The insulin receptor is a transmission blocking veterinary vaccine target for zoonotic Schistosoma japonicum

International Journal for Parasitology 42 (2012) 801–807

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Succinctus

The insulin receptor is a transmission blocking veterinary vaccine target for zoonotic Schistosoma japonicum Hong You a, Geoffrey N. Gobert a, Mary G. Duke a, Wenbao Zhang a, Yuesheng Li a,b, Malcolm K. Jones c,d, Donald P. McManus a,⇑ a

Molecular Parasitology Laboratory, Infectious Diseases Division, Queensland Institute of Medical Research, Brisbane, Queensland, Australia Hunan Institute of Parasitic Diseases, Yueyang, China Parasite Cell Biology Laboratory, Infectious Diseases Division, Queensland Institute of Medical Research, Brisbane, Queensland, Australia d The School of Veterinary Science, The University of Queensland, St. Lucia, Queensland, Australia b c

a r t i c l e

i n f o

Article history: Received 16 April 2012 Received in revised form 1 June 2012 Accepted 4 June 2012 Available online 6 July 2012 Keywords: Schistosoma japonicum Insulin receptor Ligand domain Transmission blocking veterinary vaccine

a b s t r a c t Insulin receptors have been previously identified in Schistosoma japonicum that can bind human insulin. We used the purified recombined protein of the ligand domain of S. japonicum insulin receptor 2 (SjLD2) in three independent murine vaccine/challenge trials. Compared with controls, vaccination of mice with SjLD2 resulted in a significant reduction in faecal eggs, the stunting of adult worms and a reduction in liver granuloma density in all three trials. Furthermore, in the final trial, in which mature intestinal eggs were also quantified, there was a reduction in their number. These results suggest that development of a vaccine based on rSjLD2 for preventing transmission of zoonotic schistosomiasis is feasible. Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Schistosomiasis affects 200 million people in 74 countries and is responsible directly or indirectly for hundreds of thousands of deaths annually (McManus and Loukas, 2008), and thereby represents a significant public health problem. Praziquantel is the only drug used for treatment and no anti-schistosome vaccine is currently available. In vaccine development, a reduction in worm numbers has been historically considered the ‘‘ultimate goal’’ but as schistosome eggs are central to pathology and disease transmission, a vaccine targeting parasite fecundity within the definitive host is an entirely relevant approach to vaccine design (McManus and Loukas, 2008). This strategy is additionally appropriate for Schistosoma japonicum, as this species utilises a range of mammalian reservoir hosts and the adults produce a very high egg output, compared with other schistosome species. Schistosomes exploit host hormones and nutrients for their survival; indeed, schistosomes consume their dry weight of glucose every 5 h, utilising the glucose transporter proteins, SGTP1 and SGTP4 (Skelly et al., 1994). As a key hormone, with profound effects on carbohydrate metabolism, insulin increases glucose uptake, enhances the in vitro viability of schistosome larvae (Vicogne et al., 2004), and stimulates the metabolism and development of ⇑ Corresponding author. Address: Molecular Parasitology Laboratory, Infectious Diseases Division, Queensland Institute of Medical Research, 300 Herston Road, Herston, Queensland 4006, Australia. Tel.: +61 7 33620401; fax: +61 3362 0104. E-mail address: [email protected] (D.P. McManus).

adult worms (Saule et al., 2005). This contrasts with an early report which indicated that insulin had no effect on glucose consumption by schistosomes in vitro (Clemens and Basch, 1989), but a more recent study has reinforced the importance of insulin in regulating glucose uptake in Schistosoma mansoni (Ahier et al., 2008). It remains to be fully established whether insulin modulates glucose uptake in schistosomes in a similar manner to that observed in Caenorhabditis elegans, which uses an almost identical mechanism to mammalian cells (Beall and Pearce, 2002). Recently, two types of insulin receptors, having limited (27–29%) sequence identity with the human insulin receptor (HIR), have been identified and partially characterised from both S. mansoni and S. japonicum (Khayath et al., 2007; You et al., 2010). Previous studies have also shown that host insulin can bind to the L1 sub-domains of the IRs of S. japonicum (SjIR1 and SjIR2) (You et al., 2010), S. mansoni (Khayath et al., 2007) and Echinococcus multilocularis (Konrad et al., 2003) in the yeast two-hybrid system, indicating that cellular growth and reproduction in these parasites may be dependent on host insulin. Both IRs of S. japonicum were shown to be up-regulated in schistosomula and adult worms, further emphasising their involvement in the host/parasite interaction (You et al., 2010). We have also demonstrated that a specific IR inhibitor – HNMPA (hydroxy-2-naphthalenylmethyl phosphonic acid) and antisera raised against the ligand domains of the SjIRs significantly decreased glucose uptake in adult worms of S. japonicum in vitro (You et al., 2010). These data provided good

0020-7519/$36.00 Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijpara.2012.06.002

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H. You et al. / International Journal for Parasitology 42 (2012) 801–807

evidence that the L1 sub-domains of the IRs of S. japonicum are likely schistosome vaccine targets, which we now report. In this study, the ligand domain of SjIR2, expressed in Escherichia coli and purified, was used in three independent mouse vaccination-challenge trials. A cDNA fragment (1,467 bp), referred to as SjLD2, encoding the L1, CR and L2 sub-domains (R37-C525) of SjIR2 (You et al., 2010) (GenBank Accession No. GQ214554), was amplified by PCR. The cDNA was ligated into the pET28b vector (Invitrogen, USA) and E. coli BL21 (DE3) cells (Invitrogen) were transformed with the recombinant plasmid. The recombinant (r) SjLD2 protein tested in vaccine trials 1 and 2 was purified from an E. coli lysate under denaturing conditions with 6 M guanidineHCl using His-Bind affinity resin (Novagen, USA). The rSjLD2 protein used in vaccine trial 3 was also purified from an E. coli lysate under denaturing conditions with 6 M guanidine-HCl but the protocol thereafter was modified and used a HisTrap™ FF Column (GE Health Life Science, USA) followed by a buffer exchange step prior to applying the fraction to an ion exchange column (Q Sepharose Fast Flow, GE Health Life Science). This was done to determine whether any residual endotoxin present in the purified recombinant protein affected the efficacy of the rSjLD2 vaccine. In both purification procedures, the purified rSjLD2 was assessed for quality and identity by SDS–PAGE which indicated a single band with a molecular weight of approximately 61 kDa. The yield of the purified rSjLD2 using His-Bind affinity resin was 3.0 mg/L, while the yield was reduced to 1.4 mg/L using the HisTrap™ FF Columns and buffer and ion exchange. The identity of the purified protein was confirmed by western blotting using an anti-rSjLD2 antiserum generated in mice against rSjLD2 as described previously (You et al., 2010). The purified, recombinant ligand domain of SjIR1 (SjLD1) protein was prepared as previously described (You et al., 2010). Two groups of 10 female CBA mice (6–8 weeks old) (10 vaccines, 10 controls) were used in each vaccine trial except for trial 3 where five female CBA mice were used in the control group. For trials 1 and 2, mice in the vaccinated groups were immunised i.p. with 25 lg of rSjLD2 protein in 0.1 ml of PBS homogenised with 20 lg of Quil A adjuvant (Superfos, Denmark) (Martin et al., 2002), a potent and widely used adjuvant in veterinary vaccines. The mice were boosted twice i.p. at 2 week intervals with the same dosage and challenged with 34 ± 1 S. japonicum cercariae by the abdominal skin route 2 weeks after the third injection. In vaccine trial 3, the three dosages were given s.c. using the same injection intervals and challenge method as in trials 1 and 2. In the three trials, the control groups received PBS formulated with Quil A for the primary and two adjuvant boosts by the i.p. (trials 1 and 2) or s.c. (trial 3) route. Trials 2 and 3 were blinded both for staff administering the immunizations and for those performing the analysis of results. Sera from mice in each group were collected and screened for recognition of rSjLD2 by standard ELISA. Maxisorb immunoplates (Nalge Nune International, USA) were coated overnight at 4 °C with 100 ll of 0.5 lg/ml rSjLD2 protein (prepared for vaccine trial 3) in coating buffer (100 ll/well). After three washes with 0.05% (v/v) Tween in PBS (PBST), wells were blocked with 200 ll of 5% (v/v) skim milk in PBS (SMP) and incubated for 1 h at 37 °C. The same volume of serum from all mice in a particular vaccinated or control group was pooled, mixed and the serum pool was serially diluted (from 1:200 to 1:102,400) in SMP and 100 ll in duplicate of each dilution were added to individual wells. After incubation at 37 °C for 1 h, the wells were washed with PBST (3) and 100 ll (1:2,000 dilution) of horseradish peroxidise (HRP)-conjugated sheep anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, IgA or IgM (Invitrogen) was added. After incubation at 37 °C for 1 h, the wells were washed with PBST (5), 100 ll of substrate solution [2,2-azino-di(ethyl-benzithiozolin sulfonate)] (Sigma, Castle Hill, Australia) was added and the wells were incubated at room temperature for

30 min. The O.D. was monitored at 405 nm using a Benchmark plate reader and Microplate manager (Bio-Rad, Mississauga, Canada). Data are presented as antibody endpoint titres, defined as the highest dilution of test group serum pool that yielded an average O.D. two S.D.s greater than that obtained in the absence of primary antibody. The results showed that the rSjLD2 vaccinated mice generated high levels of IgG antibodies against rSjLD2 after the third injection, which were dominated by IgG1 and IgG2a, as shown in Table 1. No IgG3, IgA and IgM responses were measurable in any of the vaccinated mouse groups in the three trials. IgG antibodies in sera from the rSjLD2-vaccinated mice also strongly cross recognised rSjLD1 with a titre of 1:12,800. Sera from control mice showed no reactivity in ELISA with either rSjLD2 or rSjLD1 in any of the three trials. Western blotting was also used to probe purified rSjLD2, the recombinant ligand domain of SjIR1 (rSjLD1) and crude extracts of adult S. japonicum collected from an infected rabbit, with anti-SjLD2 murine antiserum as previously described (You et al., 2010). Worm and egg burdens in tissues and faeces were determined in control and vaccinated mice to evaluate the vaccine efficacy of rSjLD2. The student’s t-test was employed to assess the statistical significance of differences observed using GraphPad. All results are presented as mean ± S.E. P values 6 0.05 were considered statistically significant. Six weeks post cercarial challenge, adult S. japonicum worm numbers were counted as previously described (McManus et al., 2001) and paired adult worms were selected randomly from each mouse, separated and fixed overnight at 4 °C in 4% (v/v) paraformaldehyde in PBS. The lengths of individual adult worms were measured (Abramoff et al., 2004). The liver and intestines from each mouse for all groups were processed according to standard procedures to isolate schistosome eggs (McManus et al., 2001). The collected eggs from livers or intestines were resuspended in 10 ml of 4% (v/v) paraformaldehyde in PBS. Aliquots (100 ll) were pipetted onto a microscope slide and the number of eggs in each aliquot counted using light microscopy. Faecal samples were obtained from individual mice (trials 1 and 3) or pooled from the vaccination/control groups (trial 2) for 2 days before perfusion to quantify schistosome egg output in faeces. Faeces were weighed, mixed by vortexing with 4% (v/v) paraformaldehyde in PBS and then rotated overnight at 4 °C. Eggs were concentrated in faecal matter by centrifugation at 500g for 10 min, resuspended in 4% (v/v) paraformaldehyde, filtered through 150 lm sieves, collected on 40 lm sieves, resuspended in 4% (w/v) potassium hydroxide and digested overnight. The resulting pellets were collected by centrifugation, resuspended in 4% (v/v) paraformaldehyde in PBS and eggs were counted microscopically in the faecal homogenates. To estimate liver granuloma volume density, livers of five mice from each of the vaccinated and control groups from trials 2 and 3 were selected randomly for quantitative analysis of the hepatic response to embolized eggs. For each mouse, the left anatomical lobe of each liver was fixed in 4% (v/v) formalin. Paraffin-embedded sections of these samples were prepared and stained with H&E. Slides were digitised using an Aperio Slide Scanner (Aperio, USA). The degree of liver pathology was quantified by measurement of the volume density of granulomatous response by a point-counting method, using ImageJ software Version 1.37 (National Institutes of Health, Bethesda, USA) as previously described (Bartley et al., 2008). To measure this, four regions of the same magnification and size were taken from each of two liver sections per mouse. Counts for each photograph were averaged for each group. As 59–72% of eggs are generally found in the proximal small intestine of S. japonicum-infected mice (Hirose et al., 2007), a 2 cm section of small intestine (starting 3 cm after the beginning of small intestine) was excised from each mouse within the

Table 1 Parasitological data and antibody titers of recombinant (r) SjLD2-vaccinated and control mice challenged with Schistosoma japonicum. Trial

1

Antibody endpoint titers

Number adult worms mean ± S.E.

Mean length of adult worms (mm) mean ± S.E.% reduction (P value)

Liver eggs/g/F mean ± S.E.% reduction (P value)

Liver granuloma density (%) mean ± S.E.% reduction (P value)

Intestinal eggs/ g/F mean ± SE% reduction (P value)

Control (PBS + QuilA) n = 10

IgG 1:100 IgG1 1:00 IgG2a 1:50 IgG2b 1:50 IgG 1:12,800 IgG1 1:51,200 IgG2a 1:25,600 IgG2b 1:3200 Same as PBS control group in trial 1 IgG 1:12,800 IgG1 1:25,600 IgG2a 1:12,800 IgG2b 1:1,600 Same as PBS control group in trial 1 IgG 1:12,800 IgG1 1:25,600 IgG2a 1:12,800 IgG2b 1:3,200

18 ± 1.4

(F) 15.5 ± 0.5 (M) 13.3 ± 0.4

7946 ± 637

ND

16.4 ± 2.5

(F) 9.8 ± 0.3 37% (P < 0.0001) (M) 7.6 ± 0.2 42% (P < 0.0001)

8786 ± 851ns

18.7 ± 1.8

(F) 10.68 ± 0.39 (M) 7.65 ± 0.14

19.7 ± 3.3

rSjLD2 n = 10

2a

Control (PBS + QuilA) n = 10 rSjLD2 n = 10

3a

Control (PBS + QuilA) n=5 rSjLD2 n = 10

Maturity of intestinal eggs (%) mean ± S.E.% increase " or decrease " (P value)

Faecal eggs/g/F mean ± S.E.% reduction (P value)

Stage I

Stage II

Stage III

Stage IV

Stage V

ND

ND

ND

ND

ND

ND

722 ± 118b

ND

ND

ND

ND

ND

ND

ND

238 ± 80 67% (P = 0.0033)

10,214 ± 949

48.6 ± 2.5

15354.6 ± 811

ND

ND

ND

ND

ND

203.7 ± 27.8c

(F) 8.92 ± 0.3 16% (P < 0.0001) (M) 6.76 ± 0.1% 12% (P < 0.0001)

7387.2 ± 109 28% ns (P = 0.06)

21.7 ± 2.6 55% (P < 0.0001)

11,860 ± 1515 23% (P = 0.133)

ND

ND

ND

ND

ND

85.3 ± 13.3c 58% (P = 0.003)

13.7 ± 0.5

(F) 9.83 ± 0.11 (M) 7.33 ± 0.05

8487 ± 403

37.6 ± 4.6

8604 ± 737

16 ± 1.7

9.1 ± 0.9

16 ± 1.3

38 ± 2.4

27 ± 2.2

363 ± 39b

13.9 ± 0.6

(F) 8.29 ± 0.05 16% (P = 0.002) (M) 5.9 ± 0.05 19% (P < 0.0001)

8576 ± 382ns

16.3 ± 2.645% (P < 0.0001)

9171 ± 265

26 ± 1.2ns

18 ± 0.9 190%" (P = 0.045)

22 ± 0.9ns

28 ± 1.3ns

6.6 ± 0.4 75%; (P = 0.0003)

161 ± 18b 56% (P = 0.049)

H. You et al. / International Journal for Parasitology 42 (2012) 801–807

Quil A adjuvanted immunogen

F, female worm; M, male worm; ND, not determined; n, the number of mice per group that survived the trial and were necropsied; ns, not significant. a Blinded trial. b Faecal eggs were counted using individually collected faeces from each mouse. c Faecal eggs were counted using two pooled faecal samples collected twice from each group.

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vaccinated and control groups of trial 3 to compare differences in the maturity of eggs between the two groups (oogram analysis). The section of intestine from each mouse was chopped finely and digested at 37 °C overnight with 0.4 mg/ml of Collagenase B (Sigma) with 0.2 lg/ml og penicillin and 0.4 lg/ml of streptomycin in PBS. The digestion was then centrifuged at 400g for 20 s and the pellet was washed three times with chilled PBS to remove any contaminants from the egg mixture. The intestinal egg mixture was fixed in 4% (v/v) formalin, stained and mounted on microscope slides using Prolong Gold Antifade Reagent incorporating DAPI (Invitrogen) and incubated at 4 °C overnight. The stained eggs were then visualised by fluorescence microscopy (IM1000, Leica, UK) using UV illumination to visualise nuclei. Embryonic development of intestinal eggs obtained from the rSjLD2-vaccinated and control mice was compared using a staging system (Jurberg et al., 2009), which is based on a simple relationship between the cellular content of the embryo and eggshell size. We used DAPI nucleic acid staining to classify the embryonic development and maturation of S. japonicum eggs in the murine intestine into five discrete stages shown in Fig. 1 – stage I (Fig. 1A), stage II (Fig. 1B), stage III (Fig. 1C), stage IV (Fig. 1D), stage V (Fig. 1E). We were thus able to determine the number of intestinal eggs at each of the five different developmental stages for the rSjLD2-vaccinated mice and controls in trial 3, and to compare the percentage of eggs at each stage for each mouse using the number of eggs per stage divided by the total number of eggs observed for all of the stages in an individual animal. Table 1 shows the results of the three independent vaccine trials. There were no significant (P P 0.05) changes in the mean total worm burdens or in the liver or intestinal egg numbers between the rSjLD2-vaccinated and control groups in the three trials. There were, however, significant (P 6 0.05) reductions in the vaccinated groups compared with controls in the following important parameters: (i) the mean lengths of S. japonicum adult worms from the

vaccinated mice were significantly reduced (Fig. 2); female and male worms were reduced in length by between 16–37% and 12– 42%, respectively (ii) there were significant (56–67%, P 6 0.05) reductions in faecal eggs in the vaccinated mice in the three trials (Fig. 2), (iii) as indicated above, there was almost double the average percentage of S. japonicum eggs at stage II in vaccinated animals, while there was a 75% reduction in the average percentage of eggs at stage V (mature stage) in the vaccinees in trial 3, (iv) liver granuloma volume density was reduced significantly by 45–55% in the vaccinated mice compared with controls in trials 2 and 3. Although we used a modified protocol for purification of the rSjLD2 protein used in trial 3, the general vaccine effects in all three trials were the same, indicating that any residual endotoxin present in the purified recombinant protein used in trials 1 and 2 did not have any effect on the efficacy of the rSjLD2 vaccine. In this study the vaccine efficacy of SjLD2 was assessed in parasite-challenged CBA mice with the aim of reducing host pathology and/or faecal egg numbers, thereby disrupting transmission. CBA mice were selected for the vaccine trials due to their strong splenic proliferative response and reduced suppressor T-cell response, resulting from a patent schistosome infection in this strain (Tran et al., 2006). These factors were deemed important based on the assumption that a more effective protective response would be more likely in this mouse strain than in others such as BALB/c or BL/6 mice. Previous studies have shown that the levels of the IgG1 and IgG2 antibody subclasses can be used as serological markers to indicate the induction of Th1 (IgG2) and Th2 (IgG1) responses (Mowen and Glimcher, 2004). Th1 cytokines may be beneficial in preventing schistosomiasis, targeting worms during the early stages of infection, while Th2 cytokines, which are induced by egg antigens following egg deposition in tissues during the late immune response, suppress the Th1 response (McManus and Loukas, 2008). However, studies using B-cell-deficient and cytokine-deficient mice have demonstrated that successful schistosome vaccines require

Fig. 1. Stages in the maturation of Schistosoma japonicum eggs in the murine intestine. The developmental staging (I–V) followed that of Vogal and Prata (Jurberg et al., 2009) and is based on a simple relationship between the cellular content of the schistosome embryo and eggshell size. Blue-fluorescent DAPI was used to classify the five developmental stages reflecting the density of stained nuclei shown in A–E. (A) stage I, stained nuclei occupy one third of the egg; (B) stage II, nuclei occupy one half of the egg; (C) stage III, nuclei occupy two thirds of the egg; (D) stage IV, nuclei occupy about three quarters of the egg; (E) stage V, nuclei almost fully occupy the entire interior of the egg, corresponding to the mature egg. The representative eggs and their staging shown are from a control mouse. The maturity (%) of intestinal eggs at each of the five different developmental stages for the rSjLD2-vaccinated mice and controls in trial 3 were compared using these staging characteristics (Table 1).

805

G

LD

2

on tr ol

C

15 10 5 0

2

Individual fecal eggs/female LD

Length of female worms (mm)

F

**

Groups 20

Groups

*

500

0

2

0

E

length of male worm 25

1000

500

D

0

LD

LD 2

tr ol

0

5

on tr ol

500

**

1000

10

1500

Groups

rSjLD2 Vaccinated

C

1000

1500

fecal eggs/female

Control

15

on tr ol

0

***

20

C

5

Number of eggs/g feces in each femaleLength of male worms (mm)

tr

C on

**

C on

Eggs/gfaeces/female

C

Groups

1500

LD

10

Fecal eggs burden each female

Trial 3

length of female worm 25

2

l

tr o co n

15

2

LD 2

0

***

Groups

20

LD

5

length of male worm

25

2

10

0

LD

15

5

tr ol

LD

tr on C

***

Groups

20

10

co n

2

length of male worms ol

0 25

15

ol

5

20

tr

10

***

on

Length of female worm (mm)

15

ol

B

20

Length of male worm (mm) Length of male worms (mm)

A

***

Trial 2fir

length of female worm 25

C

Trial 1

length of female worms 25

Number of eggs/g feces in each femaleLength of male worms (mm) Length of female worms (mm)

H. You et al. / International Journal for Parasitology 42 (2012) 801–807

Fig. 2. Comparison of the length of worms and faecal egg numbers from rSjDL2-vaccinated mice and controls. (A and B) The lengths (mm) of females and males, respectively; (C) the results of faecal eggs (eggs/g faeces in each female worm) in rSjLD2-vaccinated and control groups. ⁄P 6 0.05, ⁄⁄P 6 0.001, ⁄⁄⁄P value 6 0.0001. (D–G) Photomicrographs comparing the lengths of Schistosoma japonicum worms in rSjLD2-vaccinated mice and controls. Representative female worms are shown in the control group (D) and in the rSjLD2-vaccinated group (E); representative male worms are shown in the control group (F) and in the rSjLD2-vaccinated group (G).

induction of both strong Th1 and Th2 responses, each of which contribute to protection (McManus and Loukas, 2008). The detection of IgG1 and IgG2 antibody isotypes in rSjLD2-vaccinated mice indicated stimulation of both Th1 and Th2 immune responses. The antibody responses to rSjLD2 in vaccinated mice were dominated by IgG1 and IgG2a, similar to those generated with the protective S. mansoni tetraspanins (Sm-TSPs) (Tran et al., 2006). At present, we have no information regarding cellular immune responses generated by rSjLD2 and future work will address both B- and T-cell responses in rSjLD2-vaccinated mice. In the three independent vaccination/challenge trials, there was no significant reduction in the number of adult worms or in liver or intestinal egg burdens with the Quil A-adjuvant rSjLD2 vaccine. However, the highly significant (P 6 0.002) reductions in the mean lengths (37–42%) of adult worms in all three vaccinated groups indicated that although SjLD2 was unable to stimulate killing of adult worms, the vaccine was able to induce a significant retardation in the growth of male and female adult worms, presumably due to reduced glucose uptake and the subsequent starving of the adult worms. These results are supported by previous findings which showed that insulin treatment of schistosome-infected mice leads to increased worm size and an associated increase in the expression of parasite genes involved in glucose uptake (Saule et al., 2005). These previous observations also support our hypothesis that S. japonicum depends on host insulin for development and growth by sharing a similar pathway as mammalian cells in regulating glucose uptake (You et al., 2010). SjLD2 likely plays a key role in parasite sexual maturation and egg production and the rSjLD2 vaccine may inhibit this process by disrupting insulin-associated pathways. In addition to retarding the growth of male and female S. japonicum, the significant reduction (45–55%) in hepatic granuloma volume density in the vaccinated mice in the three trials indicates that the rSjLD2 vaccine also inhibited egg-induced liver granuloma formation. More importantly, the significant reduction (56–67%) in faecal eggs and the significant decrease in maturity of intestinal eggs (75%) in vaccinated animals strongly suggests the SjLD2 vaccine affected sexual development or egg production. These results support our previous transcriptomic analysis showing that host

insulin is not only essential for parasite growth, up- or down-regulating over 1,000 genes during culture in vitro, but that it also plays an important role in sexual differentiation and fecundity by activating the Mitogenic Activated Protein Kinase (MAPK) pathway (You et al., 2009). These results also further confirmed our hypothesis that SjIR2 may be involved in the development of vitelline material which provides essential nutrients and shell precursors for egg production (You et al., 2010). Although there was no significant difference in the number of intestinal eggs, the fact that mature eggs of S. mansoni are able to remain alive for many days in the intestinal wall of mice (Pellegrino et al., 1963) prior to their release into the intestinal lumen indicated it was important to compare the maturation of S. japonicum eggs in the intestines of vaccinated and control mice. The significant decrease in the percentage of mature eggs (stage V) and an increase in immature eggs (stage II) within the intestine of the rSjLD2-vaccinated mice suggests that the eggs produced by female S. japonicum from these animals had been adversely affected by the vaccine, resulting in potentially unhealthy or poorly developed eggs that may be unable to pass through the host intestine and reach the faeces, thereby contributing to the observed reduction in faecal eggs. These results are supported by a previous study showing faecal egg excretion was reduced in streptozotocin-induced diabetic mice infected with S. mansoni although worm load and total amount of eggs in the intestinal tissue were similar to control animals (Hulstijn et al., 2001). The investigation by Hulstijn and colleagues (2001) further showed that evaluation of an oogram, made from the first centimetre of the distal part of the small intestine, indicated a greater number of immature dead eggs and a lower number of mature eggs in the diabetic mice, implying that faecal egg excretion was lower in diabetic mice due to impaired egg maturation. An infection study with albino mice demonstrated that most S. japonicum eggs were recovered in the faeces (47%) and intestine (40%), with only 12% of eggs being present in the liver (Fan and Kang, 2003). Thus, given that the majority of eggs are found in the faeces and intestine and, given the critical role of faecal eggs in schistosome transmission, the reduction in both faecal eggs and the decrease in mature eggs in the intestine observed are important considerations in targeting rSjLD2 as a transmission

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blocking vaccine. Encouragingly, the significant reduction (56–67%) in the number of S. japonicum faecal eggs in the rSjLD2-vaccinated mice is higher than that obtained with many other evaluated schistosome antigens and candidate vaccine formulations which have provided at best 40–50% protection in animals using the standard readouts of reduced worm burden or egg production and viability. Nevertheless, to explore the potential long-term consequences of the rSjLD2 vaccine, further work extending the time to perfusion to 8–10 weeks using a lower number of challenge cercariae and assessing the viability of eggs isolated from both tissues and faeces from vaccinated and control mice are warranted. As well as assessing the protective efficacy of SjLD2, which is located in the vitelline cells of female S. japonicum worms and in the parenchyma of males, we tested in CBA mice the vaccine potential of rSjLD1, which is located in the sub-tegument of adult worms (You et al., 2010). However, due to the poor immune response generated, we were unable to obtain consistent results using the rSjLD1 protein, which had a tendency to degrade during the processes of expression and purification. Nevertheless, further work improving the expression/purification of rSjLD1 is currently underway in order to prevent its degradation. It is noteworthy that we were able to show high immunological cross reactivity in ELISA analysis between rSjLD1 and rSjLD2 when probed with anti-SjLD2 antiserum, suggesting the two proteins may contain similar functional epitopes located on the sub-tegument of adult S. japonicum worms. The precise immune effectors generated by the rSjLD2 vaccine need to be further investigated. The SjLD2 fusion proteins expressed in E. coli were found in inclusion body fractions and purification with His-Bind was performed under denaturing conditions using 6 M guanidine–HCl. The denaturing conditions necessary for purification may linearize the tertiary and secondary structures of the ligand domain of the IR and the changed conformation may cause partial loss of function for binding insulin and subsequent regulation of glucose uptake. In addition, the HIR has two receptor binding sites including the L1 sub-domain and the first and second type fibronectin III repeats of the receptor (Whittaker et al., 2008). We have previously shown that the L1 sub-domain of SjIR2, which is expressed as part of rSjLD2, contains an insulin binding site (You et al., 2010), but it is unknown whether there is a second insulin binding site present. The identification and characterization of other potential insulin binding sites in the SjIRs may provide additional targets for interrupting or blocking the binding between host insulin and the SjIRs. Furthermore, the selection of a suitable adjuvant and delivery system to aid in the stimulation of the appropriate immune response and increase the protective effect of vaccine antigens are critical steps on the path to the development and employment of successful schistosomal vaccines. Additional immunisation trials using the rSjLDs expressed in eukaryotic hosts, combinations of the SjLDs and/or with the use of other adjuvants may increase the protective effect of these candidate vaccines and surpass the protective effects described here. Recent surveys suggest that in China, much of the fresh water contamination with S. japonicum eggs is due to animal defecation. Furthermore, drug intervention trials have indicated that water buffaloes are the major animal reservoir host of S. japonicum, being responsible for approximately 75% of the environmental egg contamination in China (McManus and Loukas, 2008), with the average daily stool output of a water buffalo being considerably more than that of a human. As in the Chinese setting, the fact that there is up to 60% S. japonicum prevalence in buffalo (carabao) in the Philippines also suggests a major role for bovines in the transmission of S. japonicum there (Wu et al., 2010). There is 100% sequence identity in the LD2 region of the Chinese and Philippine strains of S. japonicum (data not shown), underpinning the rationale for developing a

veterinary transmission blocking vaccine based on rSjLD2 for use against S. japonicum both in China and the Philippines. In summary, we have shown in three independent pilot vaccine trials that rSjLD2 was able to induce a significant stunting of parasite growth, likely depressed egg maturity and substantially decreased faecal egg output, suggesting that development of a vaccine based on this molecule for preventing transmission of zoonotic schistosomiasis is feasible. Acknowledgements The authors thank the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, China for providing the Schistosoma japonicum cercariae, and Dr. Amber Glanfield, Hepatic Fibrosis Laboratory, Queensland Institute of Medical Research (QIMR), Australia and Patrick Driguez, Molecular Parasitology Laboratory, QIMR for providing assistance with the vaccination trials. We thank Dr. Linda Lua from the Protein Expression Facility of University of Queensland, Australian Institute for Bioengineering and Nanotechnology for protein purification. This study received financial support from the National Health and Medical Research Council, Australia. References Abramoff, M.D., Magalhaes, P.J., Ram, S.J., 2004. Image Processing with Image. J. Biophoton. Int. 7, 36–42. Ahier, A., Khayath, N., Vicogne, J., Dissous, C., 2008. Insulin receptors and glucose uptake in the human parasite Schistosoma mansoni. Parasite 15, 573–579. Bartley, P.B., Glanfield, A., Li, Y., Stanisic, D.I., Duke, M., Jones, M.K., McManus, D.P., 2008. Artemether treatment of prepatent Schistosoma japonicum induces resistance to reinfection in association with reduced pathology. Am. J. Trop. Med. Hyg. 78, 929–935. Beall, M.J., Pearce, E.J., 2002. Transforming growth factor-beta and insulin-like signalling pathways in parasitic helminths. Int. J. Parasitol. 32, 399–404. Clemens, L.E., Basch, P.F., 1989. Schistosoma mansoni: insulin independence. Exp. Parasitol. 68, 223–229. Fan, P.C., Kang, Y.C., 2003. Egg production capacity of one-pair worms of Schistosoma japonicum in albino mice. Southeast Asian J. Trop. Med. Public Health 34, 708– 712. Hirose, Y., Matsumoto, J., Kirinoki, M., Shimada, M., Chigusa, Y., Nakamura, S., Sinuon, M., Socheat, D., Kitikoon, V., Matsuda, H., 2007. Schistosoma mekongi and Schistosoma japonicum: differences in the distribution of eggs in the viscera of mice. Parasitol. Int. 56, 239–241. Hulstijn, M., Oliveira, R.M., Moura, E.G., Machado-Silva, J.R., 2001. Lower faecal egg excretion in chemically-induced diabetic mice infected with Schistosoma mansoni due to impaired egg maturation. Mem. Inst. Oswaldo Cruz 96, 393–396. Jurberg, A.D., Goncalves, T., Costa, T.A., de Mattos, A.C., Pascarelli, B.M., de Manso, P.P., Ribeiro-Alves, M., Pelajo-Machado, M., Peralta, J.M., Coelho, P.M., Lenzi, H.L., 2009. The embryonic development of Schistosoma mansoni eggs: proposal for a new staging system. Dev. Genes. Evol. 219, 219–234. Khayath, N., Vicogne, J., Ahier, A., BenYounes, A., Konrad, C., Trolet, J., Viscogliosi, E., Brehm, K., Dissous, C., 2007. Diversification of the insulin receptor family in the helminth parasite Schistosoma mansoni. FEBS J. 274, 659–676. Konrad, C., Kroner, A., Spiliotis, M., Zavala-Gongora, R., Brehm, K., 2003. Identification and molecular characterisation of a gene encoding a member of the insulin receptor family in Echinococcus multilocularis. Int. J. Parasitol. 33, 301–312. Martin, D., Rioux, S., Gagnon, E., Boyer, M., Hamel, J., Charland, N., Brodeur, B.R., 2002. Protection from group B streptococcal infection in neonatal mice by maternal immunization with recombinant Sip protein. Infect. Immun. 70, 4897–4901. McManus, D.P., Loukas, A., 2008. Current status of vaccines for schistosomiasis. Clin. Microbiol. Rev. 21, 225–242. McManus, D.P., Wong, J.Y., Zhou, J., Cai, C., Zeng, Q., Smyth, D., Li, Y., Kalinna, B.H., Duke, M.J., Yi, X., 2001. Recombinant paramyosin (rec-Sj-97) tested for immunogenicity and vaccine efficacy against Schistosoma japonicum in mice and water buffaloes. Vaccine 20, 870–878. Mowen, K.A., Glimcher, L.H., 2004. Signaling pathways in Th2 development. Immunol. Rev. 202, 203–222. Pellegrino, J., Celso, A.O., Jane, F., 1963. The oogram in the study of relapse in experimental chemotherapy of schistosomiasis mansoni. J. Parasitol. 49, 365–370. Saule, P., Vicogne, J., Delacre, M., Macia, L., Tailleux, A., Dissous, C., Auriault, C., Wolowczuk, I., 2005. Host glucose metabolism mediates T4 and IL-7 action on Schistosoma mansoni development. J. Parasitol. 91, 737–744. Skelly, P.J., Kim, J.W., Cunningham, J., Shoemaker, C.B., 1994. Cloning, characterization, and functional expression of cDNAs encoding glucose

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