Glycoengineered Outer Membrane Vesicles Displaying O-Polysaccharide Antigens Elicit Protection Against Francisella Tularensis

The O-polysaccharide antigen (O-antigen) component of lipopolysaccharide on the surface of Gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-antigens often confer protection against infection; hence, O-antigen conjugate vaccines have been designed against multiple pathogens. However, conventional methods for natural extraction or chemical synthesis of O-antigens are technically demanding, inefficient, and expensive. Furthermore, because carbohydrates are weak stimulators of the adaptive immune system, current carbohydrate-based vaccines require significant downstream processing to increase their immunogenicity.  Here, we describe a scalable glycoengineering technique where recombinant O-antigen biosynthesis is coordinated with vesiculation in lab strains of Escherichia coli to produce acellular outer membrane vesicles (OMVs) decorated with pathogen-specific O-antigens on their surfaces. Because genes required for the biosynthesis of a given O-antigen are usually located in a distinct cluster, they can be easily amplified and transferred into E. coli for efficient, large-scale production. Moreover, the use of OMVs greatly simplifies glycoconjugate assembly and purification and results in a self-adjuvanting O-antigen delivery system. Immunization of mice with engineered OMVs displaying O-antigens from Francisella tularensis subsp. tularensis Schu S4 protected against lethal challenge with F. tularensis subsp. tularensis Schu S4, demonstrating the vaccination potential of our glycoconjugate vesicles.