ChAdOx1 nCoV-19 vaccination generates spike-specific CD8+ T cells in aged mice

Abstract

Effective vaccines have reduced the morbidity and mortality caused by severe acute respiratory syndrome coronavirus-2 infection; however, the elderly remain the most at risk. Understanding how vaccines generate protective immunity and how these mechanisms change with age is key for informing future vaccine design. Cytotoxic CD8⁺ T cells are important for killing virally infected cells, and vaccines that induce antigen-specific CD8⁺ T cells in addition to humoral immunity provide an extra layer of immune protection. This is particularly important in cases where antibody titers are suboptimal, as can occur in older individuals. Here, we show that in aged mice, spike epitope-specific CD8⁺ T cells are generated in comparable numbers to younger animals after ChAdOx1 nCoV-19 vaccination, although phenotypic differences exist. This demonstrates that ChAdOx1 nCoV-19 elicits a good CD8⁺ T-cell response in older bodies, but that typical age-associated features are evident on these vaccine reactive T cells.

Keywords: Aging; CD8+ T cells; SARS-CoV-2; immunity; vaccination.

Figure 1

Advanced age negatively affects serum immunity after ChAdOx1 nCoV‐19 vaccination. Young (3 months of age) and old (22 months of age) mice were immunized with 50 μL ChAdOx1 nCoV‐19 (10⁸ infectious units) intramuscularly. At 42 days after vaccination serum samples were analyzed. (a) Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) serum neutralizing capacity expressed as reciprocal serum dilution required to inhibit pseudotyped virus entry by 50% (IC₅₀). The dotted line represents lower detection limits. (b, c) Serum anti‐RBD (b) and anti‐spike (c) immunoglobulin (Ig)G antibodies. (d) IC₅₀ versus anti‐spike IgG serum antibody. (a) Young mice, n = 8; old mice n = 7. (b, c) Young mice, n = 7; old mice n = 6. Data are representative of two individual experiments. (d) Young mice, n = 15; old mice n = 13. Data are combined from two individual experiments. (a–c) Mann–Whitney U‐tests were used. (d) Separate linear regressions were performed for young and old mice, respectively. mo, month old; RBD, receptor‐binding domain.

Figure 2

Aged mice generate comparable numbers of spike‐specific CD8⁺ T cells after ChAdOx1 nCoV‐19 vaccination. Mice were immunized with 50 μL of ChAdOx1 nCoV‐19, ChAdOx1 ovalbumin (10⁸ infectious units) or phosphate‐buffered saline intramuscularly. At the indicated timepoints, medial iliac lymph node (miLN) and spleen samples were taken for analysis. (a) Live, single, CD8⁺ T cells were identified—representative plots from miLN, 14 days after vaccination. (b) miLN and spleen spike protein tetramer (SARS‐CoV‐2 S 539–546—VNFNFNGL) binding CD8⁺ T cells were identified by flow cytometry. (c) Frequency (as a percentage of all CD8⁺ T cells) and number of spike‐protein‐specific CD8⁺ T cells was quantified in miLN and spleen. (d) Spleen spike protein binding CD8⁺ T cells were identified in both young and old mice. (e) Frequency (as a percentage of all CD8⁺ T cells) and the number of spike‐protein–specific CD8⁺ miLN T cells were quantified in 3‐ and 22‐month‐old mice at the indicated timepoints. (f) Spleen spike protein–binding CD8⁺ T cells were in both young and old mice. (g) Frequency (as a percentage of all CD8⁺ T cells) and number of spike‐protein–specific spleen CD8⁺ T cells were quantified in 3‐ and 22‐month‐old mice at the indicated timepoints. Each symbol represents a unique biological sample. Data are representative of two individual experiments. (e, g) Multiple Mann–Whitney U‐tests per row with multiple testing correction was used. SARS‐CoV‐2, severe acute respiratory syndrome coronavirus‐2. FSC‐A, forward scatter–area; FSC‐H, forward scatter–height; mo, months old; PBS, phosphate‐buffered saline; PE, phycoerythrin.

Figure 3

Splenic spike‐protein–specific CD8⁺ T cells from aged mice have increased expression of activation and exhaustion markers. Mice were immunized with 50 μL of ChAdOx1 nCoV‐19 (10⁸ infectious units) intramuscularly. At the indicated timepoints, spleen samples from 3‐ and 22‐month‐old mice were taken to analyze the frequency of various CD8⁺ T‐cell subsets by flow cytometry. (a) Spike‐specific CD44⁺ CD62L⁻ (T‐effector memory) phenotype. (b) Total CD44⁺ CD62L⁻ (T effector) phenotype. (c) Spike‐specific programmed cell death protein 1 (PD‐1) expressers. (d) Total PD‐1 expressers. (e) Spike‐specific T‐BET⁺ CXCR3⁺ (activated) phenotype. (f) Total T‐BET⁺ CXCR3⁺ (activated) phenotype. Each symbol represents a unique biological sample. Data are representative of two individual experiments. (a–f) Multiple Mann–Whitney U‐tests per row with multiple testing correction were used. FITC, fluorescein isothiocyanate; CXCR3, C–X–C chemokine receptor type 3; PD‐1, programmed cell death protein 1.

Figure 4

Medial iliac lymph node (mILN) spike‐protein–specific CD8⁺ T cells from aged mice have increased expression of activation and exhaustion markers. Mice were immunized with 50 μL of ChAdOx1 nCoV‐19 (10⁸ infectious units) intramuscularly. At the indicated timepoints, mILN samples from 3‐ and 22‐month‐old mice were taken to analyze the frequency of various CD8⁺ T‐cell subsets by flow cytometry. (a) Spike‐specific CD44⁺ CD62L⁻ (T‐effector memory) phenotype. (b) Total CD44⁺ CD62L⁻ (T effector) phenotype. (c) Spike‐specific PD‐1 expressers. (d) Total PD‐1 expressers. (e) Spike‐specific T‐BET⁺ CXCR3⁺ (activated) phenotype. (f) Total T‐BET⁺ CXCR3⁺ (activated) phenotype. Each symbol represents a unique biological sample. Data are representative of two individual experiments. (a–f) Multiple Mann–Whitney U‐tests per row with multiple testing correction were used. CXCR3, C–X–C chemokine receptor type 3; FITC, fluorescein isothiocyanate; PD‐1, programmed cell death protein 1.