key: cord-1055184-t6fa60aq
authors: Sheward, Daniel J.; Mandolesi, Marco; Urgard, Egon; Kim, Changil; Hanke, Leo; Vidakovics, Laura Perez; Pankow, Alec; Smith, Natalie L.; Dopico, Xaquin Castro; McInerney, Gerald M.; Coquet, Jonathan M.; Karlsson Hedestam, Gunilla B.; Murrell, Ben
title: Beta RBD Boost Broadens Antibody-Mediated Protection Against SARS-CoV-2 Variants in Animal Models
date: 2021-10-23
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2021.100450
sha: 2d8a728d770e10221d8599b402a2b79102ea056d
doc_id: 1055184
cord_uid: t6fa60aq

SARS-CoV-2 Variants of Concern (VOCs) with resistance to neutralizing antibodies are threatening to undermine vaccine efficacy. Vaccination and infection have led to widespread humoral immunity against the pandemic founder (Wu-Hu-1). Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet clear whether heterotypic 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 memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. We show, in macaques immunized with Wu-Hu-1 spike, that a single dose of adjuvanted beta variant receptor binding domain (RBD) protein broadens neutralizing antibody responses to heterologous VOCs. Passive transfer of plasma sampled after Wu-Hu-1 spike immunization only partially protects K18-hACE2 mice from lethal challenge with a beta variant isolate, whereas plasma sampled following heterotypic RBD boost protects completely against disease.

Challenge A number of these demonstrated high efficacy [1] [2] [3] [4] [5] , significantly reducing morbidity and mortality, and are being rolled-out globally. This first generation of vaccines all encode or deliver a spike glycoprotein derived from the pandemic founder strain, Wu-Hu-1 6 .

Driven by multiple evolutionary forces 7 , SARS-CoV-2 is evading immune responses and threatening to undermine current prevention and mitigation strategies. Globally, novel VOCs are increasingly dominating the pandemic (Fig. 1) . Of particular concern is the surge of variants harbouring spike mutations that confer resistance to prior immunity, such as 501Y.V2 (B.1.351, "beta") [8] [9] [10] [11] . This underpins the substantially reduced vaccine efficacies observed in trials in South Africa, where this variant was circulating at high frequency 12, 13 . Recently, significant numbers of vaccine breakthrough infections have been observed during infection waves dominated by the delta (B.1.617.2) variant, which also displays reduced sensitivity to neutralization [14] [15] [16] . Updated vaccines are likely required to protect against current and future mutated variants. Importantly, by the time these are rolled out, a significant proportion of the global population are likely to be seropositive from either infection, or immunization with Wu-Hu-1 based vaccines. A relevant question now is whether a single additional dose will be sufficient to induce robust neutralising antibody responses to VOCs in seropositive individuals, and whether these boosts are sufficient to confer protection. Importantly, the first exposure to a pathogen can shape future responses to mutated variants. This immunological imprinting or original antigenic sin 17 is well-described for influenza A virus, where protection is highest against the first strain encountered and diminished against those encountered later in life 18, 19 . It is crucial for the design of updated vaccines and regimens to determine if existing immunity dampens antibody responses to new VOCs, or if a heterotypic boost can efficiently recruit cross-protective memory responses.

To address this, we immunized three rhesus macaques with two doses of soluble prefusionstabilized Wu-Hu-1 spike protein (2 μg), adjuvanted with saponin-based Matrix-M™ (Novavax AB, Uppsala, Sweden), with a one-month interval between doses, mimicking an immunization schedule for approved SARS-CoV-2 vaccines. After a single dose, neutralizing antibodies were detectable against Wu-Hu-1 but not against the beta variant (Fig. 2) . Neutralizing antibody responses against Wu-Hu-1 were substantially boosted by the second immunization ("post S", GMT = 3980 at peak), and then waned over the following months (Fig. 2) , as also reported in immunized humans 20 . Notably, the beta VOC was on average 9-fold (range: 5.6 -12.2 fold) less potently neutralized (GMT = 451 at peak), consistent with the responses observed in humans following vaccination [9] [10] [11] .

Six months after their first immunization, macaques were boosted with either 2 μg (H05), 10 μg (H06), or 50 μg (H07) of soluble 501Y.V2 RBD in 50 μg Matrix-M™ adjuvant. One macaque (H05) was terminated 5 days after immunization, due to an unrelated illness that had begun prior to the third immunization, and was sampled for detailed follow-up studies of antibody specificities. The two other macaques (H06 and H07) were followed for 4 weeks. In all three animals, beta RBD efficiently boosted responses (Supp. Fig. 1 ) that potently cross-neutralized both Wu-Hu-1 and beta, with similar titers (Fig. 2a -c; Wu-Hu-1 GMT = 11795, beta GMT = 12595). This suggests that the heterotypic boost may have preferentially recruited cross neutralizing memory B cells. Indeed, heterotypic boost improved neutralization of other VOCs, including delta, alpha, and gamma, as well as the more distantly related SARS-CoV-1 (Fig. 2c) . In contrast, for macaques previously immunized with three doses of Wu-Hu-1 spike 21 , the reduced neutralization of beta (and other VOCs) compared to Wu-Hu-1 remained after the third homotypic spike immunization (Supp. Fig. 2 ).

To determine whether recovery of neutralizing antibody titers to beta afforded a biologically relevant improvement in protective immunity, mice transgenic for human ACE2 (K18-hACE2, 22 ) were passively immunized intraperitoneally (i.p.) with plasma samples taken either 2 weeks following the second spike immunization (N=8) ("post S"), or 1-2 weeks following the RBD booster immunization (N=8) ("post vRBD"). Passive immunization conferred titers approximately 10-fold lower than donor plasma (Supp. Fig. 3 ), and macaque polyclonal antibodies were not rapidly cleared following xenotransfusion with an unchallenged mouse still maintaining titers >1400 after 5 days (data not shown). Mice were then challenged intranasally with 2.4x10 6 RNA copies of either beta or 'wild-type' (encoding a spike matching Wu-Hu-1) virus (corresponding to 100 PFU of beta or 86 PFU of wild-type), and monitored for weight loss, a reliable proxy for disease severity 23 (Fig. 3) .

Across all groups, protection was strongly correlated with the neutralizing antibody titers to the challenge virus on the day of challenge (Spearman's ρ = 0.822, p<1x10 -8 , Fig. 3c ). All control mice that did not receive plasma (PBS only) succumbed to disease when challenged with either variant, showing precipitous weight loss starting around three days post-challenge ( Fig. 3a, b,d) .

Passive transfer of post S plasma conferred complete protection against weight loss following infection with WT virus (Fig. 3a , d) but not from beta ( Fig. 3b, d) , clearly demonstrating that evasion of the antibody response by this VOC was sufficient to cause disease. Notably, passive transfer of post vRBD plasma protected against both WT and beta ( Fig. 3a-d) . Analysis of lung viral loads at day 14 post-infection demonstrated heightened viral clearance in mice transfused with post vRBD plasma, in particular in mice infected with beta ( Fig. 3e ). In addition, Hematoxylin & Eosin staining was performed on lungs 14 days post-infection to assess the pathological effects of virus infection. WT and beta virus both induced signs of alveoli septa thickening and infiltration of inflammatory cells, although mice infected with beta also demonstrated a loss in general tissue architecture (Supp. Fig. 5, Fig. 3f ). Even among only the surviving mice, those transfused with post vRBD plasma displayed significantly reduced pathological scores following Beta infection than did mice that received post S plasma (Fig. 3f ).

For many licensed SARS-CoV-2 vaccines, reduced efficacy has been observed against the beta variant 12, 24, 25 . Moreover, the decay of vaccine-elicited antibody titers 20, 26 suggests that over time, protection will wane further. Consistent with reduced vaccine efficacy against beta, we show that breakthrough infection and disease occurs in K18-hACE2 mice passively immunized with plasma from rhesus macaques immunized with two doses of adjuvanted wild-type spike protein.

Cross-species passive immunization and challenge experiments have been used to show antibody-mediated protection conferred by influenza vaccination (human to mouse, 27 ) , and more recently, to characterize protection from wild-type SARS-CoV-2 (macaque to hamster, 28 ). Our data further demonstrate the utility of this approach for investigating protection against SARS-CoV-2 variants.

The ability of vaccines to broaden existing responses to new variants is still largely unclear.

Despite weak immunogenicity of soluble, monomeric RBD as a priming antigen 21 , heterotypic RBD administered as a boost elicited a potent recall response in non-human primates. This was robust to the boosting dose, and effective as low as 2 μg, possibly aided by a dose-sparing effect of Matrix-M 29 . While reduced neutralization of beta was evident following 2 doses of Wu-Hu-1 spike, both Wu-Hu-1 and beta were potently neutralized following heterotypic (beta) RBD boost. In animal challenge models, neutralizing antibodies following passive immunization represented a robust correlate of protection such that the restoration of neutralizing antibody titers to beta also translated into protective immunity.

The potent, cross-neutralizing antibody response that arises following a heterotypic boost indicates that original antigenic sin does not represent a significant barrier to the acquisition of protective immunity against current SARS-CoV-2 VOCs. This is largely consistent with recently reported results from beta spike encoding mRNA (mRNA1273.351, Moderna) booster vaccinations 30, 31 . The observation that immunization with RBD (and not whole spike) was capable of inducing robust neutralising antibody responses is particularly promising as RBD is a small, stable protein that can be rapidly synthesized and efficiently expressed. Here, in the immunized animal sampled only 5 days post vRBD boost, neutralizing titers (against both Wu-Hu-1 and beta) were already elevated, suggesting these titers are the product of a rapidly activated population of antibody secreting cells. Further, this time course indicates that successive rounds of affinity maturation likely were not required for neutralization of beta, but rather that vRBD-specific antibody responses could be boosted from the pool of existing crossneutralizing memory B cells primed by Wu-Hu-1. This is consistent with the neutralization of multiple VOCs (including alpha, gamma, and delta) with comparable potency. Crossneutralization of delta is particularly important, given the high transmissibility and global prevalence of this variant.

Taken together, these data indicate that potent, cross-neutralizing and cross-protective antibody responses can be recruited with heterotypic SARS-CoV-2 immunogens following a primary exposure, and identify soluble RBD booster immunizations as an attractive strategy to broaden vaccine protection from new SARS-CoV-2 variants.

While we show that a heterotypic RBD immunization can efficiently boost protective crossneutralizing antibody responses, these results are based on only a small number of non-human primates, and we did not characterize the development and breadth of individual antibody lineages. Furthermore, we did not have a well-controlled, homotypic boost arm for comparison. The animals in the previous study that received three doses of wild-type spike were immunized with a substantially higher dose, and received the third dose significantly sooner, preventing a direct comparison. Thus, while a heterotypic RBD boost elicits potent cross-neutralizing responses, further work is required to quantify the benefits of this approach over a third homotypic spike (or RBD) boost. 

Further information and requests for resources or reagents should be directed to and will be fulfilled by the Lead Contact, Ben Murrell (benjamin.murrell@ki.se).

All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Original data reported in this paper will be shared by the Lead Contact upon request.

All original code has been deposited at https://github.com/MurrellGroup/VOCfreq and is publicly available as of the date of publication. DOIs are listed in the key resources table.

Any additional information required to reanalyze the data reported in this work paper is available from the Lead Contact upon request.

The animal work was conducted with the approval of Stockholms Jordbruksverket (10513-2020, 18427-2019 and 10895-2020). All animal procedures were performed according to approved guidelines.

Rhesus macaques. Three female rhesus macaques (Macaca mulatta) of Chinese origin, 5-6 years old, were housed at the Astrid Fagraeus Laboratory at Karolinska Institutet. Housing and care procedures complied with the provisions and general guidelines of the Swedish Board of Agriculture. The facility has been assigned an Animal Welfare Assurance number by the Office of Laboratory Animal Welfare (OLAW) at the National Institutes of Health (NIH). The macaques were housed in groups in enriched 14 m 3 cages. They were habituated to the housing conditions for more than six weeks before the start of the experiment and subjected to positive reinforcement training in order to reduce the stress associated with experimental procedures. The macaques were weighed at each sampling. All animals were confirmed negative for simian immunodeficiency virus, simian T cell lymphotropic virus, simian retrovirus type D and simian herpes B virus.

K18-hACE2 mice. Mice transgenic for human ACE2 under control of the cytokeratin 18 (K18) promoter 22 were obtained from the Jackson Laboratory. Mice were maintained as a hemizygous line, with hACE2 transgene presence confirmed using Sanger sequencing as per the Jackson Laboratory protocol. All mice were 12-21 weeks old at the start of the study, and experiments were conducted in BSL3 facilities at the Comparative Medicine department (KM-F) J o u r n a l P r e -p r o o f at Karolinska Institutet. Mice (24 male, 20 female) were housed in individually ventilated cages, had access to food and water ad libitum, and cage enrichment included shredded cardboard and paper rolls. Cage and water changes were performed on a weekly basis and general monitoring of all mice was performed daily by technical staff.

HEK293T and HEK293T-hACE2 (Human, female) cells were cultured in a humidified 37°C incubator (5% CO 2 ) in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin, and were passaged when nearing confluency using 1X Trypsin-EDTA.

Calu-3 cells (Human, male) , a lung-derived adenocarcinoma cell line, were obtained from Jonas Klingstrom (Karolinska Institutet). Calu-3 cells were maintained in Dulbecco's Modified Eagle Medium supplemented with Ham's F-12 (Thermo Fisher Scientific), 2% Fetal Bovine Serum and 1% Penicillin/Streptomycin in a humidified 37°C incubator (5% CO 2 ) and were passaged using 0.5X Trypsin-EDTA.

Vero E6 cells (ATCC-CRL-1586, African Green Monkey) were maintained in DMEM (Gibco) supplemented with 2% fetal calf serum and 1% penicillin-streptomycin in a humidified incubator with 5% CO 2 at 37°C.

501Y.V2 RBD (encoding amino acid mutations K417N, E484K, and N501Y, and a C-terminal His-tag) was synthesized (Integrated DNA Technologies), and cloned into a mammalian expression vector (pcDNA3.1), using a Gibson Assembly Mastermix (New England Biolabs). Spike ectodomain (prefusion stabilized with 6 prolines 34 ) and RBD were produced by the transient transfection of Freestyle 293-F cells using FreeStyle MAX reagent (Thermo Fisher) or polyethylenimine (PEI), respectively. The HIS-tagged Spike ectodomain and RBD were purified from filtered supernatant using nickel IMAC resin (HisPur Ni-NTA, Thermo Fisher Scientific) followed by size-exclusion chromatography on a Superdex 200 (Cytiva) in PBS. On the day of immunization, indicated doses were mixed with 50 μg Matrix-M™ adjuvant (Novavax AB, Uppsala, Sweden) in a final inoculation volume of 800 μl.

Macaques were immunized intramuscularly (i.m.) with half of each dose administered in each quadricep. All immunizations and blood samplings were performed under sedation with 10-15 mg/kg ketamine (Ketaminol, Intervet, Sweden) administered i.m.. Blood plasma was isolated by centrifugation, and heat inactivated at 56°C for 60 minutes.

J o u r n a l P r e -p r o o f All plasma and serum samples were heat inactivated at 56°C for 60 minutes. Pseudotyped lentiviruses displaying spikes (with C-terminal truncations) from the SARS-CoV-2 pandemic founder variant (Wu-Hu-1) 35 or from variants of concern 36-38 and packaging a firefly luciferase reporter gene were generated by the co-transfection of HEK293T cells using Lipofectamine 3000 (Invitrogen) per the manufacturer's protocols. Media was changed 12-16 hours after transfection, and pseudotyped viruses were harvested at 48-and 72-hours post-transfection, clarified by centrifugation, and stored at -80°C until use. Pseudotyped viruses sufficient to generate ~50,000 relative light units (RLUs) were incubated with serial dilutions of plasma for 60 min at 37°C in a 96-well plate, and then ~15,000 HEK293T-hACE2 cells were added to each well. Plates were incubated at 37°C for 48 hours, and luminescence was then measured using Bright-Glo (Promega) per the manufacturer's protocol, on a GM-2000 luminometer (Promega).

Viral isolates expanded from clinical samples of 'wild-type' and 501Y.V2 39 were kind gifts from Jonas Klingström (Karolinska Institutet) and Alex Sigal (The African Health Research Institute) respectively. Isolates were propagated in Calu-3 cells for 72 hours and harvested from the supernatant to generate replication-competent SARS-CoV-2 stocks for animal challenge. All virus challenge stocks harboured no cell-culture adaptation mutations detectable by Sanger sequencing of the full-length spike gene (see Supp. Fig. 4 ). Viral titers were quantified by a plaque-forming assay in Vero E6 cells, as previously described 40 .

Viral RNA was quantified from clarified viral supernatant in TRIzol (ThermoFisher Scientific) and extracted using the manufacturer's protocol but with the following modifications: Total RNA was precipitated with isopropanol in the presence of Glycoblue (Thermo Fisher Scientific) coprecipitant for 45 min at -20 ० C, and RNA pellets were resuspended in warm RNase-free water.

RT-qPCR reactions were performed using the Superscript III one step RT-qPCR system with Platinum Taq Polymerase (Invitrogen) with 400 nM of each primer and 200 nM of probe. Primers and probes for the CoV-E gene target were as previously described 41 . Thermal cycling consisted of RT at 55 ० C for 10 min, denaturation at 95 ० C for 3 min, and 45 cycles of 95 ० C for 15 seconds and 58 ० C for 30 seconds. Reactions were carried out using a CFX96 Connect Real-Time PCR Detection System (Bio-Rad) following manufacturer instructions. To generate standard curves, a synthetic DNA template gBlock (Integrated DNA Technologies) was transcribed using the mMessage mMachine T7 Transcription Kit (Invitrogen) and serially diluted. For each viral stock, RT-qPCR was run on a 10-fold dilution series ranging from 1:10 to 1:10,000 to ensure linearity of the assay and avoid saturation at high copy numbers.

One day prior to challenge, K18-hACE2 mice were passively immunized with 200 μl of macaque plasma, administered intraperitoneally (i.p.) under isoflurane sedation. Each plasma sample was administered to four mice (two to be challenged with wild-type virus, and two to be challenged with 501Y.V2). The following day, mice were bled from the tail vein (to obtain serum for the characterization of neutralizing antibody titers), moved to a BSL3 facility and challenged J o u r n a l P r e -p r o o f intranasally with a standardized dose (2.4x10 6 RNA copies) of either 'wild-type' or 501Y.V2 virus stock in a total challenge volume of 40 μl in PBS. All challenges were performed under light isoflurane sedation. Weight and general body condition was monitored daily until weight loss was evident, after which mice were monitored twice daily. Throughout the experiment, weight loss, changes in general health, breathing, body movement and posture, piloerection and eye health were monitored. Mice were euthanized when they experienced weight loss of at least 20% of their starting body weight, or when movement was greatly impaired and/or they experienced difficulty breathing that was considered to reach a severity level of 0.5 on Karolinska Institutet's veterinary plan for monitoring animal health.

For plotting maps in Figure 1 , SARS-CoV-2 lineage metadata was obtained from GISAID (gisaid.org -2021-10-09 metadata release), comprising 4,197,215 genomes. Pango lineages without WHO labels were collapsed up the Pango lineage tree until a minimum of 50,000 observations for each lineage was obtained, and daily counts of genomes were aggregated at the country level. We used a multinomial regression to infer the frequencies at three selected dates, weighting the observations around the target dates using a radial basis function. Note: the lineage frequencies vary over time, but the relative advantage of one variant over another is assumed by the model to be locally constant around the target date. Inference was performed with the GLMNet.jl Julia package, using an alpha = 0.5, and maps were plotted with the Cartopy python package (https://github.com/SciTools/cartopy). Jupyter notebooks implementing this are available at https://github.com/MurrellGroup/VOCfreq.

Neutralizing antibody ID 50 titers were calculated in Prism 9 (GraphPad Software) by fitting a four-parameter logistic curve bounded between 0 and 100, and interpolating the reciprocal plasma/serum dilution where RLUs were reduced by 50% relative to control wells (N=8) in the absence of plasma.

Pulmonary pathology in blinded H&E-stained lung sections (N=89 slides in total, with a mean of 3.3 slides per mouse (range: 2-5) was scored according to a four-point scale, as described previously 42 . Briefly, normal architecture with thin alveolar septa and distinct bronchioles is scored as 0. Sections with thickened alveolar septa and reduced alveolar space are scored as 1. Thickening of the alveolar septa with reduced alveolar space and invasion of inflammatory cells is scored as 2. Sections displaying widespread loss of tissue architecture are scored as 3. The score for each animal represents the mean of the pathology scores for all sections from that animal. Representative images from each group are shown in Supp. Fig. 5 .

J o u r n a l P r e -p r o o f All statistical analyses were performed in Prism 9 (Graphpad Software) unless otherwise stated.

Differences in day 6 weights between groups were assessed using Mann-Whitney tests.

Reduction in pathology scores was assessed with a one-tailed Mann-Whitney test. Depicted symbols summarize the following: ns = p > 0.05 (not significant), * p ≤ 0.05, * * p ≤ 0.01, * * * p ≤ 0.001. The association between neutralizing antibody titers and weight loss was assessed with a Spearman's rank correlation. 

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We thank Dr. Bengt Eriksson and all personnel at Astrid Fagraeus laboratory for expert assistance with rhesus macaques. We thank Monika Àdori for assistance processing samples. We also thank Novavax, AB, Uppsala, Sweden, for generously making the Matrix-M™ adjuvant available. We gratefully thank James Voss, Deli Huang, and Jesse Bloom for reagents. We

The authors declare no competing interests J o u r n a l P r e -p r o o f