Viruses and cells

African green monkey kidney epithelial Vero-E6 cells (laboratory-passaged derivatives from ATCC CRL-1586) were grown in Dulbecco’s modified Eagle’smedium (DMEM; Gibco/Thermo Fisher) with 10% fetal bovine serum (FBS, HyClone Laboratories) and 1% anti-biotic/streptomycin (P/S, Gibco). Vero-E6-TMPRSS2 cells were purchased from SEKISUI XenoTech, LLC, and maintained in 10% FBS (HyClone Laboratories), and 1% P/S and 1 mg/ml G418 (Gibco). All cells were maintained at 37 °C with 5% CO2. The infectious clones derived USA-WA1/2020 SARS-CoV-2, ∆3678, and the BA.5 variant were generated as previously described8,19,20.

Animal studies

Female heterozygous K18-hACE2 C57BL/6J mice (strain: B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from The Jackson Laboratory and were infected intranasally (i.n.) with 2 × 103 PFU of either WT WA1 or ∆3678 infectious clone-derived viruses or were mock-infected with DPBS. Mice were between 8 and 10 weeks old at the time of initial infection and were age-matched within experimental cohorts. Intranasal challenge with 1 × 104 PFU of WT WA1 or BA.5 virus was performed on day 28 after the initial infection. For each infection, animals were anesthetized with isoflurane (Piramal) and monitored regularly until fully recovered from anesthesia. Mice were monitored daily for weight changes and illness. Mice with more than 20% weight loss were euthanized by inhalation of CO2. At day 28 post vaccination and day 4 post virus challenge, mice were euthanized by inhalation of CO2, BAL fluid, nasal washes, whole lungs, and whole spleens were collected from one subset of mice for immunological analysis. From a different subset of mice, the trachea and cranial right lobe were collected in DPBS for enumeration of viral loads via plaque assay, and the middle and caudal lobes were collected in TRIzol (Invitrogen) for RNA extraction.

Ethic statement

All animal handling was approved by the Institutional Animal Care and Use Committee (IACUC, protocol # 2103023) of the University of Texas Medical Branch and performed under animal biosafety level 3 (ABSL3) conditions at the Galveston National Laboratory in accordance with guidelines set by the IACUC.

Plaque assay

Collected lung lobes were homogenized in 1 ml of PBS at 6000 rpm for 60 s using a Roche MagNA Lyser instrument before titration. The lung homogenates were clarified by centrifugation at 15,000 × g for 3 min. Viral titers in the supernatants were enumeratedusing a standard plaque assay. Briefly, approximately 1.2 × 106 Vero-E6-TMPRSS2 cells were seeded to each well of 6-well plates and cultured at 37 °C, 5% CO2 for 16 h. The virus was serially diluted in DMEM with 2% FBS and 200 μl diluted viruses were transferred onto the monolayers. The viruses were incubated with the cells at 37 °C with 5% CO2 for 1 h. After the incubation, an overlay medium was added to the infected cells per well. The overlay medium contained MEM with 2% FBS, 1% penicillin/streptomycin, and 1% Sea-plaque agarose (Lonza, Walkersville, MD). After a 2.5-day incubation, the plates were stained with neutral red (Sigma-Aldrich) and plaques were counted on a lightbox.

Antibody ELISA

ELISA plates were coated with 100 ng/well recombinant SARS-CoV-2 RBD protein (RayBiotech) overnight at 4 °C. The plates were washed twice with PBS, containing 0.05% Tween-20 (PBS-T) and then blocked with 8% FBS for 1.5 h. Sera or BAL samples were diluted 1:100 or undiluted in blocking buffer and were added for 1 h at 37 °C. Plates were washed 5 times with PBS-T. Goat anti-mouse IgG (1:2000, 1013-05, Southern Biotech, USA), or goat anti-mouse IgG2C (1:2000, 1078-04, Southern Biotech, USA) conjugated with horseradish peroxidase (HRP) or alkaline phosphatase was added at a 1:2000 dilutions for 1 h at 37 °C followed by adding TMB (3, 3, 5, 5′- tetramethylbenzidine) peroxidase substrate (Thermo Scientific) or p-nitrophenyl phosphate (Sigma-Aldrich), and the intensity was read at an absorbance of 450 or 405 nm. To determine IgA titer, HRP -conjugated goat anti-mouse IgA (1:2000, 1040-05, Southern Biotech) was added as the secondary antibody at a 1:2000 dilution for 1 h at 37 °C, followed by adding TMB peroxidase substrate (Thermo Scientific) for about 15 min. The reactions were stopped by 1 M sulfuric acid, and the intensity was read at an absorbance of 450 nm. Binding endpoint titers were determined using a cutoff value which is negative control + 3x SD.

B cell ELISPOT

ELISPOT assays were performed as previously described21. Briefly, Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with 100 µl rSARS-CoV-2 spike protein (R&D Systems). To detect total IgA+-expressing B cells, the wells were coated with 100 µl of anti-mouse IgA capture Ab (15 µg/ml, 3865-3, Mabtech In). Cells were added in duplicates to assess total IgA ASCs or SARS-CoV-2 specific B cells. The plates were incubated overnight at 37˚C, followed by incubation with biotin-conjugated anti-mouse IgA (0.5 µg/ml, 3865-6, Mabtech In) for 2 h at room temperature, then 100 µL/well streptavidin-ALP (1:1000) was added for 1 h. Plates were developed with BCIP/NBT-Plus substrate until distinct spots emerge, washed with tap water, and scanned using an ImmunoSpot 6.0 analyzer and analyzed by ImmunoSpot software (Cellular Technology Ltd).

Intracellular cytokine staining (ICS)

Splenocytes or lung leukocytes were incubated with SARS-CoV-2 spike peptide pools (1 μg/ml, Miltenyi Biotec) for 6 h in the presence of GolgiPlug (BD Bioscience). Cells were stained with antibodies for CD3 (12-0031-81), CD4 (17-0041-82), or CD8 (11-0081-82) purchased from Thermo Fisher Scientific, fixed in 2% paraformaldehyde, and permeabilized with 0.5% saponin before adding anti-IFN-γ (12-7311-82, Thermo Fisher Scientific). Samples were processed with a C6 Flow Cytometer instrument. Dead cells were excluded based on forward and side light scatter (Supplementary Fig. 3). Data were analyzed with a CFlow Plus Flow Cytometer (BD Biosciences).

Analysis of nCounter analysis system (NanoString) data

Lung samples were homogenized in Trizol (Thermo Fisher Scientific). Total RNA was purified using the Direct-zol-96 MagBead RNA kit (Zymo) with a KingFisher Apex System (Thermo Fisher Scientific). RNA samples were normalized to 20 ng/μL and followed by analysis using the nCounter Pro Analysis System and the nSolver Analysis Software. Plots were made using R version 4.1.2. An un-adjusted p-value cutoff of 0.05 for non-paired Student’s t test of normalized nCounter reads was used to determine DEGs for the three conditions. This was due to overall sample sizes and subtetly of changes in the WT/WT vs mock/mock and ∆3678/WT vs mock/mock comparisons. An additional log2 fold change cutoff of + or – 0.6 was used to limit labeling of up-and down-regulated genes in volcano plots although the Mock/WT condition had too many genes to label. The ‘ggvenn’ package version 0.1.9 was used to create Fig. 2m. IPA (version 84978992) core analysis was performed on data (gene name, un-adjusted p-value, and fold change) from each condition, evaluating based on expression fold changes. The option “User Data Set” was used as the reference set. Data on canonical pathways for Supplementary Fig. 2 were derived from a comparison analysis. -log(p-values) and z-scores were used to generate bubble plots, using ascending p-value or descending -log(p-value) for ordering.

Statistical analysis

Values for viral load, cytokine production, antibody titers, B and T cell response experiments were compared using Prism software (GraphPad) statistical analysis and were presented as means ± SEM. P values of these experiments were calculated with a non-paired Student’s t test.

Reporting summary

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