Figure 2.

Serum IgG1 and IgG2c titres, expressed as EC50 values in mice (eight per group) subcutaneously vaccinated once with BCG, or three times at 9 day intervals with soluble recombinant Rv1626 (recRv1626), Rv1626 beads, Cpe30‐Rv1626 beads, CS.T3‐Rv1626 beads formulated with DDA or DDA alone. Three weeks after the final vaccination, the mice were euthanized and sera collected and analysed by ELISA using methods described previously by Rubio Reyes et al. (2016). Plates were incubated overnight at 4°C with 2.5 μg ml⁻¹ of soluble recombinant Rv1626 obtained using a sortase‐based tagless protein purification system as previously described (Hay et al., 2015). EC50 was calculated adjusting to a 4‐parameters logistic curve the absorbance values (450 nm) corresponding to sera dilutions. EC50 is the mid‐point of the sigmoid (c coefficient of the curve). Values were analysed using a Dunn's test of multiple comparisons following a significant Kruskal–Wallis test. Each bar represents the mean values of 8 mice ± standard error of the mean, +, significantly greater than the DDA vaccinated group; groups between brackets, significantly different (P < 0.01).