Praziquantel Treatment of Individuals Exposed to Schistosoma haematobium Enhances Serological Recognition of Defined Parasite Antigens

1108 • JID 2005:192 (15 September) • Mutapi et al.

M A J O R A R T I C L E

Praziquantel Treatment of Individuals Exposedto Schistosoma haematobium Enhances SerologicalRecognition of Defined Parasite Antigens

Francisca Mutapi,1 Richard Burchmore,2 Takafira Mduluza,3 Aude Foucher,2,a Yvonne Harcus,1 Gavin Nicoll,1

Nicholas Midzi,4 C. Michael Turner,2 and Rick M. Maizels1

1Institute for Immunology and Infection Research, Ashworth Laboratories, School of Biological Sciences, University of Edinburgh, Edinburgh,and 2Institute of Biomedical Life Sciences, Division of Infection and Immunity, University of Glasgow, Glasgow, United Kingdom; 3Departmentof Biochemistry, University of Zimbabwe, Mount Pleasant, and 4National Institute of Health Research, Causeway, Harare, Zimbabwe

Background. Schistosomiasis is a major parasitic disease affecting 1200 million people in the developingworld, and 400 million people are at risk for infection. This study aimed to identify and compare proteins recognizedby serum samples from schistosome-exposed individuals before and after curative praziquantel treatment.

Methods. Proteins recognized by pooled serum samples from Schistosoma haematobium–exposed Zimbabweanswere determined by 2-dimensional Western blotting and identified by mass spectrometry.

Results. Serum samples recognized 71 spots, which resolved to 26 different characterized proteins. Eleven ofthese proteins have not previously been shown to be immunogenic in natural human infection or in experimentalmodels of schistosomiasis, making them novel antigens in the parasite. Pretreatment serum samples recognized59 spots, which resolved to 21 different identified proteins. Posttreatment serum samples recognized an additional12 spots, which resolved to 8 different identified proteins. Of these 8 proteins, 3 had putative isoforms recognizedbefore treatment, and 5 (calreticulin, tropomyosin 1, tropomyosin 2, paramyosin, and triose phosphate isomerase)did not.

Conclusions. This study is the most comprehensive characterization of S. haematobium antigens to date anddescribes novel antigens in all schistosome species. Posttreatment results are consistent with praziquantel treatmentinducing quantitative and qualitative changes in schistosome-specific antibody responses.

Schistosomiasis is second to malaria in public health

importance [1] in tropical and subtropical countries in

Africa, the Middle East, and South America. Schisto-

soma haematobium, the causative agent of urinary schis-

tosomiasis, is primarily an African parasite and is found

in 53 countries in the Middle East and Africa, including

the islands of Madagascar and Mauritius. A recent sur-

vey of sub-Saharan Africa indicated that, of 682 million

Received 2 March 2005; accepted 11 April 2005; electronically published 5August 2005.

Potential conflicts of interest: none reported.Financial support: Medical Research Council, United Kingdom (grant G81/538);

Carnegie Trust for the Universities of Scotland; Wellcome Trust.a Present affiliation: Centre de Recherche en Infectiologie, Centre Hospitalier

Universitaire de Quebec, Pavillion CHUL, Sainte Foy, Quebec, Canada.Reprints or correspondence: Dr. Francisca Mutapi, Institute for Immunology and

Infection Research, Ashworth Laboratories, School of Biological Sciences, Uni-versity of Edinburgh, W. Mains Rd., Edinburgh EH9 3JT, United Kingdom ([email protected]).

The Journal of Infectious Diseases 2005; 192:1108–18� 2005 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2005/19206-0024$15.00

individuals, 70 million had hematuria and 32 million

had dysuria associated with S. haematobium infection

[2]. Furthermore, it was estimated that 18 million in-

dividuals had pathological changes in the bladder wall,

and 10 million individuals had hydronephrosis.

Schistosomes induce variable levels of resistance to

reinfection in humans and other animals [3–9]. The

development of naturally acquired immunity against

schistosomes is slow. This has been attributed partly to

the need for the immune system to be exposed to suf-

ficient parasite antigens and partly to effective immune

avoidance mechanisms by the parasites [10]. It is there-

fore important to characterize and study the parasite

proteins that interact with the host’s immune system

and the outcome of that interaction. The primary strat-

egy for control of schistosomiasis is treatment of in-

fected individuals with antihelminth drugs. Praziquan-

tel is widely used and is effective against the 3 primary

schistosome species affecting humans (i.e., S. mansoni,

S. japonicum, and S. haematobium), whereas oxamni-

Novel S. haematobium Antigens • JID 2005:192 (15 September) • 1109

quine is effective against S. mansoni only. Although these drugs

are effective, there is a continuing search—driven partly by

concern over the development of drug resistance and partly by

the desire for a preventative rather than a curative intervention

[11]—for alternative or complementary methods of control,

ranging from molluscides (to kill the intermediate snail host)

to vaccine development.

Several studies have identified schistosome immunogenic

proteins by screening expression libraries with serum samples

from infected or vaccinated animals [12–16]. However, there

are limitations associated with this approach: for example, it

cannot detect immunogenic epitopes arising from posttransla-

tion modifications. The proteomic approach uses native anti-

gens and can readily incorporate serological reactivity through

Western blot techniques [17]. The recent publication of S. man-

soni and S. japonicum expressed sequence tag (EST) data [18,

19] has allowed proteomic technology to be used for the iden-

tification of schistosome proteins [20] and to be systematically

applied for the first time in the identification of schistosome

antigens. We report here the application of proteomic proce-

dures to the characterization of immunogenic proteins in adult

male and female S. haematobium worms. Of the 3 primary

schistosomes that infect humans, this species is the least studied

from an immunological perspective. For example, the large field

study of 10 vaccine candidate proteins recently conducted by

the World Health Organization (WHO) focused solely on S.

mansoni [21]. At present, there is only 1 candidate vaccine an-

tigen for S. haematobium (28-kDa glutathione-S-transferase

[GST]) [22]. Because immune responses to S. haematobium

differ from those to S. mansoni [23] (and may differ from those

to S. japonicum) and because phylogenetic analyses show that

S. haematobium is more closely related to the animal schisto-

somes S. mattheei and S. bovis than to the other human schis-

tosomes [24], it is imperative to study immunogenic proteins

and acquired immunity against this important species.

The aim of this study was to identify and characterize ma-

jor immunogenic proteins for S. haematobium. Serum samples

from individuals exposed to schistosomes were used to screen

soluble extracts from adult parasites, and responses before and

after treatment with praziquantel were compared. The rationale

for this comparative analysis was that treatment with prazi-

quantel has been shown to alter schistosome-specific immune

responses, and this alteration results in qualitative and quan-

titative changes associated with resistance to infection [25–28].

We can therefore test the hypothesis that changes in antibody

responses after treatment are partly due to changes in the an-

tigen profile recognized by the immune system.

SUBJECTS, MATERIALS, AND METHODS

Parasite material. Freeze-dried adult S. haematobium solu-

ble worm antigen preparation (SWAP) was obtained from the

Theodor Bilharz Institute (Giza, Egypt). The parasite strain was

used in previous immunoepidemiological studies [29], and the

soluble fraction was used in immunological assays. To prepare

this fraction, worms were perfused in saline buffer, washed in

PBS (pH 7.4), homogenized, centrifuged to obtain the soluble

fraction, and freeze-dried in aliquots (∼5 mg/mL) that were

reconstituted with distilled water as required.

Study subjects. Serum samples were obtained from villag-

ers in the Mashonaland East province of Zimbabwe, where S.

haematobium is endemic. Only permanent inhabitants of the

study area who had never been treated for any helminth in-

fection were eligible for inclusion in the study. Permission to

conduct the study was obtained from the provincial medical

director. After an explanation of the study aims and procedures

was given to the community, an initial parasitological (using

stool and urine samples) and serological (using blood samples)

survey of all compliant participants was conducted. Stool sam-

ples were processed in accordance with the Kato Katz procedure

[30] to detect S. mansoni eggs and other intestinal helminths,

whereas the urine filtration method [31] was used to detect S.

haematobium eggs in urine samples. After collection of the sam-

ples, all participants were offered treatment with the recom-

mended dose of praziquantel (40 mg/kg of body weight). Par-

ticipants who would not accept treatment on religious grounds

or were absent on treatment days but wished to remain part of

the study cohort were classified as untreated control subjects.

Parasitological and serological samples were collected in the

same manner 12 weeks after treatment. To be included in the

study cohort, participants had to meet all of the following

criteria: (1) provide at least 2 urine and 2 stool samples on

consecutive days at both time points; (2) be negative for in-

testinal helminths, including S. mansoni, at both time points;

(3) be confirmed to be negative for S. haematobium eggs at the

second time point if they had been treated; and (4) provide a

blood sample at both time points. A total of 174 individuals

(5–42 years old) met these criteria; 112 individuals (5–42 years

old) formed the treated cohort, and 62 individuals (5–39 years

old) formed the untreated cohort. Pretreatment infection levels

were similar in the 2 cohorts (60% prevalence; mean infection

intensity, 32 eggs/10 mL of urine).

Gel electrophoresis. Two different 2-dimensional gel sep-

arations were performed in parallel: the first contained 100 mg

of SWAP (to be used for Western blotting) and the second con-

tained 200 mg of SWAP (to be used for protein identification).

Isoelectric focusing instrumentation, immobilized Ph gradient

(IPG) buffers, and related reagents were purchased from Amer-

sham, unless otherwise indicated. In the first-dimension elec-

trophoresis, the antigen was mixed with rehydration solution

(7 mol/L urea, 2 mmol/L thiourea, 4% CHAPS, 65 mmol/L

dithiothreitol [DTT], and trace bromophenol blue) and IPG

buffer (pH 3–10) to give a total sample volume of 250 mL, and

Table 1. Identities of proteins recognized by all serum samples.

Spot no. Protein name SpeciesNCBI

accession no.Hit

score pI MW

1 Fatty acid–binding protein Sm14 Schistosoma japonicum gi:16323012 267 7.82 149232 No significant hit … … … … …3 Myosin light chain S. mansoni gi:5305329 267 4.50 184614 Putative mucinlike protein Aedes aegypti gi:19335684 30 5.10 279565 Putative mucinlike protein Aedes aegypti gi:19335684 34 5.10 279566 Triose phosphate isomerase S. mansoni gi:1351281 247 7.64 28447

7 28-kDa glutathione-S-transferase S. haematobium gi:161013 500 6.76 24071

8 28-kDa glutathione-S-transferase S. haematobium gi:161013 43 6.76 240719 Phosphoglycerate kinase S. mansoni gi:556413 96 6.83 4450810 Myosin heavy chain S. mansoni gi:11276951 125 5.55 22237911 Proteasome subunit S. mansoni gi:29841012 80 5.22 2737812 14-3-3� S. mansoni gi:6649234 32 4.85 2875413 Myosin heavy chain S. mansoni gi:11276951 312 5.55 22292714 Heat-shock protein 70 S. mansoni gi:10168 245 5.40 22292715 No significant hit … … … … …16 ENSANGP00000014266 Anopheles gambiae gi:31212849 53 8.25 3903317 Phosphoglycerate kinase S. mansoni gi:556413 33 6.83 4450818 Putative mucinlike protein … gi:19335684 32 5.10 2795619 Tropomyosin 1 S. mansoni gi:42559587 710 4.62 33008

20 Tropomyosin 2 S. mansoni gi:42559587 495 4.50 33008

21 Glyceraldehyde-3-phosphate dehydrogenase S. mansoni gi:120709 240 8.16 3664022 Glyceraldehyde-3-phosphate dehydrogenase S. mansoni gi:120709 195 8.16 3664023 Fructose-1,6-bisphosphate aldolase S. mansoni gi:605647 839 7.63 3996324 Actin Strongylocentrotus purpuratus gi:224306 231 5.30 4153925 Fructose-1,6-bisphosphate aldolase S. mansoni gi:605647 68 7.63 3996326 Enolase S. mansoni gi:3023710 311 6.12 4742127 Enolase S. mansoni gi:3023710 378 6.12 4742128 Fimbrin S. mansoni gi:495668 63 6.88 7590329 Heat-shock protein 70 S. mansoni gi:10168 308 5.40 6833130 Actin 1 Aedes aegypti gi:1351866 93 5.74 41790

31 Actin S. japonicum gi:6979994 593 5.30 4199932 Actin S. japonicum gi:6979994 618 5.30 4199933 Actin Brugia malayi gi:3182894 404 5.30 4199934 Actin Helobdella triserialis gi:3319951 121 5.38 4144435 Immunophilin S. mansoni gi:561875 267 5.61 4880636 Immunophilin S. mansoni gi:561875 179 5.61 4880637 No significant hit … … … … …38 Calreticulin S. mansoni gi:477298 276 4.37 43163

39 Protein disulfide isomerase S. mansoni gi:312018 305 4.92 5446340 Enolase S. mansoni gi:3023710 203 6.12 4742141 Enolase S. mansoni gi:3023710 433 6.12 4742142 Enolase S. mansoni gi:3023710 489 6.12 4742143 Enolase S. mansoni gi:3023710 188 6.12 4742144 Putative mucinlike protein Aedes aegypti gi:19335684 33 5.10 2795645 Enolase S. mansoni gi:3023710 441 6.12 4742146 Enolase S. mansoni gi:3023710 511 6.12 4742147 Enolase S. mansoni gi:462011 79 6.12 4742148 Putative cytosol aminopeptidase S. mansoni gi:1800313 419 7.56 5689749 Putative cytosol aminopeptidase S. mansoni gi:1800313 140 7.56 5689750 No significant hit … … … … …51 No significant hit … … … … …52 No significant hit … … … … …53 ENSANGP00000019187 Anopheles gambiae gi:31198849 33 11.30 1048854 Phosphoglucomutase Crassostrea gigas gi:27525309 65 6.15 6106555 No significant hit … … … … …56 Phosphoglucomutase C. gigas gi:27525309 62 6.15 61065

(continued)


Table 1. (Continued.)

Spot no. Protein name SpeciesNCBI

accession no.Hit

score pI MW

57 Heat-shock protein 60 … gi:21634531 794 5.32 5874058 Predicted Zn-dependent peptidases Magnetococcus species gi:48833782 33 5.36 10085859 No significant hit … … … … …60 No significant hit … … … … …61 No significant hit … … … … …62 No significant hit … … … … …63 Heat-shock protein 70 S. mansoni gi:10168 515 5.40 6833164 Heat-shock protein 70 S. mansoni gi:10168 184 5.40 6833165 Heat-shock protein 70 S. mansoni gi:10168 101 5.40 6833166 Actin-binding/filaminlike protein S. mansoni gi:38683290 227 5.33 107125

67 Actin-binding/filaminlike protein S. mansoni gi:38683290 456 5.33 107125

68 Actin-binding/filaminlike protein S. mansoni gi:38683290 315 5.33 107125

69 Actin-binding/filaminlike protein S. mansoni gi:38683290 250 5.33 10712570 Paramyosin S. mansoni gi:547978 131 5.31 1000383

71 No significant hit … … … … …

NOTE. The hit score is a Mascot search engine output statistic; higher nos. give greater confidence that the protein identification is correct. Proteins withdifferent National Center for Biotechnology Information (NCBI) accession nos. but the same identification arise as a result of different peptides being used tomatch the mass spectrometry data to the entries in expressed sequence tag databases. Entries in bold represent the spots recognized by posttreatment serumsamples only. MW, molecular weight; pI, isoelectric point.

then the sample was loaded into a 13-cm gel holder with a 13-

cm gel strip (linear pH 3–10). The gel strip was rehydrated,

and the proteins were focused on an IPGPhor machine by use

of the following protocol: 12–14 h of rehydration at 20 V and

a 5-h voltage-focusing procedure (1 h at 500 V, 1 h at 1000 V,

and 3 h at 8000 V). The strips were then incubated in 5 mL

of equilibration buffer (50 mmol/L Tris, 6 mol/L urea, 2% SDS,

and 30% glycerol [pH 8.8]) containing 30 mmol/L DTT for

15 min and in equilibration buffer containing 135 mmol/L io-

doacetamide for another 15 min. Second-dimension electro-

phoresis was performed on a 12% polyacrylamide 13-cm gel

in a Hoefer SE600 system using SDS buffer. The proteins on

the gel used for protein identification were stained with Coo-

massie blue to visualize them, whereas proteins on the gel used

for Western blotting were transferred onto a nitrocellulose mem-

brane, as described below.

Immunoblotting. Proteins were transferred from the gel

onto a nitrocellulose membrane using a semidry system (Hoefer)

in transfer buffer (Invitrogen) containing 10% methanol at 30

V for 1 h. The membrane was stained with Ponceau S solution

(Sigma) to check transfer efficiency and then was blocked at

room temperature for 1 h in Tris-buffered saline (TBS) block-

ing buffer (Pierce) and 0.05% Tween 20. After blocking, the

membrane was subjected to 2 separate 10-min washes with TBS,

0.05% Tween 20, and 0.5% Triton-X 100 (TBS/TT). A pool of

pretreatment serum samples (diluted 1:100 in TBS blocking

buffer and 0.02% Tween 20) was added to the membrane, and

the membrane was incubated overnight at 4�C and then was

washed 3 times for 10 min each time in TBS/TT. Horseradish

peroxidase–conjugated rabbit anti–human IgG (Dako) was di-

luted 1:4000 in TBS blocking buffer, and 0.05% Tween 20 was

added. The membrane was incubated at room temperature for

1 h and then was washed 4 times for 10 min each time in TBS/

TT and 1 time for 10 min in TBS alone. The proteins were

visualized using the chemiluminescence product ECL Plus

(Amersham), in accordance with the manufacturer’s instructions.

Films were exposed to the blots for 5 s and then were developed,

and spots were matched to those on the Coomassie blue–stained

gel. After visualization, the membrane was stripped of the ECL

Plus reagent, secondary antibody, and serum samples, in accor-

dance with the protocol provided by the manufacturer. The same

membrane was then probed using posttreatment serum samples.

A previous assay showed that the stripping procedure removed

all proteins not directly bound to the nitrocellulose membrane,

as indicated by the lack of ECL reactivity with a stripped

membrane. This procedure did not remove any of the parasite

proteins, as evidenced by probing the same membrane with 3

serum samples successively (i.e., a pretreatment serum sample,

then a negative control serum sample, and then the same pre-

treatment serum sample). The gel electrophoresis and Western

blotting were repeated for all samples, to confirm the patterns

that were obtained.

Image analysis. Images from the Western blots were elec-

tronically scanned with Image Master 2-dimensional gel image

analysis software (version 3; Amersham) and used for matching.

Predicted matches were also visually verified. Spots on the Coo-

massie blue–stained gel that matched those on the Western blots

were excised and then were analyzed by mass spectrometry (MS).

Mass spectrometry. Plugs of 1.4 mm were excised from the

gels and were subjected to in-gel trypsin digestion in an Ettan


Figure 1. Coomassie blue–stained 2-dimensional gel showing spots matched to the Western blots. Spots on the gel were excised and identified.Molecular weight markers (in kilodaltons) are given on the right.

Spot Handing Workstation (GE Healthcare), in accordance with

standard protocols (Amersham). The resulting tryptic peptides

were solubilized in 0.5% formic acid and were fractionated by

nanoflow high-performance liquid chromatography on a C18

reverse phase column (GE Healthcare), and elution was per-

formed with a continuous linear gradient of 40% acetonitrile

for 20 min. The eluates were analyzed by online electrospray

tandem MS (MS/MS) by use of a Qstar Pulsar mass spectrom-

eter (Applied Biosystems). A 3-s survey scan preceded each MS/

MS data-collection cycle of 4 product ion scans of 3 s each,

and this gave a duty cycle of 15 s. Data were submitted for an

MS/MS ion search via the Mascot search engine (Matrix Sci-

ence), and both locally established databases for S. mansoni

EST sequences and the present nonredundant National Center

for Biotechnology Information (NCBI) database were searched.

RESULTS

Two-dimensional gel electrophoresis analysis. Two-dimen-

sional gel electrophoresis resulted in separation of S. haema-

tobium SWAP into ∼150 discrete spots that were visible after

standard staining with Coomassie blue (figure 1). Additional

spots could be detected by silver staining, but the quality of

the mass spectra obtained for identification of the proteins was

higher for the Coomassie blue–stained spots, and this technique

resulted in superior data. Moreover, no spot that was subse-

quently shown to be reactive by Western blotting failed to be

stained by Coomassie blue.

Western blot analysis. To determine which proteins were

recognized by the serum samples collected before and after pra-

ziquantel treatment, a Western blot assay was optimized on the

basis of the results of the 2-dimensional gel electrophoresis. Initial


Figure 2. Western blot analyses of serological reactivity of serum samples from the treated cohort, comparing pre- (A) and posttreatment (B) re-sponses. A, Spots reacting with serum samples collected at baseline (before treatment). Boxes represent areas where additional spots in panel B areabsent. B, Spots reacting with serum samples collected 12 weeks after treatment. Boxes highlight the additional spots recognized after treatment.Molecular weight markers (in kilodaltons) are given on the right.

assays using anti–human IgA, IgG, and IgM reagents showed that

IgG detected the maximum number of spots and that IgA and

IgM did not identify any spots that were not detected by IgG.

Therefore, for the full Western blot analysis, anti–human IgG

was used. This analysis showed that a total of 71 spots visualized

on the Coomassie blue–stained gel reacted with human serum

samples from S. haematobium–exposed individuals.

MS/MS analysis. The 71 spots identified as serologically

reactive by Western blotting were excised from the Coomassie

blue–stained gel and were subjected to in-gel trypsin digestion.

Subsequently, the tryptic peptides were analyzed by MS/MS,

and the peptide data obtained were used to search EST data-

bases. Although there are relatively few S. haematobium pep-

tide sequences available, most of the spots were successfully

matched to S. mansoni or S. japonicum proteins whose peptide

sequences are available in public databases. The identifications

made for these 71 spots are shown in table 1. The identity given

for each spot corresponds to the top hit score (the Mascot

output statistic) that had a MOWSE score 130 (MOWSE scores

are logarithmic, so that a hit score with a MOWSE score of 30

represents , a hit score with a MOWSE score of 40P p .05

represents , etc.). If the MOWSE score was !30, thenP p .005

the identification was rejected, and the spot was designated as

being a nonsignificant hit. Predicted molecular weights (MWs)

and isoelectric points (pIs) of each identified protein (not the

spot) as well as the species they come from are also given in

table 1. The majority of the spots corresponded to S. mansoni

proteins.

The MS/MS analysis revealed cases in which different spots

were derived from the same protein: for example, spots 63, 64,

and 65 are all heat-shock protein 70 (HSP70), as are spots re-

solving to the same protein but with different accession num-

bers (e.g., spots 31–34, which are all actin). Some of the rec-

ognized proteins occur as multiple isoforms differing by pIs,

MW, or both. For example, there are at least 3 GST isoforms

differing by pIs, and there are several enolase isoforms differing

by both MW and pIs. In this analysis, it is not possible to define

the precise nature of these differences, because sequence data

are not yet available from the S. haematobium orthologues.

Identity of proteins recognized by serum samples. The pro-

teins recognized included abundantly expressed proteins (as in-

dicated by the size/intensity of the spot in figure 1), such as

glyceraldehyde-3- phosphate dehydrogenase (GAPDH), and in

most, but not all, cases, the size of the spot on the Western

blot image was related to the size of the spot on the Coomas-

sie blue–stained gel. For example, spot 21 (GAPDH) in figure

1 is also a very large spot in figures 2 and 3.

Of the 71 spots recognized by the serum samples, all but 13

gave rise to protein identifications. Of the 58 identified spots, 2

were found to be ESTs whose proteins have not yet been char-


Figure 3. Western blot analyses of serological reactivity of serum samples from the untreated cohort. Serum samples were collected at the sametime points as those used for the treated cohort. A, Spots reacting with serum samples collected at baseline (before treatment in the treated cohort).Boxes represent areas where additional spots in figure 2B are absent. B, Spots reacting with serum samples collected 12 weeks after treatment inthe treated cohort. Boxes represent areas where additional spots in figure 2B are absent.

acterized, whereas the remaining 56 spots resolved to 26 different

proteins. The 26 proteins have been grouped by molecular func-

tion in table 2. They include structural/muscle proteins (which

are most numerous), enzymes (mostly components of the gly-

colytic pathway), chaperone proteins, and binding proteins. On-

ly GST has been studied in S. haematobium, whereas, to our

knowledge, the remaining 25 proteins are identified in S. hae-

matobium here for the first time. Moreover, table 2 shows 14

proteins that have not been previously shown to be immunogenic

in natural human infection with any schistosome species, and

11 of these have not previously been shown to be immunogenic

in experimental models.

Enhanced reactivity after praziquantel treatment. A com-

parative study between pretreatment and posttreatment serum

samples was conducted to determine if treatment altered re-

sponses to the proteins. The pretreatment and posttreatment

Western blot assays were conducted on the same membrane,

to exclude any variation that might arise from the use of dif-

ferent antigen preparations. These assays showed that protein

recognition patterns of serum samples from the 2 time points

differed, as is shown in figure 2. Treatment enhanced the rec-

ognition of specific proteins by serum samples. Of the 71 spots,

pretreatment serum samples recognized 59 spots representing

21 different identified proteins, as is shown in table 1. Serum

samples collected 12 weeks after treatment recognized an ad-

ditional 12 spots representing 8 identified proteins. Of the 12

additional spots, 3 had similar identities to spots of different

MWs or pIs that had been recognized by pretreatment serum

samples—for example, actin, actin-binding/filaminlike pro-

tein, and GST are likely to be different isoforms. Five proteins

(calreticulin, tropomyosin 1, tropomyosin 2, paramyosin, and

triose phosphate isomerase) were recognized only by posttreat-

ment serum samples and did not have isoforms already recog-

nized by pretreatment serum samples.

In addition to these qualitative changes, there were also

quantitative changes in protein recognition, as was indicated

by increases in the intensity of recognition for some spots after

treatment. This was most apparent in spots 1 (fatty acid–bind-

ing protein), 8 (GST), 31–34 (actin), 41–43 (enolase), and 63–

65 (HSP70).

Serum samples from untreated participants showed no

changes in protein identification patterns at the 2 time points

(baseline and 12 weeks later), as is shown in figure 3B. In

addition, at both time points, reactivity of serum samples from

untreated individuals was similar to that of serum samples from

treated individuals at the start of the study, except that they

reacted with spots 67 and 68 (actin-binding/filaminlike pro-

tein), as is shown in figures 2A and 3A.

DISCUSSION

Despite being the most prevalent and widespread schistosome

species affecting humans in Africa [1], S. haematobium is the

least studied with respect to parasite-specific immune responses

Table 2. Summary of proteins, classified by molecular function and published immunological status, recognized by serum samples.

Function, protein Life stage protein is expressed Status in experimental models Status in humans

Structure/motor activityActin All [32] NS NSActin-binding/filaminlike protein … Protective in vaccinated mice (Sm, Sj) [33] WHO vaccine candidate, recognized by human serum

samples (Sm) [21, 33]Fimbrina … NS NSMyosin light chaina … NS NSMyosin heavy chain … 62-kDa portion of molecule Sm62-IrV5 immu-

nogenic in miceSm62-IrV5 WHO vaccine candidate, recognized by human

serum samples (Sm)Paramyosin Muscle of all, also found in tegument of adult worms [32] Immunogenic in mice (Sm, Sj) [15, 34] Native protein WHO vaccine candidate, recognized by hu-

man serum samples (Sm) [21, 34]Tropomyosin 1 Muscle of all Native protein protective in mice (Sm, Sj) [35] Recognized by human serum samples [35]Tropomyosin 2 … Native protein protective in mice (Sm, Sj) [35] Recognized by human serum samples [35]

Catabolic activity (glycolysis)Fructose-1,6,-bisphosphate aldolase All, tegument of adult worms [36] Immunogenic in mice [36] NSEnolasea … NS NSGlyceraldehyde-3-phosphate dehydrogenase Tegument of adult worms [37] Immunogenic in mice (Sm) [37] WHO vaccine candidate, recognized by human serum

samples (Sm) [21]Phosphoglycerate mutasea … NS NSPhosphoglycerate kinase Surface of schitosomulae and adult worms [38] Immunogenic in mice (Sm) [39] WHO vaccine candidate, recognized by human serum

samples (Sm) [21, 39]Phosphoglucomutasea … NS NSTriose phosphate isomerase All, tegument of adult worms [40] Protective in vaccinated mice (Sm) [40] WHO vaccine candidate, recognized by human serum

samples (Sm) [21, 40]Other catalytic activity

28-kDa glutathione-S-transferase Tegument parenchyma, esophageal epithelium, and genitalorgans of adult worms [41]

Protective in vaccinated mice, cattle, andpigs (Sm, Sj, Sh) [42–45]

Leading vaccine candidate for Sh human infections, rec-ognized by human serum samples (Sm, Sj, Sh) [46]

Protein disulfide isomerasea … NS NSCytosol aminopeptidasea … NS NSProteosome subunita … NS NSZn-dependent peptidasea … NS NS

ChaperoningHeat-shock protein 60a Expressed constitutively in all [47] NS NSHeat-shock protein 70 Expressed constitutively in all Immunogenic in mice (Sm) [48] Recognized by human serum samples (Sm) [49]Immunophilin p50 … Immunogenic in rabbits [50] NS

Binding14-3-3-� All, tegument of adult worms and schitosomulae [51] Recognized by serum samples from vacci-

nated mice (Sm, Sj) [51, 52]NS

Calreticulin Cercariae and adults [53] NS Recognized by human serum samples (Sm) [53]Fatty acid–binding protein Sm14 All, basal lamella of the tegument and the gut epithelium [54] Protective in mice (Sm) [55] WHO vaccine candidate, recognized by human serum

samples (Sm) [21, 56]Other

Putative mucinlike proteina … NS NS

NOTE. A blank cell under the “Life stage protein is expressed” column means that no localization studies have been published. NS, no published study; Sh, S. haematobium; Sj, S. japonicum; Sm, S. mansoni;WHO, World Health Organization.

a No information on the protein’s immunogenicity in any schistosome species has been published. Where appropriate, the species in which immunological studies have been conducted is indicated in parentheses.


and antigen characterization. In particular, few specific antigens

have been identified or used for immunoepidemiological re-

search. The present study gives the most comprehensive analysis

to date of adult worm antigens in this species and for schis-

tosomes in general. The analysis focused on the adult stage,

which is the most long-lived developmental stage and a target

for immune elimination in S. haematobium and S. bovis [57].

Of the 150 spots visualized on the Coomassie blue–stained gel,

71 were detected by their reactivity with total IgG antibodies

in pooled serum samples. This number does not include pro-

teins recognized by a minority of serum samples and for which

reactivity could not be detected after dilution. The 13 spots

that did not have significant hits to known proteins or ESTs

(despite having been processed twice) may not be similar to

presently known proteins or may have given mass spectra that

were too unclear for identification.

Studies are now under way to identify the spots that were

not serologically recognized, particularly those that are abun-

dant in the proteome and might play an important role in host

immune evasion/modulation [58]. The recognition patterns of

the individual IgG subclasses will be investigated, because they,

together with the other isotypes, will help to characterize im-

mune responses to the antigens we have defined here.

Several proteins recognized were homologues of, or were

similar to, presently known vaccine candidates characterized in

S. mansoni and/or S. japonicum. For example, the serum sam-

ples reacted with homologues of 9 of 10 World Health Orga-

nization WHO S. mansoni vaccine candidate antigens [21] in

the S. haematobium proteome. The sequence for the remain-

ing WHO vaccine candidate antigen (PN18-cyclophilin) has

not yet been published in the literature [21], but cyclophilins

are members of the immunophilin family and are related to

the immunophilin p50 recognized by the serum samples used

in the present study.

The S. haematobium proteins that reacted with the serum

samples included those whose homologues are abundant in EST

databases of S. mansoni [18] and S. japonicum [19] as well as

those abundant in the soluble fraction of the adult S. mansoni

proteome [20]. Several of these proteins are conserved among

invertebrates, and some are vaccine candidates for other hel-

minth species. For example, paramyosin, which was first shown

to be protective against schistosomiasis [59], is a long-standing

vaccine candidate for filariasis [60], cysticercosis [61, 62], and

S. mansoni and S. japonicum infection [15, 32, 34]. Most of the

recognized proteins have been localized to the parasite tegument

in S. mansoni and S. japonicum and are therefore accessible to

the immune system. Only 1 integral tegumental protein, a ho-

mologue of S. mansoni fatty acid–binding protein Sm14, was

identified by the serum samples. This is not surprising, because

tegumental proteins are poorly soluble and were underrepre-

sented in the aqueous fraction used in the present study. For

some proteins with several isoforms that reacted with the serum

samples (e.g., GST for spots 7 and 8), the reactivity differed

between isoforms, and this suggests that the processes generating

these isoforms may alter the immunogenicity of the proteins.

Treatment with praziquantel enhanced the reactivity of se-

rum samples by increasing the number of proteins recognized

and the intensity of the recognition of proteins before treat-

ment. Most of the additional proteins recognized after treat-

ment are associated with the parasite musculature or glycolytic

metabolism, and this indicates that treatment made these pro-

teins preferentially available. This finding is consistent with the

hypothesis that treatment renders different parasite proteins

accessible to the host immune system. In the worm, prazi-

quantel induces paralysis followed by destruction of the teg-

ument, and death is believed to result from synergistic action

with the host immune system [63–65]. Experimental work in

mice has shown that praziquantel treatment exposes tegumen-

tal antigens such as actin [66, 67]. Therefore, the results of the

present study are consistent with the hypothesis that there is a

change in the antigen profile presented to the host immune

system after treatment. Previous studies have shown changes

in antibody responses to crude antigens after treatment but did

not differentiate between changes arising from different amounts

of antigen and those arising from different types of antigen

[25, 28, 68, 69]. The present study clearly shows that both

quantitative and qualitative changes in antigen recognition do

occur after treatment.

In conclusion, the present study has identified 27 S. hae-

matobium proteins that react with serum samples from a pop-

ulation of Zimbabweans exposed to the parasite. Several of these

antigens are novel in all schistosome species and require further

immunological investigation and characterization at the anti-

body and T cell response levels. The study has also shown that

treatment with praziquantel alters responses to individual pro-

teins both qualitatively (new proteins/isoforms being recog-

nized) and quantitatively (increases in reactivity with individual

proteins).

Acknowledgments

We are grateful for the cooperation of the Ministry of Health and ChildWelfare in Zimbabwe, the provincial medical director of Mashonaland East,the environmental health workers, and the residents, teachers, and school-children in Mutoko and Rusike. We are also grateful for the technicalassistance from staff at the Blair Research Institute and the technical advicefrom Rachel Curwen (University of York, United Kingdom).

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Praziquantel Treatment of Individuals Exposed to Schistosoma haematobium Enhances Serological Recognition of Defined Parasite Antigens