Testing recombinantly lipidated antigens as oral vaccines against Clostridioides difficile

One of the main bottlenecks for vaccine development is identifying suitable adjuvants. The lipid moieties of bacterial lipoproteins have attracted interest owing to their potent immunostimulatory properties. Multiple different fatty acids may be utilised by bacteria to form this lipid moiety, and dipalmitoyl and tripalmitoyl-glyceryl groups are known to be particularly potent.
Vaccinologists have exploited the lipoprotein processing machinery of Escherichia coli to lipidate chosen antigens and enhance their potency. Briefly, the antigen gene is cloned downstream of the N-terminal signal peptide of a lipoprotein. This was first demonstrated for meningococcal Factor H binding protein (FHbp) using the signal peptide of Haemophilus influenzae P4 lipoprotein. The lipidated protein elicited bactericidal antibody titres up to 10-fold higher than the non-lipidated form following subcutaneous administration in mice. Pfizer exploited this recombinant lipoprotein to develop the Trumenba vaccine which was licenced in 2014. Other studies have used the signal peptide and adjacent sequence of the meningococcal lipoprotein, Ag473, to create a fusion with the viral HPV protein and with part of toxin A of Clostridioides difficile. These lipoprotein subunit vaccines have shown significantly higher neutralizing antibody titres in mice compared to their non-lipidated counter-parts.
For vaccine manufacturing purpose, large scale production of lipoproteins by recombinant expression in E. coli remains a challenge due to typically low yields, incomplete or no lipidation and inherent challenges of purifying membrane-associated proteins from insoluble fractions. Antigens that enter culture filtrates on the other hand, are by default soluble facilitating their purification. We thus set out to identify the optimal signal peptide that can not only direct successful lipidation but also secretion of the acylated fusion protein into the extracellular milieu for facile purification.
Testing a range of different peptides of different length to which fluorescent mRaspberry was fused as a reporter, has now led to the identification of the optimal signal peptide. Currently we are testing whether the deletion of 2 specific genes in the chromosome of our E. coli expression strain will facilitate secretion and we are characterising the lipid moiety to confirm the presence of palmitic acids.