Tularemia Vaccination and BALT Figure 1. Recombinant PorB and evaluation of antibody response against F.tularensis LPS. (A) The band of rPorB detected by Coomassie gel stain is indicated by the arrow. (B) Serum was collected four weeks after vaccination, just before i.n. challenge. Antibody production was analyzed by ELISA and results are presented as nanograms (ng) of LPS-specific IgG and IgM per milliliter (ml) of serum. Black dots indicate individual mice, while bars represent mean values. Lower antibody concentrations include values between 286 and 436 ng/ml as opposed to those that were not detected (ND) in control groups. Significant differences were calculated by Mann Whitney test and are indicated by *** (P,0.001) for LPS+rPorB compared with PBS. doi:10.1371/journal.pone.0011156.g001 immunogenic efficacy of this protein to that of the known mucosal adjuvant CpG DNA. Results revealed that LPS-specific IgG and IgM induced by PorB were respectively 3 and 2-fold higher when compared with CpG DNA (data now shown). Formation of BALT after vaccination and challenge Thirty days after respiratory challenge, lungs from vaccinated mice were dissected, fixed and processed for histopathological and immunofluorescence analyses. To evaluate the inflammatory changes in the infected lungs, paraffin sections were stained with hematoxylin and eosin (H&E). The presence of lymphocyte clusters, indicative of organized lymphoid follicles, was then evaluated by light microscopy. Representative lung sections from untreated mice (Figure 3A) as well as sections from lungs of mice vaccinated intranasally with LPS alone (Figure 3B), LPS+rPorB (Figure 3, C and D) and LPS+rPorB without LVS challenge (Figure 3E) are reported. Histopathological analysis of sections revealed the presence of lymphoid follicles. Overall, no inflammation (neutrophils, edema, fibrin) was detected in the lung tissue. Lungs from unvaccinated and unchallenged control mice did not contain any inflammatory cells or visible lymphocytes (Figure 3A). Lungs from vaccinated mice that became moribund prior to day 8 post-infection did not contain any lymphoid areas (data not shown). Lung sections from all vaccinated mice presented lymphoid aggregates (Figure B–E). Nevertheless, standard hematoxylin and eosin staining did not show the extent of the organization of the follicles, thus immunofluorescence was used for this purpose. Lung sections from the group vaccinated with LPS/rPorB and challenged with LVS contained visibly organized BALT as demonstrated by B (B220+) and T cell (CD3+) infiltration (Figure 4, D and E), B220+ B cell follicles with proliferating B cell (PCNA+) (Figure 5, D and E) and B220+ B cell PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11156