Antigen-specific CD8+ T cell responses to JPT and Miltenyi peptide S protein wt and mutant pools.

Antigen-specific CD8+ T cell responses elicited by the JPT peptide S protein pools after vaccination with BioNtech (grey), Moderna (blue) or AstraZeneca (red), or one year (orange) or < 2 months (dark green) after naturally occurring infection with SARS-CoV2, or negative controls. The bar graphs show frequencies of TNF-α+, IL-2+ or IFNγ+ CD8+ T cells for wild type, and for alpha (B.1.1.7), beta (B.1.351), gamma (P.1) and delta (B.1.617.2) mutants. At least 200,000 events were counted for each individual condition.</p

Fig 1 -

A: Representative Coomassie gel electrophoresis (left panel) and silver stain (right panel) of SARS-CoV2 wild type spike RBD protein (A) and of nucleocapsid protein (B).</p

Antibody clones used for FACS analysis.

Antibody clones used for FACS analysis.</p

Quantitative spectroscopy of single molecule interaction times

Single molecule fluorescence tracking provides information at nm-scale and ms-temporal resolution about the dynamics and interaction of individual molecules in a biological environment. While the dynamic behavior of isolated molecules can be characterized well, the quantitative insight is more limited when interactions between two indistinguishable molecules occur. We address here this aspect by developing a solid theoretical foundation for a spectroscopy of interaction times, i.e. the inference of interaction constants from imaging data. The non trivial crossover between a power law to an exponential behavior of the distribution of the interaction times is highlighted here, together with the dependence of the exponential term upon the product of the microscopic reaction rates (affinity). Our approach is validated on simulated as well as experimental datasets

Antigen-specific TNF-α+ CD4+ T cell responses to JPT peptide S protein wt and mutant pools.

Antigen-specific CD4+ T cell responses elicited by the JPT peptide S protein pools after vaccination with BioNtech (grey), Moderna (blue) or AstraZeneca (red), or one year (orange) or < 2 months (dark green) after naturally occurring infection with SARS-CoV2, or negative controls. The bar graphs show frequencies of TNF-α+CD4+ T cells for wild type, and for alpha (B.1.1.7), beta (B.1.351), gamma (P.1) and delta (B.1.617.2) mutants. The figure shows results of n = 13 independent individuals after vaccination with BioNtech, n = 5 Moderna, n = 5 AstraZeneca, and n = 10 independent individuals one year and n = 7 < 2 months after natural infection with standard errors of the mean (SEM). Samples of non infected individuals tested negative (not shown in the figure to enhance legibility). At least 200,000 events were counted for each individual condition. Significance levels were tested by Kruskal-Wallis test for non-parametric values. *p < 0.05 vs. healthy controls, **p < 0.01 vs. healthy controls and also p < 0.05 vs. 1 year after infection.</p

Gating strategy of the intracellular cytokine staining.

The figure shows a representative flow cytometry gating strategy for one of the donors. Cells were gated as follows: Singlets, Cells, Live, CD3+ T cells (A) and further T cells were divided into CD4+ and CD8+ T cells and from each of the subsets IFN-γ+, TNF-α+, or IL-2+ cells were gated (B, C). Panel B shows the negative control and Panel C shows the restimulated condition with the Miltenyi peptide mix of the same representative donor.</p

Antigen-specific CD4+ T cell responses to the Miltenyi peptide S protein pool.

Antigen-specific CD4+ T cell responses elicited by the Miltenyi wt S protein peptide pool after vaccination with BioNtech (grey), Moderna (blue) or AstraZeneca (red), or one year (orange) or < 2 months (dark green) after naturally occurring infection with SARS-CoV2. In comparison, samples of healthy control subjects, who tested negative for SARS-CoV2-antibodies, are shown on the right of each panel. The bar graphs show frequencies of (A) interferon-γ (IFN-γ), (B) interleukin-2 (IL-2)- and (C) tumor necrosis factor-α (TNF-α)-positive CD4+ T cells. The figure shows results of n = 13 independent individuals after vaccination with BioNtech, n = 5 Moderna, n = 5 AstraZeneca, and n = 10 independent individuals one year and n = 7 subjects < 2 months after natural infection with standard errors of the mean (SEM). For comparison, 13 negative healthy controls were included. Significance levels were tested by Kruskal-Wallis test for non-parametric values. *p < 0.05, **p < 0.01 vs. healthy controls.</p

Serum antibodies and neutralisation.

The bar graphs show results of n = 13 independent individuals after vaccination with BioNtech (grey), n = 5 Moderna (blue), n = 5 AstraZeneca (red), and n = 10 independent individuals one year (orange), and n = 7 subjects A: Anti RBD antibody titres; B: Representative curve of a single experiment to determine IC50-values for the inhibition of biotinylated ACE2 binding to immobilized wt vs. delta mutant RBD. C: Absolute IC50-values calculated for the inhibition of biotinylated ACE2 binding to immobilized wt vs. delta mutant RBD. D: Absolute IC50-values calculated for the inhibition of biotinylated wt vs. delta mutant RBD binding to immobilized ACE2. E: Ratios between IC50-values calculated for inhibition of ACE2 binding to immobilized wt vs. delta mutant RBD. F: Ratios between IC50-values calculated for inhibition of wt vs. delta mutant RBD binding to immobilized ACE2. G: Read-out of a representative experiment showing the comparison between inhibition of biotinylated ACE2 binding to immobilized RBD with serum taken by venous sampling vs a drop of full blood. H: In additional, independent group of 14 subjects who had been fully vaccinated with BNT 162b2, we also investigated ratios between IC50 values calculated for inhibition of ACE2 binding to immobilized omicron vs. delta mutant RBD.</p

SARS-CoV2 spike protein-derived epitope peptide pools.

SARS-CoV2 spike protein-derived epitope peptide pools.</p

Antigen-specific IFN-γ CD4+ T cell responses to JPT peptide S protein wt and mutant pools.

Antigen-specific CD4+ T cell responses elicited by the JPT peptide S protein pools after vaccination with BioNtech (grey), Moderna (blue) or AstraZeneca (red), or one year (orange) or < 2 months (dark green) after naturally occurring infection with SARS-CoV2, or negative controls. The bar graphs show frequencies of IFNγ+CD4+ T cells for wild type, and for alpha (B.1.1.7), beta (B.1.351), gamma (P.1) and delta (B.1.617.2) mutants. The figure shows results of n = 13 independent individuals after vaccination with BioNtech, n = 5 Moderna, n = 5 AstraZeneca, and n = 10 independent individuals one year and n = 7 subjects < 2 months after natural infection, and n = 9 negative controls, with standard errors of the mean (SEM). Significance levels were tested with Kruskal-Wallis test for non-parametric samples. *p < 0.05 vs. healthy controls; **p < 0.01 vs. healthy controls and p < 0.05 vs 1 year after infection.</p