A third vaccination with a single T cell epitope confers protection in a murine model of SARS-CoV-2 infection

Abstract

Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8⁺ T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8⁺ T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8⁺ effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8⁺ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.

Fig. 1. Vaccines eliciting antibodies against confirmational proteins but not linear B cell epitopes are neutralizing and effective against SARS-CoV-2 infection.

a C57BL/6 mice were vaccinated subcutaneously (s.c.) on day 0, 14, and 28 with synthetic long peptides (SLPs) consisting of PADRE-coupled linear B cell epitopes adjuvanted or not with CpG and/or Inactivated Freund’s Adjuvant (IFA) (Mock, IFA: n = 5; CpG, CpG+IFA: n = 3). Spike-specific IgG kinetics in blood are shown. b Representative flow cytometry plots of PADRE-specific CD4⁺ T cells in naïve unvaccinated mice and in day 21 post-vaccinated mice. c PADRE-specific CD4⁺ T cell response on day 21 after vaccination as described in (a) (Naïve: n = 11; Mock, IFA: n = 5; CpG: n = 3; CpG+IFA: n = 13). *P = 0.048, ***P = 0.0007, ****P = <0.0001. d C57BL/6 mice were vaccinated as described in (a) with SLPs consisting of the B cell epitope (E) alone, the epitope combined (uncoupled) with the PADRE (P) peptide (E+P) or N-terminal and C-terminal PADRE coupled to the B cell epitope (P−E, E−P), and all adjuvanted with CpG and IFA (E, E+P, E−P: n = 5; P−E: n = 3). Endpoint dilutions of Spike-specific IgG in blood at day 42 after vaccination with Spike_405-422-SLP are shown. *P = 0.035, **P = 0.005, ***P = 0.0004. e–h K18-hACE2 transgenic mice were vaccinated on day 0, 14, and 28 with one or five SLP vaccines containing a PADRE-coupled linear B cell epitope adjuvanted with CpG and IFA. Four weeks after the final vaccination, mice were intranasally infected with SARS-CoV-2 (e–g, Unvaccinated: n = 9; B cell SLP 1 epitope, B cell SLP 5 epitopes: n = 10). e Spike-specific IgG kinetics in blood. f Weight loss kinetics after SARS-CoV-2 challenge. g Survival graph after SARS-CoV-2 challenge. h SARS-CoV-2 neutralizing capacity by antibodies after B cell-SLP vaccination on day 42 measured by a WT virus neutralization assay (VNA) (Unvaccinated, B cell SLP 1 epitope, n = 2; B cell SLP 5 epitopes, n = 5). i–n K18-hACE2 mice were vaccinated intradermally on day 0, 21, and 42 with a Spike-encoding DNA vaccine. i Spike-specific IgG (Unvaccinated: n = 3; Spike DNA: n = 6), j weight loss kinetics and k survival graph after SARS-CoV-2 challenge (n = 10 per group). ****P = < 0.0001. l SARS-CoV-2 neutralizing capacity by antibodies after Spike DNA vaccination on day 55 measured by a VNA. Data represented as mean ± SEM (Unvaccinated: n = 1; Spike DNA: n = 6). m Kinetics of Spike_539-546-specific CD8⁺ T cells in blood. Data represented as mean ± SEM (Unvaccinated: n = 3; Spike DNA: n = 6). n Kinetics of KLRG1⁺CD62L⁻ expression on Spike_539−546-specific CD8⁺ T cells in blood. Data represented as mean ± SEM (n = 6). One-way ANOVA with Tukey’s multiple comparison test for (c, d); log-rank test for (f, k). Source data are provided as a Source Data file.

Fig. 2. A third vaccination with a single T cell epitope protects against SARS-CoV-2 challenge.

a K18-hACE2 transgenic mice were vaccinated subcutaneously on day 0 (1^st vaccination), day 14 (2^nd vaccination), and day 28 (3^rd vaccination) with the Spike_539-546-SLP vaccine adjuvanted with CpG. The Spike_539-546-specific CD8⁺ T cell response was determined in blood in time. Five weeks after the final vaccination, vaccinated and unvaccinated mice were intranasally infected with 5000 PFU of SARS-CoV-2, and monitored for weight loss and signs of clinical discomfort. b Representative flow cytometry plots of Spike_539-546-specific CD8⁺ T cells determined in the blood circulation at day 45 by MHC class I tetramer staining. c Spike_539-546-specific CD8⁺ T cell kinetics in blood at indicated days after vaccination. Data is represented as mean ± SEM (Unvaccinated: n = 3; 1 vac: n = 10; 2 vac, 3 vac: n = 11). d Spike_539-546-specific CD8⁺ T cells in blood at day 62 after vaccination. Data represented as mean ± SEM (Unvaccinated: n = 3; 1 vac: n = 10; 2 vac, 3 vac: n = 11). Symbols represent individual mice. *P = 0.022-0.047, ****P = < 0.0001. e Weight loss kinetics in time of SARS-CoV-2 challenged K18-hACE2 transgenic mice after T cell epitope SLP vaccination. f Survival graph of SARS-CoV-2 challenged K18-hACE2 transgenic mice after T cell epitope SLP vaccination (Unvaccinated, 1 vac: n = 10; 2 vac, 3 vac: n = 11). *P = 0.0131, P = **0.0029, ***P = 0.0008. One-way ANOVA with Tukey’s multiple comparison test for (d); log-rank test for (f). Source data are provided as a Source Data file.

Fig. 3. A third vaccination with a single T cell epitope augments effector-memory and tissue-resident memory CD8⁺ T cell formation.

a Frequencies of CD69⁺ and CD69⁻ Spike_539-546-specific CD8⁺ T cells in the CD8⁺ T cell population in the spleen, liver and lungs at day 70 after 1, 2 or 3 SLP vaccinations. Data represented as mean + SEM (n = 5 per group). *P = 0.0329-0.0396, **P = 0.0065-0.0087, ***P = 0.0003, ****P = < 0.0001. b Intracellular cytokine production of CD8⁺ T cells upon stimulation with the Spike_539-546 peptide epitope in different tissues on day 70 after 1, 2 or 3 SLP vaccinations. Data represented as mean + SEM (n = 5 per group). *P = 0.0196-0.0441, **P = 0.0019–0.0079, ***P = 0.0004, ****P ≤ 0.0001. c tSNE maps describing the local probability density of Spike_539-546-specific CD8⁺ T cells stained for KLRG1, CD62L, Ly6C and CX3CR1 at day 62 after 1, 2 and 3 vaccinations. d Frequencies of KLRG1⁺CD62L⁻, CX3CR1⁺, CX3CR1⁺KLRG1⁺ and CD62L⁺KLRG1⁻ Spike_539-546-specific CD8⁺ T cells in blood in time. Data represented as mean ± SEM (1 vac: n = 11; 2 vac: n = 12; 3 vac: n = 10). Symbols represent individual mice. **P = 0.0021–0.0031, ***P = 0.0007, ****P ≤ 0.0001. e (Left) tSNE maps describing the local probability density of Spike_539-546-specific CD8⁺ T cells in spleen, liver and lungs stained at day 69 after 1, 2 and 3 vaccinations with a flow cytometry panel for CD69, CD62L, CD44, Ly6C, KLRG1, and CX3CR1. (Right) marker expression for CX3CR1, CD69, Ly6C and KLRG1. f Cell surface marker (Ly6C⁺KLRG1⁺, CX3CR1⁺KLRG1⁺, and CD62L⁺KLRG1⁻) expression and glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2NBDG) uptake of Spike_539-546-specific CD8⁺ T cells at day 69 in spleen after 1, 2 or 3 vaccinations. Data represented as mean ± SEM (n = 5 per group). *P = 0.0402, ***P = 0.001–0.004, ****P ≤ 0.0001. One-way ANOVA with Tukey’s multiple comparison test for (a, b, d, f). Source data are provided as a Source Data file.

Fig. 4. Progressive differentiation of vaccine-specific CD8⁺ T cells after booster vaccination.

C57BL/6 mice were vaccinated subcutaneously on day 0 (1^st vaccination), day 14 (2^nd vaccination), and day 28 (3^rd vaccination) with the Spike_539-546-SLP vaccine adjuvanted with CpG. a Following tSNE analysis downsampling, FlowSOM consensus metaclustering with 8 clusters as well as Wanderlust trajectory analysis was performed on 200 live CD3⁺CD8⁺CD4⁻CD19⁻ Spike_539-546 tetramer⁺ cells harvested from the spleen at day 69 after SLP vaccination. Overlay of the 8 FlowSOM clusters (left), vaccination status (middle) and Wanderlust trajectory (right) (color indication: blue, beginning/early; red, end/late) on the tSNE-map. b Expression intensity of cell surface markers (color indication: blue, low expression; yellow, high expression). c Significant clusters were selected and are shown in bar graphs. Data represented as mean ± SEM (n = 5 per group). Symbols represent individual mice. *P = 0.011–0.015, **P = 0.0043, ***P = 0.001–0.009, ****P = <0.0001. d Stacked bar graph of all 8 FlowSOM clusters per vaccination group, colors match (a). e Hierarchically clustered heatmap of phenotypes of the clusters shown in (a)—the indicated marker expression is shown per cluster as z-Score of median signal intensity per channel; blue, low expression; red, high expression. f Each of the markers used for embedding in figure (a–c) are displayed according to Wanderlust trajectory progression. g Selected markers are displayed according to Wanderlust trajectory progression. One-way ANOVA with Tukey’s multiple comparison test for (c). Source data are provided as a Source Data file.

Fig. 5. A third vaccination triggers the remigration of T_RM cells into the circulation.

a C57BL/6 mice were vaccinated with the T cell OVA_257-264-SLP vaccine adjuvanted with CpG in a prime-boost-boost regimen with 2 week intervals. Shown are the OVA_257-264-specific CD8⁺ T cell kinetics in blood at indicated days after vaccination. Data is represented as mean ± SEM (n = 8 per group). b Frequencies of KLRG1⁺CD62L⁻ OVA_257-264-specific CD8⁺ T cells in blood in time. Data represented as mean ± SEM (n = 8 per group). Symbols represent individual mice. ****P = < 0.0001. c–g 1 × 10⁴ OT-I Hobit LT CD8⁺ T cells were adoptively transferred into C57BL/6 mice. The day after, mice received the first OVA_257-264-SLP vaccination, followed by 2 boosters. After 50 days spleen and liver were analyzed by flow cytometry. c Flow cytometry plots showing the CD62L, CD69, and CD38 expression of liver OT-I YFP⁺ tdTomato⁺ CD8⁺ T cells. d Percentage of OT-I YFP and/or tdTomato positive CD8⁺ T cells in liver and spleen. Data is represented as mean + SEM (n = 5 per group). *P = 0.0144–0.0267, **P = 0.0025-0.0089, ***P = 0.0007. e Percentage of OT-I YFP⁺tdTomato⁻ (ex-T_RM) of total CD8⁺ T cells in liver and spleen. Data represented as mean ± SEM (n = 5 per group). Symbols represent individual mice. *P = 0.0267, **P = 0.0040–0.0089, ***P = 0.00070082. f Percentage of OT-I Hobit YFP⁺tdTomato⁻ (ex-T_RM) cells of total CD8⁺ T cells in blood. Data represented as mean ± SEM (n = 5 per group). Symbols represent individual mice. *P = 0.0327, **P = 0.0076–0.0082. g Phenotype of OT-I YFP⁺tdTomato⁻ (ex-T_RM) CD8⁺ T cells in the spleen. One-way ANOVA with Tukey’s multiple comparison test for (a, d–f). Source data are provided as a Source Data file.