Interleukin-17D regulates group 3 innate lymphoid cell function through its receptor CD93.

The interleukin (IL)-17 family, consisting of six members, promotes host defense but can in some context promote the development of autoimmune disease. Here, we examined the role of IL-17D, a poorly understood member in the IL-17 family. IL-17D was expressed primarily by colonic epithelial cells. Il17d-/- mice were more susceptible to acute colitis, bacterial infection and experimentally induced colon cancer than their wildtype counterparts. Il17d deficiency impaired IL-22 production by group 3 innate lymphoid cells (ILC3s) and reduced expression of IL-22-dependent antimicrobial peptides, RegIIIß and RegIIIγ, in colon tissue at steady state and in colitis; this was associated with changes in microbial composition and dysbiosis. Protein purification studies revealed that IL-17D bound not canonical IL-17 receptors, but rather CD93, a glycoprotein expressed on mature ILC3s. Mice lacking Cd93 in ILC3s exhibited impaired IL-22 production and aggravated colonic inflammation in experimental colitis. Thus, an IL-17D-CD93 axis regulates ILC3 function to preserve intestinal homeostasis.

Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge during the global pandemic and may facilitate escape from current antibody therapies and vaccine protection. Here we showed that the South African variant B.1.351 was the most resistant to current monoclonal antibodies and convalescent plasma from coronavirus disease 2019 (COVID-19)-infected individuals, followed by the Brazilian variant P.1 and the United Kingdom variant B.1.1.7. This resistance hierarchy corresponded with Y144del and 242-244del mutations in the N-terminal domain and K417N/T, E484K, and N501Y mutations in the receptor-binding domain (RBD) of SARS-CoV-2. Crystal structure analysis of the B.1.351 triple mutant (417N-484K-501Y) RBD complexed with the monoclonal antibody P2C-1F11 revealed the molecular basis for antibody neutralization and escape. B.1.351 and P.1 also acquired the ability to use mouse and mink ACE2 receptors for entry. Our results demonstrate major antigenic shifts and potential broadening of the host range for B.1.351 and P.1 variants, which poses serious challenges to current antibody therapies and vaccine protection.

Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity in COVID-19 Convalescent Individuals.

The World Health Organization has declared SARS-CoV-2 virus outbreak a worldwide pandemic. However, there is very limited understanding on the immune responses, especially adaptive immune responses to SARS-CoV-2 infection. Here, we collected blood from COVID-19 patients who have recently become virus-free, and therefore were discharged, and detected SARS-CoV-2-specific humoral and cellular immunity in eight newly discharged patients. Follow-up analysis on another cohort of six patients 2 weeks post discharge also revealed high titers of immunoglobulin G (IgG) antibodies. In all 14 patients tested, 13 displayed serum-neutralizing activities in a pseudotype entry assay. Notably, there was a strong correlation between neutralization antibody titers and the numbers of virus-specific T cells. Our work provides a basis for further analysis of protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It also has implications in developing an effective vaccine to SARS-CoV-2 infection.

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.

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.

SARS-CoV-2 hijacks neutralizing dimeric IgA for nasal infection and injury in Syrian hamsters1.

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.

The spike glycoprotein of highly pathogenic human coronaviruses: structural insights for understanding infection, evolution and inhibition.

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.

Structural basis of the IL-1 receptor TIR domain-mediated IL-1 signaling.

The cytoplasmic Toll/interleukin-1 receptor (TIR) domains of IL-1 receptors (IL-1Rs) are evolutionally conserved and essential for transmitting signals. IL-1RAcP is a shared co-receptor in the IL-1R family for signaling. Its splicing form IL-1RAcPb contains a different TIR domain and is unable to transduce NF-κB signaling. Here, we determined crystal structures of TIR domains of IL-1RAcPb and other IL-1Rs including IL-18Rß, IL-1RAPL2, and zebrafish SIGIRR (zSIGIRR). Structurally variant regions in the TIR domain important for signaling were revealed by structural comparisons. Taking advantage of the IL-1RAcP/IL-1RAcPb pair, we demonstrated that differential TIR domain determines signaling discrepancies between IL-1RAcP and IL-1RAcPb. We also proved the functional importance of two helices (αC and αD) in the structurally variable regions and pinpointed critical residues in αC and αD for signaling. These results collectively provide additional and important knowledge for fully understanding the molecular basis of IL-1R TIR domain in mediating signaling.

Structural insights into the binding of SARS-CoV-2, SARS-CoV, and hCoV-NL63 spike receptor-binding domain to horse ACE2.

Severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and human coronavirus (hCoV)-NL63 utilize ACE2 as the functional receptor for cell entry, which leads to zoonotic infection. Horses (Equus caballus) attracted our attention because the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 and SARS-CoV-2-related coronaviruses bind equine ACE2 (eACE2) with high affinity. Here we show that eACE2 binds the RBDs of these three coronaviruses and also SARS-CoV-2 variants but with lower affinities compared with human ACE2 (hACE2). Structural analysis and mutation assays indicated that eACE2-H41 accounts for the lower binding affinity of eACE2 to the RBDs of SARS-CoV-2 variants (Alpha, Beta, and Gamma), SARS-CoV, and hCoV-NL63. Pseudovirus infection assays showed that the SARS-CoV-2 Delta strain (B.1.617.2) displayed a significantly increased infection efficiency in eACE2-expressing HeLa cells. Our results reveal the molecular basis of eACE2 binding to the RBDs of SARS-CoV, SARS-CoV-2, and hCoV-NL63, which provides insights into the potential animal transmission of these ACE2-dependent coronaviruses.

Preclinical characterization of amubarvimab and romlusevimab, a pair of non-competing neutralizing monoclonal antibody cocktail, against SARS-CoV-2.

Monoclonal antibodies (mAbs) targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein have demonstrated clinical efficacy in preventing or treating coronavirus disease 2019 (COVID-19), resulting in the emergency use authorization (EUA) for several SARS-CoV-2 targeting mAb by regulatory authority. However, the continuous virus evolution requires diverse mAb options to combat variants. Here we describe two fully human mAbs, amubarvimab (BRII-196) and romlusevimab (BRII-198) that bind to non-competing epitopes on the receptor binding domain (RBD) of spike protein and effectively neutralize SARS-CoV-2 variants. A YTE modification was introduced to the fragment crystallizable (Fc) region of both mAbs to prolong serum half-life and reduce effector function. The amubarvimab and romlusevimab combination retained activity against most mutations associated with reduced susceptibility to previously authorized mAbs and against variants containing amino acid substitutions in their epitope regions. Consistently, the combination of amubarvimab and romlusevimab effectively neutralized a wide range of viruses including most variants of concern and interest in vitro. In a Syrian golden hamster model of SARS-CoV-2 infection, animals receiving combination of amubarvimab and romlusevimab either pre- or post-infection demonstrated less weight loss, significantly decreased viral load in the lungs, and reduced lung pathology compared to controls. These preclinical findings support their development as an antibody cocktail therapeutic option against COVID-19 in the clinic.

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 severe acute respiratory syndrome coronavirus 2 infection.

PURPOSE OF REVIEW: The spike glycoprotein plays a critical role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by recognizing the angiotensin converting enzyme 2 (ACE2) receptor and mediating fusion of the viral envelope with the cell membrane. It is also the major target for neutralizing antibodies and vaccines. This review summarizes recent studies on the structure and function of spike glycoprotein, which revealed the structural basis of SARS-CoV-2 infection. RECENT FINDINGS: SARS-CoV-2 spike glycoprotein, similar to those of SARS-CoV and Middle East respiratory syndrome coronavirus, spontaneously samples different prefusion states with the receptor-binding domain (RBD) adopting 'up' or 'down' conformations, and the RBD 'down' to 'up' conformational change is required for ACE2 binding. Receptor binding and spike glycoprotein priming by host proteases such as furin and transmembrane protease serine 2 induce pre to postfusion conformational changes of the spike trimer that enable membrane fusion. Interactions between SARS-CoV-2 RBD and ACE2 were elucidated at atomic resolution using high-resolution crystal structures. These structures, together with adapted and remodeled SARS-CoV-2 strains, further revealed critical residues of the spike glycoprotein for SARS-CoV-2 infection and cross-species transmission. SUMMARY: Recent studies on SARS-CoV-2 spike glycoprotein provide important structural knowledge for a better understanding of the molecular mechanisms of SARS-CoV-2 infection and cross-species transmission.

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.

Functional Relevance of Interleukin-1 Receptor Inter-domain Flexibility for Cytokine Binding and Signaling.

The interleukin 1 (IL-1) receptor family, whose members contain three immunoglobulin-like domains (D1-D3) in the extracellular region, is responsible for transmitting pleiotropic signals of IL-1 cytokines. The inter-domain flexibility of IL-1 receptors and its functional roles have not been fully elucidated. In this study, we used small-angle X-ray scattering to show that ligand-binding primary receptors and co-receptors in the family all have inherent inter-domain flexibility due to the D2/D3 linker. Variants of the IL-1RAcP and IL-18Rß co-receptors with mutated D2/D3 linkers cannot form a cytokine-receptor complex and mediate signaling. Our analysis further revealed that these mutated co-receptors exhibited a changed conformational ensemble, suggesting that loss of function is due to the alteration of receptor dynamics. Taken together, our results demonstrate that the D2/D3 linker is a critical functional determinant of IL-1 receptor and underscore the important roles of the inter-domain flexibility in cytokine/receptor binding and signaling.