Selection of positive controls and their impact on anti-drug antibody assay performance.

Development of assays to reliably identify and characterize anti-drug antibodies (ADAs) depends on positive control anti-idiotype (anti-id) reagents, which are used to demonstrate that the standards recommended by regulatory authorities are met. This work employs a set of therapeutic antibodies under clinical development and their corresponding anti-ids to investigate how different positive control reagent properties impact ADA assay development. Positive controls exhibited different response profiles and apparent assay analytical sensitivity values depending on assay format. Neither anti-id affinity for drug, nor sensitivity in direct immunoassays related to sensitivity in ADA assays. Anti-ids were differentially able to detect damage to drug conjugates used in bridging assays and were differentially drug tolerant. These parameters also failed to relate to assay sensitivity, further complicating selection of anti-ids for use in ADA assay development based on functional characteristics. Given this variability among anti-ids, alternative controls that could be employed across multiple antibody drugs were investigated as a more uniform means to define ADA detection sensitivity across drug products and assay protocols, which could help better relate assay results to clinical risks of ADA responses. Overall, this study highlights the importance of positive control selection to reliable detection and clinical interpretation of the presence and magnitude of ADA responses.

Antibodies targeting the fusion peptide on the HIV envelope provide protection to rhesus macaques against mucosal SHIV challenge.

The fusion peptide (FP) on the HIV-1 envelope (Env) trimer can be targeted by broadly neutralizing antibodies (bNAbs). Here, we evaluated the ability of a human FP-directed bNAb, VRC34.01, along with two vaccine-elicited anti-FP rhesus macaque mAbs, DFPH-a.15 and DF1W-a.01, to protect against simian-HIV (SHIV)BG505 challenge. VRC34.01 neutralized SHIVBG505 with a 50% inhibitory concentration (IC50) of 0.58 µg/ml, whereas DF1W-a.01 and DFPH-a.15 were 4- or 30-fold less potent, respectively. VRC34.01 was infused into four rhesus macaques at a dose of 10 mg/kg and four rhesus macaques at a dose of 2.5 mg/kg. The animals were intrarectally challenged 5 days later with SHIVBG505. In comparison with all 12 control animals that became infected, all four animals infused with VRC34.01 (10 mg/kg) and three out of four animals infused with VRC34.01 (2.5 mg/kg) remained uninfected. Because of the lower potency of DF1W-a.01 and DFPH-a.15 against SHIVBG505, we infused both Abs at a higher dose of 100 mg/kg into four rhesus macaques each, followed by SHIVBG505 challenge 5 days later. Three of four animals that received DF1W-a.01 were protected against infection, whereas all animals that received DFPH-a.15 were protected. Overall, the protective serum neutralization titers observed in these animals were similar to what has been observed for other bNAbs in similar SHIV infection models and in human clinical trials. In conclusion, FP-directed mAbs can thus provide dose-dependent in vivo protection against mucosal SHIV challenges, supporting the development of prophylactic vaccines targeting the HIV-1 Env FP.

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.

Zika-specific neutralizing antibodies targeting inter-dimer envelope epitopes.

Zika virus (ZIKV) is an emerging pathogen that causes devastating congenital defects. The overlapping epidemiology and immunologic cross-reactivity between ZIKV and dengue virus (DENV) pose complex challenges to vaccine design, given the potential for antibody-dependent enhancement of disease. Therefore, classification of ZIKV-specific antibody targets is of notable value. From a ZIKV-infected rhesus macaque, we identify ZIKV-reactive B cells and isolate potent neutralizing monoclonal antibodies (mAbs) with no cross-reactivity to DENV. We group these mAbs into four distinct antigenic groups targeting ZIKV-specific cross-protomer epitopes on the envelope glycoprotein. Co-crystal structures of representative mAbs in complex with ZIKV envelope glycoprotein reveal envelope-dimer epitope and unique dimer-dimer epitope targeting. All four specificities are serologically identified in convalescent humans following ZIKV infection, and representative mAbs from all four groups protect against ZIKV replication in mice. These results provide key insights into ZIKV-specific antigenicity and have implications for ZIKV vaccine, diagnostic, and therapeutic development.

Self-assembling SARS-CoV-2 spike-HBsAg nanoparticles elicit potent and durable neutralizing antibody responses via genetic delivery.

While several COVID-19 vaccines have been in use, more effective and durable vaccines are needed to combat the ongoing COVID-19 pandemic. Here, we report highly immunogenic self-assembling SARS-CoV-2 spike-HBsAg nanoparticles displaying a six-proline-stabilized WA1 (wild type, WT) spike S6P on a HBsAg core. These S6P-HBsAgs bound diverse domain-specific SARS-CoV-2 monoclonal antibodies. In mice with and without a HBV pre-vaccination, DNA immunization with S6P-HBsAgs elicited significantly more potent and durable neutralizing antibody (nAb) responses against diverse SARS-CoV-2 strains than that of soluble S2P or S6P, or full-length S2P with its coding sequence matching mRNA-1273. The nAb responses elicited by S6P-HBsAgs persisted substantially longer than by soluble S2P or S6P and appeared to be enhanced by HBsAg pre-exposure. These data show that genetic delivery of SARS-CoV-2 S6P-HBsAg nanoparticles can elicit greater and more durable nAb responses than non-nanoparticle forms of stabilized spike. Our findings highlight the potential of S6P-HBsAgs as next generation genetic vaccine candidates against SARS-CoV-2.

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.

Shark nanobodies with potent SARS-CoV-2 neutralizing activity and broad sarbecovirus reactivity.

Despite rapid and ongoing vaccine and therapeutic development, SARS-CoV-2 continues to evolve and evade, presenting a need for next-generation diverse therapeutic modalities. Here we show that nurse sharks immunized with SARS-CoV-2 recombinant receptor binding domain (RBD), RBD-ferritin (RFN), or spike protein ferritin nanoparticle (SpFN) immunogens elicit a set of new antigen receptor antibody (IgNAR) molecules that target two non-overlapping conserved epitopes on the spike RBD. Representative shark antibody variable NAR-Fc chimeras (ShAbs) targeting either of the two epitopes mediate cell-effector functions, with high affinity to all SARS-CoV-2 viral variants of concern, including the divergent Omicron strains. The ShAbs potently cross-neutralize SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1 and BA.5, and SARS-CoV-1 pseudoviruses, and confer protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model. Structural definition of the RBD-ShAb01-ShAb02 complex enabled design and production of multi-specific nanobodies with enhanced neutralization capacity, and picomolar affinity to divergent sarbecovirus clade 1a, 1b and 2 RBD molecules. These shark nanobodies represent potent immunotherapeutics both for current use, and future sarbecovirus pandemic preparation.

Extraordinary Titer and Broad Anti-SARS-CoV-2 Neutralization Induced by Stabilized RBD Nanoparticles from Strain BA.5.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a primary target of neutralizing antibodies and a key component of licensed vaccines. Substantial mutations in RBD, however, enable current variants to escape immunogenicity generated by vaccination with the ancestral (WA1) strain. Here, we produce and assess self-assembling nanoparticles displaying RBDs from WA1 and BA.5 strains by using the SpyTag:SpyCatcher system for coupling. We observed both WA1- and BA.5-RBD nanoparticles to degrade substantially after a few days at 37 °C. Incorporation of nine RBD-stabilizing mutations, however, increased yield ~five-fold and stability such that more than 50% of either the WA1- or BA.5-RBD nanoparticle was retained after one week at 37 °C. Murine immunizations revealed that the stabilized RBD-nanoparticles induced ~100-fold higher autologous neutralization titers than the prefusion-stabilized (S2P) spike at a 2 µg dose. Even at a 25-fold lower dose where S2P-induced neutralization titers were below the detection limit, the stabilized BA.5-RBD nanoparticle induced homologous titers of 12,795 ID50 and heterologous titers against WA1 of 1767 ID50. Assessment against a panel of ß-coronavirus variants revealed both the stabilized BA.5-RBD nanoparticle and the stabilized WA1-BA.5-(mosaic)-RBD nanoparticle to elicit much higher neutralization breadth than the stabilized WA1-RBD nanoparticle. The extraordinary titer and high neutralization breadth elicited by stabilized RBD nanoparticles from strain BA.5 make them strong candidates for next-generation COVID-19 vaccines.

Determination of Binding Affinity of Antibodies to HIV-1 Recombinant Envelope Glycoproteins, Pseudoviruses, Infectious Molecular Clones, and Cell-Expressed Trimeric gp160 Using Microscale Thermophoresis.

Developing a preventative vaccine for HIV-1 has been a global priority. The elicitation of broadly neutralizing antibodies (bNAbs) against a broad range of HIV-1 envelopes (Envs) from various strains appears to be a critical requirement for an efficacious HIV-1 vaccine. To understand their ability to neutralize HIV-1, it is important to characterize the binding characteristics of bNAbs. Our work is the first to utilize microscale thermophoresis (MST), a rapid, economical, and flexible in-solution temperature gradient method to quantitatively determine the binding affinities of bNAbs and non-neutralizing monoclonal antibodies (mAbs) to HIV-1 recombinant envelope monomer and trimer proteins of different subtypes, pseudoviruses (PVs), infectious molecular clones (IMCs), and cells expressing the trimer. Our results demonstrate that the binding affinities were subtype-dependent. The bNAbs exhibited a higher affinity to IMCs compared to PVs and recombinant proteins. The bNAbs and mAbs bound with high affinity to native-like gp160 trimers expressed on the surface of CEM cells compared to soluble recombinant proteins. Interesting differences were seen with V2-specific mAbs. Although they recognize linear epitopes, one of the antibodies also bound to the Envs on PVs, IMCs, and a recombinant trimer protein, suggesting that the epitope was not occluded. The identification of epitopes on the envelope surface that can bind to high affinity mAbs could be useful for designing HIV-1 vaccines and for down-selecting vaccine candidates that can induce high affinity antibodies to the HIV-1 envelope in their native conformation.

Epstein-Barr virus gH/gL has multiple sites of vulnerability for virus neutralization and fusion inhibition.

Epstein-Barr virus (EBV) is nearly ubiquitous in adults. EBV causes infectious mononucleosis and is associated with B cell lymphomas, epithelial cell malignancies, and multiple sclerosis. The EBV gH/gL glycoprotein complex facilitates fusion of virus membrane with host cells and is a target of neutralizing antibodies. Here, we examined the sites of vulnerability for virus neutralization and fusion inhibition within EBV gH/gL. We developed a panel of human monoclonal antibodies (mAbs) that targeted five distinct antigenic sites on EBV gH/gL and prevented infection of epithelial and B cells. Structural analyses using X-ray crystallography and electron microscopy revealed multiple sites of vulnerability and defined the antigenic landscape of EBV gH/gL. One mAb provided near-complete protection against viremia and lymphoma in a humanized mouse EBV challenge model. Our findings provide structural and antigenic knowledge of the viral fusion machinery, yield a potential therapeutic antibody to prevent EBV disease, and emphasize gH/gL as a target for herpesvirus vaccines and therapeutics.

Anti-viral efficacy of a next-generation CD4-binding site bNAb in SHIV-infected animals in the absence of anti-drug antibody responses.

Broadly neutralizing antibodies (bNAbs) against HIV-1 are promising immunotherapeutic agents for treatment of HIV-1 infection. bNAbs can be administered to SHIV-infected rhesus macaques to assess their anti-viral efficacy; however, their delivery into macaques often leads to rapid formation of anti-drug antibody (ADA) responses limiting such assessment. Here, we depleted B cells in five SHIV-infected rhesus macaques by pretreatment with a depleting anti-CD20 antibody prior to bNAb infusions to reduce ADA. Peripheral B cells were depleted following anti-CD20 infusions and remained depleted for at least 9 weeks after the 1st anti-CD20 infusion. Plasma viremia dropped by more than 100-fold in viremic animals after the initial bNAb treatment. No significant humoral ADA responses were detected for as long as B cells remained depleted. Our results indicate that transient B cell depletion successfully inhibited emergence of ADA and improved the assessment of anti-viral efficacy of a bNAb in a SHIV-infected rhesus macaque model.

A multispecific antibody prevents immune escape and confers pan-SARS-CoV-2 neutralization.

Despite effective countermeasures, SARS-CoV-2 persists worldwide due to its ability to diversify and evade human immunity1. This evasion stems from amino-acid substitutions, particularly in the receptor-binding domain of the spike, that confer resistance to vaccines and antibodies 2-16. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different receptor binding domain (RBD) sites17,18 into multispecific antibodies. Here, we describe multispecific antibodies, including a trispecific that prevented virus escape >3000-fold more potently than the most effective clinical antibody or mixtures of the parental antibodies. Despite being generated before the evolution of Omicron, this trispecific antibody potently neutralized all previous variants of concern and major Omicron variants, including the most recent BA.4/BA.5 strains at nanomolar concentrations. Negative stain electron microscopy revealed that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated inter-spike binding. An optimized trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2 and BA.5, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. Such multispecific antibodies decrease the likelihood of SARS-CoV-2 escape, simplify treatment, and maximize coverage, providing a strategy for universal antibody therapies that could help eliminate pandemic spread for this and other pathogens.

Vaccine-elicited murine antibody WS6 neutralizes diverse beta-coronaviruses by recognizing a helical stem supersite of vulnerability.

Immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike elicits diverse antibodies, but it is unclear if any of the antibodies can neutralize broadly against other beta-coronaviruses. Here, we report antibody WS6 from a mouse immunized with mRNA encoding the SARS-CoV-2 spike. WS6 bound diverse beta-coronavirus spikes and neutralized SARS-CoV-2 variants, SARS-CoV, and related sarbecoviruses. Epitope mapping revealed WS6 to target a region in the S2 subunit, which was conserved among SARS-CoV-2, Middle East respiratory syndrome (MERS)-CoV, and hCoV-OC43. The crystal structure at 2 Å resolution of WS6 revealed recognition to center on a conserved S2 helix, which was occluded in both pre- and post-fusion spike conformations. Structural and neutralization analyses indicated WS6 to neutralize by inhibiting fusion and post-viral attachment. Comparison of WS6 with other recently identified antibodies that broadly neutralize beta-coronaviruses indicated a stem-helical supersite-centered on hydrophobic residues Phe1148, Leu1152, Tyr1155, and Phe1156-to be a promising target for vaccine design.

A SARS-CoV-2 Spike Ferritin Nanoparticle Vaccine Is Protective and Promotes a Strong Immunological Response in the Cynomolgus Macaque Coronavirus Disease 2019 (COVID-19) Model.

The COVID-19 pandemic has had a staggering impact on social, economic, and public health systems worldwide. Vaccine development and mobilization against SARS-CoV-2 (the etiologic agent of COVID-19) has been rapid. However, novel strategies are still necessary to slow the pandemic, and this includes new approaches to vaccine development and/or delivery that will improve vaccination compliance and demonstrate efficacy against emerging variants. Here, we report on the immunogenicity and efficacy of a SARS-CoV-2 vaccine comprising stabilized, pre-fusion spike protein trimers displayed on a ferritin nanoparticle (SpFN) adjuvanted with either conventional aluminum hydroxide or the Army Liposomal Formulation QS-21 (ALFQ) in a cynomolgus macaque COVID-19 model. Vaccination resulted in robust cell-mediated and humoral responses and a significant reduction in lung lesions following SARS-CoV-2 infection. The strength of the immune response suggests that dose sparing through reduced or single dosing in primates may be possible with this vaccine. Overall, the data support further evaluation of SpFN as a SARS-CoV-2 protein-based vaccine candidate with attention to fractional dosing and schedule optimization.

Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron.

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccine-elicited immunity, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring of vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

Safety and tolerability of AAV8 delivery of a broadly neutralizing antibody in adults living with HIV: a phase 1, dose-escalation trial.

Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial ( NCT03374202 ). AAV8-VRC07 was given at doses of 5 × 1010, 5 × 1011 and 2.5 × 1012 vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1-3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml-1 in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases.

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.

Vaccine-elicited murine antibody WS6 neutralizes diverse beta-coronaviruses by recognizing a helical stem supersite of vulnerability.

Immunization with SARS-CoV-2 spike elicits diverse antibodies, but can any of these neutralize broadly? Here, we report the isolation and characterization of antibody WS6, from a mouse immunized with mRNA encoding the SARS-CoV-2 spike. WS6 bound diverse beta-coronavirus spikes and neutralized SARS-CoV-2 variants, SARS-CoV, and related sarbecoviruses. Epitope mapping revealed WS6 to target a region in the S2 subunit, which was conserved among SARS-CoV-2, MERS-CoV, and hCoV-OC43. The crystal structure at 2-Å resolution of WS6 with its S2 epitope revealed recognition to center on a conserved helix, which was occluded in both prefusion and post-fusion spike conformations. Structural and neutralization analyses indicated WS6 to neutralize by inhibiting fusion, post-viral attachment. Comparison of WS6 to other antibodies recently identified from convalescent donors or mice immunized with diverse spikes indicated a stem-helical supersite - centered on hydrophobic residues Phe1148, Leu1152, Tyr1155, and Phe1156 - to be a promising target for vaccine design. HIGHLIGHTS: SARS-CoV-2 spike mRNA-immunized mouse elicited an antibody, WS6, that cross reacts with spikes of diverse human and bat beta-coronavirusesWS6 neutralizes SARS-CoV-2 variants, SARS-CoV, and related virusesCrystal structure at 2-Å resolution of WS6 in complex with a conserved S2 peptide reveals recognition of a helical epitopeWS6 neutralizes by inhibition of fusion, post-viral attachmentWS6 recognizes a supersite of vulnerability also recognized by other recently identified antibodiesHelical supersite of vulnerability comprises a hydrophobic cluster spanning three helical turns, with acid residues framing the center turnGenetic and structural analysis indicate supersite recognition to be compatible with diverse antibody ontogenies.

A SARS-CoV-2 ferritin nanoparticle vaccine elicits protective immune responses in nonhuman primates.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 spike ferritin nanoparticle (SpFN) vaccine in nonhuman primates. High-dose (50 µg) SpFN vaccine, given twice 28 days apart, induced a Th1-biased CD4 T cell helper response and elicited neutralizing antibodies against SARS-CoV-2 wild-type and variants of concern, as well as against SARS-CoV-1. These potent humoral and cell-mediated immune responses translated into rapid elimination of replicating virus in the upper and lower airways and lung parenchyma of nonhuman primates following high-dose SARS-CoV-2 respiratory challenge. The immune response elicited by SpFN vaccination and resulting efficacy in nonhuman primates supports the utility of SpFN as a vaccine candidate for SARS-causing betacoronaviruses.

SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity.

The need for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) next-generation vaccines has been highlighted by the rise of variants of concern (VoCs) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of the prefusion SARS-CoV-2 spike (S), S1, and receptor-binding domain (RBD). These immunogens induce robust S binding, ACE2 inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID50 > 10,000) following a single immunization, whereas RBD-ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations and also show durable and potent neutralization against circulating VoCs. Passive transfer of immunoglobulin G (IgG) purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2-blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens.