key: cord-0312400-ry2voi60
authors: Szeto, Christopher; Nguyen, Andrea T.; Lobos, Christian A.; Chatzileontiadou, Dimitra S.M.; Jaya-singhe, Dhilshan; Grant, Emma J.; Riboldi-Tunnicliffe, Alan; Smith, Corey; Gras, Stephanie
title: Molecular basis of a dominant SARS-CoV-2 Spike-derived epitope presented by HLA-A*02:01 recognised by a public TCR
date: 2021-08-16
journal: bioRxiv
DOI: 10.1101/2021.08.15.456333
sha: ccab8e5d49700db6aae366d3ec6507715b7005ae
doc_id: 312400
cord_uid: ry2voi60

The data currently available on how the immune system recognizes the SARS-CoV-2 virus is growing rapidly. While there are structures of some SARS-CoV-2 proteins in complex with antibodies, which helps us understand how the immune system is able to recognise this new virus, we are lacking data on how T cells are able to recognize this virus. T cells, especially the cytotoxic CD8+ T cells, are critical for viral recognition and clearance. Here we report the X-ray crystallography structure of a T cell receptor, shared among unrelated individuals (public TCR) in complex with a dominant spike-derived CD8+ T cell epitope (YLQ peptide). We show that YLQ activates a polyfunctional CD8+ T cell response in COVID-19 recovered patients. We detail the molecular basis for the shared TCR gene usage observed in HLA-A*02:01+ individuals, providing an understanding of TCR recognition towards a SARS-CoV-2 epitope. Interestingly, the YLQ peptide conformation did not change upon TCR binding, facilitating the high-affinity interaction observed.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus which has infected over 200 mil- 36 lion people worldwide resulting in coronavirus disease 2019 (COVID- 19) , and over 4.3 million deaths (1). Despite the 37 rapid development of effective and safe vaccinations against COVID-19, the global infection rate remains high, likely 38 due to mutations within the SARS-CoV-2 virus, driven by the scale of global infections, and now vaccination, which 39 pressures the virus to select for viral mutations that facilitate immune escape. Cytotoxic T cells are vital in the control 40 and clearance of viral infections (2) (3) (4) (5) and have been shown to be an important factor of the immune response to SARS- 41 CoV-2, due their role in viral clearance and ability to recognize variants of SARS-CoV-2 (6) . CD8+ T cells typically rec- 42 ognize peptides of 8-10 amino acid long presented by human leukocyte antigen (HLA) molecules (7) . 43 44 To date, over 1000 distinct CD8+ T cell epitopes have been reported (www.iedb.org (8) ), spanning multiple SARS-CoV-45 2 proteins. These epitopes are restricted by a large range of HLA class I (HLA-I) molecules, including HLA-A*02:01, one 46 of the most prevalent HLAs in the global population (9) . Several studies have shown that HLA-A*02:01+ individuals 47 demonstrate a strong CD8+ T cell response to one such HLA-A*02:01 restricted CD8+ T cell epitope derived from the 48 Spike (S) protein of SARS-CoV-2, namely, S269-277 (YLQPRTFLL, hereafter refer as YLQ) (10) (11) (12) (13) (14) (15) (16) (17) (18) , which was characterised 49 as an immunodominant epitope (18) . 50 51 CD8+ T cells recognise the peptide-HLA complex (pHLA) though their T cell receptor (TCR). TCRs comprise an a-and 52 b-chain, composed of variable (V), joining (J), constant (C) and diversity (D; b-chain only), genes generated by somatic 53 recombination (19) . Additional diversity is introduced by the inclusion of non-template encoded (N) regions at the 54 junction of gene segments by the terminal deoxynucleotidyl transferase (tdt) enzyme, leading to incredible diversity 55 (20) . Indeed, it is estimated that there are 2x10 7 TCR combinations available in humans (21) . Within the TCR, three 56 regions of variability, termed complementarity determining regions (CDRs) exist and are responsible for TCR specificity 57 (7) . Of these, the CDR3 region, which spans the V(D)J gene segments, is the most variable (7) , and has been shown both 58 functionally and structurally to make the predominant contacts within the pHLA complex (7) . The CDR3ab loops are typically used to define peptide-specific CD8+ T cell clonotypes and the combination of these 61 clonotypes is referred to as the TCR repertoire. TCR repertoires can exhibit biases, that is a preference for particular 62 TCR a-chain Variable (TRAV) or TCR b-chain Variable (TRBV) usage (7, 22, 23) . Additionally, despite the vast array of 63 potential TCRs in any given individual, identical epitope-specific clonotypes have been described across donors. These 64 "public" TCRs are thought to have a selective advantage, or comprise predominately germline encoded sequences that 65 could be easily generated in different individuals (7, (23) (24) (25) . However, TCR repertoires are more typically private, where 66 each individual displays completely distinct TCR sequences specific for the same epitope (26-28). Understanding the 67 TCR repertoire, and in the case of public TCRs, how they interact with their pHLA molecule, is critical for a thorough 68 understanding of CD8+ T cell response towards specific epitopes. Here, we wanted to validate and dissect the CD8+ T cell response to the YLQ peptide and determine the structural 71 basis for the presentation of the YLQ peptide by HLA-A*02:01. Additionally, we aimed to provide the molecular basis 72 of the biased TCR repertoire observed in response to the YLQ epitope in COVID-19 recovered individuals in different 73 studies (16) (17) (18) . Therefore, we have selected a representative public TCR, hereafter called YLQ-SG3 TCR. We determined 74 the ternary structure of the HLA-A*02:01-YLQ peptide bound to the public YLQ-SG3 TCR and investigated the binding 75 affinity of the public TCR. 

Sequence alignment 81 The full spike proteins from the five different coronaviruses were aligned using the online alignment software Rhône-82 Alpes Bioinformatics Center (PRABI http://www.prabi.fr/) multiple sequence alignment CLUSTALW (29) . The acces-83 sion number for the sequence used were for SARS-CoV-2: YP_009724390.1, OC43: YP_009555241.1, HKU-1: AZS52618.1, 84 229E: AAG48592.1, NL63: AAS58177.1. Then the sequence aligned with the SARS-CoV-2 YLQ peptide was selected and 85 reported in Table 1 . 88 The sequence conservation of the YLQ peptide was obtained using the NCBI web site tool "Mutations in SARS-CoV-2 89 SRA Data" (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/scov2_snp) that uses the Wuhan-Hu-1 as reference se-90 quence of SARS-CoV-2 with the accession number of NC_045512.2. The web site was accessed on the 30 th of July 2021, 91 with 412,297 full sequences of the spike proteins were available. 92 93 Generation of peptide-specific CD8+ T cell lines 94 CD8+ T cell lines were generated as previously described (26, 30) . In summary, HLA typed HLA-A*02:01+ PBMCs from 95 COVID-19 recovered individuals were incubated with SARS-CoV-2 peptide pools (2µM / peptide) and cultured for 10-96 14 days in RPMI-1640 supplemented with 1x Non-essential amino acids (NEAA; Sigma), 5 mM HEPES (Sigma), 2 mM 97 L-glutamine (Sigma), 1x penicillin/streptomycin/Glutamine (Life Technologies), 50 µM 2-ME (Sigma) and 10% heat-98 inactivated (FCS; Thermofisher, Scientifix). Cultures were supplemented with 10IU IL-2 (BD Biosciences) 2-3 times 99 weekly. CD8+ T cell lines were freshly harvested and used for subsequent assays. 100 101 Intracellular cytokine assay 102 The intracellular cytokine assay was performed as previously described (26, 30) . Briefly, CD8+ T cell lines were stimu-103 lated with cognate peptide pools or 10µM individual peptides (Genscript) and incubated for 5 hours in the presence of 104 GolgiPlug (BD Biosciences), GolgiStop (BD Biosciences) and anti-CD107a-AF488 (BD Biosciences/eBioscience). Follow-105 ing incubation, cells were surface stained for 30 minutes with anti-CD8-PerCP-Cy5.5 (eBioscience/BD Biosciences), anti-106 CD4-BUV395 (BD Biosciences) anti-CD14-APCH7, CD19-APCH7 and Live/Dead Fixable Near-IR Dead Cell Stain (Life 107 Technologies). Cells were then fixed and permeabilized for 20 minutes using BD Cytofix/Cytoperm solution (BD Bio-108 sciences) and intra-cellularly stained with anti-IFN-γ-BV421, and anti-TNF-PE-Cy7(all BD Biosciences) for a further 30 109 minutes. Cells were acquired on a BD LSRFortessa with FACSDiva software. Analysis was performed using FlowJo 110 software where cytokine levels identified in the R0 control condition were subtracted from corresponding test condi-111 tions.

Protein refold, purification, crystallisation 114 The HLA-A*02:01 heavy chain and b2-microglobulin as well as both chains of the YLQ-SG3 TCR were produced using 115 bacterial expression of inclusion bodies and refolded into soluble protein (for detailed protocol see (31) ). In brief, DNA 116 plasmids encoding each recombinant protein subunit (HLA-A*02:01 a-chain, β2-microglobulin, TCR a-chain, and TCR 117 β-chain) were individually transformed into competent BL21 E. coli cells. All cells were grown separately and their in-118 clusion bodies were extracted. Soluble HLA-A*02:01-YLQ complex was produced by refolding inclusion bodies in the 119 following amounts: 30 mg of a-chain, 10 mg of β2-microglobulin and 4 mg of YLQ peptide (Genscript). Soluble YLQ-120 SG3 TCR was produced by refolding 50 mg of TCRa chain with 50 mg of TCRβ chain. The refold buffer used was 3 M 121 Urea, 0.5 M L-Arginine, 0.1 M Tris-HCl pH 8.0, 2.5 mM EDTA pH 8.0, 5 mM glutathione (reduced), 1.25 mM glutathione 122 (oxidised). The refold mixtures were separately dialysed into 10 mM Tris-HCl pH 8.0. HLA-A*02:01-YLQ was purified 123 using anion exchange chromatography (HiTrap Q, GE), whilst the YLQ-SG3 TCR was purified using anion exchange 124 followed by size exclusion chromatography (Superdex 200 16/60, GE). 125 Crystals of HLA-A*02:01-YLQ complex were obtained using the sitting-drop, vapour-diffusion method at 20 °C 126 with a protein/mother liquor drop ratio of 1:1 at 6 mg/mL in 10 mM Tris-HCl pH 8.0, 150 mM NaCl using 20% PEG3350 127 and 0.2 M NaF. YLQ-SG3 TCR was co-complexed with HLA-A*02:01-YLQ by combining both proteins at a 1:1 molar 128 ratio before purification using size exclusion chromatography (Superdex 200 10/30, GE). Crystals of YLQ-SG3 TCR-129 HLA-A*02:01-YLQ complex were obtained using the sitting-drop, vapour-diffusion method at 20°C with a pro-130 tein/mother liquor drop ratio of 1:1 at 3 mg/mL in 10 mM Tris-HCl pH 8.0, 150 mM NaCl using 20% PEG3350 and 0.05 131 M Zn-Acetate. Crystals were soaked in a cryosolution of 30% (w/v) PEG3350 diluted using mother liquor and then flash 132 frozen in liquid nitrogen. The data were collected on the MX2 beamline at the Australian Synchrotron, part of ANSTO, 133 Australia (32). 134 135 Structure determination 136 The data were processed using XDS (33) and the structures were determined by molecular replacement using the 137 PHASER program (34) from the CCP4 suite (35) using a model of HLA-A*02:01 without peptide (derived from PDB ID: 138 3GSO (36)). Manual model building was conducted using COOT (37) followed by refinement with BUSTER (38). The 139 final models have been validated and deposited using the wwPDB OneDep System and the final refinement statistics, 140 PDB codes are summarized in Table 3 . All molecular graphics representations were created using PyMOL (Schrodinger, 141 LLC, v1.7.6.3). 142 143 Stability assay 144 Thermal stability was measured using differential scanning fluorimetry, performed in a Qiagen RG6 rtPCR. HLA-145 A*02:01-YLQ was heated from 30 to 95˚C at a rate of 0.5˚C/min with excitation and emission channels set at yellow 146 (excitation of ~530 nm and detection at ~557 nm). The experiment was performed at two concentrations (5 µM and 10 147 µM) in duplicate. Each sample was dialysed in 10 mM Tris-HCl pH 8.0, 150 mM NaCl and contained a final concentra-148 tion of 10X SYPRO Orange Dye. Fluorescence intensity data was normalised and plotted using GraphPad Prism 9 (ver-149 sion 9.0.0). 150 151 Surface Plasmon resonance (SPR) 152 SPR was performed using a Biacore T200 biosensor at 25˚C. YLQ-SG3 TCR was immobilized onto a CM5 chip using 153 amine coupling, with the reference flow cell containing a negative control (M158-66 TCR (23)). The immobilization steps 154 were carried out at a flow rate of 5 µl/min in immobilization buffer 10 mM HEPES (pH 7.0), 150 mM NaCl and finally 155 blocked with Ethanolamine at 5 µl/min for 7 min. HLA-A*02:01-YLQ was injected over the chip at a range of 156 concentrations from 0.2 to 50 µM using a 1 in 2 dilution at a flow rate of 30 µl/min and in a running buffer of 10 mM 157 Tris-HCl pH 8.0, 150 mM NaCl, 1 mg/ml bovine serum albumin and 0.005% P20. All injections were run in duplicate 158 and SPR was performed twice to determine the dissociation constant between YLQ-SG3 TCR and HLA-A*02:01-YLQ 159 (n=2) using both steady state affinity measurements and kinetics data. Kinetics data was analysed using the T200 BiaE-160 valuation software, whilst steady state values were extracted using T200 BiaEvaluation software, plotted and fitted into 161 a one-site specific binding non-linear regression using Graphpad Prism (version 9.0). 162 163 165 166 The CD8+ T cell response towards the HLA-A*02:01 restricted YLQ peptide has previously been reported (10, 11, 16-167 18) , however data regarding the level of polyfunctionality associated with the CD8+ T cell response has been limited in 168 COVID-19 recovered donors. Therefore, we first tested the immunogenicity of the YLQ peptide in three COVID-19 169 recovered individuals by expanding CD8+ T cells against peptide pools including the YLQ peptide and performed an 170 intracellular cytokine staining assay to determine the immunogenicity. The CD8+ T cell response and cytokine produc-171 tion towards the YLQ peptide was variable between the COVID-19 recovered donors. CD8+ T cells from two out of 172 three donors, namely Q036 and Q042, were able to produce all four cytokines, while only double cytokine producing 173 CD8+ T cells were observed in the Q062 donor (Figure 1) . Even though the level of polyfunctionality was different 174 between the three donors, they were all able to generate a polyfunctional CD8+ T cell response specific to the YLQ 175 peptide after recovery from COVID-19. 187 We previously determined that a high level of CD8+ T cell activation towards a SARS-CoV-2 epitope derived from the 188 nucleocapsid (N105-113 or SPR peptide) was underpinned by a pre-existing and cross-reactive response (26). This pre-189 existing immunity was due to a high level of sequence identity (55-89%) between the SPR peptide from SARS-CoV-2 190 and its homologues from seasonal coronaviruses. Therefore, we questioned if the YLQ peptide was also conserved in 191 seasonal coronaviruses by aligning the spike protein sequences of SARS-CoV-2 and seasonal coronaviruses ( While the SPR peptide had up to 89% sequence identity with its seasonal coronaviruses derived homologues, the level 196 of conservation of the YLQ peptide was lower. The YLQ peptide shared only four residues with its homologues from 197 OC43 and HKU-1 b-coronaviruses, with two of those residues being primary anchor residues that will be buried in the 198 HLA cleft. The low sequence identity is in line with the lack or weak T cell activation observed in healthy individuals 199 (18) . 200 As YLQ peptide is derived from spike protein, which is relatively less conserved and more prone to mutation than other 201 SARS-CoV-2 viral proteins, we also wanted to assess the level of mutations found in the different SARS-CoV-2 isolates 202 ( Table 2) . Interestingly, this dominant T cell epitope was conserved with less than 0.5% of mutations for any of its 203 residues. In order to gain a deeper understanding of the YLQ peptide recognition by CD8+ T cells, we first refolded and 208 crystallised the HLA-A*02:01-YLQ complex and solved its structure at a high resolution ( Table 3) . The electron density 209 map was clear for the peptide, indicating a stable and rigid conformation of the YLQ peptide in the HLA-A*02:01 cleft 210 (Figure 2A-B) . The YLQ peptide bound to the HLA-A*02 :01 cleft via the canonical primary anchor of small hydrophobic residues, P2-225 Leu and P9-Leu, characteristic of HLA-A*02 :01, and an additional secondary anchor with P3-Gln (Figure 2A-B) . The 226 YLQ peptide has a series of residues with long, solvent exposed side-chains, P1-Tyr, P5-Arg, P7-Phe and P8-Leu, that 227 could potenially interact with TCRs. The side-chains were well defined in the electron density map, this is possibly due 228 to the numerous intra-peptide contacts. The only exception was the P5-Arg for which the density was partly missing 229 for the side-chain, showing high mobility ( Figure 2B ). This rigidity of the pHLA was apparent when we undertook a 230 thermal stability assay to determine the stability of the overall peptide-HLA (pHLA) complex, as this is important for 231 immunogenicity (30) . Indeed, the thermal stability of the HLA-A*02:01-YLQ complex was about 60°C, which is similar 232 to that observed for the dominant influenza derived M158-66 peptide bound to HLA-A*02:01 (23, 30). 233 234

The YLQ peptide was reported to be immunogenic in ~90% of COVID-19 recovered individuals, while only 5% of 236 healthy donors exhibited T cells specific for the peptide (18) . This shows that in the absence of an antibody response, 237 this epitope can be used as a marker of infection in HLA-A*02:01+ patients, and also that a T cell driven immune 238 response would be activated. Interestingly, three studies have reported the TCR sequences of YLQ-specific clonotypes 239 from COVID-19 recovered individuals and show an highly biased repertoire among unrelated donors ( We analysed the TCR sequences from those studies, and observed the same TCR gene usage bias, especially for the TCR 246 a-chain. The HLA-A*02:01-YLQ-specific T cells were mostly expressing a TRAV12-1 or TRAV12-2 allele for their a-247 chain, both sharing 50% sequence identity for their CDR1a and CDR2a loops. The most frequent TRBV gene expressed 248 by YLQ-specific CD8+ T cells were 2, 7-9, and 20-1 with different frequencies depending on the study ( Table 4) . 249 Interestingly, there were conserved motifs present in both a and b CDR3 loops, with a public TCR observed among 250 donors and across studies, here called the YLQ-SG3 TCR ( Table 4 ). The YLQ-SG3 TCR was composed of the TRAV12-251 2 and TRBV7-9 bias chain and contains the conserved motif within both its CDR3 loops. We therefore chose the YLQ-252 SG3 TCR to understand how T cells can engage with YLQ epitope, a SARS-CoV-2 spike-derived peptide presented by 253 the HLA-A*02:01 molecule. 254 255 256 We refolded and purified the YLQ-SG3 TCR and undertook affinity measurements by surface plasmon resonance (SPR), 257 as well as solved the structure of the YLQ-SG3 TCR in complex with the HLA-A*02:01-YLQ. 258 The SPR data shows that the YLQ-SG3 TCR binds with the HLA-A*02:01-YLQ complex with high affinity and a Kd of 259 2.09 ± 0.16 µM (Figure 3A-B) , at the high end of the affinity range observed for CD8+ TCR (7) . In addition the kinetics 260 of the interaction show a fast association (kon = 386,800 ± 25,000 M -1 s -1 ) and a fast dissociation (koff = 0.679 ± 0.001 s -1 ) 261 compare to other TCRs (27) (Figure 3A) .

We solved the structure of the YLQ-SG3 TCR in complex with the HLA-A*02:01-YLQ to better undertsand how TCRs 264 recognise SARS-CoV-2 epitope. We solved the structure at a resolution of 2.6 Å ( Table 3) with unambigous density for 265 the peptide (Figure 2C-D) . The YLQ-SG3 TCR docks diagonally above the center of the YLQ peptide with a docking angle of 73° (Figure 3B-C) , 282 within the range of other TCR-pHLA complexes (7) . The buried surface area at the interface of the TCR and HLA-283 A*02:01-YLQ was 1,809 Å, also within the range (average of 1,885 Å) (7) . Interestingly, and consistent with the strong 284 TCR bias observed for the YLQ-specific T cells, the TCR a-chain is contributing to 67% of the interaction (Figure 3B) , 285 with the CDR1/2a loops contributing to 40% of the total interactions and giving the molecular basis for the TRAV12 286 bias observed ( Table 4 ). All CDRa loops contacted the HLA-A*02:01 molecule, however, from these, only CDR1a and 287 CDR3a contacted the YLQ peptide ( The CDR1a loop streched itself above the N-terminal region of the a2-helix and forms a salt bridge with Glu166 via 296 Arg28a, as well as hydrogen bonds with the Gln155 via the Gln37a and Ser38a. In addition, the side-chain of the Gln37a 297 dips in between the HLA a2-helix and the peptide backbone to form an extensive hydrogen bond network (Figure 4A) . 298 The CDR2a sits above the a2-helix of the HLA just before the hinge region of the a2-helix, with the Ser58a forming H-299 bond with Arg157 outside the cleft, and the Tyr57 forming Van der Waals bonds with the Gln155 inside the cleft (Figure 300  4B) . The CDR3a makes limited contributions in forming contacts with the HLA molecule, with the conserved Asp109a 301 ( Table 5 ) forming a salt bridge with the Arg65 (Figure 4C ).

The YLQ-SG3 TCR b-chain has limited contact with the HLA. Both CDR1b and CDR2b loops made contacts with two 303 residues of the a1-helix and the CDR3b loop with two residues of the a2-helix ( The YLQ peptide made a significant contribution to the pHLA buried surface area at 38% and is contacted by five of the 319 CDR loops (Figure 3C and Table5), whilst the average buried surface area is only 29% for other solved TCRpMHC 320 complexes (7) . The CDR1a loop runs over half of the peptide making contacts from P1-Tyr to P5-Arg, with the side-321 chain of the Gln37a inserting itself between the peptide and a2-helix and interacting with P3-Gln, P4-Pro and P5-Arg 322 ( Figure 4D ). In the same fashion, the CDR3a loop contacts a large strecth of the YLQ peptide including P4-Pro, P5-Arg 323 and P6-Thr, and inserts a conserved CDR3a 109 DD 110 motif in between the peptide and the a1-helix of the HLA-A*02:01 324 ( Figure 4E) . The CDR1b and CDR2b loops each projected long side-chains towards the C-terminal parts of the peptide 325 surface. As as result the exposed P8-Leu is surrounded by Asn37b/Arg38b on one side and by Gln57b/Asn58b on the 326 other side. The CDR3b pushes the P5-Arg down with the Ile110b and forms a salt bridge with the Asp109b. This 327 conformation is helped by the short length of the CDR3b loop that only forms a short rigid loop due to the Pro108b. The 328 P5-Arg is surrounded by the CDR1/3a and CDR1/3b loops (Figure 4F) , and instead of wrapping the side-chain of the 329 P5-Arg with CDR loops that has been previously observed (39) , the YLQ-SG3 TCR pushes down on the P5-Arg and P6-330 Phe side-chains. This increases the contact surface between the peptide and these loops and stabilised the P5-Arg side-331 chain, yet, do not disturb the HLA-A*02:01 cleft structure (root mean square deviation of 0.22 Å). Overall the YLQ-SG3 332 TCR docks onto HLA-A*02:01-YLQ with minimal structural rearrangements, with the exception of a few residue side-333 chains. As the kinetics data from SPR shows a fast association rate (Figure 3A) , high binding affinity and moderate 334 dissociation rate. This is consistent with the larger binding interface, but minimal structural rearrangements during 335 binding. 336 337

We have described here the molecular basis of a public TCR recognizing a dominant spike-derived SARS-CoV-2 340 epitope. The structure of the YLQ peptide in the cleft of the HLA-A*02:01 molecule is a constrained and rigid peptide 341 that forms numerous intra-peptide interactions favoured by large side-chain residues. This rigidity was consistent with 342 the high thermal stability observed for the HLA-A*02:01-YLQ complex. This rigid conformation of the YLQ peptide did 343 not undergo large structural changes, beside the stabilisation of the P5-Arg, upon the YLQ-SG3 TCR docking. Despite 344 a solvent exposed P5-Arg, the side-chain of this residue was pushed down by the CDR3b loop to maximize the contact 345 between the TCR and the YLQ peptide. This resulted in a large contribution of the peptide to the pHLA surface buried 346 area of 38%, well above the average of 29% (7), highlighting the importance of the peptide in driving the interaction 347 with the YLQ-SG3 TCR. 348 The YLQ-specific T cells exhibited a bias in their TCR repertoire with frequent usage of the TRAV12 gene for the 349 a-chain. Here, we show the molecular basis behind this bias, as the TCRpHLA complex structure shows that the a-350 chain dominates this interaction, contributing to 67% of the TCR contact surface. This was mainly due to a large footprint 351 of the CDR1a (26%) on the peptide, CDR2a (14%) on the HLA, and CDR3a (19%) that binds both the peptide and the 352 HLA. 353 Interestingly, TRAV12 usage in TCRs that recognise HLA-A*02:01 has been observed in 45% of the TCRpHLA-354 A*02:01 structures solved (17/38), with 18% (7/38) using the TRAV12-2*01 gene (7) . While the TRAV12+ TCRs all used 355 their a-chains to contact the N-terminal parts of the peptide and the HLA-A*02:01 cleft, their CDR1a loops don't neces-356 sarily share the same interactions. For example, although the CDR1a loop of the CD8 (40) and 868 TCRs (41) interacts 357 similarly to the CDR1a of YLQ-SG3 TCR, the DMF5 TCR uses its CDR1a loop mainly to contact the peptide N-terminal 358 part (42) , and not the HLA. Another example is the NYE_S1 TCR, which docks with a tilt that pushes the CDR1a loop 359 away from the pHLA and does not make contact (43) . This shows, that while some common interactions between the 360 TRAV12+ TCRs and the HLA-A*02:01 are consistent between some TCRs, there is also a large variability of docking 361 modes for the same aTCR segment. 362 The malleability of docking while using the same or similar sequence between different TCR chains, show that the 363 conserved motif observed in the YLQ-specific TCRs, while not identical, could lead to the same mode of recognition of 364 the YLQ epitope. The sequence differences between these TCRs could be important to give the TCR repertoire enough 365 breadth to recognize variants of the YLQ peptide, which could be critical in recognizing emerging mutations located in 366 this region of spike. 367 The YLQ epitope has been identified as one of the dominant CD8+ T cell epitopes in individuals expressing the 368 HLA-A*02:01 allele. Information about the immune response to the YLQ epitope will be critical in understanding the 369 potential of the YLQ peptide as a targetable epitope for T cell-based therapeutics, biomarkers or vaccines against 370 COVID-19. Firstly, the YLQ peptide is highly immunogenic in most COVID-19 recovered HLA-A*02:01+ individuals, 371 and weakly or not recognized in healthy individuals (18) , so in absence of antibodies this could be used as a marker of 372 infection. Secondly, none of the current mutations reported for that region of the spike are within the Variant of Concern 373 (VOC) or of Interest (VOI). The YLQ epitope selects for biased and public TCRs (22) that could give a selective advantage 374 to HLA-A*02:01+ individuals. The public TCR exhibits a high affinity within the range of other potent anti-viral CD8+ 375 T cells (7) . And finally, we report here that in COVID-19 recovered individuals there is a polyfunctional response from 376 CD8+ T cell stimulated with the YLQ peptide. The ability of the YLQ epitope to strongly stimulate CD8+ T cells has also 377 been observed in vaccinated individuals (44) . Altogether this makes the YLQ peptide a promising target to prime and 378 boost CD8+ T cells against SARS-CoV-2 infection. 

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