Toll like-receptor agonist Pam3Cys modulates the immunogenicity of liposomes containing the tuberculosis vaccine candidate H56.

A major roadblock in the development of novel vaccines is the formulation and delivery of the antigen. Liposomes composed of a dimethyldioctadecylammonium (DDA) backbone and the adjuvant trehalose-6-6-dibehenate (TDB, termed "cationic adjuvant formulation (CAF01)", promote immunogenicity and protective efficacy of vaccines, most notably against infection with Mycobacterium tuberculosis. Specifically, the multicomponent antigen H56 delivered by CAF01 protects against tuberculosis in mice. Here we investigated whether the inclusion of immune-modulatory adjuvants into CAF01 modulates the immunogenicity of H56/CAF01 in vitro and in vivo. Based on our recent findings we selected the active sequence of the mycobacterial 19 kDa lipoprotein, Pam3Cys, which interacts with Toll like receptor 2 to induce an antimicrobial pathway. H56/CAF01-Pam3Cys liposomes were characterized for Pam3Cys incorporation, size, toxicity and activation of primary human macrophages. Macrophages efficiently take up H56/CAF01-Pam3Cys and trigger the release of significantly higher levels of TNF, IL-12 and IL-10 than H56/CAF01 alone. To evaluate the immunogenicity in vivo, we immunized mice with H56/CAF01-Pam3Cys and measured the release of IFN-γ and IL-17A by lymph node cells and spleen cells. While the antigen-specific production of IFN-γ was reduced by inclusion of Pam3Cys into H56/CAF01, the levels of IL-17A remained unchanged. In agreement with this finding, the concentration of the IFN-γ-associated IgG2a antibodies in the serum was lower than in H56/CAF01 immunized animals. These results provide proof of concept that Toll like-receptor agonist can be included into liposomes to modulate immune responses. The discordant results between the in vitro studies with human macrophages and in vivo studies in mice highlight the relevance and complexity of comparing immune responses in different species.

Identification of a novel TIF-IA-NF-κB nucleolar stress response pathway.

p53 as an effector of nucleolar stress is well defined, but p53 independent mechanisms are largely unknown. Like p53, the NF-κB transcription factor plays a critical role in maintaining cellular homeostasis under stress. Many stresses that stimulate NF-κB also disrupt nucleoli. However, the link between nucleolar function and activation of the NF-κB pathway is as yet unknown. Here we demonstrate that artificial disruption of the PolI complex stimulates NF-κB signalling. Unlike p53 nucleolar stress response, this effect does not appear to be linked to inhibition of rDNA transcription. We show that specific stress stimuli of NF-κB induce degradation of a critical component of the PolI complex, TIF-IA. This degradation precedes activation of NF-κB and is associated with increased nucleolar size. It is mimicked by CDK4 inhibition and is dependent upon a novel pathway involving UBF/p14ARF and S44 of the protein. We show that blocking TIF-IA degradation blocks stress effects on nucleolar size and NF-κB signalling. Finally, using ex vivo culture, we show a strong correlation between degradation of TIF-IA and activation of NF-κB in freshly resected, human colorectal tumours exposed to the chemopreventative agent, aspirin. Together, our study provides compelling evidence for a new, TIF-IA-NF-κB nucleolar stress response pathway that has in vivo relevance and therapeutic implications.