Primary and Recall Immune Responses to SARS-CoV-2 in Breakthrough Infection

Abstract

Breakthrough infections in SARS-CoV-2 vaccinated individuals are an ideal circumstance for the simultaneous exploration of both the vaccine-induced memory reaction to the spike (S) protein and the primary response to the membrane (M) and nucleocapsid (N) proteins generated by natural infection. We monitored 15 healthcare workers who had been vaccinated with two doses of Pfizer BioNTech BNT162b2 and were then later infected with the SARS-CoV-2 B.1.617.2. (Delta) variant, analysing the antiviral humoral and cellular immune responses. Natural infection determined an immediate and sharp rise in anti-RBD antibody titres and in the frequency of both S-specific antibody secreting cells (ASCs) and memory B lymphocytes. T cells responded promptly to infection by activating and expanding already at 2-5 days. S-specific memory and emerging M- and N-specific T cells both expressed high levels of activation markers and showed effector capacity with similar kinetics but with different magnitude. The results show that natural infection with SARS-CoV-2 in vaccinated individuals induces fully functional and rapidly expanding T and B lymphocytes in concert with the emergence of novel virus-specific T cells. This swift and punctual response also covers viral variants and captures a paradigmatic case of a healthy adaptive immune reaction to infection with a mutating virus.

Keywords: SARS-CoV-2; adaptive immune response; breakthrough infection; vaccination.

Figure 1

Antibody and B cell responses during breakthrough infection. (a) Serum anti-RBD antibody dosage (day 0, n = 12; 2–5 days, n = 6; 6–9 days, n = 11; 15–17 days, n = 5). (b) Absolute cell counts of S- and RBD-specific B cells (day 0, n = 12; 2–5 days, n = 6; 6–9 days, n = 11; 15–17 days, n = 5). Statistical significance of the comparisons is indicated by asterisks coloured according to S- or RBD-specificity. (c) Frequency of RDB-specific B cells within S-specific B cells (day 0, n = 6; 2–5 days, n = 6; 6–9 days, n = 11; 15–17 days, n = 5). Values were compared with nonparametric repeated measures Kruskall–Wallis and corrected for Dunn’s multiple comparison tests; * p < 0.05; ** p < 0.01; *** p < 0.001; (d) Frequency of the different B cell subpopulations within total B-, S- and RBD-specific cells. (RBD: Receptor Binding Domain, S: Spike, ASC: Antibody Secreting Cells, cmemSW: classical memory switched, DN1: double negative 1, DN2: double negative 2, Tr: transitional, USWmem: unswitched memory).

Figure 2

Phenotypic characterisation of B cells during breakthrough infections. Frequency of CD11c, CD80, CD95, CD86, CXCR5 and CD73 within S-specific ASC (green circles), S-specific cmemSW (red circles) and RBD-specific B cells (purple circles) (day 0, n = 6; 2–5 days, n = 6; 6–9 days, n = 11; 15–17 days, n = 5). Values were compared with nonparametric repeated measures Kruskall–Wallis and corrected for Dunn’s multiple comparison tests; * p< 0.05; ** p< 0.01. (S: Spike, RBD: Receptor Binding Domain, ASC: Antibody Secreting Cells, cmemSW: classical memory switched).

Figure 3

CD4+ and CD8+ T cell responses during breakthrough infection. (a,b) In the left panels, the graphs illustrate the median absolute cell counts of CD4+ (a) or CD8+ (b) T cells specific for S, N and M antigens as determined with the AIM assays performed at the indicated time points. The right panels display the SI for CD4+ and CD8+ antigen-specific cells. (c,d) The graphs in the left panels show absolute cell counts of IFN-γ-producing CD4+ (c) and CD8+ (d) T cells, and right panels show the SI. Non-parametric Friedman tests followed by Dunn’s post hoc tests were used to compare responses to S and non-S peptides from the same donor at the same time points. Median values obtained at each time point for S-, M- and N-specific T cells were compared with non-parametric Friedman tests followed by Dunn’s post hoc tests. Statistical significance of the comparisons is indicated by asterisks positioned over the rods coloured according to S, M or N reactivity. * p < 0.05; ** p< 0.01; *** p < 0.001; **** p < 0.0001; no symbol, not significant. (S: Spike, M: Membrane, N: Nucleocapsid, AIM: Activation Induced Markers).

Figure 4

Differentiation status of virus-specific CD4+ T cells during breakthrough infection. (a) Fractions of naïve, terminally differentiated effector memory (EMRA), effector memory (EM) and central memory (CM) within AIM+ CD4+ T cells compared with time points for each peptide S (top), M (middle) or N (bottom). (b) Frequency of naïve, EMRA, EM and CM within AIM+ CD4+ T cells compared with time points for S (top), M (middle) or N (bottom). Time points were compared with nonparametric repeated measures Krukall–Wallis and corrected for Dunn’s multiple comparison tests; lines represent median with 5 to 95th percentile. * p < 0.05; ** p < 0.01; no symbol, not significant. (S: Spike, M: Membrane, N: Nucleocapsid, AIM: Activation Induced Markers).

Figure 5

Phenotype and functionality of antigen-specific CD4+ T cells in responder subjects. (a) Frequency of different activation markers within AIM+ CD4+ T cells compared with time points for each peptide: S, M and N. Time points within each peptide were compared with nonparametric Mann–Whitney; bold lines represent the median of plotted values. * p < 0.05; ** p < 0.01; *** p < 0.001; no symbol, not significant. (b) Longitudinal analysis of absolute cell counts showing IL-2 alone (left), and IFN-γ+ and IL-2+ (right) production within CD4+ T cells for each peptide. Statistical analysis was performed as in A. (c) Longitudinal analysis of absolute cell counts of TSCM within CD4+ T cells for each peptide. Statistical analysis was performed as in (a). (S: Spike, M: Membrane, N: Nucleocapsid, AIM: Activation Induced Markers, TSCM: T Stem Cell Memory).