Cutting Edge: Mouse SARS-CoV-2 Epitope Reveals Infection and Vaccine-Elicited CD8 T Cell Responses

Mouse models of SARS-CoV-2 infection may facilitate reductionist studies to identify the kinetics and distribution of adaptive immune responses in response to primary infection, mechanisms of viral control, and evaluate and refine vaccine strategies for their ability to confer protection against intentional challenge. Human ACE2 (hACE2) transgenic mice are permissive to SARS-CoV-2 infection and acquire severe, sometimes fatal, disease that may recapitulate features of natural human infection (1).

Several lines of evidence suggest that SARS-CoV-2 T cell immunity could prove advantageous. Early robust T cell responses correlate with reduced human disease (2). Neutralizing Abs target limited motifs of the viral spike protein (3). SARS-CoV-2 variants are emerging during the pandemic, some of which escape neutralization by convalescent serum (4). T cells recognize epitopes that are more likely to be conserved among viral SARS-CoV-2 mutants and may cross-react with distinct seasonal and pandemic coronaviruses (5, 6).

The absence of a defined dominant CD8 T cell epitope is an impediment to mouse SARS-CoV-2 studies. We identified an epitope in mice, demonstrated that some human patients respond to the same epitope, and used this tool to evaluate the relationship between routes of immunization and the magnitude and distribution of memory CD8 T cells using human-relevant vaccine vectors.

For construction of DNA–SARS-CoV-2 N, a gene fragment consisting of the Kozak sequence, codons for IgE leader sequence and SARS-CoV-2 N (GenBank: QJF11943.1) were designed using SnapGene software (version 4.3.11) and further codon and RNA optimized for humans, using GenSmart optimization tool (GenScript). A 1352-bp gene block consisting of SARS-CoV-2 N together with ∼15-bp overlaps at the 5′ and 3′ ends for infusion cloning was synthesized (GenScript). pVAX1 Vector (catalog no. V26020; Thermo Fisher Scientific) was linearized with EcoR1-HF and XhoI in CutSmart buffer (New England BioLabs). SARS-CoV-2 N gene block was cloned into linearized pVAX1 using In-Fusion Cloning (Takara Bio), and resultant mix was transformed into Top10 chemically competent Escherichia coli (Thermo Fisher Scientific). Positive clones were selected on Luria–Bertani Broth kanamycin plates and validated by sequencing.

For the construction of modified vaccinia virus (MVA)-N, the SARS-CoV-2 (MN994468.1_22-Jan-2020_USA.CA) nucleocapsid open reading frame was codon optimized for vaccinia virus expression, synthesized (GenScript), and cloned into pLW-73 between the XmaI and BamHI sites under the control of the vaccinia virus modified H5 early late promoter and adjacent to the gene encoding enhanced GFP (7). MVA-N was generated using DF-1 cells. Plaques were picked for seven rounds to obtain GFP-negative recombinants, and DNA was sequenced to confirm lack of any mutations. Viral stocks were purified from lysates of infected DF-1 cells using a 36% sucrose cushion and titrated using DF-1 cells by counting PFU per milliliter. Absence of the wild-type MVA was confirmed by PCR, and MVA-N infectivity was assessed by infection of DF-1 cells and flow cytometry analysis after 36 h.

For construction of adenovirus 5 (Ad5)-N, the SARS-CoV-2 N gene (GenBank: QJF11943.1) was synthesized and subcloned into the CMV shuttle plasmid using KpnI and NotI (Blue Heron). The resultant plasmid was sequenced using CMV and polyadenylation reverse primers and sequenced. This shuttle plasmid was digested with PacI and cotransfected with the remaining adenovirus genome that contains a 2.6-kbp deletion in the E3 region of the virus into 293 cells. Viral replication was evident by day 10 posttransfection, and the virus was serially amplified to perform CsCl purification. Integrity of SARS-CoV-2 N gene in purified virus was confirmed by sequencing.

Six- to ten-week-old female C57BL/6J (B6) mice and 10-wk-old hACE2 hemizygous (stock no. 034860) mice (The Jackson Laboratory) were maintained in specific-pathogen-free conditions at the University of Minnesota and used in accordance with the Institutional Animal Care and Use Committees guidelines (8). For B6 mice, 50 μg of DNA encoding SARS-CoV-2 N was injected i.m. into the quadriceps. A total of 3 × 10⁶ PFU of MVA-N was injected i.m. or intranasally (i.n.). A total of 5 × 10¹⁰ or 1 × 10¹¹ viral particles of Ad5-N were injected i.m., i.n., or i.v. For heterologous prime-boost vaccinations, mice received a combination of the vaccinations with a minimum 30-d interval.

Transgenic mice expressing hACE2 were housed in the BSL-3 containment facility for SARS-CoV-2 challenge studies. Animals were challenged i.n. with 10⁵ PFU of SARS-CoV-2 and sacrificed 10 d later for assessment of N₂₁₉-specific CD8 T cells. SARS-CoV-2, isolate 2019-nCoV/USA-WA1/2020, NR-52281, was obtained from the Centers for Disease Control and Prevention (BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health) or Dr. V. Menachery (World Reference Center for Emerging Viruses and Arboviruses).

To discriminate intravascular from extravascular cells, we injected mice i.v. with BV605-conjugated anti-CD8α Ab, and after 3 min, mice were sacrificed and tissues were harvested. Lymphocytes were isolated as described (9, 10) and stained with Abs to CD8α (53-6.7), CD62L (MEL-14), Ly6C (HK1.4), CD127 (A7R34), CD44 (IM7), CD69 (H1.2F3), and CD103 (M290) (all from BD Biosciences, Tonbo Biosciences, BioLegend, or Affymetrix eBiosciences) and H2-D^b/N_219-227 MHC class I (MHC I) tetramers (made in-house) and ghost dye 780 (Tonbo Biosciences). For monomer preparation and all peptide-dependent assays described below, the N_219-227 peptide sequence was LALLLLDRL. Human PBMCs were stained with CD3ε (SP34-2), CD4 (OKT4), CD8 (SK1), IFN-γ (B27), and TNF-α (MAb11). Stained samples were acquired on LSRII or LSR Fortessa flow cytometers (BD Biosciences) and analyzed with FlowJo software (BD Biosciences). Cells were gated on singlets, live lymphocytes, CD8 T cells, and/or tetramer-specific cells as indicated.

PBMCs from COVID-19 hospitalized patients were acquired in accordance with institutional review board approvals (STUDY00010110 and STUDY00009458), and this study was conducted according to the principles expressed in the Declaration of Helsinki. PBMCs were stained for HLA-A*02 expression, and 10 HLA-A*02⁺ samples were stimulated with N_219-227 (LALLLLDRL) or N_222-230 (LLLDRLNQL) for 1 h, washed, and cultured in IMDM (Life Technologies) supplemented with 5% FBS (Peak Serum) and IL-2 (20 U/ml; StemCell) for 18 d, with replenishment provided every 3 d. Expanded T cell cultures were restimulated with 1 μg/ml N_219-227 or vehicle control for 12 h. Golgi stop was added in restimulated samples as per manufacturer recommendations (BD Biosciences).

We synthesized 69 15-mer overlapping peptides spanning the SARS-CoV-2 nucleoprotein as well as specific 8- to 9-mer peptides (GenScript). All peptides were reconstituted in DMSO at 10 mg/ml, and 9–10 15-mer peptides were pooled to form a total of seven pools. Splenocytes from naive or immunized B6 mice were stimulated for 12 h with overlapping peptide pools (1 μg/ml) consisting of 9–10 15-mer peptides or with individual 8- to 9-mer peptides (1 μg/ml) with Golgi stop (BD Biosciences). Unstimulated cells or cells treated with PMA/ionomycin (StemCell) were negative and positive controls.

Individual data points represent biological replicates. All statistical tests were two-tailed, and nonparametric tests were used to test for significance with a p value <0.05 considered significant. Mean and SEM are used to represent the center and dispersion.

We constructed DNA, MVA, or Ad5 vectors encoding SARS-CoV-2 nucleoprotein, which were used to successively immunize C57BL/6J mice (Fig. 1A, 1B). These vectors were chosen because each of them has been used in human clinical vaccine trials (11). We focused on the nucleoprotein because it is a prominent target of CD8 T cell responses in SARS-CoV-2–infected individuals, was the target of durable T cell immunity in those that recovered from SARS-CoV-1, is relatively conserved among emergent β coronaviruses, and mutations have been observed in the SARS-CoV-2 spike protein (4, 5, 12, 13). One week after the third immunization, splenocytes were cultured with seven pools each containing 9–10 15-mer peptides spanning the nucleoprotein proteome for 12 h. Pool no. 4 elicited production of IFN-γ and TNF-α from CD8 T cells (Fig. 1C). Individual 8- to 9-mer peptides from pool no. 4 were then similarly assessed, revealing two reactive epitopes, N_219-227 (LALLLLDRL) and N_220-227, with the former appearing dominant (Fig. 1D).

hACE2 transgenic mice on a C57BL/6J background were infected i.n. with SARS-CoV-2 (2019-nCoV/USA_WA1/2020). Ten days later, lymphocytes from spleen and lung were stained with H2-D^b/N_219-227 MHC I tetramers, revealing N₂₁₉-specific CD8 T cell responses in lung, the lung-draining mediastinal lymph node, and spleen (Fig. 2A, Supplemental Fig. 1A). A significant number of N₂₁₉-specific CD8 T cells in the lung and the mediastinal lymph node expressed CD69 and CD103, which may constitute early markers of CD8 T cell residency (Fig. 2B). Naive C57BL/6J mice served as negative controls.

Ten PBMC samples from HLA-A*02⁺ COVID-19 hospitalized patients were briefly stimulated with N_219-227 or N_222-230 (a recently defined human epitope), washed, cultured in IL-2–conditioned media for 18 d, and then restimulated with N_219-227 or N_222-230, respectively, overnight (12). CD8 T cells from 4 of 10 samples stimulated with N_219-227 expressed IFN-γ and TNF-α as determined by intracellular staining, compared with unstimulated controls (Fig. 2C, Supplemental Fig. 1B). These data indicate that N_219-227 comprises a SARS-CoV-2 epitope in both humans and H2-D^b+ mice.

Heterologous prime boosting can establish robust CD8 T cell immunity (14). To assess how route of vaccination affects CD8 T cell magnitude and distribution, heterologous immunization with SARS-CoV-2 N vectors was performed in B6 mice. Mice received i.m. or i.n. immunizations with MVA-N followed by Ad5-N. An additional DNA-N prime was also administered to a parallel cohort (Fig. 3A). The i.m. immunizations induced larger responses in blood and tissues outside of the respiratory mucosa (Fig. 3B–E). Notably, i.m. immunizations induced a robust T cell response in the brain (Fig. 3D, 3E), an extrapulmonary site of human SARS-CoV-2 infection (15). In contrast, i.n. immunization favored robust establishment of CD8 T cells in the respiratory mucosa (Fig. 3D, 3E), despite poor responses in blood and other lymphoid and nonlymphoid compartments. These data highlight that the vaccine-elicited immunity at frontline sites of infection would be difficult to compare if analysis was restricted to PBMCs. Of note, i.n. immunization was associated with greater acquisition of CD69 and CD103, residency markers associated with retention (16, 17), in most nonlymphoid tissues and the lung-draining lymph node (Fig. 3F, 3G).

Multiple heterologous immunizations present logistical challenges for manufacturing and delivery, and a single-shot immunization may provide significant advantages during an ongoing pandemic. Given the high-magnitude response detected following Ad5-N tertiary immunization, we compared primary SARS-CoV-2 N₂₁₉-specific CD8 T cell responses after single immunizations with Ad5-N delivered i.m., i.n., and also i.v., as this route has been proposed to enhance effective immunity against tuberculosis after BCG immunization (Fig. 4A) (18). The i.v. route of Ad5 induced high-magnitude SARS-CoV-2 N₂₁₉-specific CD8 T cell responses in blood (Fig. 4B). Although i.n. and i.v. immunizations both induced considerable T cell responses in pulmonary tissues in comparison with the i.m. route (Fig. 4C), i.v. vaccination elicited the greatest response in blood, lymphoid organs, and extrapulmonary tissues, including the brain, oral mucosa, and small intestine. Responses persisted through 53 d after immunization (Fig. 4D). Thus, a single i.v. immunization established a broadly distributed memory CD8 T cell response of high magnitude.

Mice are valuable for immunology research because of defined and manipulable genetics, advanced tools and reagents, low cost, and relevance to the human immune system (19). This study defined an immunodominant H2-D^b–restricted SARS-CoV-2 epitope, relevant for C57BL/6 and hACE2 transgenic mice. Additional studies will be required to determine what portion of the total SARS-CoV-2–specific CD8 T cell response is directed toward N_219-227. A defined epitope provides a convenient means for detecting Ag-specific CD8 T cell responses, including by MHC I tetramer reagents, to evaluate the magnitude, distribution, and phenotype of responses to SARS-CoV-2 infection or vaccination. This may accelerate understanding of host–pathogen interaction, the evaluation of potential T cell vaccine candidates, and controlled reductionist studies that interrogate mechanisms of protective immunity. Moreover, we define vectors and routes of immunization that establish abundant systemic immunity, respiratory immunity, or both. This work is antecedent to hACE2 mouse challenge studies to test the role of CD8 T cells in primary infection control and the potential for memory CD8 T cells to contribute to protective immunity.

We thank the individuals who provided PBMC samples, Dr. Vineet Menachery (University of Texas Medical Branch) and the World Reference Center for Emerging Viruses and Arboviruses for providing the SARS-CoV-2 virus, and those who provided assistance from the Flow Cytometry Resource and Biosafety Level 3 Program at the University of Minnesota.

The authors have no financial conflicts of interest.