The SARS-CoV-2 Omicron BA.1 spike G446S mutation potentiates antiviral T-cell recognition

Ethics statement

For the use of human specimens, all protocols involving human subjects recruited at Kumamoto University were reviewed and approved by the Institutional Review Boards of Kumamoto University (approval numbers 2074 and 477). All human subjects provided written informed consent.

Collection of human PBMCs

Human PBMCs were obtained from thirty HLA-A*24:02-positive BNT162b2 or mRNA-1273 vaccinated donors (median age: 24, range: 18–79, 67% male), five HLA-A*24:02-negative BNT162b2-vaccinated donors (median age: 24, range: 18–28, 60% Female) (Supplementary Table 1). PBMCs were purified by a density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare Life Sciences, Cat# 17-1440-03) and stored in liquid nitrogen until further use.

Cell culture

A549-ACE2/A2402 cells, the A549 cells stably expressing human ACE2 and HLA-A*24:02-IRES-GFP, were generated by retroviral transduction as previously described^13,16 and were maintained in Ham’s-F12 (Wako, Cat# 080-08565) containing 10% fetal bovine serum (FBS). C1R cells expressing HLA-A*24:02 (C1R-A2402)²⁶ and TCR-deficient Jurkat cells expressing luciferase gene (Jurkat∆-Luc) were maintained in RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11875101) containing 10% FBS.

Virus

Four clinically isolated SARS-CoV-2 lineages were used: SARS-CoV-2 Wuhan strain [SARS-CoV-2/Hu/DP/Kng/19-020 (DDBJ Accession ID: LC528232)], was provided from Kanagawa Prefectural Institute of Public Health. A B.1.617.2 (Delta) lineage [hCoV-19/Japan/TKYK01734/2021 (GISAID Accession ID: EPI_ISL_2080609)], and a B.1.1.529 (Omicron/BA.1) lineage [hCoV hCoV-19/Japan/TKYX00012/2021 (GISAID Accession ID: EPI_ISL_8559478)] were provided from Tokyo Metropolitan Institute of Public Health, Tokyo, Japan. B.1.1.529 (Omicron/BA.2) lineage [hCoV hCoV-19/Japan/TY40-385-P1/2022 (GISAID Accession ID: EPI_ISL_9595859)] were provided from National Institute of Infectious Diseases, Tokyo, Japan.

The peptide-dependent stabilization assay

The HLA binding of peptide was analyzed as previously described²⁷. Briefly, TAP-deficient C1R-A24 cells were incubated at 26 °C overnight. A total of 1 × 10⁵ cells was incubated with 1 μM β2-microglobulin (β2m) and graded concentrations of peptides in 96-well U-bottom plates. Cells were incubated at 26 °C for 1 h and then at 37 °C for 4 h. At the end of the incubation, unbound peptides were removed, and cells were stained with FITC-labeled HLA-A24-reactive mAb 17A12 (20 µg/ml) provided by Dr. Ulrich Hämmerling and analyzed by FACScan (BD, San Jose, CA, USA). The log Kd values for 50% binding were calculated from the mean fluorescence intensity (MFI) with Cell QuestTM. The log Kd values were normalized by including high-binder peptide (TYLPTNASL) and low-binder peptide (RVWESATPL) in each experiment.

Tetramer staining

SARS-CoV-2-derived peptides-loaded MHC class I tetramers were generated by QuickSwitch^TM Quant HLA-A*24:02 Tetramer Kit-PE (MBL International Corporation, Cat# TB-7302-K1) according to the manufacturer’s protocol. The rate of peptide exchange was quantitated by flow cytometry, and the tetramers, at a rate of more than 90%, were used for staining PBMCs. After treatment with a protein kinase inhibitor, Dasatinib (Cat# A10290-25, AdooQ, 50 nM) for 30 min at 37 °C, PBMCs were stained with tetramers for 30 min on ice. After tetramer staining, cells were counterstained with anti-PE unconjugated mAb (PE001, Biolegend, 1/10 dilution) for 20 min on ice and surface stained with the following antibodies: CD3 BV421 (UCHT1, 1/50 dilution), CD8 APCcy7 (RPA-T8, 1/100 dilution), CD14 PerCP/Cy5.5 (HCD14, 1/100 dilution), CD19 PerCP/Cy5.5 (HIB19, 1/100 dilution; Biolegend) was performed. Dead cells were stained with 7-aminoactinomycin D (Biolegend, Cat# 420404). After incubation for 20 min on ice, the cells were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat# 09154-85), and the levels of tetramer⁺CD8⁺ T cells were analyzed by flow cytometry using a FACS Canto II (BD Biosciences). The data obtained by flow cytometry were analyzed with FACS Diva v9.0 (BD) and FlowJo software v10 (Tree Star).

Activation-induced marker assay

An activation-induced marker assay was performed as previously described¹³. Briefly, human PBMCs were pulsed with 100 nM of the NF9 peptide (NYNYLYRLF, residues 448-456 of the SARS-CoV-2 spike protein) and the QI9 peptide (QYIKWPWYI, residues 1208-1216 of the SARS-CoV-2 spike protein) maintained in RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11875101) containing 10% FBS and 30 U/ml recombinant human IL-2 (Peprotec, Cat# 200-02) for 14 days. The HLA-A*24:02-restricted RF10 peptide (RYPLTFGWCF, residues 134-143 of the HIV Nef protein) at a concentration of 100 nM was included as a negative control. The in vitro expanded CD8⁺ T cells (i.e., T-cell lines) were restimulated with or without the peptide. After incubation at 37 °C for 24 h, the cells were washed, and surface stained with following antibodies: CD3 FITC (UCHT1, 1/100 dilution), CD8 APCcy7 (RPA-T8, 1/100 dilution), CD14 PerCP/Cy5.5 (HCD14, 1/100 dilution), CD19 PerCP/Cy5.5 (HIB19, 1/100 dilution), CD25 PEcy7 (M-A251, 1/50 dilution) and CD137 APC (4B4-1, 1/50 dilution; Biolegend). Dead cells were stained with 7-aminoactinomycin D (Biolegend, Cat# 420404). After incubation for 20 min on ice, the cells were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat# 09154-85), and the levels of protein surface expression were analyzed by flow cytometry using a FACS Canto II (BD Biosciences). The data obtained by flow cytometry were analyzed with FACS Diva v9.0 (BD) and FlowJo software v10 (Tree Star).

Plasmid construction

Plasmids expressing the SARS-CoV-2 spike proteins of the parental (D614G-bearing B.1 lineage), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Lambda (C.37 lineage) and Delta (B.1.617.2), Omicron BA.1 (B.1.1529.1), and Omicron BA.2 (B.1.1529.2) variant were prepared in our previous studies^15,28,29. Plasmids expressing the point mutants were generated by site-directed overlap extension PCR using pC-SARS2-spike D614G or SARS2-Omicron-spike as the template and the following primers are listed in Supplementary Table 3. Primers for the construction of spike derivatives, related to Figs. 2 and 3. The resulting PCR fragment was digested with KpnI and NotI and inserted into the corresponding site of the pCAGGS vector. Nucleotide sequences were determined by Genetic Analyzer 3500xL (Applied Biosystems) and the sequence data were analyzed by GENETYX v12 (GENETYX Corporation).

Intracellular cytokine staining

Intracellular cytokine staining was performed as previously described¹³. Briefly, A549-ACE2-A2402 cells (3 × 10⁵ cells) were transfected with 2 μg of plasmids expressing prototype spike or its derivatives using PEI Max (Polysciences, Cat# 24765-1) according to the manufacturer’s protocol. At 2 days post transfection, the transfectants were harvested and mixed with the T-cell lines generated from HLA-A*24:02⁺ vaccinated donors (see above) and incubated with RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11875101) containing 10% FBS, 5 μg/ml brefeldin A (Sigma-Aldrich, Cat# B7651) in a 96-well U plate at 37 °C for 5 h. The cells were washed, and surface stained with following antibodies: CD3 FITC (UCHT1, 1/100 dilution), CD8 APCcy7 (RPA-T8, 1/100 dilution), CD14 PerCP/Cy5.5 (HCD14, 1/100 dilution), CD19 PerCP/Cy5.5 (HIB19, 1/100 dilution; Biolegend). Dead cells were stained with 7-aminoactinomycin D (Biolegend, Cat# 420404). After incubation at 4 °C for 20 min, the cells were fixed and permeabilized with a Cytofix/Cytoperm Fixation/Permeabilization solution kit (BD Biosciences, Cat# 554714) and were stained with IFN-γ PE (4 S.B3, 1/100 dilution; BD). After incubation at room temperature for 30 min, the cells were washed, and levels of protein expression were analyzed by flow cytometry using a FACS Canto II (BD Biosciences) followed by analysis using FACS Diva v9.0 (BD) and FlowJo v10 software (Tree Star).

Western blotting

Transfected cells were lysed on ice for 15 min in a buffer (100 mM NaCl, 1 mM TCEP [Tris (2-carboxyethyl) phosphine hydrochloride], 2× protease inhibitor, and 10 mM HEPES; pH 7.5) containing 1% n-dodecyl-β-D-maltoside (DDM; Thermo Scientific). The resultant samples were resuspended in 1× Laemmli buffer containing 5% β-mercaptoethanol (Bio-Rad), boiled for 10 min, and subjected to protein separation by SDS-PAGE in 4–20% Mini-PROTEAN TGX precast gels (Bio-Rad) before transferred to nitrocellulose membranes (Wako). The membranes were incubated in a blocking buffer (Nacalai Tesque) for 1 h at room temperature and then mixed with primary antibodies, including rabbit anti-SARS-CoV-2 Spike (S1/S2) polyclonal antibody (1:2000; Invitrogen, Cat# PA5-112048) and mouse anti-β-actin monoclonal antibody (1:5,000; Wako, Cat# 010-27841), followed by staining with the horseradish peroxidase (HRP)-conjugated anti-rabbit (1:50,000; GE healthcare, Cat# NA934VS) and anti-mouse (1:25,000; GE healthcare, Cat# NA931VS) IgG secondary antibodies. The membrane was developed with the ImmunoStar LD enhanced chemiluminescence reagents (Wako) and visualized using ImageQuant LAS 4000 (GE Healthcare).

Jurkat reporter cell (Jurkat∆-Luc) for functional analysis of TCRs

DNA fragment of NFAT-RE-Luc2P-SV40 pro-HygroR was amplified from pGL4.3 (Promega) by PCR. The DNA fragment was cloned into Stu I/Sal I site of PiggyBac vector PB530A-2 (SBI) by Gibson assembly method. The resultant vector [PB_NFAT-RE-Luc2P-SV40 pro-HygroR] was electroporated into endogenous TCR knocked-out Jurkat cells with Transposase expression vector PB200PA-1 (SBI). To select Jurkat reporter cell (Jurkat∆-Luc) integrated with NFAT-RE-Luc2P-SV40 pro-HygroR, Hygromycin-B selection was performed at 500 μg/ml concentration for 14 days.

TCR cDNA amplification from single T cells and construction of TCR expression vector

The cryopreserved PBMCs were stained with NF9/A24 and QI9/A24 tetramers, anti-CD8 mAb (RPA-T8; Biolegend), and 7-amino-actinomycin D (7-AAD), and then tetramer⁺CD8⁺7-AAD⁻ cells were sorted into 96-well plates (NIPPON Genetics, Cat# 4ti-0770/C) by using an FACS Aria II (BD Biosciences). TCRα and TCRβ cDNA pairs were amplified from single T cells by a one-step multiplex RT-PCR method described in our previous study¹⁶. The DNA sequences of the PCR products were then analyzed by direct sequencing and the TCR repertoire by IMGT/V-QUEST (https://www.imgt.org/IMGT_vquest/vquest). The amplified TCRα and TCRβ cDNA fragments were connected to the missing constant region and linked to the blasticidin-S resistance (BlaR) gene by the Gibson assembly method with P2A ribosomal skipping sequences. Resultant TCRβ-P2A-TCRα-P2A-BlaR DNA was cloned into the PiggyBac vector (SBI, Cat# PB530A-2) by the Gibson assembly method.

TCR-sensitivity assay

The plasmid PB TCR-P2A-BlaR was electroporated into Jurkat∆-Luc with Transposase vector (SBI, Cat# PB200PA-1) using Neon® Transfection System (Thermo Fisher Scientific) under the condition 1200 v, 5 ms, five pulses. After 48 h, Jurkat∆-Luc cells stably expressing TCRs were selected with RPMI medium containing 10 μg/ml of blasticidin-S for 10-14 days. These cells were cocultured with A549-ACE2-A2402 cells expressing each spike protein at an E:T ratio of 2:1 and incubated with RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11875101) containing 10% FBS at 37 °C for 6 h. The mixture was measured for luciferase production using a luminescent substrate (Promega, Cat#E2510) by a CentroXS3 plate reader (Berthhold Technologies).

Live virus suppression assay

A549 cells expressing ACE2/A2402 (1 × 10⁴ cells) were infected with each SARS-CoV-2 lineage at an MOI of 0.1 for 120 min at 37 °C. Cells were washed and cocultured with T cells at an E:T ratio of 2:1 and 1:1. Control wells containing virus-infected targets without T cells were also included. After 72 h incubation, culture supernatant was collected and performed real-time RT-PCR. 5 μl of culture supernatant was lysed in an equal amount buffer composed of 2% Triton X-100, 50 mM KCl, 100 mM Tris-HCl (pH 7.4), 40% glycerol and 0.4 U/μl recombinant RNase inhibitor (Promega, Cat# N2615) and then incubated at room temperature for 10 min. 90 μl of RNase-free water (Nacalai tesque, Cat# 06442-95) was added, and 3 µl of diluted sample was used as the template. Real-time RT-PCR analyses for viral RNA copy number was carried out with One-Step PrimeScript™ III RT-qPCR Mix (Takara, Cat# RR600B) and reactions were performed by using LightCycler® 96 System (Roche Diagnostics GmbH, Mannheim, Germany). For the primer, Primer/Probe N2 (2019-nCoV) (Takara, Cat# XD0008) were used as follows: NIID_2019-nCOV_N_ forward, 5-AAATTTTGGGGACCAGGAAC-3; NIID_2019-nCOV_N_ reverse, 5-TGGCAGCTGTGTAGGTCAAC-3; and NIID_2019-nCoV_N_ probe, 5-FAM-ATGTCGCGCATTGGCATGGA-BHQ3. The viral RNA copy number was standardized with a Positive Control RNA Mix (2019-nCoV) (Takara, Cat#XA0142). Relative viral copy was calculated as viral RNA copy number obtained by virus-infected targets without T cells normalized to 1.

Statistics and reproducibility

Data and statistical analysis were performed using Prism 9 (GraphPad Software). For two-way comparison, the paired Wilcoxon signed-rank test (Fig. 1e), unpaired Mann–Whitney t -test (Figs. 2d, 3e and 4b, c), or unpaired Student’s t test (Figs. 3c, d and  4a) was used. For multiple-group comparisons, one-way ANOVA with multiple comparisons by Bonferroni correction (Fig. 3f) was used.

In Figs. 2c, d, 3e, and Supplementary Fig. 1e, assays were performed in triplicate. In Figs. 3c, d, f;  4a–c; and Supplementary Fig. 2a, c, assays were performed in triplicate or quadruplicate. Data are representative of two or three independent experiments. In Supplementary Figs. 1g and 2b, representative blots of three independent experiments were shown.

Reporting summary

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