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Dissecting cell surface protein diversity to enhance leptospiral vaccine efficacy.

Reference	BB/W016133/1
Principal Investigator / Supervisor	Dr Nicholas Evans
Co-Investigators / Co-Supervisors	Professor Stuart David Carter, Dr Hayley Crosby-Durrani, Professor Daniel Rigden
Institution	University of Liverpool
Department	Infection Biology & Microbiomes
Funding type	Research
Value (£)	737,023
Status	Current
Type	Research Grant
Start date	05/09/2022
End date	04/06/2026
Duration	45 months

Abstract

1) Survey vaccine candidate diversity (orthologs) across leptospire species, produce and determine any differences in seroreactivity with cattle sera panel (n=200). Subject orthologs to ELISA and thermal shift assay (TSA) to quantify binding to a variety of complement-interacting host ligands. Quantify binding of othologs to complement factor H and plasminogen of cow, dog, horse, human, mouse, rat and hamster. Generate diverse orthologs (e.g. from commensal) and quantify binding. 2) Use ortholog diversity and function with AlphaFold2 models for binding site mutagenesis including A) cofold surface proteins with host ligands in silico using AlphaFold2 and B) docking experiments of AlphaFold2 structures with host ligands (ClusPro) and C) compare structures to nearest orthologs without function. Using gene synthesis/primer directed mutagenesis produce function-restricted recombinant leptospire surface proteins (RLSPs), subject to ELISA and TSA to quantify adhesion difference and TSA to confirm no stability loss. Verify mutant RLSPs exhibit equivalent antibody titres with sera panel. 3) Use ortholog diversity and AlphaFold2 models to identify stabilising regions/mutations using FRESCO/FireProt. Produce RLSPs and verify enhanced for physiological and cold chain relevant temperatures using TSA and intrinsic fluorescence. Verify mutant RLSPs exhibit equivalent antibody titres and no function regain (ELISA). 4) Compare protective efficacy of enhanced and wild type lipidated RLSPs in hamster models of acute and chronic leptospirosis. Use select RLSPs as multivalent vaccines in acute hamster model to investigate cross-protection when challenged with different species/serovars. Clinical outcomes measures: survival, weight loss and bacterial burden (qPCR, histopathology and kidney culture to assess sterile protection). T-cells will be isolated from splenic tissue for antigen-recall assays. Anti-vaccine IgG response ELISAs and western blotting will be conducted on pooled sera.

Summary

Leptospirosis, caused by Leptospira bacteria, is a worldwide, severe infectious disease affecting several different host species including cattle, dogs and man. Globally, cattle are greatly afflicted resulting in severe economic losses, reduced food security, substantial antimicrobial use and animal to man (zoonotic) transmission. Economic cost to the UK is £22.3 million/year with much greater costs expected for tropical regions, including many low to middle income countries (LMICs), due to substantially greater disease burden and more severe disease. Current bovine leptospirosis (BL) vaccines have a limited range of specificity and require cold chain transport and storage, which is problematic in the many tropical, frequently LMIC regions with greatest disease burden. Making vaccines more broadly protective and easily accessible will increase uptake globally, decreasing global antibiotic use and antimicrobial resistance development. This is especially important for leptospirosis which is considered to be emerging/re-emerging globally and being driven by global warming and associated increases in extreme climatic events, such as flooding. Bacterial surface proteins are considered important targets to provide cross-protective and long lasting immunity against a range of Leptospira species and serovars. Immune evasion by leptospires, is considered to involve these bacteria coating themselves with host molecules. Whilst the different leptospire bacteria involved in disease are diverse, they must have near identical machinery for this immune evasion which must be present on the bacterial surface to allow for host binding and/or damage and therefore represent ideal vaccine targets. Thus, characterisation of key bacterial surface proteins, especially those involved in immune evasion and determining their mechanism of interaction should allow for development of novel vaccines or therapeutics. Recent research, mutating bacterial surface proteins to prevent binding of hostmolecules, as well as enhancing protein stability, has increased the protective ability of these bacterial components when used as vaccines. The application of such novel protein engineering has been used in the development pathway for an important human pathogen vaccine which is now licensed and can now be applied to veterinary pathogens. Here, we combine synthetic biology, artificial intelligence and in silico (bioinformatic) approaches to guide engineering of key cell surface proteins to develop a novel thermostable vaccine with broad Leptospira specificity and enhanced efficacy. This study will 1) investigate vaccine candidate diversity across leptospire species including surveying whether variants from some species exhibit adhesion preference for molecules from specific host species resulting in known host specificity and identify, whether variants from commensal (harmless) relatives lack ability to attach to host molecules, 2) use sequence diversity/conservation and differences in adhesion ability together with artificial intelligence (AI) generated structural models with in silico approaches to engineer the surface proteins to restrict host interaction which in line with recent human pathogen disease work should allow for more effective vaccines, 3) use sequence diversity together with AI generated structural models and in silico approaches to synthesise surface proteins with enhanced stability, 4) use a rodent models of disease to identify those engineered bacterial surface proteins most likely to offer protection from a range of the disease causing bacteria. Investigating BL vaccine candidates by the diverse and comprehensive methods described above, should help characterise the causal bacteria, improve understanding of the disease, substantially progress the vaccine development pipeline and/or identify novel therapeutics. Such studies are both timely and much needed to enable the prevention or even eradication of this severe, important global disease.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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