Parasitol Res (2013) 112:3653–3659 DOI 10.1007/s00436-013-3553-6

ORIGINAL PAPER

Immunization with recombinant leucine aminopeptidase showed protection against Fasciola gigantica in mice Narin Changklungmoa & Pornanan Kueakhai & Suda Riengrojpitak & Kulathida Chaithirayanon & Pannigan Chaichanasak & Narin Preyavichyapugdee & Pathanin Chantree & Veerawat Sansri & Tadashi Itagaki & Prasert Sobhon

Received: 18 June 2013 / Accepted: 22 July 2013 / Published online: 7 August 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Leucine aminopeptidase (LAP) is expressed in all stages of Fasciola gigantica and, hence, is considered as a potential vaccine candidate. In this study, we have tested a vaccine potential of LAP and the types of immune responses it elicited in vaccinated mice. Recombinant F. gigantica leucine aminopeptidase (rFgLAP) was expressed in Escherichia coli, BL21 (DE3). The imprinting control region mice subcutaneously immunized with 50 μg of rFgLAP combined with Freund's adjuvant (n=10) exhibited a significant reduction in worm recoveries when compared with non-immunized and Freund's adjuvant controls at 60.8 and 64.3 %, respectively, and both T helper (Th)1 and Th2 humoral immune responses were elicited in the hosts as reflected by the levels of IgG1 and IgG2a, with Th2 predominating. The levels of IgG1- and IgG2a-specific antibodies to rFgLAP were inversely and N. Changklungmoa : P. Kueakhai : S. Riengrojpitak Department of Pathobiology, Faculty of Science, Mahidol University, Rama VI Rd, Ratchathewi, Bangkok 10400, Thailand K. Chaithirayanon : P. Chantree : P. Sobhon (*) Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Rd, Ratchathewi, Bangkok 10400, Thailand e-mail: [email protected] P. Chaichanasak Faculty of Veterinary Medicine, Mahanakorn University of Technology, 140 Cheum-Sampan Rd, Nong Chok, Bangkok 10530, Thailand V. Sansri Faculty of Physical Therapy, Mahidol University, Phutthamonthon Sai 4 Rd, Salaya, Phutthamonthon, Nakhon Pathom 73170, Thailand N. Preyavichyapugdee Faculty of Animal Science, Silpakorn University, Phetchaburi Campus, Cha-am, Phetchaburi 76120, Thailand T. Itagaki Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan

significantly correlated with the numbers of worm recoveries. The rFgLAP-vaccinated mice showed significantly reduced levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase and liver damage. These indicated that rFgLAP has a potential as a vaccine candidate against F. gigantica, whose efficacy will be studied further in economic animals including cattle, sheep, and goat.

Introduction Fasciola gigantica is a helminth parasite that causes a liver disease, fasciolosis, in ruminants, especially cattle, goat, and sheep in the tropical regions (Sinclair 1967; Behm and Sangster 1999). The disease causes substantial economic losses, including reduction in meat and milk production. Currently, fasciolosis is controlled by a treatment with anthelmintic drugs, especially triclabendazole, and environmental management. However, a long-term use of drugs does not prevent reinfection, and incidences of drug resistance have emerged (Overend and Bowen 1995; Moll et al. 2000; Brennan et al. 2007; Devine et al. 2010). Vaccination is an alternative and, possibly, a more sustainable way to control fasciolosis. Several recombinant proteins have been tried against F. gigantica and Fasciola hepatica in various animal models, including recombinant F. gigantica glutathione S-transferase in mice (Preyavichyapugdee et al. 2008), recombinant F. hepatica saposin-like protein 2 in rabbits (Espino and Hillyer 2004), recombinant F. hepatica cathepsin L in cattle (Golden et al. 2010), recombinant F. gigantica fatty acid-binding protein in buffaloes (Nambi et al. 2005), which exhibited 70–84, 81, 48, and 36 % protection, respectively. Leucine aminopeptidase (LAP) is a member of the M1 or M17 peptidase family (McCarthy et al. 2004) that cleaves N-terminal residues from proteins and peptides. LAP is expressed in all stages of the parasite and may be involved in a wide range of functions, including tissue invasion, migration, and digestion of nutrients

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(Changklungmoa et al. 2012). Hence, LAP is a crucial molecule for the parasite's invasion and survival, and it may have a potential as a vaccine candidate against fasciolosis (Acosta et al. 2008; Jia et al. 2010; Mucha et al. 2010; Skinner-Adams et al. 2010). It was demonstrated that immunization with native F. hepatica LAP induced a high level of protection (89 %) in sheep (Piacenza et al. 1999). Acosta et al. (2008) cloned and expressed the recombinant LAP in a prokaryote vector and showed that immunization with this protein resulted in a high level of protection in rabbits. More recently, Maggioli et al. (2011) showed that immunizations of the recombinant enzyme combined with different adjuvants could induce high levels of protection up to 85 % compared with the adjuvant controls. Thus, the protective efficacy of the recombinant LAP has been amply demonstrated against F. hepatica. In this report, we have demonstrated that immunization with recombinant F. gigantica LAP (rFgLAP) elicited high levels of immune responses and protection against F. gigantica in mice, and currently, we are investigating its vaccine potential in large economic animals.

uninfected, non-immunized and infected, immunized with Freund's adjuvant (Sigma-Aldrich, St Louis, MO, USA) and infected, and immunized with 50 μg of rFgLAP with Freund's adjuvant and infected. The mice were kept in steel cages in an air-conditioned room at 22–25 °C with a light–dark cycle of 12:12 h and 50–60 % humidity, at the Central Animal Facility, Faculty of Science, Mahidol University, Thailand. The immunizations were performed with 50 μg of rFgLAP mixed with adjuvant three times at 2-week intervals by subcutaneous route. The first immunization used Freund's complete adjuvant, and the second and third immunizations used Freund's incomplete adjuvant. At 2 weeks after the third immunization, each mouse was orally infected with 30 F. gigantica metacercariae. Blood samples were taken six times at day 0 (prevaccination); 2, 4, and 6 weeks (infection); and 8 and 10 weeks (termination). Four weeks after infection (at 10 weeks), mice were anesthetized by ether inhalation, and their peritoneal cavities were opened and washed thoroughly with 0.85 % NaCl solution and examined for the worms. The livers were teased in 0.85 % NaCl solution and also examined for the worms (Preyavichyapugdee et al. 2008).

Materials and methods

Worm recovery

Expression and purification of rFgLAP

The percent of protection against the parasitic infection was expressed as the percentage of reduction in the number of worm in rFgLAP-immunized group compared with nonimmunized or Freund's adjuvant control group, which was calculated as follows: . % Protection ¼ ðA−BÞ A  100

The cDNA of FgLAP gene (accession number HQ993064) was cloned from the adult F. gigantica cDNA library. The full length of FgLAP cDNA was subcloned into the pET-30b vector and transformed into Escherichia coli BL21 (DE3). The rFgLAP was expressed at 16 °C by inducing the bacteria with isopropyl-β-D-thiogalactopyranoside, purified using nickel–nitrilotriacetic acid (Ni-NTA) affinity column under native condition, and then analyzed by 12.5 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) (Changklungmoa et al. 2012).

where A represents the mean parasite recovery from challenged non-immunized or adjuvant control groups, and B represents the mean parasite recovery from rFgLAPimmunized group.

Preparation of F. gigantica metacercariae Analyses of antibody levels The F. gigantica miracidia were produced by incubating F. gigantica eggs in fresh water about 2 weeks at 26–28 °C, and after which, the miracidia were hatched from the eggs into the water. Lymnaea ollula snails were subjected to singlemiracidium infections. About 8 weeks after infection, cercariae were shed from the snails and settled on the cellophane papers and transformed into metacercariae. The metacercariae were collected from the papers and washed several times with 0.85 % NaCl solution and kept at 4 °C until use. Experimental animals and vaccination protocol Forty-eight-week-old male imprinting control region (ICR) mice were divided into four groups as follows: non-immunized and

IgG1 and IgG2a in blood samples were analyzed by indirect ELISA. For each analysis, a 96-well plate was coated with 1 μg/ml of rFgLAP in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and incubated at 4 °C for overnight. The coated plate was washed three times with phosphatebuffered saline with Tween 20 (PBST) (0.05 % Tween 20), and nonspecific binding was blocked with 1 % bovine serum albumin at room temperature for 1 h. Then, the coated plate was washed three times with PBST. The sera, diluted at 1:50,000 with PBS, were added and incubated at room temperature for 2 h. The plate was washed three times with PBST and incubated with HRP-conjugated goat anti-mouse IgG1 and IgG2a (SouthernBiotech, Birmingham, USA), diluted at

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1:5,000, at room temperature for 1 h. Then, the plate was washed three times with PBST and incubated with 3,3′,5,5′tetramethylbenzidine (KPL, Gaithersburg, USA) at room temperature for 10 min. Finally, enzymatic reaction was stopped by adding 1 N HCl at 100 μl per well. The optical densities (OD450) were measured at 450 nm in the automatic Titertek Multiscan Spectrophotometer (Flow Laboratories, VA, USA). The experiments were performed in duplicate. The correlation between OD450 values representing the levels of IgG1 and IgG2a in mice sera at the infection (0 week), at 2 weeks, and at 4 weeks postinfection (termination) with worm recoveries was estimated by plotting the worm recoveries against the OD values of IgG1 and IgG2a.

3655 Fig. 1 The Ni-NTA affinitypurified rFgLAP was analyzed by 12.5 % SDS-PAGE and stained with Coomassie blue. The rFgLAP appeared at MW of 56 kDa. MW markers are shown on the left side

Liver enzyme assays To estimate the degree of liver damage serum levels of the liver enzymes, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and gamma-glutamyl transferase (GGT), in the pooled sera of each group were measured in triplicate using the Automatic Chemistry Analyzer (Cobas Mira, Roche, Switzerland), at prevaccination, infection, and 4 weeks postinfection. Statistical analysis Differences of the percents of protection and the levels of liver enzymes between control and rFgLAP-immunized groups were tested by Kruskal–Wallis test with one-way analysis. Correlation between the OD values, representing IgG1 and IgG2a levels, and the numbers of worm recovered were subjected to one-way analysis of variance using the SPSS 18.0 software (Spearman's rank correlation test). A p value less than 0.05 was considered as statistically significant.

64.3 % when compared with non-immunized and Freund' s adjuvant control groups (Table 1). Antibody responses The levels of IgG1 and IgG2a in the sera of controls and rFgLAP-immunized groups were measured at six intervals (0, 2, 4, 6, 8, and 10 weeks) by indirect ELISA in duplicate. The ODs, which represent the levels of rFgLAP-specific IgG1 (Fig. 2(a)) and IgG2a (Fig. 2(b)) in rFgLAP-immunized group, rose dramatically during 2 to 4 weeks and reached the highest level at 8 weeks for IgG1 and at 6 weeks for IgG2a. Thereafter, the antibody levels declined. However, Table 1 Worm recovery and percentages of worm reduction in ICR mice immunized with rFgLAP protein and challenged with 30 metacercariae of F. gigantica per mouse Group

Mice Worm recovery

Nonimmunized and infected Adjuvant and infected Immunized with rFgLAP and infected

10

4, 5, 4, 3, 9, 7, 4, 3, 5, 7 5.1±1.97

–

10

9, 8, 5, 6, 4, 5, 3, 3, 6, 7 5.6±2.01

–

10

3, 4, 1, 4, 2, 1, 0, 1, 0, 4 2.0±1.63* 60.8a, 64.3b

Results Expression of rFgLAP protein The recombinant protein, rFgLAP, was expressed in E. coli and purified by Ni-NTA affinity column. The purified rFgLAP protein was analyzed by 12.5 % SDS-PAGE, and the protein band has a molecular weight (MW) of 56 kDa (Fig. 1). This protein was used for immunizations as stated in experimental animals and vaccination protocol section. Worm recovery

Mean ± SD

*p=0.05, a significant level

After immunization with rFgLAP, there was a significant reduction (p <0.05) in the worm recoveries at 60.8 and

a

Worm reduction compared with infected control group

b

Worm reduction compared with adjuvant control group

Reduction (%)

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IgG1 at infection and 2 and 4 weeks postinfection, the r were −0.399, −0.573, and −0.399; for IgG2a, the r at the same intervals were −0.143, −0.330, and −0.262 (Fig. 3). Liver enzyme assays The level of SGOT increased above the reference range levels by 4 weeks postinfection in animals of the non-immunized and adjuvant control groups. In the rFgLAP-immunized mice, the level of SGOT also increased but remained within the reference range and was significantly different from the controls (Fig. 4(a)). The levels of SGPT remained within the reference range in all groups, but the level in rFgLAPimmunized group was significantly different from the nonimmunized and infected control group (Fig. 4(b)). GGT level did not change during the period of vaccination, and no significant difference was observed between the rFgLAPimmunized and control groups (data not shown).

Discussion

Fig. 2 The ODs representing the levels of IgG1 and IgG2a in sera of four groups of mice, including the non-immunized and uninfected (1), the non-immunized and infected (2), the Freund's adjuvant and infected (3), and the immunized with rFgLAP and Freund's adjuvant and infected (4). a Mean IgG1 levels expressed as OD450 estimated by indirect ELISA. b Mean IgG2a levels expressed as OD450. Each point of the graphs represents the average OD value of duplicated readings from 10 mice. The rFgLAP-immunized group showed significantly higher levels of IgG1 and IgG2 at 2–10 weeks when compared to control groups. Asterisk denotes a significant difference with p<0.05. Arrows indicate the time at which the infection with metacercariae was performed. Bars represent standard errors

the ODs of IgG2a at all time intervals were lower than those of IgG1 in the immunized group (Fig. 2(a, b)), while the ODs of the control groups were low over the entire period of the experiment and not significantly different from each other (Fig. 2(a, b)). Correlations between the levels (OD values) of IgG1 and IgG2a and worm recoveries Correlations between the OD values of IgG1 and IgG2a and the numbers of worm recoveries were compared in individual animals from each group. All mice of rFgLAP-immunized group showed high levels of IgG1 and IgG2 at challenge day and 2 and 4 weeks postinfection that were highly correlated with the reduced numbers of worms being recovered at the end of the experiment. The correlation coefficient (r) between the ODs and the numbers of worm recovered is as follows: for

Previous vaccine trials for F. hepatica using the recombinant F. hepatica LAP showed a high percent of protection (86.9 %) in sheep (Maggioli et al. 2011). In contrast, a recent report by Raina et al. (2011) showed no protection against F. gigantica in buffaloes immunized with rFgLAP. However, in this study, the rFgLAP was injected with Montanide 70M VG as an adjuvant, via intramuscular route in the neck and gluteal muscle, which resulted in a low level of corresponding antibody. In our study using ICR mice, rFgLAP was injected with Freund's adjuvant, and the injection was done via subcutaneous route, which resulted in rather high levels of IgG1 and IgG2a and, consequently, high percentages of protection at 60.8–64.3 %. In addition to adjuvants, different routes of antigen injections may result in different levels of the host's immune responses and, hence, the degrees of protection. Previously, our group had reported that among varied routes of immunization, intradermal and subcutaneous routes yielded considerably a better result than the intramuscular route (Preyavichyapugdee et al. 2008). This is perhaps due to the presence of more antigen-presenting cells in intradermal and subcutaneous zones which resulted in better immune responses in the hosts. Recently, it was reported that the main immune response generated against helminth infections is T helper (Th)2-like and Treg responses and that the most effective vaccines were those that could elicit a strong Th2 response (Anthony et al. 2007; Moreau and Chauvin 2010). In this study, increased levels of both IgG1 and IgG2a were observed after rFgLAP immunization, with the IgG1 levels being higher than IgG2a at all intervals, implicating the predominance of Th2 over the Th1 response in these rodent hosts, which explained the better protection results when compared

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Fig. 3 Correlations between the numbers of worm recovered and the OD values of IgG1 and IgG2a. a, c, e The levels (ODs) of IgG1 at infection (6 weeks) and 8 and 10 weeks (termination) and the numbers of worms recovered at 10 weeks. b, d, f The levels (ODs) of IgG2a at infection (6 weeks) and 8 and 10 weeks (termination) and the numbers of worms recovered at 10 weeks. The symbols represent an individual in each group of animals as follows: black filled diamond, the OD450 and numbers of worm recovered from each animals in the rFgLAPimmunized group; blue filled square, the OD450 and numbers of worm recovered from each animals in the adjuvant and infected control group; and red filled triangle, the OD450 and numbers of worm recovered from each animal in the nonimmunized and infected control group

to those obtained by Raina et al. (2011). However, to ascertain the predominance of Th2 response, estimations of levels of the cytokines directly involved in the signaling of this immune process, e.g., IL-4, IL-5, and IL-13, needed to be performed (Kapsenberg 2003; Anthony et al. 2007; Moreau and Chauvin 2010; Allen and Maizels 2011). The rises in serum levels of SGOT and SGPT, also known by other names, aspartate aminotransferase and serum alanine transaminase, are indicative of the damage to the liver parenchyma (Sandeman and Howell 1981; Edith et al. 2010), while the increased level of GGT is indicative of the bile duct damage (Ford 1974). In this experiment, serum levels of SGOT and SGPT and that of GGT in non-immunized and infected, adjuvant and infected controls, and rFgLAPimmunized groups at challenge day (6 weeks) did not show significant differences when compared with the levels in sera of the non-immunized and uninfected group. This indicated that prior injections of Freund's adjuvant and rFgLAP protein did not damage the liver and the bile duct. However, the levels of SGOT and SGPT at termination day (4 weeks postinfection) of non-immunized and infected and adjuvant and infected control groups showed significant increases when compared

with those of non-immunized and uninfected group, which indicated the liver damage by the infecting parasites. In contrast, at the termination day, the levels of SGOT and SGPT of rFgLAP-immunized group were slightly increased over the levels of all groups at the challenge day but still remained within the reference range, and they were significantly lower than those of the two control groups. On the other hand, the level of GGT exhibited no change at all time intervals, implying that the worms had not yet arrived at the bile ducts, and no damage occurred. Furthermore, no fully mature flukes were recovered from the bile ducts at the end of the experiment. The present study showed that vaccination by rFgLAP with Freund's adjuvant through the subcutaneous route showed high levels of protection at 60.8–64.3 %, and the vaccination significantly reduced liver damage when compared with control groups. In addition, the rFgLAP immunization in mice could generate the production of both IgG1 and IgG2a isotypes, implicating the mixed Th1/Th2 responses with Th2 predominating. These data indicated that rFgLAP is a plausible vaccine candidate against fasciolosis by F. gigantica. Its effectiveness and application in large economic animals, including cattle and goat, are now being studied.

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Fig. 4 The levels of liver enzymes at three intervals [at 0 week (preimmunization), at 6 weeks (challenge with metacercariae), and at 10 weeks (termination)] in four groups of mice as follows: non-immunized and uninfected (1), non-immunized and infected (2), Freund's adjuvant and infected (3), and rFgLAP-immunized and infected (4). The columns represent mean (±SD) levels of SGOT and SGPT from triplicate measurements of pool sera (n=10). The levels are expressed as in unit per liter. a The levels of SGOT are not different from each other in all groups at 0 and 6 weeks. However, at 10 weeks (termination), the SGOT level in rFgLAP-immunized and rFgLAP-infected group remained within the reference range (100–300 U/L) and is significantly lower than that in the non-immunized and infected control groups. b The levels of SGPT in rFgLAP-immunized and rFgLAP-infected group remained within the reference range (15–94 U/L) and are significantly lower than those of the non-immunized and infected group. Arrows indicate the time at which the infection with metacercariae was performed. Asterisk denotes a significant difference with p<0.05 when compared to non-immunized and infected control group

Acknowledgments This research was supported by grants from the Faculty of Science, Mahidol University, and The Thailand Research Fund to P. Sobhon.

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