New Strategy involving dendritic cell mediated immune response to improve TB vaccination

Tuberculosis (TB) represents one of the major causes of mortality from an infectious disease. The bacillus Calmette-Guérin (BCG), a live attenuated strain of Mycobacterium bovis, is the only available TB vaccine despite it doesn’t protect against some forms of this re-emerging disease. Therefore, the development of a more adequate vaccine is essential for improving tuberculosis control. It has been suggested that the limited protection conferred by BCG is partly due to his missing expression of critical immunogenic proteins. Hence, it is conceivable that Mtb antigens not expressed by BCG strains could increase the efficacy of BCG vaccination. A recently proposed strategy to improve the efficiency of a vaccine formulation is the encapsulation of antigens into nanoparticles (NPs) that can avoid their rapid clearance by host organisms and can promote their capture by specific antigen presenting cells (APCs). On the basis of these premises and considering the key role played by human dendritic cells (DCs) in the defences against TB and, more generally, in vaccination, this thesis has been aimed at the discovery of new strategies, involving human DC mediated immune response, to improve the BCG effectiveness. In particular, the study has been focused on two main goals: • investigation of the effects of Mtb antigens, HspX and ESAT-6, on human DC activity in order to verify their potential use as vaccine components (task A), • analysis of a vaccine nano-delivery system based on PLGA or pSi NPs to improve the capture of vaccine formulations by human DCs (task B). Task A. In this part of the study we found that the addition of HspX or ESAT-6 to BCG stimulated DCs does not affect DC maturation and pro inflammatory cytokine secretion. However DC stimulation with BCG and both HspX and ESAT-6 (BCG/HspX/ESAT-6) greatly enhances the expression of DC maturation markers CD83, CD86 and MHCII, and induces the release of IL-12, TNF α, IL-6, IL-23 and IL-1β from these cells. Interestingly, DC treatment with BCG/HspX/ESAT-6 improves the ability of these cells to elicit IFN-γ release and CD69 expression by CD4+ lymphocytes and NK cells as compared to DC treatment with BCG alone or with BCG plus a single antigen. Moreover, a TLR2-blocking antibody decreases IL-12 release by DCs stimulated with BCG/HspX/ESAT-6, as well as IFN-γ secretion by CD4+ lymphocytes co-cultured with these cells. Furthermore, HspX and ESAT-6 improve the capacity of BCG treated DCs to induce the expression of memory phenotype marker CD45RO in naïve CD4+ T cells. Our results indicate that BCG, HspX and ESAT-6 cooperate in enabling human DCs to induce a substantial T lymphocyte and NK cell mediated immune responses through TLR2-dependent IL-12 secretion. Therefore our findings suggest that HspX and ESAT-6 represent good candidates for improving the effectiveness of BCG vaccination. Task B. Considering their biocompatibility and their relevance in the scientific literature, two different types of NPs have been utilized: PLGA (poly (lactic-co-glycolic acid) and pSi (porous silicon). Our results show that at doses up to 0.2 mg, which are widely considered appropriate for in vitro DCs stimulation, both PLGA and pSi NPs do not affect human DC viability. However, at doses exceeding 0.4 mg, PLGA, but not pSi NPs induce DC apoptosis, indicating that pSi NPs are less toxic than PLGA NPs. Hence, the amounts of NPs used in our experiments have been chosen accordingly. In this part of the work we found that PLGA and pSi NPs are unable to modify per se the secretion of pro-inflammatory cytokines IL-12, TNF α, IL-6 and IL-23 by both resting and LPS-stimulated DCs. Interestingly, a confocal microscopy analysis reveals that both PLGA and pSi NPs are ingested by human DCs. However, PLGA NPs are more efficiently internalized by DCs than pSi NPs, indicating that PLGA NPs could be a suitable tool to be used for targeting molecules into human DCs. Take together our results suggest a new vaccination strategy against TB based on i) the combination of BCG/HspX/ESAT-6 in order to stimulate the immune system, and ii) the use of PLGA NPs to target antigens into human DCs.