Development of Melioidosis Subunit Vaccines Using an Enzymatically Inactive Burkholderia pseudomallei AhpC

Abstract

Burkholderia pseudomallei, the causative agent of melioidosis, is a facultative intracellular, Gram-negative pathogen that is highly infectious via the respiratory route and can cause severe, debilitating, and often fatal diseases in humans and animals. At present, no licensed vaccines for immunization against this CDC Tier 1 select agent exist. Studies in our lab have previously demonstrated that subunit vaccine formulations consisting of a B. pseudomallei capsular polysaccharide (CPS)-based glycoconjugate (CPS-CRM197) combined with hemolysin-coregulated protein (Hcp1) provided C57BL/6 mice with high-level protection against an acute inhalational challenge of B. pseudomallei. In this study, we evaluated the immunogenicity and protective capacity of B. pseudomallei alkyl hydroperoxide reductase subunit C (AhpC) in combination with CPS-CRM197. AhpC is a peroxiredoxin involved in oxidative stress reduction and is a potential protective antigen. To facilitate our studies and maximize safety in animals, recombinant B. pseudomallei AhpC harboring an active site mutation (AhpC^C57G) was expressed in Escherichia coli and purified using tandem nickel-cobalt affinity chromatography. Immunization of C57BL/6 mice with CPS-CRM197 combined with AhpC^C57G stimulated high-titer IgG responses against the CPS component of the glycoconjugate as well as stimulated high-titer IgG and robust interferon gamma (IFN-γ)-, interleukin-5 (IL-5)-, and IL-17-secreting T cell responses against AhpC^C57G. When challenged via an inhalational route with a high dose (~27 50% lethal doses [LD₅₀s]) of B. pseudomallei, 70% of the immunized mice survived 35 days postchallenge. Collectively, our findings demonstrate that AhpC^C57G is a potent activator of cellular and humoral immune responses and may be a promising candidate to include in future melioidosis subunit vaccines.

Keywords: Burkholderia pseudomallei; alkyl hydroperoxide reductase; capsular polysaccharide; glycoconjugate; melioidosis; subunit vaccine.

FIG 1

Physical and enzymatic analysis of B. pseudomallei AhpC and AhpC^C57G antigens. (a) Multiple-sequence alignment of B. pseudomallei (Bp), Burkholderia mallei (Bm), Burkholderia thailandensis (Bt), Burkholderia cepacia (Bc), Mycobacterium tuberculosis (Mt), Escherichia coli (Ec), Salmonella enterica serovar Typhimurium (St), and Helicobacter pylori (Hp) AhpC homologs. Conserved residues are highlighted in gray, while the N-terminal peroxidatic Cys (C_P) residue and C-terminal resolving Cys (C_R) residues are highlighted in black. (b) SDS-PAGE and SimplyBlue staining were used to assess the purity of the AhpC and AhpC^C57G preparations. Positions of the molecular weight standards (kilodaltons) are indicated on the left. (c) H₂O₂ reduction assays were used to assess the peroxidase activities associated with purified AhpC and AhpC^C57G antigens. Horseradish peroxidase (HRP) and hemolysin-coregulated protein 1 (Hcp1) were used as positive and negative controls, respectively. Bars represent the means ± standard deviation (SD) of results from individual assays conducted in duplicate. Values are representative of two independent experiments conducted on different days. *, P < 0.05.

FIG 2

Identification and characterization of T cell-restricted epitopes associated with AhpC^C57G. C57BL/6 mice (n = 12) were immunized on days 0, 21, and 35 with AhpC^C57G. Spleens (n = 4 per analyte) were harvested on day 42, and (a) IFN-γ-, (b) IL-5-, and (c) IL-17-secreting T cell responses against an AhpC^C57G peptide library (15-mers overlapping by 10 amino acids) were quantitated by ELISpot. Black dots represent the means of assays conducted in duplicate for individual mice. SFC, spot-forming cells. (d) C57BL/6 mice (n = 3) were immunized on days 0, 21, and 35 with AhpC^C57G. Spleens harvested on day 42 were combined and used to prepare three equal pools of cells. The pools were either depleted of CD8⁺ cells or CD4⁺ cells or left untreated, and IFN-γ-secreting T cell responses against peptide 2 and peptide 12 were quantitated by ELISpot. Bars represent the means ± SD from three individual experiments conducted in duplicate. *, P < 0.05.

FIG 3

Characterization of antibody responses raised against CPS-CRM197 and AhpC^C57G. C57BL/6 mice (n = 6 per group) were immunized on days 0, 21, and 35 with adjuvant only or CPS-CRM197 plus AhpC^C57G. Immune serum samples were collected on day 42. ELISAs were used to quantitate serum IgM, IgG, IgG1, IgG2b, and IgG2c titers against (a) CPS and (b) AhpC^C57G. Bars represent geometric means with 95% confidence interval (CI). Conjugate, CPS-CRM197; LOD, limit of detection. (c) Western immunoblot analysis of B. pseudomallei Bp82 and B. thailandensis E555 whole-cell lysates using pooled CPS-CRM197 plus AhpC^C57G immune serum. The positions of the molecular weight (MW) standards (kilodaltons) are indicated on the left.

FIG 4

Functional analysis of antibody responses raised against CPS-CRM197 and AhpC^C57G. C57BL/6 mice (n = 4 to 6 per group) were immunized on days 0, 21, and 35 with adjuvant only, AhpC^C57G, or CPS-CRM197 plus AhpC^C57G. Immune serum samples were collected on day 42. B. thailandensis E555 was incubated with medium only (no-serum control), pooled HI adjuvant-only immune serum, pooled HI AhpC^C57G-only immune serum, and pooled HI conjugate-plus-AhpC^C57G 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. Bars represent the means ± SD of results from three individual assays conducted in triplicate. Values are representative of three independent experiments conducted on different days. *, P < 0.05.

FIG 5

Characterization of cellular immune responses raised against AhpC^C57G. C57BL/6 mice (n = 4 per group) were immunized on days 0, 21, and 35 with CPS-CRM197 plus AhpC^C57G. Spleens were harvested on day 42, and (a) IFN-γ-, (b) IL-5-, and (c) IL-17-secreting T cell responses against AhpC^C57G, peptide 2, and peptide 12 were quantitated by ELISpot. White and black dots represent the means of assays conducted in duplicate for individual mice. Black bars represent geometric means for each group. *, (P < 0.05).

FIG 6

Protective capacity of CPS-CRM197 plus AhpC^C57G. C57BL/6 mice (n = 10 per group) were immunized on days 0, 21, and 35 with adjuvant only or CPS-CRM197 plus AhpC^C57G. Five weeks after the final boost, mice were challenged via an inhalational route with lethal doses of B. pseudomallei K96243. In the adjuvant-only group, 5 mice received 20 LD₅₀s and 5 mice received 29 LD₅₀s. In the conjugate-plus-AhpC^C57G group, 5 mice received 27 LD₅₀s and 5 mice received 28 LD₅₀s. Mice were then monitored for 35 days postchallenge, and their survival was plotted.