tularensis LPS via several systemic routes were marginally protected against intraperitoneal and intradermal challenge with type B strains [13–17]. Several proteins, including a 17-kDa protein (Tul4), a 43-kDa outer membrane protein and heat shock protein 60 have been tested for their efficacy in animal models, but they conferred minimal protection after challenge with virulent strains [18–20]. One group reported that immunization with native outer membrane proteins (OMPs) induced 50% protection against intranasal challenge with type A F. tularensis [21]. More recently, a detoxified endotoxin (from Escherichia coli) vaccine non covalently complexed with the outer membrane protein of N. meningitidis group B was also found to be partially protective against LVS and type A virulent strains [22]. Overall, research efforts to investigate novel mucosal adjuvants that potentiate the response to F. tularensis antigens have been minimal. One study reported the use of cholera toxin subunit B (CTB) as a nasal adjuvant with inactivated LVS against both LVS and virulent F. tularensis [23]. Bronchus-Associated Lymphoid Tissue (BALT) is a lymphoid structure that can be found in peribronchial, perivascular and interstitial areas of the lung. Its formation can be triggered in the lungs of mice and humans by encounter with antigen, infection or inflammation, but it is not normally present in healthy lungs of these species [24]. BALT is composed of prominent lymphocyte aggregates, often characterized by proliferating and germinal center B cells, supported by a central follicular dendritic cell network. Interfollicular T cells and dendritic cells lie underneath the follicle associated epithelium (FAE) and are located around B cell areas [25,26]. Other important constituents of this specialized lymphoid tissue are lymphatics and high endothelial venules (HEVs) expressing vascular cellular-adhesion molecule-1 (VCAM-1) [27,28]. It has been reported that similar structures were formed as a direct consequence of certain respiratory infectious diseases in experimental animal models. The influenza virus triggered formation of what is known as inducible BALT (iBALT) in mice lacking conventional lymphoid organs. It was suggested that iBALT may play an important role in protection [27,29]. Also, lungs of several other animal species infected either naturally or experimentally with a number of bacterial and viral pathogens also developed areas of organized lymphoid follicles [30–33]. Lungs of patients with pulmonary complications of Sjogren’s syndrome (SS) and rheumatoid arthritis (RA) showed areas of organized lymphoid areas, also referred to as iBALT [34]. Despite having a critical role in the modulation of local inflammatory response in mice inoculated with Influenza (JRM personal communication), the specific function of iBALT in infection and immunity still remains controversial, considering that this tissue only develops as a consequence of certain infectious diseases but not others. The present study reports the potential use meningococcal recombinant porin B (rPorB) as a putative mucosal adjuvant candidate, when delivered with F. tularensis LPS. The formation of highly organized BALT, following intranasal vaccination and subsequent bacterial challenge is also described. Results Induction of systemic antigen specific antibodies after vaccination SDS-PAGE was used to detect purified rPorB, as shown by the single band in the Coomassie gel (Figure 1A). Minimal endotoxin levels, approximately 0.036 EU per microgram, were detected in the protein preparation. After confirming the purity of rPorB, the humoral response to intranasal vaccination with the LPS+rPorB Tularemia Vaccination and BALT candidate was assessed. Blood was collected from all groups four weeks after the third immunization dose and just before intranasal challenge. Serum levels of antigen specific antibodies were measured by ELISA. Overall, mice that had received LPS+rPorB via the nasal route developed higher levels of LPS-specific immunoglobulins than control groups (Figure 1B). Sera from mice vaccinated with LPS+rPorB contained antigen specific IgG, though levels of this immunoglobulin were shown to be variable, with four mice responding more strongly than the other five (Figure 1B). The variability in IgG response did not necessarily reflect the outcome of survival following intranasal challenge. No detectable levels of IgG were present in serum from mice in the PBS, rPorB and LPS alone control groups. Levels of LPS-specific IgM detected in the serum of mice vaccinated with LPS/rPorB were overall higher than those observed in mice immunized with control substances, with a significant difference when compared with the PBS group (Figure 1B). As for IgG, mouse to mouse variability in the IgM response was also observed. Analysis of serum antigen-specific IgA, revealed that only two out of 10 mice vaccinated with LPS+rPorB had increased antibody levels when compared to the three control groups (data not shown). Nasal delivery of rPorB improved the protective capacity of LPS against F. tularensis challenge To determine the protective capacity of F. tularensis LPS with rPorB administered intranasally, we challenged groups of mice with either 105 CFU (1006 LD50) or 106 CFU (10006 LD50) of LVS via the same route. Vaccinated and control animals received the bacterial inoculum four weeks after the third immunization and their survival was monitored for 21 days. Sixty percent of the mice (6 out of 10) immunized with the LPS+rPorB were protected from challenge with 106 CFU of LVS, compared with the PBS control group that succumbed to tularemia within 8 days post- infection (Figure 2A). The poor protective capacity of LPS alone was reflected in the accelerated death of mice (Figure 2A). After infection with 105 CFU, 7 mice out of 9 (78%) of the LPS+rPorB group survived for 19 days (Figure 2A). At day 20 one additional mouse from the 105 CFU group died with a final survival rate of 67% (Figure 2A). In contrast, only 10% of inoculated with LPS alone survived after challenge with 106 CFU, and 17% survived after challenge with 105 CFU (Figure 2A). Progressive body weight loss was detected during the first 8 days after infection, and a subsequent regain, starting at day 9 post-infection, was associated with improved survival of LPS+rPorB vaccinated mice (Figure 2B). Mice that received LPS alone and survived challenge had a similar body weight recovery, although the overall survival rate was dramatically reduced compared to LPS+rPorB mice. Groups of mice receiving PBS or rPorB showed poor survival rate and never regained their initial weight. To further evaluate the efficacy of intranasal vaccination, bacterial replication in the bloodstream was analyzed. Mice (3 to 8 mice per group) immunized with LPS+rPorB or one of the control substances (PBS, LPS or rPorB alone) were bled 3 days after intranasal challenge with either 105 or 106 CFU of LVS. Figure 2C shows that mice immunized with our vaccine candidate (LPS+rPorB) had numbers of bacteria in their blood (,102 CFU per ml) approximately 100-fold lower than mock-vaccinated mice (,104 CFU; P,0.001). Bacteremia detect- ed in LPS-immunized mice was similar to that observed in the LPS+rPorB-immunized group, while bacterial levels in the blood of mice vaccinated with rPorB were closer to those found in PBS control mice (Figure 2C). A similar pattern was observed in groups of mice challenged intranasally with 105 CFU (Figure 2C). These results describe the high potential of meningococcal porin as a mucosal adjuvant. In a preliminary study we compared the PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11156