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.
Objective
With the focus of my group dedicated to testing novel mucosal vaccine platforms for their efficacy against C. difficile infection, the project will be to build on the above platform. There is currently no vaccine available against this deadly gut pathogen as intramuscular toxoid vaccines have failed human trials. Encouragingly we have shown the efficacy of the oral route to direct a mucosal and systemic immune response that affords partial protection from infection with a hypervirulent strain of C. difficile.
Methodology
In this project, using our optimal signal peptide, vaccine candidates will be acylated and purified from culture filtrates, lyophilised and encapsulated and testing for improved immunogenicity and improved protective efficacy in the C. difficile hamster model compared to their non-acylated counterparts. The immunogenicity assays to be conducted will include ELISAs and cellular assays (adherence blocking and toxin neutralisation). Challenge studies will involve monitoring well being (survival from infection) and bacterial load in faecal pellets. Histological analysis will be conducted with help from a Histopathologist. All other experiments are routinely performed in the Griffin lab.