Using C. elegans to produce proteins from parasitic nematodes for research and therapeutic use

Several species of parasitic worm infect human beings and cause debilitating diseases such as elephantiasis and river blindness. To survive the parasites need to escape the human immune system, and so they have evolved to secrete proteins that interfere with immune responses. If we could understand how these proteins work we could use the knowledge to treat diseases caused by an overactive immune system such as arthritis, eczema, asthma and irritable bowel syndrome. Towards this, we have made significant progress in understanding the mechanism of action of one of these proteins called ES-62. This progress has been made possible because we can make this protein in the laboratory from Acanthocheilonema viteae, a parasitic nematode worm that grows in gerbils. However, we need to sacrifice hundreds of lab rodents each year for this work, and it is also both expensive and time consuming. Moreover, a much larger number of animals would have to be sacrificed to produce enough ES-62 to be able to undertake a clinical trial. We propose to replace the use of the rodents by producing this protein using a free-living nematode called Caenorhabditis elegans. This worm can be grown in a lab in large amounts simply by culturing it with bacteria as a food source. C. elegans is used widely for biological research and there are established methods to introduce genes into it capable of producing proteins from other animals. This works particularly well with proteins from related animals such as other nematode worms. Furthermore we have developed a new method that should facilitate ready purification of ES-62 from C. elegans and we will check to ensure that the protein we produce has the same properties as the protein made from the parasitic worms. Our proposal is not only to directly replace laboratory animals in the manufacture of ES-62 but also to show that it is possible to produce the protein in large enough quantities to be used for therapeutic and commercial purposes. Furthermore, the technology will not be limited to manufacturing ES-62 as our strategy also has the potential to produce immunosuppressive proteins from worms that are human parasites such as those that cause the diseases referred to above. Overall our research will enable proteins from these parasites to be produced and characterised, leading to further understanding of how the immune system can be modified for therapeutic purposes.

Parasitic nematodes have evolved methods to evade the human immune system, including the secretion of proteins that suppress host inflammatory responses. We have made significant progress in understanding the properties of one such protein, ES-62, which is produced by the rodent filarial nematode Acanthocheilonema viteae. ES-62 has been shown to be protective in mouse models of inflammatory diseases such as rheumatoid arthritis and asthma, suggesting potential therapeutic use. This progress has been possible because we can maintain A. viteae in gerbils and ticks. However, we need to sacrifice hundreds of lab rodents each year for this work, and it is expensive and time consuming. Furthermore, many more animals would have to be sacrificed to produce enough ES-62 for a clinical trial. We propose to replace lab rodents by producing ES-62 in the free-living nematode Caenorhabditis elegans. This nematode can be grown in the laboratory in large amounts simply by culturing with a bacterial food source. Moreover, C. elegans can modify proteins with phosphorylcholine (PC)-N-glycan structures as found on ES-62. This modification is important for the immunomodulatory activity of ES-62. There are several established methods to make transgenics in C. elegans to direct the expression of recombinant protein. Others have used C. elegans to produce proteins from parasitic nematodes in a limited manner before but we have developed a novel strategy to improve production and purification of recombinant proteins. The resulting proteins will be tested for PC modification and immunomodulatory activity. Our proposal is not only designed to directly replace lab animals in the manufacture of ES-62 but also to show that quantities large enough for therapeutic and commercial purposes can be manufactured. Furthermore, it can be applied to producing immunosuppressive proteins from other nematode species including human parasites, therefore potentially offering further substantial benefits.

The most important impact of this research will be to replace the animals in which A. viteae have to be grown in order to make the protein ES-62. We will manufacture ES-62 using transgenic C. elegans worms, which can be easily cultured in the laboratory without the need for a host and can be frozen to make permanent stocks.

We envisage wider long-term impacts of this study in several spheres:

1. Academic research in the ES-62 field: Decreasing the time and cost of producing ES-62 will accelerate current research into this well studied protein.

2. Academic research into other parasitic proteins: This technique will enable researchers to express large quantities of proteins secreted by human parasitic nematodes, either replacing current animal models, or for nematode for which there are no animal models, allowing production of proteins with correct folding and post-translational modification in the laboratory where it was not previously possible.

3. Academic and Clinical Immunology: Acceleration of ES-62 research will enable further understanding of how parasitic nematodes modulate the immune system, and how that might be used for therapeutic purposes. There is currently great interest in this approach.

4. Biopharmaceutical Industry: Commercial production of ES-62 may be a viable business proposition, especially if recombinant ES-62 is proven to have clinical use, uncovering a huge new manufacturing market. The technology we are developing could be used for a number of protein products, and may improve yields of currently manufactured recombinant proteins. The biopharmaceutical industry is a part of the growing specialist manufacturing base of the UK.

5. Disease sufferers. Autoimmune diseases for example affect up to 10% of the population and the numbers in the UK are increasing with an ageing population. A long-term impact of this study would be to alleviate suffering from these diseases. Furthermore, the potential use of ES-62 based on mouse model studies extends to allergic diseases and intriguingly we also have some data, which suggests it could protect against the development of one of the UK's biggest killers, cardiovascular disease.

Pathways to Impact

This pilot study aims to achieve a proof of principle for our technology. As outlined in the Case for Support and Communication Plan, we will seek relevant industrial, academic and clinical partners to apply for follow-on funding from the biopharmaceutical industrial sector, BBSRC, MRC and the Technology Strategy Board. These collaborations will lead to achievement of the impacts detailed above. Communication of our success in replacing lab rodents will encourage others to use our techniques and replace more mammals currently used in medical research.