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

SARS-CoV-2 vaccines have been used worldwide to combat COVID-19 pandemic. To elucidate the factors that determine the longevity of spike (S)-specific antibodies, we traced the characteristics of S-specific T cell clonotypes together with their epitopes and anti-S antibody titers before and after BNT162b2 vaccination over time. T cell receptor (TCR) αβ sequences and mRNA expression of the S-responded T cells were investigated using single-cell TCR- and RNA-sequencing. Highly expanded 199 TCR clonotypes upon stimulation with S peptide pools were reconstituted into a reporter T cell line for the determination of epitopes and restricting HLAs. Among them, we could determine 78 S epitopes, most of which were conserved in variants of concern (VOCs). After the 2nd vaccination, T cell clonotypes highly responsive to recall S stimulation were polarized to follicular helper T (Tfh)-like cells in donors exhibiting sustained anti-S antibody titers (designated as ‘sustainers’), but not in ‘decliners’. Even before vaccination, S-reactive CD4+ T cell clonotypes did exist, most of which cross-reacted with environmental or symbiotic microbes. However, these clonotypes contracted after vaccination. Conversely, S-reactive clonotypes dominated after vaccination were undetectable in pre-vaccinated T cell pool, suggesting that highly responding S-reactive T cells were established by vaccination from rare clonotypes. These results suggest that de novo acquisition of memory Tfh-like cells upon vaccination may contribute to the longevity of anti-S antibody titers.

Abstract 23 SARS-CoV-2 vaccines have been used worldwide to combat COVID-19 pandemic. To 24 elucidate the factors that determine the longevity of spike (S)-specific antibodies, we traced the 25 characteristics of S-specific T cell clonotypes together with their epitopes and anti-S antibody 26 titers before and after BNT162b2 vaccination over time. T cell receptor (TCR) ab sequences 27 and mRNA expression of the S-responded T cells were investigated using single-cell TCR-28 and RNA-sequencing. Highly expanded 199 TCR clonotypes upon stimulation with S peptide 29 pools were reconstituted into a reporter T cell line for the determination of epitopes and 30 restricting HLAs. Among them, we could determine 78 S epitopes, most of which were 31 conserved in variants of concern (VOCs). In donors exhibiting sustained anti-S antibody titers 32 (designated as "sustainers"), S-reactive T cell clonotypes detected immediately after 2nd 33 vaccination polarized to follicular helper T (Tfh) cells, which was less obvious in "decliners". 34

Introduction 41
The pandemic COVID-19, caused by the severe acute respiratory syndrome 42 coronavirus 2 (SARS-CoV-2), has expanded worldwide [1]. Many types of vaccines have been 43 developed or in basic and clinical phases to combat infection and deterioration of 3]. Among them, messenger ribonucleic acid (mRNA) vaccines, BNT162b2/Comirnaty and 45 mRNA-1273/Spikevax, have been approved with over 90% efficacy at 2 months post-2nd dose 46 vaccination [4,5], and widely used. Pathogen-specific antibodies are one of the most efficient 47 components to prevent infection. Yet, mRNA vaccine-induced serum antibody titer is known 48 to be waning over 6 months [6,7]. Accordingly, the effectiveness of the vaccines decreases 49 over time, and thus multiple doses and repeated boosters are necessary [8]. 50 The production and sustainability of spike (S)-specific antibody could be related to 51 multiple factors, especially in the case of humans [7,9]. Among them, the characteristics of 52 SARS-CoV-2-specific T cells is critically involved in the affinity and longevity of the 53 antibodies [10][11][12]. Elucidation of the key factors of T cell responses that contribute to the 54 durable immune responses induced by vaccination would provide valuable information for the 55 vaccine development in the future. However, the relationship between antibody sustainability 56 and the types of antigen-specific T cells has not been investigated in a clonotype resolution. 57 vaccination were analyzed on clonotype level using single cell-based T cell receptor (TCR) 66 and RNA sequencing to determine their characteristics and epitopes in antibody sustainers and 67 decliners. These analyses suggest the importance of early acquisition of S-specific Tfh cells in 68 the longevity of antibodies. 69 Results 70

SARS-CoV-2 mRNA vaccine elicits transient humoral immunity 71
Blood samples were collected from a total of 43 individuals (Table 1) who had no  72 SARS-CoV-2 infection history when they received two doses of SARS-CoV-2 mRNA vaccine 73 BNT162b2. Samples were taken before and after the vaccination (Fig. 1A). Consistent with 74 the previous report [4], most participants exhibited more severe side effects after 2nd dose of 75 vaccination than 1st dose locally (Table 2) and systemically (Table 3). At 3 weeks, anti-S IgG 76 antibody titer increased in most participants. At 6 weeks, anti-S antibody titer was at its peak. 77 S antibody titer gradually decreased over 24 weeks (Fig. 1B). The antibody titer was reduced 78 by 56.8% on average. Donors of different genders or age groups showed no significant 79 difference in anti-S antibody titer (Fig. S1). The neutralization activity of the post-vaccinated 80 sera showed similar tendency with the anti-S antibody titer during the study period (Fig. 1C). 81 The above results indicate that the mRNA vaccine effectively activated humoral immune 82 responses in healthy individuals, but decreased by 24 weeks over time as reported [6,7]. 83 84 Antibody sustainers had highly expanded S-reactive Tfh clonotypes 85 To address the role of T cells in maintaining the antibody titer, we analyzed the S-86 responsive T cells in the post-vaccination samples from 8 donors, among whom 4 donors 87 showed relatively sustained anti-S antibody titer during 6 weeks to 24 weeks (reduction < 30%) 88 (sustainers, donors #8, #25, #27 and #28), while the other 4 donors showed largely declined 89 anti-S antibody titer (reduction > 80%) (decliners, donors #4, #13, #15 and #17) ( Fig. 2A and 90 Antibody sustainability did not correlate with bulk T cell responses to S protein, such as IFNg 93 production (Fig. S2C). 94 To enrich the S-reactive T cells, we labeled the peripheral blood mononuclear cells 95 (PBMCs) with a cell proliferation tracer and stimulated the PBMCs with an S peptide pool for 96 10 days. Proliferated T cells were sorted and analyzed by single-cell TCR-and RNA-97 sequencing (scTCR/RNA-seq). Clustering analysis was done with pooled samples of 3 time 98 points from 8 donors, and various T cell subtypes were identified (Fig. 2B). We found that, 99 overall, the S-reactive T cells did not skew to any particular T cell subset (Fig. 2B). However, 100 by grouping the cells from decliners and sustainers separately, we found difference in the 101 frequency of the cells within the circled population (Fig. 2C). These cells showed high Tfh 102 signature scores and expressed characteristic genes of Tfh cells (Fig. 2D), suggesting that they 103 might be circulating Tfh cells (cTfh) considering they were isolated from PBMCs. This 104 tendency became more pronounced when we selected highly expanded (top 16) clonotypes in 105 each donor (Fig. 2E). In sustainers, S-specific Tfh clusters appeared from 6 weeks (Fig. 2F), 106 suggesting that vaccine-induced Tfh cells were established immediately after 2nd vaccination. 107 108

Identification of dominant S epitopes recognized by vaccine-induced T cell clonotypes 109
To elucidate the epitopes of the highly expanded clonotypes, we reconstituted their 110 TCRs into a T cell hybridoma lacking endogenous TCRs and having an NFAT-GFP reporter 111 gene. These cell lines were stimulated with S peptides using transformed autologous B cells as 112 antigen-presenting cells (APCs). The epitopes of 53 out of 128 reconstituted clonotypes were 113 successfully determined (Fig. 3, Table 4, Figs. S3A-S3D). Epitopes of expanded Tfh cells were 114 not limited in any particular region of S protein (Fig. 3). About 72% of these epitopes conserved 115 in Delta and Omicron variants (Tables 4 and 5). Within the rest of 28% of epitopes which were 116 mutated in variants of concerns (VOCs), although some mutated epitopes located in the 117 receptor-binding domain (RBD) of VOCs lost antigenicity, recognition of most epitopes 118 outside the RBD region was maintained or rather increased in the variants (Table 5 and Figs.  119 S3E and S3F). These results suggest that the majority of S-reactive clonotypes after vaccination 120 can respond to antibody-escaping VOCs. 121 122 Identification of S epitopes and cross-reactive antigens of pre-existing T cell clonotypes 123 Before the pandemic, T cells cross-reacting to S antigen were present in the peripheral 124 blood [13][14][15][16][17]. To characterize these pre-existing S-reactive cells, we analyzed the PBMCs 125 collected from donors who consented to blood sample donation before vaccination (#4, #8, #13, 126 #15, and #17). PBMCs were stimulated with the S peptide pool for 10 days, and proliferated T 127 cells were sorted and analyzed by scTCR/RNA-seq. Similar to vaccine-induced S-reactive T 128 cells (Fig. 2B), characteristics of pre-existing S-reactive T cells were diverse (Fig. 4A). To 129 track the dynamics of cross-reactive clones after vaccination, we combined the single-cell 130 sequencing data of pre-and post-vaccinated PBMCs and analyzed the clonotypes that have 131 more than 50 cells in total (Fig. 4B). We did find some cross-reactive clonotypes that were 132 further expanded by vaccination, and most of these clonotypes had cytotoxic features, being 133 CD8 + effector memory T cells (Tem) or minor CD4 + cytotoxic T cells (CTLs). In contrast, 134 most of the cross-reactive CD4 + T cells became minor clonotypes after vaccination. 135 We also explored the epitopes of the top 16 expanded clonotypes in each pre-vaccinated 136 donor by reconstituting the TCRs into reporter cell lines. We identified 18 epitopes from S 137 protein ( Fig. 5 and Table 6) and determined some possible cross-reactive antigens. Most of 138 these cross-reactive antigens originated from environmental or symbiotic microbes (Table 6). 139 Furthermore, majority of the reactive T clonotypes showed regulatory T cell (Treg) signatures 140 (Fig. 5). Six of these 80 analyzed clonotypes could also be frequently detected in the public 141 TCR database Adaptive [23,24]. Notably, most of these clonotypes, except for one case, 142 showed comparable frequencies between pre-pandemic healthy donors and COVID-19 143 convalescent patients (Fig. 6), suggesting that these clonotypes did not expand upon SARS-144 CoV-2 infection, despite they were present before the pandemic. Thus, it is unlikely that these 145 cross-reactive T clonotypes contribute to the establishment of S-reactive T cell pools during 146 either vaccination or infection. 147

Discussion 148
Previous studies showed that Tfh function and germinal center development were 149 impaired in deceased COVID-19 patients [25] and Tfh cell number correlated with neutralizing 150 antibody [26][27][28]. Consistent with the above studies, we found that the donors having sustained 151 antibody titers between 6 to 24 weeks post-vaccination had more S antigen-responsive Tfh 152 clonotypes maintained in the periphery as a memory pool. As circulating Tfh clonotypes can 153 reflect the population of germinal center Tfh cells [29], it is possible that these maintained S-154 responsive Tfh cells contribute to the prolonged production of anti-S antibodies. These results 155 imply that Tfh polarization of S-reactive T cells in the blood after 2nd vaccination can be a 156 marker for the longevity of serum anti-S antibodies. Although monitoring of S-specific Tfh 157 cells in germinal center is ideal [30], it is currently difficult for outpatients in clinics. 158 Since the antigen used for BNT162b2 is a full-length S protein from the Wuhan-Hu-1 159 strain, it is important to estimate whether vaccine-induced Wuhan S-reactive T cells recognize 160 neutralizing antibody-evading VOCs, such as Omicron variants. Consistent with previous 161 reports [31][32][33], most of the epitopes determined in the current study were conserved in Delta 162 and Omicron (BA.1, BA.2 and BA.4/5) strains, suggesting that vaccine-induced T cells are 163 able to recognize the mutated S proteins from these variants, despite B epitopes being largely 164 mutated in these VOCs [31,32]. 165 SARS-CoV-2-recognizing T cells existed prior to exposure of the S antigens [13][14][15][16][17], 166 which is consistent with our observation with PBMCs from donors who were uninfected and 167 pre-vaccinated. Among these pre-existing S-reactive clonotypes, CD8 + cytotoxic T clonotypes 168 were expanded by the vaccination, whereas most of CD4 + T clonotypes became less dominant 169 after vaccination (Fig. 4B). Currently, the reason for the opposite tendency is unclear. In the 170 present study, we showed that pre-existing T clonotypes cross-reacting to S protein are unlikely 171 to contribute to vaccine-driven T cell immunity. This could be due to the fact that cross-reactive 172 T cells had relatively low avidity to S protein [34]. Alternatively, but not mutually exclusively, 173 considering that most of these cross-reactive T clonotypes have Treg signature (Fig. 5), they 174 could be developed to tolerate symbiotic or environmental antigens, and might be ineffective 175 to the defense against SARS-CoV-2 and thus replaced by the other effective T clonotypes 176 induced by vaccination. One exceptional pre-existing clonotype was #15-Pre_2, as they 177 vigorously expanded in COVID-19 patients (Fig. 6). This clonotype was clustered within a 178 CD4 + Tem population and cross-reactive to environmental bacteria, Myxococcales bacterium 179 (Table 6). Thus, in some particular settings, clonotypes primed by common bacterial antigens 180 might potentially contribute during infection. 181 Common cold human coronavirus (HCoV)-derived S proteins are reported as potential 182 cross-reactive antigens for pre-existing SARS-CoV-2 S-reactive T cells [15,[18][19][20]. However, 183 the highly responding SARS-CoV-2 S-reactive clonotypes in pre-vaccinated donors rarely 184 reacted with HCoV S proteins in the present study, which might be partly due to the difference 185 of cohorts or ethnicities. Instead, most of those T cells cross-reacted with environmental or 186 symbiotic bacteria. These observations suggest that these cross-reactive T cells might have 187 been developed to establish tolerance against less harmful microbes, and thus unlikely to 188 efficiently contribute to the protective viral immunity. Vaccination may induce opposite 189 tendencies on T cell clonotypes that recognize the same antigen [35], which is hardly detected 190 by the bulk T cell analyses. The current study highlights the necessity of dynamic tracing of T 191 cell responses in an epitope-specific clonotype resolution for the evaluation of vaccine-induced 192 immunity. 193 This study suggests that mRNA vaccine is potent enough to prime rare T cell clonotypes 194 that become dominant afterwards. Furthermore, we propose that the types of CD4 + T 195 clonotypes developed shortly after two doses of vaccination could be an indication of the 196 longevity of antibodies. Tfh-inducing adjuvants or Tfh-skewing epitope would be a promising 197 "directional" booster in the post-vaccine era when most people worldwide were exposed with 198 the same antigen in multiple doses within a short period. Furthermore, in addition to  CoV-2, this strategy can also be applicable for the prevention of other infectious diseases of 200 which neutralizing antibody titers are effective for protection. 201

Ethics statement and sample collection 203
This project was approved by Osaka University Institutional Review Board (IRB) (reference 204 No. 21487). 43 volunteers were enrolled in this project. Informed consent was obtained from 205 all participants before the first blood sampling. Samples (serum, whole blood, and PBMCs) 206 were collected four times at 0-7 days before 1st dose vaccination as pre-vaccination, at 14-21 207 days after 1st dose vaccination as 3 weeks sample, at 35-49 days after 1st dose vaccination as 208 6 weeks sample, and at 154-182 days after 1st dose of vaccination as 24 weeks sample. At the 209 same time of blood sampling, adverse event information was also collected from all 210 participants. PBMCs were isolated using BD vacutainer® CPT™ cell separation tube (Beckton 211 Dickinson), according to manufacturers' instructions. Isolated PBMCs were stored in the vapor 212 phase of liquid nitrogen until use. 213 214 Antibody titer determination by enzyme-linked immunosorbent assay (ELISA) 215 Serum antibody titer was measured using ELISA. Briefly, recombinant ancestral S protein 216 (S1+S2, Cell Signaling Technology; 1 µg/ml) or recombinant nucleocapsid protein 217 (Acrobiosystems; 1 µg/ml) was coated on 96-well plate at 4 °C overnight. On the second day, 218 wells were blocked with blocking buffer (PBS-T (0.05% tween®20) containing 5% skim milk) 219 for 2 h at room temperature. The sera were diluted from 10 to 31,250 folds in blocking buffer 220 and incubated overnight at 4 °C. The next day, wells were washed and incubated with 221 horseradish peroxidase (HRP)-conjugated antibodies (GE Healthcare) for 3 h at room 222 temperature. After being washed with PBS-T, wells were incubated with the peroxidase 223 chromogenic substrate 3,3'-5,5'-tetramethyl benzidine (Sigma-Aldrich) for 30 min at room 224 temperature, then the reaction was stopped by 0.5 N sulfuric acid (Sigma Aldrich). The 225 absorbance of wells was immediately measured at 450 nm with a microplate reader (Bio-Rad). 226 The value of the half-maximal antibody titer of each sample was calculated from the highest 227 absorbance in the dilution range by using Prism 8 software. The calculated antibody titer was 228 converted to BAU/ml by using WHO International Standard 20/136 (NIBSC) for ancestral S-229 specific antibody titer.

Pseudo-typed virus neutralization assay 245
The neutralizing activity of serum antibodies was analyzed with pseudo-typed VSVs as 246 previously described [37]. Briefly, Vero E6 cells stably expressing TMPRSS2 were seeded on 247 96-well plates and incubated at 37 °C for 24 h. Pseudoviruses were incubated with a series of 248 dilutions of inactivated serum for 1 h at 37 °C, then added to Vero E6 cells. At 24 h after 249 infection, cells were lysed with cell culture lysis reagent (Promega), and luciferase activity was 250 measured by Centro XS 3 LB 960 (Berthold). 251

In vitro stimulation of PBMCs 253
Cryopreserved PBMCs were thawed and washed with warm RPMI 1640 medium (Sigma) 254 supplemented with 5% human AB serum (GeminiBio), Penicillin (Sigma), streptomycin (MP 255 Biomedicals), and 2-mercaptoethanol (Nacalai Tesque). PBMCs were labeled with Cell 256 Proliferation Kit (CellTrace™ Violet, ThermoFisher) following the manufacturer's protocol 257 and were stimulated in the same medium with S peptide pool (1 μg/ml per peptide, JPT) for 10 258 days, with human recombinant IL-2 (1 ng/ml, Peprotech), IL-7 (5 ng/ml, BioLegend) and IL-259 15 (5 ng/ml, Peprotech) supplemented on day 2, day 5 and day 8 of the culture. On day 10 cells 260 were washed and stained with anti-human CD3 and TotalSeq-C Hashtags antibodies. Single cell library was prepared using the reagents from 10x Genomics following the 266 manufacturer's instructions. After reverse transcription, cDNA was amplified for 14 cycles, 267 and up to 50 ng of cDNA was used for construction of gene expression and TCR libraries. 268 Libraries were sequenced in paired-end mode, and the raw reads were processed by Cell Ranger 269 3.1.0 (10x Genomics). Doublets and empty drops were removed by using Scrublet [38] and 270 gating out the events whose main hashtag reads are less than 95% of the total hashtag reads. 271 The top 4000 highly variable genes were used for clustering. Tfh signature score was generated 272 using canonical Tfh marker genes (IL21, ICOS, CD200, PDCD1, POU2AF1, BTLA, CXCR5, 273 and CXCL13) and UMAP plots were exported using BBrowser [39]. All values with error bars are presented as the mean ± SEM. One-way ANOVA followed by 306 Turkey's post hoc multiple comparison test was used to assess significant differences in each 307 experiment using Prism 8 software (GraphPad Software). Differences were considered to be 308 significant when P value was less than 0.05. P values in Fig. 6 were calculated with t-test using 309 the "stat_compare_means" function in R.