Female, 3-month-old BALB/c mice were purchased from the Jackson Laboratory. 11 month old female BALB/c mice were purchased from Taconic Biosciences and used for experiments on aged mice. Mice were housed under specific pathogen-free conditions at Boston Children’s Hospital. All procedures were approved under the Institutional Animal Care and Use Committee (IACUC) and were performed under the supervision of the Children’s Hospital Department of Animal Resources (Protocol number 19-02-3897 R). At the University of Maryland School of Medicine, mice were housed in a biosafety level 3 facility for SARS-CoV-2 infections using all procedures approved under the IACUC (Protocol #1120004).
Mice were injected with BNT162b2 SARS-CoV-2 spike mRNA vaccine (Pfizer-BioNTech). BNT162b2 suspension (100 µg mRNA/ml) was diluted 1:5 in phosphate buffered saline (PBS) and 50 µl (1 µg) was injected intramuscularly into the caudal thigh. BNT162b2 was obtained as residual overfill volumes in used vials from the Boston Children’s Hospital vaccine clinic, using only material that would otherwise be discarded, and used within 6 hours from the time of reconstitution. Mock treatment mice received PBS alone.
SARS-CoV-2 wild-type peak and RBD expression and purification
SARS-CoV-2 Wuhan-Hu-1 full-length spike glycoprotein (M1-Q1208, GenBank MN90894) and RBD constructs (amino acid residues R319-K529, GenBank MN975262.1), both containing an HRV3C protease cleavage site, a TwinStrepTag and an 8XHisTag at C-terminus was obtained from Barney S. Graham (NIH Vaccine Research Center) and Aaron G. Schmidt (Ragon Institute), respectively. These mammalian expression vectors were used to transfect Expi293F suspension cells (Thermo Fisher) using polyethyleneimine (Polysciences). Cells were allowed to grow at 37 °C, 8% CO2 for another 5 days before harvesting for purification. Protein was purified in a PBS buffer (pH 7.4) from filtered supernatants using either StrepTactin resin (IBA) or Cobalt-TALON resin (Takara). Affinity tags were cleaved from eluted protein samples by HRV 3C protease, and tag-deleted proteins were further purified by size exclusion chromatography using a Superose 6 10/300 column (Cytiva) for full length Spike and a Superdex 75 10 /300 Increase column (Cytiva) for RBD domain in a PBS buffer (pH 7.4).
Spike protein-specific antibody concentrations were quantified in serum samples by ELISA by modification of a previously described protocol28. Briefly, 96-well flat-bottomed high-binding plates (Corning) were coated with spike protein (25 ng per well) and incubated overnight at 4 °C. Plates were washed with 0.05% Tween 20/PBS and blocked with 1% bovine serum albumin (BSA)/PBS for 1 hour at room temperature. Serum samples were serially diluted 4-fold from 1:100 to 1:1.05 × 108 and then incubated at room temperature for 2 hours. Plates were washed three times and incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated anti-mouse IgG, IgG1, and IgG2a (SouthernBiotech). Plates were washed five times and developed with tetramethylbenzidine (BD OptEIA Substrate Solution, BD Biosciences) for 5 min and then stopped with 2 NH2SO4. Optical densities (ODs) were read at 450 nm with a SpectraMax iD3 microplate reader (Molecular Devices). End point titers were calculated as the dilution that emitted an OD exceeding a 3x background. Samples with OD values below the detection limit for which no interpolation of the titer was possible were assigned an arbitrary value of 50.
hACE2-RBD Inhibition Test
The hACE2 RBD inhibition test used a modification of a previously published protocol29. Briefly, 96-well flat-bottomed high-binding plates (Corning) were coated with recombinant hACE2 (100 ng per well) (Sigma-Aldrich) in PBS, incubated overnight at 4 °C, washed three times with 0.05% Tween 20 PBS and blocked with 1% BSA PBS for 1 hr at room temperature. Serum samples were diluted 1:160, preincubated with 3 ng of wild-type RBD-Fc in 1% BSA PBS for 1 hour at room temperature and then transferred to the hACE2-coated plate. RBD-Fc without serum preincubation was added as a positive control and 1% BSA PBS without serum preincubation was added as a negative control. Plates were then washed three times and incubated for 1 hour at room temperature with HRP-conjugated anti-human IgG Fc (SouthernBiotech). Plates were washed five times and developed with tetramethylbenzidine (BD OptEIA Substrate Solution, BD Biosciences) for 5 min and then stopped with 2 NH2SO4. The OD was read at 450 nm with a SpectraMax iD3 microplate reader (Molecular Devices). Percent inhibition of RBD binding to hACE2 was calculated as: Inhibition (%) =[1 − (Sample OD value − Negative Control OD value)/(Positive Control OD value − Negative Control OD value)]× 100.
Pseudovirus Neutralization Test
The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated in an approach similar to that previously described.30.31. Briefly, the packaging plasmid psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and spike protein expressing pcDNA3.1-SARS CoV-2 SΔCT of variants were co-transfected into HEK293T cells by lipofectamine 2000 (ThermoFisher). Pseudoviruses of SARS-CoV-2 variants were generated using the WA1/2020 strain (Wuhan/WIV04/2019, GISAID accession ID: EPI_ISL_402124), B.1.617.2 (Delta, GISAID accession ID: EPI_ISL_2020950) or B.1.1. 529 (Omicron, GISAID ID: EPI_ISL_7358094.2). The supernatants containing the pseudotype viruses were collected 48 hours after transfection, which were purified by centrifugation and filtration with a 0.45 µm filter. To determine the neutralization activity of the serum samples, HEK293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 2 × 10.4 cells/well overnight. Triple serial dilutions of heat inactivated serum samples were prepared and mixed with 50 µl pseudovirus. The mixture was incubated for 1 h at 37 °C before being added to HEK293T-hACE2 cells. After 48 hours, cells were lysed in Steady-Glo Luciferase Assay (Promega) according to the manufacturer’s instructions. SARS-CoV-2 neutralization titers were defined as the sample dilution at which a 50% reduction in relative light unit (RLU) was observed relative to the mean of the virus control wells.
Splenocyte restimulation, intracellular cytokine staining and flow cytometry
Mouse spleens were mechanically dissociated and filtered through a 70 µm cell strainer. After centrifugation, the cells were treated with 1 ml of ammonium chloride-potassium lysis buffer for 2 minutes at room temperature. Cells were washed and plated in a 96-well U-bottom plate (2 x 106/well) and incubated overnight in RPMI 1640 supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml), streptomycin (100 mg/ml), 2-mercaptoethanol (55 mM), non-essential amino acids ( 60 mM), HEPES (11 mM), and L-Glutamine (800 mM) (all Gibco). The next day, SARS-CoV-2 wild-type (PM-WCPV-S-1) or Omicron (PM-SARS2-SMUT08-1) spike peptide pools (JPT) were added at 0.6 nmol/ml in the presence of anti-mouse CD28/49d (1 µg/ml, BD) and brefeldin A (5 µg/ml, BioLegend). After 6 hours of stimulation, the cells were washed twice and treated with Mouse Fc Block (BD) according to the manufacturer’s instructions. Cells were washed and stained with Aqua Live/Dead staining (Life Technologies, 1:500) for 15 minutes at room temperature. After two additional washes, cells were incubated for 30 min at 4 °C with the following Abs: anti-mouse CD44 [IM7, PerCP-Cy5.5, BioLegend #103032, 1:160]anti-mouse CD3 [17A2, Brilliant Violet 785, BioLegend #100232, 1:40]anti-mouse CD4 [RM4-5, APC/Fire 750, BioLegend 100568, 1:160] and anti-mouse CD8 [53-6.7, Brilliant UltraViolet 395, BD #563786, 1:80]. Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm kit according to the manufacturer’s instructions, and subjected to intracellular staining (30 min at 4 °C) using the following Abs: anti-mouse IFNγ [XMG1.2, Alexa Fluor 488, BioLegend #505813, 1:160]anti-mouse TNF [MP6-XT22, PE Cy7, BioLegend # 506324, 1:160]anti-mouse IL-2 [JES6-5H4, PE, BioLegend # 503808, 1:40]. Finally, cells were fixed in 1% paraformaldehyde (Electron Microscopy Sciences) for 20 min at 4 °C and stored in PBS at 4 °C until acquisition. Samples were analyzed on an LSR II (BD) flow cytometer and FlowJo v10.8.1 (FlowJo LLC).
Studying SARS-CoV-2 Omicron Challenge
Mice were anesthetized interperitoneally with 50 L of ketamine (1.3 mg/mouse) and xylazine (0.38 mg/mouse) diluted in PBS. Mice were then inoculated intranasally with 1 × 105 PFU of SARS-CoV-2 Omicron (BA.1), courtesy of Dr. Mehul Suthar, in 50 µL of PBS distributed over nostrils. Challenged mice were weighed daily. On day 2 post-infection, mice were euthanized and lungs harvested for histology, host response analysis by qPCR, and determination of viral titer by plaque assay.
SARS-CoV-2 plaque assay
The day prior to infection, 2.5 e5 VeroTMPRSS2 cells per well were seeded in 1 ml VeroTMPRSS2 media in a 12-well plate [DMEM (Quality Biological), 10% FBS (Gibco), 1% Penicillin-Streptomycin (Gemini Bio Products), and 1% L-Glutamine (Gibco)]. Tissue samples were thawed and homogenized with 1 mm beads in a Bead ruptor (Omni International Inc.) and then spun at 21,000 g for 2 minutes. A 6-point dilution curve was established by serially diluting 25 L of sample 1:10 in 225 µL of DMEM. 200 L of each dilution was then added to the cells and the plates were shaken every 15 min for 1 h at 37 °C. After 1 h, 2 ml of a semi-solid agarose overlay was added to each well [DMEM, 4% FBS, and 0.06% UltraPure agarose (Invitrogen)]. After 72 hours at 37 °C and 5% CO2plates were fixed in 2% PFA for 20 min, stained with 0.5 ml 0.05% crystal violet and 20% EtOH and washed twice with H2O prior to plaque counting. Then the titer was calculated. For tissue homogenates, this titer was multiplied by 40 based on the mean weight of the 25 mg tissue sample.
Gene expression analysis by qPCR
RNA was isolated from TRI reagent samples using phenol-chloroform extraction or column-based extraction systems (Direct-zol RNA Miniprep, Zymo Research) according to the manufacturer’s protocol. RNA concentration and purity (260/280 and 260/230 ratios) were measured by NanoDrop (Thermo Fisher Scientific). Samples with an A260/A280 ratio of <1.7 were excluded from further analysis. cDNA was prepared from purified RNA with RT2 First Strand Kit, according to the instructions of the manufacturer (Qiagen). cDNA was quantified by qPCR on a 7300 real-time PCR system (Applied Biosystems) using pre-designed SYBR Green Primers (QIAGEN) specific for Fit2 (PPM05993A), Rsad2 (PPM26539A), and Rpl13a (PPM03694A).
Statistics and reproducibility
Mouse experiments were designed to include a total of 20 mice per group and were from single experiments. Sample size and age criteria were chosen empirically based on the results of previous studies. Mice were randomly assigned to different treatment groups. No data outliers are excluded. Statistical analyzes were performed with Prism v9.0.2 (GraphPad Software). two-tailed p values <0.05 were considered significant. Normally distributed data were analyzed by one-way analysis of variance (ANOVAs) adjusted for multiple comparisons. Non-normally distributed data were log transformed and analyzed by ANOVA or analyzed by Kruskal-Wallis test corrected for multiple comparisons.
More information on research design is available in the Nature Research Reporting Summary linked to this article.