A potent and broad neutralization of SARS-CoV-2 variants of concern by DARPins.

We report the engineering and selection of two synthetic proteins-FSR16m and FSR22-for the possible treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. FSR16m and FSR22 are trimeric proteins composed of DARPin SR16m or SR22 fused with a T4 foldon. Despite selection by a spike protein from a now historical SARS-CoV-2 strain, FSR16m and FSR22 exhibit broad-spectrum neutralization of SARS-CoV-2 strains, inhibiting authentic B.1.351, B.1.617.2 and BA.1.1 viruses, with respective IC50 values of 3.4, 2.2 and 7.4 ng ml-1 for FSR16m. Cryo-EM structures revealed that these DARPins recognize a region of the receptor-binding domain (residues 456, 475, 486, 487 and 489) overlapping a critical portion of the angiotensin-converting enzyme 2 (ACE2)-binding surface. K18-hACE2 transgenic mice inoculated with B.1.617.2 and receiving intranasally administered FSR16m showed less weight loss and 10-100-fold lower viral burden in upper and lower respiratory tracts. The strong and broad neutralization potency makes FSR16m and FSR22 promising candidates for the prevention and treatment of infection by SARS-CoV-2.

Potent monoclonal antibodies neutralize Omicron sublineages and other SARS-CoV-2 variants.

The emergence and global spread of the SARS-CoV-2 Omicron variants, which carry an unprecedented number of mutations, raise serious concerns due to the reduced efficacy of current vaccines and resistance to therapeutic antibodies. Here, we report the generation and characterization of two potent human monoclonal antibodies, NA8 and NE12, against the receptor-binding domain of the SARS-CoV-2 spike protein. NA8 interacts with a highly conserved region and has a breadth of neutralization with picomolar potency against the Beta variant and the Omicron BA.1 and BA.2 sublineages and nanomolar potency against BA.2.12.1 and BA.4. Combination of NA8 and NE12 retains potent neutralizing activity against the major SARS-CoV-2 variants of concern. Cryo-EM analysis provides the structural basis for the broad and complementary neutralizing activity of these two antibodies. We confirm the in vivo protective and therapeutic efficacies of NA8 and NE12 in the hamster model. These results show that broad and potent human antibodies can overcome the continuous immune escape of evolving SARS-CoV-2 variants.

Structural basis for potent antibody neutralization of SARS-CoV-2 variants including B.1.1.529.

The rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.529 (Omicron) variant and its resistance to neutralization by vaccinee and convalescent sera are driving a search for monoclonal antibodies with potent neutralization. To provide insight into effective neutralization, we determined cryo-electron microscopy structures and evaluated receptor binding domain (RBD) antibodies for their ability to bind and neutralize B.1.1.529. Mutations altered 16% of the B.1.1.529 RBD surface, clustered on an RBD ridge overlapping the angiotensin-converting enzyme 2 (ACE2)-binding surface and reduced binding of most antibodies. Substantial inhibitory activity was retained by select monoclonal antibodies-including A23-58.1, B1-182.1, COV2-2196, S2E12, A19-46.1, S309, and LY-CoV1404-that accommodated these changes and neutralized B.1.1.529. We identified combinations of antibodies with synergistic neutralization. The analysis revealed structural mechanisms for maintenance of potent neutralization against emerging variants.

Ultrapotent antibodies against diverse and highly transmissible SARS-CoV-2 variants.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC50) 0.3 to 11.1 nanograms per milliliter; IC80 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.

A pH-dependent switch mediates conformational masking of SARS-CoV-2 spike

SARS-CoV-2 has emerged as a global pathogen, sparking urgent vaccine development efforts with the trimeric spike. However, the inability of antibodies like CR3022, which binds a cryptic spike epitope with nanomolar affinity, to neutralize virus, suggests a spike-based means of neutralization escape. Here, we show the SARS-CoV-2 spike to have 10% the unfolding enthalpy of a globular protein at physiological pH, where it is recognized by antibodies like CR3022, and up to 10-times more unfolding enthalpy at endosomal pH, where it sheds such antibodies, suggesting that the spike evades potentially neutralizing antibody through a pH-dependent mechanism of conformational masking. To understand the compatibility of this mechanism with ACE2-receptor interactions, we carried out binding measurements and determined cryo-EM structures of the spike recognizing up to three ACE2 molecules at both physiological and endosomal pH. In the absence of ACE2, cryo-EM analyses indicated lower pH to reduce conformational heterogeneity. Single-receptor binding domain (RBD)-up conformations dominated at pH 5.5, resolving into a locked all-down conformation at lower pH through lowering of RBD and refolding of a pH-dependent switch. Notably, the emerging Asp614Gly strain partially destabilizes the switch that locks RBD down, thereby enhancing functional interactions with ACE2 while reducing evasion by conformational masking.

Cryo-EM Structures of SARS-CoV-2 Spike without and with ACE2 Reveal a pH-Dependent Switch to Mediate Endosomal Positioning of Receptor-Binding Domains.

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low-pH-endosomal pathways. To understand how ACE2 binding and low pH affect spike conformation, we determined cryo-electron microscopy structures-at serological and endosomal pH-delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted "up" conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These structures provide a foundation for understanding prefusion-spike mechanics governing endosomal entry; we suggest that the low pH all-down conformation potentially facilitates immune evasion from RBD-up binding antibody.

SARS-CoV-2 S2P spike ages through distinct states with altered immunogenicity.

The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline "S2P" spike-widely employed for laboratory work and clinical studies-unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately 1 week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging; however, its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding, which affects immunogenicity, highlighting the need to monitor its integrity.

Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants.

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization1-3. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies4. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD-ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and-to our knowledge-rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

Newcastle Disease Virus-Like Particles Displaying Prefusion-Stabilized SARS-CoV-2 Spikes Elicit Potent Neutralizing Responses.

The COVID-19 pandemic highlights an urgent need for vaccines that confer protection from SARS-CoV-2 infection. One approach to an effective COVID-19 vaccine may be through the display of SARS-CoV-2 spikes on the surface of virus-like particles, in a manner structurally mimicking spikes on a native virus. Here we report the development of Newcastle disease virus-like particles (NDVLPs) displaying the prefusion-stabilized SARS-CoV-2 spike ectodomain (S2P). Immunoassays with SARS-CoV-2-neutralizing antibodies revealed the antigenicity of S2P-NDVLP to be generally similar to that of soluble S2P, and negative-stain electron microscopy showed S2P on the NDVLP surface to be displayed with a morphology corresponding to its prefusion conformation. Mice immunized with S2P-NDVLP showed substantial neutralization titers (geometric mean ID50 = 386) two weeks after prime immunization, significantly higher than those elicited by a molar equivalent amount of soluble S2P (geometric mean ID50 = 17). Neutralizing titers at Week 5, two weeks after a boost immunization with S2P-NDVLP doses ranging from 2.0 to 250 µg, extended from 2125 to 4552, and these generally showed a higher ratio of neutralization versus ELISA than observed with soluble S2P. Overall, S2P-NDVLP appears to be a promising COVID-19 vaccine candidate capable of eliciting substantial neutralizing activity.

Cryo-EM Structures Delineate a pH-Dependent Switch that Mediates Endosomal Positioning of SARS-CoV-2 Spike Receptor-Binding Domains.

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the ACE2 receptor and to facilitate virus entry. Antibodies can engage RBD but some, such as CR3022, fail to inhibit entry despite nanomolar spike affinity. Here we show the SARS-CoV-2 spike to have low unfolding enthalpy at serological pH and up to 10-times more unfolding enthalpy at endosomal pH, where we observe significantly reduced CR3022 affinity. Cryo-EM structures -at serological and endosomal pH- delineated spike recognition of up to three ACE2 molecules, revealing RBD to freely adopt the 'up' conformation. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a locked all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning and spike shedding of antibodies like CR3022. An endosomal mechanism involving spike-conformational change can thus facilitate immune evasion from RBD-'up'-recognizing antibody.