Developing and expanding the bacterial glycotoolbox for animal pathogens

Millions of animals die each year because of bacterial diseases. Despite this, and the fact that bacterial infection causes significant losses to the farming industry, the technologies designed to protect humans against bacterial infections have often not been available to combat animal disease. The biggest reason for this is cost. This proposal aims to harness genes from a bacterium that causes significant sickness and mortality in pigs. In preliminary experiments we discovered two genes from a pig pathogen called Actinobacillus pleuropneumoniae and found that they were responsible for making a sugar structure (polysaccharide) on the surface of this bacterium. We have been able to transfer these genes into a safe laboratory strain of Escherichia coli and to make a first generation of vaccine. This vaccine is based on the linkage of a protein to this sugar component. Crucially, the genes coding for this polysaccharide are present in all A. pleuropneumoniae strains. We believe that this may be a chink in the armour of this pig pathogen and would like to generate several vaccine formulations to test this theory. The method used to make this type of vaccine, called a glycoconjugate, is much cheaper than traditional techniques, which have prevented their widespread use in animals.
Our interests will also go beyond using the genes to make a vaccine against A. pleuropneumoniae. We are also going to explore if the protein responsible for making this vaccine has potential to be used as a tool to unlock current technological limitations. And make possible the assembly of novel vaccine formulations.

For the past decade we have been developing an in vivo glycosylation method in Escherichia coli termed protein glycan coupling technology that expedites the production of pure inexpensive glycoconjugate vaccines. The method relies on the cloning of a polysaccharide coding region into a safe laboratory strain, followed by the introduction of an acceptor protein. The protein and glycan are coupled by the oligosaccharyltransferase enzyme PglB. The glycoconjugate can then be purified directly from the cultured E. coli in a single step procedure. However, PglB has limitations.

PGCT has now been clearly established for the production of glycoconjugate vaccines against Shigella dysenteriae, Francisella tularensis, Staphylococcus aureus and pathogenic E. coli. However, its potential application for animal pathogens is unexplored. There are limitations to PGCT and these mean that it has yet to be developed for veterinary purposes. This proposal will exploit a newly discovered N-linked glycosylation system that the applicants have characterised from the pig pathogen Actinobacillus pleuropneumoniae that promises to overcome the current limitations of the PgB-based system currently used in PGCT. Firstly novel glycoconjugate vaccine formulations will be developed against A. pleuropneumoniae which have the potential to protect against all serotypes. In addition, multivalent vaccines will be assembled by transferring the genes used to make the A. pleuropneumoniae polysaccharide specific component into other pig pathogens, Haemophilus parasuis and Streptococcus suis.

Finally the biotechnological potential of NGT will be investigated, these experiments will ascertain if NGT is a suitable tool for glycoconjugate production and have the potential to further broaden the applicability of PGCT, hence generating a novel biotechnological tool that will facilitate a novel vaccine assembly mechanism.

The studies described in this proposal aim to generate a novel glycoconjugate vaccine and vaccine formulations against A. pleuropneumoniae. Harnessing this knowledge and in combination with our understanding of transposon mutagenesis, will also enable the first generation of multivalent vaccines by creating H. parasuis and S. suis strains capable of making the A. pleuropneumoniae specific glucose polymer within their cytoplasmic compartments.

The potential impact of an A. pleuropneumoniae vaccine capable of protecting against all serotypes alone is considerable, but the possibility of enhancing this vaccine strategy by combining it with a live attenuated strain of H. parasuis and S. suis is appealing. Through our collaboration with Professor Markus Aebi's group at ETH University, Zurich, we have been able to test the glycosylation status of proteins expressed in preliminary experiments. We have also been contacted by Malcisbo, rated 14th out of the top 100 start up companies in Switzerland. We have held lengthy discussions regarding the potential of an NGT-based glycoconjugate, and are in the process of setting up an agreement to share knowledge and genetic tools.

The second aspect of this proposal, understanding the biotechnological capability of NGT to overcome the limitations of PglB based technologies, is of particular relevance to this funding call. Should the proof-of-principle study using the S. suis capsular polysaccharide locus be successful, it could lead to the utilisation of this novel enzyme in order to tackle vaccine generation against a wide variety of pathogenic organisms for both animals and humans. To use Streptococcus pneumoniae as an example, over 90 capsule types exist but only a handful are substrates for PglB whilst the majority have glucose as their starting sugar.

As the leading group worldwide in the use of Protein Glycan Coupling Technology (PGCT), we are in frequent contact with industrial partners. The Francisella tularensis glycoconjugate vaccine generated at the LSHTM was assembled using an acceptor protein from Glycovaxyn. A Swiss spin off founded from the discovery of PglB and now the lead company in this method of biological conjugation.

Glycovaxyn and other companies with particular interest in animal vaccinology such as Zoetis, Merck and Malcisbo should provide a way to take the vaccines generated from this research to commercially viable products.

The potential impact of the research will also be realised through dissemination of our studies by publication in international peer-reviewed journals (where the applicants have a strong record) and by poster and oral presentations at major national and international meetings. The applicants have a track record of communicating the results of their research to the public (e.g. school children) and the mass media (radio broadcasts, Sky, ITV and BBC broadcasts). We also will maintain our international links in PGCT development with the Aebi and Syzmanski groups with whom we have long standing collaborations.

Exploitation of our research will continue to be delivered though the LSHTM technology transfer office, with whom the principal applicants have strong links. Two patents based on our research and PGCT has been filed and preliminary discussions have been made regarding potential licensing agreements and these will continue as and when appropriate opportunities arise.

In summary, we have demonstrated our capabilities of developing and nurturing our research relating to PGCT. We have wide collaborations internationally in both basic research laboratories and companies ranging from fledgling SME such as Malcisbo and big pharmaceutical companies such as Pfizer.