A Murine CD8+ T Cell Epitope Identified in the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein

The ongoing COVID-19 pandemic caused by SARS-CoV-2 has posed a devastating threat worldwide. The receptor-binding domain (RBD) of the spike protein is one of the most important antigens for SARS-CoV-2 vaccines, while the analysis of CD8 cytotoxic T lymphocyte activity in preclinical studies using mouse models is critical for evaluating vaccine efficacy. Here, we immunized C57BL/6 wild-type mice and transgenic mice expressing human angiotensin-converting enzyme 2 (ACE2) with the SARS-CoV-2 RBD protein to evaluate the IFN-γ-producing T cells in the splenocytes of the immunized mice using an overlapping peptide pool by an enzyme-linked immunospot assay and flow cytometry. We identified SARS-CoV-2 S395–404 as a major histocompatibility complex (MHC) class I-restricted epitope for the RBD-specific CD8 T cell responses in C57BL/6 mice.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen of human coronavirus disease 2019 (COVID-19) [1,2]. COVID-19 was declared as a pandemic by the WHO in 2020 and has inflicted tremendous negative impacts on global health, society, and economic institutions. As vaccination is considered to be one of the most effective methods for preventing the rapid global spread of this disease, many researchers and companies in the world are attempting to develop safe and effective COVID-19 vaccines [3]. Most COVID-19 vaccine candidates have focused on the SARS-CoV-2 spike protein or the receptor-binding domain (RBD) of the spike protein as the vaccine antigen [4]. The SARS-CoV-2 RBD primarily binds to angiotensin-converting enzyme 2 (ACE2) and facilitates the entry of the virus into host cells [5]. The protective efficacy of the vaccine is conferred by the induction of humoral immunity such as neutralizing antibodies and cellmediated immunity. CD8 cytotoxic T lymphocytes (CTLs) play a critical role in eliminating virus-infected cells, thereby leading to the effective clearance of viral infection. In particular, CTL activity is important to provide long-term protection from reinfection of the virus [6]. Recently, a clinical report also showed the importance of T cell immunity in COVID-19 recovery [7]. Of patients with severe COVID-19, a significantly higher expression of surface markers for T cell activation was observed in the recovering group than the severe group, whereas no significant differences were observed for neutralizing antibodies in both groups. To analyze the CTL activity induced by SARS-CoV-2 vaccines, the overlapping peptide pool for T cell stimulation was used due to the CTL epitopes of SARS-CoV-2 presently being unidentified [8][9][10]. A recent study reported SARS-CoV-2 S 526-533 as a CTL epitope in BALB/c mice, through the experimental evaluation of programming-predicted peptides [11]. C57BL/6 mice have been widely used for the evaluation of SARS-CoV-2 vaccines [8,9]. Transgenic mice with a C57BL/6 genetic background expressing human ACE2 (hACE2) are currently being used as an animal model of SARS-CoV-2 study [12][13][14][15]; however, the CTL epitope in C57BL/6 mice has not yet been identified. Additionally, many experimental approaches aimed at quantifying SARS-CoV-2-specific CTL activity have used RBD peptide pools, which are associated with limitations such as high cost and time-consuming synthesis of peptides [8,10]. Thus, finding a murine CTL epitope is important to resolve the limitations and to identify a peptide that can be used for exactly analyzing SARS-CoV-2-specific CD8 T cell activity when evaluating SARS-CoV-2 vaccine candidates in an animal model. In this study, we identified a murine CD8 T cell epitope of the SARS-CoV-2 spike RBD using C57BL/6 wild-type and hACE2-expressing transgenic (K18-hACE2) mice. To our knowledge, this is the first report to indicate that SARS-CoV-2 S 395-404 is a CD8 T cell epitope in the RBD of the SARS-CoV-2 spike protein in a C57BL/6 mouse model.

Mice
C57BL/6 wild-type (OrientBio, Gyeonggi-do, Korea) and K18-hACE2 transgenic mice (Jackson Laboratory) were housed in a specific pathogen-free facility at the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Mouse care and experiments were reviewed and conducted under guidelines of the Institutional Animal Care and Use Committee of KRIBB (KRIBB-AEC-20279).

Immunization
Six-to 10-week-old female mice were randomly distributed in groups of 3 to 5 mice and intramuscularly (i.m.) immunized with 5 µg of RBD recombinant protein combined with SAS on days 0 and 14. In a separate experiment, wild-type mice were immunized i.m. with 10 µg of OVA protein plus SAS twice, with a two-week interval. One week after the last immunization, mononuclear splenocytes were generated by mincing whole mouse spleens, followed by the lysis of red blood cells and 70-µm filtration. Splenocytes were cultured in RPMI 1640 medium plus 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Dublin, Ireland).

Flow Cytometry Intracellular Cytokine Staining
Splenocytes at 1 × 10 6 cells/well were exposed to an RBD peptide pool (2 µg/mL for each peptide) or 5 µg/mL of each peptide in the presence of a protein transport inhibitor containing brefeldin A (BD Biosciences) for 12 h at 37 • C. Intracellular cytokine staining was performed according to a protocol modified from a previous paper [8]. The treated cells were incubated with LIVE/DEAD Fixable Red Dead Cell stain (Invitrogen) for 20 min at room temperature. The cells were washed, incubated with an anti-CD16/32 antibody (2.4G2) solution to block the Fc receptor, and stained for surface markers (CD3ε, CD4, and CD8 molecules), followed by intracellular IFN-γ cytokine staining using the Foxp3/transcription factor buffer set (eBioscience). The antibodies used were anti-CD3ε BUV737 (17A2), anti-CD4 APC (RM4-5), anti-CD8α PE-Cy7 (53-6.7), and anti-IFN-γ BV650 (XMG1.2). All the antibodies were obtained from BD Biosciences. The stained cells were acquired on a FACS Aria Fusion (BD Biosciences) and were analyzed via FlowJo TM V10 (TreeStar, Ashland, OR, USA).

Statistical Analysis
All the analyses were performed using the PRISM software 9.0.0 (GraphPad, San Diego, CA, USA), and p values less than 0.05 (p < 0.05) were considered to be statistically significant.

SARS-CoV-2 S 391-405 Peptide Facilitates IFN-γ Production of CD8 T Cells in Splenocytes from Adjuvanted SARS-CoV-2 Spike RBD-Immunized Mice
To identify the CTL epitope(s) of the SARS-CoV-2 RBD, C57BL/6 mice (n = 5) were i.m. immunized twice with SARS-CoV-2 RBD recombinant protein along with SAS-composed of monophosphoryl lipid A and squalene-with a two-week interval. One week after the last immunization, an IFN-γ ELISPOT assay was performed by stimulating the splenocytes obtained from immunized mice with an RBD peptide pool of 42 synthetic RBD peptides with 15-mers overlapping by 5 aa (Table 1). As shown in Figure 1A, the number of IFN-γ SFUs was significantly increased in cells treated with 15th peptide out of 42 peptides (CFTNVYADSFVIRGD, corresponding to S 391-405 ) (202 ± 96 SFUs) compared to that in the medium control (2 ± 3 SFUs) (p < 0.0001). The level of increase provoked by treatment with the S 391-405 peptide was similar to the SFU values achieved via stimulation with the RBD peptide pool (214 ± 95 SFUs) (p < 0.0001). However, none of the other 41 individual RBD peptides induced the production of IFN-γ SFUs.

SARS-CoV-2 S395-404 Acts as the MHC Class I H-2K b /D b -Restricted Minimal CTL Epitope of SARS-CoV-2 Spike RBD
As CD8 CTLs recognize foreign antigen peptides of 8-11 aa residues, we attempted to identify the minimum length of CTL epitope that could be recognized by major histocompatibility complex (MHC) class I on CD8 T cells. Splenocytes obtained from the RBD- To confirm that S 391-405 -induced IFN-γ production was dependent on CD8 T cells but not CD4 T cells, splenocytes were subjected to intracellular IFN-γ staining. The gating strategy for flow cytometry employed is depicted in Figure 1B. In line with our expectations, the percentages of IFN-γ + CD8 T cells were almost 1.7-fold higher in the S 391-405 -treated cells (1.25 ± 0.07%) than the medium-treated cells (0.80 ± 0.25%) (p = 0.0044) ( Figure 1C). However, the percentages of IFN-γ + CD4 T cells were similar between the S 391-405 -and medium-treated cells (p = 0.5667). Next, we assessed the SARS-CoV-2 RBD-specific CTL activity using K18-hACE2 transgenic mice (C57BL/6 background), which have been reported as an animal model suitable for SARS-CoV-2 studies [14]. When stimulating splenocytes from the RBD protein-immunized K18-hACE2 transgenic mice (n = 3) with the RBD peptide pool, the IFN-γ SFU numbers were also observed to be higher in the S 391-405 peptide-stimulated cells (240 ± 64 SFUs) than in the control cells (5 ± 3 SFUs) (p = 0.0068). The extent of this finding is similar to the effect of stimulation with the RBD peptide pool (252 ± 81 SFUs) (p = 0.0053) ( Figure 1D). These results suggest that the SARS-CoV-2 S 391-405 peptide contains CTL epitope(s) in the C57BL/6 mouse model.
The SARS-CoV-2 S 526-533 peptide (GPKKSTNL) was recently reported to be the H-2K drestricted CTL epitope in BALB/c mice [11]. However, the S 526-533 -containing peptide #42 did not induce generation of IFN-γ + cells from immunized C57BL/6 mice, as shown in Figure 1A, suggesting that SARS-CoV-2 S 395-404 might be a unique CTL epitope of the SARS-CoV-2 S RBD in the C57BL/6 mouse model. Similarly to our results, some peptides, including glycoprotein 33-43 of lymphocytic choriomeningitis virus [18] and the ovalbumin peptide (OVA 257-264 ) [16], have been reported to be the H-2K b /D b -binding epitope. Further studies are needed to measure the binding affinity of SARS-CoV-2 S 395-404 peptide to MHC class I molecules.
Various domains within SARS-CoV S protein are considered SARS-CoV-2 vaccine targets. RBD domain is one of the important antigens for SARS-CoV-2 vaccines and several RBD-based vaccine candidates have been tested in clinical trials [19]. Of them, lipid nanoparticle-formulated RBD mRNA vaccine candidates are reported to induce both neutralizing antibody response and T cell activation in preclinical [20,21] and clinical fields [22]. Aluminum-adjuvanted RBD vaccine candidate is reported to increase neutralizing antibody response rather than T cell immunity in mice, rabbits and non-human primates [23]. A recombinant subunit vaccine candidate containing the RBD of SARS-CoV-2 and the Fc fragment of human lgG is shown that increase neutralizing antibody response only in a mouse model [24]. Several studies have reported that SARS-CoV-specific CD8 CTLs substantially protected against SARS-CoV infection in mice [25] and were increased in mice that were vaccinated with SARS-CoV S DNA as the vaccine antigen [26]. As an increase in CTL activity specific to SARS-CoV-2 has been reported in the peripheral blood mononuclear cells from COVID-19 convalescent patients [27], SARS-CoV-2-specific CTLs may have a protective role in SARS-CoV-2 infection. Translation of the vaccine immunity in human subjects may be difficult to accurately predict from animal models, but animal models is most useful in stratifying the vaccine candidates through careful evaluation of vaccine efficacy before clinical trials [28]. Many experimental approaches aimed at quantifying SARS-CoV-2-specific CTL activity have used RBD peptide pools having limitations such as expensive and time-consuming synthesis of peptides [8,10].
Our findings would be useful for resolving these problems and could be beneficial for evaluating SARS-CoV-2 spike RBD-specific CTL responses and studying T cell functions. Moreover, these findings may be helpful for the generation of a tetrameric MHC-peptide complex that can be used to directly quantify and visualize SARS-CoV-2 S RBD-specific T cells [29]. Taken together, the identification of SARS-CoV-2 S 395-404 as the CTL epitope may facilitate the evaluation of CTL activity of RBD-based COVID-19 vaccine candidates in a mouse model and the investigation of CTL function in SARS-CoV-2 infection.

Conclusions
Identification of the SARS-CoV-2 spike RBD-specific CTL epitope is necessary to exactly analyze SARS-CoV-2-specific CTL activity and to solve problems of costly and time-consuming peptide synthesis for peptide pools. Our finding of SARS-CoV-2 S 395-404 as the CTL epitope is helpful in the study of SARS-CoV-2-specific CTL functions and the evaluation of CTL activity of RBD-based COVID-19 vaccine candidates using a mouse model. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.