VACCINE: G. mellonella as a model to predict vaccine efficacy of bacterial pig pathogens

Actinobacillus pleuropneumoniae (APP) is a bacterium that causes lung disease in pigs. In severe cases (acute disease) animals can die within 24 hours or alternatively they can get a long lasting infection (chronic disease) which is associated with breathing problems and slow development. APP is responsible for both substantial suffering and economic losses in the worldwide pig industry. Vaccines are given to control disease but the most widely used (killed bacteria) do not prevent APP from being passed from one pig to another or protect pigs against one of the 15 types of the bacterium known. Thus there is an urgent need for new vaccines. One of the most promising vaccine types in development is the use APP strains that live in the pig, do not cause disease, and protect against infection. These so called live attenuated vaccines (LAVs) protect against many of the 15 types of APP and prevent APP from being passed from one pig to another. They consist of APP strains that have mutations in their DNA that prevent them from causing clinical disease. Finding suitable LAVs and testing whether they work involves the use of pigs - the only known natural host for APP - and\or mice. There is no simple laboratory test that can predict whether an APP mutant causes disease in pigs and will make a good LAV. In preliminary work we have, firstly, infected larvae of the wax moth (an insect) with strains of APP that do or do not cause disease in pigs. Similar results were found in the wax moth to those in pigs. Secondly, we found that giving known promising LAVs as a vaccine protects the wax moth from APP infection. Together, our results suggest that the wax moth can be used as a model to predict whether an APP mutant causes disease and will make a good LAV in pigs. In this project, we will work out what are the wax moth and bacterial factors that affect the model and how it works. Success will lead to a decrease in the time to, and costs involved in, the development of urgently needed new vaccines for APP, a substantial replacement in the use of pigs and mice in APP research throughout the world, and a greater understanding of the immune system of insects and how they fight off infection from bacteria.

Actinobacillus pleuropneumoniae (APP) is a bacterium that causes acute and chronic lung disease in pigs and is responsible for substantial suffering, deaths and economic losses worldwide. Vaccines are used to control disease, the most widely being bacterins (killed bacteria) but they do not prevent colonisation and, at best, protect pigs against a few of the 15 serotypes. There is an urgent need for new vaccines. Live attenuated vaccines (LAVs) are considered the most promising, and understanding the APP virulence mechanisms underpins their development. As there is no simple laboratory test, experiments to identify APP LAVs and determine their vaccine potential are carried out in the pig - the only known natural host of APP - and mice. In preliminary work we have found that: (1) APP mutants were attenuated for infection in Galleria mellonella (wax moth) insect larvae as they are in pigs; (2) prior administration of known promising APP LAV strains in pigs protected G. mellonella from lethal doses of APP. Collectively, these data suggest that the G. mellonella-APP (GM-APP) model can also be used to predict virulence\vaccine efficacy. However, so far. we have only tested a small number of classes of mutants and serotypes for virulence\vaccine efficacy. For the GM-APP virulence\vaccine model to be used widely the parameters affecting its applicability\reproducibility and the underlying science should be understood. In this study we will identify the parameters of both G. mellonella and APP that affect the GM-APP virulence\vaccine model and establish optimised protocols, and identify proteomic and transcriptomic G. mellonella biomarkers, that predict APP virulence\vaccine potential. Success will lead to a decrease in the time to, and costs involved in, the development of urgently needed new vaccines for APP, a substantial replacement in the use of pigs and mice in APP virulence\vaccine research and a greater understanding of insect immune defence mechanism.

There is an urgent need for effective vaccines to combat the highly contagious, and economically important, disease caused by Actinobacillus pleuropneumoniae (APP), which affects the UK and worldwide swine industry. Our research aims to decrease both the time and costs associated with the development of the new generation APP vaccines through an innovative novel approach utilising the larvae of the wax moth. Success would offer a step change in capability for combating APP, but would potentially have far reaching impact for the development of vaccines for prevention of bacterial diseases more broadly. This work therefore closely addresses the key BBSRC strategic priorities of 'Animal Health', 'Replacement, Refinement and Reduction (3Rs) in Research Using Animals' and 'Combating Antimicrobial Resistance' and, specifically, the Highlight Area of Tools and Technologies for Vaccinology.

The beneficiaries of this research and how they will benefit are as follows:

*Academic Researchers: through broad academic advancement and instigation of new research programmes.
Briefly, our findings will allow the rapid screening of APP vaccine candidates and prediction of their efficacy in pigs. Importantly, for wider uptake by the scientific community, we will determine the immune mechanisms underlying their successful application. The findings will therefore be of immediate benefit to researchers with interests in bacterial pathogenicity and immunity, vaccine design and screening, insect immunity and biomolecular research more broadly. In particular, an immediate avenue of investigation will be to confirm whether the findings and approach within this proposal have broader applicability to other pathogenic bacterial species, leading to a wider use of the wax moth vaccine toolbox in bacterial vaccine development.

* Veterinary Vaccine Companies: through exploitation and commercialisation within the antimicrobial arena.
Our research aims to provide the basis for a novel vaccine approach which should be readily exploitable by Veterinary Vaccine Companies. In the longer term, successful translation of our research into a vaccine has the potential to create wealth and economic prosperity through increased turn-over, profit and exports, and creating and safeguarding jobs for the company and workers involved. A successful vaccine overcomes the issue of antibiotic resistance, an increasing problem for APP and human and veterinary pathogens more widely.

* The 3Rs: through the substantial reduction in the use of pigs and mice in APP virulence\vaccine research in University, Research Institute and Industrial settings.

* The swine industry: through the ability to effectively treat APP.
In the longer term, successful translation of this research into a vaccine for APP will reduce medication costs, reduce pig suffering and mortality and overall reduce the significant economic impact on the swine industry of APP.

* The general public: through increasing public engagement with research.
Throughout it will be possible to engage with the general public and local schools to raise the profile of this project. This will encourage interest in biosciences research and UK innovation and provide a context to local bioscience education.

* Researchers working directly on the project: thereby contributing to a highly trained workforce.
The skills gained by the researchers working on this innovative project will be highly transferable; providing an excellent training experience to enable them to embark on a successful future career. For example, working in a comparatively new area of research provides enhanced skills and future employability, participation in outreach activities (e.g. in the 3Rs) gives experience in communicating effectively with non-specialists, and through publishing manuscripts and presenting work at conferences honing verbal and written skills.