Correlates of immune protection following cutaneous immunization with an attenuated Burkholderia pseudomallei vaccine

Abstract

Infections with the Gram-negative bacterium Burkholderia pseudomallei (melioidosis) are associated with high mortality, and there is currently no approved vaccine to prevent the development of melioidosis in humans. Infected patients also do not develop protective immunity to reinfection, and some individuals will develop chronic, subclinical infections with B. pseudomallei. At present, our understanding of what constitutes effective protective immunity against B. pseudomallei infection remains incomplete. Therefore, we conducted a study to elucidate immune correlates of vaccine-induced protective immunity against acute B. pseudomallei infection. BALB/c and C57BL/6 mice were immunized subcutaneously with a highly attenuated, Select Agent-excluded purM deletion mutant of B. pseudomallei (strain Bp82) and then subjected to intranasal challenge with virulent B. pseudomallei strain 1026b. Immunization with Bp82 generated significant protection from challenge with B. pseudomallei, and protection was associated with a significant reduction in bacterial burden in lungs, liver, and spleen of immunized mice. Humoral immunity was critically important for vaccine-induced protection, as mice lacking B cells were not protected by immunization and serum from Bp82-vaccinated mice could transfer partial protection to nonvaccinated animals. In contrast, vaccine-induced protective immunity was found to be independent of both CD4 and CD8 T cells. Tracking studies demonstrated uptake of the Bp82 vaccine strain predominately by neutrophils in vaccine-draining lymph nodes and by smaller numbers of dendritic cells (DC) and monocytes. We concluded that protection following cutaneous immunization with a live attenuated Burkholderia vaccine strain was dependent primarily on generation of effective humoral immune responses.

Fig 1

Subcutaneous immunization with Bp82 protects mice from an acutely lethal B. pseudomallei infection. C57BL/6 and BALB/c mice (n = 5) were immunized twice 10 days apart with 5 × 10⁶ CFU of Bp82 suspended in PBS. All animals were then challenged intranasally with ∼5 LD₅₀ (1.2 × 10⁴ [C57BL/6] or 5 × 10³ [BALB/c] CFU/mouse) of B. pseudomallei strain 1026b. Animals were euthanized upon reaching a predetermined euthanasia endpoint. Kaplan-Meier survival curves were generated for 60 days of survival for C57BL/6 mice (A) and BALB/c mice (B). Statistical differences in survival were determined by a log rank test. Data are pooled from two combined experiments.

Fig 2

Immunization reduces bacterial burden in vaccinated mice at 72 h after challenge. Bacterial burden in lung, spleen, and liver tissues was determined in naive and vaccinated mice (n = 5) 72 h after intranasal challenge with B. pseudomallei strain 1026b. Statistical comparisons between vaccinated and naive groups were done using a nonparametric t test. *, P < 0.05. Similar results were obtained in one additional experiment.

Fig 3

Humoral immune responses in Bp82-vaccinated mice. Serum was prepared from BALB/c and C57BL/6 mice (n = 5) vaccinated twice s.c. with Bp82, and titers of antibodies to heat-killed Bp82 were determined by endpoint dilution ELISA. Titers of total IgG (A), IgM (B), IgG1 (C), and IgG2a (D) were expressed as the reciprocal of the endpoint dilution. Significant differences (*, P < 0.05; **, P < 0.005; ***, P < 0.0001) were determined by a nonparametric Kruskal-Wallis test followed by Dunnett's multiple comparison test. Data represent pooled data from two independent experiments.

Fig 4

Cellular immune responses to Bp82 immunization. BALB/c mice (n = 5) were immunized twice s.c. with Bp82. Fourteen days after the second immunization, spleen cells from naive or vaccinated animals were restimulated in vitro with heat-killed Bp82 (2 × 10⁷ CFU/ml), lysate from Bp82 (5 μg/ml), recombinant GroEL (12.5 μg/ml), or medium alone for 3 days. Supernatants were assayed by ELISA for IFN-γ (A) and IL-17 (B) production. *, P < 0.05 using a Mann-Whitney U test. Data represent pooled data from two independent experiments.

Fig 5

Role of antibody in Bp82 vaccine protection. (A) Serum from immunized and naive animals was transferred to naive BALB/c recipient animals (n = 5 per group), which were then subjected to lethal i.n. challenge with B. pseudomallei 24 h later. Survival percentages were significantly increased in the animals that received immune serum compared to the animals that received nonimmune serum. (B) uMT⁻/⁻ and WT C57BL/6 mice (n = 5) were immunized twice s.c. with Bp82 and then challenged intranasally with B. pseudomallei 1026b, and survival times were determined. Survival was significantly reduced in uMT⁻/⁻ mice compared to WT animals. Data represent pooled data from two independent experiments.

Fig 6

Role of T cells in vaccine-induced protection against B. pseudomallei. CD8^−/− (A), CD4^−/− (B), and TCRα/β^−/− (C) and WT C57BL/6 mice were immunized twice with Bp82. Naive and immunized animals were then challenged intranasally with 1.2 × 10⁴ CFU of B. pseudomallei 1026b, and survival percentages were determined. Survival percentages for CD8^−/−, CD4^−/−, and TCRα/β^−/− mice compared to WT mice were not significantly different. Survival differences were determined using Kaplan-Meier survival curves and log rank analysis. Data are representative of two independent experiments.

Fig 7

Lymph node cellular response to Bp82 vaccination. BALB/c mice (n = 5 per group) were injected in one rear limb footpad with 1.2 × 10⁸ CFU of Bp82-gfp. Ten hours after inoculation, cells from the ipsilateral popliteal LN were collected and immunostained. Cells from the contralateral popliteal LN served as the control. (A) The appearance of GFP⁺ CD11b⁺ cells in the draining LN was determined by flow cytometry at 10 h after injection. (B) The GFP⁺ cells were further subdivided into polymorphonuclear leukocytes (PMN) (CD11b⁺ Ly6G⁺ Ly6C⁻) and monocytes (CD11b⁺ Ly6G⁻ Ly6C⁺). (C) The distribution of Bp82-gfp bacteria in relevant LN APC populations, including neutrophils (PMN), dendritic cells (DC), CD169⁺ subcapsular macrophages, and monocytes, was calculated. Data are representative of two independent experiments. Statistical differences between Bp82-injected and uninjected contralateral LN were determined by Student's two-tailed t test. **, P < 0.05. For comparisons between 4 groups (C), ANOVA was used, followed by the Tukey post hoc test.