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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 world 1 – 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 receptor 4 . 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 coronaviruses 1 – 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. High-resolution crystal structures of the receptor-binding domain of the spike protein of SARS-CoV-2 and SARS-CoV in complex with ACE2 provide insights into the binding mode of these coronaviruses and highlight essential ACE2-interacting residues.

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. Here, the authors compare the crystal structures and investigate the neutralization mechanisms of three neutralizing antibodies against SARS-CoV-2 and find that one antibody, P2C-1F11, closely mimics binding of receptor ACE2 and displays the most potent neutralizing activity in vitro, as well as conferring protection against SARS-CoV-2 infection in Ad5-hACE2-sensitized mice.

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. Here, the authors combine structural, binding, mutational in vitro and in vivo assays to characterize neutralizing antibodies derived from IGHV3-53/3-66 against SARS-CoV-2, finding one antibody, named P5A-3C8, to exhibit protective efficacy in a golden Syrian hamster model of infection while showing the emergence of mutations at position 417 of the Spike protein that confer resistance.

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. The authors identify nanobodies from immunized alpaca with broadly neutralizing activity against SARS-CoV-1, SARS-CoV-2 variants, and major sarbecoviruses. One representative nanobody binds to a highly conserved epitope on RBD and protects K18-hACE2 mice from Omicron and Delta infection.

The global outbreak of monkeypox virus (MPXV), combined with the termination of smallpox vaccination and the lack of specific antiviral treatments, raises increasing concerns. The surface proteins M1R and B6R of MPXV are crucial for virus transmission and serve as key targets for vaccine development. In this study, a panel of human antibodies targeting M1R and B6R is isolated from a human antibody library using phage display technology. Among these antibodies, A138 against M1R and B026 against B6R show the most potent broad-spectrum neutralizing activities against MPXV and Vaccinia virus (VACV). When used in combination, A138 and B026 exhibit complementary neutralizing activity against both viruses in vitro. X-ray crystallography reveales that A138 binds to the loop regions of M1R, similar to the vulnerable epitope of 7D11 on VACV L1R. By contrast, A129 targets a more cryptic epitope, primarily comprising the β-strands of M1R. Moreover, prophylactic and therapeutic administration of A138 or B026 alone provides partial protection, while combining these two antibodies results in enhanced protection against VACV in male C57BL/6 mice. This study demonstrates of a dual-targeting strategy using two different components of the virion for the prevention and treatment of MPXV infection. This study identifies monoclonal antibodies A138 and B026 targeting MPXV proteins M1R and B6R. Both antibodies exhibit neutralization of MPXV and VACV, and their combination enhances protective efficacy in mice, supporting a dual-targeting strategy.

Highly pathogenic human coronaviruses (CoV) including SARS‐CoV, MERS‐CoV and SARS‐CoV‐2 have emerged over the past two decades, resulting in infectious disease outbreaks that have greatly affected public health. The CoV surface spike (S) glycoprotein mediates receptor binding and membrane fusion for cell entry, playing critical roles in CoV infection and evolution. The S glycoprotein is also the major target molecule for prophylactic and therapeutic interventions, including neutralizing antibodies and vaccines. In this review, we summarize key studies that have revealed the structural basis of S‐mediated cell entry of SARS‐CoV, MERS‐CoV and SARS‐CoV‐2. Additionally, we discuss the evolution of the S glycoprotein to realize cross‐species transmission from the viewpoint of structural biology. Lastly, we describe the recent progress in developing antibodies, nanobodies and peptide inhibitors that target the SARS‐CoV‐2 S glycoprotein for therapeutic purposes. This review mainly focuses on structural insights into the spike glycoproteins of highly pathogenic coronaviruses, especially SARS‐CoV‐2. This includes the characteristics of the spike glycoprotein, the mechanisms of SARS‐CoV‐2 entry into host cells, the evolution strategies SARS‐CoV‐2 uses to enlarge host range and escape from host immunity, and anti‐spike therapies to fight against SARS‐CoV‐2.

Neuraminidase (NA) is a critical target for universal influenza vaccines and therapeutic antibodies, yet its antigenic landscape remains incompletely understood. Here we identify two broadly cross-protective monoclonal antibodies, CAV-F6 and CAV-F34, from influenza-infected individuals. These antibodies inhibit NA enzymatic activity across multiple subtypes and confer protection against seasonal influenza in female mouse models. Importantly, the two antibodies also neutralize emerging avian strains, including recent bovine H5N1 and H7N9 strains, both with pandemic potential. Structural studies reveal that both antibodies target conserved regions of the NA active site via HCDR3, blocking sialic acid interaction. Furthermore, we observe distinct occupancy for the two antibodies on N2 tetramer, which is likely due to differences in binding affinity. Our findings provide molecular insights into NA-targeted immunity and offer a foundation for developing broadly protective influenza vaccines and therapeutics. Influenza A virus neuraminidase (NA) is an important target for universal influenza vaccines. Here, the authors identify two monoclonal antibodies, CAV-F6 and CAV-F34, that inhibit NA activity across multiple subtypes, offering protection against seasonal and emerging avian influenza strains, thus advancing the development of broadly protective vaccines and therapeutics.

An outbreak of mpox has triggered concerns regarding the adequacy of intervention strategies. Passive immunity conferred by neutralizing antibodies exhibits potential in the prophylaxis and treatment of orthopoxvirus infections. Despite this, the investigations of effective antibody therapeutics have been hindered by the varied nature of orthopoxvirus envelope proteins and the intricate mechanisms underpinning viral invasion. Our study involves the production of six mpox virus (MPXV) envelope proteins, which are relatively conservative and considered to play a role in the neutralization process. We employed a synthetic nanobody (Nb) library to derive a broad array of specific Nbs against these viral proteins. We identified a cross-reactive Nb, termed M1R-01, which targets the M1R protein and effectively neutralizes both vaccinia virus (VACV) and MPXV. Notably, the M1R-01-based antibody strategy provided optimal protection against a lethal VACV challenge in mice. Additionally, we determined the crystal structure of the M1R–Nb complex, uncovering novel binding attributes of M1R-01 and detailed conformational epitope information. This work provides a promising candidate for the therapy and prophylaxis of orthopoxvirus infections.

DS-Cav1, SC-TM, and DS2 are distinct designer pre-fusion F proteins (pre-F) of respiratory syncytial virus (RSV) developed for vaccines. However, their immunogenicity has not been directly compared. In this study, we generated three recombinant vaccines using the chimpanzee adenovirus vector AdC68 to express DS-Cav1, SC-TM, and DS2. All three vaccines elicited robust serum binding and neutralizing antibodies following intramuscular priming and boosting. DS2 induced the strongest antibody responses, followed by SC-TM and DS-Cav1. DS2 also provided strong protection against live RSV challenge. Monoclonal antibodies (mAbs) isolated from long-lived antibody-secreting cells (ASCs) in the bone marrow six months post-immunization with AdC68-DS2 predominantly targeted site Ø as well as site II. One neutralizing antibody against site II, mAb60, conferred strong protection against live RSV infection in mice. These findings highlight the strong ability of the DS2 design in eliciting long-lived antibody responses and guide the development of next-generation RSV vaccines.

Prevention of robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) requires in vivo evaluation of IgA neutralizing antibodies. Here, we report the efficacy of receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1, B8-dIgA2 and TH335-dIgA1 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparable neutralization potency against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viral loads in lungs significantly, prophylactic intranasal B8-dIgA unexpectedly led to high amount of infectious viruses and extended damage in NT compared to controls. Mechanistically, B8-dIgA failed to inhibit SARS-CoV-2 cell-to-cell transmission, but was hijacked by the virus through dendritic cell-mediated trans-infection of NT epithelia leading to robust nasal infection. Cryo-EM further revealed B8 as a class II antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Neutralizing dIgA, therefore, may engage an unexpected mode of SARS-CoV-2 nasal infection and injury.