Development of Immunoassays for Burkholderia pseudomallei Typical and Atypical Lipopolysaccharide Strain Typing.

Teerapat Nualnoi,Michael H Norris,Christopher J Allender,David P Aucoin,Apichai Tuanyok,Paul S Keim,Erik W Settles,Paul J Brett,Mary N Burtnick

doi:10.4269/ajtmh.16-0308

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection endemic to many tropical regions. Lipopolysaccharide (LPS) is recognized as an important virulence factor used by B. pseudomallei. Isolates of B. pseudomallei have been shown to express one of four different types of LPS (typical LPS, atypical LPS types B and B2, and rough LPS) and in vitro studies have demonstrated that LPS types may impact disease severity. The association between LPS types and clinical manifestations, however, is still unknown, in part because an effective method for LPS type identification is not available. Thus, we developed antigen capture immunoassays capable of distinguishing between the LPS types. Mice were injected with B or B2 LPS for atypical LPS–specific monoclonal antibody (mAb) isolation; only two mAbs (3A2 and 5B4) were isolated from mice immunized with B2 LPS. Immunoblot analysis and surface plasmon resonance demonstrated that 3A2 and 5B4 are reactive with both B2 and B LPS where 3A2 was shown to possess higher affinity. Assays were then developed using capsular polysaccharide–specific mAb 4C4 for bacterial capture and 4C7 (previously shown to bind typical LPS) or 3A2 mAbs for typical or atypical LPS strain detection, respectively. The evaluations performed with 197 strains of Burkholderia and non-Burkholderia species showed that the assays are reactive to B. pseudomallei and Burkholderia mallei strains and have an accuracy of 98.8% (zero false positives and two false negatives) for LPS typing. The results suggest that the assays are effective and applicable for B. pseudomallei LPS typing.

Highlights

Immunoblots of LPS from various strains of Burkholderia species probed with monoclonal antibody (mAb) 4C7 demonstrated that the antibody was reactive to typical B. pseudomallei LPS type A, with no cross-reactivity to atypical LPS type B or B2 (Figure 1A)
The results suggest that the capsular polysaccharide (CPS) antigen recognized by mAb 4C4 is highly conserved in B. pseudomallei species; these results correspond with our previous studies performed on a large bacterial panel using a different CPS-specific mAb.[33]
The antigen capture immunoassay could be adapted to the lateral flow immunoassay (LFI) format. This would provide rapid LPS typing and may provide clinicians important information if it is discovered that variable LPS types correspond to changes in virulence. This is the first development of immunoassays for B. pseudomallei typical versus atypical strain typing using mAbs specific to typical and atypical LPS

Summary

Introduction

Burkholderia pseudomallei is a Gram-negative saprophytic bacillus that is the causative agent of melioidosis, which is a life-threatening infectious disease prominent in southeast Asia and northern Australia.[1,2] In the past decade, the number of melioidosis cases reported from other geographic locations such as India, China, and Brazil have increased, indicating that melioidosis is becoming a global problem.[2,3,4,5] Due to its ability to cause a severe infection that may be transmitted by aerosol, B. pseudomallei has been recognized as a potential bioterrorism agent and has been classified as a Tier 1 select agent by the Centers for Disease Control and Prevention.[6,7] Infection with B. pseudomallei results in high mortality rates that can be as high as 45%, even when medical interventions are provided.[8]. Without appropriate antibiotic administration, the mortality rate could be as high as 90%.9 The absence of a licensed vaccine for prevention of melioidosis further impedes public health success.[10]

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