key: cord-1054641-71ywsj3a
authors: Dolton, G.; Rius, C.; Hasan, M. S.; Szomolay, B.; Behiry, E.; Whalley, T.; Southgate, J. A.; COVID-19 Genomics UK Consortium,; Morin, T.; Topley, K.; Tan, L. R.; Fuller, A.; Wall, A.; Goulder, P. J.; Spiller, B.; Jones, L. C.; Connor, T. R.; Sewell, A. K.
title: Emergence of immune escape at dominant SARS-CoV-2 killer T-cell epitope
date: 2021-06-28
journal: nan
DOI: 10.1101/2021.06.21.21259010
sha: 6b55a2475823e2652c8d72023e65ee2967b84960
doc_id: 1054641
cord_uid: 71ywsj3a

The adaptive immune system protects against infection via selection of specific antigen receptors on B-cells and T-cells. We studied the prevalent CD8 killer T-cell response mounted against SARS-CoV-2 Spike269-277 epitope YLQPRTFLL via the most frequent Human Leukocyte Antigen (HLA) class I worldwide, HLA A*02. The widespread Spike P272L mutation has arisen in five different SARS-CoV-2 lineages to date and was common in the B.1.177 lineage associated with establishing the second wave in Europe. The large CD8 T-cell response seen across a cohort of HLA A*02+ convalescent patients, comprising of over 120 different TCRs, failed to respond to the P272L variant suggesting that proline 272 dominates TCR contacts with this epitope. Additionally, sizable populations (0.01%-0.2%) of total CD8 T-cells from individuals vaccinated against SARS-CoV-2 stained with HLA A*02-YLQPRTFLL multimers but failed to bind to the P272L reagent. Viral escape at prevalent T-cell epitopes restricted by high frequency HLA may be particularly problematic when vaccine immunity is focussed on a single protein such as SARS-CoV-2 Spike and provides a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlights the urgent need for monitoring T-cell escape in new SARS-CoV-2 variants.

The mammalian immune system utilises numerous highly developed mechanisms to defend against viral infection. The most sophisticated of these systems, adaptive immunity, is controlled via vast fleets of highly variable antigen-binding molecules called B-cell receptors (antibodies) or T-cell receptors (TCRs).

The advent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a novel human coronavirus and the stress on global healthcare systems caused by the associated coronavirus disease 2019 has focussed the world's attention on ways to combat this emerging infection. The mechanisms used by coronaviruses to escape from the host adaptive immune system are not well understood but are now firmly in the spotlight. The developing picture shows that neutralising antibodies, CD4 'helper' T-cells and CD8 'killer' T-cells all contribute to the control of SARS-CoV-2 and the protection offered by currently approved vaccines 1, 2 . Although research focused on antibody-mediated immunity has made up the majority of published SARS-CoV-2 immunological work to date, some evidence suggests that antibodies may play a secondary role in ultimately clearing SARS-CoV-2 infection compared to T-cells. The presence of SARS-CoV-2-specific CD4 and CD8 T-cells has been reported to correlate with reduced COVID-19 severity while neutralising antibodies in the same individuals did not 2 .

Two SARS-CoV-2 positive agammaglobulinemia patients who developed COVID-19 symptoms during the first wave of infection in Italy, required neither oxygen nor intensive care before making full recoveries 3 . The authors conclude that while an antibody-mediated response to SARS-CoV-2 might be important, it is not obligatory for overcoming disease 3 . This finding is consistent with multiple patients that developed COVID-19 while on B-cell depleting therapy across several studies, who resolved infection without the need for intensive treatment [4] [5] [6] . There are also many reports of healthy individuals successfully controlling SARS-CoV-2 infection without having detectable neutralising, or receptor-binding domain (RBD), antibodies while having prominent SARS-CoV-2-specific T-cell memory 2, [7] [8] [9] [10] . Clinical interventions using monoclonal antibodies further suggest that humoral immunity to SARS-CoV-2, although important, is not the hoped-for panacea for individuals that require intensive treatment 11, 12 .

The association of SARS-CoV-2-specific CD4 and CD8 T-cells with milder disease suggests that both Tcell subsets play a role in protective immunity 2 . Indeed, the relative scarcity of naïve T-cells in individuals over 65 years old and the connection between ageing and impaired adaptive immune responses to SARS-CoV-2 has been suggested as a major cause of severe disease 2 . Analysis of immune cells in bronchoalveolar fluid from patients with COVID-19 showed that moderate disease correlated with highly clonally expanded CD8 T-cells 13 . Acute phase SARS-CoV-2-specific T-cells displaying a highly activated cytotoxic phenotype were present in antibody-seronegative exposed family members indicating that they may be capable of eliminating infection prior to induction of humoral immunity 7 and it has been suggested that strong antibody responses but weak CD8 T-cell responses could contribute to acute COVID-19 pathogenesis and severity 1, 14 . Depletion of CD8 T-cells in convalescent non-human primates reduced the protective efficacy of natural immunity against SARS-CoV-2 rechallenge 15 suggesting CD8 T-cells in the upper respiratory tract may play a similar protective role in humans. Given the importance of CD8 T-cells . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 28, 2021. ; https://doi.org/10.1101/2021.06.21.21259010 doi: medRxiv preprint to adaptive immune protection against COVID-19, we set out to examine dominant T-cell responses to SARS-CoV-2 through the most prevalent major histocompatibility complex (MHC) allele in humans, HLA A*02 16 . HLA A*02 is an MHC class I molecule and presents processed intracellular protein antigens at the cell surface in the form of short peptides 8-10 amino acids in length for inspection by CD8 T-cells.

SARS CoV-2 infection induces T-cells that recognise peptides derived from a range of viral proteins with enrichment for those that respond to Spike, nucleocapsid, membrane, ORF1ab and ORF3a 7, 8, [17] [18] [19] .

Unbiased screening of nine HLA A*0201 + convalescent patients (CP) showed that the two biggest and most frequent CD8 T-cell responses recognised regions of the virus contained within ORF1ab residues 3,881-3,900 and Spike residues 261-280 19 . The dominant Spike epitope was narrowed down to residues 269-277 (YLQPRTFLL) and T-cells that responded to this peptide represented >1 in 10,000 total CD8 T-cells in many HLA A*0201 + CP with the majority of the nine donors using a TCR made with the TRAV-12-1 gene, suggesting a potential shared or 'public' response 19 . Another study found that YLQPRTFLL was the most frequently recognised of 13 reported HLA A*02:01-restricted epitopes (responses in 16/17 HLA A*0201 + CP studied) 10 . TCR sequencing of HLA A*02:01-YLQPRTFLL tetramer + cells revealed prominent CDR3 motifs that were shared across individuals and confirmed the general TRAV-12-1 dominance 9 . TCRβ repertoire analyses also indicates that CD8 T-cell responses to the Spike 265-277 region containing the YLQPRTFLL epitope dominate responses to Spike in both CP and individuals vaccinated with 'singleshot' Ad26.5.COV2.S vaccine in the US irrespective of HLA type 20 . We reasoned that if SARS-CoV-2 were to exhibit escape from CD8 T-cells then this would most likely first occur within a dominant T-cell response restricted by the most frequent HLA in the population. We therefore focussed our attention on residues 269-277 of the SARS-CoV-2 Spike protein and found that the most prevalent mutation in this Tcell epitope, YLQLRTFLL (P272L change indicated in bold underlined text), was not recognised by any of the >120 TCRs that responded to the founder epitope (YLQPRTFLL) across a cohort of nine HLA A*02 + CP. We also found sizeable populations of CD8 T-cells that stained with peptide-HLA A*02 multimers bearing the YLQPRTFLL peptide in a cohort of individuals that had been vaccinated against SARS-CoV-2. These cells could not be stained by reagents manufactured with the P272L variant suggesting that this variant escapes from vaccine induced T-cell responses.

The previously unprecedented genome-sequencing efforts for SARS-CoV-2 and sequencing of over 400,000 genomes in the United Kingdom to date (>45% of the global effort) has allowed detailed analyses and identification of over a thousand UK transmission lineages, with some variants associating with higher viral loads and increased transmission fitness 21, 22 . We examined the global dataset sequenced as of January 31, 2021 and performed focussed analyses on the dominant Spike epitope at residues 269-277 (Figures 1,   2 and Supplementary Figures 1) . Within our dataset, we used ancestral state reconstruction to estimate the number of independent amino acid substitutions that have occurred in this region from sequenced data . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 28, 2021. ; https://doi.org/10.1101/2021.06.21.21259010 doi: medRxiv preprint collected from the beginning of the pandemic to January 31, 2021. This analysis identified at least 24 different amino acid changes in this region, 22 of which were seen six or fewer times. The two variants resulting in the largest number of cases both occurred at position 272 (P272L and P272H). The P272H mutation occurred prior to onward transmission that resulted in 26 sequenced cases while P272L prior to onward transmission that led to an international cluster comprising over a thousand sequenced cases (Figure 2 and Supplementary Figure 1) . The P272L mutation occurred on a background of the B.1.177 lineage (Figure 1) . B.1.177 is characterised by the lineage defining Spike mutation A222V and was shown to have arisen in Spain, and to have been exported to Europe and the wider world through travel over the summer of 2020 23 

In order to study the impact of mutation in the YLQPRTFLL epitope we recruited a cohort of SARS-CoV-2 convalescent local healthcare workers during June 2020. Each had a history of COVID-19 symptoms and a positive nasopharyngeal swab for SARS-CoV-2 by PCR >28 days prior to sample collection. 15/30 donors tested (50%) were HLA A*02 + by antibody staining. Nine of the first ten HLA A*02 + CP we screened had CD8 T-cells that stained with A*02:01-YLQPRTFLL tetramer (example in Figure 3a) , confirming the widespread response to this epitope seen in previous studies 10, 19 . Bulk sorting of tetramer + populations was used to generate a T-cell line from each of the nine CP initially screened. These lines all responded to YLQPRTFLL peptide (13-53% total cells) but completely failed to respond to YLQLRTFLL peptide (Figure 3b and Supplementary Figure 3a ) despite this sequence showing improved binding to . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 28, 2021. ; https://doi.org/10.1101/2021.06.21.21259010 doi: medRxiv preprint HLA A*02 compared to the parental (Wuhan) sequence (Supplementary Figure 3b) . The ability of the YLQLRTFLL peptide to bind to HLA A*02 was confirmed during tetramer production ( Supplementary   Figure 3c) . TCR sequencing of the A*02:01-YLQPRTFLL tetramer + T-cells in each line identified 121 different TCRs across the seven patients including the public TCR chains previously identified in addition to many donor-specific TCR sequences (Figure 3c and Supplementary Figure 4) . Remarkably, these data indicate that all the TCRs failed to respond to the P272L variant sequence and suggests that the proline at residue 272 plays a critical role in recognition by all TCRs across this cohort. Failure to recognise the P272L mutant or bind to this sequence was confirmed by direct ex vivo staining of PBMC ( Figure 3A ) and by using four different CP-derived T-cell clones (Figure 4) . We further confirmed that surrogate infected cells expressing full-length Spike protein with the P272L substitution (Supplementary Figure 5) were not recognised by any of these T-cell clones (Figure 4) . We conclude that P272L has arisen on multiple occasions (appearing to show selection in the B.1.177 background) and escapes from all TCRs raised against the parental (Wuhan) sequence that is being used in all current vaccines.

We next examined responses to YLQPRTFLL in individuals who had been vaccinated against SARS-CoV-2. In total, PBMC from four HLA A*02 + healthy donors (HD) who had received a single dose of the AstraZeneca ChAdOx1 nCoV-19 vaccine 24 and a further three HD who received the Pfizer-BioNTech BNT162b2 vaccine 25 (see Materials and Methods for donor and vaccination details) were stained with peptide-HLA tetramers. All vaccinees had HLA A*02-YLQPRTFLL tetramer staining T-cells that accounted for between 0.01 and 0.2% of total CD8 T-cells ( Figure 5) . In all cases, these T-cells failed to stain with the HLA A*02-YLQLRTFLL (P272L) reagent in parallel assays, indicating that the TCR on these T-cells did not strongly engage this sequence in accordance with the CP cohort data ( Figure 5 ). We conclude that the YLQPRTFLL-specific T-cells induced by two current COVID vaccines fail to engage to the P272L variant sequence.

Emerging evidence indicates that HLA-I-restricted CD8 'killer' T-cells contribute to the control of SARS-CoV-2 and the immunity to disease offered by the currently approved vaccines 1,2 . Individual HLA-I alleles can be associated with increased likelihood of control or progression of disease with viruses such as HIV 26 .

The immune pressure of prevalent host CD8 T-cell responses against HIV results in selection of altered viral sequences and viral immune escape [27] [28] [29] . We were interested in whether there was evidence that SARS-CoV-2 might also escape from CD8 T-cells. Genome sequencing has shown extensive nonsynonymous mutations occurred within the SARS-CoV-2 genome during 2020. Some of these mutations alter viral fitness 30 while others have been shown to diminish the binding of antibodies 31 . To date, there has been limited study of T-cell escape and a broad brush-approach of examining T-cell recognition of key transmission variants failed to reveal any evidence that escape was occurring 32 . A recent report demonstrated that mutations in the RBD that enhance angiotensin converting enzyme (ACE)2 binding and . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 28, 2021. ; https://doi.org/10.1101/2021.06.21.21259010 doi: medRxiv preprint viral infectivity concomitantly reduce T-cell recognition through HLA*24:02 33 . Another study showed that some SARS-CoV-2 mutations in HLA-I-restricted epitopes resulted in lower binding to the HLA leading to reduced recognition by CD8 T-cells but there was little evidence of these mutations being disseminated 34 . It has been suggested that the L270F (YFQPRTFLL) variant might be an escape mutant 34 , but this variant was only seen six times throughout our study period, in six different locations and five different PANGO lineages. From our data, this would suggest that while this mutation does arise, there is no evidence of spread of this variant following sporadic emergence across multiple different SARS-CoV-2 lineages.

We reasoned that if escape were to occur then it would be most likely to be seen in a prevalent response through a frequent HLA. HLA A*02 is frequently carried by all human populations except those of recent African ancestry where it is present at lower levels. HLA A*02 is believed to be the most frequent HLA-I in the human population worldwide occurring in ~40% of individuals 16 . HLA A*02 is hypothesized to have become so frequent in the population due to its success at presenting well-recognised epitopes from historically dangerous pathogens, a supposition consistent with this HLA having entered the population via interbreeding with Neanderthals and Denisovans after early humans migrated from Africa 35 . A previous study used overlapping peptides from the entire SARS-CoV-2 proteome to identify that the most common HLA A*02-restricted responses in CP were to epitopes contained within peptides spanning residues 3,881-3900 of ORF1ab and 261-280 of Spike 19 . The Spike epitope was narrowed down to residues 269-277; sequence YLQPRTFLL. A further study confirmed the prevalence of CD8 T-cells that respond to YLQPRTFLL in CP that are absent in healthy donor blood taken before the spread of SARS-CoV-2 10 . We confirmed the prevalence of responses to this epitope in our cohort where responses were observed in 9/10 HLA A*02 + CP tested and all 7/7 SARS-CoV-2 vaccinees including those who had received only the first dose of a double dose schedule.

We reasoned that if escape from CD8 T-cells were to occur, then it would most likely be seen in a dominant epitope restricted by the most frequent HLA-I in the population so focussed our attention on nonsynonymous mutations in the sequence encoding YLQPRTFLL. While at least 24 different amino acid changes have occurred in this region, only two were seen in more than six sequenced cases in the period from the beginning of the pandemic to January 31 st 2021. Nine of ten of our HLA A*02 + CP cohort tested made a response to the YLQPRTFLL epitope; a response comprising >120 different TCRs in total. Remarkably, no response was seen to the P272L variant. This finding was confirmed using four CP-derived T-cell clones that included TCR α or β chains . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 28, 2021. ; https://doi.org/10.1101/2021.06.21.21259010 doi: medRxiv preprint that have previously been described in other CP cohorts 10, 19 . Responses to the YLQPRTFLL epitope in a cohort of HLA A*02 + healthy donors who had been vaccinated against SARS-CoV-2 but had never had symptoms or a positive test since January 2020 ranged from 0.01-0.2% of CD8 T-cells by peptide-HLA staining. However, all of the T-cells raised against the YLQPRTFLL epitope across our vaccinee cohort expressed TCRs that engaged the P272L very poorly and did not stain with P272L peptide-HLA tetramers.

It may seem surprising that YLQPRTFLL-specific TCRs, >120 in total across our CP cohort and likely many more in the vaccinees, all fail to engage the P272L variant when a widely polyclonal response to Tcell antigens should prevent widespread escape of this nature. The widespread failure to engage the P272L variant likely reflects the nature of the amino acid substitution involved. Substitution of a proline in the middle of an HLA-I-restricted T-cell epitope is likely to be a particularly drastic change in terms of TCR recognition as residues 4-6 in a 9-mer peptide often stand proud of the HLA-I peptide binding groove to provide an obvious focal point for host TCRs 36 . This can be especially true for the ringed imino acid proline. We have previously demonstrated that the proline at position 5 in the HLA A*02:01-restricted 10mer preproinsulin-derived epitope ALWGPDPAAA associated with type 1 diabetes is critical for recognition by patient-derived TCRs 37, 38 . Subsequent structural analyses showed how this proline stands proud like the "cherry on a cake" to provide ~70% of the binding energy between the ALWGPDPAAA preproinsulin epitope and a cognate patient-derived TCR 39, 40 . We predict that the proline at position 4 within the predominant HLA A*02-restricted SARS-CoV-2 epitope might also represent the most obvious docking point for host TCRs in the 9-mer YLQPRTFLL epitope although this awaits structural confirmation. If, as we expect, the proline at position 4 represents the major TCR docking platform this would explain why the loss of this one residue results in such widespread escape from YLQPRTFLLspecific T-cells across all donors in our study.

SARS-CoV-2 variants have been rapidly emerging throughout the current pandemic with mutants that enhance transmissibility, evade host immunity or increase disease severity being of particular concern.

Current systems for identifying variants of public health concern involve risk assessments that identify mutations that are present, and then looks for mutations that have a known biological effect in order to assess their significance. While extensive amounts of work have been undertaken to predict and characterise the likely effects of mutations in Spike on antibody recognition, the same is not true for Tcells. Current risk assessments that only consider antibody affinity and factors associated with transmissibility (e.g. mutations that improve cellular infectivity) are clearly incomplete. The range of potential effects of mutations that affect recognition by T-cells is significant, and urgently requires further study. It is clear that there was a significant outbreak that spanned Europe of a lineage carrying a mutation that we have shown has a detectable impact on T-cell recognition, which went unrecognised at the time.

Ultimately, it is unclear whether SARS-CoV-2 incorporates enough genetic plasticity to allow it to escape from humoral immune responses. However, the area targeted on the RBD by neutralising antibodies is large enough, and the range of epitopes targeted in polyclonal human sera broad enough, to ensure that no single mutation should allow complete escape from neutralisation in the majority of individuals 41 .

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 28, 2021. ; https://doi.org/10.1101/2021.06.21.21259010 doi: medRxiv preprint Likewise, the wide array of HLA across the population 16 and the broad range of epitopes responded to in COVID-19 patients 18 combine to make it unlikely that SARS-CoV-2 will completely escape from T-cell surveillance in the near future. During the early stages of the pandemic, it seems likely that alterations in SARS-CoV-2 Spike that enhance binding to ACE2 or reduce the capability of vaccine-induced, or natural, antibodies to neutralise the virus will take precedence due to their potential relevance in all transmission events. This 'shrouding effect' may well have occurred in the UK SARS-CoV-2 epidemic, although this is difficult to fully assess for two reasons. First, the B.1.1.7 lineage has been shown to have a significantly higher transmissibility compared to B.1.177 (and other circulating lineages occurring in the Autumn and Winter of 2020) 42 , and so while P272L may have conferred some advantage, this is likely to have been insufficient to prevent the lineage being replaced by B.1.1.7. Also, it is important to note that the trajectory of P272L B.1.177 isolates was generally upwards going into the Winter of 2020, however the introduction of significant non-pharmaceutical interventions in response to B.1.1.7 resulted in a strong suppression of all SARS-CoV-2 lineages in the UK. It remains to be seen whether the P272L mutant will arise and show selection in other lineages. However, since this mutation has occurred in several variants already, we predict that it will appear in future variants. Our data suggests that the 269-277 epitope of Spike is one region that should be monitored and should feature in risk assessments by public health agencies going forward.

In summary, we demonstrate that SARS-CoV-2 can readily alter its Spike protein via a single amino acid substitution so that it is not recognised by CD8 T-cells targeting the most prevalent epitope in Spike restricted by the most common HLA-I across the population. While it is not possible to directly attribute the emergence and propagation of the Spike P272L SARS-CoV-2 variant in parts of the UK where HLA A*02 is frequently expressed to CD8 T-cell-mediated selection pressure, specific focussing of immune protection on a single protein (e.g. SARS-CoV-2 Spike favoured by all currently approved vaccines 43 ) is likely to enhance any tendency for escape at predominant T-cell epitopes like YLQPRTFLL. Our demonstration that mutations that evade immunodominant T-cell responses through population-frequent HLA can readily arise and disseminate, strongly suggests that it will be prudent to monitor such occurrences and to increase the breadth of next generation SARS-CoV-2 vaccines to incorporate other viral proteins.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 28, 2021. ; https://doi.org/10.1101/2021.06.21.21259010 doi: medRxiv preprint

To characterise the distribution of mutations in the Spike epitope between positions 269-277, we performed a set of analyses making use of high quality publicly available sequence data made available by GISAID and COG-UK as of 31 January 2021 44 

We recruited six participants who had received one dose of a SARS-CoV-2 vaccine as part of the National . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. weekly. Cells were regularly tested for mycoplasma contamination (MycoAlert TM , Lonza). 

Oligonucleotides were designed to substitute the Proline to a Leucine at position 272. Non-overlapping sequences were designed so that the 5' end of the forward oligo (5' 

Peptide-HLA-A*02:01 tetramers were produced and used to stain cells as previously described 54 BioLegend) was used as described previously [55] [56] [57] . Following tetramer staining, T-cells were stained with LIVE/DEAD™ fixable violet dead stain (ThermoFisher Scientific), anti-CD3 and anti-CD8 antibody and analysed using flow cytometry as above. PBMCs were also stained with anti-CD14 (M5E2, BioLegend) and anti-CD19 (HIB19, BioLegend) pacific blue conjugated antibodies to create a 'dump' channel for dead cells.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 28, 2021. overnight at room temperature. The following day, cells were stained with anti-HLA A2 antibody (clone BB7.2, BioLegend) and incubated at 37°C for 1 h. Cells were then washed in PBS and stained with LIVE/DEAD™ fixable violet dead stain (ThermoFisher Scientific) and analysed using flow cytometry.

TCR sequencing was performed as previously described 58 . RNA extraction was carried out using the RNEasy Micro kit (Qiagen, Hilden, Germany). cDNA was synthesized using the 5′/3′ SMARTer kit (Takara Bio, Paris, France). The SMARTer approach used a Murine Moloney Leukaemia Virus (MMLV) reverse transcriptase, a 3′ oligo-dT primer and a 5′ oligonucleotide to generate cDNA templates flanked by a known, universal anchor sequence at the 5′. PCR was then set up using a single primer pair consisting of TRAC or TRBC-specific reverse primer and an anchor-specific forward primer (Takara Bio, Paris, France). All samples were used for the following PCR reaction: 2.5 μL template cDNA, 0. 5 μL High Fidelity Phusion Taq polymerase, 10 μL 5X Phusion buffer, 0.5 μL DMSO, 1 μL dNTP Mix (10 mM each) (all from ThermoFisher Scientific, UK), 1 μL of TRAC or TRBC-specific primer (10 μM), 5 μL of 10X anchor-specific UPM primer (Takara Bio, Paris, France), and nuclease-free water for a final reaction Sequence analysis was performed using MiXCR software (v3.0.7) 59 . Public TCR clonotypes were identified using the VDJdb database 60 .

Unless stated otherwise, all data were displayed using GraphPad Prism software. TCR V-J usage plots were generated using VDJ tools 61 .

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

The authors declare no competing interests in regard to this work . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 28, 2021. ; https://doi.org/10.1101/2021.06.21.21259010 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 28, 2021. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 28, 2021. ; https://doi.org/10.1101/2021.06.21.21259010 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Adaptive immunity to SARS-CoV-2 and COVID-19

Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity

Two X-linked agammaglobulinemia patients develop pneumonia as COVID-19 manifestation but recover

Anti-CD20 and COVID-19 in multiple sclerosis and related disorders: A case series of 60 patients from

COVID-19 in a MS patient treated with ocrelizumab: does immunosuppression have a protective role

B-cell depleting therapies may affect susceptibility to acute respiratory illness among patients with multiple sclerosis during the early COVID-19 epidemic in Iran

Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild COVID-19

SARS-CoV-2-derived peptides define heterologous and COVID-19-induced T cell recognition

Characterization of pre-existing and induced SARS-CoV-2-specific CD8(+) T cells

SARS-CoV-2 Epitopes Are Recognized by a Public and Diverse Repertoire of Human T Cell Receptors

REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19

A Neutralizing Monoclonal Antibody for Hospitalized Patients with Covid-19

Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19

Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses

Correlates of protection against SARS-CoV-2 in rhesus macaques

Allele frequency net database (AFND) 2020 update: gold-standard data classification, open access genotype data and new query tools

SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals

Unbiased Screens Show CD8(+) T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein

Establishment and lineage dynamics of the SARS-CoV-2 epidemic in the UK

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

Emergence and spread of a SARS-CoV-2 variant through Europe in the summer of 2020. medRxiv

Safety and immunogenicity of ChAdOx1 nCoV-19 vaccine administered in a prime-boost regimen in young and old adults (COV002): a single-blind, randomised, controlled, phase 2/3 trial

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

CD8(+) T cells in HIV control, cure and prevention

Transmission and accumulation of CTL escape variants drive negative associations between HIV polymorphisms and HLA

Positive selection of HIV-1 cytotoxic T lymphocyte escape variants during primary infection

The influence of antigenic variation on cytotoxic T lymphocyte responses in HIV-1 infection

Spike mutation D614G alters SARS-CoV-2 fitness

SARS-CoV-2 variants, spike mutations and immune escape

Negligible impact of SARS-CoV-2 variants on CD4 (+) and CD8 (+) T cell reactivity in COVID-19 exposed donors and vaccinees. bioRxiv

SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8(+) T cell responses

The shaping of modern human immune systems by multiregional admixture with archaic humans

Structural and biophysical determinants of alphabeta T-cell antigen recognition

CTLs are targeted to kill beta cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

A single autoimmune T cell receptor recognizes more than a million different peptides

Structural basis for the killing of human beta cells by CD8(+) T cells in type 1 diabetes

Computational design and crystal structure of an enhanced affinity mutant human CD8 alphaalpha coreceptor

The effect of spike mutations on SARS-CoV-2 neutralization

Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England

SARS-CoV-2 vaccines in development

Dual Molecular Mechanisms Govern Escape at Immunodominant HLA A2-Restricted HIV Epitope

An integrated national scale SARS-CoV-2 genomic surveillance network

Reconstructing ancestral character states under Wagner parsimony

Dating of the human-ape splitting by a molecular clock of mitochondrial DNA

Improving the Ultrafast Bootstrap Approximation

ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data

More tricks with tetramers: a practical guide to staining T cells with peptide-MHC multimers

Tricks with tetramers: how to get the most from multimeric peptide-MHC

T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations

Optimised peptide-MHC multimer protocols for detection and isolation of autoimmune T-cells

Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers

Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs

Peptide Super-Agonist Enhances T-Cell Responses to Melanoma

MiXCR: software for comprehensive adaptive immunity profiling

VDJdb: a curated database of T-cell receptor sequences with known antigen specificity

VDJtools: Unifying Post-analysis of T Cell Receptor Repertoires