Rationale: New vaccine approaches are needed for Pseudomonas aeruginosa, which continues to be a major cause of serious pulmonary infections. Although Th17 cells can protect against gram-negative pathogens at mucosal surfaces, including the lung, the bacterial proteins recognized by Th17 cells are largely unknown and could be potential new vaccine candidates. Objectives: We describe a strategy to identify Th17-stimulating protein antigens of Pseudomonas aeruginosa to assess their efficacy as vaccines against pneumonia.Methods: Using a library of in vitro transcribed and translated P. aeruginosa proteins, we screened for Th17-stimulating antigens by coculturing the library proteins with splenocytes from mice immunized with a live-attenuated P. aeruginosa vaccine that is protective via Th17-based immunity. We measured antibody and Th17 responses after intranasal immunization of mice with the purified proteins mixed with the Th17 adjuvant curdlan, and we tested the protective efficacy of vaccination in a murine model of acute pneumonia. Measurements and Main Results: The proteins PopB, FpvA, FptA, OprL, and PilQ elicited strong IL-17 secretion in the screen, and purified versions of PopB, FpvA, and OprL stimulated high IL-17 production from immune splenocytes. Immunization with PopB, which is a highly conserved component of the type III secretion system and a known virulence factor, elicited Th17 responses and also enhanced clearance of P. aeruginosa from the lung and spleen after challenge. PopB-immunized mice were protected from lethal pneumonia in an antibody-independent, IL-17-dependent manner. Conclusions: Screening for Th17-stimulating protein antigens identified PopB as a novel and promising vaccine candidate for P. aeruginosa.Keywords: vaccine; pneumonia; Th17; IL-17; Pseudomonas aeruginosa Pseudomonas aeruginosa is a major cause of serious, often antibiotic-resistant, lung infections in humans, particularly in patients receiving mechanical ventilation and those with cystic fibrosis (1, 2). Most P. aeruginosa vaccines developed to date, including those based on the LPS O antigen (3), the outer membrane proteins F and I (4, 5), or the type III secretion system component PcrV (6), have relied on conventional protective mechanisms-namely, antibody-mediated opsonophagocytic killing and/or antibody-mediated toxin inhibition. Although LPS O antigen-based vaccines can mediate high levels of immunity to P. aeruginosa, the protection is limited to strains having the same LPS serogroup (3). The failure of the Federal Hyperimmune Immunoglobulin Trial (7), which found no benefit in critically ill adults passively immunized with P. aeruginosa LPS O antigenspecific IgG, perhaps best illustrates that antibody-mediated protective mechanisms are not sufficient.Th17 cells have recently been shown to mediate antibodyindependent host defense against Klebsiella pneumoniae (8), although the bacterial proteins recognized by the Th17 cells in those studies were not fully characterized. In our own evaluations of live-attenuated P. ...

The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates up to four protein toxin effectors into human cells, facilitating the establishment and dissemination of infections. To discover inhibitors of this important virulence mechanism, we developed two cellular reporter assays and applied them to a library of 80,000 compounds. The primary screen was based on the dependence of the transcription of T3SS operons on the T3SS-mediated secretion of a negative regulator and consisted of a transcriptional fusion of the Photorhabdus luminescens luxCDABE operon to the P. aeruginosa exoT effector gene. Secondary assays included direct measurements of the T3SS-mediated secretion of a P. aeruginosa ExoS effector-␤-lactamase fusion protein as well as the detection of the secretion of native ExoS by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of culture supernatants. Five inhibitors in three chemical classes were demonstrated to inhibit type III secretion selectively with minimal cytotoxicity and with no effects on bacterial growth or on the type II-mediated secretion of elastase. These inhibitors also block the T3SS-mediated secretion of a YopE effector-␤-lactamase fusion protein from an attenuated Yersinia pestis strain. The most promising of the inhibitors is a phenoxyacetamide that also blocks the T3SS-mediated translocation of effectors into mammalian cells in culture. Preliminary studies of structure-activity relationships in this phenoxyacetamide series demonstrated a strict requirement for the R-enantiomer at its stereocenter and indicated tolerance for a variety of substituents on one of its two aromatic rings.The type-three secretion system (T3SS) is a complex multiprotein apparatus that facilitates the secretion and translocation of effector proteins from the bacterial cytoplasm directly into the mammalian cytosol. This complex protein delivery device is shared by more than 15 species of gram-negative human pathogens, including Salmonella spp., Shigella flexneri, Pseudomonas aeruginosa, Yersinia spp., enteropathogenic and enteroinvasive Escherichia coli, and Chlamydia spp. (23,25,43). In the opportunistic pathogen P. aeruginosa, the T3SS is the major virulence factor contributing to the establishment and dissemination of acute infections (19). Four T3SS effectors have been identified in P. aeruginosa strains: ExoS, ExoT, ExoY, and ExoU. ExoS and ExoT are bifunctional proteins consisting of an N-terminal small G-protein-activating protein (GAP) domain and a C-terminal ADP ribosylation domain, ExoY is an adenylate cyclase, and ExoU is a phospholipase (reviewed in reference 11). In studies with strains producing each effector separately, ExoU and ExoS contributed significantly to persistence, dissemination, and mortality, while ExoT produced minor effects on virulence in a mouse lung infection model, and ExoY did not appear to play a major role in the pathogenesis of P. aeruginosa (51). While not a p...

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TNF antagonists may offer therapeutic potential in solid tumors, but patients who have high serum levels of TNF-a fail to respond to infliximab, suggesting consumption of the circulating antibody and loss of transmembrane TNF-a (tmTNF-a) on tumors by ectodomain shedding. Addressing this possibility, we developed a monoclonal antibody (mAb) that binds both full-length tmTNF-a and its N-terminal truncated fragment on the membrane after tmTNF-a processing but does not cross-react with soluble TNF-a. We documented high levels of tmTNF-a expression in primary breast cancers, lower levels in atypical hyperplasia or hyperplasia, but undetectable levels in normal breast tissue, consistent with the notion that tmTNF-a is a potential therapeutic target. Evaluations in vitro and in vivo further supported this assertion. tmTNF-a mAb triggered antibodydependent cell-mediated cytotoxicity against tmTNF-a-expressing cells but not to tmTNF-a-negative cells. In tumor-bearing mice, tmTNF-a mAb delayed tumor growth, eliciting complete tumor regressions in some mice. Moreover, tmTNF-a mAb inhibited metastasis and expression of CD44v6, a prometastatic molecule. However, the antibody did not activate tmTNF-a-mediated reverse signaling, which facilitates tumor survival and resistance to apoptosis, but instead inhibited NF-kB activation and Bcl-2 expression by decreasing tmTNF-a-positive cells. Overall, our results established that tmTNF-a mAb exerts effective antitumor activities and offers a promising candidate to treat tmTNF-a-positive tumors, particularly in patients that are nonresponders to TNF antagonists. Cancer Res; 73(13); 4061-74. Ó2013 AACR.