000030262. Findings Between March 23 and May 23 of 2020, 86 COVID-19 patients with symptoms of moderate illness were recruited, and 6 patients were excluded due to not matching the inclusion criteria (patients with pneumonia through chest radiography). Among the remaining 80 patients, 40 patients were assigned to experimental group, and the others were assigned to control group to only receive standard care. Efficacy and safety were evaluated for both groups. The time of viral RNA negative conversion in experimental group (Mean, 3·80 days, 95% CI 2·07–5·53), was significantly shorter than that in control group (7·40 days, 95% CI 4·57 to 10·23) (p = 0.031), and difference between means was 3·60 days. The percentage of patients in experimental group with reversion to negative viral RNA was significantly increased compared with control group on all sampling days (every day during the 12-day observation period) (p = 0·037). For the secondary endpoint, the experimental group had a significantly shorter time until improvement was seen by CT (Mean 6·21 days, N = 38/40, 95% CI 5·11–7·31) than that in control group (8·76 days, N = 34/40, 95% CI 7·57–9·96) (p = 0.002), and difference between means was 2·55 days. No discomfort or complications during aerosol inhalation were reported to the nurses by any experimental patients. Interpretation In conclusion, we found that aerosol inhalation of IFN-κ plus TFF2 in combination with standard care is safe and superior to standard care alone in shortening the time up to viral RNA negative conversion in all clinical samples. In addition, the patients in experimental group had a significantly shortened CT imaging improvement time than those in control group. This study suggested that this combination treatment is able to facilitate clinical improvement (negative for virus, improvement by CT, reduced hospitalization stay) and thereby result in an early release from the hospital. These data support the need for exploration with a large-scale trial of IFN-κ plus TFF2 to treat COVID-19. Funding Funding was provided by the National Natural Science Foundation of China, National Major Project for Control and Prevention of Infectious Disease in China, Shanghai Science and Technology Commission, Shanghai Municipal Health Commission.

The spread of SARS-CoV-2 virus in the ongoing global pandemic has led to infections of millions of people and losses of many lives. The rapid, accurate and convenient SARS-CoV-2 virus detection is crucial for controlling and stopping the pandemic. Diagnosis of patients in the early stage infection are so far limited to viral nucleic acid or antigen detection in human nasopharyngeal swab or saliva samples. Here we developed a method for rapid and direct optical measurement of SARS-CoV-2 virus particles in one step nearly without any sample preparation using a spike protein specific nanoplasmonic resonance sensor. As low as 370 vp/mL were detected in one step within 15 min and the virus concentration can be quantified linearly in the range of 0 to 107 vp/mL. Measurements shown on both generic microplate reader and a handheld smartphone connected device suggest that our low-cost and rapid detection method may be adopted quickly under both regular clinical environment and resource-limited settings., Graphical abstract Image 1, Highlights • Rapidly and sensitively detect the whole SARS-CoV-2 viral particles with extraordinary time efficiency (

Summary COVID-19 has become a worldwide threat to humans, and neutralizing antibodies have therapeutic potential. We have purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain), and obtain 729 paired heavy and light chain fragments. Among these, 178 antibodies test positive for antigen binding, and the majority of the top 17 binders with EC50 below 1 nM are RBD binders. Furthermore, we identify 11 neutralizing antibodies, 8 of which show an IC50 within 10 nM, and the best one, 414-1, with IC50 of 1.75 nM. Through epitope mapping, we find 3 main epitopes in RBD recognized by these antibodies, and epitope B antibody 553-15 could substantially enhance the neutralizing abilities of most of the other antibodies. We also find that 515-5 could cross-neutralize the SARS-CoV pseudovirus. Altogether, our study provides 11 potent human neutralizing antibodies for COVID-19 as therapeutic candidates., Graphical Abstract, Highlights 11 neutralizing antibodies against SARS-CoV-2 targeting 3 main epitopes on RBD Epitope A antibody 414-1 shows neutralizing IC50 at 1.75 nM Epitope B antibody 553-15 can enhance the neutralizing abilities of other antibodies One neutralizing antibody, 515-5, can cross-neutralize SARS-CoV pseudovirus, Wan et al. identify 11 potent neutralizing antibodies against COVID-19 from 11 convalescent patients. These human neutralizing antibodies target 3 separate epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and epitope B antibody (553-15) can enhance the neutralizing abilities of most other antibodies.