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Induction of Protective Immunity in Cattle Against Infection with Fasciola Hepatica by Vaccination with Cathepsin L Proteinases and with Hemoglobin

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Induction of Protective Immunity in Cattle Against Infection with Fasciola Hepatica by Vaccination with Cathepsin L Proteinases and with Hemoglobin INFECTION AND IMMUNITY, Dec. 1996, p. 5066–5074 Vol. 64, No. 12 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology Induction of Protective Immunity in Cattle against Infection with Fasciola hepatica by Vaccination with Cathepsin L Proteinases and with Hemoglobin 1 1 2 2 JOHN P. DALTON, * SHARON MCGONIGLE, TIMOTHY P. ROLPH, † AND STUART J. ANDREWS School of Biological Sciences, Dublin City University, Dublin 9, Republic of Ireland,1 and Mallinckrodt Veterinary Ltd., Harefield, Uxbridge, Middlesex UB9 6LS, United Kingdom2 Received 8 July 1996/Returned for modification 9 September 1996/Accepted 2 October 1996 Two cathepsin L proteinases, cathepsin L1 and cathepsin L2, secreted by liver flukes may be involved in tissue penetration, nutrition, and protection from immune attack. To ascertain the immunoprophylactic potential of these proteinases, and of another molecule, liver fluke hemoglobin (Hb), we performed vaccine trials in cattle. In the first vaccine trial various doses of cathepsin L1 were tested. The mean protection level obtained was 53.7%. In a second vaccine trial cathepsin L1 and Hb elicited 42.5 and 43.8% protection levels, respectively, while a combination of the two molecules induced a significantly higher level of protection (51.9%). Cathepsin L2 was not examined alone; however, vaccination of cattle with a combination of cathepsin L2 and Hb elicited the highest level of protection (72.4%). The animals that received cathepsin L1-Hb or cathepsin L2-Hb showed reduced liver damage as assessed by serum glutamic dehydrogenase and gamma-glutamyl transferase levels. Furthermore, a reduced viability was observed for fluke eggs recovered from all vaccine groups. This anti-embryonation effect of vaccination was particularly evident in the group that received cathepsin L2-Hb where >98% of the eggs recovered did not embryonate to miracidia. Although all vaccine preparations induced high antibody titers which were boosted following the challenge infection, there was no correlation between antibody titers and protection. The results of these trials demonstrate that cathepsin Ls and Hb could form the basis of a molecular vaccine that would not only reduce parasite burden but would also prevent transmission of liver fluke disease. The trematode Fasciola hepatica is a causative agent of liver showed that the two enzymes differed in their specificities for fluke disease, or fascioliasis, in mammals. Liver fluke disease hydrolyzing peptide bonds (12). Therefore, the enzymes were of agricultural animals, such as cattle and sheep, has a world- termed cathepsin L1 (9, 27) and cathepsin L2 (9, 12). wide distribution and results in large economic losses in many Another antigen secreted by adult flukes into culture me- agriculture-dependent countries. Recent reports indicate that dium was isolated by McGonigle and Dalton (18). Spectromet- F. hepatica is also a major human pathogen (14). Infection is ric studies revealed that this molecule contained a heme group primarily acquired by the ingestion of vegetation on which and was a liver fluke hemoglobin (Hb) (10, 18). Hb is impor- metacercariae are encysted. Within the duodenum the meta- tant in the aerobic respiration of immature flukes within the cercariae excyst, penetrate the intestinal wall, and then migrate liver mass. However, in adult flukes, which have a largely via the peritoneal cavity to the liver. Here, the immature flukes anaerobic metabolism, it is more essential for oxygen-depen- spend 7 to 12 weeks migrating through the tissue causing dent functions such as egg production (3, 17, 30). extensive hemorrhage and fibrosis before they move into the As each of the above-described molecules may be involved bile ducts and mature to adults. Mature flukes produce nu- in processes that are crucial to the development and survival of merous eggs which are deposited on pastures in the feces. the parasite, we considered them potential targets at which a Miracidia hatch from the eggs and penetrate an intermediate molecular vaccine could be directed. In the present study we snail host, from which the infective metacercariae erupt after a report the results of vaccine trials carried out in cattle to test developmental period. the immunoprophylactic potential of cathepsin L1, cathepsin Dalton and Heffernan (11) showed, using gelatin substrate L2, and Hb, either alone or in combinations. The first trial polyacrylamide gel electrophoresis (PAGE), that when imma- demonstrated that vaccination of cattle with cathepsin L1 can ture and mature flukes are maintained in vitro they secrete induce high levels of protection (mean, 53.7%) against a het- cysteine endoproteinases into the culture medium. Several erologous challenge infection of metacercariae of F. hepatica. functions were suggested for these enzymes, including facilita- A subsequent vaccine trial confirmed the protective nature of tion of migration through host tissue (11), the acquisition of nutrient (11, 27), and evasion of host immunity (8, 11, 26). Two cathepsin L1 and showed that Hb could also elicit protective cysteine proteinases were isolated and both were characterized immune responses. Vaccination with a combination of these as having physicochemical properties in common with mam- molecules elicited a higher level of protection than did vacci- malian lysosomal cathepsin L proteinases (12, 27). Kinetic nation with either alone. Most significantly, a 72.4% protection studies with specific fluorogenic peptide substrates, however, against challenge was achieved by vaccination with cathepsin L2 and Hb. The surviving flukes in this vaccinated group were more stunted in their growth than those recovered from the * Corresponding author. Phone: 353-1-7045407. Fax: 353-1-7045412. unvaccinated control animals. Consequently, these vaccinated Electronic mail address: [email protected]. animals showed little liver damage, as assessed by their serum † Present address: Animal Health Discovery, Pfizer Central Re- glutamic dehydrogenase (GDH) and gamma-glutamyl trans- search, Pfizer Ltd., Sandwich, Kent CT13 9NJ, United Kingdom. ferase (GGT) levels. Furthermore, .98% of the eggs pro- 5066 VOL. 64, 1996 VACCINATION OF CATTLE AGAINST F. HEPATICA INFECTION 5067 duced by the flukes that did mature in these vaccinated animals did not embryonate to miracidia. MATERIALS AND METHODS Source and purification of parasite antigens. Mature F. hepatica flukes were removed from the bile ducts of infected bovine livers obtained from an abattoir in Ireland. The flukes were washed six times in phosphate-buffered saline (PBS), pH 7.3, and incubated for 16 h at 378C in RPMI 1640, pH 7.3, containing 2% glucose, 30 mM HEPES (N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid), and 25 mg of gentamycin per ml. Following incubation, the medium was removed and centrifuged at 14,900 3 g for 30 min, and the supernatant, termed excretory- secretory (ES) products, was then collected and stored at 2208C (11). F. hepatica cathepsin L1 and cathepsin L2 were isolated from the ES products and purified by gel filtration chromatography on a Sephacryl S200HR column and ion exchange chromatography on a QAE Sephadex column as previously detailed (12, 27). F. hepatica Hb was eluted in a high-molecular-sized protein peak by gel filtration on Sephacryl S200HR as described before (18). Protein concentrations were determined by the MicroBCA protein assay (Pierce, Rock- ford, Ill.). Formulation, preparation, and administration of vaccines. The purified anti- gens were dialyzed against distilled water, freeze-dried, and stored at 2208C. On the day before vaccine administration the powder was reconstituted in PBS, and the vaccines were formulated by mixing 1 ml of the reconstituted antigen with an equal volume of Freund’s complete adjuvant (FCA) or Freund’s incomplete adjuvant (FIA) (Sigma Chemical Co., Poole, United Kingdom). The mixture was emulsified by sonication (7 3 30 s bursts, duty cycle 0.7). FIG. 1. Polyacrylamide gel analysis of isolated F. hepatica ES molecules used Vaccination of cattle. Two vaccine trials were performed with cattle. All in vaccine trials. (A) SDS reducing 10% PAGE analysis of total adult fluke ES animals were fluke free and housed indoors to preclude helminth infection. In products (lane 1), purified 27-kDa cathepsin L1 (CL1) (lane 2), and purified the first trial 18 female Holstein-Friesian calves, aged 4 to 6 months, were 29.5-kDa cathepsin L2 (CL2) (lane 3); (B) non-SDS native 10% PAGE analysis randomly allocated into five groups of similar mean weight. Group 1 animals of total adult fluke ES (lane 1) and purified hemoglobin (lane 2). (n 5 4) received 10 mg of cathepsin L1 per immunization, Group 2 animals (n 5 4) received 50 mg of cathepsin L1 per immunization, Group 3 ani- mals (n 5 3) received 200 mg of cathepsin L1 per immunization, Group 4 animals (n 5 3) received 500 mg of cathepsin L1 per immunization, and Group 5 control eggs were recovered from the gall bladder, all eggs were considered in the animals (n 5 4) received 150 mg of horse spleen ferritin (HSF). Each animal calculation, and in cases where many eggs were recovered, 300 eggs were taken received a first injection of 1 ml of the vaccine preparation formulated in FCA as representative. into the biceps femoris of each hind leg on day 0. A second injection on day 28 Analysis of the antibody responses of cattle by immunoblotting. Mature F. he- (week 4) and a third on day 56 (week 7), both formulated in FIA, were given into patica ES products were separated on sodium dodecyl sulfate (SDS) reducing each side of the back between the biceps femoris and gluteus medius. 10% polyacrylamide gels for analysis of anti-cathepsin L1 and anti-cathepsin L2 In the second trial, the effects of F. hepatica cathepsin L1, cathepsin L2 and responses (12, 27) or on non-SDS native 10% polyacrylamide gels for analysis of Hb, and other combinations of these, were compared. Thirty-five male (castrated Hb responses (18) and transferred to nitrocellulose filters (0.45-mm pore size, or entire) Holstein-Freisian calves, aged 1 year, were randomly allocated into five BA85; Schleicher & Schuell) by using an Atto semidry blotting system.
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