(K) Fraction of H56+ cells in the lungs at 3, 24 and 72 h post-immunization

(K) Fraction of H56+ cells in the lungs at 3, 24 and 72 h post-immunization. prime combined with i.pulmon. pull immunization, and after parenteral or i.pulmon. prime immunization alone. We find that i.pulmon. pull immunization of mice with H56/CAF01, which are parenterally primed with H56/CAF01, substantially enhances vaccine uptake and presentation by pulmonary and splenic APCs, pulmonary endothelial cells and type I epithelial cells and induces stronger activation of dendritic cells in the lung-draining lymph nodes, compared with parenteral immunization alone, which suggests activation of both innate and memory responses. Using mass spectrometry imaging of lipid biomarkers, we further show that (i) airway mucosal N-desMethyl EnzalutaMide immunization with H56/CAF01 neither induces apparent local tissue damage nor inflammation in the lungs, and (ii) the presence of CAF01 is accompanied by evidence of an altered phagocytic activity in alveolar macrophages, evident from co-localization of CAF01 with the biomarker bis(monoacylglycero)phosphate, which is expressed in the late endosomes and lysosomes of phagocytosing macrophages. N-desMethyl EnzalutaMide Hence, our data demonstrate that innate myeloid responses differ after one and two immunizations, respectively, and the priming route and boosting route individually affect this outcome. These findings may have important implications for the design of mucosal vaccines intended for safe administration in the airways. (strains, a novel vaccine, which is more effective than the currently available Bacillus Calmette-Gurin (BCG) vaccine, is required to achieve the World Health Organizations important goal of ending the global TB epidemic by 2035 (2). In this respect, mucosal delivery via intrapulmonary (i.pulmon.) administration of subunit vaccines having excellent safety profiles (3, 4) is a promising strategy to induce protective lung-localized fate of inhaled vaccine antigens and Rabbit polyclonal to AKR7A2 adjuvants, and their safety. Innate myeloid cells include mononuclear phagocytes, monocytes, dendritic cells (DCs), and granulocytes. These cells play essential roles in pathogen clearance, initiation, regulation and resolution of inflammation, and antigen presentation (6, 7). Following repeated immunizations, i.e., prime C pull immunization strategies, there is a continuous cross-talk between innate and adaptive immune cells and vaccine components. Hence, knowledge about these events is crucial to improve the immunogenicity, protective N-desMethyl EnzalutaMide efficacy and safety of vaccines. Recent advances in the understanding of the diversity of myeloid and non-myeloid antigen-presenting cells (APCs) clearly suggest that for vaccines to induce specific immune profiles, they should be targeted to immune cell subsets capable of inducing that specific type of immune response (8, 9). For different subunit vaccines administered i.pulmon., inconsistencies exist in the immune responses they induce, and these differences may be due to factors like (i) the diversified localization of different APC subsets in the respiratory tract and the lung-draining lymph nodes (LNs), (ii) their functional differences, (iii) the size of the antigen, and (iv) the physicochemical properties of the adjuvant (10C13). Therefore, an understanding of the initial interactions taking place between the vaccine [antigen(s) and adjuvant] and the immune system is crucial for the rational design of safe vaccines, which have the capability to induce long-lasting protective immunity in the lungs (14). The subunit vaccine antigen H56 is a fusion protein composed of the antigens Ag85B, ESAT-6, and Rv2660c, and in combination with the cationic adjuvant formulation 01 (CAF01) administered parenterally, this antigen elicits a polyfunctional Th1/Th17 CD4+ T cell response and causes a significant reduction in burden N-desMethyl EnzalutaMide (15C17). CAF01 is composed of cationic liposomes based on the surfactant dimethyldioctadecylammonium (DDA) bromide and the glycolipid trehalose-6,6-dibehenate (TDB) (18). CAF01 delivers antigen and activates DCs (19), induces both humoral and cell-mediated N-desMethyl EnzalutaMide memory immune responses, and it has been tested in phase I clinical trials, demonstrating an excellent safety and immunogenicity profile (20C22). Our recent data suggests that a parenteral prime C mucosal pull immunization strategy using CAF01 can be applied to redirect immunity to mucosal tissues (23). Recently, we reported an immunization strategy comprising intramuscular (i.m.) priming followed by i.pulmon. mucosal pull immunization with the H56/CAF01 vaccine, which resulted in the induction of lung-localized, H56-specific T cells and systemic as well as lung mucosal IgA responses (24). However, the role of (i) H56/CAF01 deposition within lung tissue, (ii) CAF01 internalization by phagocytic cells, and (iii) antigen presentation in the lungs and the lung-draining LNs on the induction of immune responses after pulmonary administration are unknown. In addition, an outstanding research question is the safety of CAF01 upon pulmonary administration. Here, we applied multicolor flow cytometry to investigate H56/CAF01 uptake by innate myeloid cells and APCs in the lungs, the spleen, and the lung-draining LNs in mice after i.m. or i.pulmon. prime or i.m. prime C.