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Broad protection against clade 1 sarbecoviruses after a single immunization with cocktail spike-protein-nanoparticle vaccine – Nature Communications

Expression and purification of S proteins

DNA encoding HexaPro34 prefusion-stabilized versions of the S ectodomains of SARS-CoV-2 614D (YP_009724390.1 [https://www.ncbi.nlm.nih.gov/protein/YP_009724390.1/], residues 1–1208), Omicron BA.1 (UFO69279.1 [https://www.ncbi.nlm.nih.gov/protein/UFO69279.1/], residues 1–1205), Omicron BA.5.5 (UPI46221.1 [https://www.ncbi.nlm.nih.gov/protein/UPI46221.1/], residues 1–1203), Omicron BA.2.75.2 (UVJ48842.1 [https://www.ncbi.nlm.nih.gov/protein/UVJ48842.1/], residues 1–1205), Omicron XBB (UZS22117.1 [https://www.ncbi.nlm.nih.gov/protein/UZS22117.1/], residues 1–1204), SARS-CoV-1 Urbani (P59594 [https://www.ncbi.nlm.nih.gov/protein/P59594/], residues 1–1190), and SHC014 (AGZ48806.1 [https://www.ncbi.nlm.nih.gov/protein/AGZ48806.1/], residues 1–1191) were created by mutating the S1/S2 cleavage site from RRAR (SARS-CoV-2 and Omicron variants), SLLR (SARS-CoV-1) or SSLR (SHC014) to GSAS and by introducing six proline mutations based on the HexaPro SARS-CoV-2 S (Table S1). DNA segments encoding each HexaPro prefusion-stabilized ectodomain together with a C-terminal T4 fibritin trimerization motif, AviTag, and a his-tag were cloned into pcDNA3.1 between the NcoI and XhoI restriction sites by Gene Universal Inc. (Newark, DE). Expi293F cells (Thermo Fisher Scientific) were transfected with these plasmids using the ExpiFectamine Transfection Kit using the manufacturer’s protocol (Thermo Fisher Scientific). Six days after transfection, the cells were centrifuged at 6000 x g for 15 min, and the supernatant was removed. Then, the supernatant was dialyzed into PBS overnight and loaded onto 1 mL of HisPure Ni-NTA resin (Thermo Fisher Scientific) in a gravity flow column (G-Biosciences). The column was washed with 90 mL of binding buffer (150 mM Tris, 150 mM NaCl, 20 mM Imidazole, pH 8). Three mL of elution buffer (150 mM Tris, 150 mM NaCl, 400 mM Imidazole, pH 8) were added to the resin and allowed to incubate for 5 min. The elution buffer was then allowed to flow out of the column, and the elution process was repeated two additional times for a total eluate volume of 9 mL. The eluate was concentrated with a 10 kDa MWCO spin filter (Millipore Sigma), then purified on a Superdex 200 Increase 10/30 column in 20 mM Tris, 20 mM NaCl, pH 8 buffer. Fractions containing S protein were concentrated again and quantified using the bicinchoninic acid assay (BCA) assay (Thermo Scientific).

Expression and purification of MS2

The DNA encoding the single chain MS2 coat protein dimer was cloned into pET-28b between the Ndel and XhoI restriction sites, and an AviTag was inserted between residues 14 and 15 of the second monomer by GenScript Biotech Corporation (Piscataway, NJ)14,29. The MS2-AviTag plasmid was co-transformed with a plasmid containing BirA biotin-protein ligase into BL21(DE3) competent E. coli (New England Biolabs) according to the manufacturer’s protocol. The transformation was added to 5 mL of 2xYT media and grown overnight at 37 °C. The 5 mL starter culture was added to 1 L of 2xYT media and incubated at 37 °C and 225 rpm until the optical density reached 0.6. The culture was induced with 1 mM IPTG (Fisher BioReagents). At the same time, D-biotin (50 µM) was added, and the incubator temperature was reduced to 30 °C. The culture was incubated overnight then centrifuged at 7000 x g for 7 min to pellet the cells. The supernatant was decanted, and the pellet was then resuspended in lysis buffer containing 20 mM Tris base (pH 8), lysozyme (0.5 mg/mL), benzonase (125 units; EMD Millipore), and a quarter of a SigmaFast EDTA-free protease inhibitor cocktail tablet (Sigma Aldrich). The resuspended pellet was incubated on ice with occasional swirling for 20 min, after which sodium deoxycholate (Alfa Aesar) was added to a final concentration of 0.1% (w/v). The mixture was sonicated for 3 min at 35% amplitude with a pulse of 3 s on and 3 s off (Sonifier S-450, Branson Ultrasonics). The lysate was cooled on ice for 5 min, then sonication was repeated. The lysed cells were centrifuged at 19,000 x g for 30 min. The supernatant was collected, centrifuged again at 19,000 x g for 20 min, then diluted 3-fold in 20 mM Tris Base, pH 8. 25 mL of the lysate was loaded onto four HiScreen Capto Core columns (Cytvia) in series using an Akta Start system. MS2 was purified by washing with 20 mM Tris Base for ~5 column volumes (CVs). The entire flowthrough was collected in 1.8 mL fractions, and SDS-PAGE was used to determine purity and yield. Fractions 7–12 were typically those most enriched in MS2 while smaller impurities would concentrate in later fractions due to the CaptoCore system’s size-exclusion character. Fractions containing MS2 were pooled and concentrated to 1 mL with a 10 kDa MWCO spin filter (Millipore Sigma). MS2 was then further purified by SEC using a Superdex 200 Increase 10/30 column (Cytvia) running in an aqueous buffer (20 mM Tris, 20 mM NaCl, pH 8). Fractions from SEC were pooled and quantified by BCA assay (Thermo Scientific).

In vitro biotinylation of AviTagged MS2 and S proteins

MS2 and S proteins were biotinylated in vitro with a BirA biotin-protein ligase standard reaction kit (Avidity LLC). Following buffer exchange into 20 mM Tris, 20 mM NaCl, pH 8 buffer, the proteins were concentrated to 45 μM, then BirA and a mixture of biotin, ATP, and magnesium acetate (Biomix B) was added to the protein solution. The solution was allowed to mix overnight at 4 °C. More Biomix B was added, and the solution was mixed at 37 °C for 2 h, followed by the addition of more Biomix B and another overnight incubation at 4 °C. The protein was then purified on a Superdex 200 Increase 10/300 column (Cytiva) to remove BirA and excess biotin and quantified by a BCA assay.

Expression, refolding, and purification of streptavidin

Streptavidin (SA) was expressed, refolded, and purified essentially as previously described14,29,52,53. Briefly, DNA encoding SA (Addgene plasmid #46367, a gift from Mark Howarth) was transformed into BL21(DE3) cells (New England Biolabs) according to the manufacturer’s protocol. The transformation was split between four culture tubes containing 5 mL of 2xYT media and grown overnight at 37 °C. Each 5 mL culture was added to 1 L of 2xYT media and incubated at 37 °C until an OD of 0.6–1.0 was reached. IPTG (Fisher BioReagents) was added to a final concentration of 1 mM to induce expression, and the temperature was reduced to 30 °C. After overnight induction, the cultures were centrifuged at 7000 x g for 7 min to produce two cell pellets. Each pellet was resuspended in 50 mL of lysis buffer (50 mM Tris, 100 mM NaCl, pH 8.0) containing 1 mg/mL lysozyme (Alfa Aesar) and benzonase (500 units; EMD Millipore). The mixture was incubated with mixing for 1 h at 4 °C then homogenized. Sodium deoxycholate (Alfa Aesar) was added to a final concentration of 0.1% (w/v), then the mixture was sonicated for 3 min at 35% amplitude with a pulse of 3 s on and 3 s off. The lysate was centrifuged at 27,000 x g for 15 min, and the supernatant was removed. The pellets were again resuspended in 50 mL of lysis buffer containing 1 mg/mL lysozyme (Alfa Aesar), incubated for 30 min at 4 °C, homogenized, and sonicated. Following centrifugation, the two inclusion body pellets were washed. Each pellet was resuspended in 50 mL of wash buffer #1 (50 mM Tris, 100 mM NaCl, 100 mM EDTA, 0.5% (v/v) Triton X-100, pH 8.0), then homogenized and sonicated at 35% amplitude for 30 s. The lysate was centrifuged at 27,000 x g for 15 min, and the supernatant was discarded. This wash procedure was repeated twice for a total of 3 washes. Both pellets were then resuspended in 50 mL of wash buffer #2 (50 mM Tris, 10 mM EDTA, pH 8.0), homogenized, and sonicated at 35% amplitude for 30 s. The mixture was centrifuged at 15,000 × g for 15 min, and the supernatant was discarded. This process was repeated once more. The inclusion body pellets were then resuspended in 10 mL of resuspension buffer, and guanidine hydrochloride was added to a final concentration of 7.12 M. The mixture was stirred for 1 h at room temperature, then centrifuged for 12 min at 12,000 × g. The supernatant was transferred to a syringe and loaded onto a syringe pump, then the supernatant was added at a rate of 30 mL/h to 1 L of chilled PBS that was stirring rapidly. The solution was stirred overnight at 4 °C, then insoluble protein was pelleted out by centrifuging at 17,000 × g for 15 min. The supernatant was filtered with a 0.45-μm bottle-top filter then stirred vigorously. While stirring, ammonium sulfate was slowly added to the filtrate until a final concentration of 1.9 M was reached. At this point, protein impurities precipitate out. The solution was allowed to mix for 3 h at 4 °C then centrifuged for 15 min at 17,000 × g to remove the precipitate. The solution was filtered with a 0.45-μm bottle-top filter to further remove precipitate. While mixing, ammonium sulfate was added to bring the concentration to 3.68 M and precipitate SA. The mixture was stirred overnight at 4 °C, then the SA was pelleted by centrifuging at 17,000 × g for 15 min. The SA pellet was resuspended in 20 mL of Iminobiotin Affinity Chromatography (IBAC) binding buffer (50 mM Sodium Borate, 300 mM NaCl, pH 11.0). Five mL of Pierce Iminobiotin Agarose (Thermo Scientific) in a gravity flow column (G-Biosciences) was equilibrated with 5 column volumes of IBAC binding buffer, then the SA solution was poured over the resin. 20 column volumes of IBAC binding buffer were added to the column to wash the resin and bound SA. To elute the protein, 8 column volumes of IBAC elution buffer (20 mM Potassium Phosphate, pH 2.2) were added to the column. The eluate was dialyzed into PBS and concentrated using a 10 kDa MWCO centrifugal filter (Millipore Sigma). SA concentration was measured using UV absorbance at 280 nm.

Assembly and purification of MS2-SA VLPs

A concentrated solution of 20x molar excess SA was stirred vigorously in a 5 mL glass vial, and biotinylated MS2 was slowly added dropwise to the SA. After mixing, a Superdex 200 Increase 10/300 SEC column was used to separate the excess SA from the MS2-SA VLPs. To quantify the SA bound to the MS2 VLP, small samples were mixed with Nu-PAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) and heated at 90 °C for 30 min. After heating, the samples were loaded onto a polyacrylamide gel along with a series of SA standards with known concentrations determined using the UV absorbance at 280 nm. The intensities of the SA bands from the samples were compared to those of the standards to determine the concentration of SA. This concentration was then used in determining the stoichiometric ratio of MS2-SA to biotinylated S.

Preparation of VLP-S

Analytical SEC was used to determine the stoichiometric ratio of MS2-SA and biotinylated S. Mixtures containing 10 μg of biotinylated S protein and varying amounts of MS2-SA were run on SEC, and the ratio containing the lowest amount of MS2-SA without any excess S protein appearing on the chromatogram was chosen. MS2-SA and biotinylated S protein were mixed in this ratio, then the mixture was diluted to a final concentration of 0.12 μg S protein/μL. The VLP-S were then characterized by ELISA, DLS, SEC, and TEM.

Expression of ACE2-Fc and S-binding Antibodies

The variable region of the heavy and light chains of CR302254, S30955, and S2P656 were cloned into the TGEX-HC and TGEX-LC vectors (Antibody Design Labs), respectively, according to the manufacturer’s protocol. ACE2 (residues 1–615) was cloned into TGEX-HC. The plasmids were expressed in Expi293F cells (ThermoFisher Scientific) using the ExpiFectamine Transfection Kit (Thermo Fisher Scientific) and transfected according to the manufacturer’s protocol. After incubation for 6 days at 37 °C for, the cells were centrifuged at 6000 × g for 15 min. The supernatant was diluted in MabSelect Binding Buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.2) and passed through a 1-mL MabSelect SuRe column (Cytiva) connected to an ÄKTA Start. The column was washed, and the proteins were eluted according to the manufacturer’s protocol. Fractions containing the protein were collected and dialyzed into PBS, then the concentration of antibodies and ACE2-Fc was measured using the BCA assay.

Characterization of S and VLP-S by ELISA

VLP-S and S protein were diluted in PBS such that the concentration of S protein in each solution was 1 μg/mL. 100 μL (0.1 μg of S protein) per well of the diluted protein was then coated onto a Nunc Maxisorp 96-well plate. After a 1-h incubation, the protein solutions were discarded, and the wells were blocked with 200 μL of 5% BSA (EMD Millipore) in PBST (0.05% Tween-20) for 1 h. The wells were then washed three times with PBST. Stock solutions of primary antibodies at 0.3 mg/mL, 3.4 mg/mL, 3.8 mg/mL, and 1.9 mg/mL (by BCA) for ACE2-Fc, CR3022, S309, and S2P6 respectively were diluted 9:1500, 1:1500, 1:1500, and 1:30,000 respectively in PBST with 1% BSA, and 100 μL per well of these diluted primary antibodies were added. After 1 h, the wells were washed three times with PBST, and 100 μL of horseradish peroxidase-conjugated anti-human IgG Fc goat antibody (MP Biomedical, catalog #674171, dilution 1:5000) diluted in PBST with 1% BSA was added to all wells. The wells were washed three times with PBST after a 1-h incubation. 100 μL of TMB (Thermo Scientific) were added to each well and allowed to develop for 3 min. The reaction was stopped with 160 mM sulfuric acid, and the absorbance at 450 nm was read with a Synergy H4 plate reader (BioTek) and Gen5 2.07 software (BioTek).

SDS-PAGE

Protein samples were diluted in Nu-PAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) and heated for 30 min at 90 °C. 15 μL of protein samples and 2 μL of PageRuler Plus Prestained Protein Ladder were pipetted into the well of a 4–12% Bi-Tris gel (Invitrogen). Gels were then run in MES-SDS buffer at 4 °C for 60 min at 110 V. Afterwards, the gel was washed in DI water and stained with Imperial Protein Stain for 30 min then destained overnight. Gels were then imaged with ChemiDoc MP imaging system and Image Lab 5.2.1 software (Bio-Rad).

Analytical SEC

SEC was run using a Superdex 200 Increase 10/300 Column (Cytiva) and Unicorn 7 control system (Cytiva). 950 μL samples of either S protein or VLP-S, each containing 10 μg of S, were injected into the sample loop. The loop was flushed with PBS to inject the sample onto the column, then the protein was eluted with 1 column volume of PBS flowing at 0.5 mL/min. During elution, absorbance at 280, 210, and 205 nm was monitored.

Dynamic light scattering

VLP-S was diluted in PBS to a concentration of 0.05 μg/μL, added to a UVette (Eppendorf), and inserted into a DynaPro NanoStar Dynamic Light Scattering detector (Wyatt Technology). Ten acquisitions per sample were collected at 25 °C and displayed as % Mass with the Isotropic Spheres model.

Transmission electron microscopy

Conventional negative-stain transmission electron microscopy (NS-TEM) was performed on VLP-S variants, as described previously14,29. Briefly, 4 µl of the diluted samples were applied onto glow-discharged 200 mesh copper grids (CF200-Cu; Electron Microscopy Sciences, PA), washed with distilled water (3x) and stained in droplets of 1% phosphotungstic acid (PTA, pH 6–7) for 1 min. The grids were drained from the grid backside and then air-dried. The stained grids were imaged with a low dose of 50–60 e2, under a nominal magnification of 73 kx (pixel size of 2.0 Å), defocus of −0.5 to −2 µm, on a Talos L120C transmission electron microscope (ThermoFisher Scientific, Hillsboro, OR), operating at 120 kV. Images were acquired on a 4 K x 4 K Ceta CMOS camera (ThermoFisher Scientific, Hillsboro, OR), using SerialEM 3.8457.

Amino acid identity and phylogenetic trees

Amino acid sequences for spike proteins from all included sarbecoviruses were retrieved from GenBank (Table S2). Sequences were aligned using Clustal Omega 1.2.4 (Conway Institute, UCD Dublin), and the percentage of identical amino acids for each pair was calculated from this alignment. An RBD alignment was generated by copying the full S alignment from residues 319 through 541 inclusive of SARS-CoV-2 614D (YP_009724390.1). Maximum likelihood phylogenetic trees were generated from the RBD alignment using PhyML 3.3.20220408 (Stephane Guindon, University of Montpellier) using the LG amino acid substitution model and a maximum parsimony starting tree. Phylogenetic trees were visualized using TreeViewer 2.0.1 (Giorgio Bianchini, University of Bristol).

Cells and Virus

Virus stocks were generated on Vero E6 TMPRSS2 cells obtained from the National Institute of Infectious Diseases, Japan58 which were maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotic/antimycotic solution along with G418 (1 mg/ml). Tissue titrations were performed on Vero E6 TMPRSS2-T2A-ACE2 cells obtained from Dr. Barney Graham, NIAID Vaccine Research Center which were maintained in DMEM supplemented with 10% FBS, 10 mM HEPES (pH 7.3) and antibiotic/antimycotic solution along with puromycin (10 µg/ml) The following viruses, a prototypical ancestral isolate (SARS-CoV-2/UT-HP095-1 N/Human/2020/Tokyo), BA.1 isolate (hCoV-19/USA/WI-WSLH-221686/2021), BA.5 isolate (hCoV-19/Japan/TY41-702/2022), XBB.1 isolate (hCoV-19/USA/NY-MSHSPSP-PV73997/2022), and two recombinant SARS-like bat CoVs, SHC014 and WIV1, used in studies were provided by Ralph Baric32,59.

Animal experiments and approval

Animal studies were performed under a protocol approved by the Institutional Animal Care and Use Committee at the University of Wisconsin, Madison (protocol number V006426). Virus infections were performed under isoflurane, and all efforts were made to minimize pain. Animal studies were not blinded. Group sizes were determined based on prior virus challenge studies, and no sample-size calculations were performed to determine the power of each study.

Experimental infection of syrian hamsters

Female Syrian wild-type hamsters (4–5 weeks old, Envigo) or K18-human ACE2 homozygous transgenic hamsters60 from an established colony at UW-Madison (females, 5-6 weeks old) were used in this study. Hamsters were vaccinated with the indicated vaccine candidate (15 µg of each S protein) with adjuvant (alhydrogel, 4.5–5.5 mg; equal volume of vaccine and adjuvant) by subcutaneous inoculation. A separate group of animals was vaccinated by intramuscular inoculation in the thigh muscle with 30 µg of Pfizer-BioNTech’s bivalent vaccine (residual material stored at −80 °C 24 h or less after the vaccine was reconstituted at a university health clinic). Under isoflurane anesthesia, animals were infected by intranasal inoculation with the indicated virus isolates at a dose of 105 plaque-forming units (pfu) in 30 µl of total volume. Three days after infection, animals were humanely sacrificed by overdose of isoflurane and lung tissue and nasal turbinate samples were collected to measure the amount of virus.

Focus reduction neutralization test (FRNT)

Neutralization of all viruses was characterized by using a focus reduction neutralization test. Serial dilutions of serum from vaccinated hamsters starting at a final concentration of 1:20 were mixed with ~1000 focus-forming units (FFU) of the indicated virus/well and incubated for 1 h at 37 °C. Pooled serum from hamsters vaccinated with VLP without the S protein served as a control. The antibody-virus mixture was inoculated onto Vero E6/TMPRSS2 cells in 96-well plates and incubated for 1 h at 37 °C. An equal volume of methylcellulose solution was added to each well. The cells were incubated for 16 h at 37 °C and then fixed with formalin. After the formalin was removed, the cells were immunostained with a mouse monoclonal antibody against SARS-CoV-1/2 nucleoprotein (clone 1C7C7, Sigma-Aldrich, catalog #MA5-29982, 1:10,000 dilution), followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (ThermoFisher, catalog #31430, 1:2000 dilution). The infected cells were stained with TrueBlue Substrate (SeraCare Life Sciences) and then washed with distilled water. After cell drying, the focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology). The results are expressed as the 50% focus reduction neutralization titer (FRNT50). The FRNT50 values were calculated by using Prism 9 (Graphpad Software). Percent neutralization was calculated as (N=100%times (1-frac{{F}_{v}}{{F}_{c}})), where N is the percent neutralization, Fv is the number of foci in the presence of sera from hamsters vaccinated with VLP-S, and Fc is the number of foci in the presence of pooled sera from hamsters vaccinated with VLP control. The FRNT50 value was then calculated from the normalized percent neutralization using a four-parameter nonlinear regression in Graphpad Prism.

Antigenic cartography

Antigenic cartography was performed using the Racmacs R package (Racmacs 1.1.35 with R 4.2.1 and RStudio 2022.07.01)61. In brief, FRNT50 values were calculated from each serum sample against each virus. The dissimilarity Dij between each serum i and virus j is defined as ({D}_{{ij}}=-{log }_{2}{H}_{{ij}}+{log }_{2}{H}_{i,max }) where Hij is the FRNT50 value of serum i against virus j and Hi,max is the maximum FRNT50 value from serum i. The error function for each pair is then ({E}_{{ij}}={({D}_{{ij}}-{d}_{{ij}})}^{2}), where dij is the Euclidean distance on the two-dimensional map between serum i and virus j. For FRNT50 values below a detection threshold (e.g., <20), the error function was instead calculated as ({E}_{{ij}}=frac{{a}^{2}}{1+{e}^{-10a}}) for (a={D}_{{ij}}-1-{d}_{{ij}}). The summed error function was then minimized using conjugate gradient descent optimization, and 5000 restarts with random starting positions were used to approximate the global optimum.

Biosafety statement

Research with SARS-CoV-2 and related SARS-like viruses was performed under biosafety level 3 agriculture (BSL-3Ag) containment at the Influenza Research Institute with an approved protocol reviewed by approved the University of Wisconsin-Madison’s Institutional Biosafety Committee. The laboratory is designed to meet and exceed the standards outlined in Biosafety in Microbiological and Biomedical Laboratories (6th edition).

Statistics and reproducibility

SDS-PAGE gels of S and VLP-S (Fig. 2a, c) were conducted twice from the same preparation for each sample with similar results. For TEM imaging (Fig. 2f), at least 70 images were collected and analyzed from one VLP-S preparation per sample with similar results. Characterization of the binding of ACE2-Fc and S2P6 by ELISA (Fig. 2e) was conducted once with three technical replicates for each condition. Data are presented as the mean ± SD. For in vivo characterization of monovalent VLP-S (Fig. 3), there were seven groups with three hamsters each. Neutralization titers (Figs. 3a and 4a) were measured by focus reduction neutralization test (FRNT) conducted as a single assay using sera from each hamster. Viral titers in the lungs and nasal turbinates of Syrian hamsters immunized with VLP-S vaccines (Figs. 3b and 4b, d) 3 days after infection with BA.5 and XBB.1 variants were presented as the mean ± SD (n = 6 for BA.5 VLP-control, n = 3 for all other groups). The significance was determined by a one-way analysis of variance (ANOVA) and Dunnett post-hoc multiple comparison between groups (α = 0.05) for BA.5 variant lung and nasal titers for monotypic vaccines and by Brown-Forsythe and Welch ANOVA tests and Dunnett’s T3 multiple comparisons between groups for BA.5 variant and XBB.1 variant lung and nasal titers for cocktail vaccines. Viral titers in the lungs and nasal turbinates of hamsters immunized with Pfizer-BioNTech bivalent vaccine (Fig. 4c, e) 3 days after infection with BA.5 and XBB.1 variants were presented as the mean ± SD (n = 4). The significance was determined by a two-tailed unpaired Student’s t test. Viral titers in the lungs and nasal turbinates of hACE2 hamsters (Fig. 5) 3 days after infection with WIV1 and SHC014 viruses were presented as the mean ± SD (n = 4). Significance was determined by two-tailed unpaired Welch’s t tests for lung titers and two-tailed unpaired Student’s t tests for nasal titers. For all tests of significance, assumptions of the normality of residuals and homogeneity of variance were validated by the D’Agostino-Pearson test and the Brown-Forsythe test, respectively. All statistical analysis was carried out using Prism 9 (GraphPad).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.