In previous studies protective
antibodies that could facilitate bactericidal killing of Neisseria meningitidis (Nm) serogroup B strains were derived from immunisation with
glycoconjugates prepared from O-deacylated
lipopolysaccharide (LPS-
OH) via direct reductive amination between the reducing end of the
oligosaccharide molecule, created by treatment with
alkaline phosphatase, and amino functionalities on the
CRM(197)
carrier protein. These
glycoconjugates proved difficult to prepare because the presence of
amide linked fatty-acyl groups results in
glycolipids that are relatively insoluble and aggregate. Therefore, we have examined several strategies to prepare
glycoconjugates in order to identify a robust, consistently reproducible strategy that produces
glycoconjugates with a high loading of LPS derived
oligosaccharides. Initially we used completely deacylated LPS molecules, but lacking
phosphoethanolamine (PEtn) from the core OS as the strong basic conditions required to completely deacylate the LPS would modify the PEtn residue. We utilised a squarate linker and conjugated via the reducing end of the
carbohydrate antigen following removal of the glycosidic
phosphate to amino groups on
CRM(197), however
carbohydrate loading on the
carrier protein was low.
Glycoconjugates were then produced utilising
amidases produced by Dictyostelium discoideum (Dd), which partially remove N-linked
fatty acids from the
lipid A region of the Nm LPS molecule, which enabled the retention of the PEtn residue. LPS-
OH was treated with Dd
amidase, the reducing glycosidic
phosphate removed, and using a
cystamine linker strategy, conjugated to the
carrier protein.
Carbohydrate loading was somewhat improved but still not high. Finally, we have developed a novel conjugation strategy that targets the amino functionality created by the
amidase activity as the attachment point. The amino functionality on the PEtn residue of the inner core was protected via a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A
maleimide-
thiol linker strategy, targeting
lysine residues on the
carrier protein did not result in high loading of the
carbohydrate molecules, however when we targeted the carboxyl residues we have consistently obtained a high loading of
carbohydrate antigens per
CRM(197), which can be controlled by variation in the amount of activated
carbohydrate utilised in the conjugation reaction.