S1C). primary immunodeficiencies (1), IL-17 producing cells are also involved in host defense as our previous observations suggested that relative IL-17 deficiency might partially explain the pulmonary host defense DMAPT defect associated with either HIV infection or congenital immunodeficiency of CD4+ lymphocytes (2). Analysis of human lungs has shown that lung tissue resident memory (TRM) cells accumulate with age (3), and their protective functions have been postulated to be based on their memory to prior antigen exposures (4). Several papers have shown that lung TRM cells generated from type IL-17 expressing helper CD4+ T cells (Th17 cells) can provide prompt protection to extracellular pathogens (5, 6). Thus, elicitation of lung TRMs may be instrumental in protective immunity against bacterial pneumonia and may inform strategies towards the development of vaccines or therapeutics. Current pneumonia vaccines target polysaccharide antigens that elicit antibody responses that can be CD4+ T cell independent or dependent (7, 8). These vaccines are effective against specific vaccine serotypes, but with continued use, epidemiologic studies suggest the emergence of non-vaccine serotypes (9). Lung CD4+ T cells can recognize conserved protein antigens and thus provide broad immunity that is independent of bacterial serotype (5, 10, 11). However, much of this work has been performed with whole pathogens, heat-killed organisms, or adjuvants such as cholera toxin (CT) that have limited prospects for vaccine development due to safety concerns (12). We previously observed that intrapulmonary immunization with heat-killed induced a subset of lung CD4+ T cells that could recognize outer membrane proteins (Omps) (11). We hypothesized that recombinant bacterial Omps formulated with a mucosal Th17 adjuvant may elicit antigen-specific lung TRM cells, which could provide serotype-independent immunity in the lung. Based on prior work with influenza (13), we focused on the LTA1 adjuvant, which is derived from the A1 domain of the heat-labile toxin from elicited a robust population of antigen-specific CD4+ TRM cells that provided serotype-independent immunity to hypervirulent elicits lung TRM cells (5, 11), and a portion of these cells recognize bacterial-encoded outer membrane proteins (11), so we sought to determine if this approach could be used to develop a subunit vaccine targeting DMAPT infection. We cloned OmpX from the KP-43816 (K2) strain, due to homology with other and efficient production in to test for serotype independent immunity (11). We initially tested the mucosal adjuvant LTA1 because of its ability to elicit lung immunity and Th17 responses (13, 14). Mice were immunized twice by intratracheal oropharyngeal aspiration at three week intervals to model an inhaled vaccine schedule. One week following second immunization, mice were challenged with 104 colony forming unit (CFU) of KP-396 (Fig. 1A). Immunized mice showed a drastic reduction in lung CFU after intratracheal challenge with KP-396 that was accompanied by substantially reduced bacterial dissemination to the spleen (Fig. 1B) as well as improved survival DMAPT (Fig. 1C) and oxygen saturations (Fig. S1A). Mucosal immunization with unadjuvanted OmpX or LTA1 alone limited vaccine efficacy, (Fig. S1B), and mucosal immunization was more effective than subcutaneous immunization (Fig. S1C). H&E histology analysis of immunized mice showed perivascular and peribronchial lymphocyte accumulation consistent with the architectural features of inducible bronchus-associated lymphoid tissue (iBALT) (Fig. 1D) (15). Further evaluation showed iBALT contained B cell follicles, CD3+ T Rabbit Polyclonal to CDK10 cells, IgG producing plasma cells, follicular dendritic cells, CXCL13+ stromal cells, peripheral node addressin (PNAd)+ high endothelial venules, and lymphatic vessel endothelial receptor 1 (Lyve-1)+ lymphatic vessels (Fig. 1D). Open in a separate window Fig. 1. Intratracheal delivery of OmpX adjuvanted with LTA1 DMAPT elicits tissue resident lung CD4+ Th1 and Th17 cells.(A) Cartoon DMAPT depicting immunization schedule with OmpX+LTA1 or heat killed (ATCC 43816) followed by challenge with a KP-396 (K1 strain) of = 7C10, **** denotes P 0.0001 by Mann-Whitney U test). (C) Survival post KP-396 challenge (data were pooled from 2 independent experiment (= 6 per group). *** denotes P 0.01 by Log-rank test. (D) Cellular and structural features of iBALT in the lungs of immunized C57BL/6 mice, compared to na?ve mice, were visualized with H&E stain (left).