1. Immunology and Inflammation

Early acquisition of S-specific Tfh clonotypes after SARS-CoV-2 vaccination is associated with the longevity of anti-S antibodies

  1. Xiuyuan Lu
  2. Hiroki Hayashi
  3. Eri Ishikawa
  4. Yukiko Takeuchi
  5. Julian Vincent Tabora Dychiao
  6. Hironori Nakagami  Is a corresponding author
  7. Sho Yamasaki  Is a corresponding author
  1. Laboratory of Molecular Immunology, Immunology Frontier Research Center, Osaka University, Japan
  2. Department of Health Development and Medicine, Osaka University Graduate School of Medicine, Japan
  3. Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Japan
  4. Center for Infectious Disease Education and Research (CiDER), Osaka University, Japan
https://doi.org/10.7554/eLife.89999.4
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Cite this article
  1. Xiuyuan Lu
  2. Hiroki Hayashi
  3. Eri Ishikawa
  4. Yukiko Takeuchi
  5. Julian Vincent Tabora Dychiao
  6. Hironori Nakagami
  7. Sho Yamasaki
(2024)
Early acquisition of S-specific Tfh clonotypes after SARS-CoV-2 vaccination is associated with the longevity of anti-S antibodies
eLife 12:RP89999.
https://doi.org/10.7554/eLife.89999.4
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  3. Download .RIS
6 figures, 7 tables and 2 additional files

Figures

Figure 1 with 1 supplement
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SARS-CoV-2 mRNA vaccine elicits transient humoral immunity.

(A) Vaccination and sampling timeline of blood donors in this study. (B) Anti-S IgG titer of serum samples was determined by ELISA. Mean ± SEM (left) and individual data (right) are shown. *, p<0.05 vs. Pre, 3 weeks, 24 weeks, respectively. (C) Neutralization activity (ID50) of serum samples was determined by pseudo-virus assay. Mean ± SEM (left) and individual data (right) are shown. *, p<0.05 vs 3 weeks, 24 weeks, respectively. Wks, weeks.

Figure 1—figure supplement 1
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Humoral immune response of BNT162b2 vaccinees.

(A, B) Anti-S IgG titer from all donors at 6 weeks after vaccination was compared between male and female vaccinees (A) or among different age groups (20–39, 40–49, 50–59, 60–69) (B).

Figure 2 with 2 supplements
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Antibody sustainers had highly expanded S-reactive Tfh clonotypes.

(A) Anti-S IgG titer of serum samples from sustainers and decliners is shown individually. (B, C, E, F) UMAP projection of T cells in single-cell analysis of post-vaccinated samples collected from all donors. Each dot corresponds to a single cell and is colored according to the samples from different time points of donors. All samples together with annotated cell types (B), samples grouped by donor type (decliners and sustainers) (C), top 16 expanded clonotypes (16 clonotypes that had the most cell numbers from each donor) grouped by donor type (E), and top 16 expanded clonotypes grouped by time point and donor type (F) are shown. Tcm, central memory T cells; Tem, effector memory T cells; Treg, regulatory T cells; γδT, γδ T cells. (D) Tfh signature score and expression levels of the canonical Tfh cell markers, IL21, ICOS, PDCD1 and CD200, are shown as heat maps in the UMAP plot.

Figure 2—figure supplement 1
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Humoral and cellular immune responses of sustainers and decliners.

(A) Demographic data and magnitude of anti-S IgG titer reduction of the sustainers and decliners. Anti-S IgG titer reduction is calculated as the titers at (6 weeks – 24 weeks) /6 weeks. (B) Anti-N IgG titer of serum samples from sustainers at 24 weeks after vaccination. (C) S-specific IFNγ release from bulk CD4+ T cells (left) or CD4+ and CD8+ T cells (right) of sustainers (red) and decliners (blue) was measured using QuantiFERON.

Figure 2—figure supplement 2
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Sustainer individuals had more cells in the circled region than decliner individuals.

(A) UMAP projection of single-cell analysis of post-vaccinated samples shown in individuals. (B) The percentage of circled cells in (A) in CD4+ T cells of each individual is shown. p value was calculated using t-test.

Figure 3 with 3 supplements
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The location of S epitopes recognized by top expanded T clonotypes from post-vaccination samples.

T cell S epitopes recognized by top expanded TCR clonotypes in post-vaccinated samples from sustainers and decliners are mapped by their locations in S protein. Each short bar indicates a 15-mer peptide that activated the TCRs. Epitopes are shown in different colors according to the subsets of the T cells they activated. Relative frequencies of the T cell subsets are shown in pie charts. Numbers of identified epitopes recognized by a dominant T subset in sustainers (Tfh) are shown in blue bars. NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; HR1, heptad repeat 1; CH, central helix; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane domain.

Figure 3—figure supplement 1
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Determination of S epitopes for post-vaccinated T cell clonotypes expanded in sustainers and decliners.

(A, B) Reporter cells expressing TCR clonotypes expanded in sustainers (A) and decliners (B) were stimulated with different S peptide in the presence of APCs for overnight, and analyzed for GFP and CD69 expression. Data are representative of at least two independent experiments.

Figure 3—figure supplement 2
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Determination of restricting HLAs for post-vaccinated T cell clonotypes expanded in sustainers and decliners.

(A, B) Reporter cells expressing TCR clonotypes expanded in sustainers (A) and decliners (B) were stimulated with epitope peptides in the presence of APCs expressing various HLA alleles. Data are representative of at least two independent experiments.

Figure 3—figure supplement 3
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Determination of mutated epitope antigenicity for post-vaccinated T cell clonotypes expanded in sustainers and decliners.

(A, B) Reporter cells expressing TCR clonotypes expanded in sustainers (A) and decliners (B) were stimulated with epitope peptides from the Wuhan strain and corresponding peptides from VOCs in the presence of APCs. Data are representative of at least two independent experiments.

Figure 4
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Characteristics and dynamics of S-cross-reactive clonotypes.

(A) UMAP projection of T cells in single-cell analysis of pre-vaccinated samples from donors #4, #13, #15, #17, and #8. Each dot corresponds to a single cell and is colored according to the samples from different donors. Annotated cell types are shown. (B) Donor, name of reconstituted clonotypes, cell type, clonotype fraction in T cells from each time points, and expansion ratio of clonotypes that were found in pre-vaccinated samples and had more than 50 cells in the combined pre- and post-vaccinated sample set. For clonotypes that showed more than one type, the major type is listed in the front. The expansion ratio was calculated using the maximum cell fraction at post-vaccination points divided by the cell fraction at the pre-vaccination point of each clonotype. Clonotypes that have an expansion ratio larger than 1 are considered as expanded post-vaccination. Cell fractions at individual time points are shown as heat map. Tfr, follicular regulatory T cells; MAIT, mucosal-associated invariant T cells.

Figure 5 with 2 supplements
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The location of S epitopes of pre-existing S-reactive T cells.

S epitopes recognized by top expanded TCR clonotypes in pre-vaccinated samples are mapped by their locations in S protein. Each short bar indicates a 15-mer peptide that activated the TCRs. Epitopes are shown in different colors according to the subtypes of the T cells they activated. Relative frequencies of the T cell subtypes from all five donors are shown in the pie chart. Numbers of identified epitopes recognized by a dominant T subset of pre-existing clonotypes (Treg) from all donors are shown in green bars.

Figure 5—figure supplement 1
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Determination of S epitopes, restricting HLAs and cross-reactive epitopes for pre-existing T cell clonotypes expanded by S stimulation.

Reporter cells expressing TCRs were stimulated with 1 µg/ml of indicated S peptides in the presence of transformed B cells or HEK293T cells expressing indicated HLAs for overnight, and analyzed for GFP and CD69 expression. (A) Determination of S epitopes of T cell clonotypes. (B) Determination of restricting HLAs of T cell clonotypes. (C) Determination of cross-reactive epitopes of T cell clonotypes. Sequences of cross-reactive peptides are in Table 6. Data are representative of at least two independent experiments.

Figure 5—figure supplement 2
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The pre-existing S-reactive T cell clonotypes did not recognize HCoV epitopes.

The only two clonotypes whose epitope sequences were relatively conserved in HCoV strains, donor #8-pre_9 and pre_10, were tested for their reactivity to the similar HCoV epitope counterparts. Reporter cell lines of these clonotypes were co-cultured with indicated peptides as well as APCs, and analyzed for GFP and CD69 expression. Data are representative of at least two independent experiments.

Figure 6
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Frequencies of pre-existing S-reactive clonotypes in the public database of uninfected and infected cohorts.

TCRβ sequences of the top expanded clonotypes in pre-vaccinated samples were investigated in the Adaptive database. Frequencies of detected clonotypes are shown in box plot. Healthy, dataset from 786 healthy donors. COVID, dataset from 1485 COVID-19 patients.

Tables

Table 1
Demographic data of the participants.
Percentage (number)
Total number100% (43)
Age group
20–3939.5% (17)
40–4930.2% (13)
50–5925.6% (11)
60–694.7% (2)
Sex
Male60.5% (26)
Female39.5% (17)
Table 2
Demographic data of the reported clinical adverse effects (at injection site).
Percentage (number)
Swelling (injection site)
After 1st dose27.9% (12)
After 2nd dose51.2% (22)
Sore/pain (injection site)
After 1st dose88.4% (38)
After 2nd dose86.0% (37)
Warmth (injection site)
After 1st dose32.6% (14)
After 2nd dose41.9% (18)
Table 3
Demographic data of the reported clinical adverse effects (systemic symptoms).
Percentage (number)
Fever
After 1st dose
Mild (37.5 °C ≥)2.3% (1)
Severe (≥38.0 °C)0% (0)
After 2nd dose
Mild (37.5 °C ≥)25.6% (11)
Severe (≥38.0 °C)23.3% (10)
Fatigue
After 1st dose
Mild18.6% (8)
Severe0% (0)
After 2nd dose
Mild67.4% (29)
Severe18.6% (8)
Headache
After 1st dose
Mild7.0% (3)
Severe0% (0)
After 2nd dose
Mild32.6% (14)
Severe7.0% (3)
Chill
After 1st dose
Mild4.7% (2)
Severe0% (0)
After 2nd dose
Mild23.3% (10)
Severe9.3% (4)
Nausea
After 1st dose
Mild0% (0)
Severe0% (0)
After 2nd dose
Mild4.7% (2)
Severe0% (0)
Diarrhea
After 1st dose
Mild0% (0)
Severe0% (0)
After 2nd dose
Mild0% (0)
Severe0% (0)
Muscle pain
After 1st dose
Mild48.8% (21)
Severe0% (0)
After 2nd dose
Mild55.8% (24)
Severe4.7% (2)
Joint pain
After 1st dose
Mild4.7% (2)
Severe0% (0)
After 2nd dose
Mild25.6% (11)
Severe4.7% (2)
Table 4
TCR clonotypes expanded in post-vaccinated samples and their TCR usages, epitopes and restricting HLAs.
DonorClonotypeTRBVCDR3βTRBJTRAVCDR3αTRAJS epitope*Restricting HLA
#8Post_411–2CASSPTGTNEKLFF1–413–1CAGGADGLTF45SFSTFKCYGVSPTKL373–387†DRA-DRB1*15:02
Post_519CASSGRPEGPQHF1–520CAVLNQAGTALIF15FKIYSKHTPIN201–211DRA-DRB1*09:01
Post_611–2CASSLEGTEAFF1–15CAESRYMGRRALTF5FQFCNDPFLGVYYHK133–147DPA1*01:03-DPB1*04:02
Post_72CAGLAGVDTGELFF2–25CAERVGRRALTF5YSVLYNSASFSTFKC365–379A*24:02
Post_820–1CSATRDRRSYNEQFF2–112–2CAVLTNTGNQFYF49LLQYGSFCTQLNRAL753–767DRA-DRB1*15:02
Post_97–9CASSLLGEQYF2–722CAGAGGTSYGKLTF52KRFDNPVLPFN77–87DPA1*02:02-DPB1*05:01
Post_106–1CASSEGASNQPQHF1–512–1CVVNKGSSASKIIF3LLQYGSFCTQL753–763DRA-DRB1*15:02
Post_1220–1CSAYSIYNEQFF2–19–2CALSMNTGFQKLVF8PPAYTNSFTRGVYYP25–39DRA-DRB1*09:01
Post_1419CASRPNRGDNSPLHF1–612–1CVVSIGFGNVLHC35CSNLLLQYGSFCTQL749–763DRA-DRB1*15:02
Post_1528CASSLMGGAYGYTF1–28–6CAVRRGGSGGSNYKLTF53SKRSFIEDLLFNKVT813–827DPA1*01:03-DPB1*04:02
#25Post_77–9CAPSNANTGELFF2–212–1CVVNEADKLIF34YLQPRTFLLK269–278A*02:01
Post_1220–1CSARDVEVGSGYTF1–24CLVGPYNQGGKLIF23TGVLTESNKKFLPFQ549–563DRA-DRB1*14:54
Post_153–1CASSPLSGSSYEQYF2–712–1CVVGTDSWGKLQF24TNGTKRFDNPVLPFN73–87DPA1*02:02-DPB1*05:01/
DPA1*01:03-DPB1*05:01
#27Post_120–1CSAIAGDADTQYF2–39–2CALTSAAGNKLTF17NQFNSAIGKIQ925–935DRA-DRB1*09:01
Post_230CAWNLGGGNQPQHF1–58–2CVVSERASSYKLIF12SKRSFIEDLLFNKVT813–827DPA1*02:02-DPB1*04:02
Post_35–4CASSQGQGSYGYTF1–24CLVGDSDTGRRALTF5NFTISVTTEIL717–727DRA-DRB1*09:01
Post_57–2CASGTGSYNEQFF2–112–2CAVKRGNQGGKLIF23STEIYQAGSTPCNGV469–483DRA-DRB1*04:03
Post_76–6CASRLPGNRAQPQHF1–536/DV7CAVESGSSNTGKLIF37KSNIIRGWIFGTTLD97–111DRA-DRB4*01:03
Post_86–5CASSYSGGTVTGELFF2–241CAVGIRGNEKLTF48KVFRSSVLHST41–51DRA-DRB1*04:03
Post_920–1CSARDGQTATNEKLFF1–417CATNAGGTSYGKLTF52EIRASANLAAT1017–1027DRA-DRB1*04:03
Post_1130CAWSVKGFPSQHF1–56CALGSTSNTGKLIF37EIRASANLAAT1017–1027DRA-DRB1*04:03
Post_135–6CASSSRTGYNSPLHF1–627CAGAKGSGTYKYIF40STEIYQAGSTPCNGV469–483DRA-DRB1*04:03
Post_155–5CASSSDRNYGYTF1–212–1CVVNMVTGGYNKLIF4NFTISVTTEILPVSM717–731DRA-DRB1*09:01
Post_167–9CASSSQPGLAGVKIGNEQFF2–15CAEIPPPSNTGKLIF37ISGINASVVNIQKEI1169–1183DRA-DRB1*04:03
#28Post_53–1CASSQGGSEKLFF1–41–1CAVGGNTDKLIF34LVKNKCVNFNF533–543DRA-DRB3*03:01
Post_1012–3CASSSGRTGFGYTF1–230CGTEFGSEKLVF57VIRGDEVRQIA401–411DRA-DRB3*03:01
Post_115–8CASSLQKTTGPSYGYTF1–28–6CAVSPYTGRRALTF5SVYAWNRKRIS349–359DRA-DRB1*13:02
Post_1218CASSASVDPTEAFF1–11–1CASFTGGGNKLTF10KSTNLVKNKCVNFNF529–543DRA-DRB3*03:01
Post_147–6CASSLSGTGGTGELFF2–24CLVGDMRSGGGADGLTF45PFGEVFNATRFASVY337–351B*40:01
Post_156–2CASSYPPSGGRTGFGEAFF1–114/DV4CAMRDIGFGNVLHC35WNRKRISNCVADYSV353–367DRA-DRB4*01:03
#4Post_225–1CASTGDNYGYTF1–221CAINTGNQFYF49YYVGYLQPR265–273A*33:03
Post_107–9CASRPSGTSREQYF2–729CAGNNAGNMLTF39FIKQYGDCLGDIAAR833–847A*33:03
Post_117–9CASSTRTSGGGLSYEQYF2–73CAVNKAAGNKLTF17YSVLYNSASFSTFKC365–379A*24:02
Post_1320–1CSASIEQGDLGYTF1–223/DV6CAASIPNSGYALNF41FIKQYGDCLGDIAAR833–847DQA1*01:02-DQB1*05:03
Post_145–6CASSPGQGILEQYF2–724CAFVPLSDGQKLLF16YIKWPWYIWL1209–1218A*24:02
Post_157–3CASGIHTGELFF2–226–1CIVNNAGNMLTF39TDNTFVSGNCDVVIG1117–1131DQA1*01:02-DQB1*06:04
Post_167–6CASSPGPSEADTQYF2–31–1CAVRDGDDKIIF30KSTNLVKNKCVNFNF529–543DRA-DRB3*03:01
#13Post_137–2CASSVGQSKGKSAETQYF2–522CAVNEYSGAGSYQLTF28SKRSFIEDLLFNKVT813–827DPA1*01:03-DPB1*02:01
Post_1520–1CSAGDTASTYGYTF1–29–2CALSDGAGNKLTF17NQFNSAIGKIQ925–935DRA-DRB1*09:01
Post_1630CAWSLQGQRPQHF1–538–1CAFMKQRGGSEKLVF57FIEDLLFNKVTLADA817–831DPA1*01:03-DPB1*02:01
#15Post_112–4CASSSHRDRGVEAFF1–112–1CVVNFDRGSTLGRLYF18TRGVYYPDKVF33–43B*15:01
Post_63–1CASSQQLNTGELFF2–238–2/DV8CAYRKTSGTYKYIF40WRVYSTGSNVF633–643DRA-DRB1*15:02
Post_728CASSFPDRYYSNQPQHF1–51–2CAVRAVGGNKLVF47TRGVYYPDKVF33–43B*15:01
Post_927CASSPGHEQYF2–714/DV4CAMSPIRTYKYIF40RSVASQSIIAY685–695B*15:01
Post_113–1CASSRELISEQYF2–738–2/DV8CAYKRTSGTYKYIF40WRVYSTGSNVF633–643DRA-DRB1*15:02
Post_1228CASSSYGTSGGRAEQFF2–116CALSGGLTGGGNKLTF10LGDIAARDLICAQKF841–855DRA-DRB1*08:02
Post_1330CAWRTGQGITSPLHF1–68–2CVVNNAGNMLTF39VFKNIDGYFKIYSKH193–207DPA1*02:02-DPB1*05:01
Post_146–1CASSEAGGSGANVLTF2–69–2CALSGTGTYKYIF40KKFLPFQQFGR557–567DPA1*02:02-DPB1*05:01
Post_1627CASSLGTINTGELFF2–217CATAPAGGTSYGKLTF52IDGYFKIYSKHTPIN197–211DRA-DRB1*08:02
#17Post_46–2CASTSTARGSYNEQFF2–127CAGHSNTGNQFYF49TRFASVYAWNRKRIS345–359DRA-DRB1*08:02
Post_109CASSKTSGAYNEQFF2–19–2CALDNARLMF31FIKQYGD833–839DRA-DRB1*15:01
Post_1120–1CSARPPGGGNNEQFF2–126–2CILRDGTGANNLFF36QALNTLVKQLSSNFG957–971DRA-DRB1*08:02
Post_157–9CASSLARGNSPLHF1–638–2/DV8CAFVGSQGNLIF42AARDLICAQKFNGLT845–859DRA-DRB1*08:02
  1. *

    Overlapped epitope sequence is shown when a clonotype recognized two or three sequential peptides.

  2. †

    Number ranges indicate the location of peptides in the proteins.

Table 5
Reactivity of each clonotype to mutated epitopes in SARS-CoV-2 VOCs.
DonorClonotypeMutated epitopes in VOCsDomainResponse
#8Post_4Omicron BA.1
Omicron BA.2, 4/5		PFFTFKCYGVSPTKL*
PFFAFKCYGVSPTKL		RBD↓
↓
#8Post_5Omicron BA.1FKIYSKHTPIInon-RBD↑
#8Post_6Delta, Omicron BA.2, 4/5
Omicron BA.1		FQFCNDPFLDVYYHK
FQFCNDPFLD---HK		non-RBD↓
↓
#8Post_7Omicron BA.1
Omicron BA.2, 4/5		YSVLYNLAPFFTFKC
YSVLYNFAPFFAFKC		RBD↓
↓
#8Post_8Omicron BA1, 2, 4/5LLQYGSFCTQLKRALnon-RBD↑
#8Post_10Omicron BA1, 2, 4/5LLQYGSFCTQLKRALnon-RBD↑
#27Post_5Delta
Omicron BA.1, 2, 4/5		STEIYQAGSKPCNGV
STEIYQAGNKPCNGV		RBD↓
↓
#27Post_13Delta
Omicron BA.1, 2, 4/5		STEIYQAGSKPCNGV
STEIYQAGNKPCNGV		RBD↓
↓
#28Post_5Omicron BA.1LVKNKCVNFNFNGLKnon-RBD↑
#28Post_10Omicron BA.2, 4/5VIRGNEVSQIARBD↓
#28Post_14Omicron BA.1, 2, 4/5PFDEVFNATRFASVYRBD↓
#4Post_11Omicron BA.1
Omicron BA.2, 4/5		YSVLYNLAPFFTFKC
YSVLYNFAPFFAFKC		RBD↓
↓
#15Post_9Delta
Omicron BA.1, 2, 4/5		RRRARSVASQSIIAY
HRRARSVASQSIIAY		non-RBD↑
↑
#15Post_16Omicron BA.1IDGYFKIYSKHTPIInon-RBD→
#17Post_11Omicron BA.1, 2, 4/5QALNTLVKQLSSKFGnon-RBD↓
#17Post_15Omicron BA.1AARDLICAQKFKGLTnon-RBD↓
  1. *

    Amino acids colored red indicate mismatches compared with corresponding S epitopes of Wuhan strain.

Table 6
S-coss-reactive TCR clonotypes expanded in pre-vaccinated samples and their TCR usages, epitopes, restricting HLAs and cross-reactive epitopes in microbes other than SARS-CoV-2.
DonorClonotypeTRBVCDR3βTRBJTRAVCDR3αTRAJS epitopeRestricting HLACross-reactive antigen [species]Cross-reactive peptidePost-vaccinated expansion
#4Pre_56–6CASSYPGGGGSETQYF2–535CAGVAVQGAQKLVF54LLALHRSY
LTP241–251*		DRA-DRB1*14:54Phosphoribosyl
formylglycinamidine
cyclo-ligase
[Firmicutes bacterium]		VAEALLAVHR
SYLTP220-234†		No
#4Pre_76–6CASSYPGGSGGELFF2–221CAVENSGNTPLVF29LLALHRSY
LTP241–251		DQA1*01:04-DQB1*05:03Phosphoribosylf
ormylglycinamidine
cyclo-ligase
[Firmicutes bacterium]		VAEALLAVHR
SYLTP220-234		No
#8Pre_16–2CASRPNRGRFRGNQPQHF1–523/DV6CAGEEKETSGSRLTF58NCTFEYVSQP
FLMDL165–179		DRA-DRB1*15:02Fumarylacetoacetate
hydrolase family protein
[Alcaligenes faecalis]
Hypothetical protein
[Planctomycetales bacterium]		ASLIEYVSQP
FLLEP225-239
AAGFEYVSQ
PFSLPL533-547		No
#8Pre_26–1CASIRDRVADTQYF2–330CGTETTDSWGKLQF24RFNGIGVTQ
NV905–915		DQA1*03:02-DQB1*03:03SEL1-like repeat protein [Bacteroidaceae bacterium] ‡LGVYYFNGI
GVTQDQ236-250		No
#8Pre_327CATKGEANYGYTF1–212–3CAMSEMGTGFQKLVF8SIVRFPNI
TNL325–335		DRA-DRB1*15:02LTA synthase family protein [Dechloromonas denitrificans]LPGKSVVR
WPNITNL330-344		No
#8Pre_55–1CASSLRTGELFF2–28–1CAVNGRNTGFQKLVF8NFTISVTTEI
LPVSM717–731		DRA-DRB1*09:01Major capsid protein
[Human papillomavirus 145]
Periplasmic trehalase
[Chlamydiia bacterium]		NFTISVTTDA
GDINE350-364
LSTIVTTEIL
PVDL288-301		No
#8Pre_97–2CASAAGGTGGETQYF2–55CAETPFLSGTYKYIF40YIKWPWYIW
LGFIAG1209–1223		DRA-DRB5*01:02Spike glycoprotein
[Human coronavirus HKU1]		VKWPWYV
WLLISFSF1297-1311		No
#8Pre_106–6CASSLGQGIHEQYF2–726–1CIVERGGSNYKLTF53SKRSFIEDL
LFNKVT813–827		DPA1*01:03-DPB1*04:02Hypothetical protein, partial
[Acinetobacter baumannii]
Spike protein
[Feline coronavirus]
Spike protein
[Canine coronavirus]		GKRSAVEDL
LFNKVV204-218 GKRSAVEDL
LFNKVV980-994 GKRSAVEDLL
FNKVV977-991		No
#8Pre_144–3CASSQRQGAGDTQYF2–319CALSEAGIQGAQKLVF54IDRLITGRLQ
SLQTY993–1007		DQA1*01:03-DQB1*06:01Excinuclease ABC subunit UvrA [Lentisphaeria bacterium]VDRLITGRLE
SSRLN208-222		No
#8Pre_1520–1CSAKDRIYGYTF1–226–1CIVRSPSGSARQLTF22MIAQYTSAL
LA869–879		DRA-DRB1*15:02MATE family efflux transporter [Selenomonas noxia]ATIIAQYTSA
LLALR242-256		No
#13Pre_54–3CASSQVSTGTGITGANVLTF2–65CARRSSSASKIIF3QNVLYENQ
KLI913–923		DRA-DRB5*01:01Hypothetical protein
[Neobacillus vireti]		TNVLYENQKL
FLNLF169-183		No
#13Pre_818CASSPRAPPYEQYF2–721CAVRPAGGTGNQFYF49DKYFKNHTSP
DVDLG1153–1167		DRA-DRB1*15:01Type VI secretion
system contractile
sheath large subunit
[Salmonella enterica]		DYYFDHTSP
DVDLLG167-181		No
#13Pre_124–2CASSQEGNTEAFF1–120CGCRGGTSYGKLTF52NVTWFHAIH
VSGTNG61–75		DQA1*01:02-DQB1*06:02Dihydrofolate synthase
[Actinobaculum sp. 313]		PQRSFHAIH
VTGTNG61-75		No
#15Pre_120–1CSARDLTASAHGYTF1–217CATDAGQGGKLIF23SVTTEILP
VSM721–731		DQA1*01:03-DQB1*06:01Hypothetical protein
[Myxococcales bacterium]		PVTTEILPVSD
DPPG525-539		No
#15Pre_224–1CATSDLDQPQHF1–516CALSGYGSGYSTLTF11SVTTEILP
VSM721–731		DQA1*01:03-DQB1*06:01Hypothetical protein
[Myxococcales bacterium]		PVTTEILPVS
DDPPG525-539		No
#15Pre_36–1CASDPKNGGEQYF2–729/DV5CAASVGFGNVLHC35FKIYSKH
TPIN201–211		DRA-DRB5*01:02Uncharacterized protein
APUU_31,289 S
[Aspergillus puulaauensis]		CRAAFKLY
SKHTPVE123-137		No
#15Pre_419CASGLAGGNTGELFF2–210CVPSSGGYNKLIF4QALNTLVK
QLS957–967		DRA-DRB1*08:024-hydroxybenzoate octaprenyltransferase
[Pseudoduganella dura]		IQPLNTLVKQ
LSVAA112-126		No
#15Pre_56–5CASSAGLAGGGNTQYF2–35CAVISGSARQLTF22QALNTLV
KQLS957–967		DRA-DRB1*08:024-hydroxybenzoate octaprenyltransferase
[Pseudoduganella dura]		IQPLNTLVKQ
LSVAA112-126		No
#15Pre_62CASVGGNEQFF2–19–2CALTRFVGGATNKLIF32RTFLLKYN
ENGTITD273–287		DRA-DRB1*15:02Unnamed protein product
[Mytilus edulis]		NKKLLKYNE
NGTFIT277-291		No
#15Pre_74–1CASSHDGTPPDTQYF2–329/DV5CAAYSNYQLIW33FKIYSKHT
PIN201–211		DRA-DRB1*15:02Uncharacterized protein
APUU_31,289 S
[Aspergillus puulaauensis]		CRAAFKLYS
KHTPVE123-137		No
#15Pre_152CASSETGRGTDTQYF2–39–2CALYRGTYKYIF40LQSLQTYV
TQQLIRA1001–1015		DRA-DRB1*15:02Dyp-type peroxidase
[Acinetobacter sp.]		CTVLQTYVTQ
QLESV134-148		No
#17Pre_76–1CASSLRGAFGYTF1–235CAGHLYGGSQGNLIF42NCTFEYVSQP
FLMDL165–179		DPA1*01:03-DPB1*04:02Fumarylacetoacetate
hydrolase family protein
[Alcaligenes faecalis]
Hypothetical protein
[Planctomycetales bacterium]		ASLIEYVSQP
FLLEP225-239
AAGFEYVSQ
PFSLPL533-547		No
#17Pre_85–1CASSLNSGANVLTF2–613–1CAASIVQDQKLVF8LTPTWRVYS
TGSNVF629–643		DRA-DRB1*08:02Hypothetical protein
[Novosphingobium chloroacetimidivorans]		APGTPTWRV
YSTART277-291		No
#17Pre_145–1CASSLGAGLYNEQFF2–138–1CAFINNNAGNMLTF39QALNTLVK
QLS957–967		DRA-DRB1*08:024-hydroxybenzoate octaprenyltransferase
[Pseudoduganella dura]		IQPLNTLVKQ
LSVAA112-126		No
#17Pre_157–2CASSRTSGGTYEQYF2–725CAGQNTDKLIF34SIVRFPNI
TNL325–335		DRA-DRB1*15:01LTA synthase family protein [Dechloromonas denitrificans]LPGKSVVR
WPNITNL330-344		Yes
  1. *

    Number ranges indicate the location of peptides in the proteins.

  2. †

    Amino acids colored red indicate mismatches compared with corresponding S epitopes of Wuhan strain.

  3. ‡

    Antigen names and peptide sequences in cells with gray background indicate inactive antigens of the corresponding T clonotypes.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-Human IgG HRP (Goat polyclonal)Abcamab97175ELISA (1:5000)
AntibodyAnti-SARS-CoV-IgG WHO international Standard (Human polyclonal)NIBSC20/136ELISA (10–31250)
AntibodyAnti-human CD3-FITC (mouse monoclonal)BioLegendCat#: 300305FACS (1:100)
AntibodyTotalSeq-C anti-human Hashtags (mouse monoclonal, mixture)BioLegendCat#: 394661, etcSingle-cell sequencing (1:50)
AntibodyAnti-mouse CD69-APC (armenian hamster monoclonal)BioLegendCat#: 104513FACS (1:100)
Peptide, recombinant proteinSARS-CoV-2 Spike (trimeric)Cell Signaling Technology#65444
Peptide, recombinant proteinSARS-CoV-2 Nucleocapsid proteinACRO BiosystemsNUN-C5227
Peptide, recombinant proteinSARS-CoV-2 (Spike Glycoprotein), PepMixJPT Peptide Technologies GmbHJER-PM-WCPV-S-1–2S peptide pool
Peptide, recombinant proteinIndividual S peptideGenscripta peptide scan (15mers with 11 aa overlap) through S protein (Swiss-Prot ID: P0DTC2)
Recombinant DNA reagentpMX-IRES-rat CD2 (plasmid)Yamasaki et al., 2006retroviral vector
Cell line (Cercopithecus aethilops)Vero E6/TMPRSS2JCRB cell bank; Yoshida et al., 2021
Cell line (Mus musculus)NFAT-GFP Reporter cellMatsumoto et al., 2021T cell hybridoma lacking endogenous TCR with an NFAT-GFP reporter gene
Biological sample (Human gammaherpesvirus 4)Epstein-Barr virus (EBV)Kanda et al., 2015For B cell transformation
Software, algorithmGraphPad Prism 8GraphPad SoftwareGraphPad Prism 8

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/89999/elife-89999-mdarchecklist1-v1.pdf
Source code 1

Scripts for analysis of single-cell sequencing data.

https://cdn.elifesciences.org/articles/89999/elife-89999-code1-v1.zip

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A two-part list of links to download the article, or parts of the article, in various formats.

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  1. Xiuyuan Lu
  2. Hiroki Hayashi
  3. Eri Ishikawa
  4. Yukiko Takeuchi
  5. Julian Vincent Tabora Dychiao
  6. Hironori Nakagami
  7. Sho Yamasaki
(2024)
Early acquisition of S-specific Tfh clonotypes after SARS-CoV-2 vaccination is associated with the longevity of anti-S antibodies
eLife 12:RP89999.
https://doi.org/10.7554/eLife.89999.4