The impact of viral mutations on recognition by SARS-CoV-2 specific T cells

Summary We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.


INTRODUCTION
Evolution of SARS-CoV-2 can lead to evasion from adaptive immunity generated following infection and vaccination. Much focus has been on humoral immunity and spike protein mutations that impair the effectiveness of neutralizing monoclonal antibodies and polyclonal sera. T cells specific to conserved proteins play a significant protective role in respiratory viral infections such as influenza, particularly in broad heterosubtypic immunity (Hayward et al., 2015). T cell responses following SARS-CoV-2 infection are directed against targets across the genome and may play a role in favorable outcomes during acute infection and in immunosuppressed hosts with deficient B cell immunity (Huang et al., 2021;Peng et al., 2020;Tan et al., 2021). Although CD8+ T cells may not provide sterilizing immunity, they can protect against severe disease and limit risk of transmission, with a potentially more important role in the setting of antibody escape.
Little is known about the potential for SARS-CoV-2 mutations to impact T cell recognition. Escape from antigen-specific CD8+ T cells has been studied extensively in HIV-1 infection, where rapid intra-host evolution renders T cell responses ineffective within weeks of acute infection (Goonetilleke et al., 2009). Although these escape variants play an important role in the dynamics of chronic viral infections, the opportunities for T cell escape in acute respiratory viral infections are fewer and consequences are different. Nevertheless, several cytotoxic T-lymphocyte (CTL) escape variants have been described in influenza, such as the R384G substitution in the HLA B*08:01-restricted nucleoprotein 380-388 and B*27:05-restricted nucleoprotein 383-391 epitopes (Voeten et al., 2000). Long-term adaptation of influenza A/H3N2 has been demonstrated, with the loss of one CTL epitope every 3 years since its emergence in 1968 (Woolthuis et al., 2016).

Potential mechanisms of loss of T cell recognition
T cell escape can occur via interrupting several mechanisms: antigen processing, binding of major histocompatibility complex (MHC) to peptide, or T cell receptor (TCR) recognition of the MHC-peptide complex. Although we did not explicitly establish which of these was responsible in each case, it is likely that any partial impairment of T cell recognition is due to reduced TCR binding to MHC-peptide. Reasons for complete escape are more difficult to predict. As the anchor residues of peptide-MHC binding in A*03:01/A*11:01-restricted KTFPPTEPK 361-369 are at positions 2 and 9, T362I (position 2) may impair of Oxford, Oxford OX3 7BN, UK  (K-N) Recognition of wild-type (black) and mutant (red) peptide titrations by a polyclonal CD8+ T cell line specific for the HLA*A01:01-restricted ORF1a epitope TTDPSFLGRY 1637-1646 , using intra-cellular cytokine staining for interferon-gamma (IFNg, K), tumor necrosis factor (TNFa, L), and the degranulation factor CD107a (M), and a killing assay (N). Similar findings were seen with a T cell line generated from another donor ( Figure S4). iScience Article explain the impact of the Q213K (position 7) variant. In keeping with this, we see no significant impact of these mutations on the predicted binding affinities of epitope to MHC (Table S4). Despite a modest 4-fold decrease in predicted IC 50 for Q213K compared with wild type, FTSDYYKLY 207-215 is still a strong binder to A*01:01.
Ex vivo IFN-g ELISpots in two A*03:01 and two B*27:05 convalescent donors confirmed loss of responses to variant peptides seen with T cell lines specific to KTFPPTEPK 361-369 and QRNAPRITF 9-17 ( Figure S5). Thus, our findings using T cell lines are representative of the circulating T cell response to these epitopes and of physiological relevance. Of interest, one A*03:01 donor had low-level responses to P365S and T362I, suggesting that subdominant responses via alternative TCR are possible.
T cell receptor diversity in CD8+ T cell lines with loss of epitope recognition due to amino acid variants TCR sequencing of polyclonal CD8+ T cell lines specific for the FTSDYYKLY 207-215 epitope and B*27:05restricted QRNAPRITF 9-17 epitope was undertaken to explore whether the complete loss of T cell recognition observed was dependent on specific TCRs. A diverse range of TCRs was found in FTSDYYKLY 207-215 T cell lines from four donors (Figure 2), demonstrating that Q213K results in escape from several TCRs. Similar findings were seen in TCR data from a B*27:05-restricted QRNAPRITF 9-17 T cell line from one donor ( Figure S6). It is worth noting that our data are biased by using T cell lines generated from donors recruited early in the pandemic and therefore likely infected with ''wild-type'' viruses (i.e., lineage B or B.1 viruses) (Peng et al., 2020). Although variants that impair antigen processing or MHC-peptide binding result in irreversible loss of T cell recognition, CTLs with new TCR repertoires can overcome TCR-mediated escape variants, as has been described in HIV-1 infection (Ladell et al., 2013).

Frequency of epitope variants over time and appearance in global SARS-CoV-2 phylogeny
Many variants examined in our study were at relatively low frequency and stable prevalence at the time of writing, other than P365S in KTFPPTEPK 361-369, P1640L in TTDPSFLGRY 1637-1646 , and variants affecting the proline at position 13 in QRNAPRITF 9-17 (Table 1 and Figure 3A). We explored whether variants that result in loss of T cell recognition appeared as homoplasies in the phylogeny of SARS-CoV-2 suggestive of repeated independent selection, or whether global frequency is due mainly to the expansion of lineages after initial acquisition. Although in some cases variant frequency was dependent on a few successful lineages, P365S, Q213K, T362I, P13L, P13S, and P13T had arisen independently on several occasions including within the B.1.1.7 lineage ( Figures 3B, 3C, 3D, 3E, S7A, and S7B). It is important to emphasize that this homoplasy and our functional data do not prove selection due to T cell escape, which would require demonstration of intra-host evolution. The positions we find important for T cell recognition may be under selective pressure for reasons other than T cell immunity. A recent study has documented intra-host evolution of minority variants within A*02:01 and B*40:01 CD8+ epitopes that impair T cell recognition, although not all epitopes are dominant and very few of the variants studied were represented among the global circulating viruses (Agerer et al., 2021).

Conclusions
There is unlikely to be adequate population immunity at present to see global changes due to T cell selection akin to what has been seen in adaptation of H3N2 influenza over time (Woolthuis et al., 2016). Furthermore, polymorphism in HLA genes restricts the selective advantage of escape within one particular epitope to a relatively small proportion of the population, given the breadth in T cell responses we and others have shown. The polyclonal T cell response in a given individual is therefore unlikely to be diminished significantly by mutations present in any one circulating variant, unlike the potential impact on neutralizing antibody responses seen with mutations in the spike protein. Nevertheless, responses to many of the CTL epitopes we have studied are dominant within HLA-matched individuals across many cohorts (Peng et al., 2020). As A*03:01, A*11:01, and A*01:01 are common HLA alleles globally, loss of T cell responses to dominant epitopes such as KTFPPTEPK 361-369 and FTSDYYQLY 207-215 may be significant. Substitution of three different amino acid variants at nucleocapsid position 13 within the B*27:05-restricted QRNAP-RITF 9-17 epitope is also striking and suggests significant positive selective pressure at this site. Successful maintenance of these substitutions within some lineages also suggest that this is a position where such amino acid changes are tolerated with limited impact on the virus life cycle. A single dominant, protective B*27:05-restricted epitope has been described in HIV-1 infection, with T cell escape associated with progression to AIDS. T cell escape from a B*27:05-restricted influenza A epitope (nucleoprotein 383-391 ) has also been observed (Voeten et al., 2000). iScience Article A significant increase in sites under diversifying positive selective pressure was observed around November 2020, most notably in ORF3a, N, and S (Martin et al., 2021). As vaccine and naturally acquired population immunity increase further, the frequency of variants we have described should be monitored globally, as well as further changes arising within all immunodominant T cell epitopes. We have recently incorporated the ability to identify spike T cell epitope variants in real-time sequence data into the COG-UK mutation explorer dashboard (http://sars2.cvr.gla.ac.uk/cog-uk/). Non-spike T cell immune responses will also become increasingly important to vaccine-induced immunity as inactivated whole-virus vaccines are rolled out. Our findings demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.

Limitations of the study
We have chosen to focus on key SARS-CoV-2 immunodominant epitopes characterized early in the pandemic, and further epitopes have been identified since. It would be important to assess mutations of increasing prevalence within all immunodominant epitopes in the future to provide a comprehensive overview of potential SARS-CoV-2 T cell escape. Although our findings suggest that reduced T cell receptor binding to MHCepitope complex is likely responsible for the most striking impact of mutations on T cell responses we describe, this needs to be demonstrated experimentally. Finally, further studies are required to demonstrate the occurrence of T cell escape within individuals and establish how frequently this occurs. Given the potential for immune escape in prolonged or chronic SARS-CoV-2 infections that could give rise to new variants of concern, a focus on infections in immunocompromised individuals would be important.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  . Continued prefix of SARS-CoV-2 protein (S, spike; N, nucleocapsid), followed by wild-type amino acid, position within protein, and variant amino acid. Epiweek relates to week number since start of global SARS-CoV-2 pandemic was declared on March 11, 2020. (B-E) Phylogenies representing global SARS-CoV-2 genomes depicting the presence of epitopes variants impacting T cell responses. In each case, phylogenies represent all available variant sequences (red tips), along with a selection of non-variant sequences, which were subsampled for visualization purposes. The bar to the right of each phylogeny is annotated by main ancestral lineages only and not each individual PANGO lineage that viruses belong to. The grapevine pipeline (https://github.com/COG-UK/grapevine) was used for generating the phylogeny based on all data available on GISAID and COG-UK up until August 4, 2021.

DECLARATION OF INTERESTS
The authors have no competing interests to declare.  Figure 3. The analysis folder contains an R code and two sub-folders: input_data and output. Mutation counts obtained from COG-UK global metadata are provided in the input_data folder.
d Code and data used for plotting the global phylogenies representation are provided in Data S3 Tree visualisation, related to Figure 3. The analysis folder contains an R code and two sub-folders: input_data and output. COG-UK metadata and lists of sequences with our mutations of interest are provided in the input_data sub-folder.
d Any additional information required to re-analyze the data reported in this paper is available from the lead contact upon request.
The graphical abstract was created with Biorender.

Isolation of peripheral blood mononuclear cells
Blood from participants was collected in EDTA anticoagulant tubes, layered onto an equal volume of lymphoprep (Stemcell) in a falcon tube at room temperature, then centrifuged at 800 3 g for 20 min at room temperature with the brake off. Peripheral Blood Mononuclear Cells (PBMCs) were aspirated at the plasma:lymphoprep interface and washed twice with RPMI medium.
Isolated PBMCs were cultured in RPMI (GIBCO) with 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, 0.1 mg/mL streptomycin at 37 C in 5% carbon dioxide (CO 2 ) and used to generate polyclonal T cell lines and lymphoblastoid cell lines.
Generation of polyclonal T cell lines 1-2 million PBMCs were seeded per well in a 24-well plate in RPMI (GIBCO) with 10% (v/v) human serum, 10% (v/v) IL-2/TCGF (Helvetica healthcare), 5 ng/mL IL-7 (Biotechne). Peptides were added at 10 ug/mL. Cells were fed on day 4 or earlier if media the turned yellow and then every 4 days. On day 14, antigen-specific CD8+ T-cells were sorted with pentamer staining and CD4+ T-cells were sorted using tetramer staining. Subsequently, sorted cells were plated in a 96-well U-bottom plate with 100-1000 cells/well and fed with 200,000 irradiated allogeneic PBMCs with 50ug/mL phytohemagglutinin (PHA).