Recent advances in the production of recombinant glycoconjugate vaccines

Abstract

Glycoconjugate vaccines against bacteria are one of the success stories of modern medicine and have led to a significant reduction in the global occurrence of bacterial meningitis and pneumonia. Glycoconjugate vaccines are produced by covalently linking a bacterial polysaccharide (usually capsule, or more recently O-antigen), to a carrier protein. Given the success of glycoconjugate vaccines, it is surprising that to date only vaccines against Haemophilus influenzae type b, Neisseria meningitis and Streptococcus pneumoniae have been fully licenced. This is set to change through the glycoengineering of recombinant vaccines in bacteria, such as Escherichia coli, that act as mini factories for the production of an inexhaustible and renewable supply of pure vaccine product. The recombinant process, termed Protein Glycan Coupling Technology (PGCT) or bioconjugation, offers a low-cost option for the production of pure glycoconjugate vaccines, with the in-built flexibility of adding different glycan/protein combinations for custom made vaccines. Numerous vaccine candidates have now been made using PGCT, which include those improving existing licenced vaccines (e.g., pneumococcal), entirely new vaccines for both Gram-positive and Gram-negative bacteria, and (because of the low production costs) veterinary pathogens. Given the continued threat of antimicrobial resistance and the potential peril of bioterrorist agents, the production of new glycoconjugate vaccines against old and new bacterial foes is particularly timely. In this review, we will outline the component parts of bacterial PGCT, including recent advances, the advantages and limitations of the technology, and future applications and perspectives.

Keywords: Immunology; Pathogenesis.

Fig. 1

Traditional chemical conjugation method for the production of glycoconjugate vaccines. Multiple steps are required whereby the O-antigen must be purified from the pathogen of interest, detoxified and subject to chemical activation. In parallel, the protein must also be purified and chemically activated before protein and glycan can be conjugated. Following conjugation, further rounds of purification are necessary before vaccine can be administered

Fig. 2

Glycoengineering approach to the production of glycoconjugate vaccines. An E. coli cell is transformed with three plasmids to generate the glycoconjugate protein (GP). PGCT occurs in three stages: stage 1; Glycan expression, stage 2; Carrier protein design and expression, stage 3; Coupling. The polysaccharide is synthesised on an undecaprenol pyrophosphate lipid anchor (blue/black circle) within the cytoplasm; this is transferred to the periplasmic compartment where PglB recognises the lipid linked reducing end sugar and transfers the polysaccharide en bloc onto an acceptor-sequon (D/E-X-N-X-S/T) on the carrier protein to produce the GP. IM, inner membrane; OM, outer membrane. This figure is adapted from Cuccui et al.

Fig. 3

Glycan expression technology (GET). To express foreign sugar structures in E. coli, first the genes responsible for synthesising the glycan must be cloned. The example here is CPS I coding locus of B. pseudomallei K96243. Expression of these genes leads to synthesis and export of the foreign sugar attached to lipid A, in the absence of the native O-antigen from E. coli. a–d show Immunofluorescence microscopy of E. coli cells coated with the new sugar structures. Cells were probed with group or type-specific anti-glycan antibodies and Alexa Fluor 488 conjugated secondary antibody. A, unpublished; B, reproduced from Cuccui et al.; c, d reproduced from Kay et al.