Mucosal Delivery of Fusion Proteins with Bacillus subtilis Spores Enhances Protection against Tuberculosis by Bacillus Calmette-Guerin

Tuberculosis (TB) is the most deadly infectious disease in existence, and the only available vaccine, Bacillus Calmette-Guérin (BCG), is almost a century old and poorly protective. The immunological complexity of TB, coupled with rising resistance to antimicrobial therapies, necessitates a pipeline of diverse novel vaccines. Here, we show that Bacillus subtilis spores can be coated with a fusion protein 1 (“FP1”) consisting of Mycobacterium tuberculosis (Mtb) antigens Ag85B, ACR, and HBHA. The resultant vaccine, Spore-FP1, was tested in a murine low-dose Mtb aerosol challenge model. Mice were primed with subcutaneous BCG, followed by mucosal booster immunizations with Spore-FP1. We show that Spore-FP1 enhanced pulmonary control of Mtb, as evidenced by reduced bacterial burdens in the lungs. This was associated with elevated antigen-specific IgG and IgA titers in the serum and lung mucosal surface, respectively. Spore-FP1 immunization generated superior antigen-specific memory T-cell proliferation in both CD4+ and CD8+ compartments, alongside bolstered Th1-, Th17-, and Treg-type cytokine production, compared to BCG immunization alone. CD69+CD103+ tissue resident memory T-cells (Trm) were found within the lung parenchyma after mucosal immunization with Spore-FP1, confirming the advantages of mucosal delivery. Our data show that Spore-FP1 is a promising new TB vaccine that can successfully augment protection and immunogenicity in BCG-primed animals

The role of dendritic cells in adjuvant-induced immune responses

Dendritic cells (DCs) are sentinels of mucosal surfaces, residing directly under the epithelial layer. DCs are among the first cells that come in contact with pathogens and have the unique ability to activate T cells that subsequently can aid B cells to produce antibodies with high affinity. T and B cells constitute our immunological memory that protects us from reinfections – the basis for vaccination. Vaccines composed of purified antigens, confer high specificity but have low intrinsic immunogenicity, and require therefore an adjuvant that enhances the response. The most potent adjuvants are often toxic, and consequently a limited number of adjuvants are available for clinical use, mucosal adjuvants in particular. Therefore, a better understanding is needed concerning the interactions between adjuvants and DCs in order to unveil the mechanisms of adjuvanticity. Here we have in vivo studied the role of DCs and the characteristics of the immune response after immunization with different adjuvants. Adenoviral (Ad) vaccine vectors inducing expression of ovalbumin (OVA) at different subcellular locations were used in a mouse model in which conventional DCs (cDCs) could be depleted. We show that cDCs are required for activation of T cells although a direct transduction of cDCs by Ad-vectors is not essential. Further we determine that secreted and membrane-anchored antigens are superior at activating antigen-specific CD4+, cytotoxic CD8+ T lymphocytes as well as generating a serum IgG response compared to intracellulary expressed OVA. Cholera toxin (CT) is one of the most potent mucosal adjuvants. CT binds the ubiquitously expressed ganglioside GM1 leading to efficient uptake that in epithelial cells results in secretion of fluid into the lumen. After oral immunization with OVA and CT we find that chimeric mice lacking GM1 on hematopoietic cells, and specifically GM1-expressing DCs, fail to induce adaptive immune responses to OVA. We conclude that CT does not require the toxic epithelial cell interaction for its adjuvant activity but is dependent on direct binding of GM1 on intestinal DCs. To become plasma cells producing high affinity antibodies, B cells must undergo affinity maturation in the germinal center where they are dependent on the help of follicular helper T cells. In DC-depleted mice, we show that immunization with the adjuvant poly(I:C) and non-limiting doses of OVA generates follicular T helper cells (Tfh) and germinal centers in absence of DCs. In contrast, B cell interactions are required for a fully differentiated Tfh phenotype and the activation of a Th1 mediated T cell response is totally dependent on DCs. Strategies targeting vaccine antigens to DCs are becoming more promising as novel DC-specific receptors are being discovered. Taken together our results show great heterogeneity concerning the role of DCs in adjuvant-induced immune responses. How to modulate and take advantage of the interactions between adjuvants and DCs will be crucial knowledge in the construction of future more effective and safe vaccines

Expression of HLA-B27 causes loss of migratory dendritic cells in a rat model of spondylarthritis

<b>Objective</b><p></p> In rats transgenic for human HLA–B27 and β2-microglobulin (B27-transgenic rats), colitis and peripheral inflammation develop spontaneously. Therefore, B27-transgenic rats provide a model of spondylarthritis. Because inflammation in these rats requires CD4+ T lymphocytes and involves intestinal pathology, we hypothesized that dendritic cells (DCs) that migrate from the intestine and control CD4+ T cell differentiation would be aberrant in B27-transgenic rats. <p></p> <b>Methods</b><p></p> Migrating intestinal lymph DCs were collected via thoracic duct cannulation from B27-transgenic and control (HLA–B7–transgenic or nontransgenic) rats. The phenotypes of these DCs and of mesenteric lymph node DCs were assessed by flow cytometry. The ability of DCs to differentiate from bone marrow precursors in vitro was also assessed. <p></p> <b>Results</b><p></p> Lymph DCs showed increased activation and, strikingly, lacked the specific DC population that is important for maintaining tolerance to self-antigens. This population of DCs was also depleted from the mesenteric lymph nodes of B27-transgenic rats. Furthermore, in vitro culture of DCs from bone marrow precursors revealed a defect in the ability of B27-transgenic rats to produce DCs of the migratory phenotype, although the DCs that were generated induced enhanced interleukin-17 (IL-17) production from naive CD4+ T cells. <p></p> <b>Conclusion</b><p></p> We describe 2 different mechanisms by which HLA–B27 may contribute to inflammatory disease: increased apoptotic death of B27-transgenic DCs that normally function to maintain immunologic tolerance and enhanced IL-17 production from CD4+ T cells stimulated by the surviving B27-transgenic DCs. <p></p&gt