Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity

Abstract

Immune memory is tailored by cues that lymphocytes perceive during priming. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic created a situation in which nascent memory could be tracked through additional antigen exposures. Both SARS-CoV-2 infection and vaccination induce multifaceted, functional immune memory, but together, they engender improved protection from disease, termed hybrid immunity. We therefore investigated how vaccine-induced memory is shaped by previous infection. We found that following vaccination, previously infected individuals generated more SARS-CoV-2 RBD-specific memory B cells and variant-neutralizing antibodies and a distinct population of IFN-γ and IL-10-expressing memory SARS-CoV-2 spike-specific CD4⁺ T cells than previously naive individuals. Although additional vaccination could increase humoral memory in previously naive individuals, it did not recapitulate the distinct CD4⁺ T cell cytokine profile observed in previously infected subjects. Thus, imprinted features of SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

Keywords: COVID-19; SARS-CoV-2; adaptive immune response; human; hybrid Immunity; memory B cell; memory T cell; vaccine.

Graphical abstract

Figure 1

The greater humoral response to vaccination in SARS-CoV-2 previously infected compared with naive individuals is recovered by third vaccination (A) Timeline of blood draws from SARS-CoV-2 naive (N) and previously infected (Prev. Inf., PI) analyzed in this study relative to vaccinations. (B) Representative gating on CD19⁺CD38^lo B cells for RBD-tetramer⁺Decoy⁻ SARS-CoV-2 RBD-specific B cells from N and PI (Prev. Inf.) PBMCs at the indicated time points pre-vaccination (Pre), 1 week post-second vaccination (1w PV2), 3 months post-second vaccination (3m PV2), and 6 months post-second vaccination (6m PV2). (C) Number of RBD-specific antigen experienced (ag-exp.) B cells (CD21⁺CD27⁺ and CD21⁻CD27^+/−) in N (blue) and PI (red) PBMCs at the indicated time points. (D and E) ELISA area under the curve (AUC) for RBD-specific IgG (D) and IgA (E) plasma antibody from N and PI individuals at indicated time points. Data in (C–E) are represented both longitudinally (left) and by cross-group comparisons (right). Statistics determined by Wilcoxon matched-paired signed rank test for longitudinal analyses and multiple unpaired Mann-Whitney test for group analyses: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005, and ^∗∗∗∗p < 0.0001. Error bars represent mean and SD. See also Figure S1.

Figure S1

COVID-19 vaccination induces SARS-CoV-2 RBD-specific B cell responses in previously infected and previously naive individuals, related to Figure 1 (A) ELISA area under the curve (AUC) for RBD-specific IgG in plasma collected from individuals prior to 2020 and the SARS-CoV-2 pandemic (historical negatives [HNs], black), SARS-CoV-2 naive (N) individuals and previously infected (PI) individuals that tested PCR⁺ for SARS-CoV-2. Dashed line indicates mean + 3 SD of HN AUC values. (B) Representative flow cytometry gates for phenotyping RBD(Wu-1)- and RBD(β)-specific B cells from N and PI PBMCs. (C) Representative gating on CD19⁺CD38^loRBD-tetramer⁺Decoy⁻ cells for SARS-CoV-2 RBD-specific antigen-experienced (ag-exp.) B cells (CD21⁺CD27⁺ and CD21⁻CD27^+/−) from N and PI (Prev. Inf.) PBMCs at the indicated time points. (D) Number (left) and percent IgG⁺ or IgA⁺ (right) of RBD-specific plasmablasts (PBs, CD27⁺CD38^hi). Statistics determined by two-tailed Mann-Whitney tests: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005, and ^∗∗∗∗p < 0.0001. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2), 2 weeks post third vaccination dose (2w PV3). Error bars represent mean and SD.

Figure S2

Determination of antigen-reactive activation markers in response to SARS-CoV-2 peptide stimulation and quantification of AIM CD4⁺ T cells, related to Figure 2 (A) Representative flow cytometry plots showing the gating schematic for non-naive (nn) CD4⁺ T cells (AIM CD4⁺). (B) Boolean assessment of activation-associated markers on spike-reactive AIM CD4⁺ T cells from naive (blue) and previously infected (red) participants comparing pre-vaccine (Pre) and 1 week post two-dose mRNA vaccination (1w PV2). “+”: marker was gated; “−”: marker was excluded; “+/−”: analysis was agnostic to indicated marker. (C) Representative flow cytometry plots for AIM CD4⁺ CD69⁺CD137⁺ T cells. Dot plot overlays show cells expressing additional activation-associated markers: CD134 (top) and PD-L1 (bottom). (D) Representative flow cytometry plots for additional AIM markers CD134 and PD-L1. (E and F) Representative flow cytometry plots and summary graphs for non-naive CD69⁺CD137⁺ T cells (AIM CD4⁺) from SARS-CoV-2 naive (blue) and previously infected (red) individuals. Data in (F) are to a common antigen control peptide pool (CEFX) and represented both longitudinally (left) and by cross-group comparisons (right). (G) Total AIM CD4⁺ cell count per million CD4⁺ T cells in each SARS-CoV-2 naive (left) and previously infected (right) donor. (H) Background normalization of AIM CD4⁺ T cell data from (G) was performed by subtracting DMSO cell counts from matched CEFX, M/N, and S samples. Significance was determined by Wilcoxon matched-paired signed rank test for longitudinal analyses and multiple unpaired Mann-Whitney test for group analyses: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. Error bars represent mean and SD. Dashed lines indicate average donor background level. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2), 2 weeks post third vaccination dose (2w PV3).

Figure 2

Robust and durable CD4⁺ T cell responses to SARS-CoV-2 in both previously naive and previously infected individuals Analysis of non-naive CD4⁺CD69⁺CD137⁺ T cells (AIM CD4⁺) in SARS-CoV-2 naive (blue) and previously infected (red) individuals. (A and B) Representative flow cytometry plots and summary graphs for total AIM CD4⁺ T cells. T cell responses shown are either to SARS-CoV-2 membrane and nucleocapsid (M/N) or spike peptide pools for each donor. (C and D) Representative flow cytometry plots and summary graphs of indicated AIM CD4⁺ memory and Tfh subsets for participants shown in (A) and (B). Data in (B) and (D) are represented both longitudinally (left) and by cross-group comparisons (right). Significance was determined by Wilcoxon matched-paired signed rank test for longitudinal analyses and multiple unpaired Mann-Whitney test for group analyses: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. Error bars represent mean and SD. Dashed lines indicate average donor background level. Central memory (CM), effector memory (EM), T effector memory CD45RA⁺ (TEMRA), T follicular helper (Tfh). Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2), 2 weeks post third vaccination dose (2w PV3). See also Figure S2 and Data S1.

Figure S3

Vaccination induces enhanced variant binding and neutralizing breadth in RBD-specific MBCs and antibody in previously infected as compared with previously naive individuals, related to Figure 3 (A and B) (A) Percent naive B cells (CD21⁺CD27⁻) and (B) percent antigen-experienced (ag-exp.) B cells MBCs (CD21⁺CD27⁺ and CD21⁻CD27^+/−) of RBD-specific non-plasmablasts in N and PI PBMCs at the indicated time points. (C) Percent IgG⁺ classical MBCs (CD21⁺CD27⁺) of RBD-specific ag-exp. B cells in N and PI PBMCs at the indicated time points. (D) Representative gating on RBD(Wu-1)-specific antigen-experienced (ag-exp.) B cells for RBD(Wu-1) and RBD(β) tetramer binding to identify cross-variant specific RBD(Wu-1⁺β⁺) MBCs in N and PI PBMCs at the indicated time points. (E) Percent RBD(Wu-1⁺β⁺)-specific MBCs of RBD(Wu-1)-specific ag-exp. B cells in N and PI PBMCs at the indicated time points. (F) ELISA area under the curve (AUC) for RBD(β)-specific IgG plasma antibody from N and PI individuals at indicated time points. (G and H) (G) Percent neutralization of SARS-CoV-2(WA-1) virus by PRNT from N and PI individuals at the indicated time points and (H) correlation with RBD(Wu-1)-specific IgG AUC. Data in (A–C), (E), and (F) are represented both longitudinally (left) and by cross-group comparisons (right). Statistics determined by two-tailed Mann-Whitney tests: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005, and ^∗∗∗∗p < 0.0001. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2), 2 weeks post third vaccination dose (2w PV3). Error bars represent mean and SD.

Figure 3

Infection prior to vaccination corresponds with sustained CD21⁻CD11c⁺ MBCs and superior SARS-CoV-2 variant-neutralizing antibody (A) Representative gating on RBD-specific antigen-experienced (ag-exp.) B cells for activated MBCs (CD21⁻CD11c⁺) from N and PI PBMCs at the indicated time points. (B and C) (B) Percent and number of activated MBCs and (C) percent and number of IgG⁺ classical MBCs (CD21⁺CD27⁺) of RBD-specific ag-exp. B cells in N and PI PBMCs at the indicated time points. (D) Percent neutralization AUC of SARS-CoV-2 (WA-1) by plasma from N and PI individuals at the indicated time points by PRNT. (E) Percent neutralization of SARS-CoV-2(Delta, Δ) and SARS-CoV-2(Omicron, o) spike-pseudotyped virus by plasma from N and PI individuals at the indicated time points (left). Percent neutralization area under the curve (AUC) (right). Dashed line indicates the average percent neutralization of plasma from N individuals pre-vaccination. Data in (B) and (C) are represented both longitudinally (left) and by cross-group comparisons (right). Statistics determined by Wilcoxon matched-paired signed rank test for longitudinal analyses and multiple unpaired Mann-Whitney test for group analyses: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005, and ^∗∗∗∗p < 0.0001. Error bars represent mean and SD. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2), 2 weeks post third vaccination dose (2w PV3). See also Figure S3.

Figure 4

CD4⁺ T cell responses to SARS-CoV-2 demonstrate qualitative effector cell differences between previously naive and previously infected individuals Analysis of spike-reactive CD4⁺ T cells in SARS-CoV-2 naive (blue) and previously infected (red) individuals. (A and B) Representative flow cytometry plots, gating scheme, and summary graphs for non-naive CD4⁺CD69⁺CD137⁺ T cells (AIM CD4⁺) for the indicated T helper subsets. (C–H) Representative flow cytometry plots and summary graphs for the indicated cytokines, gated on total CD4⁺CD69⁺CD154⁺ T cells. Data in (B), (D), (F), and (H) are represented both longitudinally (left) and by cross-group comparisons (middle, right). Significance was determined by Wilcoxon matched-paired signed rank test for longitudinal analyses and multiple unpaired Mann-Whitney test for group analyses: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. Error bars represent mean and SD. Dashed lines indicate average donor background level. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2), 2 weeks post third vaccination dose (2w PV3). See also Figure S4, Figure S5, Figure S6.

Figure S4

Gating strategy, validation, and assessment of total AIM CD4⁺CD69⁺CD154⁺ T cells from ex vivo cytokine release assay, related to Figure 4 (A) Mean fluorescent intensity (MFI) of CD127 on AIM CD4⁺ T cells in previously infected (red) participants. (B) AIM gating strategy depicting removal of naive CD45RA⁺CD127⁺ T cells and CD25⁺CD127⁻ T regulatory cells. (C) Validation of CD69⁺CD154⁺ AIM cells depicting absence from T regulatory and naive compartments (left) and inclusion of all cytokine-producing cells (right). (D) Representative flow cytometry gating of non-naive (nn) AIM CD4⁺CD69⁺CD154⁺ T cells in DMSO-, M/N-, and S-stimulated memory T cells from SARS-CoV-2 naive (left, blue) and SARS-CoV-2 previously infected (right, red) participants before and 1 week after two doses of mRNA vaccine. (E and F) Summary graphs of MN- (E) and S-specific (F) nnCD4⁺ T cells. Data in (E) and (F) are represented both longitudinally (left) and by cross-group comparisons (middle, right). Lines connecting data points indicate paired samples from the same donor. Significance was determined by Wilcoxon matched-paired signed rank test for longitudinal analyses and multiple unpaired Mann-Whitney test for group analyses: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Error bars represent mean and SD. Dashed lines indicate average donor background level. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2), 2 weeks post third vaccination dose (2w PV3).

Figure S5

Cytokine production by M/N-specific AIM⁺ CD4⁺ T cells, related to Figure 4 (A–E) Summary graphs of the indicated cytokines for M/N-specific CD4⁺CD69⁺CD154⁺ T cells (AIM⁺CD4⁺) in SARS-CoV-2 naive (blue) and previously infected (red) participants. Data are represented both longitudinally (left) and by cross-group comparisons (middle, right). Significance was determined by Wilcoxon matched-paired signed rank test for longitudinal analyses and multiple unpaired Mann-Whitney test for group analyses: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Error bars represent mean and SD. Dashed lines indicate average donor background level. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2), 2 weeks post third vaccination dose (2w PV3).

Figure S6

Supplemental cytokine production by S-specific AIM⁺ CD4⁺ T cells, related to Figure 4 (A–H) Representative flow cytometry plots and summary graphs for non-naive CD4⁺CD69⁺CD154⁺ T cells (AIM⁺ CD4s) for the indicated cytokines. Data in (B) and (D) are represented both longitudinally (left) and by cross-group comparisons (middle, right). Validation of cytokine staining for cases of very low cytokine producers indicated in (E) through (H) are provided using phorbol 12-myristate 13-acetate (PMA) and ionomycin positive control (yellow). Significance was determined by Wilcoxon matched-paired signed rank test for longitudinal analyses and multiple unpaired Mann-Whitney test for group analyses: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Error bars represent mean and SD. Dashed lines indicate average donor background level. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2), 2 weeks post third vaccination dose (2w PV3).

Figure 5

Dimensionality reduction and clustering analysis of SARS-CoV-2 S-specific CD4⁺AIM⁺ T cells (A) Dimensionality reduction and phenograph-derived clustering (k = 40) overlaid for total CD4⁺AIM⁺(CD154⁺CD69⁺) T cells pooled from all participants (left). Heatmap expression of the indicated parameters over UMAP space (right). (B–P) All cells from the indicated clusters (CL) are represented in corresponding color from UMAP plot in (A) overlayed on all CD4⁺AIM⁺ T cells and showing expression of select parameters (IL-2, IFN-γ, IL-10, CXCR5, CD127, CD25, IL-4, and IL-21). Enumeration of each cluster is per 1 × 10⁶ non-naive (nn) CD4⁺ T cells in SARS-CoV-2 naive (blue) and SARS-CoV-2 previously infected (red) participants. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2). Significance was determined by multiple unpaired Mann-Whitney test for group analyses and by Wilcoxon matched-paired signed rank test for longitudinal analyses: not significant (ns), ^∗p < 0.05, ^∗∗p < 0.01.