Pulmonary mucosal immunity mediated through CpG provides adequate protection against pulmonary Mycobacterium tuberculosis infection in the mouse model. A role for type I interferon.

Toll-Like Receptor (TLR) 9 stimulation is required for induction of potent immune responses against pathogen invasion. The use of unmethylated CpG as adjuvants in vaccines provides an excellent means of stimulating adaptive immunity. Our data demonstrate that CpG-C provided prolonged immune responses in the mouse model of tuberculosis when formulated with liposomes and the Mycobacterium tuberculosis antigen ESAT-6. A reduction in the mycobacterial burden was best achieved when administered as an intranasal vaccine and was dependent on type I interferon (IFN). There was a significant difference between CpG-C inoculated wild type and IFN-αR1-/- mice, indicating that type I IFN plays a role in the immune response following CpG-C inoculation. Further analysis showed that early NK cell presence was not an absolute requirement, although elevated IFN-γ levels were detected in the lungs of mice within 48 h. The reduction in mycobacterial burden was MyD88-independent as CpG-C inoculated MyD88-/- mice showed comparable mycobacterial burdens to wild type mice with no detriment due to the lack of MyD88. Together our data show that pulmonary stimulation of TLR9 bearing antigen presenting cells resulted in the induction of protective immunity against M. tuberculosis infection that was dependent on type I IFN signaling.

Humanized NOG mice as a model for tuberculosis vaccine-induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis.

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4+ and CD8+ T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG-C, a liposome-based formulation containing the M. tuberculosis antigen ESAT-6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T-cell response. Humanized mice provide a crucial pre-clinical platform for evaluating human T-cell immune responses in vaccine development against M. tuberculosis.

Virulence of Mycobacterium tuberculosis after Acquisition of Isoniazid Resistance: Individual Nature of katG Mutants and the Possible Role of AhpC.

In the last decade, there were 10 million new tuberculosis cases per year globally. Around 9.5% of these cases were caused by isoniazid resistant (INHr) Mycobacterium tuberculosis (Mtb) strains. Although isoniazid resistance in Mtb is multigenic, mutations in the catalase-peroxidase (katG) gene predominate among the INHr strains. The effect of these drug-resistance-conferring mutations on Mtb fitness and virulence is variable. Here, we assessed differences in bacterial growth, immune response and pathology induced by Mtb strains harboring mutations at the N-terminus of the katG gene. We studied one laboratory and one clinically isolated Mtb clonal pair from different genetic lineages. The INHr strain in each pair had one and two katG mutations with significantly reduced levels of the enzyme and peroxidase activity. Both strains share the V1A mutation, while the double mutant clinical INHr had also the novel E3V katG mutation. Four groups of C57BL/6 mice were infected with one of the Mtb strains previously described. We observed a strong reduction in virulence (reduced bacterial growth), lower induction of proinflammatory cytokines and significantly reduced pathology scores in mice infected with the clinical INHr strain compared to the infection caused by its INHs progenitor strain. On the other hand, there was a subtle reduction of bacteria growth without differences in the pathology scores in mice infected with the laboratory INHr strain. Our results also showed distinct alkyl-hydroperoxidase C (AhpC) levels in the katG mutant strains, which could explain the difference in the virulence profile observed. The difference in the AhpC levels between clonal strains was not related to a genetic defect in the gene or its promoter. Cumulatively, our results indicate that the virulence, pathology and fitness of INHr strains could be negatively affected by multiple mutations in katG, lack of the peroxidase activity and reduced AhpC levels.

Heat killed Saccharomyces cerevisiae as an adjuvant for the induction of vaccine-mediated immunity against infection with Mycobacterium tuberculosis.

The use of novel vaccine delivery systems allows for the manipulation of the adaptive immune systems through the use of molecular adjuvants that target specific innate pathways. Such strategies have been used extensively for vaccines against cancer and multiple pathogens such as Mycobacterium tuberculosis. In the current study we used heat killed non-pathogenic recombinant Saccharomyces cerevisiae expressing M. tuberculosis antigen Rv1886c (fbpB, mpt59, Ag85B) as a delivery system in conjunction with its ability to stimulate innate immunity to determine its ability to induce immunity. We established that the recombinant yeast induced activated antigen specific T cells are capable of reducing the mycobacterial burden. Inoculation of the recombinant yeast after vaccination with BCG resulted in a systemic alteration of the phenotype of the immune response although this was not reflected in an increase in the reduction of the mycobacterial burden. Taken together the data suggest that heat killed yeast can induce multiple cytokines required for induction of protective immunity and can function as a vehicle for delivery of M. tuberculosis antigens in a vaccine formulation. In addition, while it can enhance the effector memory response induced by BCG, it had little effect on central memory responses.

Comparative analysis of Bacillus subtilis spores and monophosphoryl lipid A as adjuvants of protein-based mycobacterium tuberculosis-based vaccines: partial requirement for interleukin-17a for induction of protective immunity.

The development of adjuvants for vaccines has become an important area of research as the number of protein-based vaccines against infectious pathogens increases. Currently, there are a number of adjuvant-based Mycobacterium tuberculosis vaccines in clinical trials that have shown efficacy in animal models. Despite these novel adjuvants, there is still a need to design new and more versatile adjuvants that have minimal adverse side effects but produce robust long-lasting adaptive immune responses. To this end, we hypothesized that Bacillus subtilis spores may provide the appropriate innate signals that are required to generate such vaccine-mediated responses, which would be sufficient to reduce the mycobacterial burden after infection with M. tuberculosis. In addition, we compared the response generated by B. subtilis spores to that generated by monophosphoryl lipid A (MPL), which has been used extensively to test tuberculosis vaccines. The well-characterized, 6-kDa early secretory antigenic target of M. tuberculosis (ESAT-6; Rv3875) was used as a test antigen to determine the T cell activation potential of each adjuvant. Inoculated into mice, B. subtilis spores induced a strong proinflammatory response and Th1 immunity, similar to MPL; however, unlike MPL formulated with dimethyldioctadecylammonium (DDA) bromide, it failed to induce significant levels of interleukin-17A (IL-17A) and was unable to significantly reduce the mycobacterial burden after pulmonary infection with M. tuberculosis. Further analysis of the activity of MPL-DDA suggested that IL-17A was required for protective immunity. Taken together, the data emphasize the requirement for a network of cytokines that are essential for protective immunity.