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. 2011 Oct 26;29(46):8381-9.
doi: 10.1016/j.vaccine.2011年08月05日8. Epub 2011 Aug 24.

A naturally derived outer-membrane vesicle vaccine protects against lethal pulmonary Burkholderia pseudomallei infection

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A naturally derived outer-membrane vesicle vaccine protects against lethal pulmonary Burkholderia pseudomallei infection

Wildaliz Nieves et al. Vaccine. .

Abstract

Burkholderia pseudomallei, and other members of the Burkholderia, are among the most antibiotic-resistant bacterial species encountered in human infection. Mortality rates associated with severe B. pseudomallei infection approach 50% despite therapeutic treatment. A protective vaccine against B. pseudomallei would dramatically reduce morbidity and mortality in endemic areas and provide a safeguard for the U.S. and other countries against biological attack with this organism. In this study, we investigated the immunogenicity and protective efficacy of B. pseudomallei-derived outer membrane vesicles (OMVs). Vesicles are produced by Gram-negative and Gram-positive bacteria and contain many of the bacterial products recognized by the host immune system during infection. We demonstrate that subcutaneous (SC) immunization with OMVs provides significant protection against an otherwise lethal B. pseudomallei aerosol challenge in BALB/c mice. Mice immunized with B. pseudomallei OMVs displayed OMV-specific serum antibody and T-cell memory responses. Furthermore, OMV-mediated immunity appears species-specific as cross-reactive antibody and T cells were not generated in mice immunized with Escherichia coli-derived OMVs. These results provide the first compelling evidence that OMVs represent a non-living vaccine formulation that is able to produce protective humoral and cellular immunity against an aerosolized intracellular bacterium. This vaccine platform constitutes a safe and inexpensive immunization strategy against B. pseudomallei that can be exploited for other intracellular respiratory pathogens, including other Burkholderia and bacteria capable of establishing persistent infection.

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Figures

Figure 1
Figure 1. Characterization of B. pseudomallei OMVs
(A) Cryo-Transmission electron micrograph of B. pseudomallei OMVs. Purified OMVs (0.8 mg/ml) were diluted 1:10 in filtered sterile water for imaging. Image was taken using a JEOL 2010 Transmission Electron Microscope. Bar indicates 100 nm. (B) Western blot demonstrating the presence of capsular polysaccharide (CPS) in B. pseudomallei OMVs. Ten μg of two separate vaccine batches of Bp OMVs (1 and 2) were probed with monoclonal antibody 3C5 specific for B. pseudomallei CPS [33]. B. thailandensis (Bth), which lacks capsule, and B. pseudomallei 1026b (Bp) whole-cell lysates were used as negative and positive controls, respectively.
Figure 2
Figure 2. Serum IgG responses to B. pseudomallei OMVs are specific and do not require exogenous adjuvant
Mean reciprocal endpoint titers for B. pseudomallei OMV-specific serum IgG are shown for pre-immune sera, and sera obtained 3 weeks after two (1st boost) and three (2nd boost) administrations of 2.5 μg of B. pseudomallei or E. coli OMVs without exogenous adjuvant. Treatment groups (n=5 mice per group) are naïve = non-treated; Ec IN=E. coli OMV-immunized intranasally; B. pseudomallei IN=B. pseudomallei OMV-immunized intranasally; and B. pseudomallei SC=B. pseudomallei OMV-immunized subcutaneously. Asterisks indicate statistical difference of final endpoint titers compared to pre-immune titers within groups (*P < 0.05, ***P < 0.001 using a two-way ANOVA with Bonferroni’s post-test).
Figure 3
Figure 3. SC immunization with B. pseudomallei OMVs protects mice against lethal aerosol challenge
Mice (n=15 per group) were challenged with 5 LD50 of B. pseudomallei 1026b by small particle aerosol. Composite survival data from two independent experiments is shown through day 14. Mice immunized SC with B. pseudomallei OMVs were significantly protected (P < 0.001 using a log-rank Mantel-Cox survival analysis).
Figure 4
Figure 4. B. pseudomallei OMV immunization induces humoral immunity
B. pseudomallei OMV-specific serum IgG (A) and IgA (B) and E. coli OMV specific serum IgG (C) and IgA (D) were measured by ELISA. Microtiter plates were coated with 500 ng/well of purified B. pseudomallei OMVs or E. coli OMVs. Naïve = non-treated; Ec IN=E. coli OMV-immunized intranasally; Bp IN=B. pseudomallei OMV-immunized intranasally; and Bp SC=B. pseudomallei OMV-immunized subcutaneously. Horizontal line represents the median value for each group (n = 5). (*P < 0.05, **P < 0.01, ***P < 0.001 using a one-way ANOVA with Bonferroni’s post-test).
Figure 5
Figure 5. B. pseudomallei OMV immunization induces T cell memory responses
(A) Splenocytes from individual mice in each group (n = 3) were restimulated in triplicate with B. pseudomallei OMVs (2 μg) or ConA (1 μg, not shown) or left unstimulated, and cell culture supernatants were assayed in duplicate on day 3 for IFN-γ cytokine production. (**P < 0.01, ***P < 0.001 using a two-way ANOVA with Bonferroni’s post-test).

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