Tetraspanins on the surface of Schistosoma mansoni are ... - Nature

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Tetraspanins on the surface of Schistosoma mansoni are protective antigens against schistosomiasis Mai H Tran1,4, Mark S Pearson1,4, Jeffrey M Bethony2–4, Danielle J Smyth1, Malcolm K Jones1, Mary Duke1, Tegan A Don1, Donald P McManus1, Rodrigo Correa-Oliveira3 & Alex Loukas1 Schistosomes are blood-dwelling flukes that infect 200 million people worldwide and are responsible for hundreds of thousands of deaths annually1. Using a signal sequence trap, we cloned from Schistosoma mansoni two cDNAs, Sm-tsp-1 and Sm-tsp-2, encoding the tetraspanin (TSP) integral membrane proteins TSP-1 and TSP-2. We raised antibodies to recombinant TSP fusion proteins and showed that both proteins are exposed on the surface of S. mansoni. Recombinant TSP-2, but not TSP-1, is strongly recognized by IgG1 and IgG3 (but not IgE) from naturally resistant individuals but is not recognized by IgG from chronically infected or unexposed individuals. Vaccination of mice with the recombinant proteins followed by challenge infection with S. mansoni resulted in reductions of 57% and 64% (TSP-2) and 34% and 52% (TSP-1) for mean adult worm burdens and liver egg burdens, respectively, over two independent trials. Fecal egg counts were reduced by 65–69% in both test groups. TSP-2 in particular provided protection in excess of the 40% benchmark set by the World Health Organization for progression of schistosome vaccine antigens into clinical trials. When coupled with its selective recognition by naturally resistant people, TSP-2 seems to be an effective vaccine antigen against S. mansoni. Female schistosomes deposit eggs in the blood vessels surrounding the gut (S. mansoni and S. japonicum) or bladder (S. haematobium); the ensuing granulomatous response causes the symptoms associated with schistosomiasis. Schistosomes are the most important helminth in terms of human morbidity and mortality; a recent meta-analysis assigned 2–15% disability weight to this pandemic2. Current control efforts rely on anthelminthic treatment, but to sustain their effects drugs must be applied periodically and for an indefinite period of time. Moreover, high rates of reinfection after mass treatment limit strategies based on chemotherapy alone. As such, a prophylactic vaccine is the ideal method for sustainable control of schistosomiasis, alone or in combination with anthelminthic drugs3. Vaccination with infective larvae (cercariae) of S. mansoni that have been attenuated with ionizing radiation induces a high degree of protection in animals4,5. Although this model raised hopes for the

development of molecular vaccines, no single antigen has consistently induced equivalent protection, particularly when used in recombinant form (reviewed in ref. 6). An independent test funded by the World Health Organization (WHO) of six candidate antigens showed that the protection obtained with these antigens never exceeded 40%6. More recently, the transcriptome7 and genome8 of S. mansoni have been characterized, but this upsurge in genetic information has not yet been accompanied by the discovery of promising new vaccine antigens. The failure to develop an efficacious schistosome vaccine can be attributed in part to the complex immunoevasive strategies used by the parasite to avoid elimination from its intravascular environment9. Schistosomes have a unique outer syncytial surface, the tegument, that constitutes the host-parasite interface10 and therefore represents an obvious tissue to target for development of new control strategies. To identify proteins that contain membrane-targeting signals and are putatively expressed in the tegument, we used signal sequence trapping to identify two S. mansoni cDNAs of particular interest: Sm-tsp-1 and Sm-tsp-2 (ref. 11). These mRNAs encoded tetraspanins, fourtransmembrane-domain proteins homologous to surface receptors on B and T cells. Expressed sequence tags (ESTs) representing both tsp-1 and tsp-2 mRNAs are abundant in both the lung-stage larval and adult stage parasites (data not shown). The functions of schistosome tetraspanins are unknown, but some mammalian homologs associate with partner proteins in basolateral domains of the plasma membrane and function in cell-cell interactions and maintenance of cell membrane integrity12, so we reason that schistosome tetraspanins might perform a similar role in the tegument. We expressed and purified the large extracellular loop 2 (major ligand-binding domain) of TSP-1 and TSP-2 as soluble fusion proteins with E. coli thioredoxin. We then vaccinated mice with the adjuvanted fusion proteins. Antibodies to both proteins bound exclusively to the tegument of adult S. mansoni; we saw weak binding around some of the outermost nuclei that we believe belong to the tegumentary cytons10 (Fig. 1). Antiserum to the thioredoxin fusion protein did not bind to any schistosome tissues. The protein composition of the S. mansoni tegument was recently reported13,14; TSP-2 was one of relatively few integral membrane proteins to be consistently found in the tegument and not found in underlying tissues.

1Helminth Biology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, 300 Herston Road, Brisbane, Queensland 4006, Australia. 2Department of Microbiology, Tropical Medicine and Immunology, George Washington University, 2300 I Street NW, Washington DC 20037, USA. 3Centro de Pesquisas Rene ´ Rachou, Fundac¸a˜o Oswaldo Cruz (FIOCRUZ), Belo Horizonte, Minas Gerais, Brazil. 4These authors contributed equally to this work. Correspondence should be addressed to A.L. ([email protected]).

Received 7 December 2005; accepted 2 May 2006; published online 18 June 2006; doi:10.1038/nm1430

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tub

Figure 1 TSP-1 and TSP-2 are expressed in the outer tegument of adult S. mansoni. Immunofluorescence of methanol-fixed adult S. mansoni sections with anti–TSP-1, anti–TSP-2 and anti-thioredoxin sera followed by Cy2conjugated antibody to mouse Ig. Original magnifications, 63 (top three panels) and 252 (bottom three panels). Green fluorescence denotes regions where antibody has bound. We also stained sections with DAPI to label nuclei (blue). teg, tegument; tub, tubercles of the tegument; nuc, nuclei. Note fluorescence of only the tegument with antibodies to TSP-1 and TSP-2 but not antibodies to thioredoxin. Some of the nuclei immediately beneath the tegument (arrows) are probably from tegumentary cytons (see ref. 10) and are likely to represent the site of synthesis for tegument proteins before they are trafficked to the tegument syncytium; some of these putative tegumentary cyton nuclei are associated with green fluorescence.

tub teg

teg

Anti–TSP-1

Anti–TSP-2

Anti–thioredoxin

nuc nuc

teg

teg

nuc

nuc

teg

Anti–TSP-2

Because the TSPs were expressed in the tegument, we used the recombinant proteins to screen for specific antibodies in the sera of individuals from Brazil who were exposed to S. mansoni and were either (i) putatively resistant15,16 or (ii) chronically infected with the parasite. Putatively resistant individuals are resistant to infection despite years of exposure to S. mansoni. Cohorts are defined as: (i) negative over 5 years for S. mansoni infection based on fecal egg counts; (ii) never treated with anthelminthic drugs; (iii) continually exposed to infection; and (iv) maintain a vigorous cellular and humoral immune response to crude schistosome antigen preparations15–18. Levels of IgG1 and IgG3 against TSP-2 were significantly higher in sera from putatively resistant individuals than in sera from chronically infected individuals (P o 0.001 for both immunoglobulins) (Fig. 2). Control sera from unexposed individuals from either the US or Brazil did not contain detectable TSP-2–specific antibodies. None of the cohorts tested mounted substantial antibody responses to TSP-1 (data not shown). Chronically infected individuals did, however, produce moderate to high levels of antibodies (all IgG subclasses and IgE) against both soluble schistosome egg antigen and soluble adult worm antigen (data not shown)15. Notably, the antibody response mounted by the putatively resistant individuals against TSP-2 consisted exclusively of the cytophilic antibodies IgG1 and IgG3 (but not other IgG subclasses or IgE), although IgG1 and IgG3 are not the antibody isotypes commonly associated with chronic helminth infections (which are IgG4 and IgE)19. Studies in Brazil20 and Egypt21 assessed the immune responses of resistant and suscep-

Anti–thioredoxin

tible individuals to a panel of S. mansoni vaccine antigens (mostly those tested by the WHO)6; no single antigen was uniquely recognized by antibodies from resistant but not chronically infected individuals. Putatively resistant individuals produced greater amounts of interferon (IFN)-g in response to Sm14 (tested as a vaccine by the WHO) than did chronically infected individuals22; however, this association was not reported at the humoral level.

IgG1 to TSP-2

2.500

2.000

OD450 nm

Anti–TSP-1

1.500

1.000

0.500

0.000 PR

Light

Medium

Heavy

USA NEG

BRAZ NEG

USA NEG

BRAZ NEG

Patient group 1.500

IgG3 to TSP-2

1.250

1.000

Figure 2 Individuals who are putatively resistant to S. mansoni selectively recognize TSP-2, but chronically infected individuals do not. Serum IgG1 and IgG3 responses to TSP-2 from individuals exposed to S. mansoni but uninfected (putatively resistant; PR), exposed and chronically infected with different infection intensities (light, 1–99 e.p.g.; medium, 100–399 e.p.g.; heavy, Z400 e.p.g.) or unexposed blood donors from the US (USA NEG) or Brazil (BRAZ NEG). We determined antibody responses by ELISA. Dots reflect OD values of individual people and bars reflect the mean OD values of the groups. We assessed IgG2, IgG4 and IgE responses but did not detect such responses in any of the groups (data not shown).

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0.750

0.500

0.250

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LETTERS Table 1 Parasitologic data and antibody titers of mice that were vaccinated with adjuvanted recombinant TSP-1, TSP-2, thioredoxin or PBS and were challenged with S. mansoni over two independent trials Fecal eggs

Liver eggs

Adult worms Adult worms Mean ± SE

Median (% reduction)

Liver eggs Mean ± SE


Fecal eggs Mean ± SE


Adjuvanted

Antibody endpoint

Adult worms,

Immunogen

titers

range

(% reduction)

P value

(% reduction)

P values

(% reduction)

P values

22–53

37.4 ± 3

37.5

29,560 ± 5,425

26,233

ND

ND

1–48

23.2 ± 4

22

13,779 ± 3,150

11,393

ND

ND

(38%)

(41%) P ¼ 0.011

(53%)

(57%) P ¼ 0.019

14.9 ± 4 (61%)

13.0 (65%)

ND

ND


Trial 1 Control (PBS)

Ig IgG1

1:400 1:400

n¼9 TSP-1

IgG2a Ig

1:200 1:3,200,000

n ¼ 10

IgG1 IgG2a

1:1,600,000 1:400,000

TSP-2

Ig IgG1

1:3,200,000 1:1,600,000

n¼9 Trial 2

IgG2a

1:400,000

Control

Ig

1:3,200,000

(thioredoxin) n¼8

IgG1 IgG2a

1:1,600,000 1:400,000

TSP-1

Ig IgG1

1:3,200,000 1:1,600,000

n ¼ 10 TSP-2

IgG2a Ig

1:800,000 1:3,200,000

n ¼ 10

IgG1 IgG2a

1:3,200,000 1:1,600,000

1–28

9,726 ± 3,252 (67%)

P ¼ 0.001

6,534 (75%) P ¼ 0.002

68–91

73.8 ± 3

71

36,983 ± 3,232

37,666

1,700 ± 152

1,600

37–69

52.3 ± 4 (29%)

51 (28%)

18,393 ± 3,255 (50%)

16,733 (56%)

520 ± 144 (69%)

400 (75%)

0–60

34.4 ± 7

P ¼ 0.001 38

14,420 ± 3,625

P ¼ 0.004 14,566

600 ± 115

P o 0.0001 500

(53%)

(46%) P o 0.0001

(61%)

(61%) P ¼ 0.001

(65%)

(69%) P o 0.0001

Statistical analyses were performed using non-parametric Mann-Whitney U-tests on median values. ‘‘n’’ refers to the number of mice per group (from a total of 10) that survived the trial and were necropsied.

Because the TSPs were expressed on the parasite surface and TSP-2 was selectively recognized by antibodies of naturally immune individuals, we assessed their efficacies as vaccines in the mouse model of S. mansoni infection. Different strains of inbred mice have been used for schistosomiasis vaccine trials. BALB/c and BL/6 mice are considered high responders to the vaccine made from irradiated S. mansoni cercariae and have fewer worms after challenge infection than do moderate responders such as CBA mice23,24; however, infected CBA mice show a stronger splenic proliferative response and a lesser suppressor T-cell response once infections become patent than do high-responder mice25. We chose CBA/CaH mice for our vaccine trials on the assumption that a recombinant antigen that elicits a protective response in this strain would be likely to yield an even greater protective response in high-responder mice. In the vaccine trials, we immunized mice three times with adjuvant-formulated recombinant TSP-1 or recombinant TSP-2, then challenged them with S. mansoni cercariae. Control groups received adjuvanted phosphate-buffered saline (PBS; trial 1) or adjuvanted thioredoxin (trial 2). Antibody endpoint titers and parasitologic data are provided in Table 1. The antibody responses to both recombinant proteins were dominated by IgG1 and IgG2a, with endpoint titers against each of the two antigens before parasite challenge in excess of 1:1,600,000 for IgG1 and 1:400,000 for IgG2a. By necropsy (91 d after the final vaccination), titers for each of the two antigens had dropped to 1:800,000 for IgG1 and 1:200,000 for IgG2a. Mice vaccinated with either TSP antigen had significantly lower worm burdens and liver egg burdens than did control mice (Table 1). Vaccination with TSP-2 resulted in a 57% reduction in adult worm burdens and a 64% reduction in liver egg burdens compared with control animals (mean values of both trials combined). Vaccination with TSP-1 resulted in a 34% reduction in mean adult worm burdens and a 52% reduction in mean liver egg

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burdens. To assess the effects of the vaccines on reduction of parasite transmission, fecal eggs were counted (trial 2 only); feces from mice vaccinated with TSP-1 and TSP-2 had respectively 69% and 65% fewer eggs (P o 0.0001) than those from control animals. Vaccination with the TSPs significantly reduced the numbers of eggs in the liver (Table 1), the chief cause of pathology in schistosomiasis and, in our opinion, a more meaningful endpoint than adult worm burden for an antipathology vaccine. Vaccination also resulted in highly significant reductions in fecal egg outputs (Table 1), highlighting the fact that the TSP vaccines not only reduced parasite load in the host but also in the environment, thereby reducing transmission. Mathematical modeling of S. japonicum transmission dynamics in China showed that an antifecundity vaccine for the bovine reservoir host must have about 75% efficacy to ensure reduction and long-term elimination of the parasite in the human population26. Although bovines do not constitute a reservoir for S. mansoni, vaccination with either TSP-1 or TSP-2 is in the predicted range to have a substantial impact on elimination of S. mansoni schistosomiasis. Indeed, we identified an ortholog of S. mansoni TSP-2 in the ESTs of the Asian schistosome S. japonicum that shared more than 90% sequence identity over the first 116 amino acids (data not shown), indicating that a vaccine based on S. mansoni TSP-2 might also be effective against S. japonicum. Neither the protective mechanisms nor the developmental stages targeted by the TSP vaccines are known. Tsp-2 mRNA is highly upregulated in intramammalian stages of the parasite, including the lung-stage schistosomulum (data not shown)11. We believe that antibodies bind to the surface of the lung-stage parasite as well as the adult parasite in the hepatic portal system, where they opsonize the parasite for further attack by complement, antibody-dependent cellular mechanisms or both. Although their functions are unknown, it is

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LETTERS now apparent that a family of tetraspanins is expressed in the schistosome tegument. Sm23, a tetraspanin of unknown function with 31% identity to TSP-2, and one of the independently tested WHO vaccine candidates6, was immunolocalized to the tegument of S. mansoni27, although recent analyses of the most abundant proteins in the S. mansoni tegument did not detect it13. A potential function of TSPs in the tegument is immune evasion. One of the most intriguing immunoevasive mechanisms shown by schistosomes is the adsorption of host molecules, including major histocompatibility complex (MHC) molecules28, onto the tegument to mask the parasite’s nonself status. Receptors for some of these adsorbed host products have been identified29,30, but the mechanism by which MHC molecules are acquired by the parasite is unknown. Many mammalian tetraspanins complex with MHC molecules (reviewed in ref. 12) and, notably, TSP2 coimmunoprecipitates from extracts containing schistosome proteins with a protein of the same molecular weight (34 kDa) as class II heavy a chain (data not shown). We suggest that tetraspanins in the tegument of schistosomula and adult worms might be receptors for host ligands, including MHC, and that vaccination induces antibodies that interfere with the interactions between these tetraspanins and their host ligands, thereby blocking crucial immunoevasive strategies and rendering the parasite surface vulnerable to an effective immune response. The unique recognition of TSP-2 by IgG1 and IgG3 (but not IgE) antibodies from individuals who are exposed but resistant to schistosomiasis, and the vaccine efficacy of TSP-2 in mice, emphasize the potential of this molecule as a safe and effective recombinant vaccine for human schistosomiasis. METHODS Recombinant protein expression. We amplified the regions of the Sm-tsp-1 and Sm-tsp-2 cDNAs encoding extracellular loop 2 (Tyr109–Asp193 for TSP-1 and Glu107–His184 for TSP-2) by PCR with Pfu polymerase such that they fused in frame with the N-terminal E. coli thioredoxin and the C-terminal V5 and 6His epitopes encoded by the pBAD/TOPO ThioFusion plasmid (Invitrogen). We ligated cDNAs into the plasmid and transformed E. coli TOP10 cells (Invitrogen) with recombinant plasmids according to the manufacturer’s instructions. Subsequent protein expression and solubilization under native conditions were conducted as recommended by the manufacturer. We purified recombinant fusion proteins from E. coli lysates under nondenaturing conditions with Triton X-100 in the buffers using cobalt Talon affinity chromatography (BD Biosciences). The identities of the purified proteins were confirmed by western blotting with antibodies to thioredoxin and to the 6His epitopes. Immunolocalization. We fixed freshly perfused adult S. mansoni and S. japonicum in 100% methanol, embedded them in Tissue-Tek Optimal Cutting Temperature (OCT) compound (ProSciTech) and cryostatically sectioned them into 7.0-mm sections. We then immunolabeled the sections using indirect immunofluorescence as follows. We blocked sections with 5% skimmed milk powder (SMP) in PBS containing 0.1% Tween 20 and then incubated them with mouse anti–TSP-1 or anti–TSP-2 sera (1:25 dilution in SMP) followed by rabbit antibody to mouse IgG conjugated to Cy2 (Jackson ImmunoResearch; diluted 1:150 in SMP). Sections were counterstained with DAPI (Sigma; 0.1 mg/ml in PBS), which stains nuclei. We mounted the slides with DAKO mounting medium and examined them using a confocal microscope (Leica TCS SP2) fitted with a Leica DMRE camera. As negative controls we used prevaccination serum and serum raised against the thioredoxin tag alone. Assembly of cohorts. S. mansoni is endemic in the state of Minas Gerais, Brazil. We surveyed villages in the northeastern region of the state, conducting repeated-measures prospective surveys. The research team enumerated dwelling structures in each co´rrego and obtained informed consent from inhabitants using a verbal version of the standard consent form approved by the National

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Ethics Committee of Brazil. The research team then monitored water contact of the residents by previously described methods31–35. The schedule for the collection of data on water contact frequency, fecal exams and treatment over a 60-month period is shown in Supplementary Table 1 online. We determined water contact using previously described methods32,33,35. In brief, direct observation of water contact (as outlined in ref. 33) was first conducted among a subset of the study sample to determine the most frequent water contact activities, duration values of each activity and the body immersion values of each activity (amount of body in water). A survey instrument was constructed and then administered to a subset of the study sample to validate the survey (as outlined in ref. 32). Water contact frequencies were multiplied by these standardized duration and body immersion values32,35. The clinical forms of schistosomiasis were determined by physical exams performed by physicians from the Centro de Pesquisas Rene Rachou, Belo Horizonte, Brazil. No individuals with a clinical form of schistosomiasis (hepatointestinal or hepatosplenic) were included in this study because these forms of schistosomiasis are associated with profound changes in the immune response (reviewed in ref. 9). After 60 months of surveillance, we assembled two cohorts of individuals for these studies based upon the criteria in Supplementary Table 2 online. The putatively resistant cohort15,16 (n ¼ 12) we defined as resistant individuals who (i) were negative over the 5 years for S. mansoni infection, (ii) had never been treated with anthelminthic drugs, (iii) experienced continuous exposure to infection as evaluated by water contact studies and (iv) had vigorous cellular and humoral responses to crude parasite antigen extracts15–18. The chronically infected cohort we defined as infected individuals who (i) received anthelminthic treatment until negative, (ii) had continuous exposure to infection as evaluated by water contact at sites with known transmission and (iii) became reinfected with S. mansoni after anthelminthic treatment, reaching in 12 months parasite loads similar to or higher than those of the baseline infection. We stratified the chronically infected cohort by the intensity of infection based upon eggs per gram of feces (e.p.g.) as detected by the Kato Katz fecal thick smear. The infection strata were ‘light’ infection (1–99 e.p.g.; n ¼ 20), ‘medium’ infection (100–399 e.p.g.; n ¼ 20) and ‘heavy’ infection (Z400 e.p.g.; n ¼ 20). Extensive multihousehold pedigrees have been constructed in these areas and, until now, no heritable factor had been associated with putative resistance or chronic infection with S. mansoni36,37. However, previous research in the area showed a strongly heritable factor (heritability, 42%) that associated with intensity of infection37. The ethical review board of George Washington University, the ethical committee of Centro de Pesquisas Rene´ RachouFIOCRUZ and the Federal Brazilian Ethical Review Board (CONEP) reviewed and approved the study of chronically infected individuals from Americaninhas, Minas Gerais state, Brazil. The ethical review board of the Centro de Pesquisas Rene Rachou (FIOCRUZ) reviewed and approved the study of putatively resistant individuals. Indirect ELISA with human sera. We obtained serum samples from whole blood collected into siliconized tubes. We separated the serum by centrifugation at 800g for 10 min, transferred the resulting serum supernatant to sterile 1.0-ml tubes and stored it at –80 1C. We coated Nunc Maxisorp Surface 96-well plates with 0.5 mg TSP-1 or TSP-2 per well in 0.06 M NaCO3, pH 9.6, and stored them overnight at 4 1C. For IgG2 assays, 96-well plates were first adsorbed overnight at 23 1C with 100 ml per well of poly-l-lysine at 1 mg/ml in 50 mM NaCO3, pH 9.0. We then washed the plates with PBS, added crude antigen and incubated the plates in the manner described above. We washed the plates five times with PBS and then blocked for 1 h with PBS containing 1% FCS at 23 1C. We then washed the plates five times with PBS containing 0.05% Tween 20 (PBST). We diluted serum samples 1:100 in PBST and added 100 ml per well in duplicate to a plate. We incubated the plates overnight at 4 1C and then washed them five times with PBST as before. We added 100 ml of the following dilutions of horseradish peroxidase–conjugated human protein–specific antibodies (Zymed) to each well: 1:5,000 of IgG1; 1:1,000 of IgG2, IgG3 and IgG4; and 1:800 of IgE. We incubated the plates for 1 h at 23 1C and then washed them ten times with PBST. We then added 100 ml per well of ortho-phenylenediamine (OPD, Sigma) containing 0.03% hydrogen peroxide. We developed the plates for 30 min in the dark and stopped the reaction with 50 ml per well of 30% H2SO4. The optical density (OD) was measured at 492 nm on an automated ELISA reader (Molecular Devices).

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LETTERS To standardize the assay conditions, we included the following negative control groups on each plate: (i) combined sera of six uninfected individuals from the United States; (ii) combined sera of six egg-negative individuals without a history of S. mansoni infection from Belo Horizonte, Minas Gerais, Brazil; (iii) single serum controls from four egg-negative US volunteers who had not traveled to areas in which schistosomiasis is endemic. We diluted negative control sera 1:100 in PBST and added 100 ml per well to each plate in duplicate. We repeated assays if negative sera had a coefficient of variation 410% between plates, and we rejected duplicates on a single plate if they differed by a coefficient of variation of 410% from each other. We conducted each assay for a specific isotype during one run, using the same stocks of PBS, PBST, PBS with 1% FCS and OPD developer, to ensure standard conditions across plates. We tested each plate for correlations (r ¼ 0.08) in OD reading by well position. Assay plates in a single run were also tested for correlations (r Z 0.01) in OD reading by the serial position of the plate during reading. Vaccination of mice with recombinant proteins. Recombinant TSP-1 and TSP-2 (25 mg per dose in 25 ml) were formulated with an equal volume of either Freund complete (prime) or Freund incomplete (two boosts) adjuvants. We immunized two groups of ten female CBA/CaH mice with adjuvanted TSP-1 or TSP-2, respectively. We immunized control groups of ten mice with either PBS (trial 1) or 25 mg of recombinant E. coli thioredoxin (trial 2); both PBS and thioredoxin immunogens were formulated with Freund complete (prime) and incomplete (two boosts) adjuvants. We boosted mice twice at 2-week intervals and challenged with 120 S. mansoni cercariae by abdominal skin penetration38. At –2, 40 (pre–parasite challenge) and 89 (post-challenge) d after immunization, we took serum samples to assess antibody responses. The Animal Ethics Committee of The Queensland Institute of Medical Research approved the animal studies in 2004. Mouse serology. We screened sera from mice in each vaccine group for recognition of their corresponding recombinant immunogen by ELISA. Microtiter plates (Greiner, Microlon high-binding plates) were coated with either TSP-1 or TSP-2 at a final concentration of 3.0 mg/ml in 0.06 M NaCO3, pH 9.6. After incubating for 16 h at 23 1C, we then blocked plates with 5% SMP for 2 h at 23 1C. We serially diluted mouse sera (pooled for trial 1 and individual samples for trial 2) in PBST from 1:100 to 1:6,400,000 and 100 ml of each dilution was added to wells. We allowed antibodies to bind for 1 h at 23 1C before addition of horseradish peroxidase–conjugated goat antibody to mouse Ig (Silenus) for 1 h at 23 1C. We performed three washes with PBST after each incubation step. Peroxidase activity was detected using ABTS substrate (Chemicon) and the OD was monitored at 405 nm using a Benchmark plate reader and Microplate manager (Bio-Rad). Data are reported as antibody endpoint titers, which we defined as the highest dilution of test group sera that yielded an average OD three standard deviations greater than that obtained in the absence of primary antibody (PBS). Necropsy and estimation of worm and egg burdens in liver and feces. Seven weeks after challenge, all mice were killed with CO2 and necropsied to determine worm and liver egg burdens. Mice were perfused with PBS from the mesenteric veins and the numbers of male and female adult parasites were counted. We removed mouse livers, weighed them and digested them overnight at 37 1C with 10 ml each of 5% potassium hydroxide. For each group, we measured total adult worm burdens and liver egg burdens and calculated the reductions as a percentage of the parasite burdens in the control group. We determined fecal egg counts by collecting pooled feces from mice in each group over a 24-h period, starting on day 48 after infection. We homogenized an equal amount of fecal material (0.5 g) from each group by vortexing in PBS and then rotating overnight at 4 1C. We pelleted each homogenate by centrifugation at 500g for 10 min, resuspended it in 50 ml of PBS and filtered it through a 600-mm mesh followed by a 250-mm mesh. We pelleted the filtered material by centrifugation at 500g for 10 min and resuspended it in 10 ml of PBS. The number of eggs in a 100-ml aliquot was counted ten times and averaged, and the e.p.g. was calculated. Statistical analyses. We analyzed the human antibody responses to recombinant TSP-2 using a Student t-test. For all vaccine trial data, we used nonparametric Mann-Whitney U-tests because the sample sizes were too small to

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determine normal distribution. We compared the medians of each individual test group (TSP-1 or TSP-2) with the control groups for each analysis. We used SPSS version 13 for all statistical analyses. Accession codes. GenBank: Sm-tsp-1 cDNA, AF521093; Sm-tsp-2 cDNA, AF521091. Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS We thank M. Smout for assistance with figure preparations, W. Schroeder for technical assistance and D. Diemert and P. Hotez for comments on the manuscript and discussions. This work was supported by grants from the National Health and Medical Research Council of Australia (NHMRC) and The Wellcome Trust. A.L. is the recipi of an R. Douglas Wright Career Development Award from NHMRC. M.S.P. was supported by an Australian Postgraduate Award. J.M.B. is supported by an International Research Scientist Award from Fogarty International Center, US National Institutes of Health. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.nature.com/naturemedicine/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. World Health Organization. Prevention and control of schistosomiasis and soiltransmitted helminthiasis: report of a WHO expert committee (World Health Organization, Geneva, 2002). 2. King, C.H., Dickman, K. & Tisch, D.J. Reassessment of the cost of chronic helmintic infection: a meta-analysis of disability-related outcomes in endemic schistosomiasis. Lancet 365, 1561–1569 (2005). 3. Bergquist, N.R., Leonardo, L.R. & Mitchell, G.F. Vaccine-linked chemotherapy: can schistosomiasis control benefit from an integrated approach? Trends Parasitol. 21, 112–117 (2005). 4. Minard, P., Dean, D.A., Jacobson, R.H., Vannier, W.E. & Murrell, K.D. Immunization of mice with cobalt-60 irradiated Schistosoma mansoni cercariae. Am. J. Trop. Med. Hyg. 27, 76–86 (1978). 5. Stek, M.F., Minard, P., Dean, D.A. & Hall, J.E. Immunization of baboons with Schistosoma mansoni cercariae attenuated by gamma irradiation. Science 212, 1518–1520 (1981). 6. Bergquist, N.R. & Colley, D.G. Schistosomiasis vaccines: research to development. Parasitol. Today 14, 99–104 (1998). 7. Verjovski-Almeida, S. et al. Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni. Nat. Genet. 35, 148–157 (2003). 8. El-Sayed, N.M., Bartholomeu, D., Ivens, A., Johnston, D.A. & LoVerde, P.T. Advances in schistosome genomics. Trends Parasitol. 20, 154–157 (2004). 9. Pearce, E.J. & MacDonald, A.S. The immunobiology of schistosomiasis. Nat. Rev. Immunol. 2, 499–511 (2002). 10. Jones, M.K., Gobert, G.N., Zhang, L., Sunderland, P. & McManus, D.P. The cytoskeleton and motor proteins of human schistosomes and their roles in surface maintenance and host-parasite interactions. Bioessays 26, 752–765 (2004). 11. Smyth, D. et al. Isolation of cDNAs encoding secreted and transmembrane proteins from Schistosoma mansoni by a signal sequence trap method. Infect. Immun. 71, 2548–2554 (2003). 12. Levy, S. & Shoham, T. The tetraspanin web modulates immune-signalling complexes. Nat. Rev. Immunol. 5, 136–148 (2005). 13. van Balkom, B.W. et al. Mass spectrometric analysis of the Schistosoma mansoni tegumental sub-proteome. J. Proteome Res. 4, 958–966 (2005). 14. Braschi, S. & Wilson, R.A. Proteins exposed at the adult schistosome surface revealed by biotinylation. Mol. Cell. Proteomics (2005). 15. Correa-Oliveira, R. et al. The human immune response to defined immunogens of Schistosoma mansoni: elevated antibody levels to paramyosin in stool-negative individuals from two endemic areas in Brazil. Trans. R. Soc. Trop. Med. Hyg. 83, 798–804 (1989). 16. Correa-Oliveira, R., Caldas, I.R. & Gazzinelli, G. Natural versus drug-induced resistance in Schistosoma mansoni infection. Parasitol. Today 16, 397–399 (2000). 17. Viana, I.R. et al. Comparison of antibody isotype responses to Schistosoma mansoni antigens by infected and putative resistant individuals living in an endemic area. Parasite Immunol. 17, 297–304 (1995). 18. Viana, I.R. et al. Interferon-gamma production by peripheral blood mononuclear cells from residents of an area endemic for Schistosoma mansoni. Trans. R. Soc. Trop. Med. Hyg. 88, 466–470 (1994). 19. Hoffmann, K.F., Wynn, T.A. & Dunne, D.W. Cytokine-mediated host responses during schistosome infections; walking the fine line between immunological control and immunopathology. Adv. Parasitol. 52, 265–307 (2002). 20. Ribeiro de Jesus, A. et al. Human immune responses to Schistosoma mansoni vaccine candidate antigens. Infect. Immun. 68, 2797–2803 (2000). 21. Al-Sherbiny, M. et al. In vitro cellular and humoral responses to Schistosoma mansoni vaccine candidate antigens. Acta Trop. 88, 117–130 (2003).

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