key: cord-0748865-rxeat3zy
authors: Lang-Meli, Julia; Luxenburger, Hendrik; Wild, Katharina; Karl, Vivien; Oberhardt, Valerie; Salimi Alizei, Elahe; Graeser, Anne; Reinscheid, Matthias; Roehlen, Natascha; Reeg, David B.; Giese, Sebastian; Ciminski, Kevin; Götz, Veronika; August, Dietrich; Rieg, Siegbert; Waller, Cornelius F.; Wengenmayer, Tobias; Staudacher, Dawid; Huzly, Daniela; Bengsch, Bertram; Kochs, Georg; Schwemmle, Martin; Emmerich, Florian; Boettler, Tobias; Thimme, Robert; Hofmann, Maike; Neumann-Haefelin, Christoph
title: SARS-CoV-2-specific T-cell epitope repertoire in convalescent and mRNA-vaccinated individuals
date: 2022-04-28
journal: Nat Microbiol
DOI: 10.1038/s41564-022-01106-y
sha: 3e5b567f2311b353e72ae5e005368a8e87bb8a14
doc_id: 748865
cord_uid: rxeat3zy

Continuously emerging variants of concern (VOCs) sustain the SARS-CoV-2 pandemic. The SARS-CoV-2 Omicron/B.1.1.529 VOC harbours multiple mutations in the spike protein associated with high infectivity and efficient evasion from humoral immunity induced by previous infection or vaccination. By performing in-depth comparisons of the SARS-CoV-2-specific T-cell epitope repertoire after infection and messenger RNA vaccination, we demonstrate that spike-derived epitopes were not dominantly targeted in convalescent individuals compared to non-spike epitopes. In vaccinees, however, we detected a broader spike-specific T-cell response compared to convalescent individuals. Booster vaccination increased the breadth of the spike-specific T-cell response in convalescent individuals but not in vaccinees with complete initial vaccination. In convalescent individuals and vaccinees, the targeted T-cell epitopes were broadly conserved between wild-type SARS-CoV-2 variant B and Omicron/B.1.1.529. Hence, our data emphasize the relevance of vaccine-induced spike-specific CD8(+) T-cell responses in combating VOCs including Omicron/B.1.1.529 and support the benefit of boosting convalescent individuals with mRNA vaccines.

We first mapped the overall SARS-CoV-2-specific CD8 + T-cell response against a set of 43 previously described immunodominant SARS-CoV-2-specific CD8 + T-cell epitopes (Supplementary Table 2 ) restricted by common human leukocyte antigen (HLA) class I alleles [1] [2] [3] [4] [5] [6] in epitope-specific T-cell cultures followed by interferon-γ (IFN-γ) staining. In agreement with their association with a mild COVID-19 course, CD8 + T-cell responses in convalescent individuals targeted most epitopes distributed over all viral proteins, with spike-specific epitopes not being dominant (Fig. 1a , left column). In vaccinees, in contrast and as expected, CD8 + T-cell responses were predominantly directed against spike epitopes (Fig. 1a , right column and Extended Data Fig. 1a ). Few CD8 + T-cell responses targeted non-spike epitopes, with the HLA-B*07/N 105-113 epitope being the main target. For this epitope, cross-recognition by T cells against common cold coronaviruses has been suggested previously [8] [9] [10] . Individual spike-specific CD8 + T-cell epitopes were more often targeted in vaccinees compared to convalescent individuals; the spike-specific CD8 + T-cell repertoire also appeared broader in vaccinees compared to convalescent individuals. When we compared the corresponding viral sequences between wild-type (WT) SARS-CoV-2 variant B and Omicron/B.1.1.529, only a single tested optimal CD8 + T-cell epitope was affected by viral variation in subvariants BA. 1 and BA.2 (Fig. 1a, red and Supplementary 

Spike-specific T-cell epitope repertoire. To analyse the spike-specific CD8 + T-cell response in convalescent individuals versus vaccinees in more detail, we assessed these responses using overlapping peptides (OLPs) spanning the whole spike protein. For all positive responses, we evaluated the OLP for the described optimal epitopes restricted by the HLA class I alleles expressed by the respective individual. If no matching optimal epitopes were previously described, we performed an in silico analysis to predict the most likely HLA class I restriction and optimal epitope. Using this comprehensive approach, we identified an overall substantially broader repertoire of spike-specific CD8 + T-cell responses in vaccinees ( Fig. 1b (second panel) and Extended Data Fig. 1b (second panel)) compared to convalescent individuals (Fig. 1b (first panel) and Extended Data Fig. 1b (first panel) ). Indeed, in convalescent individuals, no HLA class I allele restricted more than two spike-specific CD8 + T-cell epitopes, while several HLA class I alleles restricted five or more spike-specific CD8 + T-cell epitopes in vaccinees. In addition, we detected more spike-specific CD8 + T-cell responses per individual in vaccinees compared to convalescent individuals (Extended Data Fig. 1c) . Hence, the increased breadth of the spike-specific CD8 + T-cell response in vaccinees was evident at the individual and population levels.

We also analysed the CD4 + T-cell response using spike-spanning OLPs as described above. In contrast to the CD8 + T-cell response, the spike-specific CD4 + T-cell response showed a more limited repertoire of targeted epitopes after vaccination compared to infection (Fig. 1b, bottom) . In particular, fewer spike-specific CD4 + T-cell epitopes were restricted by single HLA class II alleles (Fig. 1b, bottom) and fewer CD4 + T-cell responses were detectable per individual (Extended Data Fig. 1d ) in vaccinees compared to convalescent individuals. Therefore, the spike-specific CD4 + T-cell repertoire was limited with regard to the individual-and population-based DRB1*01 (n = 3) DRB1*03 (n = 2) DRB1*04 (n = 2) DRB1*11 (n = 3) DRB1*13 (n = 1) DRB1*15 (n = 7) DQB1*02 (n = 3) All patients (n = 16) DRB1*01 (n = 1) DRB1*03 (n = 3) DRB1*04 (n = 6) DRB1*11 (n = 7) DRB1*13 (n = 3) DRB1*15 (n = 5) DQB1*02 (n = 6) All patients (n = 

CD4 + T-cell response in vaccinees. Of note, the CD4 + and CD8 + spike-specific T-cell epitope repertoire was relatively stable over time in both vaccinees and convalescent individuals (Extended Data Fig. 2 ). Importantly, the fewer targeted spike-specific CD4 + T-cell epitopes in vaccinees exhibited high conservation between WT variant B and Omicron/B.1.1.529 SARS-CoV-2 (subvariants BA.1 and BA.2) as it is also the case for most targeted epitopes in convalescent individuals, similar to spike-specific CD8 + T-cell epitopes ( Table 3 ). While the differential responsiveness of CD4 + versus CD8 + T cells to peptide stimulation may limit comparison between the two T-cell subsets, mRNA vaccination appears to particularly broaden and thus increase a CD8 + T-cell response that targets conserved spike epitopes (Extended Data Fig. 1e ).

Booster effect on spike-specific T-cell epitope repertoire. Next, to assess the effect of boosting vaccination-or infection-induced T-cell responses by mRNA vaccination on the spike-specific CD8 + T-cell repertoire, we again used overlapping spike peptides to map spike-specific CD8 + and CD4 + T-cell responses in longitudinally followed vaccinees getting their third vaccine dose (Pfizer/BioNTech mRNA vaccine; n = 7; Supplementary Table 1 ) and convalescent individuals who received an mRNA booster vaccination (n = 3; Supplementary Table 1 ). After the third mRNA vaccination, we observed a similarly broad and spike cross-recognizing CD8 + T-cell response and similarly limited but still spike cross-recognizing CD4 + T-cell response compared to the completed initial immunization with two vaccine doses ( 

To investigate whether the observed broader spike-specific CD8 + T-cell repertoire after mRNA vaccination may also be beneficial for potentially emerging future SARS-CoV-2 VOCs beyond Omicron/B.1.1.529, we analysed the T-cell response targeting highly conserved selective sweep regions in SARS-CoV-2 that were identified by Kang et al. 11 in convalescent individuals versus vaccinees. Selective sweep regions mediate per definition an evolutionary advantage; therefore, it is very likely that newly emerging SARS-CoV-2 VOCs also harbour high conservation within these regions. Four different selective sweep regions have so far been described in the spike protein of SARS-CoV-2 (ref. 11 ) that also exhibit, as expected, a high degree of amino acid homology among the already evolved SARS-CoV-2 VOCs. For example, complete homology is present in VOC Delta, only one point mutation is present in VOC 

Alpha or Beta, two mutations in VOC Gamma, seven point mutations in VOC Omicron/B. Fig. 4a,b) . Importantly, compared to convalescent individuals more vaccinees showed spike-specific CD8 + T-cell responses targeting epitopes within the highly conserved selective sweep regions indicating a spike-specific CD8 + T-cell response with focused targeting of highly conserved regions after vaccination (Fig. 2c) . A similarly focused spike-specific CD4 + T-cell response was not evident after vaccination (Fig. 2d) . Hence, a broadly spike cross-recognizing CD8 + T-cell response is induced after mRNA vaccination that may be also reactive towards emerging SARS-CoV-2 VOCs in the future beyond Omicron/B.1.1.529.

In conclusion, our data indicate that (1) convalescent individuals target a variety of SARS-CoV-2-specific CD8 + T-cell epitopes over the complete SARS-CoV-2 proteome with spike-specific CD8 + T-cell responses being non-dominant; (2) in contrast to the CD4 + T-cell response, CD8 + T-cell responses in vaccinees are focused on a broader repertoire of highly conserved spike-specific CD8 + T-cell epitopes leading to an increased cross-recognizing potential; (3) 

Patients were recruited at the Freiburg University Medical Center between August 2019 and January 2022. Written informed consent was obtained from all participants. The study was conducted according to federal guidelines and local ethics committee regulations and the Declaration of Helsinki (first revision). The study was approved by the ethics committee of the University of Freiburg (nos. 21-1135 and 21-1372). Table 1 ). All patients were confirmed to have tested positive for SARS-CoV-2 using PCR with reverse transcription from an upper respiratory tract (nose and throat) swab tested at an accredited laboratory. The degree of COVID-19 severity was identified according to recommendations from the World Health Organization. Moreover, 16 individuals (all testing negative for anti-N-IgGs, (Mikrogen)) were screened 2-4 weeks after the second dose of mRNA vaccination (Pfizer/BioNTech BNT162) and 7 of the same individuals 2-4 weeks after the third mRNA vaccination (Pfizer/ BioNTech BNT162). Three additional individuals were analysed who had a mild course of COVID-19 and were vaccinated once with the mRNA vaccine (Pfizer/ BioNTech BNT162). The median age of vaccinated donors (n = 16) was 36 years; the median age of donors with a history of natural SARS-CoV-2 infection (n = 19) was also 36 years. The sex ratio of vaccinated donors was M/F 10/6; in donors with a history of natural SARS-CoV-2 infection, it was M/F 11/8. Participants did not receive any compensation for participating in the study.

Peripheral blood mononuclear cell isolation. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples anticoagulated with density gradient centrifugation (Pancoll Separation Medium; PAN Biotech) and subsequently stored at −80 °C. Frozen PBMCs were thawed in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin and 1.5% HEPES buffer 1 mol l −1 (complete medium; all additives from Thermo Fisher Scientific) until further usage.

A total of 180 OLPs spanning the SARS-CoV-2 spike sequence (GenBank accession no. MN908947.3) were synthesized as 18-mer sliding by 7 amino acids and thus overlapping by 11 amino acids with a free amine NH 2 terminus and a free acid COOH terminus with standard Fmoc chemistry and a purity of >70% (Genaxxon Bioscience). Since two OLPs contained the two amino acid residues with sequence modification in the 'stabilized' mRNA spike vaccine (K986P, V987P), these peptide variants were also synthesized and used, resulting in a total of 182 OLPs. In addition, 60 predescribed SARS-CoV-2-specific optimal CD8 + T-cell epitopes were synthesized. In the figures and Supplementary Table 2 , we display only those 43 optimal epitopes that were tested in at least 3 HLA-matched individuals of each cohort (convalescent and vaccinated).

In vitro expansion and intracellular IFN-γ staining using overlapping peptides or predescribed optimal CD8 + T-cell epitopes. In vitro expansion with OLPs or optimal epitopes was performed as follows: 20% of the PBMCs were stimulated with a pool of all 182 SARS-CoV-2 spike OLPs or all 60 optimal epitopes (10 μg ml −1 ) for 1 h at 37 °C, washed and cocultured with the remaining PBMCs in RPMI medium supplemented with 20 U ml −1 recombinant interleukin-2 (IL-2). On day 10, intracellular IFN-γ staining was performed with pooled OLPs (45 pools with 4 OLPs each). Therefore, cells were restimulated with OLP pools (50 μM), dimethyl sulfoxide as negative control or phorbol 12-myristate 13-acetate and ionomycin as positive control in the presence of brefeldin A and IL-2. After 5 h of incubation at 37 °C, cells were stained for surface markers (CD8 + , CD4 + ; Via-Probe) and intracellular markers (IFN-γ). Subsequently, on days 12-14 single OLPs of positive pools and HLA-matched optimal CD8 + T-cell epitopes were tested by intracellular cytokine staining. Viral amino acid sequences of positive individual OLPs were analysed for predescribed minimal epitopes 3, 5, 6, [12] [13] [14] or the best HLA-matched predicted candidate using the Immune Epitope Database (IEDB, https://www.iedb.org/; we used two prediction algorithms, ANN 4.0 and NetMHCpan EL 4.123, for 8-mer, 9-mer and 10-mer peptides with a half maximal inhibitory concentration of <500 nM). The major histocompatibility complex class I (MHC class I) binding predictions were made using the IEDB analysis resource ANN aka NetMHC v4.0 tool or the IEDB analysis resource NetMHCpan v.4.0 tool. The MHC class II binding predictions were made using the IEDB recommended 2.22 analysis resource consensus tool (smm/nn/sturniolo). 11 . The SARS-CoV-2 VOC amino acid sequences were aligned to selective sweep regions and peptides were mapped to the spike protein to identify peptides that localize to the selective sweep regions.

Statistical analysis was performed with Prism 9 (GraphPad Software). Statistical significance was assessed by two-tailed Mann-Whitney U-test, two-sided Wilcoxon matched-pairs signed-rank test and Spearman correlation; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Sequences of the tested epitopes, sequences of the spike overlapping peptides used as well as a list summarizing all CD8 + and CD4 + T-cell responses to overlapping peptides are available at the community repository Open Science Framework (https://www.cos.io/products/osf) and can be found via https://osf.io/zbk6q/. All requests for additional raw and analysed data and materials will be promptly Articles NATuRE MICROBIOlOgy reviewed by the University of Freiburg Center for Technology Transfer to verify if the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in the paper were generated as part of clinical examination and may be subject to patient confidentiality. Any data and materials that can be shared will be released via a material transfer agreement. Source data are provided with this paper.

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Sequences of tested epitopes, sequences of used spike overlapping peptides as well as a list summarizing all CD8+ and CD4+ T cell responses to overlapping peptides are available in the community repository "Open Science Framework" and can be found via https://osf.io/zbk6q/. Additional raw and analyzed data and materials include pseudonymized patient data that may be subject to confidentiality. Requests of this data are promptly reviewed by the University of Freiburg Center for Technology Transfer to verify if the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in the

Unbiased screens show CD8 + T cells of COVID-19 patients recognize shared epitopes in SARS-CoV-2 that largely reside outside the spike protein

Identification of cross-reactive CD8 + T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants

SARS-CoV-2-specific CD8 + T cell responses in convalescent COVID-19 individuals

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

SARS-CoV-2 genome-wide T cell epitope mapping reveals immunodominance and substantial CD8 + T cell activation in COVID-19 patients

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

SARS-CoV-2 human T cell epitopes: adaptive immune response against COVID-19

CD8 + T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope cross-react with selective seasonal coronaviruses

CD8 + T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity

An immunodominant NP 105-113 -B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease

A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation

A sequence homology and bioinformatic approach can predict candidate targets for immune responses to SARS-CoV-2

SARS-CoV-2 epitopes are recognized by a public and diverse repertoire of human T cell receptors

Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases

Any data and materials that can be shared will be released via a Material Transfer Agreement. Materials and Correspondence: Christoph Neumann-Haefelin: christoph.neumann-haefelin@uniklinik-freiburg.de Maike Hofmann: maike.hofmann@uniklinik-freiburg

Patients were recruited and patient material was banked at the University Hospital Freiburg; inclusion criteria were: (1) 16 individuals that received a prime and boost vaccination with the mRNA vaccine bnt162b2/Comirnaty, (2) 19 acutely infected and convalescent individuals following a mild course of SARS-CoV-2 infection, SARS-CoV-2 infection was confirmed by positive PCR testing from oropharyngeal swab and/or SARS-CoV-2 spike IgG positive antibody testing. No sample size calculations were performed. 16 vaccinated health care workers gave informed consent and were available to donate blood samples. Therefore

Flow cytometry analysis: 19 convalescent individuals following a mild course of SARS-CoV-2 infection were analyzed up to 7 months after infection (3 outliers up to 12 months). Moreover, 16 individuals were screened 2-4 weeks after first mRNA boost vaccination (Pfizer/BioNTech, bnt162b2) and 7 of the same individuals 2-4 weeks after second boost vaccination (Pfizer/BioNTech, bnt162b2) Three individuals were analyzed who had a mild course of SARS-CoV-2 infection and were vaccinated once with mRNA vaccine

Randomization Vaccinated donors and donors with a history of natural SARS-CoV-2 infection were selected based on availability and HLA-typing. The covariates age and gender are well-documented: Median age of vaccinated donors was 36 years, donors with a history of natural SARS-CoV-2 infection was 36 years. The gender ratio of vaccinated donors was m/f: 10/6, donors with a history of natural SARS

Blinding Non-objective parameters were not included in the study design and standardized analyses were applied. Thus, blinding was not considered necessary since biased analysis can be excluded

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SK-1, 1:200), BD, Cat#345775 anti-CD4-efluor450 (RPA-T4, 1:250), eBioscience

Standardized analysis in different cohorts, antibody titration on PBMCs including unstained controls, comparisons of different antibody clones and conjugates and validated by publications: CD4: antibody titration on PBMCs; control clones SK3; using B cells as negative control CD8: antibody titration on PBMCs; control clone GHI/75; using B cells as negative control IFNɣ, clone 25723.11: antibody titration on PBMCs; control clone 4S.B3; validated with respect to differential expression of activated and non-activated T cell subpopulations Viability Dye was titrated on PBMCs

The gender ratio of vaccinated donors was m/f: 10/6, donors with a history of natural SARS-CoV-2 infection was m/f: 11/8. Recruitment Vaccinated donors as well as SARS-CoV-2-infected and SARS-CoV-2-convalescent patients were recruited at the University Hospital Freiburg; self-selection bias or other biases can be excluded since several people were included in the recruitment. Samples were banked and retrospectively selected

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Cryopreserved isolated human PBMCs were thawed and prepared for flow cytometry or in vitro expansion described in the of SARS-CoV-2-specific T cells are low

Gating strategy Lymphocytes gated on FSC-A and SSC-A, Doublet exclusion on FSC-H and FSC-W, Exclusion of dead cells

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We thank all donors for participating in the current study and the FREEZE-biobank-Center for Biobanking of the Freiburg University Medical Center and Medical Faculty for support. This study was supported by grants from the German Federal Ministry of Education and Research (no. 01KI2077 to G. 

Articles NATuRE MICROBIOlOgy Extended Data Fig. 1 | CD8 + and CD4 + T cell responses targeting spike-specific epitopes. (a) Intensity (sum interferon-γ production of all CD8 + T cell responses per patient) to optimal CD8 + T cell epitopes. (b) Number and location of spike-specific CD8 + and CD4 + T cell responses to overlapping peptides (OLP) in SARS-CoV-2 convalescents and vaccinees after two doses of Pfizer/BioNTech mRNA vaccine. Epitopes with amino acid sequence variations in omicron BA.2 are marked in blue. Number of CD8 + (c,e) and CD4 + (d,e) T cell responses targeting spike-specific epitopes per individual in convalescents (n = 19) and vaccinees after two doses of Pfizer/BioNTech mRNA vaccine (n = 16). Numbers of tested individuals (per HLA allotype and in total) and % of total T cell responses targeting variant epitopes are indicated. Median and range are depicted, statistical analysis was performed with two-sided Mann-Whitney-Test. NTD:N-Terminal Domain; RBD: receptor-binding domain; FCS: furin cleavage site; wt: epitope conserved between ancestral and omicron SARS-CoV-2; mut: epitope mutated in omicron compared to ancestral SARS-CoV-2.