JMIR Publications

ABSTRACT

Background:

BCG immunisation has been associated with a reduction in Mycobacterium (M.) tuberculosis infection. BCG immunisation has been shown to enhance innate immunity via an effect on Nucleotide oligomerisation domain (NOD) receptors. The reduction of Mycobacterium tuberculosis infection by BCG can be explained by an enhancing effect on innate immunity. Hypotheses: • BCG immunisation can prevent infection with M. tuberculosis • Prevention of infection occurs via stimulation of NOD-2 and toll like receptors (TLR)- 2. • The effect of BCG immunisation on prevention of infection with M. tuberculosis can be enhanced by giving stimulators of NOD-2 and TLR-2.

Objective:

Objective are to establish: 1. To confirm the validity of a mouse model of a partial infection with M. tuberculosis. 2. To investigate whether BCG immunisation can reduce infection with M. tuberculosis. 3. To investigate whether stimulators of NOD-2, TLR-2 or dectin-1 receptors can enhance an effect of BCG on reduction of infection with M. tuberculosis. 4. To investigate the influence of T-suppressor cells on a reduced reactivity in the gamma interferon release assay results in M. tuberculosis exposed BCG vaccinated mice. 5. To investigate whether a reduction of infection with M. tuberculosis in BCG immunised mice and in mice injected with enhancers of innate immunity is associated with a significant increase in markers of enhancement of innate immunity like H3K4 trimethylation of monocyte DNA and increase in Ly6Chigh and CD11b positive monocytes in uninfected mice. 6. To investigate whether a reduction of infection with M. tuberculosis is associated with an increased intracellular expression of TNF and Interferon gamma mRNA levels in monocytes in uninfected mice. 7. To establish whether inhibition of epigenetic programming can reduce the effect of BCG immunisation and stimulators of innate immunity on M. tuberculosis infection and markers of activation of innate immunity.

Methods:

Experimental protocols to confirm hypotheses To detect the influence of immunisation on infection rates the ultra-low dose (ULD) infection model is used: Groups of female adult C57BL/6 mice are exposed to ultra-low dose M. tuberculosis aerosol. Mice vaccinated with BCG and exposed after 6 weeks to ultra- low dose of M. tuberculosis and mice not vaccinated are compared in infection rate by cultures of lung homogenates and interferon gamma release assay. If a reduced infection rate by BCG immunisation is confirmed the experiment is repeated by giving BCG combined simultaneously or in time sequence with the enhancers of innate immunity murabutide or beta-glycan. In addition the influence of murabutide or beta-glycan alone on infection rates is investigated. To quantify the contribution of innate immunity levels of TNF and IFN gamma expression and histone H3 K4me3 trimethylation and concentrations of monocytes with features of activation of innate immunity as defined by the Ly6Chigh as well as CD11b positive phenotype in immunized versus unimmunized infected and uninfected mice in the various immunisation protocols is compared and the experiments repeated with prior application of the inhibitors of epigenetic programming of innate immunity histone methyltransferase inhibitor 5’-deoxy-5’-methylthio-adenosine and histone acetyl transferase inhibitor epigallocatechin-3-gallate. The influence of BCG on innate immunity is further corroborated by a prospective observational study in human infants.

Results:

Evidence supporting the hypothesis: • The first experiments using derivatives of MDP to enhance early immunity in the C57BL/6 mouse strain (7 weeks old mice) used 300 micrograms per mouse of oil-associated 6-0-mycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (mycol-MDP) 50/50 mixed with Freund’s incomplete adjuvant suspended with 0.9% sodium chloride solution with 0.2% Tween aiming at a final oil concentration of 3% and given intravenously. Comparison of colony forming unit (CFU) count in the lungs 3 weeks after aerosol challenge with Mycobacterium bovis of groups (n=5) between groups receiving mycol-MDP in oil emulsion (see above) versus controls (n=5) receiving only oil emulsion showed a significantly lower CFU count of 94.5 x106 (SD 22.0) in cases versus controls with 204.0 X 106 (SD 77.6) [20]. It is important to note that after elimination of T-cells in this model (by irradiation and thymectomy) a reduction of CFU in lungs of mice treated with mycol-MDP persisted albeit without statistical significance, which was possibly related to the small number of animals used. This result confirmed the findings of a previous study of the same group in the mouse strain C3H/He. • The BCG primed increased TNF release by monocytes has been shown to be related to effects of epigenetic programming in form of stimulation of trimethylation of histone H3 at lysine 4 (H3K4). Establishment of innate immunity in monocytes could hereby be inhibited by use of inhibitors of epigenetic programming. Evidence against the hypothesis: • The most important alternative hypothesis, which could be advanced to explain the apparently reduced infection rate on gamma interferon release assay testing in BCG immunised humans or mice is clonal imprinting (previously termed “original antigenic sin” phenomenon) where previous exposure to an antigen (in this case BCG) leads to reinforcement of the immune reaction to this antigen on exposure of the immune-system to a similar antigen (M. tuberculosis) containing this previous antigen (in this case BCG) rather than a reaction to the new antigen (M. tuberculosis specific epitopes) not contained in the antigen mixture of the previous exposure. This process has been found to be dependent on the action of the cytokine IL-10. IL-10 is hereby produced by non-antigen specific T-suppressor cells [23] thus postulated to reduce interferon gamma release in M. tuberculosis exposed individuals in the gamma interferon release assays if there was a previous exposure to BCG. This alternative hypothesis can be tested by elimination of T-suppressor cells. • The protective effect of BCG immunisation against infection with M. tuberculosis is reduced in low income countries but its effect against other infections, which is more consistent with enhancement of innate immunity, is considerable. This is more supportive of the hypothesis that the reduction of infection with M. tuberculosis is also due to T-effector cells, which can be influenced by malnutrition and helminth infection triggered regulatory T-cell activation and not due to effects of BCG on innate immunity. The conclusion would therefore be that innate immunity can actually not be enhanced by antigens similar to BCG derived substances, which can stimulate NOD-2 or TLR-2.

Conclusions:

Potential impact of confirmation of hypotheses Demonstration of a reduction of M. tuberculosis infection by enhancement of innate immunity could show a new approach to improving vaccine efficacy against this pathogen. Clinical Trial: Not applicable