Novel targets for vaccine development and immunotherapy to combat Staphylococcus aureus and other pathogens

Lead Research Organisation: University of Sheffield
Department Name: Molecular Biology and Biotechnology


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Technical Summary

Antibiotic resistant, pathogenic bacteria are a constant and increasing threat. An important alternative to antibiotics is disease prevention by vaccination and the use of therapeutic antibodies. The project will develop a new class of vaccine/antibody targets against the superbug Staphylococcus aureus and other infectious microbes. The targets are those which are essential for the life of the cell and so the bacterium cannot do without them. Our BBSRC funded work has identified suitable novel targets in S. aureus and has shown them to be accessible to the immune system. A patent has been filed by the University of Sheffield to protect this finding. For further exploitation and commercialisation the essential components need to be produced and their potential as vaccine constituents examined. Concomitant with scientific advances a commercial structure will be established to allow the full potential of the area to be realised.


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Description Summary
There is a crucial need for the development of new methods for the control of important pathogenic bacteria such as drug resistant strains of Staphylococcus aureus (e.g. MRSA). Work leading up to the project (BBSRC grant EGH16083) identified the possibility of a range of novel targets for an immunological approach. The project developed these targets to the point of proof of principle, showing that immunization with a peptide, designed against a membrane protein, could protect against S. aureus infection. This opens up a range of novel targets, which may be exploited for vaccination or for the production of monoclonal antibodies. Based on the findings a full investment proposal was drawn up and used to successfully obtain funding to establish a new University spinout company. Absynth Biologics Ltd was launched on 1st February 2007 and now employs both of the researchers from the FoF grant.

The project stemmed from a BBSRC Exploiting Genomics grant (EGH16083) in the area of analysis of essential gene products of Staphylococcus aureus. S. aureus is a major human pathogen of increasing importance due to the spread of antibiotic resistant strains such as MRSA (Methicillin Resistant S. aureus). MRSA and other drug resistant organisms have been dubbed the "Superbugs". Our EGH grant characterised several novel essential genes of unknown function and as part of that approach revealed that a subclass of them were membrane associated with loops or domains outside the cytoplasmic membrane. These are potentially accessible to the immune system and so may constitute possible targets for immunotherapy/prophylaxis. Our initial studies had shown that immunization with specific polypeptides led rabbit serum to have bactericidal properties on S. aureus. The aim of the 12-month project was to develop the field to allow commercialisation of our results.

The aims of the project were to produce a proof of principle, that our range of novel targets was suitable for immunological prophylaxis/therapy and to commercialize this area to the point of licensing or spinout.


Scientific Objectives
1. Full range of target proteins identified by bioinformatic analysis.
2. Production of carrier protein linked peptides and recombinant proteins.
3. Production and testing of polyclonal sera and specific monoclonal antibodies for antibacterial and protective effects.
4. Protection studies to establish targets as vaccine components.

Commercial Objectives
1. Identification and analysis of potential commercial partners.
2. Development of structure and proposal to interested parties.
3. Development of business plan and research and development programme dependent on the results of the current investigations.

Results and Discussion

Scientific Objectives
1. Full range of target proteins identified by bioinformatic analysis.
The basis for choice of target was that it should be potentially accessible to the immune system and be important for the ability of S. aureus to cause disease. This meant that all targets would be present in all strains and be highly conserved. Based on our knowledge of components important to S. aureus (mostly identified as part of EGH16083) 9 individual proteins were chosen. These were mostly integral membrane proteins, or those which had predicted domains outside the cytoplasmic membrane. A combination of bioinformatics programmes were used to deduce their likely protein topology. Using conditional lethal mutant analysis the essential nature of 6 of these proteins has been shown (3 by us and 3 by other groups). One target, PheP, was chosen as we found it to be to be required for pathogenesis in every animal model used to date (mouse abscess, Drosophila, mouse septic arthritis and zebrafish). PheP is an integral membrane protein which functions as a predicted phenylalanine transporter.

2. Production of carrier protein linked peptides and recombinant proteins.
The bioinformatics analysis revealed that 5 of the targets had sufficient nonmembrane associated regions that they could be produced as recombinant protein in E. coli. Of these 4 were successfully cloned and overexpressed. Recombinant proteins were produced with His-tags to aid purification. All 4 polypeptides were produced in sufficient quantities (2mg) and purified to allow immunization and the production of antibodies.
The bioinformatics analysis revealed for several integral membrane proteins, the presence of predicted loops, which would be exposed on the outer face of the cytoplasmic membrane. For some proteins multiple predicted loops were present. Based on this information peptides (10-18 amino acids) were designed representing the exposed loops. In total 14 peptides were designed based on 4 proteins. All the peptides were produced linked to the carrier protein KLH (for immunization) and BSA (for testing antibody response).

3. Production and testing of polyclonal sera and specific monoclonal antibodies for antibacterial and protective effects.
The recombinant polypeptides and carrier-linked peptides were used for the production of polyclonal and monoclonal antibodies (MAbs). So far polyclonal antibodies were made against 5 targets. MAb production has been attempted for 5 targets. Problems were encountered with the low immunogenicity of several of the peptide targets. This has been recently circumvented by the use of peptide dendrimers for presentation of the antigens. Our current success is exemplified by protein YneS. We have previously proven YneS to be essential in S. aureus and have made predictions as to its location and topology (Fig. 1).

In order to test if the YneS predicted loops are antibody accessible in whole cells, 2 carrier-linked peptides have been made (731, 733; Fig. 1). Immunization of rabbits has led to the production of antisera, which recognises the cognate peptides, as shown by Western blot (Fig. 2A). Also the sera react with native S. aureus proteins present in purified membranes (Fig. 2B).

To determine the ability of antibodies to reach their membrane-associated target FACS analysis was used. This showed that antibodies specific for YneS can bind specifically to whole cells (Fig. 3). This also verifies the predicted YneS topology (Fig. 1).
Peptides 731 and 733 were used for the production of MAbs that will react with YneS. Only peptide 731 proved to be useful for MAb production resulting in 3 IgG and 13 IgM clones. The specificity of the MAbs has been verified using alternative carrier-linked peptides. MAbs have been produced and purified in sufficient quantities to allow assessment of their activity on whole, live cells of S. aureus and for use in passive transfer experiments to determine if they can prevent S. aureus infection.

4. Protection studies to establish targets as vaccine components.
Recombinant polypeptides and carrier-linked peptides have been used in a series of immunization experiments to determine protection against S. aureus infection. The animal model used for the experiments is the well-established murine septic arthritis model in collaboration with Prof. Andrej Tarkowski (University of Gotheborg). Mice have been immunized and then challenged with S. aureus. So far 2 polypeptides and 5 peptides have been used. A major problem has been the lack of immunogenicity of the peptides and in most cases the mice have not raised a significant response. This problem is being solved by the use of peptide dendrimers (see above). However, using a peptide designed against a proposed extramembranous loop of PheP a response (5-40 fold control levels in sera) has been obtained. This led to a significant protection against infection (Fig. 4). Thus PheP is produced during infection and is accessible to the immune system. This is an exciting proof of principle and means that antibodies can reach the membrane of S. aureus. This opens up the possibility of a much larger range of targets, which are currently being evaluated.

Commercial Objectives
1. Identification and analysis of potential commercial partners
Commercial development has been carried out in partnership with Biofusion to establish the market potential of the field and the project findings. This included discussions and presentations to scientific and clinical consultants and seeking the advice of several independent clinicians on the area and the medical need. A market analysis was carried out to establish current players and products and those under development. This revealed that although several products were under development the only 2 that had got to Phase III trials had failed. The market has been estimated for a prophylactic vaccine and therapeutic vaccine against S. aureus as $1.8billion and $2.6billion pa. respectively (Datamonitor, 2004).

2. Development of structure and proposal to interested parties
There are 2 products in development based on our finding during the FoF project. These are a vaccine and prophylactic/therapeutic antibodies against S. aureus. The University currently has 2 filed patents to protect our findings (UK Patent Application No: 0505949.8 and 0409559.2). Given the level of scientific success of the project, gaining proof of principle of our approach it was deemed practical to develop either a licensing agreement with a major pharmaceutical company or to launch a spinout company. A market assessment was carried out and the most suitable approach was found to be the development of a business plan and to attempt to attract seed funding. All the preliminary research, on which the technical plan was based, was carried out by Dr Jorge Garcia-Lara and Mr Jagath Kasturiariachchi. They were both employed on the FoF grant and so to maintain skills, experience and continuity, funds had to be obtained by the end of BBSRC funding.

3. Development of business plan and research and development programme dependent on the results of the current investigations
A full investment proposal was written and developed (39 pages), based on the FoF research. This had many sections (summary, market and statistics, background, the invention, potential major advantages, competition, operational plan, funding recommendation, specific identified risks) and appendices (Staphylococcus aureus, market statistics, clinician's viewpoints, biographies, intellectual property summary, competition, technical milestones, product development, forecasted cash requirement). The business plan was written with the objective of creating a new University spinout company with an initial 2-year technical plan. The investment proposal was presented to the board of Biofusion in November 2006 with a requirement of £325K over 2 years for the technical plan. The proposal was approved and Absynth Biologics Ltd was launched in February 2007 with Simon Foster and Jorge Garcia-Lara as the two founding academics. The business is currently implementing the technical plan and we are actively seeking pharmaceutical company partners for developments into the clinic. The company has already identified several areas for expansion into different organisms, targets and approaches (alone and in partnership).
Exploitation Route Spinout company formed
Sectors Pharmaceuticals and Medical Biotechnology