Tularemia Vaccination and BALT Figure 4. Detection of B and T cell infiltrates in lungs of vaccinated mice 30 days post-challenge. (A) Representative lung from na¨ıve mouse as a negative control; (B) Mediastinal lymph node from LPS+rPorB/challenged mouse as an internal positive control; (C) Lung from LPS vaccinated/challenged mouse; (D, E) Lung from LPS/rPorB vaccinated/challenged mouse and (F) lung from LPS+PorB vaccinated/unchallenged mouse. Cell surface markers used were B220 for B cells (green- Alexa 488) and CD3 epsilon for visualizing T cells (red- Alexa 549). Cell nuclei were stained with 49-6-Diamino-2-phenylindole (DAPI-blue). Representative pictures were taken with a 206 objective. doi:10.1371/journal.pone.0011156.g004 lenged mice. A significant increase in CCL21 concentration in lung lysates from vaccinated mice was found (Figure 8B). Analysis of CXCL13 production in lung lysates and serum did not reveal any noteworthy differences between na¨ıve and vaccinated mice (data not shown). Chemokine analysis is not reported for the LPS- vaccinated mice because only one mouse survived in the group, and data analyzed at day 30 could not be included in the study. Discussion The present work reports the use of meningococcal PorB as a potential mucosal adjuvant when delivered with F. tularensis LPS, a poorly immunogenic antigen. A high percentage of mice vaccinated with LPS and PorB were significantly protected against lethal respiratory LVS infection when compared with the control groups, and presented well-organized bronchus-associated lym- phoid tissue (BALT) in peribronchial and perivascular areas of their lungs. The vaccine candidate was administered via the intranasal route, an attractive alternative to conventional paren- teral formulations against respiratory pathogens. We are aware that LVS is an attenuated strain of F. tularensis, and that challenge should ideally also be conducted with virulent type A and B strains. Nevertheless, it should be noted that very few studies have been able to show effective protection against virulent F. tularensis strains after administration of LPS-based or other subunit vaccines. Poor protection is associated with the rapid induction of fatal disease in mice challenged with extremely low infection doses. In addition to this experimental difficulty, virulent strains of F.tularensis cause fulminant systemic disease in the mouse, which does not optimally mimic human tularemia. Overall, the selection of a vaccine candidate able to confer successful protection against virulent F. tularensis has proven to be difficult over the years, and is beyond the purpose of the present work, which mostly shows the potential of a bacterial porin protein as a mucosal candidate and its ability to enhance BALT development. Following intranasal immunization of mice, meningococcal recombinant PorB significantly improved the protective capacity of Ft-LPS, as shown by the increased survival rate of C57BL/6 mice (67% and 60%) challenged intranasally with 105 (1006 LD50) and 106 CFU (10006 LD50) of LVS, respectively. LPS administered alone conferred less than 20% protection from respiratory challenge, confirming the results we previously obtained in a subcutaneous (s.c.) vaccination model [12]. In the s.c. model, LPS+PorB induced slightly higher protection (70%) than that observed in our i.n. model, but it should be noted that BALB/c mice were used instead of the current C57BL/6 strain [12], and that genetic background can have an impact on vaccine efficacy and disease outcome. The reason for selecting C57BL/6 for this study is based on our interest in elucidating the role of Toll- like receptors (TLR) in the innate immune response against Francisella and vaccine candidates against tularemia, as most TLR knock-out mouse strains are available in this genetic background. The high level of protection observed was associated with progressive weight recovery and a pronounced decrease in bacteremia (10 to 100-fold) in the LPS+rPorB group. This result contrasted with control groups, in particular mice that were mock- vaccinated with PBS that exhibited considerable disseminated disease. Interestingly, mice vaccinated with LPS alone also showed PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11156