The SARS-CoV-2 Omicron BA.1 spike G446S potentiates HLA-A*24:02-restricted T cell immunity

Abstract

Although the Omicron variant of the SARS-CoV-2 virus is resistant to neutralizing antibodies, it retains susceptibility to cellular immunity. Here, we characterized vaccine-induced T cells specific for various SARS-CoV-2 variants and identified HLA-A*24:02-restricted CD8 ⁺ T cells that strongly suppressed Omicron BA.1 replication. Mutagenesis analyses revealed that a G446S mutation, located just outside the N-terminus of the cognate epitope, augmented TCR recognition of this variant. In contrast, no enhanced suppression of replication was observed against cells infected with the prototype, Omicron BA.2, and Delta variants that express G446. The enhancing effect of the G446S mutation was lost when target cells were treated with inhibitors of tripeptidyl peptidase II, a protein that mediates antigen processing. These results demonstrate that the G446S mutation in the Omicron BA.1 variant affects antigen processing/presentation and potentiates antiviral activity by vaccine-induced T cells, leading to enhanced T cell immunity towards emerging variants.

SciScore for 10.1101/2022.04.17.488095: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: 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). Consent: All human subjects provided written informed consent.
Sex as a biological variable	67% male), five HLA-A*24:02-negative BNT162b2-vaccinated donors (median age: 24, range: 18-28, 60% Female) (Supplementary Table 1).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After tetramer staining, cells were counterstained with anti-PE unconjugated …

SciScore for 10.1101/2022.04.17.488095: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: 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). Consent: All human subjects provided written informed consent.
Sex as a biological variable	67% male), five HLA-A*24:02-negative BNT162b2-vaccinated donors (median age: 24, range: 18-28, 60% Female) (Supplementary Table 1).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After tetramer staining, cells were counterstained with anti-PE unconjugated mAb (Cat# 408104, Biolegend) for 20 min on ice and surface stained with the following antibodies: CD3 BV421 (UCHT1), CD8 APCcy7 (RPA-T8), CD14 PerCP/Cy5.5 (HCD14), CD19 PerCP/Cy5.5 (HIB19; Biolegend) was performed.	anti-PE suggested: None CD3 suggested: None
After incubation at 37°C for 24 h, the cells were washed, and surface stained with following antibodies: CD3 FITC (UCHT1), CD8 APCcy7 (RPA-T8), CD14 PerCP/Cy5.5 (HCD14), CD19 PerCP/Cy5.5 (HIB19), CD25 PEcy7 (M-A251) and CD137 APC (4B4-1; Biolegend)	UCHT1 suggested: None HCD14 suggested: None HIB19 suggested: None CD25 suggested: None CD137 suggested: None
The cells were washed, and surface stained with following antibodies: CD3 FITC (UCHT1), CD8 APCcy7 (RPA-T8), CD14 PerCP/Cy5.5 (HCD14), CD19 PerCP/Cy5.5 (HIB19; Biolegend)	CD14 suggested: None CD19 suggested: None
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 (1:2,000; Invitrogen) and mouse anti-β-actin monoclonal antibody (1:5,000; Wako), followed by staining with the horseradish peroxidase (HRP)-conjugated anti-rabbit (1:50,000; GE healthcare) and anti-mouse (1:25,000; GE healthcare) IgG secondary antibodies.	anti-SARS-CoV-2 suggested: None S1/S2 suggested: None anti-β-actin suggested: None anti-rabbit suggested: None anti-mouse suggested: None
Experimental Models: Cell Lines
Sentences	Resources
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 described15, 19 and were maintained in Ham’s-F12 (Wako, Cat# 080-08565) containing 10% fetal bovine serum (FBS).	A549-ACE2/A2402 suggested: None
Briefly, TAP-deficient C1R-A24 cells were incubated at 26 °C overnight.	C1R-A24 suggested: RRID:CVCL_S990)
To select Jurkat reporter cell (JurkatΔ-Luc) integrated with NFAT-RE-Luc2P-SV40 pro-HygroR, Hygromycin-B selection was performed at 500 ug/ml concentration for 14 days.	Jurkat suggested: None
After 48 h, JurkatΔ-Luc cells stably expressing TCRs were selected with RPMI medium containing 10 μg/ml of blasticidin-S for 10-14 days.	JurkatΔ-Luc suggested: None
These cells were cocultured with A549-ACE2-A2402 cells expressing each spike protein an E:T ratio of 2:1 and incubated with RPMI 1640 medium (Thermo Fisher Scientific, Cat#	A549-ACE2-A2402 suggested: None
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.	A549 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
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.	SARS-CoV-2 Wuhan suggested: None
Recombinant DNA
Sentences	Resources
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 listed in Supplementary Table 3.	pC-SARS2-spike suggested: None
The resulting PCR fragment was digested with KpnI and NotI and inserted into the corresponding site of the pCAGGS vector.	pCAGGS suggested: RRID:Addgene_127347)
Jurkat reporter cell (JurkatΔ-Luc) for functional analysis of TCRs: DNA fragment of NFAT-RE-Luc2P-SV40 pro-HygroR was amplified from pGL4.3 (Promega) by PCR.	pGL4.3 suggested: None
Software and Algorithms
Sentences	Resources
The data obtained by flow cytometry were analyzed with FlowJo software (Tree Star).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
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)38”.	IMGT/V-QUEST suggested: (IMGT/V-QUEST, RRID:SCR_010749)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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The SARS-CoV-2 Omicron BA.1 spike G446S potentiates HLA-A*24:02-restricted T cell immunity

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