Development of capsular polysaccharide-based glycoconjugates for immunization against melioidosis and glanders

Abstract

Burkholderia pseudomallei and Burkholderia mallei, the etiologic agents of melioidosis and glanders, respectively, cause severe disease in humans and animals and are considered potential agents of biological warfare and terrorism. Diagnosis and treatment of infections caused by these pathogens can be challenging and, in the absence of chemotherapeutic intervention, acute disease is frequently fatal. At present, there are no human or veterinary vaccines available for immunization against these emerging/re-emerging infectious diseases. One of the long term objectives of our research, therefore, is to identify and characterize protective antigens expressed by B. pseudomallei and B. mallei and use them to develop efficacious vaccine candidates. Previous studies have demonstrated that the 6-deoxy-heptan capsular polysaccharide (CPS) expressed by these bacterial pathogens is both a virulence determinant and a protective antigen. Consequently, this carbohydrate moiety has become an important component of the various subunit vaccines that we are currently developing in our laboratory. In the present study, we describe a reliable method for isolating CPS antigens from O-polysaccharide (OPS) deficient strains of B. pseudomallei; including a derivative of the select agent excluded strain Bp82. Utilizing these purified CPS samples, we also describe a simple procedure for covalently linking these T-cell independent antigens to carrier proteins. In addition, we demonstrate that high titer IgG responses can be raised against the CPS component of such constructs. Collectively, these approaches provide a tangible starting point for the development of novel CPS-based glycoconjugates for immunization against melioidosis and glanders.

Keywords: Burkholderia mallei; Burkholderia pseudomallei; capsular polysaccharide; glycoconjugate; immunization; vaccine.

Figure 1

Elution profile of column purified B. pseudomallei CPS. Acid hydrolyzed BP2683 CPS was loaded onto a Sephadex G-50 column and eluted with PBS. Carbohydrate positive fractions eluting near the column void volumes (V_o) were collected and pooled for further use.

Figure 2

Structural analysis of B. pseudomallei CPS. (A) ¹H NMR and (B) ¹³C NMR spectra were obtained for column purified BP2683 CPS using a Varian Inova-500 MHz spectrometer. For reference purposes, the chemical structure of the monosaccharide making up the CPS homopolymer is inset in panel A. dH, 6-deoxy-heptose; M, mannose; OAc, O-acetyl.

Figure 3

Generalized scheme for the conjugation of activated B. pseudomallei CPS to carrier proteins.

Figure 4

Physical analysis of B. pseudomallei CPS-cBSA glycoconjugates. (A) SDS-PAGE and Coomassie Blue staining was used to confirm the covalent linkage of BP2683 CPS to cBSA. The positions of protein molecular size standards are indicated on the left. (B) Western immunobloting was also used to assess the antigenicity of the chemically activated/coupled BP2683 CPS. The CPS + cBSA lane represents unconjugated controls. CPS was detected using the 3C5 mAb. Lanes were loaded with similar amounts of protein or carbohydrate to facilitate direct comparisons.

Figure 5

Characterization of murine immune responses against the CPS2B1 conjugate. (A) ELISA was used to determine the total serum IgG response of mice immunized with CPS2B1 or the unconjugated controls. Individual symbols represent a single mouse while black bars indicate geometric means. ^*, P < 0.05. (B) Western immunoblotting was used to assess the reactivity of pooled anti-CPS2B1 immune serum (n = 6) with B. pseudomallei 1026b (Bp) and B. mallei ATCC 23344 (Bm) whole cell lysates. The positions of protein molecular size standards are indicated on the left.

Figure 6

Opsonophagocytic uptake of B. mallei by RAW 264.7 murine macrophages. Bacterial uptake in the presence of DMEM alone (no serum), pooled HI anti-cBSA immune serum (cBSA; n = 6) or pooled HI anti-CPS2B1 immune serum (CPS2B1; n = 6) was quantitated at 3 h post-infection. Values represent the means ± SD of three independent experiments. ^*, P < 0.05.