Burkholderia pseudomallei ΔtonB Δhcp1 Live Attenuated Vaccine Strain Elicits Full Protective Immunity against Aerosolized Melioidosis Infection

C57BL/6 mice were intranasally vaccinated with a three-dose regimen of PBS or strain PBK001 at 2-week intervals and then challenged with 6.9 to 11.6 LD₅₀ of B. pseudomallei K96243, 21 days after the last immunization. Following challenge with B. pseudomallei K96243, mice were monitored for survival and changes in body weight for 27 days postinfection (dpi). At the end of the study, mice vaccinated with strain PBK001 exhibited 100% survival, whereas nine of ten PBS-vaccinated mice succumbed to infection by day 4 and the tenth mouse died on day 21, as shown in Fig. 1A. On day 27 dpi, the lungs, livers, and spleens of surviving mice (n = 10) were harvested and processed for CFU enumeration (n = 7) and histopathology evaluation (n = 3). No bacteria were detected in all three organs evaluated from five animals; however, some bacteria (limit of detection, <10 CFU/organ) were detected in the organs from two of the PBK001-vaccinated mice (Fig. 1B). The survival study and bacterial load determinations indicated that immunization with strain PBK001 protected mice from aerosolized melioidosis and significantly reduced the level of B. pseudomallei infection in the three organs analyzed.

Sera from individual PBS and PBK001-vaccinated mice were analyzed for antibody responses to irradiated B. pseudomallei K96243 whole-cell lysate (WCL). Total IgG and IgG2a and IgG1 subclasses were determined using indirect ELISA. The sera from individual mice that received a prime and two boosts of PBK001 vaccine showed significantly higher reciprocal endpoint titers (mean ± standard errors of the means [SEM]) for IgG (46,720 ± 12,730), IgG2a (4,520 ± 2,464), and IgG1 (231 ± 73) compared to PBS-vaccinated mice (Fig. 2A to C). In addition, the IgG2a/IgG1 ratio was calculated to be 19.5 for the PBK001 vaccine. PBK001 vaccination generated high levels of B. pseudomallei-specific IgG and higher levels of IgG2a, indicative of Th1-biased immune response.

To assess the role of cellular immune responses in PBK001 vaccine-induced protection, the spleens of PBS- and PBK001-vaccinated mice were collected on day 21 after the last vaccination. Single-cell suspensions of splenocytes were seeded and restimulated with BSA or heat-killed B. pseudomallei K96243 WCL for 72 h. Splenocytes from PBK001-vaccinated mice significantly increased production of IFN-γ, TNF-α, and IL-17A after stimulation with specific antigen (B. pseudomallei WCL) compared to nonspecific stimuli (BSA) (Fig. 3). Consistent with nonspecific innate immune activation by WCL, some IFN-γ was generated from spleen cells from PBS-vaccinated mice in response to antigen. These nonspecific responses were significantly lower than the antigen-specific response observed with splenocytes from the PBK001-vaccinated mice (P = 0.0273). Significant activation of TNF-α by WCL was observed in splenocytes from both PBS and PBK001 groups, though further increased production was observed in the vaccinated group. The nonspecific TNF-α production is consistent with activation of splenic macrophage innate immunity via pathogen recognition receptor binding of WCL components. These results demonstrated that the PBK001 vaccine induced an antigen-specific cellular response by production of effector cytokines characteristic of Th1 (IFN-γ) and Th17 (IL-17A) immune responses.

To determine the protective role of either CD4⁺ or CD8⁺ T cells in PBK001-immunized mice, vaccinated animals were depleted of individual T cell populations prior to challenge with B. pseudomallei. Two weeks after the last vaccination, mice received an i.p. injection with either isotype control or with antibodies specific to murine CD4 or CD8 T cells, 3 days before challenge (day −3) or on the day of challenge (day 0), and depletion was continued every 3 days after challenge (days +3, +6, +9, and +12). The depletion efficiencies were confirmed by flow cytometric analysis of CD4⁺ and CD8⁺ T cell populations in an age- and gender-matched group of noninfected mice. The results demonstrate that the depletion protocol resulted in 99.78 to 100% depletion of CD4⁺ and 98.9 to 99.5% of CD8⁺ T cells in the lung, spleen, and peripheral blood (Fig. 4A).

As predicted by the percent animal survival (Fig. 4B), mice that received PBS succumbed to infection at 3 days postinfection (dpi), while PBK001-immunized mice without T cell depletion (isotype control) showed 100% survival at 16 dpi. The survival of PBK001-CD4⁺ and PBK001-CD8⁺ depleted mice showed 60% and 100% survival on 16 dpi, respectively. At the end of study, the lungs, livers, and spleens of surviving mice were harvested and processed for CFU enumeration to determine the effect T cell depletion had on controlling the bacterial burden. Depletion of CD4⁺ T cells increased bacterial burden in liver compared to isotype control and CD8⁺ depleted mice (Fig. 4C), though overall liver burden was very low. These data showed a moderate contribution of CD4⁺ T cells to cell-mediated immunity against B. pseudomallei, while CD8⁺ T cells do not appear to contribute significantly to vaccine-induced protection.

To determine the extent of tissue damage, gross pathology and histological analysis of tissues (lung, liver, and spleen) from surviving mice (n = 3) exposed to aerosolized B. pseudomallei K96243 were evaluated at 27 dpi. Gross pathological observation of all the organs analyzed indicated normal size and unremarkable pathology (data not shown). For histopathology, the numbers of inflammatory foci from three mice were quantified by counting 10 fields of ×40 magnification per organ and reported as means ± standard deviations (SD). Representative images of H&E-stained lungs, livers, and spleens from PBK001-vaccinated mice are shown in Fig. 5. Lung sections showed areas of lymphocyte-rich inflammation surrounding the bronchoalveolar tree estimated as 3 ± 1.4 foci per ten ×40 magnification fields. Liver sections also showed multifocal infiltrates of mixed inflammatory cells (1 ± 0.63) (lymphocyte rich) occasionally associated with apoptotic hepatocytes. Spleen sections showed minimal to unremarkable pathological damage. Prominent periarteriolar lymphoid sheaths (PALS) and increased hematopoiesis were found in the spleens of surviving animals. Overall, the histopathologic analysis showed minimal change in the organs of surviving vaccinated mice after aerosol challenge, supporting our findings that vaccination prevents pathogen persistence and therefore causing minimal pathological changes in target organs.

This study describes a safe and attenuated B. pseudomallei ΔtonB Δhcp1 mutant vaccine strain that generated full protection against a lethal dose of aerosolized melioidosis, producing almost complete sterilized immunity. The value of this vaccine may increase if protection against different B. pseudomallei strains and cross-protective properties against B. mallei infection can be confirmed.

All animals in this study were handled in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were housed in microisolator cages under pathogen-free conditions, provided with rodent feed and water ad libitum, and maintained on a 12-h light cycle in an animal biosafety level 3 (ABSL3) laboratory, and all experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch (protocol 0503014D) and the Animal Care and Use Review Office of the Department of the Army.

The bacterial strains used in this study are listed in Table 1. Escherichia coli was grown on Luria-Bertani (LB) agar or in LB broth containing 50 μg/ml of kanamycin. B. pseudomallei K96243 and B. pseudomallei K96243 Δhcp1 (CLH010) strains were grown on LB with 4% glycerol (LBG) agar for 48 h and shaken in LBG broth for 12 h. B. pseudomallei ΔtonB Δhcp1 mutant was grown in low-salt (0.5% NaCl) LB agar supplemented with 200 μM FeSO₄·7H₂O or low-salt LB broth for 16 h. Bacteria on agar plates were incubated at 37°C. Bacterial stocks were maintained in 20% glycerol and stored in −80°C freezer. All manipulations of B. pseudomallei strains were conducted in a CDC-approved and registered biosafety level 3 (BSL3) or CDC/USDA-approved and registered animal biosafety level 3 (ABSL3) laboratory facility at the University of Texas Medical Branch, and all experiments were performed in guidance with standard select agent operating practices.

We employed a similar methodology used to construct the B. mallei ΔtonB Δhcp1 (CLH001) live attenuated vaccine to create the B. pseudomallei ΔtonB Δhcp1 (PBK001) vaccine strain. The PBK001 strain was constructed using a select agent compliant suicide vector allelic exchange system described previously (55). First, the B. pseudomallei K96243 Δhcp1 (CLH010) strain was obtained using donor strain E. coli S17-1 λpir containing the plasmid pMo130-ΔBPSS1498 (provided by Mary Burtnick, University of Nevada at Reno). This plasmid created an unmarked in-frame deletion of hcp1 (23). Five hundred microliters of E. coli donor strain were mixed with 500 μl of B. pseudomallei K96243, centrifuged, and resuspended in 100 μl of 10 mM MgSO₄ as a mix or individually for controls. Each conjugation was spotted (25 μl) on LBG agar and incubated for 8 to 16 h at 37°C. Selection of merodiploids was performed by resuspending the mixed reactions in Dulbecco’s phosphate-buffered saline (DPBS), and the diluted solutions were plated on LBG agar containing 250 μg/ml of kanamycin to select for integration and 30 μg/ml of polymyxin B to select against the donor E. coli strain. The plates were incubated for 48 to 96 h at 37°C. Merodiploids were screened by exposing the plates to 0.45 M pyrocatechol spray solution and selecting for yellow colonies. Counterselection was performed by subculturing merodiploids in 2× yeast extract-tryptone (2×YT) broth without salt for 4 h and then plating on 2×YT agar without salt and supplemented with 15% sucrose for 48 to 72 h. The resulting colonies were then screened with pyrocatechol, and nonyellow colonies were screened by PCR to confirm deletion of hcp1. Mutants positive by PCR were then sequenced to confirm the mutation. Strain PBK001 was constructed by using strain CLH010 (Δhcp1) and introducing the tonB mutation using pMo130ΔtonB as described previously (19). Mutant construction was performed as described above except that LBG agar was supplemented with 200 μM FeSO₄. The Δhcp1 mutant was confirmed using primers as previously described (26), and the ΔtonB mutation was then confirmed by PCR, followed by sequencing using the following primers: forward primer, GAA TTG CTG ATC GGA TTC GT; reverse primer, TCC GTA GCT TTG CAT TTC CT.

Female 6- to 8-week-old C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA, USA) and acclimated in the ABLS3 laboratory for 5 days before vaccination. Anesthetized mice (n = 20) were administered with three intranasal (i.n.) vaccination regimens of 50 μl PBS or 1.5 × 10⁴ CFU of strain PBK001 at 2-week intervals (days −21, −35, and −49 prechallenge). Three weeks after the second boost (day 0), mice were challenged via aerosol with B. pseudomallei K96243 using a nebulizer concentration of ∼4.45 × 10⁷ CFU/ml. Briefly, the UTMB aerobiology facility utilized a Biaera AeroMP aerosol management platform housed within the IsoGARD class III glovebox, a nebulizer, a stainless steel dilution/delivery line, a rodent exposure chamber, a relative humidity/temperature transducer, and an impinger. Mice were placed into nose-only restraint tubes, transferred to stainless steel boxes, and loaded into rodent exposure chamber. Two groups of mice (n = 20) were exposed to bacteria via a three-jet nozzle collision nebulizer for 15 min. Nebulizers containing appropriate concentrations of B. pseudomallei K96243 in 10 ml of LB and samples collected from a SKC Biosampler containing 20 ml of LBG were diluted and plated to determine the presented dose (Dp). Mice received Dp of 1,070 and 1,780 CFU of B. pseudomallei K96243 from runs 1 and 2 (10 mice per run). Animal weight and survival were monitored for 27 days after aerosol challenge.

Serum samples were collected from individual mice before and 2 weeks after the last vaccination. Briefly, whole blood was collected via retro-orbital bleeding. The blood was stored in Microvette tubes without an anticoagulant and incubated at room temperature for 30 min to permit clotting before centrifugation. Mouse serum samples were irradiated, and sterility was verified by plating on LBG with FeSO₄. Irradiated serum from PBS- or PBK001-vaccinated C56BL/6 mice were used to determine B. pseudomallei-specific IgG and subclasses (IgG2a and IgG1) endpoint titers by indirect ELISA. The 96-well high binding microplates were coated with 10 μg/ml of irradiated B. pseudomallei K96243 in DPBS at 4°C overnight. The wells were washed twice with washing buffer (0.05% Tween 20 in 1× DPBS) and incubated with blocking buffer (0.1% Tween 20, 1% BSA, 1× DPBS) for 2 h at room temperature (RT). After blocking, plates were washed twice. Twofold dilutions of sera were made with sample diluent, added to triplicate wells, and then incubated at RT for 2 h. After the microplates were washed three times, goat anti-mouse IgG, IgG2a, or IgG1 antibody (Southern Biotech) (diluted 1:5,000) was added to each well and then incubated for 2 h. The plates were washed four times before the addition of tetramethylbenzidine (TMB) substrate solution (Invitrogen). After 15 min, 100 μl of stop solution (2 N H₂SO₄) was added, and the wells were read at 450 and 570 nm using a microplate reader (Biotek). The results were reported as the reciprocal of the highest titer giving an optical density (OD) reading of at least mean plus 2 SD of baseline sera. All assays were performed in triplicate, and results were shown as the mean reciprocal endpoint titer.

Spleens were collected from vaccinated mice 21 days after the last vaccination, and single-cell suspensions were isolated by passage through a 100-μm nylon cell strainer (BD Falcon) and then treated with 1× red blood cell (RBC) lysis buffer (Invitrogen). Splenocytes were seeded in 24-well plates at a concentration of 1.5 × 10⁶ cells/ml and restimulated with 2 mg/ml BSA (negative control), heat-killed B. pseudomallei K96243 WCL (1 × 10⁷ CFU/ml), or Dynabead mouse T-activator CD3/CD28 (positive control) (BD Bioscience) at 37°C and 5% CO₂ for 72 h. Cell culture supernatants were collected and analyzed for production of secreted IFN-γ, IL-17A, and TNF-α production using ELISA kits according to the manufacturer’s instruction (Invitrogen).

Female, 6- to 8-week-old C57BL/6 mice (n = 20; 5 mice/group) were vaccinated using a prime and two-boost regimen with strain PBK001 or PBS as described above. The depletion was performed 2 weeks after the last vaccination using rat IgG2b isotype control (LTF-2) (catalog no. BE0090), rat anti-mouse CD4 (GK1.5) (catalog no. BE0003-1), or rat anti-mouse CD8 (YTS 169.4) (catalog no. BE0117) purchased from BioXcell. Mice were administered with 500 μg of MAb intraperitoneally (i.p.) 3 days before infection and 250 μg on the day of infection. Depletion was maintained by further administration of 250 μg of MAb every 3 days postinfection. The mice were monitored for 16 days, and then their organs were collected and plated to evaluate bacterial burden. The peripheral blood (retro-orbital), lungs, and spleens were collected from a separate but matched group of mice (2 mice/group) to allow assessment of the protocol over the period of the depletion. The efficiency of depletion was confirmed by flow cytometry analysis 24 h after staining. Animals were monitored, weighed, and recorded until the end of experiment. Flow cytometry analysis was performed on 0.1-ml portions of blood samples transferred to Microvettes coated with lithium heparin. Peripheral blood cells or single-cell tissue suspensions were incubated with Fc block (catalog no. 553142; BD Bioscience) for 5 min to block non-antigen-specific binding of immunoglobulins. APC-conjugated anti-mouse CD3 (17A2), PE-conjugated anti-mouse CD4, and PerCP-Cyanine 5.5-conjugated anti-mouse CD8α purchased from eBioscience were used for surface marker analysis of CD3, CD4, and CD8 T cells, respectively. RBCs were lysed using RBC lysis buffer. Surface-stained samples were fixed with 2% ultrapure formaldehyde diluted in PBS for 48 h before flow cytometry analysis using a BD Fortessa LSR II flow cytometer, and results were analyzed using FCS Express 6 (Glendale, CA, USA).

The lungs, livers, and spleens from surviving mice were harvested for CFU enumeration (n = 7) and histopathology analysis (n = 3). Organs from seven mice were homogenized in PBS, serially diluted, and plated on LBG agar. The colonies were counted after 48 h of incubation at 37°C. Organs from three mice were collected, fixed in 10% formalin, embedded in paraffin, and then cross-sectioned before hematoxylin and eosin (H&E) staining. The stained tissue sections were examined in a blinded fashion by a pathologist.

Analyses were performed using GraphPad Prism7 software (La Jolla, CA). Survival differences were compared using Kaplan-Meier survival curves, followed by a log rank test. A nonparametric t test (Mann-Whitney test) was used to analyze the significant difference between two groups, while one-way analysis of variance (ANOVA) was used for multiple group comparison, followed by Dunn’s multiple means or Turkey’s comparison test.