Network analysis uncovers the communication structure of SARS-CoV-2 spike protein identifying sites for immunogen design.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has triggered myriad efforts to understand the structure and dynamics of this complex pathogen. The spike glycoprotein of SARS-CoV-2 is a significant target for immunogens as it is the means by which the virus enters human cells, while simultaneously sporting mutations responsible for immune escape. These functional and escape processes are regulated by complex molecular-level interactions. Our study presents quantitative insights on domain and residue contributions to allosteric communication, immune evasion, and local- and global-level control of functions through the derivation of a weighted graph representation from all-atom MD simulations. Focusing on the ancestral form and the D614G-variant, we provide evidence of the utility of our approach by guiding the selection of a mutation that alters the spike's stability. Taken together, the network approach serves as a valuable tool to evaluate communication "hot-spots" in proteins to guide design of stable immunogens.

Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine.

Coronavirus vaccines that are highly effective against current and anticipated SARS-CoV-2 variants are needed to control COVID-19. We previously reported a receptor-binding domain (RBD)-sortase A-conjugated ferritin nanoparticle (scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected non-human primates (NHPs) from SARS-CoV-2 WA-1 infection. Here, we find the RBD-scNP induced neutralizing antibodies in NHPs against pseudoviruses of SARS-CoV and SARS-CoV-2 variants including 614G, Beta, Delta, Omicron BA.1, BA.2, BA.2.12.1, and BA.4/BA.5, and a designed variant with escape mutations, PMS20. Adjuvant studies demonstrate variant neutralization titers are highest with 3M-052-aqueous formulation (AF). Immunization twice with RBD-scNPs protect NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protect mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect animals from multiple different SARS-related viruses. Such a vaccine could provide broad immunity to SARS-CoV-2 variants.

An antibody from single human VH-rearranging mouse neutralizes all SARS-CoV-2 variants through BA.5 by inhibiting membrane fusion.

SARS-CoV-2 Omicron subvariants have generated a worldwide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of vaccination-induced antibodies. To manage Omicron subvariants and prepare for new ones, additional means of isolating broad and potent humanized SARS-CoV-2 neutralizing antibodies are desirable. Here, we describe a mouse model in which the primary B cell receptor (BCR) repertoire is generated solely through V(D)J recombination of a human VH1-2 heavy chain (HC) and, substantially, a human Vκ1-33 light chain (LC). Thus, primary humanized BCR repertoire diversity in these mice derives from immensely diverse HC and LC antigen-contact CDR3 sequences generated by nontemplated junctional modifications during V(D)J recombination. Immunizing this mouse model with SARS-CoV-2 (Wuhan-Hu-1) spike protein immunogens elicited several VH1-2/Vκ1-33-based neutralizing antibodies that bound RBD in a different mode from each other and from those of many prior patient-derived VH1-2-based neutralizing antibodies. Of these, SP1-77 potently and broadly neutralized all SARS-CoV-2 variants through BA.5. Cryo-EM studies revealed that SP1-77 bound RBD away from the receptor-binding motif via a CDR3-dominated recognition mode. Lattice light-sheet microscopy-based studies showed that SP1-77 did not block ACE2-mediated viral attachment or endocytosis but rather blocked viral-host membrane fusion. The broad and potent SP1-77 neutralization activity and nontraditional mechanism of action suggest that it might have therapeutic potential. Likewise, the SP1-77 binding epitope may inform vaccine strategies. Last, the type of humanized mouse models that we have described may contribute to identifying therapeutic antibodies against future SARS-CoV-2 variants and other pathogens.

Cryo-EM structures of SARS-CoV-2 Omicron BA.2 spike.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sub-lineage has gained in proportion relative to BA.1. Because spike (S) protein variations may underlie differences in their pathobiology, here we determine cryoelectron microscopy (cryo-EM) structures of the BA.2 S ectodomain and compare these with previously determined BA.1 S structures. BA.2 receptor-binding domain (RBD) mutations induce remodeling of the RBD structure, resulting in tighter packing and improved thermostability. Interprotomer RBD interactions are enhanced in the closed (or 3-RBD-down) BA.2 S, while the fusion peptide is less accessible to antibodies than in BA.1. Binding and pseudovirus neutralization assays reveal extensive immune evasion while defining epitopes of two outer RBD face-binding antibodies, DH1044 and DH1193, that neutralize both BA.1 and BA.2. Taken together, our results indicate that stabilization of the closed state through interprotomer RBD-RBD packing is a hallmark of the Omicron variant and show differences in key functional regions in the BA.1 and BA.2 S proteins.

Structural diversity of the SARS-CoV-2 Omicron spike.

Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor-binding domain (RBD) and neutralizing antibody epitope presentation, affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.

mRNA-encoded HIV-1 Env trimer ferritin nanoparticles induce monoclonal antibodies that neutralize heterologous HIV-1 isolates in mice.

The success of nucleoside-modified mRNAs in lipid nanoparticles (mRNA-LNP) as COVID-19 vaccines heralded a new era of vaccine development. For HIV-1, multivalent envelope (Env) trimer protein nanoparticles are superior immunogens compared with trimers alone for priming of broadly neutralizing antibody (bnAb) B cell lineages. The successful expression of complex multivalent nanoparticle immunogens with mRNAs has not been demonstrated. Here, we show that mRNAs can encode antigenic Env trimers on ferritin nanoparticles that initiate bnAb precursor B cell expansion and induce serum autologous tier 2 neutralizing activity in bnAb precursor VH + VL knock-in mice. Next-generation sequencing demonstrates acquisition of critical mutations, and monoclonal antibodies that neutralize heterologous HIV-1 isolates are isolated. Thus, mRNA-LNP can encode complex immunogens and may be of use in design of germline-targeting and sequential boosting immunogens for HIV-1 vaccine development.

A glycan cluster on the SARS-CoV-2 spike ectodomain is recognized by Fab-dimerized glycan-reactive antibodies

The COVID-19 pandemic caused by SARS-CoV-2 has escalated into a global crisis. The spike (S) protein that mediates cell entry and membrane fusion is the current focus of vaccine and therapeutic antibody development efforts. The S protein, like many other viral fusion proteins such as HIV-1 envelope (Env) and influenza hemagglutinin, is glycosylated with both complex and high mannose glycans. Here we demonstrate binding to the SARS-CoV-2 S protein by a category of Fab-dimerized glycan-reactive (FDG) HIV-1-induced broadly neutralizing antibodies (bnAbs). A 3.1 A resolution cryo-EM structure of the S protein ectodomain bound to glycan-dependent HIV-1 bnAb 2G12 revealed a quaternary glycan epitope on the spike S2 domain involving multiple protomers. These data reveal a new epitope on the SARS-CoV-2 spike that can be targeted for vaccine design. Highlights: Fab-dimerized, glycan-reactive (FDG) HIV-1 bnAbs cross-react with SARS-CoV-2 spike.3.1 A resolution cryo-EM structure reveals quaternary S2 epitope for HIV-1 bnAb 2G12.2G12 targets glycans, at positions 709, 717 and 801, in the SARS-CoV-2 spike.Our studies suggest a common epitope for FDG antibodies centered around glycan 709.

Glycans on the SARS-CoV-2 Spike Control the Receptor Binding Domain Conformation

The glycan shield of the beta-coronavirus (ß-CoV) Spike (S) glycoprotein provides protection from host immune responses, acting as a steric block to potentially neutralizing antibody responses. The conformationally dynamic S-protein is the primary immunogenic target of vaccine design owing to its role in host-cell fusion, displaying multiple receptor binding domain (RBD) 'up' and 'down' state configurations. Here, we investigated the potential for RBD adjacent, N-terminal domain (NTD) glycans to influence the conformational equilibrium of these RBD states. Using a combination of antigenic screens and high-resolution cryo-EM structure determination, we show that an N-glycan deletion at position 234 results in a dramatically reduced population of the 'up' state RBD position. Conversely, glycan deletion at position N165 results in a discernable increase in 'up' state RBDs. This indicates the glycan shield acts not only as a passive hinderance to antibody meditated immunity but also as a conformational control element. Together, our results demonstrate this highly dynamic conformational machine is responsive to glycan modification with implications in viral escape and vaccine design.

Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike mutations enable increased transmission and antibody resistance. We combined cryo-electron microscopy (cryo-EM), binding, and computational analyses to study variant spikes, including one that was involved in transmission between minks and humans, and others that originated and spread in human populations. All variants showed increased angiotensin-converting enzyme 2 (ACE2) receptor binding and increased propensity for receptor binding domain (RBD)-up states. While adaptation to mink resulted in spike destabilization, the B.1.1.7 (UK) spike balanced stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 (Brazil) and B.1.351 (South Africa) variants to neutralizing antibodies. Our studies revealed allosteric effects of mutations and mechanistic differences that drive either interspecies transmission or escape from antibody neutralization.

In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.

Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

Cold sensitivity of the SARS-CoV-2 spike ectodomain.

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its receptor binding domain in two conformations, the receptor-accessible 'up' or receptor-inaccessible 'down' states. Here we report that the commonly used stabilized S ectodomain construct '2P' is sensitive to cold temperatures, and this cold sensitivity is abrogated in a 'down' state-stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.

D614G Mutation Alters SARS-CoV-2 Spike Conformation and Enhances Protease Cleavage at the S1/S2 Junction.

The severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein is the target of vaccine design efforts to end the coronavirus disease 2019 (COVID-19) pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic and are now the dominant form worldwide. Here, we explore S conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryoelectron microscopy (cryo-EM) structures reveal altered receptor binding domain (RBD) disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the RBDs in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, which harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 S conformational landscape and allostery and have implications for vaccine design.

D614G Spike Mutation Increases SARS CoV-2 Susceptibility to Neutralization.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein acquired a D614G mutation early in the pandemic that confers greater infectivity and is now the globally dominant form. To determine whether D614G might also mediate neutralization escape that could compromise vaccine efficacy, sera from spike-immunized mice, nonhuman primates, and humans were evaluated for neutralization of pseudoviruses bearing either D614 or G614 spike. In all cases, the G614 pseudovirus was moderately more susceptible to neutralization. The G614 pseudovirus also was more susceptible to neutralization by receptor-binding domain (RBD) monoclonal antibodies and convalescent sera from people infected with either form of the virus. Negative stain electron microscopy revealed a higher percentage of the 1-RBD "up" conformation in the G614 spike, suggesting increased epitope exposure as a mechanism of enhanced vulnerability to neutralization. Based on these findings, the D614G mutation is not expected to be an obstacle for current vaccine development.

Controlling the SARS-CoV-2 spike glycoprotein conformation.

The coronavirus (CoV) spike (S) protein, involved in viral-host cell fusion, is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available ß-CoV S-protein structures. Despite an overall similarity in domain organization, we found that S-proteins from different ß-CoVs display distinct configurations. Based on this analysis, we developed two soluble ectodomain constructs for the SARS-CoV-2 S-protein, in which the highly immunogenic and mobile receptor binding domain (RBD) is either locked in the all-RBDs 'down' position or adopts 'up' state conformations more readily than the wild-type S-protein. These results demonstrate that the conformation of the S-protein can be controlled via rational design and can provide a framework for the development of engineered CoV S-proteins for vaccine applications.

Glycans on the SARS-CoV-2 Spike Control the Receptor Binding Domain Conformation.

The glycan shield of the beta-coronavirus (ß-CoV) Spike (S) glycoprotein provides protection from host immune responses, acting as a steric block to potentially neutralizing antibody responses. The conformationally dynamic S-protein is the primary immunogenic target of vaccine design owing to its role in host-cell fusion, displaying multiple receptor binding domain (RBD) 'up' and 'down' state configurations. Here, we investigated the potential for RBD adjacent, N-terminal domain (NTD) glycans to influence the conformational equilibrium of these RBD states. Using a combination of antigenic screens and high-resolution cryo-EM structure determination, we show that an N-glycan deletion at position 234 results in a dramatically reduced population of the 'up' state RBD position. Conversely, glycan deletion at position N165 results in a discernable increase in 'up' state RBDs. This indicates the glycan shield acts not only as a passive hinderance to antibody meditated immunity but also as a conformational control element. Together, our results demonstrate this highly dynamic conformational machine is responsive to glycan modification with implications in viral escape and vaccine design.

A glycan cluster on the SARS-CoV-2 spike ectodomain is recognized by Fab-dimerized glycan-reactive antibodies.

The COVID-19 pandemic caused by SARS-CoV-2 has escalated into a global crisis. The spike (S) protein that mediates cell entry and membrane fusion is the current focus of vaccine and therapeutic antibody development efforts. The S protein, like many other viral fusion proteins such as HIV-1 envelope (Env) and influenza hemagglutinin, is glycosylated with both complex and high mannose glycans. Here we demonstrate binding to the SARS-CoV-2 S protein by a category of Fab-dimerized glycan-reactive (FDG) HIV-1-induced broadly neutralizing antibodies (bnAbs). A 3.1 Å resolution cryo-EM structure of the S protein ectodomain bound to glycan-dependent HIV-1 bnAb 2G12 revealed a quaternary glycan epitope on the spike S2 domain involving multiple protomers. These data reveal a new epitope on the SARS-CoV-2 spike that can be targeted for vaccine design. HIGHLIGHTS: Fab-dimerized, glycan-reactive (FDG) HIV-1 bnAbs cross-react with SARS-CoV-2 spike.3.1 Å resolution cryo-EM structure reveals quaternary S2 epitope for HIV-1 bnAb 2G12.2G12 targets glycans, at positions 709, 717 and 801, in the SARS-CoV-2 spike.Our studies suggest a common epitope for FDG antibodies centered around glycan 709.