Double-hit approach for novel glycoconjugates combining cytoplasmic glycoengineering and selective chemistry

Chemical conjugation to carrier proteins has been traditionally used to improve polysaccharides immunogenicity and to overcome the limitations of T-independent antigens, including lack of immunological memory and efficacy in infants. A double-hit approach, meaning that both polysaccharide and carrier protein belong to the same pathogen, may be particularly useful for targeting bacterial species with large glycan variability. Recently, bacterial protein glycosylation has been exploited to obtain glycosylated proteins in E. coli cytoplasm. In our work we have combined cytoplasmic glycoengineering and chemical conjugation for the development of novel selective glycoconjugates, with the aim to preserve the immunogenicity of the protein chosen as carrier. The potential protective protein MrkA, the major component of Klebsiella pneumoniae type 3 fimbriae, was successfully modified with a lactose moiety in E. coli. K. pneumoniae K2 K-antigen and O1v1 O-antigen were then covalently linked to MrkA at the level of this unique sugar handle and tested in vivo. Immune response against MrkA and sugars was evaluated in animal models. This work contributes to expand the application of the glycoengineering technology for the development of effective glycoconjugate vaccines.