Discordant Antigenic Properties of Soluble and Virion SARS-CoV-2 Spike Proteins.

Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2.

Extraction of the CDRH3 sequence of the mouse antibody repertoire selected upon influenza virus infection by subtraction of the background antibody repertoire.

Historically, antibody reactivity to pathogens and vaccine antigens has been evaluated using serological measurements of antigen-specific antibodies. However, it is difficult to evaluate all antibodies that contribute to various functions in a single assay, such as the measurement of the neutralizing antibody titer. Bulk antibody repertoire analysis using next-generation sequencing is a comprehensive method for analyzing the overall antibody response; however, it is unreliable for estimating antigen-specific antibodies due to individual variation. To address this issue, we propose a method to subtract the background signal from the repertoire of data of interest. In this study, we analyzed changes in antibody diversity and inferred the heavy-chain complementarity-determining region 3 (CDRH3) sequences of antibody clones that were selected upon influenza virus infection in a mouse model using bulk repertoire analysis. A decrease in the diversity of the antibody repertoire was observed upon viral infection, along with an increase in neutralizing antibody titers. Using kernel density estimation of sequences in a high-dimensional sequence space with background signal subtraction, we identified several clusters of CDRH3 sequences induced upon influenza virus infection. Most of these repertoires were detected more frequently in infected mice than in uninfected control mice, suggesting that infection-specific antibody sequences can be extracted using this method. Such an accurate extraction of antigen- or infection-specific repertoire information will be a useful tool for vaccine evaluation in the future. IMPORTANCE: As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.

Antibody targeting of conserved sites of vulnerability on the SARS-CoV-2 spike receptor-binding domain.

Given the continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs), immunotherapeutics that target conserved epitopes on the spike (S) glycoprotein have therapeutic advantages. Here, we report the crystal structure of the SARS-CoV-2 S receptor-binding domain (RBD) at 1.95 Å and describe flexibility and distinct conformations of the angiotensin-converting enzyme 2 (ACE2)-binding site. We identify a set of SARS-CoV-2-reactive monoclonal antibodies (mAbs) with broad RBD cross-reactivity including SARS-CoV-2 Omicron subvariants, SARS-CoV-1, and other sarbecoviruses and determine the crystal structures of mAb-RBD complexes with Ab246 and CR3022 mAbs targeting the class IV site, WRAIR-2134, which binds the recently designated class V epitope, and WRAIR-2123, the class I ACE2-binding site. The broad reactivity of class IV and V mAbs to conserved regions of SARS-CoV-2 VoCs and other sarbecovirus provides a framework for long-term immunotherapeutic development strategies.

Cryptic-site-specific antibodies to the SARS-CoV-2 receptor binding domain can retain functional binding affinity to spike variants.

IMPORTANCE: Multiple SARS-CoV-2 variants of concern have emerged and caused a significant number of infections and deaths worldwide. These variants of concern contain mutations that might significantly affect antigen-targeting by antibodies. It is therefore important to further understand how antibody binding and neutralization are affected by the mutations in SARS-CoV-2 variants. We highlighted how antibody epitope specificity can influence antibody binding to SARS-CoV-2 spike protein variants and neutralization of SARS-CoV-2 variants. We showed that weakened spike binding and neutralization of Beta (B.1.351) and Omicron (BA.1) variants compared to wildtype are not universal among the panel of antibodies and identified antibodies of a specific binding footprint exhibiting consistent enhancement of spike binding and retained neutralization to Beta variant. These data and analysis can inform how antigen-targeting by antibodies might evolve during a pandemic and prepare for potential future sarbecovirus outbreaks.

The solution structure of the heavy chain-only C5-Fc nanobody reveals exposed variable regions that are optimal for COVID-19 antigen interactions.

Heavy chain-only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities than conventional antibodies. To address the challenge of SARS-CoV-2 coronavirus, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 virus neutralization potency. The fusion protein C5-Fc comprises two C5 domains attached to a glycosylated Fc region of a human IgG1 antibody and shows therapeutic efficacy in vivo. Here, we have characterized the solution arrangement of the molecule. Two 1443 Da N-linked glycans seen in the mass spectra of C5-Fc were removed and the glycosylated and deglycosylated structures were evaluated. Reduction of C5-Fc with 2-mercaptoethylamine indicated three interchain Cys-Cys disulfide bridges within the hinge. The X-ray and neutron Guinier RG values, which provide information about structural elongation, were similar at 4.1 to 4.2 nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modeling based on Monte Carlo simulations resulted in 72,737 and 56,749 physically realistic trial X-ray and neutron structures, respectively. From these, the top 100 best-fit X-ray and neutron models were identified as representative asymmetric solution structures, similar to that of human IgG1, with good R-factors below 2.00%. Both C5 domains were solvent exposed, consistent with the functional effectiveness of C5-Fc. Greater disorder occurred in the Fc region after deglycosylation. Our results clarify the importance of variable and exposed C5 conformations in the therapeutic function of C5-Fc, while the glycans in the Fc region are key for conformational stability in C5-Fc.

Combined Antibodies Evusheld against the SARS-CoV-2 Omicron Variants BA.1.1 and BA.5: Immune Escape Mechanism from Molecular Simulation.

The Omicron lineage of SARS-CoV-2, which was first reported in November 2021, has spread globally and become dominant, splitting into several sublineages. Experiments have shown that Omicron lineage has escaped or reduced the activity of existing monoclonal antibodies, but the origin of escape mechanism caused by mutation is still unknown. This work uses molecular dynamics and umbrella sampling methods to reveal the escape mechanism of BA.1.1 to monoclonal antibody (mAb) Tixagevimab (AZD1061) and BA.5 to mAb Cilgavimab (AZD8895), both mAbs were combined to form antibody cocktail, Evusheld (AZD7442). The binding free energy of BA.1.1-AZD1061 and BA.5-AZD8895 has been severely reduced due to multiple-site mutated Omicron variants. Our results show that the two Omicron variants, which introduce a substantial number of positively charged residues, can weaken the electrostatic attraction between the receptor binding domain (RBD) and AZD7442, thus leading to a decrease in affinity. Additionally, using umbrella sampling along dissociation pathway, we found that the two Omicron variants severely impaired the interaction between the RBD of SARS-CoV-2's spike glycoprotein (S protein) and complementary determining regions (CDRs) of mAbs, especially in CDR3H. Although mAbs AZD8895 and AZD1061 are knocked out by BA.5 and BA.1.1, respectively, our results confirm that the antibody cocktail AZD7442 retains activity against BA.1.1 and BA.5 because another antibody is still on guard. The study provides theoretical insights for mAbs interacting with BA.1.1 and BA.5 from both energetic and dynamic perspectives, and we hope this will help in developing new monoclonals and combinations to protect those unable to mount adequate vaccine responses.

Structural basis for cross-group recognition of an influenza virus hemagglutinin antibody that targets postfusion stabilized epitope.

Plasticity of influenza virus hemagglutinin (HA) conformation increases an opportunity to generate conserved non-native epitopes with unknown functionality. Here, we have performed an in-depth analysis of human monoclonal antibodies against a stem-helix region that is occluded in native prefusion yet exposed in postfusion HA. A stem-helix antibody, LAH31, provided IgG Fc-dependent cross-group protection by targeting a stem-helix kinked loop epitope, with a unique structure emerging in the postfusion state. The structural analysis and molecular modeling revealed key contact sites responsible for the epitope specificity and cross-group breadth that relies on somatically mutated light chain. LAH31 was inaccessible to the native prefusion HA expressed on cell surface; however, it bound to the HA structure present on infected cells with functional linkage to the Fc-mediated clearance. Our study uncovers a novel non-native epitope that emerges in the postfusion HA state, highlighting the utility of this epitope for a broadly protective antigen design.

Targeting the Spike Receptor Binding Domain Class V Cryptic Epitope by an Antibody with Pan-Sarbecovirus Activity.

Novel therapeutic monoclonal antibodies (MAbs) must accommodate comprehensive breadth of activity against diverse sarbecoviruses and high neutralization potency to overcome emerging variants. Here, we report the crystal structure of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) in complex with MAb WRAIR-2063, a moderate-potency neutralizing antibody with exceptional sarbecovirus breadth, that targets the highly conserved cryptic class V epitope. This epitope overlaps substantially with the spike protein N-terminal domain (NTD) -interacting region and is exposed only when the spike is in the open conformation, with one or more RBDs accessible. WRAIR-2063 binds the RBD of SARS-CoV-2 WA-1, all variants of concern (VoCs), and clade 1 to 4 sarbecoviruses with high affinity, demonstrating the conservation of this epitope and potential resiliency against variation. We compare structural features of additional class V antibodies with their reported neutralization capacity to further explore the utility of the class V epitope as a pan-sarbecovirus vaccine and therapeutic target. IMPORTANCE Characterization of MAbs against SARS-CoV-2, elicited through vaccination or natural infection, has provided vital immunotherapeutic options for curbing the COVID-19 pandemic and has supplied critical insights into SARS-CoV-2 escape, transmissibility, and mechanisms of viral inactivation. Neutralizing MAbs that target the RBD but do not block ACE2 binding are of particular interest because the epitopes are well conserved within sarbecoviruses and MAbs targeting this area demonstrate cross-reactivity. The class V RBD-targeted MAbs localize to an invariant site of vulnerability, provide a range of neutralization potency, and exhibit considerable breadth against divergent sarbecoviruses, with implications for vaccine and therapeutic development.

Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus-cell interface.

The interaction between the receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 and the peptidase domain of the human angiotensin-converting enzyme 2 (ACE2) allows the first specific contact at the virus-cell interface making it the main target of neutralizing antibodies. Here, we show a unique and cost-effective protocol using Drosophila S2 cells to produce both RBD and soluble human ACE2 peptidase domain (shACE2) as thermostable proteins, purified via Strep-tag with yields >40 mg L-1 in a laboratory scale. Furthermore, we demonstrate its binding with KD values in the lower nanomolar range (independently of Strep-tag removal) and its capability to be blocked by serum antibodies in a competition ELISA with Strep-Tactin-HRP as a proof-of-concept. In addition, we assess the capacity of RBD to bind native dimeric ACE2 overexpressed in human cells and its antigen properties with specific serum antibodies. Finally, for completeness, we analyzed RBD microheterogeneity associated with glycosylation and negative charges, with negligible effect on binding either with antibodies or shACE2. Our system represents an accessible and reliable tool for designing in-house surrogate virus neutralization tests (sVNTs), enabling the rapid characterization of neutralizing humoral responses elicited against vaccines or infection, especially in the absence of facilities to conduct virus neutralization tests. Moreover, our biophysical and biochemical characterization of RBD and shACE2 produced in S2 cells lays the groundwork for adapting to different variants of concern (VOCs) to study humoral responses elicited against different VOCs and vaccine formulations.

Broad Sarbecovirus Neutralizing Antibodies Obtained by Computational Design and Synthetic Library Screening.

Members of the Sarbecovirus subgenus of Coronaviridae have twice caused deadly threats to humans. There is increasing concern about the rapid mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has evolved into multiple generations of epidemic variants in 3 years. Broad neutralizing antibodies are of great importance for pandemic preparedness against SARS-CoV-2 variants and divergent zoonotic sarbecoviruses. Here, we analyzed the structural conservation of the receptor-binding domain (RBD) from representative sarbecoviruses and chose S2H97, a previously reported RBD antibody with ideal breadth and resistance to escape, as a template for computational design to enhance the neutralization activity and spectrum. A total of 35 designs were purified for evaluation. The neutralizing activity of a large proportion of these designs against multiple variants was increased from several to hundreds of times. Molecular dynamics simulation suggested that extra interface contacts and enhanced intermolecular interactions between the RBD and the designed antibodies are established. After light and heavy chain reconstitution, AI-1028, with five complementarity determining regions optimized, showed the best neutralizing activity across all tested sarbecoviruses, including SARS-CoV, multiple SARS-CoV-2 variants, and bat-derived viruses. AI-1028 recognized the same cryptic RBD epitope as the parental prototype antibody. In addition to computational design, chemically synthesized nanobody libraries are also a precious resource for rapid antibody development. By applying distinct RBDs as baits for reciprocal screening, we identified two novel nanobodies with broad activities. These findings provide potential pan-sarbecovirus neutralizing drugs and highlight new pathways to rapidly optimize therapeutic candidates when novel SARS-CoV-2 escape variants or new zoonotic coronaviruses emerge. IMPORTANCE The subgenus Sarbecovirus includes human SARS-CoV, SARS-CoV-2, and hundreds of genetically related bat viruses. The continuous evolution of SARS-CoV-2 has led to the striking evasion of neutralizing antibody (NAb) drugs and convalescent plasma. Antibodies with broad activity across sarbecoviruses would be helpful to combat current SARS-CoV-2 mutations and longer term animal virus spillovers. The study of pan-sarbecovirus NAbs described here is significant for the following reasons. First, we established a structure-based computational pipeline to design and optimize NAbs to obtain more potent and broader neutralizing activity across multiple sarbecoviruses. Second, we screened and identified nanobodies from a highly diversified synthetic library with a broad neutralizing spectrum using an elaborate screening strategy. These methodologies provide guidance for the rapid development of antibody therapeutics against emerging pathogens with highly variable characteristics.

Accelerating drug target inhibitor discovery with a deep generative foundation model.

Inhibitor discovery for emerging drug-target proteins is challenging, especially when target structure or active molecules are unknown. Here, we experimentally validate the broad utility of a deep generative framework trained at-scale on protein sequences, small molecules, and their mutual interactions-unbiased toward any specific target. We performed a protein sequence-conditioned sampling on the generative foundation model to design small-molecule inhibitors for two dissimilar targets: the spike protein receptor-binding domain (RBD) and the main protease from SARS-CoV-2. Despite using only the target sequence information during the model inference, micromolar-level inhibition was observed in vitro for two candidates out of four synthesized for each target. The most potent spike RBD inhibitor exhibited activity against several variants in live virus neutralization assays. These results establish that a single, broadly deployable generative foundation model for accelerated inhibitor discovery is effective and efficient, even in the absence of target structure or binder information.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Searching for common ground.

The conserved coronavirus fusion peptide is a target of broadly neutralizing antibodies.

Potent Therapeutic Strategies for COVID-19 with Single-Domain Antibody Immunoliposomes Neutralizing SARS-CoV-2 and Lip/cGAMP Enhancing Protective Immunity.

The worldwide spread of COVID-19 continues to impact our lives and has led to unprecedented damage to global health and the economy. This highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. We modified a single-domain antibody, SARS-CoV-2 VHH, to the surface of the liposomes. These immunoliposomes demonstrated a good neutralizing ability, but could also carry therapeutic compounds. Furthermore, we used the 2019-nCoV RBD-SD1 protein as an antigen with Lip/cGAMP as the adjuvant to immunize mice. Lip/cGAMP enhanced the immunity well. It was demonstrated that the combination of RBD-SD1 and Lip/cGAMP was an effective preventive vaccine. This work presented potent therapeutic anti-SARS-CoV-2 drugs and an effective vaccine to prevent the spread of COVID-19.

Exploring the role of framework mutations in enabling breadth of a cross-reactive antibody (CR3022) against the SARS-CoV-2 RBD and its variants of concern.

Cross-reactive and broadly neutralizing antibodies against surface proteins of diverse strains of rapidly evolving viral pathogens like SARS-CoV-2 can prevent infection and therefore are crucial for the development of effective universal vaccines. While antibodies typically incorporate mutations in their complementarity determining regions during affinity maturation, mutations in the framework regions have been reported as players in determining properties of broadly neutralizing antibodies against HIV and the Influenza virus. We propose an increase in the cross-reactive potential of CR3022 against the emerging SARS- CoV-2 variants of concern through enhanced conformational flexibility. In this study, we use molecular dynamics simulations, in silico mutagenesis, structural modeling, and docking to explore the role of light chain FWR mutations in CR3022, a SARS-CoV anti-spike (S)-protein antibody cross-reactive to the S-protein receptor binding domain of SARS-CoV-2. Our study shows that single substitutions in the light chain framework region of CR3022 with conserved epitopes across SARS-CoV strains allow targeting of diverse antibody epitope footprints that align with the epitopes of recently-categorized neutralizing antibody classes while enabling binding to more than one strain of SARS-CoV-2. Our study has implications for rapid and evolution-based engineering of broadly neutralizing antibodies and reaffirms the role of framework mutations in effective change of antibody orientation and conformation via improved flexibility.Communicated by Ramaswamy H. Sarma.

Antibodies targeting the neuraminidase active site inhibit influenza H3N2 viruses with an S245N glycosylation site.

Contemporary influenza A H3N2 viruses circulating since 2016 have acquired a glycosylation site in the neuraminidase in close proximity to the enzymatic active site. Here, we investigate if this S245N glycosylation site, as a result of antigenic evolution, can impact binding and function of human monoclonal antibodies that target the conserved active site. While we find that a reduction in the inhibitory ability of neuraminidase active site binders is measurable, this class of broadly reactive monoclonal antibodies maintains protective efficacy in vivo.

Reduced B cell antigenicity of Omicron lowers host serologic response.

The SARS-CoV-2 Omicron variant evades most neutralizing vaccine-induced antibodies and is associated with lower antibody titers upon breakthrough infections than previous variants. However, the mechanism remains unclear. Here, we find using a geometric deep-learning model that Omicron's extensively mutated receptor binding site (RBS) features reduced antigenicity compared with previous variants. Mice immunization experiments with different recombinant receptor binding domain (RBD) variants confirm that the serological response to Omicron is drastically attenuated and less potent. Analyses of serum cross-reactivity and competitive ELISA reveal a reduction in antibody response across both variable and conserved RBD epitopes. Computational modeling confirms that the RBS has a potential for further antigenicity reduction while retaining efficient receptor binding. Finally, we find a similar trend of antigenicity reduction over decades for hCoV229E, a common cold coronavirus. Thus, our study explains the reduced antibody titers associated with Omicron infection and reveals a possible trajectory of future viral evolution.

Protective anti-gB neutralizing antibodies targeting two vulnerable sites for EBV-cell membrane fusion.

Epstein-Barr virus (EBV) infects more than 90% of the world's adult population and accounts for a significant cancer burden of epithelial and B cell origins. Glycoprotein B (gB) is the primary fusogen essential for EBV entry into host cells. Here, we isolated two EBV gB-specific neutralizing antibodies, 3A3 and 3A5; both effectively neutralized the dual-tropic EBV infection of B and epithelial cells. In humanized mice, both antibodies showed effective protection from EBV-induced lymphoproliferative disorders. Cryoelectron microscopy analyses identified that 3A3 and 3A5 bind to nonoverlapping sites on domains D-II and D-IV, respectively. Structure-based mutagenesis revealed that 3A3 and 3A5 inhibit membrane fusion through different mechanisms involving the interference with gB-cell interaction and gB activation. Importantly, the 3A3 and 3A5 epitopes are major targets of protective gB-specific neutralizing antibodies elicited by natural EBV infection in humans, providing potential targets for antiviral therapies and vaccines.

Antibody-mediated immunity to SARS-CoV-2 spike.

Despite effective spike-based vaccines and monoclonal antibodies, the SARS-CoV-2 pandemic continues more than two and a half years post-onset. Relentless investigation has outlined a causative dynamic between host-derived antibodies and reciprocal viral subversion. Integration of this paradigm into the architecture of next generation antiviral strategies, predicated on a foundational understanding of the virology and immunology of SARS-CoV-2, will be critical for success. This review aims to serve as a primer on the immunity endowed by antibodies targeting SARS-CoV-2 spike protein through a structural perspective. We begin by introducing the structure and function of spike, polyclonal immunity to SARS-CoV-2 spike, and the emergence of major SARS-CoV-2 variants that evade immunity. The remainder of the article comprises an in-depth dissection of all major epitopes on SARS-CoV-2 spike in molecular detail, with emphasis on the origins, neutralizing potency, mechanisms of action, cross-reactivity, and variant resistance of representative monoclonal antibodies to each epitope.

Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes.

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.