Human participants

Participants who met the following criteria were included in the study: age 18 or older, a SARS-CoV-2 infection and/or a scheduled COVID-19 vaccination, and the ability and willingness to provide informed consent. Candidates who were under the age of 18 or were unwilling or unable to provide informed consent were excluded from the study. Enrollment started on August 2023 and is expected to be completed by August 2023. The study population consisted of residents across the Chicago area. Adults of different ages, races, and ethnicities were included in the study. Participants were deidentified by assigning them a 4-letter study code to be used for the duration of the study. Participants who were considered to be exposed before vaccination had a positive RT-PCR test for SARS-CoV-2 any time prior to vaccination. Blood was collected by phlebotomy using BD Vacutainer 10 mL tubes containing sodium heparin. Anticoagulated blood was added to LeucoSep tubes (Greiner Bio), and plasma was separated by density gradient centrifugation. To protect the individual’s identity, all samples were labeled with their assigned 4-letter study code and stored in the principal investigator’s laboratory freezers.

Mice, vaccinations, infections, and challenges

Six- to 8-week-old C57BL/6, BALB/c, A/J mice were used. For VSV-SARS-2 spike vaccinations, k18-hACE2 (on C57BL/6 background) mice were used. All mice were purchased from The Jackson Laboratory (approximately half of the mice were males and half were females) and housed at Northwestern University’s Center for Comparative Medicine (CCM) or the UIC. Mice were immunized intramuscularly (50 μL per quadriceps) with Ad5-SARS-CoV-2 spike (109 PFU); VSV expressing SARS-CoV-2 spike protein (VSV-SARS-CoV-2 spike; 107 PFU); mRNA-based vaccine encoding SARS-CoV-2 spike protein (mRNA-SARS-CoV-2 spike; 5 μg); SARS-CoV-2 “whole-spike” protein (SARS-CoV-2 spike; 100 μg with 1:5 Adju-Phos, InvivoGen); SARS-CoV-2 RBD protein (100 μg with 1:5 Adju-Phos); γ-irradiated SARS-CoV-2 (inactivated SARS-CoV-2; 2.5 × 105 PFU); and MVA expressing SARS-CoV-1 spike protein (MVA-SARS-CoV-1 spike; 107 PFU). The vaccine doses were chosen empirically on the basis of prior studies by us and others (5, 14–22).

We obtained Ad5-SARS-CoV-2 spike from the University of Iowa Viral Vector Core (VVC-U-7643, Iowa City, Iowa, USA); VSV-SARS-CoV-2 spike from Sean Whelan (Washington University in St. Louis, St. Louis, Missouri, USA); and MVA-SARS-CoV-1 spike from the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, (NR-623, originally developed by Bernard Moss at the NIH (5). We obtained Ad5-SARS-CoV-2 nucleocapsid from David Masopust (University of Minnesota, Minneapolis, Minnesota, USA); this vector has been used in prior studies (15, 23).

We synthesized mRNA vaccines encoding for the codon-optimized SARS-CoV-2 spike protein from the strain USA-WA1/2023. Constructs were purchased from Integrated DNA Technologies (IDT) and contained a T7 promoter site for in vitro transcription of mRNA. The sequences of the 5′- and 3′-UTRs were identical to those in a Dengue virus mRNA vaccine documented in a previous publication (19). mRNA was synthesized from linearized DNA with T7 In Vitro Transcription Kits, following the manufacturer’s protocol. RNA was generated with pseudouridine in place of uridine with the Incognito mRNA Synthesis Kit (CellScript, catalog C-ICTY110510). A 5′ cap-1 structure and a 3′ poly-A tail were enzymatically added. mRNA was encapsulated into lipid nanoparticles using the NanoAssemblr Benchtop system (Precision NanoSystems). mRNA was dissolved in Formulation Buffer (catalog NWW0043, Precision NanoSystems) and run through a laminar flow cartridge with GenVoy ILM (catalog NWW0041, Precision NanoSystems) encapsulation lipids at a flow ratio of 3:1 (RNA: GenVoy-ILM), with a total flow rate of 12 mL/min, to produce mRNA–lipid nanoparticles (mRNA-LNPs). These mRNA-LNPs were characterized for encapsulation efficiency and mRNA concentration via the RiboGreen assay using the Quant-iT RiboGreen RNA Assay Kit (catalog R11490, Invitrogen, Thermo Fisher Scientific).

SARS-CoV-2 spike and RBD proteins used for vaccinations were produced by Sergii Pshenychnyi at Northwestern’s University Recombinant Protein Production Core using the following plasmids produced under HHSN272201400008C and obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), NIH: vector pCAGGS containing the SARS-related coronavirus 2; Wuhan-Hu-1 spike glycoprotein gene (soluble, stabilized); NR-52394 and RBD; and NR-52309. Protein vaccines were administered with 1:5 Adju-Phos (InvivoGen).

Inactivated SARS-CoV-2 was obtained from BEI Resources, NIAID, NIH (SARS-related coronavirus 2, isolate USA-WA1/2023, γ-irradiated, NR-52287). MHV-1 was purchased from the American Type Culture Collection (ATCC, VR-261), and OC43 was received from BEI Resources, NIAID, NIH (NR-52725). MHV-A59 was a gift from Susan Weiss (University of Pennsylvania, Philadelphia, Pennsylvania, USA).

For OC43 and MHV challenges, mice were infected intranasally (25 μL/nostril) with OC43 (2 × 106 PFU) or mouse hepatitis virus (MHV-1/MHV-A59; 106 PFU).

For SARS-CoV-2 challenges, mouse-adapted SARS-CoV-2 (MA10) was provided by Ralph Baric (University of North Carolina, Chapel Hill, North Carolina, USA) (24). SARS-CoV-2 (MA10) was propagated and titered on Vero-E6 cells (ATCC, CRL1586). BALB/c mice were anesthetized with isoflurane and challenged via intranasal inoculation with 8 × 103 foci-forming units (FFU SARS-CoV-2 (MA10). Lungs were isolated from mice 5 days after infection and homogenized in PBS. RNA was extracted from lung homogenate using a Zymo Research Quick-RNA 96 Kit (R1052). Viral genomes were quantified via quantitative RT-PCR with the N1 Primer/Probe Kit from Integrated DNA Technologies (IDT, catalog 10006713).

Protein-specific ELISA (SARS-CoV-2 spike, RBD, nucleocapsid; SARS-CoV-1 spike; OC43 spike)

Antigen-specific total antibody titers were measured by ELISA as described previously (16, 25). Briefly, 96-well, flat-bottomed MaxiSorp plates (Thermo Fisher Scientific) were coated with 1 μg/mL of the respective protein for 48 hours at 4°C. Plates were washed 3 times with wash buffer (PBS plus 0.05% Tween 20). Blocking was performed with blocking solution (200 μL PBS plus 0.05% Tween 20 plus 2% BSA) for 4 hours at room temperature. Six microliters of sera (plasma for human ELISAs) was added to 144 μL blocking solution in the first column of the plate, 1:3 serial dilutions were performed until row 12 for each sample, and plates were incubated for 60 minutes at room temperature. Plates were washed 3 times with wash buffer followed by addition of HRP-conjugated goat anti–mouse IgG secondary antibody (SouthernBiotech; diluted in blocking solution 1:5000) at 100 μL/well and incubated for 60 minutes at room temperature. For the ELISAs with human plasma samples, goat anti–human IgG (H+L) conjugated to HRP at 1:1000 (Jackson ImmunoResearch) was used. After washing the plates 3 times with wash buffer, 100 μL/well SureBlue Substrate (SeraCare) was added for 1 minute. The reaction was stopped using 100 μL/well KPL TMB Stop Solution (SeraCare). Absorbance was measured at 450 nm using a Spectramax Plus 384 (Molecular Devices). In all ELISA plots, the y axis indicates the endpoint titer (the sera or plasma dilution at which absorbance was greater than 2 times the average for the negative controls human pre-2023 plasma or mouse-naive sera). SARS-CoV-2 spike and RBD proteins used for ELISAs were produced by Sergii Pshenychnyi and Irina Shepotinovskaya at the Northwestern Recombinant Protein Production Core using the following plasmids produced under HHSN272201400008C and obtained from BEI Resources, NIAID, NIH: vector pCAGGS containing the SARS-related coronavirus 2; Wuhan-Hu-1 spike glycoprotein gene (soluble, stabilized); NR-52394 and RBD; and NR-52309. SARS-CoV-2 nucleocapsid protein was obtained through BEI Resources, NIAID, NIH (NR-53797). SARS-CoV-1 spike protein was also obtained through BEI Resources, NIAID, NIH (NR-722). OC43 spike protein was purchased from Sino Biological (40607-V08B).

Virus-specific ELISAs (OC43; MHV-1)

Virus-specific ELISAs were performed as described earlier (16, 25). In brief, 96-well, flat-bottomed MaxiSorp plates (Thermo Fisher Scientific) were coated with 100 μL/well of the respective viral lysate (OC43-, or MHV-1–infected cell lysates) diluted 1:10 in PBS for 48 hours at room temperature. Plates were washed 3 times with wash buffer (PBS plus 0.5% Tween 20) followed by blocking with blocking solution (200 μL/well PBS plus 0.2% Tween 20 plus 10% FCS) for 2 hours at room temperature. Five microliters of sera (plasma for human ELISAs) was added to 145 μL blocking solution in the first column of the plate, and 1:3 serial dilutions were performed until row 12 for each sample followed by incubation at room temperature for 90 minutes. Plates were washed 3 times with wash buffer, followed by addition of 100 μL/well HRP-conjugated goat anti–mouse IgG (SouthernBiotech), diluted 1:5000 in blocking solution. Plates were incubated for 90 minutes at room temperature. Goat anti–human IgG (H+L) conjugated to HRP (1:1000; Jackson ImmunoResearch) was used when ELISA was performed with human samples. After washing the plates 3 times with wash buffer, 100 μL/well SureBlue Substrate (SeraCare) was added for 8 minutes. The reaction was stopped using 100 μL/well KPL TMB Stop Solution (SeraCare). Absorbance was measured at 450 nm using a Spectramax Plus 384 (Molecular Devices).

Virus propagation

OC43 was propagated in a 80%–90% confluent monolayer of HCT-8 cells (ATCC, CCL-244) in T175 flasks at a MOI of 0.01, diluted in 5 mL RPMI supplemented with 2% FBS, 1% penicillin/streptomycin, and 1% l-glutamine. Infected cells were incubated at 33°C for 2 hours in a humidified 5% CO2 incubator. After incubation, the flasks were supplemented with 20 mL 2% RPMI and incubated for 5 days at 33°C in a CO2 incubator. MHV-A59 and MHV-1 were expanded in 17CL-1 cells (gift from Susan Weiss, University of Pennsylvania, Philadelphia, Pennsylvania, USA) following a previously published protocol (26).

OC43 and MHV quantification by plaque assay

For MHV quantification, 106 cells/well L2 cells (gift from Susan Weiss, University of Pennsylvania) were seeded onto 6-well plates in 10% DMEM (10% FBS, 1% penicillin/streptomycin, and l-glutamine). After 2 days, when cells reached approximately 100% confluence, the media were removed. Ten-fold serial dilutions of viral stock or homogenized lung were prepared in 1% DMEM (1% FBS, 1% penicillin/streptomycin, and l-glutamine), added to the wells, and incubated at 37°C for 1 hour, with gentle rocking of the plates every 10 minutes. After incubation, 3.5 mL 1% agarose diluted 1:1 with 20% 2X-199 media (2X-199 media supplemented with 20% FBS, 1% penicillin/streptomycin, and l-glutamine) was overlaid onto the monolayer, and the plates were incubated at 37°C in 5% CO2 for 2 days. On day 2, the agar overlay was gently removed, and the monolayer was stained with 1% crystal violet for 15 minutes. After staining, the crystal violet was aspirated, plates were washed once with 2 mL water per well, and then dried to visualize plaques. Quantification of OC43 stocks for challenge studies was similar to the quantification of MHV-A59 (26), except that 5 mL agar overlay was added on an infected monolayer of L2 cells and incubated at 33°C in a CO2 incubator for 5–6 days. The monolayer was stained with 1% crystal violet, and plaques were quantified by manual counting. For viral load quantification in lung, tissue was collected in round-bottomed 14 mL tubes (Falcon) containing 2 mL 1% FBS DMEM. Tissues were ruptured using a Tissue Ruptor Homogenizer (QIAGEN). Homogenized tissues were clarified using a 100 μm strainer (USA Scientific Inc.) to remove debris, and clarified tissue lysates were used for the plaque assay.

Quantification of OC43 by RT-PCR

Lungs were isolated from mice and homogenized in 1% FBS DMEM. RNA was extracted from lung homogenate using a PureLink Viral RNA/DNA Mini Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions. OC43 viral loads in lungs were determined using 1-step quantitative real-time RT-PCR. RT-PCR was performed using OC43-nucleocapsid–specific TaqMan primers and a probe labeled with a 5′-FAM reporter dye and a 3′-BHQ quencher (IDT) and an AgPath-ID One-Step RT-PCR kit (AgPath AM1005, Applied Biosystems) on an ABI QuantStudio 3 platform (Thermo Fisher Scientific). Each sample was tested in duplicate in 25 μL reactions containing 12.5 μL of a 2× RT-PCR buffer, 1 μL 25× RT-PCR enzyme mix provided with the AgPath kit, 0.5 μL (450 nM) forward primer, 0.5 μL (450 nM) reverse primer, 0.5 μL (100 nM) probe, and 10 μL RNA. In parallel, each sample was also tested for the β-actin gene as an internal control to verify RNA extraction quality using mouse β-actin–specific TaqMan primers and probe labeled with 5′-FAM and 3′-BHQ (IDT). Thermal cycling involved reverse transcription at 45°C for 10 minutes and denaturation at 95°C for 15 minutes, followed by 45 cycles of amplification (15 seconds at 95°C and 1 minute at 60°C.) To avoid cross-contamination, single-use aliquots were prepared for all reagents including primers, probes, buffers, and enzymes.

Quantification of SARS-CoV-2 by RT-PCR

Lungs were isolated from mice and homogenized in PBS. RNA was extracted from lung homogenate using a Zymo Research Quick-RNA 96 Kit (R1052). Viral genomes were quantified via RT-PCR with the TaqMan RNA-to-Ct One-Step Kit (Thermo Fisher Scientific, catalog 4392653) and primer/probe sets with the following sequences: forward, 5′-GACCCCAAAATCAGCGAAAT-3′, reverse, 5′-TCTGGTTACTGCCAGTTGAATCTG-3′, probe, 5′-ACCCCGCATTACGTTTGGTGGACC-3′ (IDT, catalog 10006713). A SARS CoV-2 copy number control was obtained from BEI Resources, NIAID, NIH (NR-52358) and used to quantify SARS-CoV-2 genomes.

Reagents, flow cytometry, and equipment

Dead cells were gated out using LIVE/DEAD fixable dead cell stain (Invitrogen, Thermo Fisher Scientific). The SARS-CoV-2 spike overlapping peptide pools obtained from BEI Resources, NIAID, NIH (NR-52402) were used for intracellular cytokine staining. Biotinylated MHC class I monomers (Kb VL8) were obtained from the NIH tetramer facility at Emory University (Atlanta, Georgia, USA). Cells were stained with fluorescence-labeled antibodies against CD44 (IM7 on Pacific blue, BioLegend, catalog 103020); CD8α (53-6.7 on PerCP-Cy5.5, BD Pharmingen, catalog 551162); IFN-γ (XMG1.2 on APC, BD Pharmingen, catalog 554413); and Vβ11 (RR3-15 on FITC, BioLegend, catalog 125905). Fluorescence-labeled antibodies were purchased from BD Pharmingen, except for anti-CD44, which was from BioLegend. Flow cytometric samples were acquired with a BD FACSCanto II or a BD LSR II and analyzed using FlowJo software (Tree Star).

SARS-CoV-2 pseudovirus neutralization assays

A SARS-CoV-2 pseudovirus was generated by transfection of HEK-293T cells (ATCC, CRL-1573) with a pCAGGS vector expressing the SARS-CoV-2 spike glycoprotein (BEI Resources, NIAID, NIH: NR-52310). Twenty-four hours later, transfected cells were infected with VSVΔG*G-GFP at a MOI of 0.5. After 24 hours, GFP foci were visualized, and the supernatant was harvested and passed through a 0.45 μM filter. This SARS-CoV-2 pseudovirus was concentrated using an Amicon Ultra-15 filter (UFC910024, MilliporeSigma) and then stored at –80°C. Titers were measured by infecting HEK-293T-hACE2 cells (BEI Resources, NIAID, NIH, NR-52511) and counting GFP foci under a fluorescence microscope after 24 hours.

The SARS-CoV-2 pseudovirus neutralization assay was performed by mixing serial dilutions of MVA-SARS-CoV-1 immune mouse sera (or naive sera) with 200 FFU SARS-CoV-2 pseudovirus in a 96-well plate and incubated for 2 hours. After incubation, 100 μL of the sera-virus mixture was transferred to a 96-well half-area plate containing HEK-293T-hACE2 cells. The next day, GFP foci were counted in each well under a fluorescence microscope.

MHC class I binding predictions

The MHC class I binding predictions were made on May 17, 2023, using the Immune Epitope Database (IEDB) analysis resource tool NetMHCpan, version 4.1 (http://tools.iedb.org/mhci/) (27).

scTCR-Seq data acquisition and analysis

C57BL/6 mice were immunized intramuscularly with 109 PFU Ad5-SARS-2 spike, and on day 28, splenic CD8+ T cells were MACS sorted using negative selection (STEMCELL Technologies). Purified CD8+ T cells were stained with Kb VL8, LIVE/DEAD stain, and flow cytometry antibodies against CD8 and CD44. Live, CD8+, CD44+, and Kb VL8+ cells were FACS sorted to approximately 99% purity on a FACS Aria Cytometer (BD Biosciences) and delivered to the Northwestern University NU-Seq core for scTCR-Seq using the Chromium NextGem 5′ v2 kit (10X Genomics). Once the library was sequenced, the output file in BCL format was converted to fastq files and aligned to the mouse genome in order to generate a matrix file using the Cell Ranger pipeline. These upstream quality control (QC) steps were performed by Ching Man Wai and Matthew Schipma at the Northwestern University NUSeq Core (Evanston, Illinois, USA). TCR analyses were performed using the scRepertoire package (28). Only cells expressing both TCRα and TCRβ chains were selected. For cells with more than 2 TCR chains, only the top 2 expressed chains were used. scTCR-Seq accession data were deposited in the NCBI’s Gene Expression Omnibus (GEO) database (GEO GSE173567; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE173567).

Adoptive plasma transfers

C57BL/6 mice received 50 μL heat-inactivated human plasma from different human donors (before vaccination, after vaccination, before 2023, or SARS-CoV-2 convalescent). Each mouse received plasma from 1 different human donor. On the next day, mice were infected intranasally with 5 × 107 PFU OC43. Lungs were harvested on day 5 after infection and ruptured using a Tissue Ruptor Homogenizer (QIAGEN). Viral loads were quantified by RT-PCR as described above.

Statistics

Statistical analyses were performed using a Mann-Whitney U test, a paired t test, a 1-way ANOVA with multiple comparisons, or a paired Wilcoxon test. Dashed lines in ELISA/plaque assay figures represent the LOD. Data were analyzed using GraphPad Prism (GraphPad Software).

Study approval

Human specimens. All protocols used for participant recruitment, enrollment, blood collection, sample processing, and immunological assays with human samples were approved by the IRB of Northwestern University (STU00212583). All participants voluntarily enrolled in the study by signing an informed consent form after receiving detailed information about the clinical study.

Mouse studies. All mouse experiments were performed with approval from the IACUCs of Northwestern University and the UIC (study approval nos. IS00003258 and 20-107). All mouse experiments with BL2 agents were performed with approval from the IACUC of Northwestern University. SARS-CoV-2 infections of mice were performed at the UIC following BL3 guidelines, with approval from the UIC’s IACUC.