Highly conserved, non-human-like, and cross-reactive SARS-CoV-2 T cell epitopes for COVID-19 vaccine design and validation

Abstract

Natural and vaccine-induced SARS-CoV-2 immunity in humans has been described but correlates of protection are not yet defined. T cells support the SARS-CoV-2 antibody response, clear virus-infected cells, and may be required to block transmission. In this study, we identified peptide epitopes associated with SARS-CoV-2 T-cell immunity. Using immunoinformatic methods, T-cell epitopes from spike, membrane, and envelope were selected for maximal HLA-binding potential, coverage of HLA diversity, coverage of circulating virus, and minimal potential cross-reactivity with self. Direct restimulation of PBMCs collected from SARS-CoV-2 convalescents confirmed 66% of predicted epitopes, whereas only 9% were confirmed in naive individuals. However, following a brief period of epitope-specific T-cell expansion, both cohorts demonstrated robust T-cell responses to 97% of epitopes. HLA-DR3 transgenic mouse immunization with peptides co-formulated with poly-ICLC generated a potent Th1-skewed, epitope-specific memory response, alleviating safety concerns of enhanced respiratory disease associated with Th2 induction. Taken together, these epitopes may be used to improve our understanding of natural and vaccine-induced immunity, and to facilitate the development of T-cell-targeted vaccines that harness pre-existing SARS-CoV-2 immunity.

Fig. 1. Predicted SARS-CoV-2 T-cell epitopes are antigenic ex vivo in COVID-19 convalescent donors but not healthy donors.

a Convalescent (n = 15) and pre-SARS-CoV-2 pandemic donors (naive, n = 10) were stimulated with the total peptide pool consisting of our 32 predicted epitopes and IFNγ-producing cells were measured by Fluorospot assay. Open circles identify responses to low-dose restimulation at 0.313 μg/mL per peptide; closed circles are stimulations at 10 μg/mL per peptide. Dotted horizontal line indicates positivity criteria at SFC/10⁶ splenocytes = 25; solid horizontal line indicates threshold of detection for assay (t = 2.058, d.f. = 23). b IFNγ responses to individual peptides were also assessed, identifying the breadth of response in individual donors (t = 2.113, d.f. = 23), c the frequency of responses to unique peptides within the cohort (vertical lines denote 20% of each cohort), d and the depth of response indicated by the frequency of IFNγ-producing, epitope-specific cells (vertical lines indicate positivity criteria at SFC/10⁶ splenocytes = 25). Error bars depict SD. *p < 0.05.

Fig. 2. SARS-CoV-2 experienced individuals exhibit variable immune recall responses ex vivo.

a Significant responses to individual peptides (combined per source antigen) identify three distinct immunotype cohorts within covalescent donors (donor notations: *pneumonia, **hospitalized, non-ICU). b Correlation of cumulative T-cell responses and age according to gender. Male cohort (n = 7, R² = 0.7571, p = 0.0109). Female cohort (n = 8, R² = 0.00001, p = 0.9934). Mild subset of female cohort (n = 6, R² = 0.7034, p = 0.0368).

Fig. 3. Antigen-specific T-cell expansion increases responses in COVID-19 convalescents and uncovers pre-existing SARS-CoV-2 immunity in healthy donors.

a PBMCs of convalescent (n = 15) and naive donors (n = 10) were restimulated with the total peptide pool following an 8-day expansion culture and IFNγ-producing cells were measured by Fluorospot assay. Dotted horizontal line indicates positivity criteria at SFC/10⁶ splenocytes = 25, solid horizontal line indicates threshold of detection for assay (p = 0.8216, t = 0.2281, d.f. = 23). b IFNγ responses to individual peptides were also assessed, identifying the breadth of response in individual donors (p = 0.1424, t = 1.571, d.f. = 23), c the frequency of responses to unique peptides within the cohort (vertical lines denote 20% of each cohort), and d the depth of response indicated by the frequency of IFNγ-producing, epitope-specific cells (vertical lines indicate positivity criteria at SFC/10⁶ splenocytes = 25). Error bars depict SD.

Fig. 4. EPV-CoV-19 immunization stimulates strong type 1-skewed T-cell responses in HLA-DR3 transgenic mice.

Eight days post boost, murine splenocytes were isolated and assayed for epitope-specific recall responses. Cells were plated in dual IFNγ/IL-4 Fluorospot plates and restimulated with peptide pools for 48 h. a Representative images and spot counts are shown for both cytokines (n = 3). b IFNγ SFC counts were normalized to 1 × 10⁶ cells and adjusted by background subtraction, and c IFNγ SI index was determined by calculating the fold change of individual restimulation replicates over background. d IL-4 SFC and e SI were similarly calculated. Dotted horizontal lines denote positivity criteria of SFC > 25 and SI > 5, respectively. Solid horizontal lines denote threshold of detection for assay. f From the reported IFNγ and IL-4 stimulation indexes, we calculated the IFNγ : IL-4 ratio of each restimulation replicate to model the overall skewing of the immune response, identifying a sharply type 1 skewed phenotype in all vaccinated animals. Dotted horizontal lines identify 40, 100, and 1000-fold skewing of type 1/type 2 response. Solid horizontal line identifies hypothetical balanced response (Th1 = Th2). Error bars depict SD. Saline n = 3, pICLC n = 5, EPV-CoV-19_LD n = 5, EPV-CoV-19_HD n = 4, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5. EPV-CoV-19 immunization stimulates type 1-skewed memory CD4⁺ and CD8⁺ T cells in HLA-DR3 transgenic mice.

Splenocytes were restimulated with a vaccine-matched peptide pool for 6 h in the presence of brefeldin A and monensin. Following incubation, cells were stained for surface markers, fixed and permeabilized, stained for intracellular markers, and expression of markers was recorded by flow cytometry. Memory CD4⁺ T cells and CD8⁺ T cells were assessed for IFNγ, IL-4, or IL-5-production (both frequency in parent T-cell population and mean fluorescence intensity (MFI) of cytokines). a Representative images of type 1 and type 2 skewed, epitope-specific memory T-cell populations are shown. b The fold increase of epitope-specific responses (over anti-CD28 stimulated controls) identify vaccine-specific induction of IFNγ, but not c IL-4 or d IL-5. e From ICS generated data, we calculated the fold increase of IFNγ or IL-4- and/or IL-5-producing cells with peptide restimulation, and used the ratio of type 1-to-type 2 responses to model Th-skewing and Tc-skewing in vaccinated animals. Error bars depict SD. Saline n = 3, pICLC n = 5, EPV-CoV-19_LD n = 5, EPV-CoV-19_HD n = 4, *p < 0.05, *p < 0.01, ***p < 0.001.