key: cord-0785822-hb9b8seu
authors: Titov, A.; Shaykhutdinova, R.; Shcherbakova, O. V.; Serdyuk, Y. V.; Sheetikov, S. A.; Zornikova, K. V.; Maleeva, A. V.; Khmelevskaya, A.; Dianov, D. V.; Shakirova, N. T.; Malko, D. B.; Shkurnikov, M.; Nersisyan, S.; Tonevitsky, A.; Khamaganova, E. G.; Ershov, A. V.; Osipova, E. Y.; Nikolaev, R. V.; Pershin, D. E.; Vedmedskia, V. A.; Maschan, M.; Ginanova, V. R.; Efimov, G. A.
title: Immunogenic epitope panel for accurate detection of non-cross-reactive T cell response to SARS-CoV-2
date: 2021-12-13
journal: nan
DOI: 10.1101/2021.12.12.21267518
sha: 5756ab51a2a9ab8d69c9ac22988ca02561b5c2a8
doc_id: 785822
cord_uid: hb9b8seu

The ongoing COVID-19 pandemic calls for more effective diagnostic tools, and T cell response assessment can serve as an independent indicator of prior COVID-19 exposure while also contributing to a more comprehensive characterization of SARS-CoV-2 immunity. In this study, we systematically assessed the immunogenicity of 118 epitopes with immune cells collected from multiple cohorts of vaccinated, convalescent, and healthy unexposed and SARS-CoV-2 exposed donors. We identified seventy-five immunogenic epitopes, 24 of which were immunodominant. We further confirmed HLA restriction for 49 epitopes, and described association with more than one HLA allele for 14 of these . After excluding two cross-reactive epitopes that generated a response in pre-pandemic samples, we were left with a 73-epitope set that offers excellent diagnostic specificity without losing sensitivity compared to full-length antigens, which evoked a robust cross-reactive response. We subsequently incorporated this set of epitopes into an in vitro diagnostic 'Corona-T-test' which achieved a diagnostic accuracy of 95% in a clinical trial. When applied to a cohort of asymptomatic seronegative individuals with a history of prolonged SARS-CoV-2 exposure, this test revealed a lack of specific T cell response combined with strong cross-reactivity to full-length antigens, indicating that abortive infection had occurred in these individuals.

The COVID-19 pandemic has posed a considerable challenge for healthcare systems worldwide, necessitating the rapid development of novel diagnostic tools. RT-PCR is the gold standard assay for confirming COVID-19 infection, while serology tests are commonly used for retrospective diagnosis, assessment of vaccination efficiency, and measuring the stability of immune protection over time. Nevertheless, estimating the actual rate of infection is complicated because many infections are asymptomatic, and up to 15% of patients do not develop a humoral immune response to infection [1] [2] [3] T cell response can offer an independent metric of SARS-CoV-2-specific immunity in the aftermath of either COVID-19 4-8 or vaccination [9] [10] [11] [12] [13] . It has been demonstrated that IgG titers strongly correlate with [14] [15] [16] and that antibodies can provide protection even in the absence of T cells both in animal models 17, 18 and in prospective human studies 16, 19 . Other studies have suggested that cellular immunity has a role in the context of suboptimal humoral response 16, 18, 19 or at the early stages after vaccination before seroconversion 20,21 .

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 It has also become clear that the humoral response gradually fades and may no longer be detectable six months post-infection 22, 23 or -vaccination 24 , whereas T cells persist long after exposure 23, 25, 26 . Indeed, T cell responses have remained detectable for up to 17 years after infection with SARS-CoV-1 27 .

However, the detection of SARS-CoV-2-specific T cell response is hindered by the relatively frequent occurrence of false-positive responses in non-SARS-CoV-2-exposed individuals due to cross-reactivity to other coronaviruses 4, 7, 28 . The role of this response remains controversial; some studies report that such low-affinity cross-reactive responses may contribute to a poor prognosis 29 , while others have demonstrated that pre-existing memory T cells rapidly respond upon vaccination 30 and that the expansion of cross-reactive T cells is associated with mild disease 31 and may explain asymptomatic infections 32 .

Nevertheless, it is important to distinguish between cross-reactive and COVID-19-specific T cell responses. To date, several kits for in vitro detection of T cell response have been proposed based on ELISpot/Fluorospot technology 33-36 , high-throughput sequencing (HTS)based detection of T cell receptor (TCR) sequences 37 and measuring cytokine production in whole blood 38 . Most of these exploit custom peptide sets that were bioinformatically selected to minimize cross-reactivity, but to the best of our knowledge, these were not experimentally validated on pre-pandemic samples.

Previous studies have predicted 39 and experimentally confirmed 37,40-42 numerous SARS-CoV-2 T cell epitopes. Some researchers have focused on the properties of individual epitopes, such as the diversity and repertoires of specific T cell receptors and structural aspects of epitope recognition 7,43-48 . Others have aimed at characterizing the response to sets of epitopes 26, [48] [49] [50] . Nevertheless, the employment of different assays hinders direct comparison, and the limited cohort size and number of epitopes tested per study have left essential questions pertaining to the immunodominance of individual epitopes-and for some epitopes, their HLA restriction-unresolved.

Understanding patterns of immunodominance of SARS-CoV-2 epitopes could guide future vaccine development. For example, ORF1ab-and ORF3a-derived epitopes seem to be more immunogenic than other components of the viral proteome-including the S glycoprotein-in individuals bearing HLA-A*01:01, which is common in the European population 37, 40, 49 . Moreover, although several studies have demonstrated that the total magnitude of CD8 + and CD4 + T cell response in people vaccinated with existing S proteinbased vaccines is on par with or even surpasses that of patients who have recovered from infection [9] [10] [11] 13 , it remains unclear whether the spectrum of recognized epitopes is the same . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 4 in both groups. Given that increased diversity of recognized epitopes is known to correlate with better outcomes in some other viral infections, such as with hepatitis B virus 51 , it is important to profile T cell immunity-including the landscape of recognized epitopes-in vaccinated individuals and patients after natural infection with SARS-CoV-2.

In the present study, we aimed to systematically characterize a pre-selected set of 118 SARS-CoV-2 epitopes presented by common HLA-I and -II alleles. In sharp contrast to full-length antigens, the selected epitopes did not induce a response in pre-pandemic samples, with the exception of two HLA-II-restricted peptides. We confirmed immunogenicity for 75 epitopes, and HLA restriction for 49 of them, including nine HLA-I epitopes that had previously displayed ambiguous binding. We further demonstrated that seven epitopes are presented by more than one HLA allele. 26 epitopes were immunodominant, meaning they were identified in at least 50% of patients with the restricting HLA allele. Based on these findings, we designed the ELISpot-based in vitro diagnostic 'Corona-T-test', which is designed for specific detection of COVID-19-or vaccine-induced-but not cross-reactive-T cell response to SARS-CoV-2. This test demonstrated 95% accuracy in a clinical trial of 69 vaccinated individuals, 50 COVID-19 convalescent patients (CPs), and 101 unexposed donors. We subsequently used this test to study a cohort of asymptomatic seronegative individuals with a history of prolonged SARS-CoV-2 exposure, and observed a lack of specific T cell response and substantial cross-reactivity to full-length antigens, indicating abortive infection in this cohort. Finally, we demonstrated that individuals vaccinated with the two-component Gam-COVID-Vac adenoviral vaccine (Sputnik V) maintained a strong, broad, and diverse CD8 + T cell response at a median of 66 days after the first injection, with a significantly higher number of recognized S-derived epitopes but negligible CD4 + reactivity in comparison to CPs.

To assemble a set of peptides, we collected available information on SARS-CoV-2 T cell epitopes 7, 37, [40] [41] [42] 48, 50 , as depicted in Fig.1A . The selected peptides were derived from both structural and non-structural SARS-CoV-2 proteins, based on their high immunogenicity in CPs and low immunogenicity in non-exposed individuals as well as their predicted binding to one or several common HLA alleles across the European population.

The final set included 94 putative HLA-I binders (i.e., MHC-I peptides) and 24 putative HLA-. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 doi: medRxiv preprint 5 II binders (i.e., MHC-II peptides), where each peptide was predicted to bind on average to four HLA-I and five HLA-II alleles, respectively (Table S1, Fig. 1A) . We did not observe a difference in the homology scores of the selected peptides and a set of other SARS-CoV-2 immunogenic epitopes annotated in the Immune Epitope Database (IEDB) relative to common cold coronaviruses ( Fig. S1A-D) . To estimate the theoretical coverage of the population for this set of peptides, we evaluated the frequency distribution of the restricting HLA alleles among HLA-typed individuals in the local bone marrow donor registry (n = 2,210). Only a single person (0.05%) had none of the alleles that were predicted to present these MHC-I or MHC-II peptides (Fig. 1B) , indicating the designed peptide set offers sufficient predictive sensitivity.

We compared the specificity for pools of peptides spanning the full length of the various SARS-CoV-2 structural proteins-S, nucleoprotein (N), and membrane protein (M)with that for the MHC-I and -II peptide sets using a cohort of pre-pandemic healthy donor samples (HD-2019; n = 52) and measuring the interferon ɣ (IFNɣ) response by ELISpot. Ten donors produced a positive (≥7 spots) T cell response to any of the peptide pools (S, N, or M) (Fig.1C ) or to recombinant S protein. Three of these donors also had a positive response to the set of MHC-II peptides (Fig. S1E) . Using matrix pools (see Methods), we identified two cross-reactive MHC-II peptides (RWY from N protein and IED from S protein; Fig. 1C , S1F; sequences are given in Table S1 ). The high frequency of cross-reactive responses induced by the S, N, and M peptide pools or by recombinant S protein makes these targets ill-suited for measuring SARS-CoV-2-specific T cell response.

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Table S1 ) for the MHC-I (left) and -II (right) sets. The distribution of the peptides according to the number of HLA that they bind is shown at top. The x-axis displays the number of predicted binding alleles per peptide. The y-axis shows the percentage of peptides that bind to a given number of alleles. Numbers below the SARS-CoV-2 genome schematic indicate the number of peptides derived from each gene. (B) The number of HLA class I (left) and II (middle) alleles alone or in combination (right) that are predicted to bind at least one peptide from the set per individual among 2,210 donors from the bone marrow registry. (С) Antigen response among our healthy donors 2019 (HD2019) cohort (n = 52). Two cross-reactive peptides from the MHC-II peptides are marked with red arrows. Dots represent the mean of two duplicates with negative control subtracted. The positive threshold (7 spots) is indicated by the dotted line. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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We next analyzed the response to S, N, and M peptides and MHC-I and MHC-II peptides in cohorts of COVID-19 convalescent patients (n = 51, CP) and Sputnik Vvaccinated individuals (n = 45, Vac). Full information on these cohorts is provided in Table   S2 . We excluded the two cross-reactive peptides identified above (RWY and IED) to create a new 'MHC-II crosspeptides' set; the initial MHC-II set will subsequently be referred to as 'MHC-II cross + '. In agreement with recently published data 13,35 , we observed that Vac individuals demonstrated a greater response to S peptides than CP, while the response to N and M peptides in Vac was non-existent. Both cohorts demonstrated comparable responses to the MHC-I set ( Fig. 2A) , although peptides derived from S protein accounted for only 27% of that set.

Surprisingly, individuals in the Vac group demonstrated a significantly weaker response to MHC-II peptides in comparison to CP ( Fig. 2A) . We hypothesized that the MHC-II peptide set, although it included multiple S-derived peptides, was skewed toward immunogenic peptides from the other antigens, N and M. We tested this by selecting the eight CP donors with the strongest response to the MHC-II peptides (>50 spots) and calculating the ratio of the number of spots in wells containing peptides from the MHC-II crossset to the number in wells containing MHC-II peptides derived from S protein (excluding IED peptide). We observed a detectable response to the latter set in just one donor, accounting for ~8% of the response to MHC-II crosspeptides (Fig S2A) . This suggests that S-derived MHC-II epitopes might be non-immunogenic or evoke a lowfrequency T cell response that is barely detectable without ex vivo expansion. We also observed negligible response in the Vac cohort to the recombinant S protein ( Fig. 2A) in comparison to CP, where this response was more strongly correlated with the response to MHC-II peptides than to MHC-I peptides (Fig. S2B) . This probably reflects the predominant presentation of the recombinant protein by MHC-II pathway. We tested whether time influenced the antigen response within our sampling period, and did not observe significant association with the recombinant S protein, MHC-I, or MHC-II peptides ( Fig. S2C-H) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 8 demonstrated even greater responses to the MHC-I + II set in comparison to S peptides. We next assessed the contribution of cross-reactive peptides IED and RWY to the total response (Fig. S3B) . We observed a significant impact of these peptides (> 30 spots) in three CP donors, but this response was only present in patients with high reactivity to other MHC-II peptides. Patients with low responses to these cross-reactive peptides demonstrated virtually no difference between MHC-II cross + and crosssets, and we therefore do not expect lower sensitivity due to the exclusion of the IED and RWY peptides.

We next examined the effect of the presence of a particular HLA on the magnitude of response to different antigens (Fig. 2B) . For CP donors, we observed a very strong association between the presence of HLA-A*01:01 and the number of spots in response to MHC-I peptides. Indeed, most of the HLA-A*01:01-restricted peptides, which we confirmed as immunodominant, were derived from viral ORFs and thus could not evoke a response in the Vac cohort. This suggests that HLA-A*01:01 may be associated with increased response to ORF-derived epitopes (Fig. S3С) . For the other HLA alleles, we observed significant variability in the responses. As an example, CP donors p2037 and p2034, who had detectable responses to MHC-I peptides (17.5 and 30 spots), exclusively carried A*02:01 out of all the HLA-I alleles that were confirmed to present immunogenic peptides (i.e., 'confirmed HLA'). In contrast, three other CPs bearing A*02:01 alongside other confirmed HLA-I alleles demonstrated only a negligible response (0; 5; and 7 spots). The increased response to MHC-II peptides that we observed in carriers of DRB1*11:01 may be associated with higher immunogenicity of some peptides in the context of this particular allele. We also compared differences in the prevalence of common HLA alleles (>10% phenotypic prevalence among bone marrow donors) between groups and observed that the CP cohort included fewer patients with B*07:02/C*07:02 (Fig S3D) in comparison to either bone marrow donors or the Vac cohort. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint protein-derived peptides is sufficient for detecting T cell response here (Fig. 3A) . In contrast, the MHC-I + II peptides discriminated CP from HD-2019 better than any of the peptide pools covering full-length antigens (Fig. 3B) , providing the same sensitivity (94%) with better specificity (S: 88%, cutoff 8 spots vs. MHC-I + II: 94%, cutoff 7.5 spots) and a higher AUC (0.99 vs. 0.97). In the CP cohort, the lack of specificity was more prominent when we analyzed the sum of spots in wells with M, N, and S peptides (Fig. 3C) ; this . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint reduced specificity could not be fully mitigated by application of the logistic regression model based on the spot values for the S, N, and M peptides. However, the wide confidence intervals of AUC values do not allow us to assess statistical significance for these differences. 

In order to systematically analyze the immunogenicity and HLA restriction of each epitope in our set, we performed a short-term memory T cell expansion assay. Peripheral blood mononuclear cells (PBMCs) from each donor were stimulated with the complete set of MHC-I and -II peptides, split to five 'Expansion pools'. To reduce the number of individual peptides tested in each assay, we assessed the response to smaller pools of ~5 peptides each using IFNɣ ELISA. When a response to one of these 'ELISA pools' was detected, the expansion was individually tested for reactivity to the individual peptides in that pool, although only peptides that bound to that individual's HLA alleles were tested. Table S3 summarizes all of the epitopes that were tested as part of ELISA pools or individually. We observed at least one response to 59 of 94 MHC-I epitopes, and at least two responses to 47 epitopes. The latter epitopes were selected for HLA association analysis. Using the Fisher exact test, we identified the association with a particular HLA for 36 of the 47 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint immunogenic MHC-I epitopes (Fig. 4A ). Of those, 22 epitopes were exclusively immunogenic in carriers of the associated HLA allele, with no "off-HLA" response. For four peptides, one off-HLA response was detected, and ten peptides demonstrated more than one off-HLA response. For this latter set of peptides, we searched for a second allele association that covered most of the off-HLA responses (Fig. S4A) . For the remaining 11 peptides that did not demonstrate association with a particular HLA allele, we sought associations with a combination of two HLA alleles. We identified such a combination for two peptides (ADA and KAY, Fig S4A) . We observed no significant association for the remaining nine peptides, suggesting that they may bind three or more alleles (e.g., NRY presumably binds B*08:01, C*04:01, and C*06:02; Table S3 ) or are insufficiently immunogenic. The SSP peptide could not be unambiguously assigned, as it demonstrated the association with either C*04:01 ( Fig. 4A) or B*35:02 and B*35:01 (Fig S4A) . We also detected two MHC-I epitopes within predicted MHC-II epitopes.

We observed at least one response for 16 of 24 MHC-II epitopes. For the 13 epitopes demonstrating at least two CD4 + responses, we could not detect any apparent association with a single HLA-II allele (Fig. S4B) . After searching for associations with a combination of two HLA alleles, several peptides still demonstrated multiple off-HLA responses ( Fig. S4B) , suggesting an association with three or even four HLA alleles. For these peptides, we present the final HLA association assignment that explained most of the responses (Fig. 4B) . Statistics and association patterns are presented in Table S3 . HLA alleles with a validated association with at least one immunogenic peptide are referred to as "confirmed HLA." Moreover, 19 MHC-I and five MHC-II epitopes were immunodominant, in that they were recognized in at least 50% of patients with the restricting HLA alleles. The most dominant MHC-I epitopes from S protein were YLQ/A*02:01 and KCY/A*03:01, with nearly 100% response; in contrast, KCY produced a response in only four out of 12 A*11:01 carriers. We also noted that QYI generated higher immunogenicity with A*23:01 in comparison to the already observed association with A*24:01 (Rowntree, 2021). Among ORF-derived epitopes, TTD/A*01:01, HTT/A*01:01, and KTI/A*30:01 elicited the most frequent response. ATE/A*11:01, SPR/B*07:02, and KTF/A*03:01/A*30:01 from N protein also showed a dominant response. In contrast to KCY, KTF was equally immunogenic both in A*03:01 and A*30:01. Assessment of immunodominance for MHC-II epitopes was hindered by their promiscuous binding, but IED, GAV, TSR, LSY, and IGY may be considered immunodominant in the context of all of the HLAs they bound. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint Immune response in vaccinated was skewed towards MHC-I epitopes Unsurprisingly, expanded T cells from the CP cohort recognized a higher median number of MHC-I epitopes (four) than those from Vac donors (two), where the response was limited to S protein (Fig. 4C) . However, this decrease in the number of recognized epitopes was not accompanied by a decrease in overall response magnitude (see Fig. 2A 

suggesting a more robust response per epitope. At the same time, the number of recognized MHC-I epitopes from S protein was significantly higher in the Vac cohort (two) than in CP (one) (Fig. 4C ). In contrast, the Vac cohort exhibited significantly fewer recognized MHC-II epitopes per person, both for all MHC-II epitopes (median 3 vs. 0) or Sderived epitopes (median 1 vs. 0; Fig. 4D ). Using flow cytometry, we confirmed that most of the responses to S-derived epitopes were mediated by CD8 + rather than CD4 + cells ( Fig.   4E, Fig. S3C ). Indeed, the profile of recognized MHC-II epitopes was clearly different in Vac and skewed towards the recognition of LQT and QQL peptides, predominantly by CD8 + T cells (Fig. S3C) . We observed a strong prevalence of either B*40:01 or B*44:03 among the CD8 + responders to QQL (four out of five). AEIRASANL (a 9-mer derived from the 15-mer QQL) was predicted to be a strong binder for both alleles, which may explain CD8 + reactivity to this peptide. All responders to this peptide belonged to the Vac cohort, although the frequency of B*44:03/B*40:01 was similar in the CP group (Table S2) . Among eight responders (including six Vac) to the LQT epitope, we observed seven carriers of either A*23:01 or A*24:02. We believe these responses are due to the LQT-derived 9-mer TYVTQQLI, which is predicted as a strong binder for both alleles but seems to be more immunogenic in A*23:01 (tree our of seven responses) than in A*24:02 (four out of 23 responses). One more MHC-I epitope evoked response in two Vac individuals, but in none of the CP individuals (VRF/B*13:02) despite the similar number of B13:02 carriers. These data suggest that the observed higher number of recognized MHC-I S protein-derived epitopes per person in Vac compared to CP is either a consequence of a wider repertoire of recognized MHC-I S-derived epitopes or better detection with our readout due to a higher frequency of specific T cells.

In contrast, although T cells specific to S protein-derived MHC-II peptides could not be detected with ELISpot among PBMCs from either Vac or CP individuals (see Fig. 2A , S2A), they were readily detected after ex vivo expansion in CP but not in Vac samples (Fig.   4D ). We did not observe any difference in the prevalence of frequent HLA-II alleles between . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint 13 CP and Vac groups (Fig. S3D) , explaining the striking difference in CD4 + T cell response.

We hypothesize that the frequency of S protein-specific CD4 + T cells in the Vac cohort is lower in comparison to CP. And in contrast to MHC-I epitopes, the absence of the other immunogens besides S protein does not widen the CD4 + response to the S protein, at least within the given timeframe of sampling. We also observed responses to non-S-derived epitopes in 10 Vac donors. In two, we observed response exclusively to cross-reactive RWY or SPR epitopes, while eight responded to two or more non-S protein epitopes, suggesting prior infection. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint 14 plots shows the number of responses in donors without the indicated HLA. For HLA-I, the association between the response and a single HLA allele is shown; for HLA-II, the best associations (including associations with several HLAs) are shown. Fisher exact test, p-values < 0.05 were considered significant (exact p-values are specified in Table S4 ). (C, D) Number of MHC-I (C) and MHC-II (D) epitopes from S or any viral protein recognized per individual for the CP and Vac cohorts. (E) Flow cytometric analysis of the phenotype of T cells responding to MHC-II peptides. Plot shows the difference in the % of CD4 + or CD8 + IFNɣ + T cells between peptide-stimulated cells and negative controls.

We designed and manufactured an ELISpot-based in vitro diagnostic 'Corona-T-test' for detection of SARS-CoV-2-specific T cells, which included the above-identified combination of peptides (designated here as "MHC-I + II_IVD"; see Methods). We performed a clinical trial in which we enrolled three independent cohorts of vaccinated (Vac_trial, n = 69), convalescent (CP_trial, n = 50), and healthy but unvaccinated individuals (HD-2021, n = 101). We measured the T cell response in parallel with the Corona-T-test and by stimulation with MHCI+II_IVD peptides followed by intracellular IFNɣ staining. As expected, intracellular INFɣ produced a low signal-to-noise ratio, and multiple patients with a robust ELISpot response were not positive based on intracellular IFNɣ staining (Fig. S5A) .

Using flow cytometry, we confirmed that the CD8 + response to MHC-I + II_IVD was higher than the CD4 + response in the Vac_trial and CP_trial cohorts (Fig. S5B) , which is consistent with our previous ELISpot data (Fig. S3A) . We did not observe a correlation between sampling time and humoral or T cell response in the Vac_trial cohort ( Fig. S5C-E) , which is probably due to the short timeframe that had elapsed since the boost vaccination (7-21 days). For CP individuals, we observed a weak association between sampling time and T cell or humoral response (Fig. S5E-F) . Consistent with the previous results, our Corona-Ttest demonstrated high overall accuracy in discriminating HD-2021 from Vac_trial (AUC = 0.98) and CP_trial (AUC = 0.98) (Fig. 5A, B) individuals, with 96.4% sensitivity, 93.5% specificity, and 95% accuracy. Several false positives in the HD-2021 might be explained either by the enrolment of asymptomatic convalescents without detectable antibody response or by the presence of cross-reactive epitopes in our MHCI+II_IVD peptide set.

HLA-genotyping of the responders in the HD-2021 cohort did not reveal any obvious HLA bias (e.g., high B*07:02 prevalence), most likely excluding the possibility that a single crossreactive epitope caused the occasional false-positive responses. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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We also HLA-genotyped the non-responders (below the grey zone in Fig. 5A ) from the Vac_trial and CP_trial cohorts to assess possible biases and identify HLA alleles associated with weak response. Among two non-responders in Vac_trial, we observed only 1-2 confirmed HLA alleles presenting epitopes with modest immunogenicity. Two CP_trial non-responders bore three confirmed HLA alleles, which potentially bound 13 and six confirmed peptides, respectively. This is comparable with the median number of recognized epitopes per person in the CP group (Fig. 4C, D) . Thus, we expect that lower responses, at least in CP individuals, are an intrinsic property rather than a consequence of lacking confirmed HLA alleles. In line with this hypothesis, we observed only modest correlation between the number of confirmed HLA-I alleles and the response to MHC-I peptides in the Vac_trial (r = 0.52, p = 0.0004) cohort, and a number of confirmed HLA-II alleles with MHC-II crossin the CP_trial (r = 0.4, p = 0.007) cohort. We examined the distribution of the population according to the number of confirmed HLA alleles, and observed that up to 2.8% of Vac_trial-but only 0.1% of CP_trial-lacked any of the confirmed HLA alleles (Fig. 5C, 

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint Healthy SARS-CoV-2-exposed individuals demonstrated strong

During June-July 2020, we recruited an additional cohort of healthy exposed (HE) individuals (n = 37) who were in close contact with COVID-19 patients but did not report any flu-like symptoms and remained IgG-and IgM-negative. The median time of contact was 22 days (Q1 = 10 days, Q3 = 29 days). In comparison to HD-2019, HE individuals demonstrated significantly higher responses to M and S peptides but not to MHC-I and MHC-II crosspeptides (Fig 6 A, B) . For CP individuals, in contrast, we observed high concordance of positive responses (≥ seven spots) to M, N, or S peptides and MHC-I + II peptides (Fig. S2A) , with only 2/51 demonstrating a discordant response. Finally, we observed a significant cross-reactive response to the RWY and IED epitopes in the HE cohort that was not seen in the Vac cohort ( Fig. 6C and Fig. S3B) . These results show that the HE cohort is substantially different from both CP and HD-2019, suggesting the presence of a cross-reactive T cell response that might have prevented the development of symptomatic disease and T cell priming with other SARS-CoV-2 epitopes from our set. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. Difference in responses to MHC-II peptides before and after exclusion of two cross-reactive peptides in HE (Wilcoxon test, p = 0.0045).

Systematic study of the immunogenicity of SARS-CoV-2 T cell epitopes opens the door to vaccine optimization and the utilization of T cell response as an independent measure of immune protection. In the present study, we observed a significant decrease of B*07:02/C*07:02 haplotype frequency in the CP cohort compared to donors from the bone marrow registry, vaccinated and healthy exposed. This HLA bias may be explained by the partial protection of B*07:02 carriers mediated by the T cell response induced by seasonal coronaviruses. At least one epitope presented by this allele is known to be cross-reactive 41,44 and immunodominant. Interestingly B*07:02 is a single HLA allele that is characterized by a consistent decrease in the number of predicted epitopes over the course of SARS-CoV-2 evolution 52 . We have also selected a set of immunogenic and putatively non-crossreactive SARS-CoV-2 peptides that are predicted to be presented by common HLA-I and -II alleles. Based on analysis of IFNɣ response in 52 pre-pandemic samples (HD-2019), we identified two cross-reactive MHC-II epitopes. Other epitopes (SPR, KLW, and LLY) that have subsequently been proven to be cross-reactive by others 32,41,44,53 did not produce a measurable response in our assay, and this could possibly be explained by the low peripheral frequency of specific T cells. In support of that hypothesis, HLAs presenting these cross-reactive epitopes were not enriched in a small subgroup of HD-2021 who were positive based on our Corona-T-test. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint 19 response to the full-length structural proteins, and this can probably be explained to a large extent by the presence of seven confirmed A*01:01 epitopes-including six from nonstructural proteins, five of them immunodominant-within the MHC-I + II set. This underlines the notion that in A*01:01 carriers, the majority of the CD8 + T cell response might be focused beyond structural proteins, including S protein. Alongside the recent data that the T cell response to early-expressed non-structural proteins may result in protection from infection 32 , this provides the rationale for designing vaccines that contain such immunodominant peptides. We did not observe an increased response for other HLAs, presenting multiple epitopes (e.g. eight confirmed and three immunodominant epitopes for A*02:01).

T cells from Vac individuals recognized significantly more S protein-derived MHC-I epitopes than those from CP donors, while the total number of recognized MHC-I epitopes from any antigen was, unsurprisingly, higher in the latter group. Alongside the lack of a significant difference between these groups in the magnitude of the response to MHC-I peptides, this allows us to assume higher frequency-and potentially, clonal diversity-of S protein-specific CD8 + T cells in vaccinated individuals, resulting from focusing of the response on a single antigen. However, this effect was not replicated in CD4 + T cells, and the number of recognized S protein MHC-II epitopes per individual was significantly lower in Vac than in CP. It should be noted that multiple studies have suggested comparable CD8 + and CD4 + T cell responses at least four weeks after the first dose of vaccine [9] [10] [11] 13 . Accordingly, we believe that the negligible PBMC response to recombinant S protein ( Fig.   2A ) and low detection rate of S-protein-specific CD4 + T cells after ex vivo expansion (Fig.   4D , S4C) are associated with late sampling time (median 66 days after the first dose of vaccine). We believe that we did not observe a correlation between sampling time and response to S protein (Fig. S2D ) because at the earliest sampling time, the peripheral abundance of S-protein-specific CD4 + T cells was already below the quantification limit of the ELISpot platform. This may indicate earlier contraction of vaccine-induced S-proteinspecific CD4 + T cells, or their diminished proliferative potential compared to CD8 + T cells.

We used the MHC-I + II peptides identified here as the basis for the ELISpot-based Corona-T-test. This test exhibited 95% accuracy in detecting SARS-CoV-2-specific T cell response in a blinded clinical trial. Intracellular IFNɣ staining performed in parallel was less sensitive, but allowed us to confirm our previous finding that the measured IFNɣ response was largely mediated by CD8 + cells. HLA genotyping of non-responders from the Vac_trial and CP_trial cohorts did not show an association between negative response and insufficient coverage of HLA alleles by the epitopes in the set. Instead, this lack of specific T . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint 20 cells was explained by individual variation in the immune response. The rare responders in the HD-2021 group did not have an increased incidence of HLAs presenting the small number of cross-reactive peptides in the MHC-I set, and it is therefore most likely that this group included some seronegative individuals after asymptomatic infection.

HLA restriction was previously reported for most immunogenic MHC-I epitopes (Table S1 ). Nevertheless, due to the large number of immunized donors, we detected some additional-and often less immunodominant-restricting HLAs (e.g., KCY/A*11:01, QUI/A*23:01, and KTF/A*30:01). Several immunogenic epitopes were not, to the best of our knowledge, assigned to their HLA before (e.g., FQP, GRL, SSP, QQQ, FCN, FLL, RYR, VRF, and LQT-derived TYVTQQLI), or at least not with statistical confirmation of their HLA restriction. Five epitopes with two or more detected responses in our study were not reported in previous high throughput screenings 42, 49, 54, 55 , or were tested only within peptide pools and not individually 37 . In contrast to MHC-I epitopes, MHC-II epitopes were mostly promiscuous in their HLA-binding (Fig. 4B) . Most of the CD4 + response was focused on non-S-protein epitopes. Compared to the most comprehensive screening performed to date date 49 , we identified an additional 11 MHC-II epitopes, and statistically confirmed HLA associations for nine of them. Five precise epitopes were predicted from immunogenic peptides 50,56 ; six others were annotated in IEDB epitopes of SARS-CoV-1. We also confirmed two MHC-I epitopes within MHC-II epitopes.

Although there are several kits aimed at detecting SARS-CoV-2-specific T cells, we believe that the strategy of epitope selection is critical for discriminating pre-existing crossreactive immunity from specific immunity. Since we confirmed that the MHC-I + II peptide set can accurately discriminate SARS-CoV-2-induced and cross-reactive immunity, we next characterized the HE cohort of individuals without clinical or laboratory signs of COVID-19 after prolonged contact with COVID-19 patients. Surprisingly, we saw virtually no response (1 of 37) to MHC-I + II peptides, versus relatively frequent and robust responses to S, N, and M peptides, as well as significant responses to the cross-reactive IED and RWY peptides.

The discordant responses in this group contrasted sharply with the CP cohort, in which responses to full-length antigens correlated strongly with response to the MHC-II set (Fig.   S2B ). This is best explained by the protective effect of pre-existing cross-reactive T cell immunity in this cohort. Notably, we initially recruited two patients with a low-level IgM anti-S protein response, who were subsequently excluded using a more sensitive commercial kit.

These donors, however, demonstrated an easily detectable MHC-I + II response (18 and 54 spots), suggesting that the reported peptide set could even be used to diagnose asymptomatic CP with a negligible humoral response. Based on these results, we believe . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 that the described set of SARS-CoV-2 epitopes offers a sensitive and specific tool for the detection of COVID-19 or vaccination-induced (but not cross-reactive) T cell response. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 2 Infection and Development of Anamnestic Immune Responses in T Cell-Depleted Rhesus is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 the ELISPOT Technological Platform as Part of Anti-Epidemic Measures Against the New is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The following describes donors for the non-clinical-trial component of this study. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint COVID-19 patients was collected into EDTA tubes (Sarstedt, Germany) and subjected to Ficoll (Paneco, Russia) density gradient centrifugation (400 x g, 30 min). Isolated PBMCs were washed with PBS containing 2 mM EDTA twice, counted, cryopreserved in 7% DMSO and 93% heat-inactivated fetal bovine serum (Capricorn Scientific, Germany), and stored in liquid nitrogen until used in the assays. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 PepTivator SARS-CoV-2 Prot_S1, and PepTivator SARS-CoV-2 Prot_S+. For the positive control we used 40 ng/mL PMA (P8139-1MG, Sigma; USA) and 400 ng/mL calcium ionophore (C7522-1MG, Sigma; USA), with 0.02% isopropanol plus 0.02% DMSO as a negative control. After 18-20 hours of incubation at 37 °C, 5% CO2, the plates were developed according to the manufacturer's guidelines. Spots were counted using the CTL ImmunoSpot Analyzer (CTL, USA). T cell responses were considered positive when more than seven spots (mean of duplicates) were detected after subtracting the negative control.

Samples with >17 spots in the negative control or < 50 spots in the positive control were excluded from the analysis.

PBMCs from cryotubes or blood cryobags were thawed, rested 4-24 hours in CTLtest medium, counted, and used for rapid in vitro expansion. 2-4 x 10 6 cells per well were plated in 24-well plates in RPMI 1640 culture medium supplemented with 10% normal human A/B serum, 1 mM sodium pyruvate, 2 mM GlutaMAX Supplement (Thermo Fisher Scientific, USA), 25 ng/mL IL-7, 40 ng/mL IL-15, and 50 ng/mL IL-2 (Miltenyi Biotec, Germany) at a volume of 1 mL/well. The initial full set of peptides (final concentration of each = 10 μM) were added on day 0. On day 3, 1 mL of supplemented medium without peptides was added to each well (final volume 2 mL). Half of the medium was replaced on days 5, 7, 10. On days 10 and 11, an aliquot of cell suspension was used for anti-IFNɣ ELISA with pooled peptides and individual peptides respectively. On day 13, cells were sampled for flow cytometry analysis. In order to maintain detectable IFNɣ secretion, a quarter of the medium was replaced with supplemented medium on days 11 and 13.

After 10 days of expansion, an aliquot of cell culture was washed twice in 1.5 mL of PBS, then transferred to AIM-V medium (Thermo Fisher Scientific, USA), plated at 10 5 cells per well in 96-well plates, and incubated overnight (12-16 hours) with the peptide pools. 0.04% DMSO and 0.04% isopropanol was used as a negative control, with 40 ng/mL PMA and 400 ng/mL calcium ionophore as positive control. On day 11, the culture medium was collected and tested for IFNɣ as described below in the "Anti-IFNɣ ELISA" section. On days 11-12, we stimulated the cells as described above individually with each peptide (2 μM) from the pools. Only peptides predicted to bind to each individual's HLA were tested. Finally, on day 13, we stimulated the MHC-II peptide-expanded cells with the MHC-II peptides that generated a positive response with ELISA for flow cytometry experiments. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Anti-IFNɣ ELISA 96-well high-protein-binding ELISA plates (Shanghai Meikang Biological Project, KH-M-02, China) were coated with 100 μL per well of 0.01 mg/mL anti-IFNɣ antibody (Hytest, clone GF1) in 100 mM bicarbonate/carbonate (pH 9.6). After 14 h, the plates were washed once with 250 mL PBS + 0.1% Tween 20 (PBST) and blocked with 100 mL of 1% hydrolyzed casein (XEMA, Russia) in PBS for 3 h at room temperature, dried, and stored sealed at 4 ˚С.

Culture plates were centrifuged for 3 min at 700 x g, and 100 µL of the medium was transferred to the ELISA plates. Plates were incubated for 1.5 h at 37˚C on a rocking platform. The plates were washed thrice with PBST, and then 100 µL of 0.1 µg/mL biotinylated anti-IFNɣ antibody (R&D Systems, USA) was added and incubated for one hour at 37˚C on a rocking platform. The plates were washed thrice with PBST, and then 100 µL of Streptavidin-HRP (XEMA, Russia) was added and incubated for 0.5 h at 37˚C on a rocking platform. Finally, the plates were washed five times with PBST, and 100 µL of 3,3',5,5'tetramethylbenzidine substrate (XEMA, Russia) was added to each well. 15 min later, 100 µL of 1 M H2SO4 was added as a stop solution, and the optical density (OD) was measured at 450 nm on a MultiScan FC (Thermo Fisher Scientific, USA) instrument. Each plate included standards corresponding to 0, 15, 120, and 7,700 pg/mL of IFNɣ to control the sensitivity and linearity.

Test wells with peptides where the ratio OD450_test_well/OD450_negative control ≥ 1.25 and the difference OD450_test_well -OD450_negative control ≥ 0.08 were considered positive. Peptides with a ratio between 1.25 and 1.5 were tested again up to three times as biological replicates to ensure the accuracy of their response. Peptides with two or three positive results were considered positive. If such peptide was not repeatedly tested, it was considered negative. If ELISA results conflicted with flow cytometry data (for MHC-II peptides), the peptide was considered non-immunogenic.

After 13 days of expansion, an aliquot of cell suspension was washed twice in 1.5 mL of PBS, then resuspended in AIM-V medium with 1.0 µg/mL brefeldin A (GolgiPlug, BD Biosciences; USA). Cells were plated at 10 5 per well in a 96-well polypropylene V-bottom plate and incubated at 37 ˚С for ~5 h with MHC-II peptides that were identified as positive in the previous ELISA assay. 0.04% DMSO and 0.04% isopropanol were used as a negative control, and 40 ng/mL PMA and 400 ng/mL calcium ionophore were used as positive . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Star, Ashland, OR). The percentage of IFNɣ-positive cells was calculated in the CD3 + CD8 + and CD3 + CD4 + gate. The difference or ratio (fold-change) of the percentage of IFNɣ-positive cells incubated with peptide and with negative control was calculated. Peptides with a ratio >2 were considered positive for CD4 + recognition, or with a ratio >3 for CD8 + recognition due to a higher background % of CD8 + IFNɣ + cells. If the total number of CD8 + cells was lower than 5000 cells, this well was not analyzed for the CD8 + response. In case of conflicting results in ELISA and flow cytometry the peptide was considered negative.

ELISA kits for the detection of anti-RBD IgG (K153, National Research Center for Hematology, Russia) and SARS-CoV-2-IgМ-EIA-BEST (D-5502, Vector Best, Russia) for the detection of IgM antibodies to full-length S protein were used according to the manufacturers' instructions.

To assemble the control peptide pool, IEDB was queried for epitopes with positive MHC binding and a minimum of two positive T cell assays using 'Severe acute respiratory syndrome-related coronavirus' as 'Organism' on 11 October 2021. MHC-I and MHC-II peptides used in this study were excluded from the IEDB epitope pool. Alignments were performed using best global-local alignment by the 'pairwiseAlignment' (bioPython) function for four ORFs shared by all five strains: orf1ab, S, N, and M, allowing amino acid substitutions with similar biochemical properties (1, 2) and low penalties for gap opening and extension (-0.5) . The segments of the alignments corresponding to the given SARS-CoV-2 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint 34 peptide were further aligned again with high gap penalties (-10/-1) followed by calculation of the similarity score 57,58 nand identity score.

We selected the HLA list based on the most-presented HLA among the CP cohort: Fig. 1A . Thereafter, we searched for the exact MHC-II epitopes from those peptides and predicted their binding to the selected HLA-II epitopes assigned to SARS-CoV-1 in IEDB and four peptides with predicted high binding promiscuity (> 7 HLA-II alleles). HLA binding was predicted using both NetMHCIIpan 3.2 59 and Neon MHC2 60 for MHC-II peptides using standard thresholds. Strong binders, weak binders, or peptides with discordant NetMHCIIpan and Neon MHC2 predictions were considered as potential binders and were tested in donors bearing those HLAs.

. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Table S1 ).

For the first step of epitope identification in anti-IFNɣ ELISA 5*(~5) = 26 standard mixes (ELISA-pools, Table S1 ) corresponding to the composition of the five standard mixes for T cell expansion.

All data comparisons were performed using GraphPad Prism 8, Python 3.2, and FlowJo 10 software. Statistical analyses were performed using Spearman correlation and is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint 36 individuals without this HLA were compared with Fisher exact test, with p-values < 0.05 considered significant.

The detection of SARS-CoV-2-specific T cells was performed by Corona-T-testa single-color enzymatic ELISpot kit for IFNɣ detection produced by the National Research Center for Hematology. After washing cells with sterile RPMI-1640 media twice, cells were counted in a hemocytometer (Sysmex XS-1000i, Sysmex Corporation, Japan) and resuspended in serum-free AIM-V + AlbuMAX BSA (Thermo Fisher Scientific, USA) medium to a concentration of 6 x 10 6 /mL, and then 3 x 10 5 cells were loaded per well. The SARS-CoV-2 antigens (MHC-I + II _IVD) were used at a final concentration of 1 μM/mL. Phytohemagglutinin (PHA) was used as a positive control at a concentration of 10 μg/mL.

The final volume of each well was 150 μL. All manipulations with cells and antigen dilutions were performed in serum-free media in sterile conditions. We used four wells for each PBMC sample: negative control stimulated with AIM-V medium, SARS-CoV-2-antigeninduced stimulation in duplicate, and positive control with PHA stimulation. Plates were incubated 16-18 hours at 37 °C, 5% CO2. The next day, the assay was developed according to the manufacturer's guidelines, with spots counted using the automated ImmunoSpot Series 5 UV Analyzer (CTL, USA) using automated software. Results were considered valid if the number of spots was < 10/well in negative controls and > 100/well in positive controls. Non-specific activation in negative controls was subtracted from the average of the two stimulated sample wells. Responses with < 4 spots were considered negative and > 6.5 spots were considered positive. Grey zone samples with 4-6.5 spots were considered inconclusive, requiring repeated testing, and such samples (n = 6) were excluded from the accuracy analysis.

For flow cytometry assessment, PBMCs were separated as described previously and rested overnight at 7.5 x 10 6 cells/mL in AIM-V + AlbuMAX (BSA) medium at 37°C with 5% CO2. The next day, 200 μL of cell suspension was added to each well of a 96-well polypropylene V-bottom plate. All samples received 0.25 μL of brefeldin A at the beginning of incubation. For the immune stimulation, 1 μM MHC-I + II_IVD peptides was added. 1.5 μg/mL PHA (Capricorn Scientific GmbH, Germany) was used as a positive control. Nonstimulated cultures were used to assess spontaneous intracellular levels of cytokines. The cells were incubated for 4 hours at 37 °C, 5% CO2 and then transferred into 12 x 75 mm . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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plastic tubes at 1.5 x 10 6 cells per tube and washed with 2 mL MACS PBS/EDTA Buffer without Ca 2+ or Mg 2+ (Miltenyi Biotec, Germany) with 0.5% heat-inactivated MACS BSA (Miltenyi Biotec, Germany) , PBS/BSA/EDTA. The cells were centrifuged at 350 x g for 5 min and then stained. Cell surface staining of T cells was done in 0.1 mL PBS/BSA/EDTA for 15 min with FITC-conjugated anti-CD3 (clone SK7, BD Biosciences, USA), PE-Cy7 anti-CD8 (clone SK1, BD Biosciences, USA), VioGreen anti-CD4 (clone REA623, Miltenyi Biotech, Germany), and 7-amino-actinomycin D (7-AAD, Miltenyi Biotech, Germany) in the dark at room temperature. Fixation and permeabilization were performed with BD Cytofix/Cytoperm according to the manufacturer's protocol, and intracellular staining was carried out for 30 min in the dark at room temperature with APC anti-IFNɣ (clone B27, BD Biosciences, USA).Cells were analyzed on a CytoFLEX (Beckman Coulter) flow cytometer. Instrument performance was monitored daily with CytoFLEX Daily QC Fluorospheres (Beckman Coulter, USA). Individual populations were gated according to forward scatter, side scatter, and specific markers, and the data were subsequently analyzed with CytExpert software (Beckman Coulter, USA). Dot plots representing anti-CD3 versus anti-IFNɣ were carried out to establish CD3 + IFNɣ bright lymphocyte gates. Identical dot plots were generated for CD8 + IFNɣ bright and CD4 + IFNɣ bright cells. Typically, 300,000 events were acquired in the gating CD3 + region. Non-specific activation in unstimulated controls was subtracted from stimulated samples to account for specific activation. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.12.21267518 doi: medRxiv preprint