Identification and Characterization of Novel Melioidosis Vaccine Candidates
Burkholderia pseudomallei, the causative agent of melioidosis, is a CDC Tier 1 select agent. This facultative intracellular, Gram-negative pathogen 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 melioidosis exist. We have previously demonstrated that a co-formulation of B. pseudomallei hemolysin co-regulated protein 1 (Hcp1) with a capsular polysaccharide-based glycoconjugate (CPS-CRM197) could be used to immunize C57BL/6 mice against a lethal inhalation challenge with B. pseudomallei. Additionally, we showed that 100% of the vaccinated mice survived for 35 days post-challenge and that 70% of those mice exhibited sterilizing immunity. The aim of this study was to identify and characterize potential vaccine candidates to add to our existing glycoconjugate vaccine. In our first approach, through a review of the literature we identified AhpC as a potential protective antigen. AhpC is a constitutively expressed peroxiredoxin involved in oxidative stress reduction. We first inactivated AhpC by introducing a glycine substitution at Cys57 (AhpCC57G), which is the peroxidatic cysteine residue critical for AhpC activity. We immunized C57BL/6 mice with AhpCC57G and found AhpCC57G stimulated high-titer IgG and robust IFN-γ, IL-5, and IL-17 secreting T cell responses. When we performed a high dose lethal inhalational challenge with B. pseudomallei, 70% of mice vaccinated with AhpCC57G co-formulated with CPS-CRM197 survived 35 days post-challenge. Additionally, we tested AhpCC57G in combination with CPS-CRM197 and Hcp1 in a trivalent formulation. In an inhalational challenge with B. pseudomallei, 100% of mice vaccinated with the trivalent formulation survived 43 days post-challenge.In our second approach, we utilized an in vitro culture system that promotes the expression of proteins that, similar to Hcp1, are upregulated when B. pseudomallei is grown in media devoid of iron. Using a select agent excluded B. pseudomallei strain that lacks both O-polysaccharide and CPS expression, bacterial cell pellets grown under iron rich and limited conditions were exposed to sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin) and then lysed to facilitate the affinity capture of biotinylated surface antigens. As expected, analysis of the antigens by SDS-PAGE indicated that only a subset of the B. pseudomallei proteome had been isolated, and that some of these antigens were differentially expressed. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) data dependent analysis (DDA) we identified an initial 246 proteins expressed under iron limiting conditions for use as potential melioidosis vaccine candidates. Evaluation of a select few of these proteins identified antigens BopE, BipD, and GltI that were seroreactive with patient immune serum. Further investigation of BopE, determined that BopE stimulated strong humoral and cellular immune responses in combination with CPS-CRM197 but provided poor protection against a lethal inhalational challenge with B. pseudomallei. Additionally, utilizing LC-MS/MS data independent analysis (DIA) we identified a total of 159 proteins significantly upregulated in an iron-limited environment. We used bioinformatic and immunoinformatic approaches to down-select our list of 159 antigens to 45 promising potential vaccine candidates with desirable physiochemical and immunogenic properties.
AhpC, Burkholderia pseudomallei, Iron, Melioidosis, Surface Proteins, Vaccine
Networked Digital Library of Theses and Dissertations