Synthetic coevolution reveals adaptive mutational trajectories of neutralizing antibodies and SARS-CoV-2 (preprint)

The Covid-19 pandemic showcases a coevolutionary race between the human immune system and SARS-CoV-2, mirroring the Red Queen hypothesis of evolutionary biology. The immune system generates neutralizing antibodies targeting the SARS-CoV-2 spike protein's receptor binding domain (RBD), crucial for host cell invasion, while the virus evolves to evade antibody recognition. Here, we establish a synthetic coevolution system combining high-throughput screening of antibody and RBD variant libraries with protein mutagenesis, surface display, and deep sequencing. Additionally, we train a protein language machine learning model that predicts antibody escape to RBD variants. Synthetic coevolution reveals antagonistic and compensatory mutational trajectories of neutralizing antibodies and SARS-CoV-2 variants, enhancing the understanding of this evolutionary conflict.

Neutralisation sensitivity of the SARS-CoV-2 BA.2.87.1 variant (preprint)

Against the backdrop of the rapid global takeover and dominance of BA.1/BA.2 and subsequently BA.2.86 lineages, the emergence of a highly divergent SARS-CoV-2 variant warrants characterization and close monitoring. Recently, another such BA.2 descendent, designated BA.2.87.1, was detected in South Africa. Here, we show using spike-pseudotyped viruses that BA.2.87.1 is less resistant to neutralisation by prevailing antibody responses in Sweden than other currently circulating variants such as JN.1. Further we show that a monovalent XBB.1.5-adapted booster enhanced neutralising antibody titers to BA.2.87.1 by almost 4-fold. While BA.2.87.1 may not outcompete other currently-circulating lineages, the repeated emergence and transmission of highly diverged variants suggests that another large antigenic shift, similar to the replacement by Omicron, may be likely in the future.

Multi-compartmental diversification of neutralizing antibody lineages dissected in SARS-CoV-2 spike-immunized macaques (preprint)

The continued evolution of SARS-CoV-2 underscores the need to understand qualitative aspects of the humoral immune response elicited by spike immunization. Here, we combined monoclonal antibody (mAb) isolation with deep B cell receptor (BCR) repertoire sequencing of rhesus macaques immunized with prefusion-stabilized spike glycoprotein. Longitudinal tracing of spike-sorted B cell lineages in multiple immune compartments demonstrated increasing somatic hypermutation and broad dissemination of vaccine-elicited B cells in draining and non-draining lymphoid compartments, including the bone marrow, spleen and, most notably, periaortic lymph nodes. Phylogenetic analysis of spike-specific monoclonal antibody lineages identified through deep repertoire sequencing delineated extensive intra-clonal diversification that shaped neutralizing activity. Structural analysis of the spike in complex with a broadly neutralizing mAb provided a molecular basis for the observed differences in neutralization breadth between clonally related antibodies. Our findings highlight that immunization leads to extensive intra-clonal B cell evolution where members of the same lineage can both retain the original epitope specificity and evolve to recognize additional spike variants not previously encountered.

Full-spike deep mutational scanning helps predict the evolutionary success of SARS-CoV-2 clades (preprint)

SARS-CoV-2 variants acquire mutations in spike that promote immune evasion and impact other properties that contribute to viral fitness such as ACE2 receptor binding and cell entry. Knowledge of how mutations affect these spike phenotypes can provide insight into the current and potential future evolution of the virus. Here we use pseudovirus deep mutational scanning to measure how >9,000 mutations across the full XBB.1.5 and BA.2 spikes affect ACE2 binding, cell entry, or escape from human sera. We find that mutations outside the receptor-binding domain (RBD) have meaningfully impacted ACE2 binding during SARS-CoV-2 evolution. We also measure how mutations to the XBB.1.5 spike affect neutralization by serum from individuals who recently had SARS-CoV-2 infections. The strongest serum escape mutations are in the RBD at sites 357, 420, 440, 456, and 473--however, the antigenic impacts of these mutations vary across individuals. We also identify strong escape mutations outside the RBD; however many of them decrease ACE2 binding, suggesting they act by modulating RBD conformation. Notably, the growth rates of human SARS-CoV-2 clades can be explained in substantial part by the measured effects of mutations on spike phenotypes, suggesting our data could enable better prediction of viral evolution.

Sensitivity of BA.2.86 to prevailing neutralising antibody responses (preprint)

A new SARS-CoV-2 variant, designated BA.2.86, has recently emerged with over 30 spike mutations relative to its parental BA.2, raising questions about its degree of resistance to neutralising antibodies. Using a spike-pseudotyped virus model we characterise neutralisation of BA.2.86 by clinically relevant monoclonal antibodies and by two cohorts of serum sampled from Stockholm, including both a recent cohort, and one sampled prior to the arrival of XBB in Sweden.

Vaccination of SARS-CoV-2-infected individuals expands a broad range of clonally diverse affinity-matured B cell lineages (preprint)

Vaccination of SARS-CoV-2 convalescent individuals generates broad and potent antibody responses. Here, we isolated 459 spike-specific monoclonal antibodies (mAbs) from two individuals who were infected with an early ancestral strain of SARS-CoV-2 and later boosted with mRNA-1273. We characterized mAb genetic features by sequence assignments to the donors’ personal immunoglobulin genotypes and assessed antibody neutralizing activities against ancestral SARS-CoV-2, Beta, Delta, and Omicron variants. The mAbs used a broad range of immunoglobulin heavy chain (IGH) V genes in the response to all sub-determinants of the spike examined, with similar characteristics observed in both donors. IGH repertoire sequencing and B cell lineage tracing at longitudinal time points revealed extensive evolution of SARS-CoV-2 spike-binding antibodies from acute infection until vaccination five months later. These results demonstrate that highly polyclonal repertoires of affinity-matured memory B cells were efficiently recalled by vaccination, providing a basis for the potent antibody responses observed in convalescent persons following vaccination.

MIGH-T: A Multi-Parametric and High-Throughput Platform for Host-Virus Binding Screens (preprint)

Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to the pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here we describe MIGH-T, a multi-parametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform was validated by blocking SARS-CoV-2 spike particles with nanobodies and IgGs from human serum samples.

Omicron sublineage BA.2.75.2 exhibits extensive escape from neutralising antibodies (preprint)

Several sublineages of omicron have emerged with additional mutations that may afford further antibody evasion. Here, we characterise the sensitivity of emerging omicron sublineages BA.2.75.2, BA.4.6, and BA.2.10.4 to antibody-mediated neutralisation, and identify extensive escape by BA.2.75.2. BA.2.75.2 was resistant to neutralisation by Evusheld (tixagevimab + cilgavimab), but remained sensitive to bebtelovimab. In recent serum samples from blood donors in Stockholm, Sweden, BA.2.75.2 was neutralised, on average, five-fold less potently than BA.5, representing the most neutralisation resistant variant evaluated to date. These data raise concerns that BA.2.75.2 may effectively evade humoral immunity in the population.

Evasion of neutralizing antibodies by Omicron sublineage BA.2.75 (preprint)

Towards the end of 2021, SARS-CoV-2 vaccine effectiveness was threatened by the emergence of the Omicron clade (B.1.1.529), with more than 30 mutations in spike. Recently, several sublineages of Omicron, including BA.2.12.1, BA.4, and BA.5, have demonstrated even greater immune evasion, and are driving waves of infections across the globe. One emerging sublineage, BA.2.75, is increasing in frequency in India and has been detected in at least 15 countries as of 19 July 2022. Relative to BA.2, BA.2.75 carries nine additional mutations in spike. Here we report the sensitivity of the BA.2.75 spike to neutralization by a panel of clinically-relevant and pre-clinical monoclonal antibodies, as well as by serum from blood donated in Stockholm, Sweden, before and after the BA.1/BA.2 infection wave. BA.2.75 does not show greater immune evasion than the currently-dominating BA.5 in our set of serum samples, and exhibits moderate susceptibility to tixagevimab and cilgavimab that form a widely used monoclonal antibody cocktail (Evusheld).

Selection analysis identifies unusual clustered mutational changes in Omicron lineage BA.1 that likely impact Spike function (preprint)

Among the 30 non-synonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (i) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (ii) interactions of Spike with ACE2 receptors, and (iii) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron over all previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

Structural basis of Omicron neutralization by affinity-matured public antibodies (preprint)

The SARS-CoV-2 Omicron Variant of Concern (B.1.1.529) has spread rapidly in many countries. With a spike that is highly diverged from that of the pandemic founder, it escapes most available monoclonal antibody therapeutics and erodes vaccine protection. A public class of IGHV3-53-using SARS-CoV-2 neutralizing antibodies typically fails to neutralize variants carrying mutations in the receptor-binding motif, including Omicron. As antibodies from this class are likely elicited in most people following SARS-CoV-2 infection or vaccination, their subsequent affinity maturation is of particular interest. Here, we isolated IGHV3-53-using antibodies from an individual seven months after infection and identified several antibodies capable of broad and potent SARS-CoV-2 neutralization, extending to Omicron without loss of potency. By introducing select somatic hypermutations into a germline-reverted form of one such antibody, CAB-A17, we demonstrate the potential for commonly elicited antibodies to develop broad cross-neutralization through affinity maturation. Further, we resolved the structure of CAB-A17 Fab in complex with Omicron spike at an overall resolution of 2.6 angstroms by cryo-electron microscopy and defined the structural basis for this breadth. Thus, public SARS-CoV-2 neutralizing antibodies can, without modified spike vaccines, mature to cross-neutralize exceptionally antigenically diverged SARS-CoV-2 variants.

Variable loss of antibody potency against SARS-CoV-2 B.1.1.529 (Omicron) (preprint)

The recently-emerged SARS-CoV-2 B.1.1.529 variant (Omicron) is spreading rapidly in many countries, with a spike that is highly diverged from the pandemic founder, raising fears that it may evade neutralizing antibody responses. We cloned the Omicron spike from a diagnostic sample which allowed us to rapidly establish an Omicron pseudotyped virus neutralization assay, sharing initial neutralization results only 13 days after the variant was first reported to the WHO, 8 days after receiving the sample. Here we show that Omicron is substantially resistant to neutralization by several monoclonal antibodies that form part of clinical cocktails. Further, we find neutralizing antibody responses in pooled reference sera sampled shortly after infection or vaccination are substantially less potent against Omicron, with neutralizing antibody titers reduced by up to 45 fold compared to those for the pandemic founder. Similarly, in a cohort of convalescent sera prior to vaccination, neutralization of Omicron was low to undetectable. However, in recent samples from two cohorts from Stockholm, Sweden, antibody responses capable of cross-neutralizing Omicron were prevalent. Sera from infected-then-vaccinated healthcare workers exhibited robust cross-neutralization of Omicron, with an average potency reduction of only 5-fold relative to the pandemic founder variant, and some donors showing no loss at all. A similar pattern was observed in randomly sampled recent blood donors, with an average 7-fold loss of potency. Both cohorts showed substantial between-donor heterogeneity in their ability to neutralize Omicron. Together, these data highlight the extensive but incomplete evasion of neutralizing antibody responses by the Omicron variant, and suggest that increasing the magnitude of neutralizing antibody responses by boosting with unmodified vaccines may suffice to raise titers to levels that are protective.

Conserved recombination patterns across coronavirus subgenera (preprint)

Recombination contributes to the genetic diversity found in coronaviruses and is known to be a prominent mechanism whereby they evolve. It is apparent, both from controlled experiments and in genome sequences sampled from nature, that patterns of recombination in coronaviruses are non-random and that this is likely attributable to a combination of sequence features that favour the occurrence of recombination breakpoints at specific genomic sites, and selection disfavouring the survival of recombinants within which favourable intra-genome interactions have been disrupted. Here we leverage available whole-genome sequence data for six coronavirus subgenera to identify specific patterns of recombination that are conserved between multiple subgenera and then identify the likely factors that underlie these conserved patterns. Specifically, we confirm the non-randomness of recombination breakpoints across all six tested coronavirus subgenera, locate conserved recombination hot- and cold-spots, and determine that the locations of transcriptional regulatory sequences are likely major determinants of conserved recombination breakpoint hot-spot locations. We find that while the locations of recombination breakpoints are not uniformly associated with degrees of nucleotide sequence conservation, they display significant tendencies in multiple coronavirus subgenera to occur in low guanine-cytosine content genome regions, in non-coding regions, at the edges of genes, and at sites within the Spike gene that are predicted to be minimally disruptive of Spike protein folding. While it is apparent that sequence features such as transcriptional regulatory sequences are likely major determinants of where the template-switching events that yield recombination breakpoints most commonly occur, it is evident that selection against misfolded recombinant proteins also strongly impacts observable recombination breakpoint distributions in coronavirus genomes sampled from nature.

Antigen receptor stimulation drives selection against pathogenic mtDNA variants that dysregulate lymphocyte responses (preprint)

Pathogenic mitochondrial (mt)DNA molecules can exhibit heteroplasmy in single cells and cause a range of clinical phenotypes, although their contribution to immunity is poorly understood. Here, in mice carrying heteroplasmic C5024T in mt-tRNA Ala – that impairs oxidative phosphorylation – we found a reduced mutation burden in peripheral T and B memory lymphocyte subsets, compared to their naïve counterparts. Furthermore, selection diluting the mutation was induced in vitro by triggering T and B cell antigen receptors. While C5024T dysregulated naïve CD8 + T cell respiration and metabolic remodeling post-activation, these phenotypes were partially ameliorated by selection. Analogous to mice, peripheral blood memory T and B lymphocyte subsets from human MELAS (Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like episodes) patients – carrying heteroplasmic A3243G in mt-tRNA Leu – displayed a reduced mutation burden, compared to naïve cells. In both humans and mice, mtDNA selection was observed in IgG + antigen-specific B cells after SARS-CoV-2 Spike vaccination, illustrating an on-going process in vivo . Taken together, these data illustrate purifying selection of pathogenic mtDNA variants during the oxidative phosphorylation checkpoints of the naïve-memory lymphocyte transition. Highlights In human MELAS patients (A3243G in mt-tRNA Leu ) and a related mouse model (C5024T in mt-tRNA Ala ), T and B memory subsets displayed a reduced mtDNA mutation burden compared to their naïve counterparts. Selection was observed in antigen-specific IgG + B cells after SARS-CoV-2 Spike protein vaccination. T and B cell antigen receptor stimulation triggered purifying selection in vitro , facilitating mechanistic studies of mtDNA selection. Heteroplasmic pathogenic mutations in mtDNA dysregulated metabolic remodeling after lymphocyte activation and reduced macrophage OXPHOS capacity.

Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies (preprint)

Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 g/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

Driving potent neutralization of a SARS-CoV-2 Variant of Concern with a heterotypic boost (preprint)

The emergence of SARS-CoV-2 Variants of Concern (VOCs) with mutations in key neutralizing antibody epitopes threatens to undermine vaccines developed against the pandemic founder variant (Wu-Hu-1). Widespread vaccine rollout and continued transmission are creating a population that has antibody responses of varying potency to Wu-Hu-1. Against this background, it is critical to assess the outcomes of subsequent booster vaccination with variant antigens. It is not yet known whether such heterotypic vaccine boosts would be compromised by original antigenic sin, where pre-existing responses to a prior variant dampen responses to a new one, or whether the primed memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. Here, we show that a single adjuvanted dose of receptor binding domain (RBD) protein from VOC 501Y.V2 (B.1.351) drives an extremely potent neutralizing antibody response capable of cross-neutralizing both Wu-Hu-1 and 501Y.V2 in rhesus macaques previously immunized with Wu-Hu-1 spike protein.

The emergence and ongoing convergent evolution of the N501Y lineages coincides with a major global shift in the SARS-CoV-2 selective landscape (preprint)

The emergence and rapid rise in prevalence of three independent SARS-CoV-2 '501Y lineages', B.1.1.7, B.1.351 and P.1, in the last three months of 2020 has prompted renewed concerns about the evolutionarily capacity of SARS-CoV-2 to adapt to both rising population immunity and public health interventions such as vaccines and social distancing. Viruses giving rise to the different 501Y lineages have, presumably under intense natural selection following a shift in host environment, independently acquired multiple unique and convergent mutations. As a consequence all have gained epidemiological and immunological properties that will likely complicate the control of COVID-19. Here, by examining patterns of mutations that arose in SARS-CoV-2 genomes during the pandemic we find evidence of a major change in the selective forces acting on immunologically important SARS-CoV-2 genes (such as N and S) that likely coincided with the emergence of the 501Y lineages. In addition to involving continuing sequence diversification, we find evidence that a significant portion of the ongoing adaptive evolution of the 501Y lineages also involves further convergence between the lineages. Our findings highlight the importance of monitoring how members of these known 501Y lineages, and others still undiscovered, are convergently evolving similar strategies to ensure their persistence in the face of mounting infection and vaccine induced host immune recognition.

Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2 (preprint)

Isolation and characterization of antibodies in COVID-19 patients has largely focused on memory B cells, however it is the antibody-secreting plasma cells that are directly responsible for the production of serum antibodies, which play a critical role in controlling and resolving SARS-CoV-2 infection. To date there is little known about the specificity of plasma cells in COVID-19 patients. This is largely because plasma cells lack surface antibody expression, which complicates their screening. Here, we describe a technology pipeline that integrates single-cell antibody repertoire sequencing and high-throughput mammalian display screening to interrogate the specificity of plasma cells from 16 convalescent COVID-19 patients. Single-cell sequencing allows us to profile antibody repertoire features in these patients and identify highly expanded clonal lineages. Mammalian display screening is employed to reveal that 37 antibodies (out of 132 candidates) derived from expanded plasma cell clonal lineages are specific for SARS-CoV-2 antigens, including antibodies that target the receptor binding domain (RBD) with high affinity and exhibit potent neutralization of SARS-CoV-2. One Sentence SummarySingle-cell antibody repertoire sequencing and high-throughput screening identifies highly expanded plasma cells from convalescent COVID-19 patients that produce SARS-CoV-2-specific antibodies capable of potent neutralization.

Seropositivity in blood donors and pregnant women during 9-months of SARS-CoV-2 transmission in Stockholm, Sweden (preprint)

In Sweden, social restrictions to contain SARS-CoV-2 have to date primarily relied upon voluntary adherence to a set of recommendations and strict lockdowns/regulations have not been enforced, potentially affecting viral dissemination. To understand the levels of past SARS-CoV-2 infection in the Stockholm population before the start of mass vaccinations, healthy blood donors and pregnant women (n=5,100) were sampled at random between 14th March 2020-28th February 2021. All individuals (n=200/sampling week) were screened for anti-SARS-CoV-2 spike (S) trimer- and RBD-specific IgG responses and the results were compared with those from historical controls (n=595). Data were modelled using a probabilistic Bayesian framework that considered individual responses to both viral antigens. We found that after a steep rise at the start of the pandemic, the seroprevalence trajectory increased more steadily (over summer) in approach to the winter second-wave of infections, approaching 15% of all adults surveyed by mid-December 2020. The population seropositivity rate again increased more rapidly as cases rose over the winter period. By the end of February 2021, [~]19% ([~]one-in-five) in this study group tested seropositive. Notably, 96% of random seropositive samples screened (n=56), displayed virus neutralizing responses, with titers comparable to those engendered by recently approved mRNA vaccines, supporting that milder infections generally provoke a competent B cell response. These data offer baseline information about the level of seropositivity in this group of active adults in the Stockholm metropolitan area following a full year of SARS-CoV-2 transmission and prior to the introduction of vaccines. Structured abstractO_ST_ABSObjectivesC_ST_ABSSweden did not enforce social lockdown in response to the SARS-CoV-2 pandemic. Therefore, we sought to determine the proportion of seropositive healthy, active adults in Stockholm, the countrys most populous region. Random sampling (of blood donors and pregnant women) was carried out during the first year following virus emergence in the country and prior to vaccination of the general adult population - allowing for an estimate of seroprevalence in response to natural infection. DesignIn this cross-sectional prospective study, otherwise-healthy blood donors (n=2,600) and pregnant women(n=2,500) were sampled at random for consecutive weeks (at four intervals) between 14th March and 28th February 2021. Sera from all participants and a cohort of historical controls (n=595) were screened for IgG responses against trimers of the SARS-CoV-2 spike (S) glycoprotein and the smaller receptor-binding domain (RBD). As a complement to standard analytical approaches, a probabilistic (cut-off-independent) Bayesian framework that assigns likelihood of past infection was used to analyze data over time. The study was carried out in accordance with Swedish Ethical Review Authority: registration number 2020-01807. SettingHealthy participant samples were selected from their respective pools at random through Karolinska University Hospital. ParticipantsNone of the participants were symptomatic at sampling. No additional metadata was available from the samples. ResultsBlood donors and pregnant women showed a similar seroprevalence. After a steep rise at the start of the pandemic, the seroprevalence trajectory increased steadily in approach to the winter second-wave of infections, approaching 15% of all individuals surveyed by 13th December 2020. By the end of February 2021, when deaths were in decline and at low levels following their winter peak, 19% of the population tested seropositive. Notably, 96% of seropositive healthy donors screened (n=56) developed neutralizing antibody responses at titers comparable to, or higher than those observed in clinical trials of SARS-CoV-2 spike mRNA vaccination, supporting that mild infection engenders a competent B cell response. ConclusionsThese data indicate that in the year since the start of community transmission, seropositivity levels in metropolitan Stockholm had reached approximately one-in-five persons, providing important baseline seroprevalence information prior to the start of vaccination.

SARS-CoV-2 protein subunit vaccination elicits potent neutralizing antibody responses (preprint)

The outbreak and spread of SARS-CoV-2 (Severe Acute Respiratory Syndrome coronavirus 2), the cause of coronavirus disease 2019 (COVID-19), is a current global health emergency and a prophylactic vaccine is needed urgently. The spike glycoprotein of SARS-CoV-2 mediates entry into host cells, and thus is a target for neutralizing antibodies and vaccine design. Here we show that adjuvanted protein immunization with SARS-CoV-2 spike trimers, stabilized in prefusion conformation 1, results in potent antibody responses in mice and rhesus macaques with neutralizing antibody titers orders of magnitude greater than those typically measured in serum from SARS-CoV-2 seropositive humans. Neutralizing antibody responses were observed after a single dose, with exceptionally high titers achieved after boosting. Furthermore, neutralizing antibody titers elicited by a dose-sparing regimen in mice were similar to those obtained from a high dose regimen. Taken together, these data strongly support the development of adjuvanted SARS-CoV-2 prefusion-stabilized spike protein subunit vaccines.