T‐cell responses to SARS‐CoV‐2 Omicron spike epitopes with mutations after the third booster dose of an inactivated vaccine

Abstract The rapidly spreading severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) Omicron variant contains more than 30 mutations that mediate escape from antibody responses elicited by prior infection or current vaccines. Fortunately, T‐cell responses are highly conserved in most individuals, but the impacts of mutations are not clear. Here, we showed that the T‐cell responses of individuals who underwent booster vaccination with CoronaVac were largely protective against the SARS‐CoV‐2 Omicron spike protein. To specifically estimate the impact of Omicron mutations on vaccinated participants, 16 peptides derived from the spike protein of the ancestral virus or Omicron strain with mutations were used to stimulate peripheral blood mononuclear cells (PBMCs) from the volunteers. Compared with the administration of two doses of vaccine, booster vaccination substantially enhanced T‐cell activation in response to both the ancestral and Omicron epitopes, although the enhancement was slightly weakened by the Omicron mutations. Then, the peptides derived from these spike proteins were used separately to stimulate PBMCs. Interestingly, compared with the ancestral peptides, only the peptides with the G339D or N440K mutation were detected to significantly destabilize the T‐cell response. Although more participants need to be evaluated to confirm this conclusion, our study nonetheless estimates the impacts of mutations on T‐cell responses to the SARS‐CoV‐2 Omicron variant.


| INTRODUCTION
The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern Omicron (B.1.1.529), which has more than 30 mutations in the spike protein, has raised an alarm related to the control of coronavirus disease 2019. [1][2][3][4][5] Recent data have shown that the induction of a neutralizing antibody response against the Omicron variant by either vaccines or infection is drastically reduced, [5][6][7][8][9][10][11][12] while T-cell responses are largely preserved. 5,[13][14][15][16] The T-cell response to the Omicron variant is based on dozens of peptide epitopes; several groups have performed computational analyses and found that even for the Omicron strain with more than 30 mutations,~80% of the antigenic peptides recognized by T cells are identical to those of original ancestral strain. 14,[17][18][19] As most of the peptides in the overlapping peptide pools previously used to stimulate immune cells are the same as those of the ancestral strain, the impact of mutations on T cells might be underestimated. [13][14][15][16] There are three important reasons to interrogate whether the

| Ex vivo interferon-γ enzyme-linked immunospot assays
Enzyme-linked immunospot (ELISpot) assays were performed with PBMCs collected after the administration of two or three vaccine doses as previously described. 22 The assays were performed using Human IFN-γ ELISpotPRO (ALP) plates (Mabtech 3420-2AST-10) following the manufacturer's instructions. In brief, after five washes with phosphate-buffered saline (PBS) and blocking with cell culture medium for 30 min, approximately 2 × 10 5 PBMCs were added to each well. After an hour, mixed peptides or one peptide (Supporting Information:

| Multiplex cytokine profiling
The cytokine responses in the culture supernatants of PBMCs Analysis was performed using the online analysis software LEGEN-Dplex, which distinguishes between the 12 different analytes on the basis of bead size and internal dye.

| Data analysis and statistics
Flow cytometry data were analyzed using CytExpert (2.3.0.84). ELISpot plates were counted using an ELISpot Reader (AID) using the same suggested system settings for all plates. The concentrations of cytokines were analyzed with the online analysis software LEGENDplex. Statistical analyses were performed using Prism version 6 (GraphPad). Significance was analyzed by a paired parametric t-test or an unpaired two-sample parametric t-test with Welch's correlation.

| RESULTS
We collected PBMCs from 20 healthy vaccinated individuals Week 2, the percentage of activated memory T cells was not significantly different between the Omicron-and ancestral strain peptide-stimulated groups ( Figure 1C). Nevertheless, at the Week 4 timepoint, a statistically significant decrease was measured in the Omicron-stimulated group compared with the ancestral strain peptide-stimulated group (p = 0.0356, paired t-test). When we divided the participants by the age of 40 and compared the groups, the differences for both activated T cells and activated memory T cells remained nonsignificant ( Figure 1D,E). Overall, the frequency of AIM + spike-specific T cells was clearly enhanced for both the Omicron and ancestral strains after the third vaccine dose, but the Omicron mutations did weaken this enhancement.
To further investigate which mutation in the Omicron spike protein evades the T-cell response to SARS-CoV-2 epitopes, eight peptides with 17 mutations were used to separately stimulate PBMCs collected at Week 0 and Week 4. Based on sample availability, PBMCs from 14 or 16 participants were used in the ELISpot assay to detect IFN-γ-secreting cells (Figure 2A). Interestingly, compared with the ancestral peptides, only the peptides with the G339D or N440K mutation were found to significantly change the number of IFN-γ SFUs (Figure 2A). The finding that most mutations did not change T-cell reactivity was consistent with the calculation that HLA binding was well conserved for the majority of the epitopes with mutations. 14 Surprisingly, even for the peptides with three or five mutations, no significant difference in the number of SFUs was observed by paired comparison with the ancestral peptides. Notably, the number of SFUs stimulated by only a single peptide was very small, generally less than 100 per 10 6 PBMCs. T-cell responses to a single type of peptide are quite weak in most individuals, which may be the reason that significant differences were seldom detected. The small number of SFUs was suspected to be a consequence of HLA restriction.
Interestingly, the G339D mutation increased the number of SFUs, while the N440K mutation decreased this number (Figure 2A

| DISCUSSION
Encouragingly, as the vast majority of T cell epitopes are fully conserved, ancestral SARS-CoV-2-specific T cells are expected to cross-recognize the Omicron variant, which has been shown by our data and several recent works. [13][14][15][16] Previous studies reported that a third vaccine dose showed a satisfactory safety profile and induced a stronger immune response to SARS-CoV-2 and variants of concern. [26][27][28][29] Our study provides further evidence that after two doses of an inactivated vaccine, a third booster vaccination substantially enhanced T-cell responses to the spike proteins of both the ancestral strain and the Omicron variant, although the enhancement was slightly weakened by Omicron mutations, especially G339D and N440K.
The prevalence of the mutation G339D is the highest among those mutations in the receptor-binding domain region, which is 96.6% with a χ 2 test value of 0.115. 30 The single mutations G339D and N440K allow escape from a subset of neutralizing antibodies, 7 which indicates that G339D and N440K may also change the binding affinity between T-cell epitopes and the major histocompatibility complex and consequently increase the transmissibility and infectivity of the Omicron variant. We computationally assessed the predicted binding affinity of ancestral and Omicron peptides used in ELISpot assays for 12 Chinese common HLA alleles 31 using NetMHCIIpan-4.0. 32 The predicted binding affinity and ELISpot results were not consistent. More powerful tools, such as cryoelectron microscopy-based structure analysis, may be needed to further understand the impacts of mutations on the binding affinity of T cell epitopes. All authors read and approved the final manuscript.