Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine

Animals and immunizations

The study protocol and all veterinarian procedures were approved by the Bioqual IACUC per a memorandum of understanding with the Duke IACUC, and were performed based on standard operating procedures. Macaques studied were housed and maintained in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited institution in accordance with the principles of the National Institutes of Health. All studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health in BIOQUAL (Rockville, MD). BIOQUAL is fully accredited by AAALAC and through OLAW, Assurance Number A-3086. All physical procedures associated with this work were done under anesthesia to minimize pain and distress in accordance with the recommendations of the Weatherall report, “The use of non-human primates in research.” Teklad 5038 Primate Diet was provided once daily by animal size and weight. The diet was supplemented with fresh fruit and vegetables. Fresh water was given ad libitum. All monkeys were maintained in accordance with the Guide for the Care and Use of Laboratory Animals.

Cynomolgus macaques were on average 8–9 years old and ranged from 2.75 to 8 kg in body weight. Male and female macaques per group were balanced when availability permitted. Studies were performed unblinded. The immunogens were formulated with adjuvants and given intramuscularly in the right and left quadriceps. In the adjuvant study (Fig. 1 and Supplementary Fig. 1), cynomolgus macaques were immunized for twice with 100 μg of RBD-scNP or recombinant soluble RBD with 5 μg of 3M-052 aqueous formulation admixed with 500 μg of alum in PBS. In the fourth study, macaques were divided into 8 groups (n = 5 per group) as following: 1) control group: no immunization; 2) immunogen alone group: 100 μg of RBD-scNP; 3) RBD-scNP + 3M-052-Alum group: 100 μg of RBD-scNP + 5 μg of 3M-052 in aqueous formulation + 500 μg of Alum (i.e. aluminum ion); 4) 3M-052-Alum alone group: 5 μg of 3M-052 in aqueous formulation + 500 μg of Alum; 5) RBD-scNP + Alum group: 100 μg of RBD-scNP + 500 μg of Alum; 6) Alum alone group: 500 μg of Alum; 7) RBD-scNP + 3M-052-AF group: 100 μg of RBD-scNP + 5 μg of 3M-052 in aqueous formulation; 8) 3M-052-AF alone group: 5 μg of 3M-052 in aqueous formulation. In the RBD-, NTD-, and S2P-scNP study (Fig. 2 and Supplementary Fig. 2), cynomolgus macaques (n = 5) were immunized for three times with 100 μg of RBD-scNP, NTD-scNP and S2P-scNP adjuvanted with 5 μg of 3M-052 aqueous formulation admixed with 500 μg of alum in PBS. In the prime-boost study (Fig. 3 and Supplementary Fig. 3), cynomolgus macaques (n = 5) were were immunized twice with 50 μg of S-2P mRNA-LNP (encoding the transmembrane spike protein stabilized with K986P and V987P mutations) and boosted once with 100 μg of RBD-scNP, NTD-scNP and S2P-scNP adjuvanted with 5 μg of 3M-052 aqueous formulation admixed with 500 μg of alum in PBS. In the last RBD-scNP 2× immunization study (Fig. 4 and Supplementary Fig. 4), cynomolgus macaques (n = 5) were immunized for two times with 100 μg of RBD-scNP adjuvanted with 5 μg of 3M-052 aqueous formulation admixed with 500 μg of alum in PBS.

SARS-CoV-2 viral challenge

For SARS-CoV-2 challenge, 10⁵ plaque-forming units (PFU) of SARS-CoV-2 virus Isolate USA-WA1/2020, or 2 × 10⁵ PFUs of SARS-CoV-2 Beta or Delta variants, were diluted in 4 mL and were given by 1 mL intranasally and 3 mL intratracheally on Day 0. Biospecimens, including nasal swabs, BAL, plasma, and serum samples, were collected before immunization, after every immunization, before challenge, 2 days post-challenge and 4 days post-challenge. Animals were necropsied on Day 4 post-challenge, and lungs were collected for histopathology and immunohistochemistry (IHC) analysis.

Recombinant protein production

The coronavirus ectodomain proteins were produced and purified as previously described^10,12,46,47. S-2P was stabilized by the introduction of 2 prolines at amino acid positions 986 and 987. Plasmids encoding SARS-CoV-2 and other coronavirus S-2P (Genscript) were transiently transfected in FreeStyle 293-F cells (Thermo Fisher) using Turbo293 (SpeedBiosystems) or 293Fectin (ThermoFisher). All cells were tested monthly for mycoplasma. The constructs contained an HRV 3C-cleavable C-terminal twinStrepTagII-8×His tag. On day 6, cell-free culture supernatant was generated by centrifugation of the culture and filtering through a 0.8-μm filter. Protein was purified from filtered cell culture supernatants by StrepTactin resin (IBA) and by size-exclusion chromatography using Superdex 200 (RBD and NTD) or Superose 6 (S-2P and ferritin) column (GE Healthcare) in 10 mM Tris pH = 8500 mM NaCl. ACE2-Fc was expressed by transient transfection of Freestyle 293-F cells. ACE2-Fc was purified from cell culture supernatant by HiTrap protein A column chromatography and Superdex200 size-exclusion chromatography in 10 mM Tris pH8,150 mM NaCl. SARS-CoV-2 RBD and NTD were produced as previously described^10,47.

RBD-scNP, NTD-scNP, and S2P-scNP were produced by conjugating SARS-CoV-2 RBD to H. pylori ferritin nanoparticles using Sortase A as previously described¹⁰. Briefly, SARS-CoV-2 Wuhan strain RBD, NTD or S-2P (with a C-terminal foldon trimerization motif) was expressed with a sortase A donor sequence LPETGG encoded at its C terminus. C-terminal to the sortase A donor sequence was an HRV-3C cleavage site, 8×His tag and a twin StrepTagII (IBA). The proteins were expressed in Freestyle 293-F cells and purified by StrepTactin affinity chromatography and Superdex 200 or Superose 6 size-exclusion chromatography. Helicobacter pylori ferritin particles were expressed with a pentaglycine sortase A acceptor sequence encoded at its N terminus of each subunit. For affinity purification of ferritin particles, 6×His tags were appended C-terminal to a HRV3C cleavage site. Ferritin particles with a sortase A N-terminal tag were buffer exchanged into 50 mM Tris, 150 mM NaCl, 5 mM CaCl₂, pH 7.5. Then 180 μM SARS-CoV-2 RBD was mixed with 120 μM of ferritin subunits and incubated with 100 μM of sortase A overnight at room temperature. Following incubation, conjugated particles were isolated from free ferritin or free RBD/NTD/S-2P by size-exclusion chromatography using a Superose 6 16/60 column. Western blots of reduced RBD, NTD, or Spike ectodomain scNPs were performed to determine the approximate amount of coronavirus protein within the 100 μg immunogen dose. Mouse polyclonal antisera against ferritin was used to blot for ferritin that was either unconjugated or conjugated to coronavirus proteins. Bio-Rad ImageDoc system was used to quantify the pixels of the ferritin conjugate band and unconjugated ferritin band. The percentage of conjugated ferritin was determined as the pixels of the conjugated ferritin divided by the sum of the pixels for the ferritin conjugate band and unconjugated ferritin band multiplied by 100 percent. The average percentages of conjugated ferritin were 80%, 70%, and 94.1%, for RBD, NTD, or Spike ectodomain respectively. We approximate the micrograms of RBD, NTD, or Spike ectodomain per 100 micrograms of immunogen to be 86, 55, and 51 micrograms respectively.

Negative-stain electron microscopy

The RBD, NTD, or S-2P nanoparticle protein at about 1–5 mg ml−1 concentration that had been flash-frozen and stored at −80 °C was thawed in an aluminum block at 37 °C for 5 min; then 1–4 μl of RBD, NTD, or S-2P nanoparticle was diluted to a final concentration of 0.1 mg ml−1 into room-temperature buffer containing 150 mM NaCl, 20 mM HEPES pH 7.4, 5% glycerol and 7.5 mM glutaraldehyde. After 5 min of cross-linking, excess glutaraldehyde was quenched by adding sufficient 1 M Tris pH 7.4 stock to give a final concentration of 75 mM Tris and incubated for 5 min. For negative stain, carbon-coated grids (EMS, CF300-cu-UL) were glow-discharged for 20 s at 15 mA, after which a 5-μl drop of quenched sample was incubated on the grid for 10–15 s, blotted and then stained with 2% uranyl formate. After air drying, grids were imaged with a Philips EM420 electron microscope operated at 120 kV, at 82,000× magnification and images captured with a 2k × 2k CCD camera at a pixel size of 4.02 Å.

Processing of negative-stain images

The RELION 3.0 program was used for all negative-stain image processing. Images were imported, CTF-corrected with CTFFIND and particles were picked using a nanoparticle template from previous 2D class averages of nanoparticles alone. Extracted particle stacks were subjected to 2 or 3 rounds of 2D class averaging and selection to discard junk particles and background picks.

mRNA-LNP vaccine production

The S-2P mRNA was designed based on the SARS-CoV-2 spike (S) protein sequence (Wuhan-Hu-1) and encoded the full-length S with K986P and V987P amino acid substitutions. Production of the mRNA was performed as described earlier^48,49. Briefly, the codon-optimized S-2P gene was synthesized (Genscript) and cloned into an mRNA production plasmid. A T7-driven in vitro transcription reaction (Megascript, Ambion) using linearized plasmid template was performed to generate mRNA with 101 nucleotide long poly(A) tail. Capping of the mRNA was performed in concert with transcription through addition of a trinucleotide cap1 analog, CleanCap (TriLink) and m1Ψ-5’-triphosphate (TriLink) was incorporated into the reaction instead of UTP. Cellulose-based purification of S-2P mRNA was performed as described⁵⁰. The S-2P mRNA was then tested on an agarose gel before storing at −20 °C. The cellulose-purified m1Ψ-containing S-2P mRNA was encapsulated in LNPs using a self-assembly process as previously described wherein an ethanolic lipid mixture of ionizable cationic lipid, phosphatidylcholine, cholesterol and polyethylene glycol-lipid was rapidly mixed with an aqueous solution containing mRNA at acidic pH⁵¹. The RNA-loaded particles were characterized and subsequently stored at 80 °C at a concentration of 1 mg/ml.

Antibody binding ELISA

For binding ELISA, 384-well ELISA plates were coated with 2 μg/mL of antigens in 0.1 M sodium bicarbonate overnight at 4 °C. Plates were washed with PBS +0.05% Tween 20 and blocked with blocked with assay diluent (PBS containing 4% (w/v) whey protein, 15% Normal Goat Serum, 0.5% Tween-20, and 0.05% Sodium Azide) at room temperature for 1 h. Plasma or mucosal fluid were serially diluted threefold in superblock starting at a 1:30 dilution. Nasal fluid was started from neat, whereas BAL fluid was concentrated ten-fold. To concentrate BAL, individual BAL aliquots from the same macaque and same time point were pooled in 3-kDa MWCO ultrafiltration tubes (Sartorious, catalog # VS2091). Pooled BAL was concentrated by centrifugation at 3200 × g for 30 min or until volume was reduced by a factor of 10. The pool was then aliquoted and frozen at −80 °C until its use in an assay. Serially diluted samples were added and incubated for 1 h, followed by washing with PBS-0.1% Tween 20. HRP-conjugated goat anti-human IgG secondary Ab (SouthernBiotech, catalog# 2040-05) was diluted to 1:10,000 and incubated at room temperature for 1 h. These plates were washed four times and developed with tetramethylbenzidine substrate (SureBlue Reserve- KPL). The reaction was stopped with 1 M HCl, and optical density at 450 nm (OD₄₅₀) was determined.

ACE2 and neutralizing antibody blocking assay

ELISA plates were coated as stated above with 2 μg/mL recombinant ACE-2 protein or neutralizing antibodies, then washed and blocked with 3% BSA in 1x PBS. While assay plates blocked, plasma or mucosal samples were diluted as stated above, only in 1% BSA with 0.05% Tween-20. In a separate dilution plate spike-2P protein was mixed with the antibodies at a final concentration equal to the EC50 at which spike binds to ACE-2 protein. The mixture was incubated at room temperature for 1 h. Blocked assay plates were then washed and the antibody-spike mixture was added to the assay plates for a period of 1 h at room temperature. Plates were washed and a polyclonal rabbit serum against the same spike protein (nCoV-1 nCoV-2P.293F) was added for 1 h, washed and detected with goat anti rabbit-HRP (Abcam catalog # ab97080) followed by TMB substrate. The extent to which antibodies were able to block the binding spike protein to ACE-2 or neutralizing antibodies was determined by comparing the OD of antibody samples at 450 nm to the OD of samples containing spike protein only with no antibody. The following formula was used to calculate percent blocking: blocking % = (100 − (OD sample/OD of spike only)*100).

Pseudotyped SARS-CoV-2 neutralization assay

Neutralization of SARS-CoV-2 Spike-pseudotyped virus was performed by adopting an infection assay described previously⁵² with lentiviral vectors and infection in 293T/ACE2.MF (the cell line was kindly provided by Drs. Mike Farzan and Huihui Mu at Scripps). Cells were maintained in DMEM containing 10% FBS and 50 µg/ml gentamicin. An expression plasmid encoding codon-optimized full-length spike of the Wuhan-1 strain (VRC7480), was provided by Drs. Barney Graham and Kizzmekia Corbett at the Vaccine Research Center, National Institutes of Health (USA). Mutations were introduced into VRC7480 either by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit from Agilent Technologies (Catalog # 210518), or were created by spike gene synthesized by GenScript using the spike sequence in VRC7480 as template. All mutations (D614G, Omicron BA.1, BA.2, BA.2.12.1, BA.4/BA.5 and PMS20) were confirmed by full-length spike gene sequencing by Sanger Sequencing, using Sequencher and SnapGene for sequence analyses. Pseudovirions were produced in HEK 293T/17 cells (ATCC cat. no. CRL-11268) by transfection using Fugene 6 (Promega, Catalog #E2692). Pseudovirions for 293T/ACE2 infection were produced by co-transfection with a lentiviral backbone (pCMV ΔR8.2) and firefly luciferase reporter gene (pHR’ CMV Luc)⁵³. Culture supernatants from transfections were clarified of cells by low-speed centrifugation and filtration (0.45 µm filter) and stored in 1 ml aliquots at −80 °C. A pre-titrated dose of virus was incubated with 8 serial 3-fold or 5-fold dilutions of mAbs in duplicate in a total volume of 150 µl for 1 h at 37 °C in 96-well flat-bottom poly-L-lysine-coated culture plates (Corning Biocoat). Cells were suspended using TrypLE express enzyme solution (Thermo Fisher Scientific) and immediately added to all wells (10,000 cells in 100 µL of growth medium per well). One set of 8 control wells received cells + virus (virus control) and another set of 8 wells received cells only (background control). After 66–72 h of incubation, medium was removed by gentle aspiration and 30 µL of Promega 1x lysis buffer was added to all wells. After a 10-min incubation at room temperature, 100 µl of Bright-Glo luciferase reagent was added to all wells. After 1–2 min, 110 µl of the cell lysate was transferred to a black/white plate (Perkin-Elmer). Luminescence was measured using a PerkinElmer Life Sciences, Model Victor2 luminometer. Neutralization titers are the serum dilution (ID₅₀/ID₈₀) at which relative luminescence units (RLU) were reduced by 50% and 80% compared to virus control wells after subtraction of background RLUs. Negative neutralization values are indicative of infection-enhancement. Maximum percent inhibition (MPI) is the reduction in RLU at the highest mAb concentration tested.

Another protocol was used to test plasma neutralization against pseudoviruses of SARS-CoV-2 WA-1 strain and variants. Human codon-optimized cDNA encoding SARS-CoV-2 spike glycoproteins of various strains were synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamHI and XhoI sites. Pseudovirions were produced by co-transfection of Lenti‐X 293T cells with psPAX2(gag/pol), pTrip-luc lentiviral vector and pcDNA 3.1 SARS-CoV-2-spike-deltaC19, using Lipofectamine 3000. The supernatants were collected at 48 h after transfection and filtered through 0.45-μm membranes and titrated using HEK293T cells that express ACE2 and TMPRSS2 protein (293T-ACE2-TMPRSS2 cells). For the neutralization assay, 50 μl of SARS-CoV-2 spike pseudovirions were pre-incubated with an equal volume of medium containing serum at varying dilutions at room temperature for 1 h, then virus-antibody mixtures were added to 293T-ACE2-TMPRSS2 cells in a 96-well plate. After a 3-h incubation, the inoculum was replaced with fresh medium. Cells were lysed 24 h later, and luciferase activity was measured using luciferin. Controls included cell-only control, virus without any antibody control and positive control sera. Neutralization titers are the serum dilution (ID50 or ID80) at which relative luminescence units (RLU) were reduced by 50% or 80%, respectively, compared to virus control wells after subtraction of background RLUs.

Live SARS-CoV-2 neutralization assays

The SARS-CoV-2 virus (Isolate USA-WA1/2020, NR-52281) was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH. SARS-CoV-2 Micro-neutralization (MN) assays were adapted from a previous study⁵⁴. In short, sera or purified Abs are diluted two-fold and incubated with 100 TCID50 virus for 1 h. These dilutions are used as the input material for a TCID50. Each batch of MN includes a known neutralizing control Ab (Clone D001; SINO, CAT# 40150-D001). Data are reported as the concentration at which 50% of input virus is neutralized. A known neutralizing control antibody is included in each batch run (Clone D001; SINO, CAT# 40150-D001). GraphPad Prism was used to determine ID₅₀ values.

Spike protein-expressing cell antibody binding assay

The cell antibody binding assay was performed as previously described (Pino et al., 2021). Briefly, target cells were derived by transfection with plasmids designed to express the SARS-CoV-2 D614 Spike protein with a c-terminus flag tag (kindly provided by Dr. Farzan, Addgene plasmid no. 156420 (Zhang et al., 2020)). Cells not transfected with any plasmid (mock transfected) were used as a negative control condition. After resuspension, washing and counting, 1 × 10⁵ Spike-transfected target cells were dispensed into 96-well V-bottom plates and incubated with six serial dilutions of macaque plasma starting at 1:50 dilution. Mock transfected cells were used as a negative infection control. After 30 min incubation at 37 °C, cells are washed twice with 250 μL/well of PBS, stained with vital dye (Live/Dead Far Red Dead Cell Stain, Invitrogen) to exclude nonviable cells from subsequent analysis, washed with Wash Buffer (1%FBS-PBS; WB), permeabilized with CytoFix/CytoPerm (BD Biosciences), and stained with 1.25 µg/mL anti-human IgG Fc-PE/Cy7 (Clone HP6017; Biolegend) and 5 µg/mL anti-flag-FITC (clone M2; Sigma Aldrich) in the dark for 20 min at room temperature. After three washes with Perm Wash (BD Biosciences), the cells were resuspended in 125 μL PBS-1% paraformaldehyde. Samples were acquired within 24 h using a BD Fortessa cytometer and a High Throughput Sampler (HTS, BD Biosciences). Data analysis was performed using FlowJo 10.8.0 software. A minimum of 50,000 total events were acquired for each analysis. Gates were set to include singlet, live, flag+ and IgG+ events. All final data represent specific binding, determined by subtraction of non-specific binding observed in assays performed with mock-transfected cells.

Antibody-dependent NK cell degranulation assay

Cell-surface expression of CD107a was used as a marker for NK cell degranulation, a prerequisite process for ADCC (Ferrari et al., 2011), was performed as previously described (Pino et al., 2021). Briefly, target cells were either Vero E6 cells after a 2 day-infection with SARS-CoV-2 USA-WA1/2020 or 293T cells 2-days post transfection with a SARS-CoV-2 S protein (D614) expression plasmid. NK cells were purified from peripheral blood of a healthy human volunteer, from the External Quality Assurance Program Oversight Laboratory (EQAPOL)⁵⁵, in compliance with Institutional Review Board protocols approved by Duke University Medical Center. NK cells were by negative selection (Miltenyi Biotech), and were incubated with target cells at a 1:1 ratio in the presence of diluted plasma or monoclonal antibodies, Brefeldin A (GolgiPlug, 1 μl/ml, BD Biosciences), monensin (GolgiStop, 4 μl/6 mL, BD Biosciences), and anti-CD107a-FITC (BD Biosciences, clone H4A3) in 96-well flat bottom plates for 6 h at 37 °C in a humidified 5% CO2 incubator. NK cells were then recovered and stained for viability prior to staining with CD56-PECy7 (BD Biosciences, clone NCAM16.2), CD16-PacBlue (BD Biosciences, clone 3G8), and CD69-BV785 (Biolegend, Clone FN50). Flow cytometry data analysis was performed using FlowJo software (v10.8.0). Data is reported as the % of CD107A+ live NK cells (gates included singlets, lymphocytes, aqua blue-, CD56+ and/or CD16+, CD107A+). All final data represent specific activity, determined by subtraction of non-specific activity observed in assays performed with mock-infected cells and in absence of antibodies.

Intracellular cytokine staining (ICS) assay

Cryopreserved PBMC were thawed and rested 4 h at 37 °C in a 5% CO₂ environment. PBMC were then incubated for 6 h in the presence of either RPMI containing 10% fetal bovine serum (unstimulated), Staphylococcus enterotoxin B (SEB) as positive control, or pool peptide spanning the entire SARS-CoV-2 spike protein. All cultures contained a protein transport inhibitor, monensin (Golgi Plug; Becton, Dickinson and Company), and 1 μg/ml of anti-CD49d (Becton, Dickinson and Company, Cat# 340976). Cultured cells were then stained with a cell viability marker and pre-titered quantities of antibodies against CD3/CD4/CD8/CD45RA/ICOS/CCR7/CXCR3/PD-1/CXCR5/CD69/CD154/IL-2/IFN-g/TNF-a/IL-4/IL-21/IL-13/IL-17A. Samples with at least 1,000 viable CD4+ or CD8+ T cells were included. Samples were analyzed on a LSR II instrument (Becton, Dickinson and Company, Franklin Lakes, NJ) using FlowJo v10.8.1 software.

Viral RNA extraction and subgenomic mRNA quantification

SARS-CoV-2 E gene and N gene subgenomic mRNA (sgRNA) was measured by a one-step RT-qPCR adapted from previously described methods^56,57. To generate standard curves, a SARS-CoV-2 E gene sgRNA sequence, including the 5’UTR leader sequence, transcriptional regulatory sequence (TRS), and the first 228 bp of E gene, was cloned into a pcDNA3.1 plasmid. For generating SARS-CoV-2 N gene sgRNA, the E gene was replaced with the first 227 bp of N gene. The recombinant pcDNA3.1 plasmid was linearized, transcribed using MEGAscript T7 Transcription Kit (ThermoFisher, catalog # AM1334), and purified with MEGAclear Transcription Clean-Up Kit (ThermoFisher, catalog # AM1908). The purified RNA products were quantified on Nanodrop, serial diluted, and aliquoted as E sgRNA or N sgRNA standards.

A QIAsymphony SP (Qiagen, Hilden, Germany) automated sample preparation platform along with a virus/pathogen DSP midi kit. RNA extracted from animal samples or standards were then measured in Taqman custom gene expression assays (ThermoFisher). For these assays we used TaqMan Fast Virus 1-Step Master Mix (ThermoFisher, catalog # 4444432) and custom primers/probes targeting the E gene sgRNA (forward primer: 5′ CGA TCT CTT GTA GAT CTG TTC TCE 3′; reverse primer: 5′ ATA TTG CAG CAG TAC GCA CAC A 3′; probe: 5′ FAM-ACA CTA GCC ATC CTT ACT GCG CTT CG-BHQ1 3′) or the N gene sgRNA (forward primer: 5′ CGA TCT CTT GTA GAT CTG TTC TC 3′; reverse primer: 5′ GGT GAA CCA AGA CGC AGT AT 3′; probe: 5′ FAM-TAA CCA GAA TGG AGA ACG CAG TGG G-BHQ1 3′). RT-qPCR reactions were carried out on CFX384 Touch Real-Time PCR System (Bio-Rad) using a program below: reverse transcription at 50 °C for 5 min, initial denaturation at 95 °C for 20 s, then 40 cycles of denaturation-annealing-extension at 95 °C for 15 s and 60 °C for 30 s. Standard curves were used to calculate E or N sgRNA in copies per ml; the limit of detections (LOD) for both E and N sgRNA assays were 12.5 copies per reaction or 150 copies per mL of BAL/nasal swab.

Histopathology

Lung specimen from nonhuman primates were fixed in 10% neutral buffered formalin, processed, and blocked in paraffin for histological analysis. All samples were sectioned at 5 µm and stained with hematoxylin-eosin (H&E) for routine histopathology. Sections were examined under light microscopy using an Olympus BX51 microscope and photographs were taken using an Olympus DP73 camera. Samples were scored by a board-certified veterinary pathologist in a blinded manner. The representative images are to characterize the types and arrangement of inflammatory cells, while the scores show the relative severity of the tissue section.

Immunohistochemistry (IHC)

Staining for SARS-CoV-2 antigen was achieved on the Bond RX automated system with the Polymer Define Detection System (Leica) used per manufacturer’s protocol. Tissue sections were dewaxed with Bond Dewaxing Solution (Leica) at 72 °C for 30 min then subsequently rehydrated with graded alcohol washes and 1× Immuno Wash (StatLab). Heat-induced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1 (Leica), heated to 100 °C for 20 min. A peroxide block (Leica) was applied for 5 min to quench endogenous peroxidase activity prior to applying the SARS-CoV-2 antibody (1:2000, GeneTex, GTX135357). Antibodies were diluted in Background Reducing Antibody Diluent (Agilent). The tissue was subsequently incubated with an anti-rabbit HRP polymer (Leica) and colorized with 3,3’-Diaminobenzidine (DAB) chromogen for 10 min. Slides were counterstained with hematoxylin.

Mouse immunization and challenge

Eleven-month-old female BALB/c mice were purchased from Envigo (#047) and were used for the SARS-CoV, SARS-CoV-2 WA-1, SARS-CoV-2 B.1.351, and RsSHC014-CoV protection experiments. The study was carried out in accordance with the recommendations for care and use of animals by the Office of Laboratory Animal Welfare (OLAW), National Institutes of Health and the Institutional Animal Care and Use Committee (IACUC) of University of North Carolina (UNC permit no. A-3410-01). Animals were housed in groups of five and fed standard chow diets. Virus inoculations were performed under anesthesia and all efforts were made to minimize animal suffering. Mice were intramuscularly immunized with 10 μg RBD-scNP formulated with 3M-052-Alum or GLA-SE. For the SARS-CoV-2 WA-1 and RsSHC014 study, mice were immunized on week 0 and 2, and challenged on week 7. For the SARS-CoV-2 B.1.351 and SARS-CoV study, mice were immunized on week 0 and 4, and challenged on week 6. All mice were anesthetized and infected intranasally with 1 × 10⁴ PFU/ml of SARS-CoV MA15, 1 × 10⁴ PFU/ml of SARS-CoV-2 WA1- MA10 or B.1.351-MA10, 1 × 10⁴ PFU/ml RsSHC014, which have been described previously^{15,45,58,59,60}. Mice were weighted daily and monitored for signs of clinical disease, and selected groups were subjected to daily whole-body plethysmography. For all mouse studies, groups of n = 10 mice were included per arm of the study. Lung viral titers and weight loss were measured from individual mice per group.

Biocontainment and biosafety

Studies were approved by the UNC Institutional Biosafety Committee approved by animal and protocols in the Baric laboratory. All work described here was performed with approved standard operating procedures for SARS-CoV-2 in a biosafety level 3 (BSL-3) facility conforming to requirements recommended in the Microbiological and Biomedical Laboratories, by the U.S. Department of Health and Human Service, the U.S. Public Health Service, and the U.S. Center for Disease Control and Prevention (CDC), and the National Institutes of Health (NIH).

Statistics analysis

Data were plotted using Prism GraphPad 8.0. Two-sided Wilcoxon rank sum exact test was performed to compare differences between groups with p-value < 0.05 considered significant using SAS 9.4 (SAS Institute, Cary, NC). The Benjamini-Hochberg correction⁶¹ was used to adjust the p-values for multiple comparisons.

Reporting summary

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