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In Depth

Main project

A universal bacterial chassis for vaccination purposes based on Mycoplasma pneumoniae

Annually, infections caused by Mycoplasma species in poultry, cows, and pigs result in multimillion losses in the USA and Europe. There is no effective vaccination against many Mycoplasmas that infect pets, humans and farm animals (e.g. Mycoplasma bovis cow infection). Furthermore, most Mycoplasmas are difficult to grow in axenic culture, requiring a complex media that includes animal serum. Consequently, even in those cases in 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.

 

Based on our extensive system biology knowledge of M. pneumoniae and on cutting-edge synthetic biology methodologies, we will design a universal Mycoplasma chassis that can be deployed as single or multi-vaccine in a range of animal hosts. We envision an iterative workflow that is (whole-cell) model-driven and relies on a range of genome-editing and transplantation tools, circuit (re-)design and chassis plug-in as well as on assessment of vaccine performance in pigs in an industrial setting. The chassis will be free of virulence determinants from M. pneumoniae and will be optimized for fast growth in a serum-free medium. Using this chassis, we will express heterologous antigens from one or more pathogens (i.e. Mycoplasma and virus) and biological adjuvants to create a targeted vector vaccine.

 

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). Last but not least, we will ensure that foreseeable risks are avoided, all ethical issues are handled in a transparent manner, and that our results and their implications are disseminated effectively and communicated efficiently with the European public.

    

Pig on a farm

Subprojects

Optimization of Large-Scale Production

A hallmark of the synthetic biology is the model-driven development of circuits, chassis and processes. We will link and embed the dynamic model of central metabolism into a whole-cell constraint-based framework. By incorporating the dynamic metabolic model, we will be able to study the effects of synthetic gene circuits designed to increase growth of M. pneumoniae. Furthermore, we will use the model to identify and test new medium components that could enhance growth rate. The model will be experimentally validated in dedicated bioreactor growth experiments under defined sets of conditions. Measurement of well-chose fluxes will allow capturing the main metabolic features. We will use the genome-scale metabolic models developed to algorithmically generate minimal media “candidate” formulations. We will deploy a series of constraint-based analysis methods to systematically query and ascertain possible flux distributions and genetic construction that can possibly lead to improved growth rates without loss of the other desired functions. The ranked configurations will be assessed under a number of criteria for fitness, feasibility, gain, etc. for subsequent implementation.

Chassis Engineering

The main objective of MycoSynVac is to rationally design a non-virulent M. pneumoniae to obtain a universal chassis optimized to grow in a serum-free medium in bioreactors. We have identified main virulence and pathogenicity factors in M. pneumoniae. We will identify other putative pathogenic and virulence factors by comparing the essentiality of all M. pneumoniae genes with the sequence variability of 22 clinical isolates of M. pneumoniae. These genes will be validated by cell infection assays. M. pneumoniae has an 8-hour doubling time similar to that of other species used for vaccination. However, there are other Mycoplasma species that divide faster. Decreasing the doubling time will significantly improve the industrial efficiency per fermentor. We will follow three approaches to increase growth rate.

Vaccine Design

We will modify existing methods of genome transplantation used for Mycoplasma species. The major challenge in the transplantation procedure is to avoid the destruction of the incoming genome by the specific endonucleases expressed in the recipient cell. Although in MycoSynVac we will work only with strains whose genome has been sequenced, we will develop a pipeline that could be used for any new strain or target species. Adhesins play a crucial role in the primary steps employed by Mycoplasmas while interacting with their host eukaryotic cells using specific mammalian membrane receptors. The physical association of Mycoplasmas with the host cell surface is the basis for the development and persistence of disease, as well as for triggering an immune response. We will do a genome comparison analysis of available Mycoplasma species to identify all putative adhesin genes, and then select those from two target Mycoplasma species. We will replace the main M. pneumoniae adhesins by the counterparts from the three species and test the adhesion and infection properties in in vitro cell culture and/or tracheal assays. Using the genome engineering tools, we will clone and surface-express the selected chimaeric proteins and adjuvants in the chassis. We will then check by western and immunofluorescence if these are recognized by the serum of infected animals.

Exploitation

We will analyze how to bring the product to market after efficacy of the vaccine vector has been shown in studies. We will follow a strict plan  regarding steps and dependencies including RACI (Responsible, Accountable, Consulted, Informed) and SIPOC (Suppliers, Inputs, Process, Output and Customers) of the whole process, going from idea/lead to product. There are quite a number of unmet needs from a Mycoplasma vaccine perspective. The first is to have an effective vaccine against mastitis. Specifically, these should be against M. bovis, the most common cause of Mycoplasma mastitis and estimated to be responsible for about 50% or more of the cases of mastitis caused by mycoplasma, as well as against M. bovigenitalium, M. canadense, M. californicum and M. alkalescens, which also affect cattle. Another gap is an efficacious vaccine against contagious agalactia, caused by M. agalactiae, a disease of sheep and goats that is characterized by mastitis, arthritis and keratoconjunctivitis and with devastating effect on the Mediterranean sheep and goat dairy industry. In addition to vaccines, the chassis generated here could have other uses, such as for instance to generate a delivery system for therapeutical applications (e.g. in cell reprogramming, cell therapy, for antibiotics, etc.).

 

Responsible Research and Innovation

In MycoSynVac we aim to carry out the research and technical development in a responsible manner, following the concept of RRI (Responsible Research and Innovation). To do so we have two partner institutions on board who focus particularly on potential bioethical issues, organize a series of focus groups and Science Cafes with members of the public, hold interviews with relevant stakeholders, produce adequate science communication material (short documentary film, a science game, high school teaching package etc) and prepare material for later risk assessment of the intended products. The feedback we got from our numerous public dialogue events have been presented and discussed within the project consortium to raise awareness within the scientific community about how MycoSynvac is seen by citizens and consumers. 

 

Impact

Environmental and social impacts

An effective vaccine for livestock will increase animal welfare, decrease disease management expenses and reduce the environmental footprint of food production.

Mycoplasma infection in animals cause pain and can lead to stunted development or even death. For this reason, preventing the disease can help to improve animal welfare. Furthermore, healthy animals can increase profit for farmers and the food industry, because healthy animals produce greater yields. Moreover, happy animals lead to happy consumers, because consumers are becoming increasingly aware of animal welfare in food production.

An effective vaccine means sanitary cull of infected animals can be prevented or reduced and thus prevent economic losses for farmers. Any potential efficiency gains in the production chain are also likely to benefit consumers in terms of price. Moreover, MycoSynVac is in favor of promoting accessibility and adoption rates in less well-off regions, where a successful vaccine would have the biggest impact on both animal and human welfare, and where adoption of new technologies is vital if agriculture is to remain competitive.

Last but not least, decreasing Mycoplasma infection rates may have important beneficial effects for the environment and human health. An effective vaccine would indeed help to limit the widespread use of antibiotics in livestock and its consequences. Antibiotics used in animals end up in our food, which alter our microbiota and affects our health, and are also a major cause for the development of antibiotic resistance, which is one of the biggest threats to global health.
An effective vaccine would thus represent an important scientific breakthrough. Biosafety risks and ethical concerns are assessed to ensure that MycoSynVac derived products are safe and socially acceptable. MycoSynVac also pays attention to hopes and concerns by citizens and aims to transparently communicate relevant information to the public.

More information in the ethical framework section

Impact on reinforcing cooperation of industry with academia

The work that should be carried out during the project requires a strong collaboration between industrial and academic leaders. This will lead to valuable strategic links that will aid translational research and its industrial development.

At the same time, the project also improves innovation capacity and integration of new knowledge, especially for two of the project partners: MSD and ATG. Both would be able to improve their services, include new products on their pipeline and become or continue being a major player in the field. 

Impact on synthetic biology

The expected impacts on synthetic biology are related to our work on:

  • Developing a Whole-cell mode​​

  • Design of a bacterial cellular chassis​

  • Genome engineering