Infection with wild-type SARS-CoV-2 elicits broadly neutralizing and protective antibodies against omicron subvariants.

The omicron variants of SARS-CoV-2 have substantial ability to escape infection- and vaccine-elicited antibody immunity. Here, we investigated the extent of such escape in nine convalescent patients infected with the wild-type SARS-CoV-2 during the first wave of the pandemic. Among the total of 476 monoclonal antibodies (mAbs) isolated from peripheral memory B cells, we identified seven mAbs with broad neutralizing activity to all variants tested, including various omicron subvariants. Biochemical and structural analysis indicated the majority of these mAbs bound to the receptor-binding domain, mimicked the receptor ACE2 and were able to accommodate or inadvertently improve recognition of omicron substitutions. Passive delivery of representative antibodies protected K18-hACE2 mice from infection with omicron and beta SARS-CoV-2. A deeper understanding of how the memory B cells that produce these antibodies could be selectively boosted or recalled can augment antibody immunity against SARS-CoV-2 variants.

Broadly neutralizing and protective nanobodies against SARS-CoV-2 Omicron subvariants BA.1, BA.2, and BA.4/5 and diverse sarbecoviruses.

As SARS-CoV-2 Omicron and other variants of concern (VOCs) continue spreading worldwide, development of antibodies and vaccines to confer broad and protective activity is a global priority. Here, we report on the identification of a special group of nanobodies from immunized alpaca with potency against diverse VOCs including Omicron subvariants BA.1, BA.2 and BA.4/5, SARS-CoV-1, and major sarbecoviruses. Crystal structure analysis of one representative nanobody, 3-2A2-4, discovers a highly conserved epitope located between the cryptic and the outer face of the receptor binding domain (RBD), distinctive from the receptor ACE2 binding site. Cryo-EM and biochemical evaluation reveal that 3-2A2-4 interferes structural alteration of RBD required for ACE2 binding. Passive delivery of 3-2A2-4 protects K18-hACE2 mice from infection of authentic SARS-CoV-2 Delta and Omicron. Identification of these unique nanobodies will inform the development of next generation antibody therapies and design of pan-sarbecovirus vaccines.

Structural basis of a two-antibody cocktail exhibiting highly potent and broadly neutralizing activities against SARS-CoV-2 variants including diverse Omicron sublineages.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), especially the latest Omicron, have exhibited severe antibody evasion. Broadly neutralizing antibodies with high potency against Omicron are urgently needed for understanding the working mechanisms and developing therapeutic agents. In this study, we characterized the previously reported F61, which was isolated from convalescent patients infected with prototype SARS-CoV-2, as a broadly neutralizing antibody against all VOCs including Omicron BA.1, BA.1.1, BA.2, BA.3 and BA.4 sublineages by utilizing antigen binding and cell infection assays. We also identified and characterized another broadly neutralizing antibody D2 with epitope distinct from that of F61. More importantly, we showed that a combination of F61 with D2 exhibited synergy in neutralization and protecting mice from SARS-CoV-2 Delta and Omicron BA.1 variants. Cryo-Electron Microscopy (Cryo-EM) structures of the spike-F61 and spike-D2 binary complexes revealed the distinct epitopes of F61 and D2 at atomic level and the structural basis for neutralization. Cryo-EM structure of the Omicron-spike-F61-D2 ternary complex provides further structural insights into the synergy between F61 and D2. These results collectively indicated F61 and F61-D2 cocktail as promising therapeutic antibodies for combating SARS-CoV-2 variants including diverse Omicron sublineages.

Deep learning guided optimization of human antibody against SARS-CoV-2 variants with broad neutralization.

SignificanceSARS-CoV-2 continues to evolve through emerging variants, more frequently observed with higher transmissibility. Despite the wide application of vaccines and antibodies, the selection pressure on the Spike protein may lead to further evolution of variants that include mutations that can evade immune response. To catch up with the virus's evolution, we introduced a deep learning approach to redesign the complementarity-determining regions (CDRs) to target multiple virus variants and obtained an antibody that broadly neutralizes SARS-CoV-2 variants.

A Potent and Protective Human Neutralizing Antibody Against SARS-CoV-2 Variants.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge and spread around the world, antibodies and vaccines to confer broad and potent neutralizing activity are urgently needed. Through the isolation and characterization of monoclonal antibodies (mAbs) from individuals infected with SARS-CoV-2, we identified one antibody, P36-5D2, capable of neutralizing the major SARS-CoV-2 variants of concern. Crystal and electron cryo-microscopy (cryo-EM) structure analyses revealed that P36-5D2 targeted to a conserved epitope on the receptor-binding domain of the spike protein, withstanding the three key mutations-K417N, E484K, and N501Y-found in the variants that are responsible for escape from many potent neutralizing mAbs, including some already approved for emergency use authorization (EUA). A single intraperitoneal (IP) injection of P36-5D2 as a prophylactic treatment completely protected animals from challenge of infectious SARS-CoV-2 Alpha and Beta. Treated animals manifested normal body weight and were devoid of infection-associated death up to 14 days. A substantial decrease of the infectious virus in the lungs and brain, as well as reduced lung pathology, was found in these animals compared to the controls. Thus, P36-5D2 represents a new and desirable human antibody against the current and emerging SARS-CoV-2 variants.

Antibody neutralization of SARS-CoV-2 through ACE2 receptor mimicry.

Understanding the mechanism for antibody neutralization of SARS-CoV-2 is critical for the development of effective therapeutics and vaccines. We recently isolated a large number of monoclonal antibodies from SARS-CoV-2 infected individuals. Here we select the top three most potent yet variable neutralizing antibodies for in-depth structural and functional analyses. Crystal structural comparisons reveal differences in the angles of approach to the receptor binding domain (RBD), the size of the buried surface areas, and the key binding residues on the RBD of the viral spike glycoprotein. One antibody, P2C-1F11, most closely mimics binding of receptor ACE2, displays the most potent neutralizing activity in vitro and conferred strong protection against SARS-CoV-2 infection in Ad5-hACE2-sensitized mice. It also occupies the largest binding surface and demonstrates the highest binding affinity to RBD. More interestingly, P2C-1F11 triggers rapid and extensive shedding of S1 from the cell-surface expressed spike glycoprotein, with only minimal such effect by the remaining two antibodies. These results offer a structural and functional basis for potent neutralization via disruption of the very first and critical steps for SARS-CoV-2 cell entry.

Potent and protective IGHV3-53/3-66 public antibodies and their shared escape mutant on the spike of SARS-CoV-2.

Neutralizing antibodies (nAbs) to SARS-CoV-2 hold powerful potentials for clinical interventions against COVID-19 disease. However, their common genetic and biologic features remain elusive. Here we interrogate a total of 165 antibodies from eight COVID-19 patients, and find that potent nAbs from different patients have disproportionally high representation of IGHV3-53/3-66 usage, and therefore termed as public antibodies. Crystal structural comparison of these antibodies reveals they share similar angle of approach to RBD, overlap in buried surface and binding residues on RBD, and have substantial spatial clash with receptor angiotensin-converting enzyme-2 (ACE2) in binding to RBD. Site-directed mutagenesis confirms these common binding features although some minor differences are found. One representative antibody, P5A-3C8, demonstrates extraordinarily protective efficacy in a golden Syrian hamster model against SARS-CoV-2 infection. However, virus escape analysis identifies a single natural mutation in RBD, namely K417N found in B.1.351 variant from South Africa, abolished the neutralizing activity of these public antibodies. The discovery of public antibodies and shared escape mutation highlight the intricate relationship between antibody response and SARS-CoV-2, and provide critical reference for the development of antibody and vaccine strategies to overcome the antigenic variation of SARS-CoV-2.

A Highly Automated Mobile Laboratory for On-site Molecular Diagnostics in the COVID-19 Pandemic.

BACKGROUND: Infectious disease outbreaks such as the COVID-19 (coronavirus disease 2019) pandemic call for rapid response and complete screening of the suspected community population to identify potential carriers of pathogens. Central laboratories rely on time-consuming sample collection methods that are rarely available in resource-limited settings. METHODS: We present a highly automated and fully integrated mobile laboratory for fast deployment in response to infectious disease outbreaks. The mobile laboratory was equipped with a 6-axis robot arm for automated oropharyngeal swab specimen collection; virus in the collected specimen was inactivated rapidly using an infrared heating module. Nucleic acid extraction and nested isothermal amplification were performed by a "sample in, answer out" laboratory-on-a-chip system, and the result was automatically reported by the onboard information platform. Each module was evaluated using pseudovirus or clinical samples. RESULTS: The mobile laboratory was stand-alone and self-sustaining and capable of on-site specimen collection, inactivation, analysis, and reporting. The automated sampling robot arm achieved sampling efficiency comparable to manual collection. The collected samples were inactivated in as short as 12 min with efficiency comparable to a water bath without damage to nucleic acid integrity. The limit of detection of the integrated microfluidic nucleic acid analyzer reached 150 copies/mL within 45 min. Clinical evaluation of the onboard microfluidic nucleic acid analyzer demonstrated good consistency with reverse transcription quantitative PCR with a κ coefficient of 0.979. CONCLUSIONS: The mobile laboratory provides a promising solution for fast deployment of medical diagnostic resources at critical junctions of infectious disease outbreaks and facilitates local containment of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) transmission.

Bat and pangolin coronavirus spike glycoprotein structures provide insights into SARS-CoV-2 evolution.

In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the "down" conformation, indicating they are more prone to adopt the receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. We further identified critical residues in the RBD underlying different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes. These results collectively indicate that tight RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.

Robust SARS-CoV-2 infection in nasal turbinates after treatment with systemic neutralizing antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a burst in the upper respiratory portal for high transmissibility. To determine human neutralizing antibodies (HuNAbs) for entry protection, we tested three potent HuNAbs (IC50 range, 0.0007-0.35 µg/mL) against live SARS-CoV-2 infection in the golden Syrian hamster model. These HuNAbs inhibit SARS-CoV-2 infection by competing with human angiotensin converting enzyme-2 for binding to the viral receptor binding domain (RBD). Prophylactic intraperitoneal or intranasal injection of individual HuNAb or DNA vaccination significantly reduces infection in the lungs but not in the nasal turbinates of hamsters intranasally challenged with SARS-CoV-2. Although postchallenge HuNAb therapy suppresses viral loads and lung damage, robust infection is observed in nasal turbinates treated within 1-3 days. Our findings demonstrate that systemic HuNAb suppresses SARS-CoV-2 replication and injury in lungs; however, robust viral infection in nasal turbinate may outcompete the antibody with significant implications to subprotection, reinfection, and vaccine.

Human neutralizing antibodies elicited by SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global health emergency that is in urgent need of intervention1-3. The entry of SARS-CoV-2 into its target cells depends on binding between the receptor-binding domain (RBD) of the viral spike protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2)2,4-6. Here we report the isolation and characterization of 206 RBD-specific monoclonal antibodies derived from single B cells from 8 individuals infected with SARS-CoV-2. We identified antibodies that potently neutralize SARS-CoV-2; this activity correlates with competition with ACE2 for binding to RBD. Unexpectedly, the anti-SARS-CoV-2 antibodies and the infected plasma did not cross-react with the RBDs of SARS-CoV or Middle East respiratory syndrome-related coronavirus (MERS-CoV), although there was substantial plasma cross-reactivity to their trimeric spike proteins. Analysis of the crystal structure of RBD-bound antibody revealed that steric hindrance inhibits viral engagement with ACE2, thereby blocking viral entry. These findings suggest that anti-RBD antibodies are largely viral-species-specific inhibitors. The antibodies identified here may be candidates for development of clinical interventions against SARS-CoV-2.

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.

A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from December 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an atomic level, we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural analysis identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analysed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.