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A new approach for developing a less immunosuppressive tuberculosis vaccine

Final Report Summary - IMMUNOVACTB (A new approach for developing a less immunosuppressive tuberculosis vaccine)

Infections due to Mycobacterium tuberculosis cause over two million deaths each year. A major problem in combating tuberculosis is insufficient efficacy of the current vaccine, M. bovis BCG. The insufficient efficacy is due to the fact that BCG induces the Th2 cytokines IL-4 and the immunosuppressive cytokine IL-10, apart from the protective Th1 cytokines IL-12 and INF?. Current research data suggest that two mycobacterial glycolipids - lipoarabinomannan (LAM) and phenolic glycolipid (PGL) - play an important role in this immunosuppression.

The IMMUNOVACTB consortium aimed to design less immunosuppressive BCG strains, lacking PGL and / or (part of) the mannose cap to find a new strategy to overcome these known problems of inefficacy. The project attempted to isolate BCG strains that lack the LAM mannose cap (or parts thereof) and / or cannot produce PGL.

During the first year of the project, the consortium was able to construct the capless LAM, the PGL and the double mutant BCG strains. Currently, the in vivo testing of the mutant strains by our consortium partner in Porto is underway. The structure of the glycolipids has been determined by a combination of analytical procedures, including NMR analyses on the native molecules and chemical degradations coupled to analyses by gas chromatography and capillary electrophoresis. We have found that deletion of the capA gene resulted in both mycobacterial species in the complete abrogation of mannose caps biosynthesis.

The project has now identified Rv1635c (capA) to be a mannosyltransferase gene involved in cap synthesis and characterised M. marinum and M. bovis BCG capless mutants in vitro and in vivo. Furthermore, they have identified another gene (capB) also involved in cap biosynthesis. A second DC-SIGN ligand on the Mycobacterial surface, i.e. PIM6 has been identified.

Subsequently, a knockout was made in BCG and it was demonstrated that in the pimE knockout of BCG, DC-SIGN did not react with PIMs anymore. Also a double knockout pimE-capA was constructed in BCG. However, in analogy with the mannose cap of LAM, also PIM6 does not dominate the interaction of Mycobacteria with host cells. Finally, the project has identified a third DCSIGN ligand, i.e. capsular alpha glucan and have demonstrated its immunomodulatory properties. The capless mutant, the PGL mutant and the double mutant proliferate equally well in vitro in cultured macrophages and respond to the same extent to the bacteriostatic effects of IFN? and 5 TNF as compared to the wild-type BCG. In vitro analysis of capless BCG has shown an increased ability to trigger cytokine release from antigen presenting cells.

It is hoped that the new and improved insight will also be gained into how the immune system is manipulated by mycobacterial glycolipids, with the aim of developing an improved M. bovis BCG vaccine.