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The Use of Reverse Vaccinology in the Design and Construction of Nano-glycoconjugate Vaccines against Burkholderia pseudomallei .

Burkholderia pseudomallei (Bpm ) is a Gram negative, facultative intracellular pathogen that causes the disease melioidosis in humans and other mammals. Respiratory infection with B. pseudomallei leads to a fulminant and often fatal disease. It has previously been shown that glycoconjugate vaccines can provide significant protection against lethal challenge; however, the limited number of known Burkholderia antigens has slowed progress towards vaccine development. The objective of this study was to identify novel antigens and evaluate their protective capacity when incorporated into a nano-glycoconjugate vaccine platform. First, an in silico approach to identify antigens with strong predicted immunogenicity was developed. Protein candidates were screened and ranked according to predicted subcellular localization, transmembrane domains, adhesive properties and ability to interact with Major Histocompatibility Complex (MHC) I and II. From these in silico predictions, we identified seven "high priority" proteins that demonstrated seroreactivity with anti- Bpm murine sera and convalescent human melioidosis sera, providing validation of our methods. Two novel proteins, together with Hcp1, were linked to lipopolysaccharide (LPS) and incorporated onto the surface of a gold nanoparticle (AuNP). Animals receiving AuNP-glycoconjugate vaccines generated high protein- and polysaccharide-specific antibody titers. Importantly, immunized animals receiving the AuNP-FlgL-LPS alone or as a combo demonstrated up to 100% survival and reduced lung colonization following lethal challenge with Bpm. Together, this study provides a rational approach to vaccine design that can be adapted for other complex pathogens, and provides rationale for further pre-clinical testing of AuNP-glycoconjugate in animal models of infection.

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