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. 2021 Nov 22;12:6774. doi: 10.1038/s41467-021-27029-6

Fig. 3. Immunogenicity of the H56:IC31 vaccine.

Fig. 3

Effect of H56:IC31 administered at two doses (day 84 and day 140) on vaccine-induced immunity in the H56:IC31 group (blue; n = 12) and controls (gray, n = 12) (Hypothesis 2, Table 3). a, b, c and d depicts data from Fluorospot analysis of thawed PBMCs, isolated at day 84 and 154 (n for H56:IC31/n for controls) and stimulated with H56 fusion protein, Ag85B and ESAT-6* in the presence of anti-CD28 (0.1 mg/ml) for 17 h. Cytokine+ was defined as the sum of IFNγ and IL-2 responses (total IFNγ plus total IL-2 minus duo IFNγ/IL-2). a Cytokine+ T-cells in response to a H56 fusion protein at day 84 (n: 12/10) and day 154 (n: 11/10), b Ag85B and ESAT-6 summed at day 84 (n: 12/10) and day 154 (n: 11/10). c Longitudinal Cytokine+ T-cell responses to H56 from day 0 to day 238. d Individual trajectories of Cytokine+ T-cell responses to Ag85B and ESAT-6 stratified by intervention. e, f Depicts data from flow cytometry analysis with intracellular staining (ICS) of 1 ml peripheral whole blood (WB) incubated for 12 h (Brefeldin A added after 7 h) with Ag85B, ESAT-6, and Rv2660c* in the presence of anti-CD28 and anti-CD49d (0.1 mg/ml) within 75 min of sampling at day 84 and day 154. Cytokine+ was defined as the sum of IFNγ, IL-2 and TNFα responses (total IFNγ plus duo IL-2/TNFα plus single IL-2 plus single TNFα). e Cytokine+ CD4 T-cells in response to Ag85B, ESAT-6, and Rv2660c summed at day 84 (n: 12/10) and day 154 (n: 11/10). f Boolean gating was used to create CD4 T-cell subpopulations defined by their co-production of cytokines in response to Ag85B and ESAT-6 at day 84 (n: 12/10) and day 154 (n: 11/10). g Longitudinal log transformed anti-H56 IgG serum levels from day 0 (n: 12/10) to day 238 (n: 10/6) analyzed by in-house ELISA. *Stimulants in Fluorospot and ICS as follows: the H56 fusion protein at 5 μg/ml, the Ag85B, ESAT-6, and Rv2660c (15mer overlapping individual peptide pools >80–85% purity at 2 μg/ml). Boxplots are shown with median, interquartile ranges (IQR) and minimum/maximum values. Line plots are shown with IQR. Median regression with treatment as only independent explanatory factor was applied for immunogenicity read-outs pre-assigned a hierarchical order to compensate for lack of multiple testing adjustments. Effect estimates of primary priority and post-hoc analyses of secondary priority, 95% confidence intervals based on 1000 bootstrap replications and corresponding two-sided p-values depicted in Table 3 and in (a, b, e, g).