In-vivo expressed Mycobacterium tuberculosis antigens recognised in three mouse strains after infection and BCG vaccination

Abstract

Novel tuberculosis (TB)-vaccines preferably should (i) boost host immune responses induced by previous BCG vaccination and (ii) be directed against Mycobacterium tuberculosis (Mtb) proteins expressed throughout the Mtb infection-cycle. Human Mtb antigen-discovery screens identified antigens encoded by Mtb-genes highly expressed during in vivo murine infection (IVE-TB antigens). To translate these findings towards animal models, we determined which IVE-TB-antigens are recognised by T-cells following Mtb challenge or BCG vaccination in three different mouse strains. Eleven Mtb-antigens were recognised across TB-resistant and susceptible mice. Confirming previous human data, several Mtb-antigens induced cytokines other than IFN-γ. Pulmonary cells from susceptible C3HeB/FeJ mice produced less TNF-α, agreeing with the TB-susceptibility phenotype. In addition, responses to several antigens were induced by BCG in C3HeB/FeJ mice, offering potential for boosting. Thus, recognition of promising Mtb-antigens identified in humans validates across multiple mouse TB-infection models with widely differing TB-susceptibilities. This offers translational tools to evaluate IVE-TB-antigens as diagnostic and vaccine antigens.

Fig. 1. Most IVE-TB antigens are recognised by cells from Mtb infected mice with differences among mouse strains, organs and cytokines.

a C57BL/6 (BL6), BALB/c (BALB) and C3HeB/FeJ (C3H) mice were challenged with 1 × 10⁴ CFU Mtb-lux strain intratracheally and killed at an early (5 weeks) or later (9–12 weeks) stage of Mtb infection. Uninfected naïve control mice were killed and tested in parallel. Pooled cells from lungs, mediastinal lymph nodes (medLN) or spleens were stimulated with single or fusion Mtb antigens or with positive control stimuli ConA and PWM. The concentrations of IFN-γ, TNF-α and IL-17 were measured in the cell supernatants after 3 days of stimulation and corrected for background. b Heatmaps of the specific Mtb antigen responses for each cytokine are shown. Colour gradients indicate the cytokine concentration, while asterisks indicate significant differences between the naïve and the Mtb challenged groups. White *p-value < 0.05, computed with Kruskal–Wallis test; black asterisk marks differences that remained significant after multiple test correction (q-value < 0.1, i.e., FDR-adjusted p-values of Mann–Whitney.test). c 3D plot of the Variable Importance in Projection (VIP) scores obtained from each PLS-DA model computed on the basis of the following known classes: (i) time points (i.e., naïve mice vs. mice killed at early or late point after Mtb infection); (ii) mouse strains (i.e., C57BL/6 (BL6), BALB/c (BALB) and C3HeB/FeJ (C3H) mice); (iii) organs (i.e., lungs, mediastinal lymph nodes or spleens); (iv) cytokines (i.e., IFN-γ, TNF-α and IL-17). Antigens with a VIP score >1 were considered to have an above average influence on the model and are depicted by solid dots. Antigens with a VIP score >1 for the cytokine PLS-DA model are depicted by solid red dots. Antigens with a VIP score <1 are depicted by empty dots and their names are omitted in the 3D plot.

Fig. 2. IVE-TB antigen-specific responses after Mtb infection and BCG vaccination in C3HeB/FeJ (C3H) mice.

a C3HeB/FeJ mice were either challenged intranasally with H37Rv-Mtb strain (10⁵ CFU) (10 mice), vaccinated subcutaneously with BCG (5 mice) or left untreated (5 mice). After 6 weeks, splenocytes were harvested and stimulated per mouse for 6 days with a set of eighteen Mtb antigens or positive controls (ConA, PPD, bead disrupted BCG (BCG bub) and Mtb lysate), or negative control (HPV16E6 recombinant protein). The IFN-γ concentrations detected in the unstimulated samples were subtracted from the stimulated samples for each condition within each mouse. b IFN-γ production after antigen stimulation in splenocytes from naïve unimmunized mice (left panel), and BCG immunised versus Mtb infected mice (right panel). Dots display the median IFN-γ production. The (Rv) codes of the antigens are indicated if the median IFN-γ production exceeded 500 pg/ml in both BCG immunised and Mtb infected mice. The horizontal dotted line indicates the cut-off at 500 pg/ml, which is twice the median IFN-γ production found in response to the negative control in BCG immunised and Mtb infected mice (right panel). c IFN-γ responses against Mtb antigens in splenocytes from naïve, BCG immunised and Mtb infected mice are shown. Dots represent a single mouse (10 mice in the Mtb infected group and 5 mice in the naïve and BCG immunised group in total) and bars indicate the median IFN-γ response of each group. The Mtb challenged and BCG immunised groups were compared to the naïve group using the Kruskal–Wallis test and a p-value < 0.05 was considered significant. Asterisk marks differences that remained significant after multiple test correction (q-value < 0.1, i.e., FDR-adjusted p-values of Mann–Whitney test).