Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane.

The entry of SARS-CoV-2 into host cells depends on the refolding of the virus-encoded spike protein from a prefusion conformation, which is metastable after cleavage, to a lower-energy stable postfusion conformation1,2. This transition overcomes kinetic barriers for fusion of viral and target cell membranes3,4. Here we report a cryogenic electron microscopy (cryo-EM) structure of the intact postfusion spike in a lipid bilayer that represents the single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membrane-interacting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.

Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane.

Entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells depends on refolding of the virus-encoded spike protein from a prefusion conformation, metastable after cleavage, to a lower energy, stable postfusion conformation. This transition overcomes kinetic barriers for fusion of viral and target cell membranes. We report here a cryo-EM structure of the intact postfusion spike in a lipid bilayer that represents single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membraneinteracting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.

Immune recall improves antibody durability and breadth to SARS-CoV-2 variants.

Key features of immune memory are greater and faster antigen-specific antibody responses to repeat infection. In the setting of immune-evading viral evolution, it is important to understand how far antibody memory recognition stretches across viral variants when memory cells are recalled to action by repeat invasions. It is also important to understand how immune recall influences longevity of secreted antibody responses. We analyzed SARS-CoV-2 variant recognition; dynamics of memory B cells; and secreted antibody over time after infection, vaccination, and boosting. We find that a two-dose SARS-CoV-2 vaccination regimen given after natural infection generated greater longitudinal antibody stability and induced maximal antibody magnitudes with enhanced breadth across Beta, Gamma, Delta and Omicron variants. A homologous third messenger RNA vaccine dose in COVID-naïve individuals conferred greater cross-variant evenness of neutralization potency with stability that was equal to the hybrid immunity conferred by infection plus vaccination. Within unvaccinated individuals who recovered from COVID, enhanced antibody stability over time was observed within a subgroup of individuals who recovered more quickly from COVID and harbored significantly more memory B cells cross-reactive to endemic coronaviruses early after infection. These cross-reactive clones map to the conserved S2 region of SARS-CoV-2 spike with higher somatic hypermutation levels and greater target affinity. We conclude that SARS-CoV-2 antigen challenge histories in humans influence not only the speed and magnitude of antibody responses but also functional cross-variant antibody repertoire composition and longevity.

The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans.

In Streptococcus mutans, we find that the histidine kinase WalK possesses the longest C-terminal tail (CTT) among all 14 TCSs, and this tail plays a key role in the interaction of WalK with its response regulator WalR. We demonstrate that the intrinsically disordered CTT is characterized by a conserved tryptophan residue surrounded by acidic amino acids. Mutation in the tryptophan not only disrupts the stable interaction, but also impairs the efficient phosphotransferase and phosphatase activities of WalRK. In addition, the tryptophan is important for WalK to compete with DNA containing a WalR binding motif for the WalR interaction. We further show that the tryptophan is important for in vivo transcriptional regulation and bacterial biofilm formation by S. mutans. Moreover, Staphylococcus aureus WalK also has a characteristic CTT, albeit relatively shorter, with a conserved W-acidic motif, that is required for the WalRK interaction in vitro. Together, these data reveal that the W-acidic motif of WalK is indispensable for its interaction with WalR, thereby playing a key role in the WalRK-dependent signal transduction, transcriptional regulation and biofilm formation.

Structural and functional impact by SARS-CoV-2 Omicron spike mutations.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), bearing an unusually high number of mutations, has become a dominant strain in many countries within several weeks. We report here structural, functional, and antigenic properties of its full-length spike (S) protein with a native sequence in comparison with those of previously prevalent variants. Omicron S requires a substantially higher level of host receptor ACE2 for efficient membrane fusion than other variants, possibly explaining its unexpected cellular tropism. Mutations not only remodel the antigenic structure of the N-terminal domain of the S protein but also alter the surface of the receptor-binding domain in a way not seen in other variants, consistent with its remarkable resistance to neutralizing antibodies. These results suggest that Omicron S has acquired an extraordinary ability to evade host immunity by excessive mutations, which also compromise its fusogenic capability.

Preserved recognition of Omicron spike following COVID-19 messenger RNA vaccination in pregnancy.

BACKGROUND: SARS-CoV-2 infection is associated with enhanced disease severity in pregnant women. Despite the potential of COVID-19 vaccines to reduce severe disease, vaccine uptake remained relatively low among pregnant women. Just as coordinated messaging from the Centers for Disease Control and Prevention and leading obstetrics organizations began to increase vaccine confidence in this vulnerable group, the evolution of SARS-CoV-2 variants of concerns, including the Omicron variant, raised new concerns about vaccine efficacy because of their ability to escape vaccine-induced neutralizing antibodies. Early data point to a milder disease course following infection with the Omicron variant in vaccinated individuals. Thus, these data suggest that alternate vaccine-induced immunity beyond neutralization may continue to attenuate Omicron variant-induced disease, such as Fc-mediated antibody activity. OBJECTIVE: This study aimed to test whether vaccine-induced antibodies raised during pregnancy continue to bind to and leverage Fc receptors to protect against variants of concern including the Omicron variant. STUDY DESIGN: The receptor binding domain or whole spike-specific antibody isotype binding titers and Fc gamma receptor binding directed toward variants of concern, including the Omicron variant, were analyzed in pregnant women after receiving the full dose regimen of either the Pfizer/BioNTech BNT62b2 (n=10) or Moderna mRNA-1273 (n=10) vaccination using a multiplexing Luminex assay. RESULTS: Reduced isotype recognition of the Omicron receptor binding domain was observed following administration of either vaccine with relatively preserved, albeit reduced, recognition of the whole Omicron spike by immunoglobulin M and G antibodies. Despite the near complete loss of Fc receptor binding to the Omicron receptor binding domain, Fc receptor binding to the Omicron spike was more variable but largely preserved. CONCLUSION: Reduced binding titers to the Omicron receptor binding domain aligns with the observed loss of neutralizing activity. Despite the loss of neutralization, preserved, albeit reduced, Omicron spike recognition and Fc receptor binding potentially continue to attenuate disease severity in pregnant women.

Omicron variant Spike-specific antibody binding and Fc activity are preserved in recipients of mRNA or inactivated COVID-19 vaccines.

The Omicron variant of SARS-CoV-2 has been shown to evade neutralizing antibodies elicited by vaccination or infection. Despite the global spread of the Omicron variant, even among highly vaccinated populations, death rates have not increased concomitantly. These data suggest that immune mechanisms beyond antibody-mediated virus neutralization may protect against severe disease. In addition to neutralizing pathogens, antibodies contribute to control and clearance of infections through Fc effector mechanisms. Here, we probed the ability of vaccine-induced antibodies to drive Fc effector activity against the Omicron variant using samples from individuals receiving one of three SARS-CoV-2 vaccines. Despite a substantial loss of IgM, IgA, and IgG binding to the Omicron variant receptor binding domain (RBD) in samples from individuals receiving BNT162b2, mRNA-1273, and CoronaVac vaccines, stable binding was maintained against the full-length Omicron Spike protein. Compromised RBD binding IgG was accompanied by a loss of RBD-specific antibody Fcγ receptor (FcγR) binding in samples from individuals who received the CoronaVac vaccine, but RBD-specific FcγR2a and FcγR3a binding was preserved in recipients of mRNA vaccines. Conversely, Spike protein-specific antibodies exhibited persistent but reduced binding to FcγRs across all three vaccines, although higher binding was observed in samples from recipients of mRNA vaccines. This was associated with preservation of FcγR2a and FcγR3a binding antibodies and maintenance of Spike protein-specific antibody-dependent natural killer cell activation. Thus, despite the loss of Omicron neutralization, vaccine-induced Spike protein-specific antibodies continue to drive Fc effector functions, suggesting a capacity for extraneutralizing antibodies to contribute to disease control.

Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant.

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report the structure, function, and antigenicity of its full-length spike (S) trimer as well as those of the Gamma and Kappa variants, and compare their characteristics with the G614, Alpha, and Beta variants. Delta S can fuse membranes more efficiently at low levels of cellular receptor angiotensin converting enzyme 2 (ACE2), and its pseudotyped viruses infect target cells substantially faster than the other five variants, possibly accounting for its heightened transmissibility. Each variant shows different rearrangement of the antigenic surface of the amino-terminal domain of the S protein but only makes produces changes in the receptor binding domain (RBD), making the RBD a better target for therapeutic antibodies.

Structure of SARS-CoV-2 spike protein.

The COVID-19 (coronavirus disease 2019) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to loss of human life in millions and devastating socio-economic consequences worldwide. The disease has created urgent needs for intervention strategies to control the crisis and meeting these needs requires a deep understanding of the structure-function relationships of viral proteins and relevant host factors. The trimeric spike (S) protein of the virus decorates the viral surface and is an important target for development of diagnostics, therapeutics and vaccines. Rapid progress in the structural biology of SARS-CoV-2 S protein has been made since the early stage of the pandemic, advancing our knowledge on the viral entry process considerably. In this review, we summarize our latest understanding of the structure of the SARS-CoV-2 S protein and discuss the implications for vaccines and therapeutics.

Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant.

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report here structure, function and antigenicity of its full-length spike (S) trimer in comparison with those of other variants, including Gamma, Kappa, and previously characterized Alpha and Beta. Delta S can fuse membranes more efficiently at low levels of cellular receptor ACE2 and its pseudotyped viruses infect target cells substantially faster than all other variants tested, possibly accounting for its heightened transmissibility. Mutations of each variant rearrange the antigenic surface of the N-terminal domain of the S protein in a unique way, but only cause local changes in the receptor-binding domain, consistent with greater resistance particular to neutralizing antibodies. These results advance our molecular understanding of distinct properties of these viruses and may guide intervention strategies.