Development of Subunit Vaccines That Provide High-Level Protection and Sterilizing Immunity against Acute Inhalational Melioidosis

Abstract

Burkholderia pseudomallei, the etiologic agent of melioidosis, causes severe disease in humans and animals. Diagnosis and treatment of melioidosis can be challenging, and no licensed vaccines currently exist. Several studies have shown that this pathogen expresses a variety of structurally conserved protective antigens that include cell surface polysaccharides and cell-associated and cell-secreted proteins. Based on those findings, such antigens have become important components of the subunit vaccine candidates that we are currently developing. In the present study, the 6-deoxyheptan capsular polysaccharide (CPS) from B. pseudomallei was purified, chemically activated, and covalently linked to recombinant CRM197 diphtheria toxin mutant (CRM197) to produce CPS-CRM197. Additionally, tandem nickel-cobalt affinity chromatography was used to prepare highly purified recombinant B. pseudomallei Hcp1 and TssM proteins. Immunization of C57BL/6 mice with CPS-CRM197 produced high-titer IgG and opsonizing antibody responses against the CPS component of the glycoconjugate, while immunization with Hcp1 and TssM produced high-titer IgG and robust gamma interferon-secreting T cell responses against the proteins. Extending upon these studies, we found that when mice were vaccinated with a combination of CPS-CRM197 and Hcp1, 100% of the mice survived a lethal inhalational challenge with B. pseudomallei Remarkably, 70% of the survivors had no culturable bacteria in their lungs, livers, or spleens, indicating that the vaccine formulation had generated sterilizing immune responses. Collectively, these studies help to better establish surrogates of antigen-induced immunity against B. pseudomallei as well as provide valuable insights toward the development of a safe, affordable, and effective melioidosis vaccine.

Keywords: Burkholderia pseudomallei; Hcp1; capsule; glycoconjugate; immunity; inhalation; melioidosis; mouse; protection; vaccines.

FIG 1

Synthesis and physical analysis of CPS-CRM197. (a) Basic conjugation strategy used to couple purified B. pseudomallei CPS to recombinant CRM197. (b) SDS-PAGE and Coomassie blue staining were used to assess the covalent linkage of CPS to CRM197 in PBS or BB. Samples were drawn from the reaction mixtures on days 0, 1, 3, 7, and 10. Day 0 represents unconjugated controls. All lanes were loaded with equal amounts of protein to facilitate direct comparisons. The positions of the protein molecular weight standards (molecular weights × 10³) are indicated on the left.

FIG 2

Characterization of antibody titers raised against CPS-CRM197, Hcp1, and TssM. C57BL/6 mice (n = 6 per group) were immunized on days 0, 21, and 35 with adjuvant only (Alhydrogel/CpG), conjugate only, Hcp1 only, TssM only, conjugate plus Hcp1, or conjugate plus TssM. Immune serum samples were collected for testing on day 42. ELISAs were used to quantitate serum IgM, IgG, IgG1, and IgG2b titers against (a) CPS, (b) Hcp1, and (c) TssM. Bars represent geometric means with 95% confidence intervals. “Conjugate” is CPS-CRM197; LOD, limit of detection.

FIG 3

Functional analysis of antibody responses raised against CPS-CRM197, Hcp1, and TssM. C57BL/6 mice (n = 6 per group) were immunized on days 0, 21, and 35 with adjuvant only (Alhydrogel/CpG), conjugate only, Hcp1 only, TssM only, conjugate plus Hcp1, and conjugate plus TssM. Immune serum samples were collected on day 42. B. pseudomallei K96243 was incubated with (a) medium only (no-serum control), pooled HI adjuvant (Alhydrogel/CpG)-only immune serum, pooled HI conjugate-only immune serum, pooled HI Hcp1-only immune serum, pooled HI conjugate plus Hcp1-immune serum, or (b) medium only (no-serum control), pooled HI adjuvant (Alhydrogel/CpG)-only immune serum, pooled HI conjugate-only immune serum, pooled HI TssM-only immune serum, or pooled HI conjugate plus TssM immune serum. Following incubation for 1 h, opsonized bacteria were added to RAW 264.7 murine macrophage monolayers. Uptake was quantitated at 3 h postinfection. Reported values represent mean results ± standard deviations for three individual assays conducted in triplicate. Figures are representative of at least three independent experiments conducted on different days. ns, not significant; *, P < 0.05.

FIG 4

Characterization of cellular immune responses raised against CPS-CRM197, Hcp1, and TssM. C57BL/6 mice (n = 4 per group) were immunized on days 0, 21, and 35 with adjuvant only (Alhydrogel/CpG), conjugate only, Hcp1 only, TssM only, conjugate plus Hcp1, or conjugate plus TssM. Spleens were harvested on day 42, and IFN-γ-secreting T cell responses against (a) CPS-CRM197, (b) Hcp1, and (c) TssM were quantitated in an ELISpot assay. Black dots represent the mean results of assays conducted in duplicate for individual mice. Black bars represent geometric means for a group. ns, not significant; *, P < 0.05.

FIG 5

Protective capacities of the subunit vaccine formulations tested in this study. C57BL/6 mice (n = 9 to 10 mice per group) were immunized on days 0, 21, and 35 with adjuvant only (Alhydrogel/CpG), conjugate only, Hcp1 only, TssM only, conjugate plus Hcp1, or conjugate plus TssM. Five weeks after the final boost, mice were challenged via the inhalational route with ∼10 LD₅₀ of B. pseudomallei K96243. (a) Mice were monitored for 35 days postchallenge, and their survival was plotted. (b) Significance for survival was determined using a log rank (Mantel-Cox) test. (c) At the end of the study, survivors were culled (n = 10 for conjugate plus Hcp1; n = 8 for conjugate plus TssM; n = 6 for conjugate only), organs were removed, and bacterial loads were determined. Individual mice were designated according to the numbers next to the colored dots.

FIG 6

Histopathological analysis of mouse tissues following a lethal inhalational challenge with B. pseudomallei. Following termination of the challenge study, lungs, livers, and spleens were harvested from five mice (numbers 1 to 5) of the 10 survivors that had been immunized with CPS-CRM197 plus Hcp1. The tissues were fixed and stained with H&E. Images are representative of all 5 mice. Original magnification, ×400.