Unbiased Screens Show CD8+ T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein

Abstract

Developing effective strategies to prevent or treat coronavirus disease 2019 (COVID-19) requires understanding the natural immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8⁺ T cells of COVID-19 patients. In total, we identified 3-8 epitopes for each of the 6 most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8⁺ T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8⁺ T cell immunity to SARS-CoV-2.

Keywords: CD8+ T cells; COVID-19; SARS-CoV-2; T-Scan; immunity; immunodominant epitopes; novel coronavirus; seasonal coronaviruses; spike protein; vaccine.

Graphical abstract

Figure 1

T-Scan Approach for Comprehensive Mapping of the Memory CD8⁺ T Cell Response to SARS-CoV-2 (A) Overview of the T-Scan antigen discovery screen. (B) Design of the ORFeome-wide coronavirus antigen library. (C) Example SARS-CoV-2 ORFeome-wide T-Scan screen data for a convalescent COVID-19 patient (top panel) and a healthy control individual (bottom panel). Each circle represents a single 61-aa SARS-CoV-2 protein fragment, with the x axis showing the position of each fragment in the concatenated SARS-CoV-2 ORFeome. The y axis shows the performance of the fragment in the screen, calculated as the ratio of sorted target cells expressing the protein fragment in relation to the unsorted target library. The right panels show the performance of the 60 positive control protein fragments derived from CMV, EBV, and influenza.

Figure 2

Discovery and Validation of Shared SARS-CoV-2 Epitopes Presented on HLA-A^∗02:01 (A) T-Scan screen data for nine HLA-A^∗02:01 COVID-19 patients. Each circle corresponds to a 20-aa segment of the SARS-CoV-2 ORFeome, with the x axis indicating the position of the segment in the ORFeome (gaps added for display purposes). The y axis shows the mean performance of all library fragments spanning the given 20-aa segment, calculated as described in Figure 1C. Results for each patient are denoted with different colors. (B) Screen data for the KLW epitope (KLWAQCVQL). The boxplots represent the screen enrichments of all fragments in the library that contain the KLW epitope. Data for the nine HLA-A^∗02:01 COVID-19 patient screens are shown in blue, two healthy control individual HLA-A^∗02:01 screens are shown in gray, and five HLA-A^∗03:01 COVID-19 patient screens are shown in red. (C) Collapsed screen data for six identified shared epitopes. Each boxplot shows the aggregate enrichment of one epitope in each of the nine screened HLA-A^∗02:01 patients (black dots) and two healthy control individuals (blue dots). The y axis shows the mean enrichment of all fragments in the library containing the given epitope. The dashed line indicates the mean enrichment, and a dotted line indicates the mean enrichment + 2 SD of all A^∗02:01 epitopes in all negative controls. (D) IFN-γ ELISA validation of identified epitopes. HLA-A^∗02:01 target cells were pulsed with 1 μM peptide and incubated with memory CD8⁺ T cells from four HLA-A^∗02:01 COVID-19 patients. The y axis shows the concentration of IFN-γ secreted by T cells from each patient (black dot) in the presence of each peptide compared with the no-peptide control. Data are the means of two technical replicates and representative of two independent experiments. (E) Tetramer staining of memory CD8⁺ T cells reactive to six shared HLA-A^∗02:01 epitopes. Memory CD8⁺ T cells from 27 HLA-A^∗02:01 COVID-19 patients (black dots), one healthy HLA-matched control individual (blue dot), and three MHC-mismatched control individuals (orange dots) were stained with tetramers loaded with each of the six identified epitopes. The y axis indicates the percentage of tetramer-positive memory CD8⁺ cells. (F) Correlation of T-Scan screen performance and cognate T cell frequency as determined by tetramer staining. Each circle indicates the performance of one epitope in one of the nine screened HLA-A^∗02:01 patients. The x axis shows the aggregate performance of the epitope in the T-Scan screen, calculated as the average enrichment of all fragments containing that epitope. The y axis shows the frequency of tetramer-positive memory CD8⁺ T cells recognizing each epitope. (G and H) Recognition of the three most common HLA-A^∗02:01 epitopes across COVID-19 patients on the basis of (G) screening data (n = 9) or (H) tetramer staining (n = 27). For (G), patients were considered positive for an epitope when the aggregate performance of the epitope in the screen data exceeded a set threshold (mean + 2SD of the enrichment of all of the SARS-CoV-2 fragments in the healthy control individuals). For (H), patients were considered positive for an epitope when 0.05% or more of memory CD8⁺ T cells were positive by tetramer staining.

Figure 3

Discovery and Validation of Shared SARS-CoV-2 Epitopes Presented on HLA-A^∗01:01, HLA-A^∗03:01, HLA-A^∗11:01, HLA-A^∗24:02, and HLA-B^∗07:02 (A) Collapsed T-Scan screen data for shared epitopes identified for each analyzed MHC allele. Each boxplot shows the aggregate enrichment of one epitope in each of the five COVID-19 patients (black dots) screened for the listed allele. The y axis shows the mean enrichment of all fragments in the library containing the given epitope. Full epitope sequences are listed in Table 1. (B) IFN-γ ELISA validation of identified epitopes. Memory CD8⁺ T cells from four COVID-19 patients positive for each prioritized MHC allele were incubated with MHC-matched target cells pulsed with 1 μM peptide. The y axis shows the concentration of IFN-γ secreted by T cells from each patient (black dot) in the presence of each peptide compared with a no-peptide control. Data are the means of two technical replicates and representative of two independent experiments. (C) Recognition of the three most common epitopes for each prioritized MHC allele across five COVID-19 patients. Patients were considered positive for an epitope when the aggregate performance of the epitope in the screen data exceeded a set threshold (mean + 2SD of the enrichment of all of the SARS-CoV-2 fragments in the healthy controls).

Figure 4

Shared Epitopes Span the SARS-CoV-2 ORFeome and Are Recognized by TCRs with Common Features (A) Distribution of shared CD8⁺ T cell epitopes across the SARS-CoV-2 genome. Each bar represents one validated shared epitope, with the x axis showing its position in the SARS-CoV-2 ORFeome, the color indicating its MHC restriction, and the height of the bar indicating the percentage of MHC-matched patients recognizing the epitope. Patients were considered positive for an epitope when the aggregate performance of the epitope in the screen data exceeded a threshold (mean + 2SD of the enrichment of all SARS-CoV-2 fragments in the healthy controls). For clarity, overlapping epitopes are plotted as adjacent bars. (B) TCR alpha variable (TRAV) gene usage in tetramer⁺ T cells across patients. The height of each box corresponds to the number of T cells within the clonotype. Blue corresponds to the conserved TRAV gene for a specific epitope, and red corresponds to all other TRAV genes. (C) The magnitude of detected memory CD8⁺ T cell response correlates negatively with time from diagnosis to blood draw. The p value and correlation coefficient r were calculated using Pearson correlation. (D) Magnitude of detected memory T cell response in patients with mild, moderate, and severe disease. For (C) and (D), the magnitude of T cell response indicates the total fraction of memory CD8⁺ T cells that stained positive with tetramers for one of the six identified HLA-A^∗02:01 epitopes (KLW, YLQ, LLY, ALW, LLL, and YLF) for 27 HLA-A^∗02:01 patients.

Figure 5

Minimal Cross-reactivity of SARS-CoV-2-Reactive Memory CD8⁺ T Cells with Other Coronaviruses (A) Screen data compared across coronavirus ORFeomes. Each graphic shows the collective reactivity to one coronavirus genome (SARS-CoV-2, SARS-CoV-1, OC43, HKU1, NL63, or 229E) detected in the 34 T-Scan screens performed. Each circle corresponds to a 20-aa stretch of the coronavirus ORFeome, with the x axis indicating the position of the stretch in the ORFeome. The y axis shows the mean performance of all of the library fragments spanning the given 20-aa stretch, calculated as described in Figure 1C. Results include nine HLA-A^∗02:01 patients, five HLA-A^∗03:01 patients, five HLA-A^∗01:01 patients, five HLA-A^∗11:01 patients, five HLA-A^∗24:02 patients, and five HLA-B^∗07:02 patients. For visualization, the positions of the conserved ORF1ab, S, M, E, and N proteins were aligned across all ORFeomes. (B) Alignment of the KLW epitope across coronavirus genomes. The alignment shows the region of each coronavirus genome corresponding to the SARS-CoV-2 HLA-A^∗02:01 KLW epitope. The boxplots show the aggregate screen performance of all fragments containing each epitope variant for nine HLA-A^∗02:01-positive COVID-19 patients (black dots) and two HLA-A^∗02:01-positive healthy controls (blue dots). (C) Alignment of the SPR epitope across coronavirus genomes. The alignment shows the region of each coronavirus genome corresponding to the SARS-CoV-2 HLA-B^∗07:02 epitope. The boxplots show the aggregate screen performance of all fragments containing each epitope variant for five HLA-B^∗07:02-positive COVID-19 patients (black dots).