Evaluation of two different vaccine platforms for immunization against melioidosis and glanders

Abstract

Burkholderia pseudomallei and the closely related species, Burkholderia mallei, produce similar multifaceted diseases which range from rapidly fatal to protracted and chronic, and are a major cause of mortality in endemic regions. Besides causing natural infections, both microbes are Tier 1 potential biothreat agents. Antibiotic treatment is prolonged with variable results, hence effective vaccines are urgently needed. The purpose of our studies was to compare candidate vaccines that target both melioidosis and glanders to identify the most efficacious one(s) and define residual requirements for their transition to the non-human primate aerosol model. Studies were conducted in the C57BL/6 mouse model to evaluate the humoral and cell-mediated immune response and protective efficacy of three Burkholderia vaccine candidates against lethal aerosol challenges with B. pseudomallei K96243, B. pseudomallei MSHR5855, and B. mallei FMH. The recombinant vaccines generated significant immune responses to the vaccine antigens, and the live attenuated vaccine generated a greater immune response to OPS and the whole bacterial cells. Regardless of the candidate vaccine evaluated, the protection of mice was associated with a dampened cytokine response within the lungs after exposure to aerosolized bacteria. Despite being delivered by two different platforms and generating distinct immune responses, two experimental vaccines, a capsule conjugate + Hcp1 subunit vaccine and the live B. pseudomallei 668 ΔilvI strain, provided significant protection and were down-selected for further investigation and advanced development.

Keywords: Burkholderia mallei; Burkholderia pseudomallei; aerosol; cellular immunity; humoral immunity; immune response; mice; vaccine.

Figure 1

Overview of the immunization and challenge strategy for direct comparison of Burkholderia vaccine candidates that were challenged with Bp K96243. The numbers with the degree sign (°) denote vaccine prime and consecutive boost(s). The experiments that included mice challenged with Bm FMH or Bp MSHR5855 followed a similar timeline.

Figure 2

Survival curves of vaccinated C57BL/6 mice challenged with Bp K96243. Mice (n = 10/group) were exposed to 3.4 LD₅₀ (1.35 × 10³ CFU) by the aerosol route and monitored for 60 days post challenge. The PBS control represented truly naïve mice and the adjuvant control confirmed that protection was not afforded to the mice due to non-specific immune stimulation associated with alhydrogel and CpG injections.

Figure 3

The recovery of bacteria as determined by CFU from blood, spleens and lungs of C57BL/6 mice 3 days post-challenge with Bp K96243. The samples were from six mice/group. The left axis represents CFU/ml (blood) or CFU/g (lung and spleen). The individual points represent one animal, and the baseline points indicate the remaining survivors with no detectable CFU. The diamond-shaped symbol and vertical line are the geometric mean and standard deviation, respectively. The limits of detection were 100 CFU/ml blood and 5 CFU/g spleen or lung homogenate.

Figure 4

Cytokine analysis of lung homogenates from vaccinated mice collected 3 days after challenge with a 3.4 LD₅₀ dose of Bp K96243. Cytokine levels are depicted as pg/ml homogenate supernatant.

Figure 5

(A) Survival curves of vaccinated and control C57BL/6 mice (n = 20/group) challenged with Bm FMH by the aerosol route. Mice were exposed to 4 LD₅₀ or 11 LD₅₀ in two separate aerosol exposures of ten mice per group. (B) Survival curves of vaccinated and control C57BL/6 mice (n = 20/group) challenged with Bp MSHR5855 by the aerosol route. Mice were exposed to inhaled doses of 11 LD₅₀ or 12 LD₅₀ in two separate aerosol exposures of ten mice per group.

Figure 6

(A) The recovery of bacteria as determined by CFU counts from blood, spleens and lungs of C57BL/6 mice 3 days post-challenge with Bm FMH. The combined results for both challenges are shown (n = 10–11 mice/vaccine group). (B) The recovery of bacteria as determined by CFU counts from blood, spleens and lungs of C57BL/6 mice 3 days post-challenge with Bp MSHR5855. The combined results for both challenges are shown (n = 12 mice/group). The left axis represents CFU/ml (Blood) or CFU/g (Lung and Spleen). The individual points represent one animal, and the baseline points indicate the remaining survivors with no detectable CFU. The diamond-shaped symbol and vertical line are the GM and GSD, respectively. The limits of detection were ~10 CFU/ml (blood) and 5 CFU/g (organs).