Persistent memory despite rapid contraction of circulating T Cell responses to SARS-CoV-2 mRNA vaccination

Abstract

Introduction: While antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined.

Methods: Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD).

Results: In two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination.

Discussion: Overall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

Keywords: SARS-CoV-2; SARS-CoV-2 mRNA vaccines; T cell memory; T cells; cellular immunity; elispot; intracellular cytokine staining.

Figure 1

Transience of peripheral blood SARS-CoV-2 spike-specific CD8+ T cells elicited by mRNA vaccination compared to natural infection, as assessed by IFN-γ ELISpot. Spike-specific CD8+ T cells were assayed by IFN-γ ELISpot assay using pooled overlapping peptides.  (A, B) Serial CD8+ T cell responsesc against spike (open circles) and IgG responses against the spike RBD (Xs) are plotted for two SARS-CoV-2-naïve persons who received the BNT162b2 vaccine. The X-axis starts with the first vaccine dose, and the timing of the second dose is indicated by an arrow. (C) Serial CD8+ T cell responsesc against spike (closed squares) and IgG responses against the spike RBD (Xs) are plotted for a SARS-CoV-2-naïve person who received the ChAdOx1-S vaccine. The X-axis starts with the first vaccine dose, and the timing of the second dose is indicated by an arrow. (D) CD8+ T cell spike-specific responses are plotted for 25 persons who were SARS-CoV-2-naïve after the first vaccine dose with BNT162b2 (16 persons, 20 data points, circles) or mRNA-1273 (9 persons, 9 data points, triangles).  (E) CD8+ T cell spike-specific responses are plotted for 24 persons who were SARS-CoV-2-naïve after the second vaccine dose with BNT162b2 (15 persons, 20 data points, circles) or mRNA-1273 (9 persons, 9 data points, triangles).  (F) CD8+ T cell spike-specific responses are plotted for 45 COVID-19-recovered persons according to time after symptom onset (diamonds).

Figure 2

Example of intracellular cytokine staining for CD4⁺ and CD8⁺ T cell responses against SARS-CoV-2 spike. PBMC from a person 13 days after symptom onset of COVID-19 were cultured in the absence or presence of overlapping 15-mer synthetic peptides spanning the SARS-CoV-2 spike protein and assessed for production of IFN-γ, IL-2, IL-10 (not shown) and IL-4 (not shown) by intracellular cytokine staining and flow cytometry. Controls included cells cultured without peptides and PMA-ionomycin stimulated cells.

Figure 3

CD4+ and CD8+ T cell responses against spike measured by IFN-γ intracellular cytokine staining after mRNA SARS-CoV-2 vaccination versus natural infection. Background-subtracted values are plotted for CD4+ and CD8+ T cell spike-specific IFN-γ productiondetermined as shown in Figure 2 .  (A) CD4+ T cell responses are plotted for 22 persons vaccinated with BNT162b2 (18 points from 16 persons, circles) or mRNA-1273 (7 points from 6 persons, triangles). Time points ranged from 13 to 235 days after the second vaccine dose. Only one response was detectable above 0.01% frequency. (B) CD8+ T cell responses measured in parallel are plotted for the same 22 persons in (A) Only one response was detectable above 0.01% frequency. (C) CD4+ T cell responses are plotted for 25 COVID-19-recovered persons ranging from 15 to 49 days after symptom onset. 17/25 (68.0%) had responses greater than 0.01%. (D) CD8+ T cell responses are plotted for the same 25 persons in (C) Again, 17/25 (68.0%) had responses greater than 0.01%. (E) The frequencies of responding CD4+ and CD8+ T cells from (C, D) are compared, demonstrating Pearson correlation r2 = 0.66, p<0.00001.

Figure 4

PBMC cultured with the mRNA-1273 vaccine in vitro reveal enrichment of spike-specific memory CD4+ and CD8+ T cell responses . An example is shown for detection of spike-specific T cells (as described in Figure 2. ) in PBMCs from a SARS-CoV-2-naïve person who had completed vaccination with mRNA-1273 128 days prior. Top row: The PBMC were directly tested for T cell reactivity against spike. Bottom row: Prior to testing, the PBMC were cultured with the addition of mRNA-1273 vaccine for 14 days before testing for spike-specific T cells.

Figure 5

Vaccine-elicited spike-specific memory CD4+ and CD8+ T cells are persistent. In parallel to Figure 3 panels A and B, the same PBMC from 22 vaccinees were assessed for spike-specific T cell memory responses as shown in Figure 4 . (A) 22/25 (88.0%) vaccinees had detectable spike-specific CD4+ T cell memory responses of greater than 0.01% frequency (14/18 BNT162b2 vaccinees, circles, and 7/7 mRNA-1273 vaccinees, triangles). (B) 18/25 (76.0%) vaccinees had detectable CD8+ T cell memory responses greater than 0.01% frequency (15/18 BNT162b2 vaccinees, circles, and 4/7 mRNA-1273 vaccinees, triangles). (C) The frequencies of spike-specific memory CD4+ and CD8+ T cells after in vitro enrichment are compared, demonstrating Pearson correlation r2 = 0.49, p<0.0001.