Summary
Leptospirosis is a worldwide, severe infectious disease affecting several different species including man and ruminants, particularly in LMIC countries. Globally, cattle are the most severely afflicted in terms of numbers, resulting in severe economic losses, food security impact and substantial antimicrobial use, as well as increased zoonotic spread. Bovine leptospirosis (BL) vaccines are available, although there are several constraints inhibiting use in LMIC countries where most of the disease burden is present. Current BL vaccines have a limited range of specificity, only last for a short duration and require cold chain transport and storage which is problematic in many tropical, frequently LMIC, regions with substantial disease burden.
Bacterial outer membrane proteins (OMPs) are considered an important target to provide cross-protective and long lasting immunity against a range of Leptospira species and serovars. Immune evasion by spirochetes (such as leptospires) is considered to utilise adhesion of host molecules to the bacterial cell surface. Recent research, mutating OMPs to prevent binding to host molecules has increased immune efficacy of OMP vaccines for bacteria. We have identified several OMP amino acids key for adhesion to the host for a thermostable leptospire OMP which already has demonstrated some protective efficacy. Here, through mutating OMP proteins we aim to develop a novel thermostable vaccine with broad Leptospira specificity and enhanced efficacy and safety. Vaccines are considered key mechanisms to reduce AMR. Making vaccines more broadly protective, easily accessible and financially affordable can only increase uptake globally, especially in LMIC countries, therefore decreasing antibiotic use and AMR.
Project outcomes
We were able to generate de novo specific vaccine candidate genes within expression plasmids using cutting edge gene synthesis technology at the University of Liverpool (UoL). These genes are novel as we designed them so that the proteins they encode no longer attach to host skin or host wound healing molecules. This design was informed using previous collected data, which demonstrates a natural diversity of binding to different host molecules by different bacterial strains. It is anticipated that the production of these mutated vaccine components should enable better access of the proteins to the host immune system on vaccination.
These vaccine candidate genes were used to produce the putative vaccine components through expression in Escherichia coli as insoluble aggregates. These aggregates were refolded and purified so that pure protein was produced. Initial stability assays included investigations into maximum protein concentration as well as repeating previous work showing that when using electrophoretic mobility-shift assays that the thermostable protein only unfold after boiling in chaotropic 8M urea for 1 hour.
We have developed structural models for the novel leptospiral proteins. We have identified that the amino acids identified as key for binding to host molecules, were for the most part determined as surface exposed.
We also investigated the diversity of the thermostable vaccine candidates using phylogenetic trees to better understand how many variants might be needed to make a cross-protective vaccine. When comparing sequence diversity rom various cattle relevant serovars and species from across the world there were six distinct clusters suggesting that, dependent upon future immunological studies into cross-reactivity, that a hexavalent vaccine might be needed to ensure efficacy in LMIC cattle.
A global panel of cattle serum has been collected. The protein OMPs have been screened for antibody (IgG1 and IgG2) seroreactivity, using ELISA verifying interaction with the host immune system. Purified OMPs were subjected to ELISA-based functional assays to investigate loss of host binding (ongoing).
Vaccine candidates were produced at UoL and transported to Afrigen in South Africa to undertake stability testing in adjuvant formulations. Electrophoretic mobility-shift assay were used to determine the stability as a function of temperature and time (ongoing).
Further protein was produced at UoL and detergent exchange completed into different high purity detergents and transferred to the University of Manchester for crystal trials. Three different detergents were used for two different vaccine candidate preparations which were trialled under a wider variety of crystallisation conditions. No crystals were identified after initial incubation at room temperature although after subsequent refrigeration some conditions are now more suggestive of enabling crystals containing birefringent flecks.
We have carried out knowledge exchange and skills transfer to the LMIC vaccine developer, Afrigen. Using a number of meetings we described the protein production and lab techniques used involving both University of Liverpool and University of Kelantan (Malaysia). Further meetings have occurred between all groups to determine what funding might be applied for subsequently and we have plans for applications to both Innovate (UK) and the BBSRC for funds to enable future collaborative work across all groups.