Immune responses in Omicron SARS-CoV-2 breakthrough infection in vaccinated adults

Abstract

The SARS-CoV-2 Omicron variant has more than 15 mutations in the receptor binding domain of the Spike protein enabling increased transmissibility and viral escape from antibodies in vaccinated individuals. It is unclear how vaccine immunity protects against Omicron infection. Here we show that vaccinated participants at a super-spreader event have robust recall response of humoral and pre-existing cellular immunity induced by the vaccines, and an emergent de novo T cell response to non-Spike antigens. Individuals with Omicron SARS-CoV-2 breakthrough infections have significantly increased activated SARS-CoV-2 wild type Spike-specific cytotoxic T cells, activated follicular helper (T_FH) cells, functional T cell responses, boosted humoral responses, and rapid release of Spike and RBD-specific IgG⁺ B cell plasmablasts and memory B cells into circulation. Omicron breakthrough infection affords significantly increased de novo memory T cell responses to non-Spike viral antigens. Concerted T and B cell responses may provide durable and broad immunity.

Fig. 1. Cytotoxic cellular immunity during Omicron and Delta BTI.

CD8⁺ T cells in Omicron or Delta BTI, vaccinated healthy donors (HD) or SARS-CoV2 WT convalescents were compared for their phenotypes (a–d) or functions in flow cytometry-based assays (e, f). a Characterization of Spike-, and CMV-specific CD8⁺ T cells. Anti-Spike Dextramer (red) and anti-CMV tetramer staining (blue) were combined with deep immune-phenotyping by flow cytometry and overlaid on total CD8 T cells (grey). Representative examples of single individual are shown. See Methods for peptides and peptide: HLA multimers. b Ex vivo immune phenotype of SARS-CoV-2 Spike-specific CD8⁺ T cells. A cold to hot heatmap represents the scaled frequency of each individual marker expressed by antigen-specific CD8⁺ T cells. Patient, HLA for A and B alleles, time post-infection (early acute <10 d, median acute for Day 10–11 or late acute >11 d) and marker subsets are indicated in the top three rows and in the leftmost column respectively. c Signature of Spike-specific CD8 T cells. The identification of specific markers is based on the fold change and significance in comparison to the phenotype of Spike-specific CD8 T cells from HD and is visualized by red dots on the volcano plot. d Quantification of SARS-CoV-2 Spike and non-Spike-specific CD8⁺ T cells defined by peptide: HLA multimers in Omicron and Delta BTI. Mann–Whitney test (two-sided) P values are shown. e Functionality of Spike-specific CD8⁺ T cells. PBMCs were stimulated overnight in vitro by overlapping SARS-CoV-2 Spike (wild type, WT) peptides or left unstimulated (US) and stained to assess the induction of activation markers. IFNγ and/or TNF responding cells or dual expression of IFN-γ, CD137 and TNF is shown. Wilcoxon test (two-sided) P values are shown for comparison between unstimulated and peptides stimulated samples. f Functionality of SARS-CoV-2 specific CD8⁺ T cells. Response over background of CD8⁺ T cells to the stimulation in vitro by Spike (WT) peptides, left; middle: pooled overlapping SARS-CoV-2 membrane (M) and nucleoprotein (N) peptides, and ORF peptides (See Methods and Table 2)—and right: the whole proteome of WT, 88 peptides from SARS-CoV-2 (S, M, N, E, O), see “Methods”. IFN-γ and/or TNF responding cells are shown. Mann–Whitney test P (two-sided) values are shown. g Biplot showing in vitro responses of CD8⁺ T cells to non-Spike peptide pools vs. Spike-peptides. Quadrants are labeled as described: infected and nonvaccinated (I⁺V⁻), non-infected and vaccinated (I⁻V⁺); or infected and vaccinated (I⁺V⁺). See also Supplementary Figs. 3, 4. Source data are provided as a Source Data file.

Fig. 2. Th cellular immunity during Omicron and Delta BTI.

Omicron or Delta BTI vaccinated healthy donors or SARS-CoV2 WT convalescents were analyzed to quantify their frequency and characterize the phenotypes of CD4⁺ T_FH cells (a–c) or to assess their functions (e, f). a Mass Cytometry profile of CXCR5⁺CD4⁺ Follicular helper T cells. Visualization by tSNE-plot of clusters automatically identified by Phenograph in total T_FH from concatenated files of vaccinated HD, Omicron BTI, and Delta BTI (see Methods and Supplementary Fig. 6 legend for details). b The absolute frequency of T_FH CD4 T cells in PBMCs for all BTI patients. c Frequency of T_FH cells expressing memory (CD95) or activation markers (HLA-DR), PD-1, and chemokine receptors (CCR4, CCR6, CXCR3). d Functional responses of SARS-CoV-2 specific CD4⁺ T cells. The specific responding CD4⁺ T cells are shown similarly as in Fig. 1f. e Biplot showing in vitro responses of CD4⁺ T cells to non-Spike peptide pools vs. Spike-peptides as described: infected and nonvaccinated (I⁺V⁻), non-infected and vaccinated (I⁻V⁺); or infected and vaccinated (I⁺V⁺). Mann–Whitney test P values are shown for (b–d). See also Supplementary Fig. 6.

Fig. 3. Humoral and B cell immunity during Omicron and Delta breakthrough infection.

a Identification of B cells that bind Spike but not RBD (Spike⁺RBD⁻ B cells) or bind both Spike and RBD (Spike⁺RBD⁺ B cells) in total B cells from three individuals: Healthy Donor (HD), Omicron BTI and Delta BTI. b Quantification of Spike-binding and RBD-binding B cells. Left: Absolute frequency of Spike-binding and RBD-binding B cells. Middle: serum levels of anti-RBD IgG (BAU/ml). Right: Relative frequency of anti-RBD to spike-specific B cells. Ratio of RBD-binding B cells (see top region in a.)/total Spike-binding B cells is shown. c Biplot showing ex vivo profile of Spike⁺RBD⁻ and Spike⁺RBD⁺ B cells during BTI. Top: frequency of CD38 expression in Spike⁺RBD⁻ vs Spike⁺RBD⁺ B cells. Bottom: frequency of CD38^HiCD71⁺ plasmablasts in Spike⁺RBD^- vs Spike⁺RBD⁺ B cells. HD (blue), Omicron BTI (green) and Delta BTI (red) are shown. d Phenotype of Spike-binding B cells. Visualization by tSNE-plot of selected markers as indicated (bottom two rows) in total B cells and location of Spike-binding (Spike⁺) B cells (top row) in HD, Omicron BTI, Delta BTI and aggregated donors (20 000 cells from two individuals per group, total = 120 000 cells). Each marker is visualized by a cold to hot heatmap. e Quantification of anti-Spike antibody secreting cells (ASC). The frequency of specific markers for ASC among B cells (CD38, IRF4, CD71, BLIMP-1) are shown for Total B cells (blue) and Spike-binding B cells. f Characterization of Spike-binding B cells. A heat-plot shows the phenotype of Spike-specific B cells in the three groups. The normalized frequency of each marker is displayed and automatic hierarchical clustering of Spike-binding B cells for each patient is shown. Mann–Whitney test P values are shown for (b) and (e). See also Supplementary Fig. 7.