In Depth

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 difticult to grow in axenic culture, 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.

Based on our extensive systems 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 en 'sion 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 perhormanoe pigs in an industrial setting. The chassis will be free of virulence determinants from M. pneumoniae and will be optimized forfast 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 adjullants 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.



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.

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.

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.

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.).