key: cord-0858480-hc4zu9t5
authors: Naranbhai, V.; Nathan, A.; Kaseke, C.; Berrios, C.; Khatri, A.; Choi, S.; Getz, M. A.; Tano-Menka, R. K. K.; Ofoman, O.; Gayton, A. C.; Senjobe, F.; St. Denis, K.; Lam, E. C.; Garcia-Beltran, W. F.; Balazs, A. B.; Walker, B. D.; Iafrate, A. J.; Gaiha, G. D.
title: T cell reactivity to the SARS-CoV-2 Omicron variant is preserved in most but not all prior infected and vaccinated individuals
date: 2022-01-05
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2022.01.04.21268586
sha: b0cc256aa17a811e88732483c41aa7402c136bd7
doc_id: 858480
cord_uid: hc4zu9t5

The SARS-CoV-2 Omicron variant (B.1.1.529) contains mutations that mediate escape from infection and vaccine-induced antibody responses, although the extent to which these substitutions in spike and non-spike proteins affect T cell recognition is unknown. Here we show that T cell responses in individuals with prior infection, vaccination, both prior infection and vaccination, and boosted vaccination are largely preserved to Omicron spike and non-spike proteins. However, we also identify a subset of individuals (~21%) with a >50% reduction in T cell reactivity to the Omicron spike. Evaluation of functional CD4+ and CD8+ memory T cell responses confirmed these findings and reveal that reduced recognition to Omicron spike is primarily observed within the CD8+ T cell compartment. Booster vaccination substantially enhanced T cell responses to Omicron spike. In contrast to neutralizing immunity, these findings suggest preservation of T cell responses to the Omicron variant, although with reduced reactivity in some individuals.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (B.1.1.529), first identified in November 2021, has been the cause of a new surge of infections globally (Viana et al., 2021) . With as many as 36 substitutions in the viral spike protein and 59 mutations in total throughout its genome, Omicron has been found to evade neutralization by infection-and vaccine-induced antibodies with unprecedented frequency (Garcia-Beltran et al., 2021a; Hoffmann et al., 2021) and escape neutralization by most therapeutic monoclonal antibodies (Ikemura et al., 2021; VanBlargan et al., 2021) . Additional booster vaccine doses partially compensate for this effect (Garcia-Beltran et al., 2021b; Hoffmann et al., 2021) , but the durability of such protective antibody response remains to be determined. Thus, whether additional arms of the adaptive immune response, namely T cell responses, can augment protection against Omicron infection and disease is of considerable interest and has implications for predicting the course of future SARS-CoV-2 variants.

In individuals with previous SARS-CoV-2 infection and vaccinees, robust T cell responses are quantitatively and qualitatively associated with milder outcomes (Rydyznski Moderbacher et al., 2020) . Early induction of antigen-specific CD4 + T cells following vaccination is associated with coordinated generation of antibody and CD8 + T cell responses (Painter et al., 2021) . Previous studies have also shown a key role for CD8 + T cells in mitigating COVID-19 disease severity and inducing long-term immune protection. Mild COVID-19 disease is associated with increased clonal expansion of CD8 + T cells in bronchoalveloar lavage fluid (Liao et al., 2020) , robust CD8 + T-cell reactivity to SARS-CoV-2 epitopes (Peng et al., 2020; Sekine et al., 2020) , and rapid CD8 + T cell-mediated viral clearance (Tan et al., 2021) . In addition, depletion of CD8 + T cells from convalescent macaques reduced protective immunity (McMahan et al., 2020) .

Given that T cells can target regions across the SARS-CoV-2 proteome and are not limited solely to the spike protein, it is perhaps not unexpected that prior SARS-CoV-2 variants were able to escape neutralizing antibody responses (Garcia-Beltran et al., 2021a) but not T cells (Geers et al., 2021) . Thus, in light of the emergence of the All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi. org/10.1101 org/10. /2022 Omicron variant, we sought to determine the extent to which mutations in the variant spike and non-spike proteins affect CD4 + and CD8 + T cell reactivity.

Utilizing samples from prior SARS-CoV-2 infected, vaccinated, and both prior infected and vaccinated individuals, we found that circulating effector T cell responses and both CD4 + and CD8 + memory T cell responses were generally preserved to the Omicron variant. However, distinct from previous variants of concern (VOC), such as Delta, a subset of individuals had reduced effector and memory T cell recognition to the Omicron spike protein relative to wildtype spike, with a particularly noticeable effect on spikespecific CD8+ T cell memory responses. Booster doses enhanced the magnitude of responses to wildtype and Omicron spike, although did not completely mitigate the comparatively reduced T cell reactivity to Omicron in individual participants. These findings therefore have important implications in ascertaining the role of immune responses in morbidity and mortality due to Omicron and may inform the development variant-specific and variant-resistant second-generation vaccines. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi. org/10.1101 org/10. /2022 

Massachusetts sampled prior to vaccination, after primary series vaccination, and/or after receipt of additional 'booster' doses. Study groups were stratified by prior infection (confirmed by anti-nucleocapsid antibody testing) and vaccination status (Table S1 ). In total, we studied 101 samples from 76 donors ( Figure 1A ). The median age was 45 years (range 37-60 years) and 64% were female. Of the previously infected individuals, we included 11 unvaccinated, 12 vaccinated sampled after initial vaccination series, and 13 vaccinated and sampled after booster doses. Among individuals without previous infection, we included 10 unvaccinated, 24 sampled after initial vaccination series, and 31 vaccinated and sampled after booster doses. Samples were obtained at a median 220 (range 130-286) days after primary series vaccination or 10 days (range 8-54) after additional booster doses. The primary analysis of host, vaccine, and variant variables that affect T cell responses was by multivariate regression.

To assess the total (CD4 + and CD8 + ) effector T cell response, we performed an IFN-γ ELISpot following stimulation with pooled overlapping 15mer peptides spanning the full length of the wildtype, Delta (B.1.617.2), or Omicron (B.1.1.529) spike protein and the non-spike SARS-CoV-2 structural and accessory proteins (nucleocapsid, membrane, enveloped and open reading frame 3A, i.e. NC/M/E/3A) from wildtype and Omicron. The evaluated peptides span the full-length of spike: relative to wildtype, 27.3% (86/315) spike peptides were unique to Omicron and 8.6% (27/315) to Delta. For the NC/M/E/3A pools, the evaluated peptides span the full-length of the respective proteins: relative to wildtype, 10.1% (24/237) of the peptides were unique to Omicron (Table S2 ). In the primary multivariate analysis of T cell reactivity (Table S3) , the magnitude of effector T cell responses to spike and non-spike proteins did not vary by variant, and was not affected by age, sex and primary vaccine series. However, examination of individual responses reveals specific patients in whom responses to the Omicron spike were All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 reduced by >50% ( Figure 1B , denoted in red). Prior infection, duration after primary series vaccination, and receipt of an additional 'booster' dose were independently associated with magnitude of response (Table S3 ).

The median effector T cell reactivity against wildtype and Omicron spike (in SFU/10 6 PBMC) was 152 and 114 for individuals with prior infection; 43 and 42 for individuals after primary series vaccination (without prior infection); and 311 and 315 for individuals with prior infection after primary series vaccination ( Figure 1C ). In comparison, the median effector T cell reactivity against delta spike (in SFU/10 6 PBMC) was 155 for individuals with prior infection; 34 for individuals after primary series vaccination (without prior infection); and 277 for individuals with prior infection after primary series vaccination ( Figure 1D ). Regardless of variant, prior infection was associated with a higher magnitude of effector T cell responses (0.55 log 10 SFU/10 6 PBMC higher response, 95%CI 0.38-0.72, p<0.001). Effector T cell responses declined modestly over time (-0.02 log 10 SFU/10 6 PBMC lower response per week, 95%CI -0.05,0.00, p=0.028). Neither age nor sex influenced responses, and in this analysis primary vaccine type was not associated with differences in responses (Table S3) . Surprisingly, 21.2% (10/47) of participants with prior infection and/or vaccination had a >50% (0.3log 10 ) reduction in T cell response to Omicron spike (denoted in red in Figure 1C ), with 12.7% of participants (6/47) having a >70% (0.5 log 10 ) reduction. In contrast, only 9.7% (4/41) of participants with prior infection and/or vaccination had a >50% (0.3log 10 ) reduction in overall effector T cell response to Delta spike. Thus, while T cell responses induced by prior infection and/or vaccination are broadly cross-reactive at a population level, a distinct subset of individuals have substantially reduced T cell recognition of the mutated Omicron spike protein.

In contrast to spike-specific T cell responses, T cell reactivity against wildtype and Omicron NC/M/E/3A was preserved in all individuals with prior infection (with or without subsequent vaccination). The median effector T cell reactivity against wildtype and Omicron NC/M/E/3A (in SFU/10 6 PBMC) was 275 and 220 for individuals with prior infection; 1 and 0 for individuals after primary series vaccination (without prior infection); All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint and 160 and 237 for individuals with prior infection and after primary series vaccination ( Figure 1E ). In individuals with prior infection or prior infection and vaccination, the magnitude of reactivity towards NC/M/E/3A was correlated with that of spike for wildtype and Omicron peptides ( Figure S1 ).

Individuals with prior infection demonstrated higher T cell responses to spike, suggesting that repeated exposure to antigen may potentially enhance cross-reactive T cell responses. We therefore assessed the impact of booster vaccination on T cell reactivity by IFN-γ ELISpot. Similar to the evaluation of pre-boost samples, overall effector T cell responses towards wildtype and Omicron spike across our study participants were comparable post-booster ( Figure 2B ). Moreover, receipt of a booster dose was associated with a 1.1log 10 SFU/10 6 PBMC increase (95% CI 0.91-1.2, p<0.001) in the magnitude of T cell response (Table S3) , with specific fold increases of 20.1 against wildtype and 20.4 against Omicron in 25 participants with paired sampling ( Figure 2C ). However, even after booster vaccination, 9.1% (4/44) participants still demonstrated >50% reduced reactivity to Omicron spike relative to wildtype. Overall, the frequency of >50% reduced effector T cell responses to the Omicron variant was more frequent than to Delta in 85 individual sample points with both measures (Fisher's exact p-value 0.023, Table S4 ).

To assess the cross-reactivity of functional CD4 + and CD8 + memory T cell responses to Omicron, we performed a 6-day carboxyfluorescein succinimidyl ester (CFSE) proliferation assay ( Figure S2 ) on samples from individuals who were vaccinated and/or previously infected and/or received booster vaccine doses (n = 33 participants) using overlapping wildtype or Omicron spike peptide pools. We felt it was important to utilize this assay given that antigen-specific proliferation has been strongly associated with functional T cell responses and cytotoxicity (Migueles et al., 2002 (Migueles et al., , 2008 Ndhlovu et al., All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint 2013). The patient cohort studied here was a subset of that used for the IFN-γ ELISpot assay, wherein 11 samples were from vaccinated individuals, 13 from previously infected and vaccinated individuals, and nine from vaccinated and boosted individuals (five of whom were also previously infected). A paired t-test demonstrated that while the magnitude of proliferative spike-specific CD4 + T cell responses did not vary by variant, proliferative CD8+ T cell responses to Omicron spike were decreased compared to wildtype in previously infected, vaccinated participants (p = 0.009) and across all study participants (p < 0.005), which is further illustrated by examination of individual patient responses ( Figure 3A , denoted in red). CD4 + proliferative responses remained largely cross-reactive to Omicron spike, with only 12% (4/33) of individuals with prior infection and/or vaccination and/or booster showing a >50% (0.3log 10 ) reduction ( Figure 3B ). A larger proportion of 39% of individuals (13/33) exhibited a decreased CD8 + T cell proliferative response to Omicron spike ( Figure 3C ). A multivariate regression analysis revealed that neither age nor sex influenced CD4 + or CD8 + T cell responses but proliferative CD8 + responses tended to be lower for Omicron vs wildtype after adjusting for all covariates and were significantly increased by booster doses (Table S5 ). These data indicate that the reduced reactivity in a subset of individuals to the Omicron spike protein is primarily observed in the CD8 + T cell compartment, although this can be enhanced with booster vaccination.

Within this cohort of individuals, we recently reported markedly reduced neutralization of Omicron following primary series vaccination, which was overcome by additional (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint SFU/10 6 PBMC (the maximal response detected among unvaccinated individuals without prior infection), 4/4 prior infected vaccinated individuals and 8/15 (no prior infection) vaccinated individuals who had low neutralization had measurable T cell responses. However, 38.9% (7/18) of individuals vaccinated with the primary series without prior infection demonstrated T cell reactivity and neutralization of the Omicron variant beneath the above-described threshold.

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint 1 0

antigens subject to mutation. In this report, we evaluated whether existing anti-SARS-CoV-2 T cell responses are cross-reactive towards the Omicron variant or differ in comparison to wildtype and the Delta variant. We found that, in aggregate, the magnitude of circulating effector T cell responses towards Omicron spike and non-spike structural proteins were conserved across variants and enhanced by additional booster vaccine doses. However, examination of individual responses revealed that a distinct proportion of individuals with prior infection and/or vaccination have substantially reduced T cell reactivity to Omicron (but not Delta). Further evaluation of the spikespecific CD4+ and CD8+ memory T cell compartment revealed a significant difference in CD8+ T cell proliferation in response to Omicron spike relative to wildtype. These findings are consistent with the modestly higher degree of antigen diversity in Omicron, although remain somewhat unexpected given that the vast majority of the spike protein remains largely sequence conserved (97.2%, i.e. 1237/1273 amino acids unchanged).

Therefore, in some individuals, it is possible that Omicron variation may mediate escape from specific HLA-restricted T cell responses induced by prior infection and vaccination.

In sum, T cell reactivity to the SARS-CoV-2 Omicron variant was preserved in most but not all prior infected and vaccinated individuals. that are critical for neutralization by antibodies (Greaney et al., 2021) . In contrast, we found that T cell reactivity was relatively preserved in most individuals against Omicron, and in many individuals with undetectable omicron neutralizing antibody responses, effector T cell responses were measurable. Previous studies have identified an association between T cell immunity and mild COVID-19 disease (Peng et al., 2020; Rydyznski Moderbacher et al., 2020; Tan et al., 2021) . In addition, in animal models of SARS-CoV-2 (McMahan et al., 2020), T cell responses appear to be important in 1 1 reducing disease acquisition and severity. Thus, the high frequency of preserved T cell responses against Omicron suggests that T cell responses may be responsible for vaccine effectiveness (and also from natural infection) against severe outcomes from Omicron infection that appear higher than predicted by absent or lower neutralization.

Prior SARS-CoV-2 infection, despite being remote, was associated with higher T cell effector and memory responses than vaccination alone, and responses were directed against both spike and non-spike proteins in contrast to being focused solely on spike.

This may reflect the impact of distinct antigen kinetics and multiple antigen exposures during infection leading to qualitatively different responses in comparison to vaccination.

The preservation of T cell reactivity to non-spike structural and accessory proteins in all individuals is likely due to the substantially reduced number of mutations within NC/M/E/3A relative to spike, suggesting that these proteins may be highly attractive for second-generation COVID-19 vaccines. In particular, vaccine strategies that induce robust memory and effector T cell responses alongside antibody responses which are collectively targeted against conserver, variant-resistant sites (Meyers et al., 2021; Nathan et al., 2021) may yield more durable T cell immunity capable of providing broad protection against future variants.

Collectively, these data provide insight into the immune mechanisms that may account for clinical observation of Omicron pathophysiology and demonstrate the contribution of vaccine boosters to enhancing cellular immunity to SARS-CoV-2 variants. These findings also support continued evaluation of second-generation vaccine approaches that induce robust T cell responses that target both variant spike and non-spike antigens in order to overcome current and future SARS-CoV-2 evolution.

Our study has some noteworthy limitations. First, this is a study of dynamic immune responses which have distinct kinetics but timing of sampling was constrained to ~6 months after primary series vaccination and sooner after booster doses. Secondly, although we included individuals with prior infection, primary series vaccination and All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint 1 2 booster vaccines with the three vaccines deployed in the USA (mRNA1273, BNT162b2 and Ad26.COV2.S), these groups cannot comprehensively capture the variables that may plausibly affect reactivity such as the variant with which individuals were infected, the wide variety of vaccines deployed globally, and the differences in the timing of additional doses. Thirdly, we employed IFN-γ ELISPOT and proliferation assays to estimate T cell responses. While these assays are highly sensitive for functionally relevant T cell responses, additional assays, such as the activation induced marker assay (Grifoni et al., 2020) or intracellular cytokine staining following peptide stimulation could also be utilized. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint

We thank Shiv Pillai, MD PhD for excellent advice on this manuscript. We thank Anand Dighe, MD, Andrea Nixon, BS, and the MGH Core Laboratory for excellent assistance with clinical SARS-CoV-2 serology testing. This work was supported by the Peter and Ann Lambertus Family Foundation. This study was supported by NIH grants P01 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. 

G.D.G has filed patent application PCT/US2021/028245.

We worked to ensure gender balance in the recruitment of human subjects. We worked to ensure ethnic or other types of diversity in the recruitment of human subjects. We worked to ensure that the study questionnaires were prepared in an inclusive way. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community. Table S1 ). 101 PBMC samples from 76 individuals were studied. 25 individuals provided samples prior to and after receipt of additional booster vaccine doses. Total (CD4 + and CD8 + ) effector T cell reactivity to SARS-CoV-2 overlapping peptide pools from wildtype, Omicron or Delta spike and from wildtype or Omicron non-spike structural and accessory proteins (nucleocapsid/membrane/envelope/ORF3A, i.e. NC/M/E/3A) was assessed by IFN-g ELISpot (the number is shown for each group). CD4 + and CD8 + memory T cell response All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint to wildtype or Omicron spike was assessed in a subset of participants by CFSE-based proliferation assay (see Figure 3 ). Numbers for each group are shown in parentheses. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Gaurav D. Gaiha (ggaiha@mgh.harvard.edu). All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint 2 0

All requests for resources and reagents should be directed to and will be fulfilled by the lead contact. All reagents will be made available on request after completion of a Materials Transfer Agreement.

All data supporting the findings of this study available within the paper and available from the lead contact upon request.

Review Board (protocol 2020P001081). Consenting ambulatory adults in Chelsea, Massachusetts were enrolled in a study of immune responses and sampled in mid-2020 or December 2021. Demographic data, information regarding prior SARS-CoV-2 testing, symptoms, and exposure was collected as was vaccine related information. Prior infection was defined by positive anti-nucleocapsid antibody testing on the Roche Elecsys SARS-CoV-2 assay performed at the MGH clinical laboratory and absence of prior positive SARS-CoV-2 PCR testing. Rates of infection in the Chelsea community were high during early SARS-CoV-2 waves (Naranbhai et al., 2021) and most participants in this study had been infected in the initial waves of infection. The duration from receipt of the final dose of the primary series (first Ad26.COV2.S, or second BNT-162b2 or mRNA-1273) and duration post booster dose were collected and included as covariates as was age and sex. Samples from unvaccinated participants were obtained in 2020 (pre-Omicron period) and the remaining samples of vaccinated, prior infected and vaccinated and boosted individuals were obtained between December 3, 2021 and December 13, 2021. In total, we include 101 samples from 76 individuals; 25 individuals provided pre-booster and post-booster samples.

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint 1 Blood was collected in heparin tubes and processed within 4 hours of collection.

Peripheral blood mononuclear cells (PBMC) were isolated by density gradient sedimentation using Lymphocyte Separation Media (Corning) as per the manufacturer's instructions and cryopreserved in freezing media consisting of heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich) containing 10% DMSO and stored in liquid nitrogen until use.

Complete overlapping 15mer Spike, Nucleocapsid, Membrane, and Envelope peptides (15mer peptide overlapping by 11 amino acids) from the SARS-CoV-2 Omicron (B.1.1.529) variant were synthesized on an automated robotic peptide synthesizers (AAPPTEC, 396 Models MBS, Omega and Apex) by using Fmoc solid-phase chemistry (Behrendt et al., 2016) on 2-chlorotrityl chloride resin (Chatzi et al., 1991) . The Cterminal amino acids were loaded using the respective Fmoc-Amino Acids in the presence of DIEA. Unreacted sites on the resin were blocked using methanol, DIEA and DCM (15:5:80 v/v). Subsequent amino acids were coupled using optimized (to generate peptides containing more than 90% of the desired full-length peptides) cycles consisting of Fmoc removal (deprotection) with 25% Piperidine in NMP followed by coupling of Fmoc-AAs using HCTU/NMM activation. Each deprotection or coupling was followed by several washes of the resin with DMF to remove excess reagents. After the peptides were assembled and the final Fmoc group removed, peptide resin was then washed with dimethylformamide, dichloromethane, and methanol three times each and air dried.

Peptides were cleaved from the solid support and deprotected using odor free cocktail (TFA/triisopropyl silane/water/DODT; 94/2.5/2.5/1.0 v/v) for 2.5h at room temperature (Teixeira et al., 2002) . Peptides were precipitated using cold methyl tertiary butyl ether (MTBE). The precipitate was washed 2 times in MTBE, dissolved in a solvent (0.1% trifluoroacetic acid in 30%Acetonitrile/70%water) followed by freeze drying. Peptides were characterized by Ultra Performance Liquid Chromatography (UPLC) and Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS). All peptides were dissolved initially in 100% DMSO at a concentration of 40 mM, prior to dilution at All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint the appropriate concentration to create protein-specific peptide pools in RPMI-1640 medium.

Peptide pools of 15mer sequences (overlapping by 11 amino acids) covering the full length of wildtype spike, nucleocapsid, membrane, envelope and ORF3A were obtained 

All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 PBMCs were suspended at 1 x 10 6 /mL in PBS and incubated at 37 o C for 20 min with 0.5 uM carboxyfluorescein succinimidyl ester (CFSE; Life Technologies). After the addition of serum and washes with PBS, cells were resuspended at 1 x 10 6 /mL and plated into 96-well U-bottom plates (Corning) at 200 uL volumes. Peptide pools were added at a final concentration of 0.25 ug/mL. On day 6, cells were harvested, washed with PBS + 2% Fetal Bovine Serum, and stained with anti-CD3-PE-Cy7 (clone SK7; BioLegend), anti-CD8 APC (clone SK1; BioLegend), anti-CD4 BV711 (clone RPA-T4;

BioLegend) and LIVE/DEAD violet viability dye (Life Technologies). Cells were washed and fixed in 2% paraformaldehyde, prior to flow cytometric analysis on a BD LSR II (BD Biosciences). A positive response was defined as one with a percentage of CD3 + CD8 + or CD3 + CD4 + CFSE low cells at least 1.5x greater than the highest of two negativecontrol wells and greater than 0.2% CD8 + or CD4 + CFSE low cells in magnitude following background subtraction. For graphical analyses, responses are plotted at a value of 0.1% CD8 + or CD4 + CFSE low cells.

Neutralization data is from our recent study in a subset of individuals described here and previously reported (Garcia-Beltran et al., 2021b) . In brief, pseudovirus neutralization titer 50 (pNT50) was calculated by taking the inverse of the serum concentration that achieved 50% neutralization of SARS-CoV-2 pseudotyped lentivirus particles entry into ACE2 expressing 293T cells (a gift from Michael Farzan). We introduced mutations corresponding to the SARS-CoV-2 variants of concern by site directed mutagenesis and confirmed clones by sequencing.

The primary statistical analysis shown in Table S3 and S5 was a multivariate regression modelling T cell response (log 10 CFU/10 6 PBMC) as the response variable, and age, sex, peptide pool, prior infection, vaccine type, duration from vaccination as covariates.

To compare proportions of individuals we used a fishers-exact test. Analyses were performed in R and figures rendered in GraphPad Prism. A p-value of < 0.05 was considered significant. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. Representative gating strategy for identification of proliferating CD3 + CD4 + and CD3 + CD8 + CFSE low T cells in response to peptide pools of interest. The gate establishing the frequency of CFSE low CD4 + or CD8 + cells was chosen based on minimizing responses in two negative-control (DMSO) wells and verified using positive control (CD3/CD28) wells. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. A B E All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 5, 2022. ; https://doi.org/10.1101/2022.01.04.21268586 doi: medRxiv preprint

Advances in Fmoc solid-phase peptide synthesis

2-Chlorotrityl chloride resin: Studies on anchoring of Fmoc-amino acids and peptide cleavage

Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity

mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant

COVID-19-neutralizing antibodies predict disease severity and survival

SARS-CoV-2 variants of concern partially escape humoral but not T cell responses in COVID-19 convalescent donors and vaccine recipients

An antibody-escape calculator for mutations to the SARS-CoV-2 receptor-binding domain

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

The Omicron variant is highly resistant against antibody-mediated neutralization -implications for control of the COVID-19 pandemic

SARS-CoV-2 Omicron variant escapes neutralization by vaccinated and convalescent sera and therapeutic monoclonal antibodies

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

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

Highly conserved, non-human-like, and cross-reactive SARS-CoV-2 T cell epitopes for COVID-19 vaccine design and validation

HIVspecific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors

Lytic granule loading of CD8+ T cells is required for HIV-infected cell elimination associated with immune control

Comparative immunogenicity and effectiveness of mRNA-1273, BNT162b2 and Ad26.COV2.S COVID-19 vaccines

Structure-guided T cell vaccine design for SARS-CoV-2 variants and sarbecoviruses

High-dimensional immunomonitoring models of HIV-1-specific CD8 T-cell responses accurately identify subjects achieving spontaneous viral control

Rapid induction of antigen-specific CD4+ T cells is associated with coordinated humoral and cellular immunity to SARS-CoV-2 mRNA vaccination

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19

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

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19. Cell

Early induction of functional SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients

The use of DODT as a non-malodorous scavenger in Fmoc-based peptide synthesis

An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes neutralization by several therapeutic monoclonal antibodies

Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa