Effects of SARS-CoV-2 Omicron BA.1 Spike Mutations on T-Cell Epitopes in Mice

T-cell immunity plays an important role in the control of SARS-CoV-2 and has a great cross-protective effect on the variants. The Omicron BA.1 variant contains more than 30 mutations in the spike and severely evades humoral immunity. To understand how Omicron BA.1 spike mutations affect cellular immunity, the T-cell epitopes of SARS-CoV-2 wild-type and Omicron BA.1 spike in BALB/c (H-2d) and C57BL/6 mice (H-2b) were mapped through IFNγ ELISpot and intracellular cytokine staining assays. The epitopes were identified and verified in splenocytes from mice vaccinated with the adenovirus type 5 vector encoding the homologous spike, and the positive peptides involved in spike mutations were tested against wide-type and Omicron BA.1 vaccines. A total of eleven T-cell epitopes of wild-type and Omicron BA.1 spike were identified in BALB/c mice, and nine were identified in C57BL/6 mice, only two of which were CD4+ T-cell epitopes and most of which were CD8+ T-cell epitopes. The A67V and Del 69-70 mutations in Omicron BA.1 spike abolished one epitope in wild-type spike, and the T478K, E484A, Q493R, G496S and H655Y mutations resulted in three new epitopes in Omicron BA.1 spike, while the Y505H mutation did not affect the epitope. These data describe the difference of T-cell epitopes in SARS-CoV-2 wild-type and Omicron BA.1 spike in H-2b and H-2d mice, providing a better understanding of the effects of Omicron BA.1 spike mutations on cellular immunity.


Introduction
The SARS-CoV-2 Omicron variant was first detected in South Africa and Botswana in November 2021 and was listed by the WHO as a variants of concern (VOC) [1]. Omicron has since expanded to more than 100 subfamilies, such as BA.1, BA.2, BA.2.12.1, BA.4 and BA.5, and become globally dominant. Omicron subvariants BQ.1, BQ.1.1 and XBB are rapidly circulating in the United States, France, Singapore and India [2]. XBB.1.5 has become the dominant strain in the United States and is highly likely to cause the next wave of global coronavirus infections [3].

Screening of T-Cell Epitopes by ELISpot
Two-dimensional matrix pools were designed for screening of the T-cell epitopes. The overlapping peptides of the WT and Omicron BA.1 spike were screened and further verified with individual peptides. The 316 overlapping peptides were coded, mixed in 70 matrix peptide pools ( Figure S1) and detected using an IFNγ ELISpot kit (Mabtech, Nacka Strand, Sweden). ELISpot plates were coated overnight at 4 • C with 5 µg/mL of anti-mouse IFNγ antibody. The antibody-coated plates were washed five times with sterile PBS and blocked with complete RPMI1640 medium for 2 h at room temperature. After blocking, 100 µL of splenocyte suspension (2 × 10 6 cells/mL) containing matrix peptide pools (1 µg/mL) or individual peptide (10 µg/mL) were added to each well. A 'no peptide' negative control was included in all assays. The plates were incubated for 18-24 h at 37 • C/5% CO 2 . Following incubation, the wells were washed five times with PBS. Biotinylated anti-mouse IFNγ was added to each well at a concentration of 1 µg/mL and incubated for 2 h at room temperature. Following three washes, streptavidin-horseradish peroxidase was added to each well and incubated for 1 h. After five washes with PBS, the colorimetric reactions were developed using 3,3 ,5,5 -tetramethylbenzidine as a substrate. Upon visualization of the spots, the reaction was stopped by rinsing in tap water. Membranes were allowed to dry overnight in the dark; then, spots were counted with an AT-Spot 3200 (SinSage Technology, Beijing, China). Results were expressed as the number of spot-forming cells (SFCs) per 10 6 splenocytes and considered positive if the magnitude of the response was SFCs > 50 and the magnitude of the positive was 2-fold greater than the control well.

Intracellular Cytokine Staining
An intracellular cytokine staining (ICS) assay was conducted as previously described [27]. Briefly, the splenocytes were stimulated for 6 h at 37 • C with 1 µg/mL of peptide pools, 10 µg/mL of a selected single peptide or the same volume of DMSO as the background control and with BD GolgiStop TM to block cytokine secretion. A positive control with ionomycin and PMA was included in each assay. Following stimulation, the cells were washed and stained with Near-IR viability dye (Thermo Fisher Science, Waltham, MA, USA) for 20 min to exclude dead cells from data analysis. After one wash with PBS, the splenocytes were incubated with a mixture of antibodies against lineage markers, including anti-CD3 PerCP-Cy5.5 (clone 17A2), anti-CD4 Alexa Fluor 700 (clone RM4-5) and anti-CD8 FITC (clone 5H10-1). After one wash with PBS, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences, San Diego, CA, USA), washed with Perm/Wash buffer (BD Biosciences, San Diego, CA, USA) and incubated with anti-IFNγ PE (clone XMG1.2). The cells were washed successively with Perm/Wash buffer and PBS, an data were acquired on a FACS Canto TM (BD Biosciences, San Diego, CA, USA).

Ad5-Spike-WT and Ad5-Spike-BA.1 Induced Robust but Different Cellular Immune Responses in Mice
More than 30 amino acid mutations were identified within the spike of the Omicron BA.1 variant ( Figure 1A). In order to test the immune response caused by the mutations, BALB/c and C57BL/6 mice were vaccinated with Ad5-Spike-WT or Ad5-Spike-BA.1, and the cellular immune response of the vaccine candidates against both WT and Omicron BA.1 spike were detected by IFNγ ELISpot and ICS assays. Both the Ad5-Spike-WT and Ad5-Spike-BA.1 induced robust IFNγ responses specific to WT and Omicron BA.1 spike ( Figure 1B-G). In BALB/c mice, Ad5-Spike-WT induced more IFNγ spots specific to WT spike, and Ad5-Spike-BA.1 induced more IFNγ spots specific to Omicron BA.1 spike ( Figure 1B). These differences were reflected in a higher CD4 + IFNγ response specific to WT spike in mice vaccinated with Ad5-Spike-WT and a higher CD8 + IFNγ response specific to Omicron BA.1 spike in mice vaccinated with Ad5-Spike-BA.1 ( Figure 1C,D). However, in C57BL/6 mice, both Ad5-Spike-WT and Ad5-Spike-BA.1 induced more IFNγ spots specific to Omicron BA.1 spike ( Figure 1E). Furthermore, a higher CD8 + IFNγ response specific to Omicron BA.1 spike ( Figure 1F) and a higher CD4 + IFNγ response specific to WT spike ( Figure 1G) were induced by Ad5-Spike-WT. The difference in the cellular immune response induced by Ad5-Spike-WT and Ad5-Spike-BA.1 indicates that the mutations of the spike caused changes in T-cell epitopes.

Characterization of the CD8 + and CD4 + T-Cell Epitopes
To further determine which T-cell subsets were activated by these peptides, the splenocytes were stimulated with a single peptide, and the IFNγ secretion the antigen-specific CD8 + and CD4 + T cells was examined using an ICS assay. For BALB/c mice, W61-75 and W353-367 stimulated CD4 + T cells, while W269-283, W501-515, W505-519, W521-535, W525-539, W529-543, W533-547 and W1049-1063 stimulated CD8 + T cells (Figures 3A and S6). However, all the responsive peptides in Omicron BA.1 spike were recognized as CD8 + T cell epitopes ( Figures 3B and S6). For C57BL/6 mice, the CD4 + T-cell-specific peptide in WT spike was W61-75; the CD8 + T-cell-specific peptides in WT spike were W257-271, W261-275, W389-403, W505-519, W509-523, W533-547, W537-551 and W741-755 ( Figures 3C and S7); and all the responsive peptides in Omicron BA.1 spike were CD8 + T-cell-specific ( Figures 3D and S7). Taken together, these results demonstrate that both WT To further identify the exact short epitopes recognized by CD8 + T cells within th overlapping 15-mer peptides, we predicted the potential 9-mer epitopes for MHC-I bind ing in the Immune Epitope Database (http://tools.iedb.org/mhci/, accessed on 8 Augus 2022) as determined by IFNγ- ELISpot (Figures 4, S8 and S9). A total of three H-2D d -, two H-2K d -, four H-2L d -, four H-2D b -and four H-2K b -restricted epitopes were identified (Tabl 1). Two I-E d -restricted epitopes and one I-A b -restricted epitope with 15 amino acids wer also identified (Table 1). For the T-cell epitopes of WT spike, W61-75 and W505-513 in volved Omicron BA.1 mutations, and the rest of the epitopes were unaffected in BALB/ mice, while only W61-75 was involved in Omicron BA.1 mutations in C57BL/6 mice. Thi suggests that most of the T-cell epitopes in BALB/c and C57BL/6 mice are highly preserved among WT and Omicron BA.1. We also identified some T-cell epitopes only present in Omicron BA.1 spike, including O475-483, O486-494 and O502-510 in BALB/c mice and O645-653 in C57BL/6 mice, which were derived from Omicron BA.1 spike mutations. To further identify the exact short epitopes recognized by CD8 + T cells within the overlapping 15-mer peptides, we predicted the potential 9-mer epitopes for MHC-I binding in the Immune Epitope Database (http://tools.iedb.org/mhci/, accessed on 8 August 2022) as determined by IFNγ-ELISpot (Figure 4, Figures S8 and S9). A total of three H-2D d -, two H-2K d -, four H-2L d -, four H-2D b -and four H-2K b -restricted epitopes were identified (Table 1). Two I-E d -restricted epitopes and one I-A b -restricted epitope with 15 amino acids were also identified ( Table 1). For the T-cell epitopes of WT spike, W61-75 and W505-513 involved Omicron BA.1 mutations, and the rest of the epitopes were unaffected in BALB/c mice, while only W61-75 was involved in Omicron BA.1 mutations in C57BL/6 mice. This suggests that most of the T-cell epitopes in BALB/c and C57BL/6 mice are highly preserved among WT and Omicron BA.1. We also identified some T-cell epitopes only present in Omicron BA.1 spike, including O475-483, O486-494 and O502-510 in BALB/c mice and O645-653 in C57BL/6 mice, which were derived from Omicron BA.1 spike mutations.

The Effect of Spike Mutations on T-Cell Epitopes
T-cell epitopes of WT spike were compared with those identified in Omicron BA.1 spike in BALB/c and C57BL/6 mice. The epitopes involving Omicron BA.1 spike mutations are summarized in Figure 5A. Those peptides were further verified in the mice vaccinated with Ad5-Spike-WT and Ad5-Spike-BA.1 to determine the effect of amino acid mutation on the epitopes. In Ad5-Spike-WT-immunized BALB/c and C57BL/6 mice, W61-75 was the core sequence of dominant CD4 + T-cell epitopes of WT spike but was unresponsive in Ad5-Spike-BA.1-immunized mice, whereas O61-73, the corresponding peptide of W61-75 in Omicron BA.1 spike, was not responsive in either Ad5-Spike-WT-or Ad5-Spike-BA.1immunized mice ( Figure 5B,C), implying that the A67V and Del 69-70 mutation abrogated this T-cell epitope. On the other hand, Omicron BA.1 spike mutations formed new epitopes that were not present in WT spike. The T478K and E484A mutations in peptide W478-486 resulted in a new H-2L d epitope, O475-483, and the Q493R and G496S mutations in W489-497 resulted in a new H-2L d epitope, O486-494, in BALB/c mice ( Figure 4B). Similarly, the H655Y mutation in W648-656 resulted in a new peptide, O645-653, which induced T-cell response in C57BL/6 mice vaccinated with Ad5-Spike-BA.1 ( Figure 5C). Furthermore, some mutations had no effect on the T-cell responses, for example, there was no difference in the induction of T-cell responses between W505-513 and O502-510, indicating that Y505H mutation did not affect the T-cell epitope ( Figure 5B). in W489-497 resulted in a new H-2L d epitope, O486-494, in BALB/c mice ( Figure 4B). Similarly, the H655Y mutation in W648-656 resulted in a new peptide, O645-653, which induced T-cell response in C57BL/6 mice vaccinated with Ad5-Spike-BA.1 ( Figure 5C). Furthermore, some mutations had no effect on the T-cell responses, for example, there was no difference in the induction of T-cell responses between W505-513 and O502-510, indicating that Y505H mutation did not affect the T-cell epitope ( Figure 5B).

Discussion
In this study, we found that the cellular immune responses of Ad5-Spike-BA.1 in BALB/c and C57BL/6 mice were robust but differed from those of Ad5-Spike-WT, and the T-cell epitopes of the spike in WT and Omicron BA.1 variant were identified.
Nine MHC-I-restricted epitopes and two MHC-II-restricted epitopes in BALB/c mice, as well as eight MHC-I-restricted epitopes and one MHC-II-restricted epitope in C57BL/6 mice, were identified in the current study. MHC-I-restricted epitopes account for the majority, which is consistent with the T-cell response of the Ad5-vectored SARS-CoV-2 vaccine in mice, i.e., that CD8 + T cells dominate the spike-specific response [27]. This phenomenon was also reported in the ChAdOx1-vectored SARS-CoV-2 vaccine and mRNA vaccines [28][29][30]. However, those vaccines elicited a robust CD4 + T-cell response in humans [7,31,32]. The difference in T-cell response between mice and humans may be due to differences in the immune system and the diversity of MHC molecules between species.
There are more than 30 mutations on the Omicron BA.1 spike, almost half of which are located in the RBD. These mutations in the spike largely attenuated the protective ability of humoral immunity developed against previous strains. However, several studies suggested that in donors who were vaccinated with wild-type spike or infected by previous strains, the T-cell responses against the Omicron variant were largely maintained. At the epitope level, T-cell epitopes of wild-type SARS-CoV-2 are considerably preserved across major Omicron subvariants [33][34][35]. The currently circulating XBB.1.5, BF.7 and BQ.1 variants exhibit stronger humoral immune evasion than BA.1, but it does not have more mutations against the T-cell epitope in mice.
Antigen recognition by T cells involves the interaction of three classes of molecules: MHC, T-cell epitopes (antigenic peptides) and T-cell receptors. T-cell epitopes generally have two or more sites that bind to specific MHC molecules, and these amino acids bind to MHC molecules via hydrogen bonds and are then recognized by T-cell receptors. BALB/c and C57BL/6 mice possess H-2K/D/L d and H-2K/D b MHC class I molecules, respectively, and no identical MHC-I-restricted epitope of the SARS-CoV-2 Spike was found between the two mice; however, an identical MHC-II-restricted epitope of SARS-CoV-2 spike was identified. This I-A/E d -and I-A b -restricted epitope, W61-75, has also been described to show T-cell responsiveness in humans [24]. Peptides that bind to MHC class II molecules contain an internal sequence of 7 to 10 amino acids that provides the major contact points, and the class II pocket shows fewer restricted amino acid sequence preferences. Furthermore, the hydrogen bonds between the backbone of the peptide and the class II molecule are distributed throughout the binding site rather than being clustered predominantly at the ends of the site. It is likely that the internal 7~10 amino acids of W61-75 can provide major contact points that can interact well with MHC-II molecules of both mice and humans.
Mutations at different locations on the epitope have different effects on the function of the T-cell epitopes. For example, the Y505H mutation, which is localized on the first amino acid position of the peptide, does not affect the T-cell response. However, T478K, E484A, Q493R and G496S mutations form two new epitopes in BALB/c mice, and the H655Y mutation forms one new epitope in C57BL/6 mice. It is likely that those mutations contribute important anchor residues to the epitopes. Some mutations cause T-cell epitope inactivation, A67V and Del 69-70 resulted in the incapacitation of W61-75 in both BALB/c and C57BL/6 mice in this study, as well as in humans [24]. Similarly, one P272L mutation of wild-type CD8 + T-cell epitopes, Spike269-277 YLQPRTFLL, allows the virus to evade T-cell responses in the HLA A*02 convalescent patients and individuals vaccinated against SARS-CoV-2 [36].
Some CD8 + T-cell epitopes identified in our work have been previously reported in mice infected with SARS-CoV-2 or vaccinated with the SARS-CoV-2 Spike RBD subunit vaccine [37,38]. The conserved Spike539-546 VNFNFNGL and Spike511-518 VVLSFELL are common CD8 + T-cell epitopes within SARS-CoV-2 and SARS-CoV and were also found in the C57BL/6 mice and BALB/c mice vaccinated with Ad5-Spike-WT and Ad5-Spike-BA.1 in our work. Spike539-546 VNFNFNGL was validated to be fully protective in K18-ACE2 mice infected with SARS-CoV-2 [39]. This suggests that these CD8 + T-cell epitopes identified by viral infection or vaccination with different types of vaccine were consistent and that these conserved T-cell epitopes play an important role in cross protection against SARS-CoV-like strains.
Previous infection usually negatively impacts the response to a subsequent infection with pathogens sharing cross-reactive antigens. The initial immune background places restrictions on the antibody response induced by a subsequent SARS-CoV-2 variant [40][41][42], but it seems to show limited impact on recalling CD8 + T-cell responses [43]. The spike mutation of Omicron BA.1 induced two new MHC-I-restricted epitopes in BALB/c mice and one new MHC-I-restricted epitope in C57BL/6 mice. It is likely that a bivalent SARS-CoV-2 vaccine containing Omicron spike may induce a broader T-cell response. However, the T-cell response against the new Omicron epitopes after previous exposure or vaccination with WT spike was not tested in this study, and the effect of Omicron spike mutations on T-cell epitopes in humans needs to be studied.
In summary, we identified the T-cell epitopes of SARS-CoV-2 WT and Omicron BA.1 spike in BALB/c and C57BL/6 mice, and the results serve contribute to our understanding of the effects of Omicron BA.1 spike mutations on T-cell immunity.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/v15030763/s1, Figure S1: Arrangement of two-dimensional peptide matrix; Figure S2. Mapping of WT spike-specific T-cell epitopes in BALB/c mice; Figure S3. Mapping of Omicron BA.1 spike-specific T-cell epitopes in BALB/c mice; Figure S4. Mapping of WT spikespecific T-cell epitopes in C57BL/6 mice; Figure S5. Mapping of Omicron BA.1 spike-specific T-cell epitopes in C57BL/6 mice; Figure S6. Representative flow cytometric plots of the responsive peptides identified in BALB/c mice; Figure S7. Representative flow cytometric plots of the responsive peptides identified in C57BL/6 mice; Figure S8. Identification of the exact CD8 + T-cell epitopes in BALB/c mice; Figure S9. Identification of the exact CD8 + T-cell epitopes in C57BL/6 mice. Informed Consent Statement: Not applicable.
Data Availability Statement: All data required to interpret the data are provided in the main document or the Supplemental Materials. Further data are available from the corresponding author upon reasonable request.