Tularemia Vaccination and BALT Figure 5. Detection of proliferating B cells within BALT of vaccinated mice 30 days post-challenge. (A) 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 mice. B cells were detected with B220 (green- Alexa 488) and PCNA was used to identify proliferating cells (red- Alexa 549). Areas of mixed color overlap indicate proliferation of B cells (see arrows). 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.g005 decreased levels of bacteremia. One plausible explanation for this phenomenon is that other mechanisms other than bacterial clearance, including lung inflammation or injury, might be contributing to the difference in survival but not in bacterial loads between LPS- and LPS+rPorB-vaccinated mice. Serum from mice vaccinated with LPS+rPorB showed an overall increased produc- tion of antigen-specific antibodies, compared with antibody levels in sera from the LPS-, rPorB- and PBS-immunized groups, which presented low levels of class switched, LPS-specific immunoglo- blulins. For both antigen specific IgG and IgM we observed that some mice were more responsive than others, indicating variability in the humoral response to LPS administered in combination with rPorB. Nasal vaccination is known to induce serum IgG due to the capacity of mucosal immune cells (e.g. DCs, B cells) to activate and effectively mediate contact with systemic immune sites [35]. In our model, mice presented variable levels of antigen specific antibodies and the higher amounts did not necessarily correlate with protection from challenge. One plausible explanation is that despite the low levels detected, these antibodies may still have a higher affinity for our antigen, and this may potentially be associated with the protective immune response observed. Relative affinity assays will be conducted in future studies. Another possibility is that antibodies are produced and rapidly consumed locally, explaining the containment of the infection in the lung. Other factors, like cellular immunity, might also be playing a crucial role in conferring protection against respiratory challenge. We were able to detect dendritic cells (DCs), follicular dendritic cells (FDCs) and macrophages within the lymphoid structures of the murine lungs, suggesting a role for this cell type in cell mediated immunity in response to our intranasal vaccine and challenge regimen. Recently, another study reported a role for DCs in iBALT homeostasis and induction of antibody responses [36]. In this study we report the development of organized lymphoid structures forming in peribronchial and perivascular areas of lungs following intranasal vaccination and challenge in a murine model of experimental tularemia. In a previous report, organized lymphoid areas defined as inducible BALT (iBALT) developed in the lungs of C57BL/6 mice following infection with the Influenza virus [27]. Lymphoid aggregates were also observed in various other animal species after infection with Mycobacterium tuberculosis, Pseudomonas aeruginosa and Mycoplasma spp. [30–32]. In human lungs, organized lymphoid areas have been described in patients with pulmonary complications of autoimmune diseases including rheumatoid arthritis and Sjogren syndrome [34], or other chronic lung conditions like hypersensitivity bronchiolitis and pan-bronchiolitis [37,38]. In Francisella tularensis infection studies, one group found that LVS administered via the aerosol route induced medium sized lymphoid aggregates in murine lungs [39]. The authors hypothesized that these clusters might either be residual inflammatory foci, or Francisella-specific T cells to be recalled upon re-infection [39]. Similarly, our group described the appearance of peribronchial lymphoid aggregates in mice that spontaneously recovered from experimentally induced tularemia 50 days after intranasal infection with 104 CFU (106 LD50) of LVS [40]. In the present model, we observed that lungs of mice PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11156