Use of Reverse Vaccinology in the Design and Construction of Nanoglycoconjugate Vaccines against Burkholderia pseudomallei.

is a Gram-negative, facultative intracellular pathogen that causes the disease melioidosis in humans and other mammals. Respiratory infection with 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 antigens has slowed progress toward vaccine development. The objective of this study was to identify novel antigens and evaluate their protective capacity when incorporated into a nanoglycoconjugate vaccine platform. First, an 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) class I and class II. From these predictions, we identified seven "high priority" proteins that demonstrated seroreactivity with anti- 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 with 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 combination demonstrated up to 100% survival and reduced lung colonization following a lethal challenge with Together, this study provides a rational approach to vaccine design that can be adapted for other complex pathogens and provides a rationale for further preclinical testing of AuNP glycoconjugate in animal models of infection.