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Engineering of Mycoplasma pneumoniae as a broad-spectrum animal vaccine

Periodic Reporting for period 4 - MycoSynVac (Engineering of Mycoplasma pneumoniae as a broad-spectrum animal vaccine)

Reporting period: 2018-10-01 to 2020-03-31

Annually, infections caused by Mycoplasma species in poultry, cows, and pigs result in multimillion Euro losses in the USA and Europe. There is no effective vaccination against many Mycoplasmas that infect pets, humans and farm animals. Furthermore, most Mycoplasmas are difficult to grow, requiring a complex media that includes animal serum. Consequently, even in those cases for which effective vaccines are available (namely, M.hyopneumoniae in pigs and M.gallisepticum and M.synoviae in poultry), the production process of the vaccines is challenging.
The main aim of this project is to design a universal Mycoplasma chassis that can be deployed as single- or multi-vaccine in a range of animal hosts. Specifically in this project, we will target the development of attenuated and/or inactivated vaccine(s) against two Mycoplasma pathogens: M. hyopneumoniae (pigs) and M. bovis (cattle), and a combined one against M. hyopneumoniae and PRSSV virus (pigs).
To achieve this overarching goal, the MycoSynVac project has the following specific objectives: Vaccine Design, Chassis Engineering, and Optimization of Large-Scale Production, all this taking into account the future exploitation of the technology developed and facing ethical concerns that synthetic biology can awaken.
WUR developed a whole-cell dynamic model framework of the metabolism of M. pneumoniae and built further upon M. pneumoniae models to develop a genome-scale, constraint-based model of M. hyopneumoniae for vaccine optimization. WUR also deployed their metabolic model(s) to assess, at genome scale, the metabolic capabilities of a series of Mycoplasmas with the purpose of designing a tailored vaccine portfolio.
CRG developed two genome engineering methods for M. pneumoniae. Resulting from this, we produced a non-pathogenic chassis that can survive in the mice lung as long as the WT Mycoplasma pneumoniae that does not trigger lung lesions and has a reduced inflammation response. This chassis can express on its surface heterologous proteins and protein adjuvants for vaccination. We have determined which genes are essential for survival in the mice lung and cannot be removed from the chassis.
ICL designed, optimised and tested their biosafety circuits in the WT strain, which show a good killing efficiency when the circuits are activated. Also, integrating redundancy improves the biosafety system. Very promising results have been obtained in the pairwise competitions that challenged the different biosafety systems developed when tested in vivo using mice models. The biosafety circuits were also transformed into the chassis strains. Here the circuits had to be re-optimised and not all cases succeeded. The best strain generated is a CV2 with two copies of the re-optimised circuits KS1.
INRA developed a method called Genomic transfer-Recombinase-Mediated Cassette Exchange (GT-RMCE). This new technology enables to perform large-scale genomic modifications at specific loci on M. pneumoniae. It allows inserting, replacing large DNA fragments and deleting genes and could be extended to introduce point mutations. All these new tools allowed to inactivate known virulence factors of M. pneumoniae and lead to building of a vaccine chassis prototypes.
ATG completed the selection of candidate peptides to be included into chimeric proteins for vaccine production, 44 were selected for Mycoplasma hyopneumoniae and 23 for Mycoplasma bovis. Additionally the analysis of 64 PRRSV strains with the peptide microarray approach allowed the selection of 20 further candidate peptides for this virus. The peptides were combined into 8 chimeric proteins for M. hyopneumoniae, 6 for M. bovis and 4 for PRRSV.
MSD performed 8 animal experiments in large food animals to test the various vaccines that were generated in the project. Expression of heterologous antigens on the surface of an attenuated M. pneumoniae chassis grown in a serum free medium, is safe in food animals as inactivated vaccine and results in seroconversion to all antigens exposed. Preliminary results also indicate that the chassis variants may be safe as live attenuated vaccine as the bacteria cannot be detected in other body sites than the injection site. Additional animal experiments would be necessary to reach a conclusion on whether these vaccines are efficacious.
The work done by UCPH on the social concerns has also established that while in general there is a broad acceptance of the use of a synthetic livestock vaccine in Europe, there are significantly different perceptions of such a vaccine between countries. Their internal dialogue activities helped our researchers to reflect on how their work might be perceived by other relevant stakeholders.
Biofaction developed a large variety of different types of public engagement ranging from personal interactions at Science Cafes, High School Visits and Public Science Communication Events to online activities such as 5 character animation videos, a short science documentary film and the science game ’Battle for Cattle’ for web and mobile devices.
The MycoSynVac project was highly ambitious, using a really disruptive approach to vaccine production: a universal chassis for vaccination based on M. pneumoniae able to grow efficiently and reproducibly in a defined serum-free medium. The consortium brought together cutting-edge knowledge and resources on Mycoplasma, modeling, synthetic biology, antigen epitope determination and vaccine production and commercialization as well as other important areas such as dissemination and ethical aspects. The main impacts arising from the project focus in 4 main areas: synthetic biology, relationship industry-academia, public engagement and innovation capacity of the European arena. MycoSynVac had a positive impact on all the partners:
- For CRG, MycoSynVac facilitated the creation of a spin-off company called Pulmobiotics, which aims to treat the respiratory disease Ventilator Associated Pneumonia (VAP) in humans by using the chassis as delivery system.
- MSD had access to new technologies developed in ATG that were used by MSD for MycoSynVac unrelated research. They also gained insights in biology of mycoplasmas and synthetic biology, allowing MSD to keep its leadership position in the vaccine market.
- INRA increased collaborations with leading European Labs and with companies active in the field of animal health. Also, they gained awareness of social aspects thanks to UCPH and Biofaction.
- WUR gained insights into host-microbe interactions and strengthened its positioning in the agro-food & health area.
- ICL was able to patent the development of their biosafety circuits.
- ATG was able to develop new methods for analyzing hundreds of surface antigens and viral protein families on high-density peptide microarrays for antibody response in search for best epitopes. The cooperation with MSD and CRG and the integration of their data sets allowed them to make valuable experiences in planning, running and evaluating data analysis pipelines.
- UCPH developed competences to do social science studies across borders in Europe and to expand their approach to applied empirical ethics.
- Biofaction gained additional visibility (e.g. for science animation clips and science game development) and established additional contacts to science communication channels (YouTube channels, podcasts, etc.).
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