Developing a 'Validation Portfolio' to Exploit Key Virulence Proteins in Fasciola Species for Parasite Control

Liver fluke impose a major impact on global economies through the reduction of animal welfare and agricultural productivity. Whilst Fasciola hepatica is most common in temperate regions, Fasciola gigantica is one of the most important parasites of ruminants in the tropics. Although current control measures rely on anthelmintics, their use is financially unsustainable in developing countries and is further undermined by increasing drug resistance, leaving profound deficiencies in liver fluke control. Although a number of putative vaccine target candidates have been tested in small and large animals and shown some success, none have provided protection levels adequate for commercialization. Motivated by recent successes in RNAi and proteomics methods developed in our laboratories, this project proposes to optimise, validate and interface gene silencing and proteomic platforms for various liver fluke life stages, and to exploit these technologies as a drug/vaccine target validation portfolio. To do this, we propose the generation of a F. gigantica transcriptome to kick-start a reverse-chemotherapy/reverse-vaccinology programme whereby the superfamilies of leading virulence proteins (control targets) in fluke are probed for gene variation across pooled geographical isolates and via sampling of liver fluke adults and juveniles; variation qualified using transcriptomics would be quantified using subproteomic methods. Target proteins present in the majority of pooled geographical isolates and individuals would be validated using an integrated gene silencing/proteomics platform to select the most promising candidates. The leading validated candidates (i.e. population-abundant candidates that are functionally important and which are not compensated for by other pathways) would be tested for vaccine candidature in animals challenged with F. gigantica infections. These efforts will provide validated vaccine candidates/drug targets for future control strategies.

The tropical liver fluke parasite Fasciola gigantica is one of the most important worm infections of livestock in Asia and Africa. The disease inflicts very significant losses especially in livestock in India, with infection levels reaching 55% in some regions. Recently-estimated costs for liver fluke induced losses associated with livestock in India fall within the range of US$1.95-4.78 billion per year. This is a huge burden to the largely agricultural Indian economy and directly impacts the productivity of large and small farm holdings alike, negatively impacting individual farmers and their families. Liver fluke infected buffalo show reduction in general health, weight gain, feed conversion efficiency and reproduction. The infective worm stage encysts on vegetation such that host animals inadvertently consume the cysts during grazing. Once in the gut, the juvenile worms hatch, burrow through the gut wall and penetrate the liver, ending up as adult worms in bile ducts where they feed on host blood, significantly diminishing the health of the animal and greatly reducing agricultural productivity. In the developed world liver fluke is mainly controlled using the chemical triclabendazole (TCBZ). This drug is an Achilles heel of liver fluke control as it is the only drug that kills both adult and pathogenic juveniles. New control strategies are urgently needed as anthelmintic resistance in parasitic worm populations is spreading globally, and agricultural communities in many parts of the developing world simply cannot afford short-lived, anthelmintic-based treatment options. Therefore, there is an urgent need to develop a cost effective, single-treatment control strategy based on vaccination. Numerous vaccine trials have been completed in liver fluke, many showing some success, but none have provided protection levels adequate for immediate commercialization. The problem is that since most leading vaccine candidates exist in large, related groups of proteins within parasites, it has been impossible to determine which is the best candidate from the group such that up until now, these decisions have not been subject to rigorous validation. To this end, this international collaborative project between laboratories in India and the UK will, for the first time, incorporate new fast throughput nucleotide and protein technologies to validate the candidate proteins and thereby assess if these leading vaccine candidates can be used to treat liver fluke parasites in different parts of the world, a key to successful commercial production. Once a vaccine target is confirmed as widely present in liver fluke populations, we will use another new technology called reverse genetics that can switch-off (or silence) a target in a parasite, and then confirm if this target is essential for parasite survival. If confirmed as important to parasite survival, we will subsequently use yeast laboratory cultures to produce the candidates for vaccine trials in livestock in India. Since this is a multi-discipline and multi-laboratory project, we have formed an International Science Advisory Board (ISAB) in order to manage and co-ordinate progress and to trouble-shoot. Members of the ISAB include a representative from a large Animal Pharmaceutical Company, a parasitologist with many years of industrial experience, a leading world authority on liver fluke vaccination and a representative from the industrial Biotechnology sector in India. The programme addresses the mission of the BBSRC, including treatment of diseases of livestock, application of new technologies and international collaboration. Validated trial vaccine(s) from this programme would form the basis for commercial development for treating liver fluke disease, and our technology strategy is directly transferable to other helminth parasites of animal and humans.