To begin to address this issue in an animal model of acute infection, we established that immunologically naive BALB/c mice challenged i.p. with 2 × 10⁷ CFU resulted in death by day 4–6, while i.p. immunization with 1 × 10⁵ heat killed (HK) bacteria provided partial protection against a subsequent challenge. Two independent experiments resulted in similar findings of 40% survival for HK-vaccinated mice with a mean survival time (MST) of 8 days versus 4 days in naïve mice (Fig. 1). The administration of vaccines for B. mallei during an outbreak would mandate relatively rapid onset of protection for human or veterinary use. Based on non-routine use and vaccine implementation in the course of an outbreak, a 14 day window was chosen for assessment of protection. Our results indicate that HK vaccination can afford partial protection to an otherwise lethal challenge of B. mallei by the i.p. route.

To dissect the cellular basis for protection mediated by HK vaccination, 13 days after immunization with HK bacteria (day -1), and at day of challenge, mice were dosed with antibodies to deplete CD4⁺, CD8⁺ or B220⁺ cells. Antibody depletion of CD4⁺, CD8⁺, or B220⁺ cells in these mice was confirmed by flow cytometric analysis with depletion efficiencies for CD4, CD8, and B220 populations at 99.7%, 96%, and 95%, respectively, relative to mice treated with isotype control monoclonal antibodies (data not shown). Our results demonstrated decreased survival rates in B220 (p = 0.3418), CD4⁺ (p = 0.5417) and CD8⁺ (p = 0.4684) antibody depleted mice, compared to isotype control antibody, a finding that indicated a possible role for vaccine induced antibody production. When challenged with 2 × 10⁷ CFU/mouse by the i.p. route, loss of T cells resulted in reduced survival (50%) relative to the non-specific isotype control (Fig. 2). In contrast to the loss of T cells, depletion of B220⁺ cells resulted in 100% mortality relative to the non-specific isotype control (Fig. 2). To further evaluate the necessity of these effector cells in providing protection following HK vaccination, relatively resistant C57BL/6 mice, deficient in mature B-cells (μMT), CD4 T-cells (CD4^-/-) or CD8 T-cells (CD8^-/-) were subjected to an identical HK vaccination and challenge regimen. Mature, B-cell-deficient mice demonstrated a 50% decreased survival (p = 0.0888) compared to the wild-type mice with an MST of 35.5 days (Fig. 3). CD4^-/- and CD8^-/- mice exhibited a 60% (p = 0.1343) and 0% reduced survival, respectively (Fig. 3).

Similar studies were performed to determine the role of IFN-γ or TNF-α in acute infection in BALB/c mice immunized with HK bacteria. Six hours before challenge, mice were dosed with antibodies that neutralize IFN-γ or TNF-α. Individual depletion of either TNF-α (p = 0.0145) or IFN-γ (p = 0.0446) resulted in 100% mortality with an MST of 3 and 2 days, respectively, compared to the HK-vaccinated isotype control mice (Fig. 4). In contrast, 40% of HK-vaccinated, isotype control mice survived to at least 12 days post-challenge (Fig 4). To further evaluate the host TNF-α response during an established B. mallei chronic infection, we infected 12 BALB/c mice by the i.p. route with 1 × 10⁶ CFU B. mallei. One animal was terminally ill on day 37 post-infection. On day 42 post-infection, the remaining 11 mice were dosed with either anti-TNF-α (n = 6), or control mAb (AFRC Mac 49) (n = 5). No further deaths were observed in the control mAb-treated mice. Rapid mortality was observed in the anti-TNF-α-treated group, with all mice dying within 7 days of treatment (p = 0.0023) relative to the isotype-treated controls (Fig. 5).

Complement mediated uptake assays were performed to evaluate opsonization. Results indicated enhanced bacterial uptake in J774A.1 phagocytes inoculated with serum treated B. mallei (p = .0082), compared to B. mallei alone, while heat-inactivated serum produced uptake percentages similar to those prior to serum addition (Fig. 6). Taken together, these results imply an active role for complement components in the uptake of organism by macrophages.

We further characterized the ability of HK vaccination to induce a predominant IgG isotype by determining IgG2a/IgG1 ratios in i.p. and i.n. vaccinated BALB/c mice. Pre (day 14 post vaccination) and post (day 2 post infection) exposure serum samples were obtained and evaluated for IgG isotype concentrations (Table 1). No appreciable differences in IgG pre-exposure levels were seen when comparing i.n. to i.p. vaccination. In addition, cobra venom factor-treated animals showed no significant differences to non-cobra venom factor-treated animals in IgG pre-exposure (challenge) levels. Conversely, isotype switching in the cobra venom factor treated animals was enhanced in post-exposure serum IgG2a (Table 1).

B. mallei strain ATCC 23344 (China 7) was cultured on Luria-Bertani agar supplemented with 4% glycerol (LB+4%G) agar plates for 48 h at 37°C. Isolated colonies were sub-cultured to LB+4%G broth, and cultures were incubated at 37°C until optical density readings at 600 nm (OD₆₀₀) reached an exponential phase of growth. Bacteria were pelleted by centrifugation, washed and re-suspended in sterile 1× phosphate-buffered saline (PBS, pH 7.4) to obtain the desired CFU/ml. To obtain HK inoculums, bacterial suspensions were incubated at 85°C for 3 h and stored at 4°C until use. The absence of live B. mallei organisms in the HK preparations was confirmed after plating 10% of the total inoculums (v/v) and incubating these at 37°C for 48 h. All procedures were performed under a class II biosafety cabinet in a biosafety level 3 laboratory. Female, 6- to 8-week-old, BALB/c mice (n = 5–7) were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, Indiana). Female, 6- to 8-week-old, C57BL/6 mice deficient in mature B-cells (μMT), CD4 T-cells (CD4-/-) and CD8 T-cells (CD8-/-) and wild-type mice were obtained from The Jackson Laboratory (Bar Harbor, Maine).

BALB/c and C57BL/6 mice were grouped and vaccinated with 0.5 μg of HK B. mallei (without adjuvant) by i.p. injection using a 25-gauge syringe. Two weeks post HK vaccination mice were injected i.p. with 2 × 10⁷ CFU/100 μl of live B. mallei (~20 LD₅₀) 18. Complement depleted animals were challenged with 2.5 × 10⁴ CFU/50 μl (~0.25 LD₅₀) by intranasal (i.n.) route. Aliquots from the inoculums were plated to confirm the infecting dose. All procedures and animal protocols used in this study were approved by the Biosafety and IACUC committees at UTMB and conducted in either BSL-3 or ABSL-3 laboratories.

Acute in vivo cell/cytokine depletion was performed with monoclonal rat anti-mouse CD4 (GK1.5), CD8α (53-6.7) or B220 (RA3-6B2) obtained from R&D Systems, Inc. (Minneapolis, MN) by methods similar to those we have previously described 19. Functional grade purified rat anti-mouse interferon-gamma (IFN-γ, AN-18) was obtained from eBioscience (San Diego, CA) and purified anti-mouse tumor necrosis factor (TNF-α, MP6-XT3) from BD Pharmingen (San Diego, CA). IFN-γ and TNF-α antibodies were injected i.p. 6 h prior to challenge, 200 μg per mouse in 200 μl PBS or at later time points as indicated. Rat IgG isotype control was obtained from Southern Biotech (Birmingham, AL) and administered i.p. on day of challenge, 200 μg/mouse. Rat anti-mouse CD4, CD8α and B220 were injected i.p. twice, 1 day prior to challenge and on day of challenge, with an equivalent dosage sufficient to deplete T or B cells from 6 × 10⁸ bone marrow cells per injection. The efficiency of depletion at time of infection for CD4⁺, CD8⁺, and B220⁺ cells was confirmed by flow cytometry analysis immediately prior to infection.

Mice, six to seven per group, were vaccinated i.p. with 1 × 10⁵ CFU of nonviable B. mallei cell preparation in a total volume of 0.1 ml. Two weeks later, 24 h and 1 h before challenge, complement depleted mice were treated i.p. with 12.5 units total cobra venom factor (Quidel Corporation Speciality Products, San Diego, CA) in 0.1 ml of PBS. Complement depletion was confirmed prior to challenge by micro-titer hemolytic complement activity (CH₅₀) assay as previously described 20.

J774A.1 cells were seeded (5 × 10⁵) onto Corning costar 24 well plates (Corning, NY) with DMEM and incubated overnight at 37°C with 5% CO₂. Bacterial suspensions were incubated at 37°C for 45 minutes supplemented with 2% mouse serum from Sigma-Aldrich (St. Louis, MO.), heat inactivated mouse serum (56°C 30 minutes), or bacteria alone and then added at an MOI of 10:1 to J774A.1 cells in triplicate. Inoculated wells were centrifuged at 800 g for 2 minutes and incubated for 2 hours at 37°C with 5% CO₂ followed by a PBS wash (×2) and 2 hour incubation with 250 μg/ml kanamycin. Wells were washed twice with PBS and lysed with 0.1% Triton X-100, followed by serial 10-fold dilutions plated on LBG plates and incubated at 37°C for 2 days. Colony forming units were enumerated and uptake expressed as a percentage of initial inoculating dose ± SEM.

Flow cytometric analysis was performed on 0.1-ml blood samples transferred to micro centrifuge tubes containing 90 μl of acid citrate dextrose (ACD) solution. Red blood cells were lysed using ACK-lysing buffer (Biosource International, Inc., Camarillo, CA) according to the manufacturer's instruction. Antibodies used for analysis of surface markers included: FITC-conjugated rat anti-mouse CD45R/B220 (RA3-6B2, BD Pharmingen San Diego, CA) for B cells; FITC-conjugated rat anti-mouse CD8α (53-6.7) and CD4 (GK1.5, BD Pharmingen, San Diego, CA) for CD8⁺ or CD4⁺ cells, respectively. Samples evaluated for CD4⁺ and CD8α⁺ cells were also incubated with biotin-conjugated hamster anti-mouse CD3e (145-2C11) monoclonal antibody (BD Pharmingen, San Diego, CA) and subsequently with streptavidin APC Cy7. Isotype-matched, non-specific controls were assayed in parallel (BD Pharmingen, San Diego, CA). Surface staining was performed according to previously published protocols 21. Following cell staining, the samples were fixed with 2% buffered paraformaldehyde overnight prior to analysis by flow cytometry. Samples were analyzed using a FACSCalibur flow cytometer with BD CellQuest Pro software.

Immunoglobulin subclass IgG1 and IgG2a titers in mice were determined by a whole bacterial cell ELISA performed in 96-well, Immulon 2 HB, round-bottom plates (Dynex Technologies). B. mallei antigen was diluted in 0.1 M carbonate buffer (pH 9.5) and 50 μl of diluted cells placed into wells. Plates were stored overnight at 4°C. The plates were washed with washing solution (1× PBS, 0.05% Tween 20), and incubated with 100 μl of blocking solution (1× PBS, 1% bovine serum albumin, 0.05% Tween 20) for 1 h at 37°C. Dilutions of mouse sera were made with blocking solution in duplicate and plates were incubated for 1 h at 37°C. Following incubation, plates were washed and 50 μl of anti-Ig-horseradish peroxidase subclass conjugate, diluted accordingly to manufacturer's instructions (Southern Biotechnology Associates, Inc. Birmingham, Ala.), was added to each well and incubated for 1 h at 37°C. After washing, 50 μl of 2,2'-azino-di-(3-ethylbenzthizoline)-6-sulfonate (ABTS) peroxidase substrate (KPL, Inc., Gaithersburg, Maryland) was added to each well and plates incubated for 25 min at room temperature. The amount of bound antibody was determined colorimetrically by absorbance at 405 nm.

Survival curves were calculated by Kaplan Meier survival analysis with log-rank tests between groups using GraphPad Prism (V.4.03 for windows). Statistical analysis was generally performed with the paired Student's t-test. P value ≤ 0.05 was considered significant.