Else Marie Agger

The mycobacterial cord factor trehalose-6,6-dimycolate (TDM) and its synthetic analog trehalose-6,6-dibehenate (TDB) are potent adjuvants for Th1/Th17 vaccination that activate Syk-Card9 signaling in APCs. In this study, we have further investigated the molecular mechanism of innate immune activation by TDM and TDB. The Syk-coupling adapter protein FcRgamma was essential for macrophage activation and Th17 adjuvanticity. The FcRgamma-associated C-type lectin receptor Mincle was expressed in macrophages and upregulated by TDM and TDB. Recombinant Mincle-Fc fusion protein specifically bound to the glycolipids. Genetic ablation of Mincle abolished TDM/TDB-induced macrophage activation and induction of T cell immune responses to a tuberculosis subunit vaccine. Macrophages lacking Mincle or FcRgamma were impaired in the inflammatory response to Mycobacterium bovis bacillus Calmette-Guérin. These results establish that Mincle is a key receptor for the mycobacterial cord factor and controls the Th1/Th17 adjuvanticity of TDM and TDB.

Improved vaccines capable of promoting long-term cellular immunity are urgently required for a number of diseases that remain global health problems. In the present study, we demonstrate that a tuberculosis subunit vaccine, Ag85B-ESAT-6/CAF01 (where ESAT-6 is early secreted antigenic target of 6 kDa and CAF01 is cationic adjuvant formulation 01), induces very robust memory CD4 T cell responses that are maintained at high levels for >1 year postvaccination. This long-term, vaccine-induced memory response protects against a challenge with Mycobacterium tuberculosis at levels that are comparable to or better than those of bacillus Calmette-Guérin. Characterization of the CD4 memory T cells by multicolor flow cytometry demonstrated that the long-lived memory population consisted almost exclusively of TNF-alpha(+)IL-2(+) and IFN-gamma(+)TNF-alpha(+)IL-2(+) multifunctional T cells. In addition, memory cells isolated >1 year postvaccination maintained a strong, vaccine-specific proliferative potential. Long-term memory induced by the BCG vaccine contained fewer multifunctional T cells and was biased toward effector cells mainly of the TNF-alpha(+)IFN-gamma(+)-coexpressing subset. Ag85B-ESAT-6/CAF01 vaccination very efficiently sustained multifunctional CD4 T cells that accumulated at the site of infection after M. tuberculosis challenge, whereas the response in unvaccinated animals was characterized by CD4 effector T cells. Our data demonstrate that adjuvanted subunit vaccines can promote long-term protective immune responses characterized by high levels of persisting multifunctional T cells and that the quality and profile of this response is sustained postinfection.

It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis, but that only 10% of infected people break down with the disease. In the remaining 90% the infection remains clinically latent. In the present study, the immune mechanisms controlling the latent phase of tuberculosis infection were evaluated in a mouse model of latency and reactivation. Mice aerosol-infected with M. tuberculosis were treated with anti-mycobacterial drugs resulting in very low, stable bacterial numbers (<500 CFU in the spleen and lung) for 10-12 weeks followed by reactivation of the disease with increasing bacterial numbers. During latency, pathological changes in the lung had almost completely resolved and lymphocyte number and turnover were at the pre-infection level. The CD4 subset was highly active during the acute phase of infection and could be detected by intracellular staining for IFN-gamma as well as after antigen-specific stimulation with mycobacterial antigens. The CD8 subset was not involved in the acute stage of infection, but this subset was active and produced IFN-gamma during the latent phase of infection. In vivo depletion of T cell subsets supported these findings with a 6-7-fold increase in bacterial numbers in the lung following anti-CD4 treatment during the acute phase, while anti-CD8 treatment did not have an effect. The opposite was found during the latent phase where anti-CD8 treatment as well as anti-IFN-gamma treatment both resulted in a 10-fold increase in bacterial numbers in the lung, while anti-CD4 treatment induced only a modest change.

Tests based on tuberculin purified protein derivative (PPD) cannot distinguish between tuberculosis infection, Mycobacterium bovis BCG vaccination, or exposure to environmental mycobacteria. The present study investigated the diagnostic potential of two Mycobacterium tuberculosis-specific antigens (ESAT-6 and CFP10) in experimental animals as well as during natural infection in humans and cattle. Both antigens were frequently recognized in vivo and in vitro based on the induction of delayed-type hypersensitivity responses and the ability to induce gamma interferon production by lymphocytes, respectively. The combination of ESAT-6 and CFP10 was found to be highly sensitive and specific for both in vivo and in vitro diagnosis. In humans, the combination had a high sensitivity (73%) and a much higher specificity (93%) than PPD (7%).