Terry W. Spithill

Progress in Development of Liver Fluke Vaccines

Infection of ruminants by Fasciola spp continues to cause large economic losses worldwide. Recent results from several laboratories have demonstrated that animals can be significantly protected against infection by vaccination with defined Fasciola antigens. Apart from reducing fluke burdens, some vaccines can elicit a concurrent reduction in parasite egg production. The expectation of a commercially feasible vaccine that might also reduce parasite transmission in the field is now realistic, although major hurdles still exist. Here, Terry Spithill and John Dalton review the results of several recent vaccine trials and discuss the future prospects for vaccine development.

Evaluation of antigens of Fasciola gigantica as vaccines against tropical fasciolosis in cattle

Vaccine trials were conducted in Brahman cross cattle evaluating the efficacy of 4 native antigens purified from adult Fasciola gigantica flukes, and 1 recombinant F. gigantica antigen, as vaccines against tropical fasciolosis. The antigens tested were native glutathione S-transferase, cathepsin L, paramyosin, fatty acid binding protein (FABP), and a recombinant FABP expressed in E. coli, and were formulated in 1 or more of several adjuvants (Quil A, Squalene Montanide 80, MF59-100, Auspharm, NAGO, polylactoglycolide microspheres, Algammulin, DEAE, Freunds). Vaccination induced low, moderate or high antibody titres to the various antigens which were dependent on the adjuvant. Low but significant reductions in fluke burdens (31%, P < 0.026) and fluke wet weight (36%, P < 0.041) were only observed in cattle vaccinated with the native FABP in Freunds adjuvant. There was no correlation between total antibody titres to FABP and protection. The protection observed in cattle vaccinated with native FABP of F. gigantica supports the notion that this class of proteins is a useful target for protection of animals against Fasciola and extends the efficacy of FABPs to the tropical liver fluke. This is the first report of vaccination of cattle against F. gigantica with a purified protein.

Protection of cattle against Fasciola hepatica infection by vaccination with glutathione S-transferase

Glutathione S-transferase (GST) from the liver fluke Fasciola hepatica was assessed as a vaccine immunogen in cattle in a number of immunological adjuvants. Significant reductions in fluke burdens (49-69%) were only observed in cattle vaccinated with GST in Quil Alsqualene Montanide (SM) and PLG microspheres in SM but there was no correlation between anti-GST IgG titres and protection. In separate experiments, animals vaccinated with GST in Quil AlSM were still significantly protected (48%, P < 0.05) 6 months after boosting and no significant differences in protection were seen when the metacercarial challenge was given over 1 month instead of as a single bolus. Inhibition of GST enzyme activity in vitro by cattle antisera did not correlate with reduced fluke burdens.

THE SENSITIVITY AND SPECIFICITY OF TWO METHODS FOR DETECTING FASCIOLA INFECTIONS IN CATTLE

Counts of Fasciola spp. eggs in faeces and measurements of antibody concentration to the excretory/secretory antigens of Fasciola spp. by ELISA were related to the numbers of flukes in the livers of 92 cattle killed in the abattoirs of Hanoi City, Vietnam. In this population, about 22% of the cattle had no flukes, another 22% had between 1 and 10 flukes, 44% between 11 and 100 flukes and 12% had more than 100 flukes in their livers. Of the 14 animals less than 2 years of age, only three were infected. At 2 years of age the mean number of flukes per liver was 10 whereas at 3 years and older, the mean varied between 60 and 80 flukes. Prevalence of infection was 78.3%. No eggs of Fasciola spp. were detected in the faeces of one third of infected cattle and 60% of the counts were less than 100 eggs per gram. The sensitivity of the egg counting method was 66.7% and specificity 100%, overall accuracy was 73.9%. Corresponding values for the ELISA method were 86.1, 70 and 82.6%, respectively. The positive and negative predictive values for the egg counting method were 100 and 45.5% and for the ELISA method were 91.2 and 58.3%, respectively.

The Evolution of Enzyme Specificity in Fasciola spp.

Fasciola spp., commonly known as liver fluke, are significant trematode parasites of livestock and humans. They secrete several cathepsin L-like cysteine proteases, some of which differ in enzymatic properties and timing of expression in the parasites life cycle. A detailed sequence and evolutionary analysis is presented, based on 18 cathepsin L-like enzymes isolated from Fasciola spp. (including a novel clone identified in this study). The enzymes form a monophyletic group which has experienced several gene duplication events over the last ~135 million years, giving rise to the present-day enzymatic repertoire of the parasite. This timing of these duplications appears to correlate with important points in the evolution of the mammalian hosts. Furthermore, the dates suggest that Fasciola hepatica and Fasciola gigantica diverged around 19 million years ago. A novel analysis, based on the pattern of amino acid diversity, was used to identify sites in the enzyme that are predicted to be subject to positive adaptive evolution. Many of these sites occur within the active site cleft of the enzymes, and hence would be expected to lead to differences in substrate specificity. Using homology modeling, with reference to previously obtained biochemical data, we are able to predict S2 subsite specificity for these enzymes: specifically those that can accommodate bulky hydrophobic residues in the P2 position and those that cannot. A number of other positions subject to evolutionary pressure and potentially significant for enzyme function are also identified, including sites anticipated to diminish cystatin binding affinity.

Cloning and Expression of the Major Secreted Cathepsin B-Like Protein from Juvenile Fasciola hepatica and Analysis of Immunogenicity following Liver Fluke Infection

ABSTRACT The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.

Immunological approaches for the control of fasciolosis

The immunological relationship between liver flukes and their mammalian hosts is being unravelled by in vivo and in vitro studies. Vaccine studies in cattle and sheep with purified antigens (fatty acid binding protein, FABP; glutathione S-transferase, GST; cathepsin L, CatL; hemoglobin) have shown that high reductions in worm burdens (31-72%) and egg production (69-98%) can be achieved, raising the realistic possibility that immunological control of Fasciola infection is a commercially achievable goal. Combination vaccines may also be feasible since a cocktail of CatL and hemoglobin elicits a significant 72% protection in cattle. Analysis of immune responses to Fasciola during infection in ruminants suggests that chronic infection correlates with a type 2 helper T cell response, implying that type 1 helper T cell responses are down-regulated in fasciolosis. Recent results studying the resistance of Indonesian Thin Tail (ITT) sheep to F. gigantica have shown that this breed exhibits high innate (or rapidly acquired) resistance to infection and acquires a higher level of resistance after a primary challenge. Initial studies suggest that the resistance of ITT sheep to F. gigantica may be determined by a major gene. Merino sheep also acquire resistance to F. gigantica. In contrast, ITT and Merino sheep do not exhibit resistance to F. hepatica. These results suggest that there are fundamental differences between these two species of Fasciola in the biology of their interaction with the sheep immune system. In vitro studies on immune mechanisms of killing of juvenile fluke have shown that juvenile larvae of F. hepatica are susceptible to antibody-dependent killing by activated rat macrophages in vitro which is mediated by nitric oxide. Future studies on the immune effector mechanisms expressed by resistant sheep which control infection by F. gigantica will lead to new knowledge which may allow the design of more effective vaccines for fasciolosis.

Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens.

Liver flukes produce cathepsin B and cathepsin L in their excretory-secretory material. These proteases are proposed to be key virulence factors for parasite infection, and are therefore targets for vaccination. Cathepsin B is predominately released in the juvenile stage of the life cycle, while different cathepsin Ls are released throughout the cycle. Three proteases (cathepsin L5, cathepsin L1g and cathepsin B) were expressed in yeast from cDNA clones isolated from adult, metacercariae and newly excysted juvenile flukes respectively. Each was used singly or in combination to vaccinate rats that were subsequently challenged with Fasciola hepatica metercercariae. Each protein induced an immune response, and all groups vaccinated with recombinant protein yielded significantly fewer and smaller flukes than the control group. Maximal protection of 83% was seen in the group vaccinated with cathepsin B and cathepsin L5 in combination.

Identification of species, strains and clones of Leishmania by characterization of kinetoplast DNA minicircles

Molecular analysis of kinetoplast deoxyribonucleic acid (kDNA) minicircles has permitted the genotypic characterization of pathogenic isolates of Leishmania species. The apparent size in agarose gels of unit-length minicircles released by EcoRI digestion of kDNA networks is not conserved during speciation in this genus since the minicircles of strains and clones of L. major are smaller (710 base pairs, bp) than those found in certain strains of L. mexicana subspecies (820 bp), L. donovani (825, 865 bp) or L. tropica (900, 930 bp). EcoRI-cut minicircles within any one species of Leishmania are heterogeneous in mobility during electrophoresis in acrylamide gels. Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of closely related clones and strains within a given species. Southern blot hybridization reveals that overall minicircle sequence homology is conserved among clones and strains of one species (L. major or L. tropica) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species-specific diagnostic test for the identification of Leishmania isolates. The analysis of kDNA from two L. tropica strains isolated at 14 year intervals from a patient with leishmaniasis recidivans has shown that the two strains are closely related, suggesting that the individual suffered the cutaneous disease as a result of a resurgence of the same parasite which caused the initial infection. The differences in the properties of kDNA from the L. tropica and L. major strains studied support the taxonomic separation of L. tropica and L. major into distinct species.

Liver fluke vaccines in ruminants: strategies, progress and future opportunities

The development of a vaccine for Fasciola spp. in livestock is a challenge and would be advanced by harnessing our knowledge of acquired immune mechanisms expressed by resistant livestock against fluke infection. Antibody-dependent cell-mediated cytotoxicity directed to the surface tegument of juvenile/immature flukes is a host immune effector mechanism, suggesting that antigens on the surface of young flukes may represent prime candidates for a fluke vaccine. A Type 1 immune response shortly after fluke infection is associated with resistance to infection in resistant sheep, indicating that vaccine formulations should attempt to induce Type 1 responses to enhance vaccine efficacy. In cattle or sheep, an optimal fluke vaccine would need to reduce mean fluke burdens in a herd below the threshold of 30-54 flukes to ensure sustainable production benefits. Fluke infection intensity data suggest that vaccine efficacy of approximately 80% is required to reduce fluke burdens below this threshold in most countries. With the increased global prevalence of triclabendazole-resistant Fasciolahepatica, it may be commercially feasible in the short term to introduce a fluke vaccine of reasonable efficacy that will provide economic benefits for producers in regions where chemical control of new drug-resistant fluke infections is not viable. Commercial partnerships will be needed to fast-track new candidate vaccines using acceptable adjuvants in relevant production animals, obviating the need to evaluate vaccine antigens in rodent models.