The prior existence of human ACE2 protein–expressing mice used to study SARS-CoV and the rapid development of mouse-adapted virus strains have allowed the study of SARS-CoV-2 in mice, even as we are still learning about its natural pathology in humans. With myriad genetically altered strains on the C57BL/6 background and the abundance of immunological reagents available to interrogate its immune responses, the C57BL/6 mice may provide useful insight into the immunology of SARS-CoV-2 infection and vaccination. To conduct more detailed studies on their T cell responses to vaccines and infection, the epitopes eliciting those responses must be characterized in further detail. In this study, we mapped CD8 T cell epitopes within the receptor binding domain of the SARS-CoV-2 spike protein in C57BL/6 mice. Our study identified five major CD8 T cell epitopes in immunized C57BL/6 mice, including one, VVLSFELL, presented by H-2Kb and common between SARS-CoV and SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as one of the most devastating pandemics in a century. The global response to this threat has been swift, leading to the development of multiple safe and efficacious vaccines (13).

The primary target for SARS-CoV-2 vaccine studies in humans is the spike (S) protein (4), the surface protein on coronaviruses essential for Ab-mediated neutralization of viral particles. Although the two mRNA-based vaccines now approved by emergency use authorization by the U.S. Food and Drug Administration elicit strong Ab responses, they also elicit CD8 T cell responses to the S protein (5, 6), as do S-encoding adenoviral vectors of other leading vaccine candidates (7, 8). Future studies may define the protective effect of CD8 T cell responses, especially in the latter. Indeed, in a recent study of COVID-19 patients, CD4 and CD8 T cell responses were independently associated with less severe disease (9).

Despite the availability of multiple mouse models of disease, mechanistic studies into the roles for T cells in vaccine-mediated protection and immunity derived from natural infection have been hampered by limited knowledge of the SARS-CoV-2 Ags targeted by CD8 T cells. To define the epitopes contained within the receptor binding domain (RBD) of the S protein, we used a subunit vaccine platform composed of recombinant RBD protein Ag in combination with an adjuvant containing agonistic anti-CD40 Ab and the TLR3 agonist poly(I:C) (10). With peptide stimulation and subsequent cytokine staining, we identified five major and two minor CD8 T cell epitopes in immunized C57BL/6 mice. Furthermore, we defined the MHC class I (MHC-I) restriction as H-2Kb for a peptide epitope that is shared between both SARS-CoV and SARS-CoV-2.

All experiments involving mice were conducted following protocols approved by the University of Colorado Institutional Animal Care and Use Committee according to guidelines provided by the Association for Assessment and Accreditation of Laboratory Animal Care. C57BL/6 mice were obtained from the Jackson Laboratory and were subsequently bred in specific pathogen–free facilities at the University of Colorado Anschutz Medical Campus. Experiments were performed in 6- to 12-wk-old female mice. Mice were immunized via tail vein injection with 100 μg or 200 μg, of SARS-CoV-2 S RBD protein plus adjuvant. SARS-CoV-2 RBD protein (Wuhan-Hu-1; GenBank identifier: MT380724.1) was expressed by transfection of Expi293 cells with a His-tagged vector (a gift from F. Krammer, Icahn School of Medicine at Mount Sinai, New York, NY) (11) and subsequently purified from cell culture supernatants by the University of Colorado Cell Technologies Shared Resource. Immunizations were adjuvanted with 40 μg of poly(I:C) (Invivogen) and 40 μg of anti-CD40 (clone FGK4.5; Bio X Cell). Vaccines were made immediately prior to immunization and injected in a total volume of 200 μl.

To determine the MHC-I restriction of SARS-CoV-2 peptide epitopes shown to induce RBD-specific CD8 T cell responses in immunized mice, we employed the murine TAP-deficient RMA-S lymphoma cell line, which is derived from C57BL/6 mice (12, 13). RMA-S cells were cultured overnight at 27°C to stabilize unloaded MHC-I H-2Db and H-2Kb on the cell surface. RMA-S cells containing peptide-empty H-2Db and H-2Kb were coincubated with the indicated CD8 T cell peptides at 10 μM for 5 h at 37°C. We tested the 15-mer peptides representing the five major SARS-CoV-2 RBD epitopes revealed in these studies (S1–14,319, S337–351, S401–415, S477–491, S505–519/509–523) and the 8-mer S511–518. The following peptides with known H-2Kb, H-2Db, and H-2Kb/H-2Db restriction, respectively, were included as controls: OVA257–264, LCMV NP396–404, and LCMV GP33–41. After 5 h at 37°C, MHC-I stabilization was quantified by flow cytometry using anti-mouse Abs directed against H-2Kb (clone AF6-14-8) and H-2Db (clone 28-14-8).

Seven days after immunization, single-cell suspensions generated from spleens were subjected to ACK RBC lysis and counted using a Vi-CELL automated cell counter (Beckman Coulter). For in vitro stimulation assays, 1 × 106 cells were incubated with 1 μg/ml peptide and 3 μg/ml brefeldin A for 5 h at 37°C in complete media (RPMI 1640 containing 10% FBS, 10 mM HEPES, 0.1 mM β-ME, 0.1 mM nonessential amino acids, 0.1 mM sodium pyruvate, 2 mM l-glutamine, and penicillin–streptomycin). After stimulation, cells were surfaced-stained with BV421 CD8α (clone 53.67; BioLegend), FITC CD4 (GK1.5; BioLegend), PE-Cy7 B220 (clone RA3-6B2; Tonbo Biosciences), and a fixable viability dye (Ghost Dye Red 780; Tonbo Biosciences) for 10 min at room temperature. After staining for surface Ags, cells were fixed and permeabilized with Foxp3 fixation/permeabilization buffers (Tonbo Biosciences) for 15 min at room temperature. After fixation and permeabilization, cells were washed in perm/wash buffer and stained for intracellular cytokines using APC IFN gamma (XMG1.2; Tonbo Biosciences) and PE TNF-α (MP6-XT22; BD Biosciences) diluted in perm/wash buffer for 30 min at room temperature. After a final wash, flow cytometry data were acquired on a four-laser (405, 488, 561, 638 nm) CytoFLEX S Flow Cytometer (Beckman Coulter), and analysis was performed using FlowJo (version 10.7.1; BD Biosciences).

Crude preparations of 58 peptides covering the SARS-CoV-2 S RBD protein (GenBank identifier: MT380724.1), derived from isolate Wuhan-Hu-1, were generated (ChinaPeptides), comprising 15-mer peptides overlapping by 11 aa. Highly purified (>96% purity) VVSLFELL peptide was also prepared (ChinaPeptides).

Prism (version 9.01, GraphPad) was used to plot data and perform one-way ANOVA tests with Dunnett multiple comparisons test to compare all values to stimulation with an irrelevant peptide (HSVgB498–504).

One week following vaccination via i.v. injection with 100 μg of purified, rSARS-CoV-2 RBD protein adjuvanted with poly(I:C) and anti-CD40, splenic CD4 and CD8 T cells from C57BL/6 mice were evaluated by ex vivo peptide restimulation and subsequent intracellular cytokine staining for IFN-γ and TNF-α and flow cytometric analysis. Cells were stimulated using a peptide library of 15-mers, overlapping by 11 aa, covering the entire RBD protein (Table I). No CD4 T cell responses to RBD peptides were revealed for C57BL/6 mice by this analysis; however, several major CD8 T cell epitopes were identified. Five peptides were determined to generate statistically significant IFN-γ responses in a one-way ANOVA analysis, including S1–14,319, S337–351, S401–415, S477–491, and S505–519/509–523 (Fig. 1A). The latter sequences, spanning S505–523, aligned with a previously identified SARS-CoV CD8 T cell epitope, VVLSFELL (14). Using this same 8-mer sequence, S511–518 (511*) was determined to be the minimal epitope for SARS-CoV-2 (Fig. 1A). Two additional minor epitopes were confirmed in an experiment in which the Ag dose was increased to 200 μg (Fig. 1B). In this experiment, the three strongest epitopes each elicited IFN-γ production in roughly 3% of CD8 T cells each, whereas S529–343 and S389–403 elicited significant but relatively modest CD8 T cell responses at ∼0.3% of CD8 T cells. Representative flow cytometry plots show that most of the CD8 T cells responding to peptide restimulation stain positive for both IFN-γ and TNF-α, with negligible background cytokine production in negative control wells (stimulated with HSVgB498–505) (Fig. 1C).

FIGURE 1.

Epitope mapping of CD8 T cell responses to SARS-CoV-2 RBD protein in C57BL/6 mice. Five mice were immunized with RBD protein plus adjuvant, and their spleens were harvested one week later. (A) The percentage of CD8 T cells staining for IFN-γ after a 5-h incubation with individual 15-mer peptides spanning SARS-CoV-2 RBD. Responses that were significantly greater than those induced by an irrelevant peptide (HSVgB498–504), as determined by Dunnett multiple comparisons test. (B) The percentage of CD8 T cells staining for IFN-γ for the six potential minor epitopes and three of the major epitopes identified in (A) in mice immunized with 200 μg of RBD plus adjuvant. (C) Representative intracellular IFN-γ and TNF-α staining. Cells were pregated on lymphocytes, singlets, live cells, and CD8+CD4B220. *p < 0.01.

FIGURE 1.

Epitope mapping of CD8 T cell responses to SARS-CoV-2 RBD protein in C57BL/6 mice. Five mice were immunized with RBD protein plus adjuvant, and their spleens were harvested one week later. (A) The percentage of CD8 T cells staining for IFN-γ after a 5-h incubation with individual 15-mer peptides spanning SARS-CoV-2 RBD. Responses that were significantly greater than those induced by an irrelevant peptide (HSVgB498–504), as determined by Dunnett multiple comparisons test. (B) The percentage of CD8 T cells staining for IFN-γ for the six potential minor epitopes and three of the major epitopes identified in (A) in mice immunized with 200 μg of RBD plus adjuvant. (C) Representative intracellular IFN-γ and TNF-α staining. Cells were pregated on lymphocytes, singlets, live cells, and CD8+CD4B220. *p < 0.01.

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Table I.

Amino acid sequences for peptides used in in vitro cytokine stimulation assays

Spike Protein Amino Acid NumbersSequence
1–14, 319 MFVFLVLLPLVSSQR 
5–14, 319–323 LVLLPLVSSQRVQPT 
9–14, 319–327 PLVSSQRVQPTESIV 
14, 323–331 SQRVQPTESIVRFPN 
321–335 QPTESIVRFPNITNL 
325–339 SIVRFPNITNLCPFG 
329–343 FPNITNLCPFGEVFN 
333–347 TNLCPFGEVFNATRF 
337–351 PFGEVFNATRFASVY 
341–355 VFNATRFASVYAWNR 
345–359 TRFASVYAWNRKRIS 
349–363 SVYAWNRKRISNCVA 
353–367 WNRKRISNCVADYSV 
357–371 RISNCVADYSVLYNS 
361–375 CVADYSVLYNSASFS 
365–379 YSVLYNSASFSTFKC 
369–383 YNSASFSTFKCYGVS 
373–387 SFSTFKCYGVSPTKL 
377–391 FKCYGVSPTKLNDLC 
381–395 GVSPTKLNDLCFTNV 
385–399 TKLNDLCFTNVYADS 
389–403 DLCFTNVYADSFVIR 
393–407 TNVYADSFVIRGDEV 
397–411 ADSFVIRGDEVRQIA 
401–415 VIRGDEVRQIAPGQT 
405–419 DEVRQIAPGQTGKIA 
409–423 QIAPGQTGKIADYNY 
413–427 GQTGKIADYNYKLPD 
417–431 KIADYNYKLPDDFTG 
421–435 YNYKLPDDFTGCVIA 
425–439 LPDDFTGCVIAWNSN 
429–443 FTGCVIAWNSNNLDS 
433–447 VIAWNSNNLDSKVGG 
437–451 NSNNLDSKVGGNYNY 
441–455 LDSKVGGNYNYLYRL 
445–459 VGGNYNYLYRLFRKS 
449–463 YNYLYRLFRKSNLKP 
453–467 YRLFRKSNLKPFERD 
457–471 RKSNLKPFERDISTE 
461–475 LKPFERDISTEIYQA 
465–479 ERDISTEIYQAGSTP 
469–483 STEIYQAGSTPCNGV 
473–487 YQAGSTPCNGVEGFN 
477–491 STPCNGVEGFNCYFP 
481–495 NGVEGFNCYFPLQSY 
485–499 GFNCYFPLQSYGFQP 
489–503 YFPLQSYGFQPTNGV 
493–507 QSYGFQPTNGVGYQP 
497–511 FQPTNGVGYQPYRVV 
501–515 NGVGYQPYRVVVLSF 
505–519 YQPYRVVVLSFELLH 
509–523 RVVVLSFELLHAPAT 
513–527 LSFELLHAPATVCGP 
517–531 LLHAPATVCGPKKST 
521–535 PATVCGPKKSTNLVK 
525–539 CGPKKSTNLVKNKCV 
529–541, 2xH KSTNLVKNKCVNFHH 
533–541, 6xH LVKNKCVNFHHHHHH 
HSVgB 498–505 SSIEFARL 
511*–518 VVLSFELL 
Spike Protein Amino Acid NumbersSequence
1–14, 319 MFVFLVLLPLVSSQR 
5–14, 319–323 LVLLPLVSSQRVQPT 
9–14, 319–327 PLVSSQRVQPTESIV 
14, 323–331 SQRVQPTESIVRFPN 
321–335 QPTESIVRFPNITNL 
325–339 SIVRFPNITNLCPFG 
329–343 FPNITNLCPFGEVFN 
333–347 TNLCPFGEVFNATRF 
337–351 PFGEVFNATRFASVY 
341–355 VFNATRFASVYAWNR 
345–359 TRFASVYAWNRKRIS 
349–363 SVYAWNRKRISNCVA 
353–367 WNRKRISNCVADYSV 
357–371 RISNCVADYSVLYNS 
361–375 CVADYSVLYNSASFS 
365–379 YSVLYNSASFSTFKC 
369–383 YNSASFSTFKCYGVS 
373–387 SFSTFKCYGVSPTKL 
377–391 FKCYGVSPTKLNDLC 
381–395 GVSPTKLNDLCFTNV 
385–399 TKLNDLCFTNVYADS 
389–403 DLCFTNVYADSFVIR 
393–407 TNVYADSFVIRGDEV 
397–411 ADSFVIRGDEVRQIA 
401–415 VIRGDEVRQIAPGQT 
405–419 DEVRQIAPGQTGKIA 
409–423 QIAPGQTGKIADYNY 
413–427 GQTGKIADYNYKLPD 
417–431 KIADYNYKLPDDFTG 
421–435 YNYKLPDDFTGCVIA 
425–439 LPDDFTGCVIAWNSN 
429–443 FTGCVIAWNSNNLDS 
433–447 VIAWNSNNLDSKVGG 
437–451 NSNNLDSKVGGNYNY 
441–455 LDSKVGGNYNYLYRL 
445–459 VGGNYNYLYRLFRKS 
449–463 YNYLYRLFRKSNLKP 
453–467 YRLFRKSNLKPFERD 
457–471 RKSNLKPFERDISTE 
461–475 LKPFERDISTEIYQA 
465–479 ERDISTEIYQAGSTP 
469–483 STEIYQAGSTPCNGV 
473–487 YQAGSTPCNGVEGFN 
477–491 STPCNGVEGFNCYFP 
481–495 NGVEGFNCYFPLQSY 
485–499 GFNCYFPLQSYGFQP 
489–503 YFPLQSYGFQPTNGV 
493–507 QSYGFQPTNGVGYQP 
497–511 FQPTNGVGYQPYRVV 
501–515 NGVGYQPYRVVVLSF 
505–519 YQPYRVVVLSFELLH 
509–523 RVVVLSFELLHAPAT 
513–527 LSFELLHAPATVCGP 
517–531 LLHAPATVCGPKKST 
521–535 PATVCGPKKSTNLVK 
525–539 CGPKKSTNLVKNKCV 
529–541, 2xH KSTNLVKNKCVNFHH 
533–541, 6xH LVKNKCVNFHHHHHH 
HSVgB 498–505 SSIEFARL 
511*–518 VVLSFELL 

These data suggest a promiscuity of the peptide VVLSFELL (S511–518) for MHC of multiple haplotypes, in this study eliciting responses in C57BL/6 mice, and in another recent publication, S511–525 elicited responses in BALB/c mice immunized with a DNA-based vaccine encoding the S protein (15). Using the MHC-I peptide-binding prediction tool NetH2pan (16), the only 8- to 14-mer peptides predicted to bind H-2Kd or H-2Dd within S511–525 are VVLSFELL and VVVLSFELL (S510–518), which are both predicted to strongly bind H-2Dd. Interestingly, the 9-mer VVVLSFELL is also predicted to bind to H-2Db. To determine whether this epitope was restricted to H-2Kb and/or H-2Db, we performed a cell-based MHC-I stabilization assay. RMA-S cells were interrogated with the 15-mers S1–14,319, S337–351, S401–415, S477–491, S505–519, and S509–523, as well as the minimal 8-mer S511–518. RMA-S cells are deficient in the expression of the TAP peptide transporter, critical for stabilizing MHC-I through peptide loading in the endoplasmic reticulum. This results in little to no MHC-I expression on the cell surface at 37°C (12). However, when RMA-S cells are cultured at 27°C, empty H-2Db and H-2Kb MHC-I molecules accumulate on the cell surface. The addition of peptides able to bind to either Kb or Db, followed by shifting the cells to 37°C, permits identification of the MHC-I molecules (i.e., Kb, Db, or both) stabilized on the cell surface. Staining with Abs specific for H-2Kb and H-2Db indicated that the 8-mer VVLSFELL (S511–518) was clearly restricted to H-2Kb (Fig. 2). In contrast, S505–519, and S509–523, which contain the S511–518 8-mer as well as the 9-mer VVVLSFELL, appeared to stabilize H-2Db, as predicted, with S509–523 stabilizing both Kb and Db. Results for the 15-mer peptides covering the remaining major epitopes were less clear, with the exception of S477–491, which also stabilized H-2Db. It is not surprising that the RMA-S assay was unable to define the restriction for every 15-mer, as it is likely a less sensitive measure of peptide binding than the cytokine staining of activated T cells, known to react to picomolar quantities of peptide-bound MHC (17). However, use of the MHC-I peptide-binding prediction tool NetH2pan (16) indicated a likely VFLVLLPL epitope binding H-2Kb within S1–14,319, an NATRFASV epitope binding H-2Kb in S337–351, and a STPCNGVEGF epitope binding H-2Db in S477–491.

FIGURE 2.

Determination of peptide MHC-I restriction. H-2Kb or H-2Kb staining of RMA-S cells 5 h after incubation with the indicated peptide. For both graphs, the x-axis intersects the y-axis at the average geometric mean fluorescence intensity value for controls without peptide.

FIGURE 2.

Determination of peptide MHC-I restriction. H-2Kb or H-2Kb staining of RMA-S cells 5 h after incubation with the indicated peptide. For both graphs, the x-axis intersects the y-axis at the average geometric mean fluorescence intensity value for controls without peptide.

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The relative durability of the Ab responses to SARS-CoV-2 infection has been controversial, with initial studies reporting a dramatic early decline in titers that may leave patients susceptible to reinfection (18). More recent, much larger studies, however, indicate that neutralizing Ab titers persist for at least five months postinfection (19). In line with these data, preliminary studies suggest the risk of reinfection remains very low and is associated with asymptomatic disease (20). Yet, whether or not Ab responses ultimately demonstrate long-term durability, cellular immune responses are likely an important determinant of prolonged protection.

COVID-19 patients show T cell reactivity toward multiple proteins, including membrane (M), nucleocapsid (N), and nonstructural proteins (21); in fact, one recent study identified an epitope within the nucleocapsid, N219–227, shared by both mouse (H-2Db) and human (HLA-A2) T cells (22). However, in serum isolated from PCR-confirmed SARS-CoV-2–positive patients, the primary target for neutralizing Ab is the S protein, with epitope specificity of neutralization directed against both the S protein RBD, and the S protein N-terminal domain (23). As such, the S protein may experience greater pressure to mutate from one virus strain to another; thus, the T cell epitopes identified within S, are more likely to be unique to SARS-CoV-2 than those from other structural proteins. Indeed, the sequence identity between SARS-CoV and SARS-CoV-2 is 91% for both the membrane (M) and nucleocapsid (N) proteins, whereas it is only 76% for S, and 73% for the RBD. Despite this, we identified one epitope shared by the two viruses within the RBD, S511–518. Two of the five major epitopes (S337–351 and S401–415) had high sequence homology but were not known to the authors to be previously described epitopes for SARS-CoV. In addition, we identified two unique CD8 T cell epitopes: the sequence homology at S1–14,319 and S477–491 is only 50% and 40%, respectively, between SARS-CoV and SARS-CoV-2. Although the minimal epitope within S1–14,319, could comprise a hybrid peptide between the signal peptide and the RBD, not seen in natural infection, this is unlikely, given that NetH2pan predictions only predict MHC-I binding for S1–8, and S3–10.

The combination of both conserved and unique epitopes within the RBD of the S protein may foster future investigations into serial infections using SARS-CoV and SARS-CoV-2 in either mouse-adapted coronavirus strains or human ACE2–expressing C57BL/6 mice. During infection, CD8 T cell responses to additional structural and nonstructural proteins will undoubtedly also arise, as recently reported for the nucleocapsid protein (22), and each may contribute to viral control. Moreover, infection may elicit CD8 T cell responses to these epitopes to various degrees compared with what we have reported in this study for vaccination, especially as CD8 T cells responding to immunogenic epitopes within other proteins compete for immunodominance. Nonetheless, we expect one or more of these epitopes to be involved in the infectious response, and we hope the data reported in this study will be a useful resource, reducing the financial and practical threshold for new studies of SARS-CoV-2 infection or vaccination in mice.

We thank Lori Sherman and the University of Colorado Cancer Center Cell Technologies Shared Resource for producing the SARS-CoV-2 spike RBD protein. We also thank Timothy Davis and the Peptide Core Facility at the University of Colorado Anschutz for technical assistance.

This work was supported by funds from the University of Colorado School of Medicine and the National Institutes of Health, National Institute of Allergy and Infectious Diseases Grants AI148919, AI066121, and AI126899 (to R.M.K.).

Abbreviations used in this article

     
  • COVID-19

    coronavirus disease 2019

  •  
  • MHC-I

    MHC class I

  •  
  • RBD

    receptor binding domain

  •  
  • S

    spike

  •  
  • SARS-CoV-2

    severe acute respiratory syndrome coronavirus 2

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R.M.K. is a founder of ImmuRx, a vaccine company for which intellectual property is based on the combined TLR agonist/anti-CD40 immunization platform. The other authors have no financial conflicts of interest.