stimulating B and T cells, sustaining local antibodies for prolonged periods of time [48,49]. Spleens and livers from immunized and subsequently challenged mice also did not contain any viable F. tularensis. This may suggest that immune responses initiated in the lungs are also able to control disseminated infection, leading to bacterial clearance from peripheral tissues. It should be noted that the studies we mentioned above addressed potential BALT persistence in viral infection, while in the case of bacteria lymphoid structures have mainly been associated with chronic disease, as in the case of M. tuberculosis and P. aeruginosa [30,32]. In our F. tularensis vaccine/challenge model it is difficult to exactly conclude what contributes to BALT maintenance over time, though its development in lungs of mice surviving tularemia suggests that it may play a role in the induction of local immunity and protection in our model of experimental tularemia. In the present work, we demonstrate the efficacy of F. tularensis LPS, combined with meningococcal PorB against experimental tularemia. For future studies it would be optimal to test the adjuvanticity of this protein when combined with other promising antigen candidates, and test their efficacy against LVS and virulent strains of F. tularensis. We also report the association of lung BALT structures with mouse survival following vaccination and subse- quent respiratory infection. Additional studies are required to reveal the mechanisms involved in BALT formation and its active role in the induction of local and systemic protection against pulmonary Francisella infection. Materials and Methods Bacterial strains and growth conditions F. tularensis subsp. holarctica live vaccine strain (LVS- ATCC 29684) was obtained from the CDC, Fort Collins, CO. For mouse challenge, LVS was grown on chocolate agar for 72 hours and bacterial suspensions were prepared by resuspending colonies in PBS at an OD600 of 0.3 [12,40]. Purification methods Lipopolysaccharide (LPS) was isolated from the LVS strain using the hot phenol method as previously described [12]. Purification of recombinant PorB (rPorB) was performed based on a well established protocol [50]. E. coli BL21(lDE3) DompA, obtained from Dr. Milan Blake at the Rockefeller University, was grown on a LB agar plate containing 50 mg/ml kanamycin and incubated overnight at 37uC. Colonies from the plate were inoculated into 10 ml of liquid medium consisting of supplement- ed M9 minimal media grown overnight at 37uC. Subsequent growth was induced with IPTG for 3 hours at 37uC. After centrifugation the bacterial pellet was resuspended in 3 ml TEN buffer. PMSF and lysozyme were added, followed by deoxycholate and the suspension was then placed in a 37uC water bath. DNase I was then added to the mixture. The lysate was centrifuged at 10,0006g for 20 min at 4uC. The pellet was resuspended in 5 ml TEN buffer and then sonicated. Zwittergent (10%, wt/vol) was added to the mixture, sonicated for 10 min and loaded onto Sephacryl S-300 gel filtration columns previously equilibrated with 100 mM Tris, 200 mM NaCl, 10 mM EDTA, pH 8.0. The flow rate of the column was set at 0.8 ml/min and 10 ml fractions were collected. Fractions containing protein as determined by measure- ment of OD280 were analyzed by SDS-PAGE. Preparation of proteosomes and SDS-PAGE Recombinant PorB proteosomes were prepared as previously described [51]. Briefly, fractions containing porin were pooled and precipitated with 80% (v/v) ethanol and held at 220uC overnight. Tularemia Vaccination and BALT The precipitate was centrifuged at 15,0006g for 20 min, the pellet was resuspended in 10 ml 10 mM Hepes buffer containing 10% D-octyl-glucoside (DOG), pH 7.2 and dialyzed extensively against PBS containing 0.02% sodium azide [51]. The protein concen- tration was determined using a BCA protein assay kit (Pierce) and tested for endotoxin by Pyrotell (Associates of Cape Cod, East Falmouth, MA) with a sensitivity of 0.06 endotoxin units per milliliter (EU/ml). Fractions containing protein as determined by measurement of OD280 were analyzed by SDS-PAGE. PreciseTM Protein Gels 4%–20% were used in Tris-HEPES-SDS buffer (Pierce Biotechnology, Inc.) and were stained with standard Coomassie (Bio-Rad, Inc. Hercules, CA). Mice Seven week old female C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained within the Laboratory Animal Science Center (LASC) at Boston University. All experimental procedures were in accordance with institutional animal care and use committee. Intranasal vaccination and challenge Groups of five to ten mice were immunized by the intranasal (i.n.) route three times at two-week intervals. Mice were anesthetized via the i.p. route with ketamine HCl (Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (Lloyd Laboratories, Shenandoah, IA), and vaccinated with LVS-LPS, LVS-LPS with neisserial rPorB, rPorB alone, or PBS in a total volume of 20 ml (a quantity of 10 mg for each substance). Three to ten mice per group, were bled by the submandibular vein after the last vaccination, to detect the production of LPS-specific immuno- globulins. Four weeks after the last immunization, mice were challenged intranasally with either 105 or 106 CFU of LVS as described before [12]. All animals were closely observed until completely awake from anesthesia. Survival and changes in body weight were recorded for up to 21 days after infection. For lung tissue analysis, survivors were humanely euthanized 30 days post- challenge. Data reported in this study are representative of three independent experiments. Antibody and chemokine assays Mouse serum and lungs were processed for the detection of immune markers. Analysis of LPS-specific serum IgG, IgM and IgA was performed in pre-challenge serum by enzyme-linked immunosorbent assay (ELISA) as previously reported [12,40]. Briefly, plate wells were coated with LPS from F. tularensis LVS (0.25 mg/ml), incubated at 37uC and stored overnight at 4uC. Sera were diluted starting from 1:50, added to the previously coated wells, and incubated at 37uC. Alkaline phosphatase-conjugated anti-mouse IgG, IgM or IgA (Sigma, St. Louis, MO) were added. After washing, the color was developed with one-step p-nitrophenyl phosphate (Pierce, Rockford, IL) and the optical density at 405 nm was measured on an ELx800 reader (Bio-Tek Instruments, Inc., Winooski, VT). Colorimetric values were converted to nanograms/milliliter, according to the standard curves generated for IgG, IgM and IgA. Levels of the CCL21 chemokine were measured in both serum and lung lysates of vaccinated/challenged mice by ELISA using the DuoSet Development System kit specific for CCL21/6Ckine (R&D Systems, Minneapolis, MN). Histopathological and morphometric analyses of lymphocytic infiltration All histopathological procedures were conducted as previously described [40]. Briefly, lungs were removed aseptically 30 days post challenge, and inflated with 10% buffered formalin through PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e11156