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Schistosoma: Cross-reactivity and antigenic community among different species
Experimental Parasitology 111 (2005) 182–190 www.elsevier.com/locate/yexpr
Schistosoma: Cross-reactivity and antigenic community among diVerent species S. Losada a, N. Chacón a, C. Colmenares a, H. Bermúdez a, A. Lorenzo a, J.P. Pointier b, A. Theron b, B. Alarcón de Noya a, O. Noya a,¤ a
Sección de Biohelmintiasis, Instituto de Medicina Tropical, Escuela de Medicina “Luis Razetti,” Facultad de Medicina, Universidad Central de Venezuela, Apartado 47623, Los Chaguaramos 1041-A, Caracas, Venezuela b UMR 5555 CNRS-UP, Parasitologie fonctionnelle et évolutive, CBETM, Université Via Domitia, 52 Av. Paul Alduy, 66860 Perpignan, France Received 25 February 2005; received in revised form 22 July 2005; accepted 26 July 2005 Available online 12 September 2005
Abstract It is not unusual to Wnd common molecules among diVerent species of the genus Schistosoma. When those molecules are antigenic, they may be used in immunodiagnosis and vaccines, but they could also be applied to taxonomic and evolutionary studies. To study cross-reactivity and antigenic community among diVerent species of schistosomes, plasmas from laboratory animals infected with Schistosoma bovis, S. guineensis, S. rodhaini, S. haematobium, and four strains of S. mansoni were evaluated with a crude extract of adult worms of S. mansoni by Western blot. Using the multiple antigen blot assay, plasmas from these infected animals were exposed to a selected group of synthetic peptides from Sm28GST, Sm28TPI, Sm elastase, Sm97, Sm32, Sm31, and Sm Cathepsin L. The results presented herein demonstrate diVerential cross-reactivity and antigenic community among the Mansoni and Haematobium groups of schistosomes, which is of relevance as an additional new tool for phylogenetic studies of schistosomes as well as for diagnosis and vaccine purposes. 2005 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Schistosomes; Cross-reactivity; Antigens; Peptides; Taxonomy; WB, Western blot; MABA, multiple antigen blot assay; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Sm28GST, Sm glutathione S-transferase; Sm28TPI, Sm28 triose phosphate isomerase; Sm97, Sm paramyosin; Sm32, Sm asparaginyl endopeptidase; Sm31, Sm Cathepsin B; SAWA, soluble adult worm antigen; PBS, phosphate buVer saline; PMSF, phenylmethylsulphonylXuoride; EDTA, ethyl-diamine-tetra-acetic acid; IMT, Instituto de Medicina Tropical
1. Introduction The sharing of molecules among organisms is an expected Wnding since there are several functional molecules, such as enzymes, hormones, receptors, etc., that are conserved during the extremely long process of evolution. However, this has special relevance for the identiWcation of molecules with potential for diagnosis or vaccine studies. In 1976, Smith et al. demonstrated crossimmunity to Schistosoma mansoni and S. haematobium *
Corresponding author. Fax: +58212 6930454. E-mail addresses: [email protected] (S. Losada), noyaoo@ yahoo.com (O. Noya). 0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.07.007
in hamsters, suggesting common antigens on the surface of young schistosomula in both species. Bahghat et al. (2001) found that immunoglobulin G against cercarial secretions cross-reacted among S. mansoni, S. haematobium, and S. japonicum. Similarly, Rege et al. (1992) demonstrated the cross-reactivity of cystein proteinases from S. mansoni and S. haematobium. Other enzymes, like cathepsin D, show immunological similarity not only among S. mansoni and S. japonicum but also with bovine cathepsin D (Valdivieso et al., 2003). Molecular biology techniques have allowed the application of antigenic community in systematics and phylogeny, so it is conceivable that together with the utilization of isoenzymes as phenotypic markers, certain molecules or
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fragments that behave as antigens could be used for that purpose, as is the case of cercarial elastase, which is encoded by a functionally conserved gene family across diVerent species of schistosomes (Salter et al., 2002). Antigenic community is also present between parasites and their hosts. That is the case of Schistosoma and Biomphalaria, where several molecules such as tropomyosin have been characterized (Dissous et al., 1990). Cross-reactivity in serologic tests between S. mansoni and other parasites such as Fasciola hepatica is well known (Aronstein et al., 1985). Similarly, infected patients with hookworms and Ascaris lumbricoides developed cross-reactive antibodies against S. mansoni, which are able to kill schistosomula in the presence of complement (Correa-Oliveira et al., 1988). The demonstration of the antigenic community and cross-reactivity could eventually be used for diagnosis, the development of vaccines, and the study of systematics or phylogeny. The aim of this study was to investigate this antigenic community amongst diVerent schistosome species, through the recognition of an adult worm crude extract of Schistosoma mansoni by plasma of rodents infected with S. bovis, S. guineensis, S. rodhaini, S. haematobium, and several strains of S. mansoni by Western blot (WB). We also studied the recognition of peptides derived from proteins from S. mansoni, including Sm28GST, Sm28TPI, Sm31, Sm32, Sm elastase, Sm97, and Sm cathepsin L, by those plasmas from infected animals using the multiple antigen blot assay (MABA) (Noya and Alarcón de Noya, 1998). These proteins were selected since they have been shown to be relevant for either diagnosis or for vaccines against schistosomiasis (Noya et al., 2001; Noya et al., 2003; Valdivieso et al., 2003).
2. Materials and methods 2.1. Animals Outbred white mice (SWISS OF1) and gerbils (Meriones umguiculatus) were infected with diVerent species of Schistosoma (Table 1). Infection was achieved by indi-
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vidual immersion of mice/gerbils in plastic jars containing 50 ml of water (paddling method) containing cercariae of each specie of parasite. Plasmas were obtained from intracardiac puncture with heparinized syringes before hepatic perfusion (Duvall and Dewitt, 1967) under penthotal intraperitoneal injection. Sodium azide was added to plasma from these animals and afterwards they were stored at ¡70 °C. Non-infected mice and gerbils were also sacriWced and their plasmas used as controls. Animals were managed following international principles regarding the Guide for the Care and Use of Laboratory Animals (1996). 2.2. Preparation of the soluble adult worm antigen Adult worms from S. mansoni (JL Venezuelan strain, provided by Dr. I.M. Cesari from the Instituto Venezolano de Investigaciones CientíWcas)-infected mice were collected after hepatic perfusion and used to prepare SAWA-JL. BrieXy, worms were homogenized in PBS with protease inhibitors (1 mM PMSF and 1 mM EDTA) in ice bath and centrifuged at 12,000g for 2 h at 4 °C (Noya et al., 1995). Supernatant was used as soluble adult worm antigen (SAWA). 2.3. SDS–PAGE and Western blot The antigenic community between S. mansoni (JL Venezuelan strain) and the rest of the Schistosoma species or strains was studied by WB. A crude extract of the SAWA-JL was run by 10% SDS–PAGE mini gels and electro-transferred antigens onto nitrocellulose were exposed to diVerent plasmas as described by Laemmli (1970) and Towbin et al. (1979). Nitrocellulose strips transferred with SAWA were incubated with animal plasmas (1:100) and later with anti-mouse total IgG conjugated to horseradish peroxidase (Sigma) (1:2000). A chemiluminescent substrate of the enzyme was used (Super Signal, Pierce) and the luminescence was recorded in photographic Wlm (HyperWlm, Amersham) (Noya et al., 1995). The bands were analyzed with a MultiAnalyst 1.1 software (Bio-Rad, CA, USA).
Table 1 Animals infected with diVerent Schistosoma species Host
Number
Schistosoma species
Schistosoma strains
Infected/No. cercariae
Weeks of infection
Outbred mice Outbred mice Outbred mice Outbred mice Outbred mice Outbred mice Outbred mice Gerbils Gerbils
9 13 12 10 4 8 10 12 7
S. bovis S. rodhaini S. guineensis S. mansoni S. mansoni S. mansoni S. mansoni S. haematobium S. mansoni
— — — Murid from Guadeloupe Human from Guadeloupe Brazil Human JL from Venezuela — Human from Guadaloupe
400 230 140 170 170 170 100 450 200
12 13 13 15 15 16 8 20 9
SD, standard deviation.
Average number § SD of adult worms recovered 15.6 § 9.9 44.8 § 34.2 58.1 § 32.8 63.2 § 48.1 19 § 19.2 53.4 § 38.6 22.5 § 10.2 58.1 § 18.9 88.7 § 38.7
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2.4. Chemical peptide synthesis
3. Results
Non-overlapping peptides corresponding to the whole sequences of Sm31 (GI: 160950 NCBI) and Sm32 (GI: 729709 NCBI) and some regions of the Sm28GST (GI: 121700 NCBI), Sm28TPI (GI: 1351281 NCBI), Sm elastase (GI: 116116 NCBI), Sm97 (GI: 547978 NCBI), cecarial Sm cathepsin L (GI: 3023456 NCBI) and adult Sm cathepsin L (GI: 630486 NCBI) (Table 2) were selected based mainly on hydrophylicity, using the predictive algorithm described by Hoops and Woods (1981). Some of the sequences corresponded to regions surrounding the active site of the enzymes. Peptides were solid-phase synthesized as described by MerriWeld (1963) and modiWed by Houghten et al. (1986). All these proteins except one (Sm97, paramyosin) are enzymes; their aminoacid sequences were obtained from Swiss Protein Data Bank. Peptides were synthesized with cysteine–glycine and glycine–cysteine aminoacids in the amino and carboxy terminus, respectively, to obtain polymerizable peptides that are usually more reactive than monomeric peptides (Noya et al., 2003a). Quality control of each peptide was achieved by mass spectrometry and HPLC by molecular exclusion. The resulting purity was at least 65%.
3.1. Recognition of antigenic molecules from SAWA-JL by Western blot
2.5. MABA This multiple dot blot technique was used to evaluate the immunogenicity of the peptides (Noya and Alarcón de Noya, 1998). Peptides were absorbed individually at 10 g/ml in 28 parallel rows onto a nitrocellulose membrane. Afterwards, 2 mm wide non-fat milk blockedstrips were cut perpendicularly to the sensitized lines of peptides, to expose all the peptides simultaneously to immune mice plasmas (1:100), anti-mouse IgG-horseradish peroxidase (Sigma) (1:2000) and developed with Super Signal (Pierce) in HyperWlm (Amersham). Positive reactions are visually seen as black squares and only moderate and strong reactions were taken into account.
The recognition patterns by WB of S. mansoni adult worm molecules by plasmas from animals infected with three S. mansoni strains, S. rodhaini, S. guineensis, and S. bovis are shown in Figs. 1A–C, and those from gerbils infected with S. haematobium are shown in Fig. 1C. The frequencies of reactivity to each molecule are summarized in Table 3. The common antigens recognized by all infected animals corresponded to the 56, 74, and 84 kDa molecules of SAWA-JL. Within the Mansoni group, the human and murine strains from Guadeloupe in mice shared 10/12 molecules with the Venezuelan strain, while the Brazilian strain recognized 9/12. The S. mansoni JL-infected mice and S. mansoni Guadeloupean-infected gerbils both recognized 6/12, while S. rodhaini recognized 7/12 (Table 3). Within the Haematobium group, S. guineensis, S. bovis, and S. haematobium showed a strong recognition to SAWA-JL (8/12, 10/12, and 6/12, respectively). S. haematobiuminfected gerbils showed a distinct pattern of bands when exposed to S. mansoni SAWA-JL (Fig. 1C). There is a unique band of 28 kDa identiWed only by mice and gerbils infected with S. mansoni, but not by S. haematobium infected gerbils (Fig. 1C). It was noticed that not all molecules were consistently recognized by all infected animals from each group, but the strongest reactivities in terms of intensity were observed in the case of mice infected with both S. mansoni Guadeloupean strains and S. rodhaini (Figs. 1A and B). 3.2. Recognition of synthetic peptides by MABA MABA was performed to evaluate cross-reactivity among these species using polymerizable synthetic peptides derived from a group of selected proteins. An example is shown in Fig. 2. Table 4 summarizes the
Table 2 Synthetic peptides derived from relevant antigenic molecules of Schistosoma mansoni used for exploring antigenic community among schistosome species Molecules
Peptides synthesized (IMT-)a
Sequence (aa)
References
Sm 31
156, 158, 160, 162, 164, 166, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 2, 4, 6, 8, 10, 12, 14, 16, 18, 70, 72, 89, 22, 64, 24, 26, 28, 66, 30, 32, 34, 36
1–340 aa (in polymeric peptides of 20 aa each) 1–429 aa (in polymeric peptides of 20 aa each)
Klinkert et al. (1989)
232, 234 54, 215 645–648 649–652 627–629 630, 631
115–131, 190–211 194–203, 142–154 9–28, 27–46, 101–121,186–205 19–44, 89–112, 137–165, 195–227 165–181, 88–94, 106–127 203–216, 572–582
Sm 32 Sm28GST Sm28TPI Cathepsin L Sm (adult worm) Cathepsin L Sm (cercariae) Elastase Sm Sm97 (Paramyosin)
IMT, Instituto de Medicina Tropical; aa, aminoacids. a Sequential number assigned during peptide synthesis.
CaVrey et al. (2000) and Klinkert et al. (1987) Balluol et al. (1987) Harn et al. (1992) Smith et al. (1994) Michel et al. (1995) Newport et al. (1988) Lanar et al. (1986)
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Fig. 1. (A) Western blot of Schistosoma mansoni-adult worm antigen (SAWA) exposed to plasmas from mice infected with diVerent S. mansoni strains. (B) Western blot of JL Schistosoma mansoni-soluble adult worm antigen (SAWA) exposed to plasmas from mice infected with diVerent species of Schistosoma. (C) Western blot with JL Schistosoma mansoni-adult worm antigen (SAWA) exposed to plasmas from gerbils infected with S. mansoni, gerbils infected with S. haematobium and mice infected with Venezuelan JL S. mansoni strain.
reacting peptides from three (Sm32, Sm31, and Sm28GST) out of the eight molecules evaluated in this study (Table 2). Weak reactivities were not considered. There was a noteworthy absence of reactivity against peptides from Sm28 TPI, cathepsin L (adult and cercariae), elastase, and paramyosin, which were selected on the basis of antigenicity by predictive algorithms. All of the S. mansoni infected animals (mice and gerbils) had a similar pattern of recognition with the excep-
tion of the Sm32 derived peptides, in which there was great variability. The Brazilian strain only reacted with IMT-26 and -64, the Guadeloupean murine strain with IMT-14 and -26, and the Guadeloupean human strain infected-mice plasmas reacted with all the peptides except the IMT-24. Gerbils infected with S. mansoni Guadeloupean human strain did not recognize any Sm32 peptide but did so with Sm31 IMT-180. The S. mansoni strain from Brazil had in common with both
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Table 3 Frequency of recognition of Schistosoma mansoni (JL strain) SAWA molecules by plasmas from diVerent Schistosoma species or strains by Western blot kDa
Freq Mansoni group
125 110 100 84 74 64 56 42 32 28 22 19
Haematobium group
S. mansoni Brazil (mice)
S. mansoni Guadeloupe (hum) (mice)
S. mansoni Guadeloupe (mur) (mice)
S. mansoni JL (mice)
S. mansoni Guadeloupe (hum) (gerbils)
S. rhodaini (mice)
S. guineensis (mice)
S. bovis (mice)
S. haematobium (gerbils)
2/8 0/8 0/8 6/8 6/8 3/8 3/8 6/8 7/8 6/8 2/8 0/8
4/4 0/4 0/4 3/4 4/4 4/4 4/4 3/4 4/4 2/4 1/4 2/4
8/10 0/10 0/10 7/10 8/10 6/10 6/10 10/10 9/10 2/10 2/10 2/10
5/10 0/10 0/10 7/10 7/10 0/10 1/10 0/10 1/10 9/10 0/10 0/10
0/7 0/7 0/7 2/7 0/7 7/7 3/7 0/7 0/7 6/7 2/7 6/7
13/13 0/13 0/13 9/13 11/13 10/13 11/13 0/13 11/13 13/13 0/13 0/13
7/9 0/9 0/9 9/9 9/9 9/9 6/9 0/9 7/9 6/9 0/9 3/9
5/9 4/9 4/9 4/9 5/9 0/9 1/9 4/9 4/9 1/9 0/9 5/9
0/11 0/11 0/11 3/11 3/11 8/11 5/11 0/11 0/11 0/11 5/11 9/11
kDa, molecular weight in kilodaltons; hum, from human; mur, from mice; Freq, frequency of recognition (ratio of the number of animals that recognized each antigen/the total number of animals evaluated). IMT 2 4 6 8 10 12 14 16 18 70 72 89 22 64 24 26 28 66 30 32 34 36
Negative controls
S. guineensis -infected mouse plasmas
S. rhodaini -infected mouse plasmas
Human Guadeloupean S. mansoni infected-mouse plasmas
Murid Guadeloupean S. mansoni infected-mouse plasmas
Fig. 2. MABA showing recognition of synthetic peptides derived from Sm32 by plasmas of mice infected with Schistosoma guineensis, S. rodhaini, and two strains of Guadelupean S. mansoni.
S. mansoni strains from Guadeloupe (in mice) high reactivity to the Sm31 peptides IMT-176, 178, and 180, and to the peptide IMT-232 from Sm28GST. S. mansoni Venezuelan JL strain recognized all the three Sm31 peptides and weakly recognized Sm32 peptides. S. rodhainiinfected mice showed recognition of peptides from the two molecules, Sm32 (IMT-14, 22, 26, and 64) and Sm 31 (IMT-176 and 180). Peptide IMT-178 could be used as speciWc marker of S. mansoni infections in mice, while IMT-180 was the only peptide speciWcally recognized by the entire Mansoni group in spite of a weak reactivity observed in only one gerbil infected with S. haematobium. From the Haematobium group, only S. guineensis infected mice recognized S. mansoni synthetic peptides from Sm32 (IMT-14, 24, and 26) and from Sm-31 (IMT176). Plasmas of the hosts infected with S. bovis and
S. haematobium were non-reactive with all peptides assayed with the exception of one gerbil that recognized peptide IMT-180. Fig. 3 integrates the results obtained by WB with the SAWA-JL from S. mansoni and by MABA with synthetic peptides.
4. Discussion The sharing of molecules able to elicit immune responses between parasites and their vectors and among diVerent species of parasites from various genera, families, or phyla, is known as antigenic community, which is responsible for antigenic cross-reactivity. The advantage of antigenic community is that drugs or vaccines can be simultaneously eVective against diVerent
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Table 4 Ratios of the reactive infected mice plasmas by MABA vs the total number of animals evaluated in each case (IMT, Instituto de Medicina Tropical) Schistosoma Mansoni group mansoni Peptides derived Mice/S. Mice/ proteins from S. mansoni mansoni S.mansoni proteins IMT Brazil Guadeloupe mur
Haematobium group Mice/ S.mansoni Guadeloupe hum
Mice/ S.mansoni Venezuelan hum JL
Gerbils/ Mice/ Mice/ Mice/ Gerbils/ S.mansoni S.rhodaini S.guineensis S.bovis S.haematobium Guadeloupe hum
Sm32
14 22 24 26 28 64
— — — 6/8 — 4/8
6/10 — — 7/10 — —
2/4 1/4 — 4/4 3/4 3/4
2/5 — — 2/5 — 1/5
— — — — — —
13/13 9/13 — 13/13 — 6/13
12/12 — 6/12 12/12 — —
— — — — — —
— — — — — —
Sm31
176 178 180
6/8 6/8 6/8
6/10 7/10 7/10
2/4 4/4 3/4
3/5 4/5 3/5
— — 5/7
12/12 — 6/12
12/12 — —
— — —
— 1/11
232
7/7
8/8
4/4
2/5
—
—
—
—
—
Sm28-GST
Bold: Strong signal; non-bold: moderate signal; ( ) Recognizes only S. mansoni species; ( ) Recognizes only Mansoni group; (—) no recognition.
Fig. 3. Molecules and peptides recognized by plasmas of animals infected with diVerent species of Schistosoma. (IMT: Instituto de Medicina Tropical).
species or genera of parasites. However, the non-speciWc reactions are a disadvantage for immunodiagnostic tests (Alarcón de Noya and Colmenares, 2001). Previous studies (Murare et al., 1992) have shown a high degree of serological cross-reactivity between sera from mice infected with diVerent schistosome species and egg homogenates by ELISA. In the same work, complete immunological cross-reactivity of geographically distinct S. mansoni isolates (Puerto Rico, Brazil,
Egypt, and Kenya) was also observed. Agnew et al. (1989) found high cross-reactivity between S. bovis, S. haematobium, and S. mansoni antibodies and antigens by immunoprecipitation and a greater degree of homology between S. haematobium and S. bovis egg antigens was demonstrated by ELISA. They resemble each other more closely than either resembles S. mansoni. Cesari et al. (1998) found that the alkaline phosphatase from S. mansoni adults may be considered a species-speciWc
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antigen that can be used for immunodiagnosis of S. mansoni infection when sera from individuals infected with S. haematobium, S. guineensis, and S. japonicum were assayed. It was demonstrated by WB that the patterns of recognition of antigens from S. mansoni SAWA-JL by plasmas from animals infected with other Schistosoma species (S. rodhaini, S. guineensis, and S. bovis) were similar, except for S. haematobium, which showed a clearly diVerent pattern, since it did not recognize the 28, 32, 42, 100, 110, and 125 kDa molecules. The design of the experiment does not allow the identiWcation of the molecules recognized by the diVerent groups of animals. However, based on the pattern, the molecular weight and the correspondence with the recognition of the respective synthetic peptides, we suggest that the broad antigen of 28 kDa may correspond to the 31–32 kDa doublet described by Klinkert et al. (1987, 1989) and to the 36 kDa antigen described by Noya et al. (1995). Interestingly, there is correspondence between the WB and the results obtained with synthetic peptides by MABA. Animals infected with S. haematobium did not recognize this doublet and/or any of the peptides derived from these two molecules, with the exception of 1 out of 11 gerbils, which recognized peptide IMT-180, in contrast to the rest of the species studied. Molecules of 100 and 110 kDa were only recognized by sera of mice infected with S. bovis and could be considered speciWc markers of infection with this specie; they might be of potential value for diagnosis of this veterinary infection. The fact that S. haematobium was maintained in gerbils could be responsible for the diVerences observed. Furthermore, in the case of gerbils infected with S. mansoni, the pattern of recognition to S. mansoni antigens by WB was not similar to mice, which is coherent with the results obtained with the peptides by MABA. These Wndings suggest that the diVerences observed with S. haematobium were not only due to the parasite antigenic composition but also to the host species. Nevertheless, the fact that the 28 kDa molecule was recognized by mice and gerbils infected with S. mansoni and not by S. haematobium allows the diVerentiation of the two species. Other factors that might inXuence these results are: parasite load, initial inoculums, time of infection, and mice strains. All these elements should be considered when analyzing the variability between the diVerent groups of infected animals. The reactivity of plasmas from S. guineensis- and S. bovis-infected mice by WB against SAWA-JL appeared antigenically more similar to the species of the Mansoni group than to S. haematobium. Within the Mansoni species, we expected a stronger reactivity, but it was particularly weak. It is possible that, since some of these molecules are excretory–secretory products, in the case of homologous parasite strains the antibodies might be bound with a higher aYnity as circulating immune
complexes (Riggione et al., 1996). In contrast, the reactivity obtained with S. rodhaini was stronger than that observed with the S. mansoni strains and this remains to be explained. It is noteworthy that the S. mansoni-JL infected mice were exposed to fewer cercariae and had only 8 weeks post-infection (Table 1) compared to the Guadeloupean strains and S. rodhaini infected mice. Due to the severity of the infection with the JL strain, it is not possible to maintain mice for longer. Based on the crossreactivity observed with the WB, it is evident how diYcult is to discriminate between the diVerent species in terms of diagnosis. Therefore, the speciWcity detected by these methods does not appear to be suYcient to distinguish between S. mansoni strains from diVerent regions. The analysis with synthetic peptides by MABA was aimed at to discriminating at an epitopic level among the diVerent species. The lack of reactivity against peptides from other proteins (Sm28TPI, Cathepsin L, elastase, and paramyosin) could be due to weak antigenicity and it is possible that we were not able to imitate the original conformation of these epitopes or they might be discontinuous epitopes, which was not explored in this study. There is a high community between certain speciWc peptides within the S. mansoni strains, such as IMT-178 and 180 from Sm31 and IMT-232 from GST28. Moreover, peptides IMT-178 and 232 could be used to detect Mansoni species. It is also relevant that the peptide IMT-232 is the one proposed by Wolowczuk et al. (1991), who used it in a MAP-8 construction as a vaccine candidate. Even though it is well known that there is a very high homology between the sequence of the IMT232 peptides from S. mansoni, S. bovis, and S. haematobium, the plasmas from S. haematobium and S. bovisinfected animals failed to recognize IMT-232 or any of the other S. mansoni peptides, suggesting no cross-reactions between these distant species, at least at the level of the linear structure of this protein. The three Sm31 peptides (IMT-176, 178, and 180) were strongly recognized by the Mansoni group. These results are in accordance with previous studies that have demonstrated the diagnostic value of these three peptides in humans (Noya et al., 2001). Only peptide IMT176 showed cross-reactivity with mice infected with S. guineensis. The possibility of designing a multi species schistosome vaccine will depend on the identiWcation of common and conserved sequences of antigens shared between the species evaluated in this study. Previous results obtained by our group and others have shown the protective value of GST-28 (Xu et al., 1993) and Sm32 (Chlichlia et al., 2002; Wasilewski et al., 1996) molecules. The results obtained in this study would predict that peptides from these two molecules would only protect against Mansoni group infections. Based on egg morphology, snail host speciWcity and other life-history characteristics, African schistosomes
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are traditionally divided into the S. mansoni group and the S. haematobium group (Fig. 3) (Rollinson et al., 1997). Recent molecular phylogenetic studies of the Schistosomatidae (Lockyer et al., 2003; Morgan et al., 2003a,b), have detailed the interrelationships among Schistosoma species and corroborated that S. mansoni and S. rodhaini cluster together, as do the species of the S. haematobium group. The diVerential cross-reactivity and antigenic community obtained with the techniques used in this study clearly discriminated the S. mansoni/S. rodhaini group from the S. haematobium/S. bovis group. Furthermore, there is Weld evidence of the production of hybrids between the S. mansoni and S. rodhaini groups, demonstrating the close phylogenetic origin of these two species (Morgan et al., 2003a,b). However, the fact that the reactivity of S. guineensis appeared antigenically more similar to the species of the S. mansoni group than to the S. haematobium/S. bovis group was unexpected and remains to be explored. S. guineensis, previously considered as the Lower Guinea strain of S. intercalatum, has been recently recognized and described as a distinct species from S. intercalatum (Zaire strain) (Pagès et al., 2003). Phylogenic analysis of three mitochondrial genes demonstrated that this new species is closer to S. bovis and S. curassoni (Kane et al., 2003). The fact that S. haematobium needs to be maintained in gerbils and not in mice is additional biological evidence of the relative phylogenic distance from the other species, conWrming the validity of our results obtained with synthetic peptides. In general, we conclude that peptides can more clearly reveal the progressive distance between the diVerent schistosome species; it is probable that Wner deWnition is reached at the epitopic level, reinforcing their role as tools for taxonomic and evolutionary studies. Therefore, it is important to select other synthetic peptides that might increase the discriminatory capacity between the diVerent species of schistosomes. Since by using WB we were not able to discriminate the diVerent schistosome groups and species, it may be necessary to use more reWned techniques such as 2D electrophoresis to separate overlapping molecules by their isoelectric point (O’Farrell, 1975). We expect to continue the evaluation of antigen preparations from each specie and strain with plasma from S. haematobium, S. guineensis, S. rhodaini, and S. bovis-infected mice to corroborate the results obtained herein.
Acknowledgments We are very grateful to EcosNord-Fondo Nacional de Ciencia y Tecnología (FONACIT) Program for allowing the exchange between researchers, including traveling expenses for international meeting presentations. This work was partially supported by FONACIT Project No. S1-2000000564. We thank Anne Rognon for
189
her contribution to the maintenance of life cycles of the schistosome species.
References Alarcón de Noya, B., Colmenares, C., 2001. Comunidad Antigénica y Reactividad cruzada: su repercusión en el diagnóstico y tratamiento de enfermedades parasitarias. Especial referencia a Esquistosomiasis. Archivos Venezolanos de Farmacología y Terapéutica 20, 163–171. Agnew, A.M., Murare, H.M., Lucas, S.B., DoenhoV, M.J., 1989. Schistosoma bovis as an immunological analogue of S. haematobium. Parasite Immunology 11, 329–340. Aronstein, W.S., Dalton, J.P., Weiss, J.B., Strand, M., 1985. IdentiWcation and characterization of a major Schistosoma mansoni glycoprotein antigen cross-reactive with Fasciola hepatica. American Journal of Tropical Medicine and Hygiene 34, 879–888. Balluol, J.M., Sondermeyer, P., Dreyer, D., Capron, M., Grzych, J.M., Pierce, R.J., Carvallo, D., Lecocq, J.P., Capron, A., 1987. Molecular cloning of a protective antigen of schistosomes. Nature 326, 149– 153. Bahghat, M., Francklow, K., DoenhoV, M.J., Li, Y.L., Ramzy, R.M., Kirsten, C., Ruppel, A., 2001. Infection induces antibodies against the cercarial secretions, but not against the cercarial elastases of Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, and Trichobilharzia ocellata. Parasite Immunology 23, 557–565. CaVrey, C.R., Mathieu, M.A., GaVney, A.M., Salter, J.P., Sajid, M., Lucas, K.D., Franklin, C., Bogyo, M., McKerrow, J.H., 2000. IdentiWcation of a cDNA encoding and active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris. FEBS Letters 466, 244–248. Cesari, I.M., Ferrer, A., Kombila, M., Richard, E., Decam, C., Li-Shu, Q., Bout, D., Richard-Lenoble, D., 1998. SpeciWcity of the solid phase alkaline phosphatase immunocapture assay for the diagnosis of human Schistosoma mansoni infection. Transactions of the Royal Society of Tropical Medicine & Hygiene 92, 38–39. Correa-Oliveira, R., Dusse, L.M.S., Viana, I.R.C., Colley, D.G., Carvalho, O.S., Gazzinelli, G., 1988. Human antibody response against schistosomal antigens. Antibodies from patients with Ancylostoma, Ascaris lumbricoides, or Schistosoma mansoni infections react with schistosome antigens. American Journal of Tropical Medicine and Hygiene 38, 348–355. Chlichlia, K., Bahgat, M., Ruppel, A., Schirrmacher, V., 2002. DNA vaccination with asparaginyl endopeptidase (Sm32) from the parasite Schistosoma mansoni: anti-fecundity eVect induced in mice. Vaccine 20, 439–447. Dissous, C., Torpier, G., Duvaux-Miret, O., Capron, A., 1990. Structural homology from the human trematode Schistosoma mansoni and its intermediate host Biomphalaria glabrata. Molecular and Biochemical Parasitology 43, 245–256. Duvall, R.H., Dewitt, W.B., 1967. An improved perfusion technique for recovering adult schistosomes from laboratory animals. American Journal of Tropical Medicine and Hygiene 16, 483–486. Guide for the Care and Use of Laboratory Animals. Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council, National Academy Press, Washington, DC, 1996 (www.nap.edu/openbook/0309053773/html). Harn, D.A., Gu, W., Oligino, L.D., Mitsuyama, M., Gebremichael, A., Richter, D., 1992. A protective monoclonal antibody speciWcally recognizes and alters the catalytic activity of schistosome triosephosphate isomerase. Journal of Immunology 148, 562–567. Hoops, T.P., Woods, K.R., 1981. Prediction of protein antigenic determinants from aminoacid sequences. Proceedings of the Natural Academy of Science, USA 78, 3824–3828.
190
S. Losada et al. / Experimental Parasitology 111 (2005) 182–190
Houghten, R.A., DeGraw, S.T., Bray, M.K., HoVmann, S.R., Frizzel, N.D., 1986. Simultaneous multiple peptide synthesis: the rapid preparation of large number of discrete peptides for biological, inmunological and methodological studies. BioTechniques 4, 522–528. Kane, R.A., Southgate, V.R., Rollinson, D., Littlewood, D.T., Lockyer, A.E., Pagès, J.R., Tchuem Tchuente, L.A., Jourdane, J., 2003. A phylogeny based on three mitochondrial genes supports the division of Schistosoma guineensis into two separate species. Parasitology 127, 131–137. Klinkert, M.Q., Ruppel, A., Beck, E., 1987. Cloning of diagnostic 31/32 kilodalton antigens of Schistosoma mansoni. Molecular and Biochemical Parasitology 25, 247–255. Klinkert, M.Q., Felleisen, R., Link, G., Ruppel, A., Beck, E., 1989. Primary structures of Sm31/32 diagnostic proteins of Schistosoma mansoni and their identiWcation as proteases. Molecular and Biochemical Parasitology 33, 113–122. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lanar, D.E., Pearce, E.J., James, S.L., Sher, A., 1986. IdentiWcation of paramyosin as schistosome antigen recognized by intradermally vaccinated mice. Science 234, 593–596. Lockyer, A.E., Olson, P.D., Ostergaard, P., Rollinson, D., Johnston, D.A., Attwood, S.W., Southgate, V.R., Horak, P., Snyder, S.D., Le, T.H., Agatsuma, T., McManus, D.P., Carmichael, A.C., Naem, S., Littlewood, D.T., 2003. The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland, 1858. Parasitology 126, 203–224. MerriWeld, R.B., 1963. Solid-phase peptide syntesis. The syntesis of a tetra peptide. Journal of American Chemistry Society 85, 2149– 2455. Michel, A., Ghoneim, H., Resto, M., Klinkert, M.Q., Kunz, W., 1995. Sequence, characterization and localization of a cysteine proteinase cathepsin L in Schistosoma mansoni. Molecular and Biochemical Parasitology 73, 7–18. Morgan, J.A.T., DeJong, R.J., Kazibwe, F., Mkoji, G.M., Loker, E.S., 2003a. A newly-identiWed lineage of Schistosoma. International Journal for Parasitology 33, 977–985. Morgan, J.A.T., DeJong, R.J., Lwambo, N.J., Mungai, B.N., Mkoji, G.M., Loker, E.S., 2003b. First report of a natural hybrid between Schistosoma mansoni and S. rodaini. The Journal of Parasitology 89, 416–418. Murare, H.M., Dunne, D.W., Bain, J., DoenhoV, M.J., 1992. Schistosoma mansoni: control of hepatotoxicity and egg excretion by immune serum in infected immunosuppressed mice is schistosome species-speciWc, but not S. mansoni strain-speciWc. Experimental Parasitology 75, 329–339. Newport, G.R., McKerrow, J.H., Hedstrom, R., Petitt, M., McGarrigle, L., Barr, P.J., Agabian, N., 1988. Cloning of the proteinase that facilitates infection by schistosome parasites. Journal of Biological Chemistry 263, 13179–13184. Noya, O., Fermín, Z., Alarcón de Noya, B., Losada, S., Colmenares, C., Hermoso, T., 1995. Humoral immune responses of children with chronic schistosomiasis. Isotype recognition of adult worm antigens. Parasite Immunology 17, 319–328. Noya, O., Alarcón de Noya, B., 1998. The multiple antigen blot assay (MABA): a simple immunoenzymatic technique for simultaneous screening of multiple antigens. Immunology Letters 63, 53–56. Noya, O., Alarcón de Noya, B., Ballén, D., Bermúdez, H., Bout, D., Hoebeke, J., 2001. Immunogenicity of synthetic peptides from the
Sm31 antigen (cathepsin B) of the Schistosoma mansoni adult worms. Parasite Immunology 23, 567–573. Noya, O., Alarcón de Noya, B., Guzmán, F., Bermúdez, H., 2003. Immunogenicity of Sm32 synthetic peptides derived from the Schistosoma mansoni adult worm. Immunology Letters 88, 1–9. Noya, O., Patarroyo, M.E., Guzmán, F., Alarcón de Noya, B., 2003a. Immunodiagnosis of parasitic diseases with synthetic peptides. Current Protein and Peptide Science 4, 299–308. O’Farrell, P.H., 1975. High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250, 4007–4021. Pagès, J.R., Jourdane, J., Southgate, V.R., Tchuem Tchuente, L.A., 2003. Reconnaissance de deux espèces jumelles au sein du taxon Schistosoma intercalatum Fisher, 1934, agent de la schistosomose humaine rectale en Afrique. Description de Schistosoma guineensis n. sp. In Taxonomie, Ecologie et Evolution des Métazoaires Parasites. Tome II. Eds. Combes C. & Jourdane J. Universitaires de Perpignan. pp. 139–146, 380 pp. Rege, A.A., Wang, W., Dresden, M.H., 1992. Cystein proteinases from Schistosoma haematobium adult worms. The Journal of Parasitology 78, 16–23. Riggione, F., Pulido, M., Noya, O., 1996. Plasmodium falciparum: surface modiWcations of infected erythrocytes from clinical isolates. Parasitological Research 82, 490–496. Rollinson, D., Kaukas, A., Johnston, D.A., Simpson, A.J.G., Tanaka, M., 1997. Some molecular insights into schistosome evolution. International Journal for Parasitology 27, 11–28. Salter, J.P., Choe, Y., Albrecht, H., Franklin, C., Lim, K.C., Craik, C.S., McKerrow, J.H., 2002. Cercarial elastase is encoded by a functionally conserved gene family across multiple species of schistosomes. Journal of Biological Chemistry 277, 24618–24624. Smith, M.A., Clegg, J., Webbe, G., 1976. Cross-immunity to Schistosoma mansoni and S. haematobium in the hamster. Parasitology 73, 53–64. Smith, A.M., Dalton, J.P., Clough, K.A., Kilbane, C.L., Harrop, S.A., Hole, N., Brindley, P.J., 1994. Adult Schistosoma mansoni express cathepsin L proteinase activity. Molecular and Biochemical Parasitology 67, 11–19. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the Natural Academy of Science, USA 76, 4350–4354. Valdivieso, E., Bermúdez, H., Hoebeke, J., Noya, O., Cesari, I.M., 2003. Immunological similarity between Schistosoma and bovine cathepsin D. Immunology Letters 89, 81–88. Wasilewski, M., Lim, K., Phillips, J., McKerrow, J., 1996. Cysteine protease inhibitors block schistosome hemoglobin degradation in vitro and decrease worm burdens and egg production in vivo. Molecular and Biochemical Parasitology 81, 179–189. Wolowczuk, I., Auriault, C., Bossus, M., Boulanger, D., Gras-Masse, H., Mazingue, C., Pierce, R., Grezel, D., Ried, G., Tartar, A., Capron, A., 1991. Antigenicity and immunogenicity of a multiple peptidic construction of the Schistosoma mansoni Sm28-GST antigen in rat, mouse and monkey. Journal of Immunology 146, 1987– 1995. Xu, C., Verwaerde, C., Gras-Masse, H., Fontaine, J., Bossus, M., Trottein, F., Wolowczuk, I., Tartar, A., Capron, A., 1993. Schistosoma mansoni 28 kDa Glutation S-transferase and immunity against parasite fecundity and eggs viability. Journal of Immunology 50, 940– 949.
Schistosoma: Cross-reactivity and antigenic community among diVerent species S. Losada a, N. Chacón a, C. Colmenares a, H. Bermúdez a, A. Lorenzo a, J.P. Pointier b, A. Theron b, B. Alarcón de Noya a, O. Noya a,¤ a
Sección de Biohelmintiasis, Instituto de Medicina Tropical, Escuela de Medicina “Luis Razetti,” Facultad de Medicina, Universidad Central de Venezuela, Apartado 47623, Los Chaguaramos 1041-A, Caracas, Venezuela b UMR 5555 CNRS-UP, Parasitologie fonctionnelle et évolutive, CBETM, Université Via Domitia, 52 Av. Paul Alduy, 66860 Perpignan, France Received 25 February 2005; received in revised form 22 July 2005; accepted 26 July 2005 Available online 12 September 2005
Abstract It is not unusual to Wnd common molecules among diVerent species of the genus Schistosoma. When those molecules are antigenic, they may be used in immunodiagnosis and vaccines, but they could also be applied to taxonomic and evolutionary studies. To study cross-reactivity and antigenic community among diVerent species of schistosomes, plasmas from laboratory animals infected with Schistosoma bovis, S. guineensis, S. rodhaini, S. haematobium, and four strains of S. mansoni were evaluated with a crude extract of adult worms of S. mansoni by Western blot. Using the multiple antigen blot assay, plasmas from these infected animals were exposed to a selected group of synthetic peptides from Sm28GST, Sm28TPI, Sm elastase, Sm97, Sm32, Sm31, and Sm Cathepsin L. The results presented herein demonstrate diVerential cross-reactivity and antigenic community among the Mansoni and Haematobium groups of schistosomes, which is of relevance as an additional new tool for phylogenetic studies of schistosomes as well as for diagnosis and vaccine purposes. 2005 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Schistosomes; Cross-reactivity; Antigens; Peptides; Taxonomy; WB, Western blot; MABA, multiple antigen blot assay; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Sm28GST, Sm glutathione S-transferase; Sm28TPI, Sm28 triose phosphate isomerase; Sm97, Sm paramyosin; Sm32, Sm asparaginyl endopeptidase; Sm31, Sm Cathepsin B; SAWA, soluble adult worm antigen; PBS, phosphate buVer saline; PMSF, phenylmethylsulphonylXuoride; EDTA, ethyl-diamine-tetra-acetic acid; IMT, Instituto de Medicina Tropical
1. Introduction The sharing of molecules among organisms is an expected Wnding since there are several functional molecules, such as enzymes, hormones, receptors, etc., that are conserved during the extremely long process of evolution. However, this has special relevance for the identiWcation of molecules with potential for diagnosis or vaccine studies. In 1976, Smith et al. demonstrated crossimmunity to Schistosoma mansoni and S. haematobium *
Corresponding author. Fax: +58212 6930454. E-mail addresses: [email protected] (S. Losada), noyaoo@ yahoo.com (O. Noya). 0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.07.007
in hamsters, suggesting common antigens on the surface of young schistosomula in both species. Bahghat et al. (2001) found that immunoglobulin G against cercarial secretions cross-reacted among S. mansoni, S. haematobium, and S. japonicum. Similarly, Rege et al. (1992) demonstrated the cross-reactivity of cystein proteinases from S. mansoni and S. haematobium. Other enzymes, like cathepsin D, show immunological similarity not only among S. mansoni and S. japonicum but also with bovine cathepsin D (Valdivieso et al., 2003). Molecular biology techniques have allowed the application of antigenic community in systematics and phylogeny, so it is conceivable that together with the utilization of isoenzymes as phenotypic markers, certain molecules or
S. Losada et al. / Experimental Parasitology 111 (2005) 182–190
fragments that behave as antigens could be used for that purpose, as is the case of cercarial elastase, which is encoded by a functionally conserved gene family across diVerent species of schistosomes (Salter et al., 2002). Antigenic community is also present between parasites and their hosts. That is the case of Schistosoma and Biomphalaria, where several molecules such as tropomyosin have been characterized (Dissous et al., 1990). Cross-reactivity in serologic tests between S. mansoni and other parasites such as Fasciola hepatica is well known (Aronstein et al., 1985). Similarly, infected patients with hookworms and Ascaris lumbricoides developed cross-reactive antibodies against S. mansoni, which are able to kill schistosomula in the presence of complement (Correa-Oliveira et al., 1988). The demonstration of the antigenic community and cross-reactivity could eventually be used for diagnosis, the development of vaccines, and the study of systematics or phylogeny. The aim of this study was to investigate this antigenic community amongst diVerent schistosome species, through the recognition of an adult worm crude extract of Schistosoma mansoni by plasma of rodents infected with S. bovis, S. guineensis, S. rodhaini, S. haematobium, and several strains of S. mansoni by Western blot (WB). We also studied the recognition of peptides derived from proteins from S. mansoni, including Sm28GST, Sm28TPI, Sm31, Sm32, Sm elastase, Sm97, and Sm cathepsin L, by those plasmas from infected animals using the multiple antigen blot assay (MABA) (Noya and Alarcón de Noya, 1998). These proteins were selected since they have been shown to be relevant for either diagnosis or for vaccines against schistosomiasis (Noya et al., 2001; Noya et al., 2003; Valdivieso et al., 2003).
2. Materials and methods 2.1. Animals Outbred white mice (SWISS OF1) and gerbils (Meriones umguiculatus) were infected with diVerent species of Schistosoma (Table 1). Infection was achieved by indi-
183
vidual immersion of mice/gerbils in plastic jars containing 50 ml of water (paddling method) containing cercariae of each specie of parasite. Plasmas were obtained from intracardiac puncture with heparinized syringes before hepatic perfusion (Duvall and Dewitt, 1967) under penthotal intraperitoneal injection. Sodium azide was added to plasma from these animals and afterwards they were stored at ¡70 °C. Non-infected mice and gerbils were also sacriWced and their plasmas used as controls. Animals were managed following international principles regarding the Guide for the Care and Use of Laboratory Animals (1996). 2.2. Preparation of the soluble adult worm antigen Adult worms from S. mansoni (JL Venezuelan strain, provided by Dr. I.M. Cesari from the Instituto Venezolano de Investigaciones CientíWcas)-infected mice were collected after hepatic perfusion and used to prepare SAWA-JL. BrieXy, worms were homogenized in PBS with protease inhibitors (1 mM PMSF and 1 mM EDTA) in ice bath and centrifuged at 12,000g for 2 h at 4 °C (Noya et al., 1995). Supernatant was used as soluble adult worm antigen (SAWA). 2.3. SDS–PAGE and Western blot The antigenic community between S. mansoni (JL Venezuelan strain) and the rest of the Schistosoma species or strains was studied by WB. A crude extract of the SAWA-JL was run by 10% SDS–PAGE mini gels and electro-transferred antigens onto nitrocellulose were exposed to diVerent plasmas as described by Laemmli (1970) and Towbin et al. (1979). Nitrocellulose strips transferred with SAWA were incubated with animal plasmas (1:100) and later with anti-mouse total IgG conjugated to horseradish peroxidase (Sigma) (1:2000). A chemiluminescent substrate of the enzyme was used (Super Signal, Pierce) and the luminescence was recorded in photographic Wlm (HyperWlm, Amersham) (Noya et al., 1995). The bands were analyzed with a MultiAnalyst 1.1 software (Bio-Rad, CA, USA).
Table 1 Animals infected with diVerent Schistosoma species Host
Number
Schistosoma species
Schistosoma strains
Infected/No. cercariae
Weeks of infection
Outbred mice Outbred mice Outbred mice Outbred mice Outbred mice Outbred mice Outbred mice Gerbils Gerbils
9 13 12 10 4 8 10 12 7
S. bovis S. rodhaini S. guineensis S. mansoni S. mansoni S. mansoni S. mansoni S. haematobium S. mansoni
— — — Murid from Guadeloupe Human from Guadeloupe Brazil Human JL from Venezuela — Human from Guadaloupe
400 230 140 170 170 170 100 450 200
12 13 13 15 15 16 8 20 9
SD, standard deviation.
Average number § SD of adult worms recovered 15.6 § 9.9 44.8 § 34.2 58.1 § 32.8 63.2 § 48.1 19 § 19.2 53.4 § 38.6 22.5 § 10.2 58.1 § 18.9 88.7 § 38.7
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2.4. Chemical peptide synthesis
3. Results
Non-overlapping peptides corresponding to the whole sequences of Sm31 (GI: 160950 NCBI) and Sm32 (GI: 729709 NCBI) and some regions of the Sm28GST (GI: 121700 NCBI), Sm28TPI (GI: 1351281 NCBI), Sm elastase (GI: 116116 NCBI), Sm97 (GI: 547978 NCBI), cecarial Sm cathepsin L (GI: 3023456 NCBI) and adult Sm cathepsin L (GI: 630486 NCBI) (Table 2) were selected based mainly on hydrophylicity, using the predictive algorithm described by Hoops and Woods (1981). Some of the sequences corresponded to regions surrounding the active site of the enzymes. Peptides were solid-phase synthesized as described by MerriWeld (1963) and modiWed by Houghten et al. (1986). All these proteins except one (Sm97, paramyosin) are enzymes; their aminoacid sequences were obtained from Swiss Protein Data Bank. Peptides were synthesized with cysteine–glycine and glycine–cysteine aminoacids in the amino and carboxy terminus, respectively, to obtain polymerizable peptides that are usually more reactive than monomeric peptides (Noya et al., 2003a). Quality control of each peptide was achieved by mass spectrometry and HPLC by molecular exclusion. The resulting purity was at least 65%.
3.1. Recognition of antigenic molecules from SAWA-JL by Western blot
2.5. MABA This multiple dot blot technique was used to evaluate the immunogenicity of the peptides (Noya and Alarcón de Noya, 1998). Peptides were absorbed individually at 10 g/ml in 28 parallel rows onto a nitrocellulose membrane. Afterwards, 2 mm wide non-fat milk blockedstrips were cut perpendicularly to the sensitized lines of peptides, to expose all the peptides simultaneously to immune mice plasmas (1:100), anti-mouse IgG-horseradish peroxidase (Sigma) (1:2000) and developed with Super Signal (Pierce) in HyperWlm (Amersham). Positive reactions are visually seen as black squares and only moderate and strong reactions were taken into account.
The recognition patterns by WB of S. mansoni adult worm molecules by plasmas from animals infected with three S. mansoni strains, S. rodhaini, S. guineensis, and S. bovis are shown in Figs. 1A–C, and those from gerbils infected with S. haematobium are shown in Fig. 1C. The frequencies of reactivity to each molecule are summarized in Table 3. The common antigens recognized by all infected animals corresponded to the 56, 74, and 84 kDa molecules of SAWA-JL. Within the Mansoni group, the human and murine strains from Guadeloupe in mice shared 10/12 molecules with the Venezuelan strain, while the Brazilian strain recognized 9/12. The S. mansoni JL-infected mice and S. mansoni Guadeloupean-infected gerbils both recognized 6/12, while S. rodhaini recognized 7/12 (Table 3). Within the Haematobium group, S. guineensis, S. bovis, and S. haematobium showed a strong recognition to SAWA-JL (8/12, 10/12, and 6/12, respectively). S. haematobiuminfected gerbils showed a distinct pattern of bands when exposed to S. mansoni SAWA-JL (Fig. 1C). There is a unique band of 28 kDa identiWed only by mice and gerbils infected with S. mansoni, but not by S. haematobium infected gerbils (Fig. 1C). It was noticed that not all molecules were consistently recognized by all infected animals from each group, but the strongest reactivities in terms of intensity were observed in the case of mice infected with both S. mansoni Guadeloupean strains and S. rodhaini (Figs. 1A and B). 3.2. Recognition of synthetic peptides by MABA MABA was performed to evaluate cross-reactivity among these species using polymerizable synthetic peptides derived from a group of selected proteins. An example is shown in Fig. 2. Table 4 summarizes the
Table 2 Synthetic peptides derived from relevant antigenic molecules of Schistosoma mansoni used for exploring antigenic community among schistosome species Molecules
Peptides synthesized (IMT-)a
Sequence (aa)
References
Sm 31
156, 158, 160, 162, 164, 166, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 2, 4, 6, 8, 10, 12, 14, 16, 18, 70, 72, 89, 22, 64, 24, 26, 28, 66, 30, 32, 34, 36
1–340 aa (in polymeric peptides of 20 aa each) 1–429 aa (in polymeric peptides of 20 aa each)
Klinkert et al. (1989)
232, 234 54, 215 645–648 649–652 627–629 630, 631
115–131, 190–211 194–203, 142–154 9–28, 27–46, 101–121,186–205 19–44, 89–112, 137–165, 195–227 165–181, 88–94, 106–127 203–216, 572–582
Sm 32 Sm28GST Sm28TPI Cathepsin L Sm (adult worm) Cathepsin L Sm (cercariae) Elastase Sm Sm97 (Paramyosin)
IMT, Instituto de Medicina Tropical; aa, aminoacids. a Sequential number assigned during peptide synthesis.
CaVrey et al. (2000) and Klinkert et al. (1987) Balluol et al. (1987) Harn et al. (1992) Smith et al. (1994) Michel et al. (1995) Newport et al. (1988) Lanar et al. (1986)
S. Losada et al. / Experimental Parasitology 111 (2005) 182–190
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Fig. 1. (A) Western blot of Schistosoma mansoni-adult worm antigen (SAWA) exposed to plasmas from mice infected with diVerent S. mansoni strains. (B) Western blot of JL Schistosoma mansoni-soluble adult worm antigen (SAWA) exposed to plasmas from mice infected with diVerent species of Schistosoma. (C) Western blot with JL Schistosoma mansoni-adult worm antigen (SAWA) exposed to plasmas from gerbils infected with S. mansoni, gerbils infected with S. haematobium and mice infected with Venezuelan JL S. mansoni strain.
reacting peptides from three (Sm32, Sm31, and Sm28GST) out of the eight molecules evaluated in this study (Table 2). Weak reactivities were not considered. There was a noteworthy absence of reactivity against peptides from Sm28 TPI, cathepsin L (adult and cercariae), elastase, and paramyosin, which were selected on the basis of antigenicity by predictive algorithms. All of the S. mansoni infected animals (mice and gerbils) had a similar pattern of recognition with the excep-
tion of the Sm32 derived peptides, in which there was great variability. The Brazilian strain only reacted with IMT-26 and -64, the Guadeloupean murine strain with IMT-14 and -26, and the Guadeloupean human strain infected-mice plasmas reacted with all the peptides except the IMT-24. Gerbils infected with S. mansoni Guadeloupean human strain did not recognize any Sm32 peptide but did so with Sm31 IMT-180. The S. mansoni strain from Brazil had in common with both
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Table 3 Frequency of recognition of Schistosoma mansoni (JL strain) SAWA molecules by plasmas from diVerent Schistosoma species or strains by Western blot kDa
Freq Mansoni group
125 110 100 84 74 64 56 42 32 28 22 19
Haematobium group
S. mansoni Brazil (mice)
S. mansoni Guadeloupe (hum) (mice)
S. mansoni Guadeloupe (mur) (mice)
S. mansoni JL (mice)
S. mansoni Guadeloupe (hum) (gerbils)
S. rhodaini (mice)
S. guineensis (mice)
S. bovis (mice)
S. haematobium (gerbils)
2/8 0/8 0/8 6/8 6/8 3/8 3/8 6/8 7/8 6/8 2/8 0/8
4/4 0/4 0/4 3/4 4/4 4/4 4/4 3/4 4/4 2/4 1/4 2/4
8/10 0/10 0/10 7/10 8/10 6/10 6/10 10/10 9/10 2/10 2/10 2/10
5/10 0/10 0/10 7/10 7/10 0/10 1/10 0/10 1/10 9/10 0/10 0/10
0/7 0/7 0/7 2/7 0/7 7/7 3/7 0/7 0/7 6/7 2/7 6/7
13/13 0/13 0/13 9/13 11/13 10/13 11/13 0/13 11/13 13/13 0/13 0/13
7/9 0/9 0/9 9/9 9/9 9/9 6/9 0/9 7/9 6/9 0/9 3/9
5/9 4/9 4/9 4/9 5/9 0/9 1/9 4/9 4/9 1/9 0/9 5/9
0/11 0/11 0/11 3/11 3/11 8/11 5/11 0/11 0/11 0/11 5/11 9/11
kDa, molecular weight in kilodaltons; hum, from human; mur, from mice; Freq, frequency of recognition (ratio of the number of animals that recognized each antigen/the total number of animals evaluated). IMT 2 4 6 8 10 12 14 16 18 70 72 89 22 64 24 26 28 66 30 32 34 36
Negative controls
S. guineensis -infected mouse plasmas
S. rhodaini -infected mouse plasmas
Human Guadeloupean S. mansoni infected-mouse plasmas
Murid Guadeloupean S. mansoni infected-mouse plasmas
Fig. 2. MABA showing recognition of synthetic peptides derived from Sm32 by plasmas of mice infected with Schistosoma guineensis, S. rodhaini, and two strains of Guadelupean S. mansoni.
S. mansoni strains from Guadeloupe (in mice) high reactivity to the Sm31 peptides IMT-176, 178, and 180, and to the peptide IMT-232 from Sm28GST. S. mansoni Venezuelan JL strain recognized all the three Sm31 peptides and weakly recognized Sm32 peptides. S. rodhainiinfected mice showed recognition of peptides from the two molecules, Sm32 (IMT-14, 22, 26, and 64) and Sm 31 (IMT-176 and 180). Peptide IMT-178 could be used as speciWc marker of S. mansoni infections in mice, while IMT-180 was the only peptide speciWcally recognized by the entire Mansoni group in spite of a weak reactivity observed in only one gerbil infected with S. haematobium. From the Haematobium group, only S. guineensis infected mice recognized S. mansoni synthetic peptides from Sm32 (IMT-14, 24, and 26) and from Sm-31 (IMT176). Plasmas of the hosts infected with S. bovis and
S. haematobium were non-reactive with all peptides assayed with the exception of one gerbil that recognized peptide IMT-180. Fig. 3 integrates the results obtained by WB with the SAWA-JL from S. mansoni and by MABA with synthetic peptides.
4. Discussion The sharing of molecules able to elicit immune responses between parasites and their vectors and among diVerent species of parasites from various genera, families, or phyla, is known as antigenic community, which is responsible for antigenic cross-reactivity. The advantage of antigenic community is that drugs or vaccines can be simultaneously eVective against diVerent
S. Losada et al. / Experimental Parasitology 111 (2005) 182–190
187
Table 4 Ratios of the reactive infected mice plasmas by MABA vs the total number of animals evaluated in each case (IMT, Instituto de Medicina Tropical) Schistosoma Mansoni group mansoni Peptides derived Mice/S. Mice/ proteins from S. mansoni mansoni S.mansoni proteins IMT Brazil Guadeloupe mur
Haematobium group Mice/ S.mansoni Guadeloupe hum
Mice/ S.mansoni Venezuelan hum JL
Gerbils/ Mice/ Mice/ Mice/ Gerbils/ S.mansoni S.rhodaini S.guineensis S.bovis S.haematobium Guadeloupe hum
Sm32
14 22 24 26 28 64
— — — 6/8 — 4/8
6/10 — — 7/10 — —
2/4 1/4 — 4/4 3/4 3/4
2/5 — — 2/5 — 1/5
— — — — — —
13/13 9/13 — 13/13 — 6/13
12/12 — 6/12 12/12 — —
— — — — — —
— — — — — —
Sm31
176 178 180
6/8 6/8 6/8
6/10 7/10 7/10
2/4 4/4 3/4
3/5 4/5 3/5
— — 5/7
12/12 — 6/12
12/12 — —
— — —
— 1/11
232
7/7
8/8
4/4
2/5
—
—
—
—
—
Sm28-GST
Bold: Strong signal; non-bold: moderate signal; ( ) Recognizes only S. mansoni species; ( ) Recognizes only Mansoni group; (—) no recognition.
Fig. 3. Molecules and peptides recognized by plasmas of animals infected with diVerent species of Schistosoma. (IMT: Instituto de Medicina Tropical).
species or genera of parasites. However, the non-speciWc reactions are a disadvantage for immunodiagnostic tests (Alarcón de Noya and Colmenares, 2001). Previous studies (Murare et al., 1992) have shown a high degree of serological cross-reactivity between sera from mice infected with diVerent schistosome species and egg homogenates by ELISA. In the same work, complete immunological cross-reactivity of geographically distinct S. mansoni isolates (Puerto Rico, Brazil,
Egypt, and Kenya) was also observed. Agnew et al. (1989) found high cross-reactivity between S. bovis, S. haematobium, and S. mansoni antibodies and antigens by immunoprecipitation and a greater degree of homology between S. haematobium and S. bovis egg antigens was demonstrated by ELISA. They resemble each other more closely than either resembles S. mansoni. Cesari et al. (1998) found that the alkaline phosphatase from S. mansoni adults may be considered a species-speciWc
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S. Losada et al. / Experimental Parasitology 111 (2005) 182–190
antigen that can be used for immunodiagnosis of S. mansoni infection when sera from individuals infected with S. haematobium, S. guineensis, and S. japonicum were assayed. It was demonstrated by WB that the patterns of recognition of antigens from S. mansoni SAWA-JL by plasmas from animals infected with other Schistosoma species (S. rodhaini, S. guineensis, and S. bovis) were similar, except for S. haematobium, which showed a clearly diVerent pattern, since it did not recognize the 28, 32, 42, 100, 110, and 125 kDa molecules. The design of the experiment does not allow the identiWcation of the molecules recognized by the diVerent groups of animals. However, based on the pattern, the molecular weight and the correspondence with the recognition of the respective synthetic peptides, we suggest that the broad antigen of 28 kDa may correspond to the 31–32 kDa doublet described by Klinkert et al. (1987, 1989) and to the 36 kDa antigen described by Noya et al. (1995). Interestingly, there is correspondence between the WB and the results obtained with synthetic peptides by MABA. Animals infected with S. haematobium did not recognize this doublet and/or any of the peptides derived from these two molecules, with the exception of 1 out of 11 gerbils, which recognized peptide IMT-180, in contrast to the rest of the species studied. Molecules of 100 and 110 kDa were only recognized by sera of mice infected with S. bovis and could be considered speciWc markers of infection with this specie; they might be of potential value for diagnosis of this veterinary infection. The fact that S. haematobium was maintained in gerbils could be responsible for the diVerences observed. Furthermore, in the case of gerbils infected with S. mansoni, the pattern of recognition to S. mansoni antigens by WB was not similar to mice, which is coherent with the results obtained with the peptides by MABA. These Wndings suggest that the diVerences observed with S. haematobium were not only due to the parasite antigenic composition but also to the host species. Nevertheless, the fact that the 28 kDa molecule was recognized by mice and gerbils infected with S. mansoni and not by S. haematobium allows the diVerentiation of the two species. Other factors that might inXuence these results are: parasite load, initial inoculums, time of infection, and mice strains. All these elements should be considered when analyzing the variability between the diVerent groups of infected animals. The reactivity of plasmas from S. guineensis- and S. bovis-infected mice by WB against SAWA-JL appeared antigenically more similar to the species of the Mansoni group than to S. haematobium. Within the Mansoni species, we expected a stronger reactivity, but it was particularly weak. It is possible that, since some of these molecules are excretory–secretory products, in the case of homologous parasite strains the antibodies might be bound with a higher aYnity as circulating immune
complexes (Riggione et al., 1996). In contrast, the reactivity obtained with S. rodhaini was stronger than that observed with the S. mansoni strains and this remains to be explained. It is noteworthy that the S. mansoni-JL infected mice were exposed to fewer cercariae and had only 8 weeks post-infection (Table 1) compared to the Guadeloupean strains and S. rodhaini infected mice. Due to the severity of the infection with the JL strain, it is not possible to maintain mice for longer. Based on the crossreactivity observed with the WB, it is evident how diYcult is to discriminate between the diVerent species in terms of diagnosis. Therefore, the speciWcity detected by these methods does not appear to be suYcient to distinguish between S. mansoni strains from diVerent regions. The analysis with synthetic peptides by MABA was aimed at to discriminating at an epitopic level among the diVerent species. The lack of reactivity against peptides from other proteins (Sm28TPI, Cathepsin L, elastase, and paramyosin) could be due to weak antigenicity and it is possible that we were not able to imitate the original conformation of these epitopes or they might be discontinuous epitopes, which was not explored in this study. There is a high community between certain speciWc peptides within the S. mansoni strains, such as IMT-178 and 180 from Sm31 and IMT-232 from GST28. Moreover, peptides IMT-178 and 232 could be used to detect Mansoni species. It is also relevant that the peptide IMT-232 is the one proposed by Wolowczuk et al. (1991), who used it in a MAP-8 construction as a vaccine candidate. Even though it is well known that there is a very high homology between the sequence of the IMT232 peptides from S. mansoni, S. bovis, and S. haematobium, the plasmas from S. haematobium and S. bovisinfected animals failed to recognize IMT-232 or any of the other S. mansoni peptides, suggesting no cross-reactions between these distant species, at least at the level of the linear structure of this protein. The three Sm31 peptides (IMT-176, 178, and 180) were strongly recognized by the Mansoni group. These results are in accordance with previous studies that have demonstrated the diagnostic value of these three peptides in humans (Noya et al., 2001). Only peptide IMT176 showed cross-reactivity with mice infected with S. guineensis. The possibility of designing a multi species schistosome vaccine will depend on the identiWcation of common and conserved sequences of antigens shared between the species evaluated in this study. Previous results obtained by our group and others have shown the protective value of GST-28 (Xu et al., 1993) and Sm32 (Chlichlia et al., 2002; Wasilewski et al., 1996) molecules. The results obtained in this study would predict that peptides from these two molecules would only protect against Mansoni group infections. Based on egg morphology, snail host speciWcity and other life-history characteristics, African schistosomes
S. Losada et al. / Experimental Parasitology 111 (2005) 182–190
are traditionally divided into the S. mansoni group and the S. haematobium group (Fig. 3) (Rollinson et al., 1997). Recent molecular phylogenetic studies of the Schistosomatidae (Lockyer et al., 2003; Morgan et al., 2003a,b), have detailed the interrelationships among Schistosoma species and corroborated that S. mansoni and S. rodhaini cluster together, as do the species of the S. haematobium group. The diVerential cross-reactivity and antigenic community obtained with the techniques used in this study clearly discriminated the S. mansoni/S. rodhaini group from the S. haematobium/S. bovis group. Furthermore, there is Weld evidence of the production of hybrids between the S. mansoni and S. rodhaini groups, demonstrating the close phylogenetic origin of these two species (Morgan et al., 2003a,b). However, the fact that the reactivity of S. guineensis appeared antigenically more similar to the species of the S. mansoni group than to the S. haematobium/S. bovis group was unexpected and remains to be explored. S. guineensis, previously considered as the Lower Guinea strain of S. intercalatum, has been recently recognized and described as a distinct species from S. intercalatum (Zaire strain) (Pagès et al., 2003). Phylogenic analysis of three mitochondrial genes demonstrated that this new species is closer to S. bovis and S. curassoni (Kane et al., 2003). The fact that S. haematobium needs to be maintained in gerbils and not in mice is additional biological evidence of the relative phylogenic distance from the other species, conWrming the validity of our results obtained with synthetic peptides. In general, we conclude that peptides can more clearly reveal the progressive distance between the diVerent schistosome species; it is probable that Wner deWnition is reached at the epitopic level, reinforcing their role as tools for taxonomic and evolutionary studies. Therefore, it is important to select other synthetic peptides that might increase the discriminatory capacity between the diVerent species of schistosomes. Since by using WB we were not able to discriminate the diVerent schistosome groups and species, it may be necessary to use more reWned techniques such as 2D electrophoresis to separate overlapping molecules by their isoelectric point (O’Farrell, 1975). We expect to continue the evaluation of antigen preparations from each specie and strain with plasma from S. haematobium, S. guineensis, S. rhodaini, and S. bovis-infected mice to corroborate the results obtained herein.
Acknowledgments We are very grateful to EcosNord-Fondo Nacional de Ciencia y Tecnología (FONACIT) Program for allowing the exchange between researchers, including traveling expenses for international meeting presentations. This work was partially supported by FONACIT Project No. S1-2000000564. We thank Anne Rognon for
189
her contribution to the maintenance of life cycles of the schistosome species.
References Alarcón de Noya, B., Colmenares, C., 2001. Comunidad Antigénica y Reactividad cruzada: su repercusión en el diagnóstico y tratamiento de enfermedades parasitarias. Especial referencia a Esquistosomiasis. Archivos Venezolanos de Farmacología y Terapéutica 20, 163–171. Agnew, A.M., Murare, H.M., Lucas, S.B., DoenhoV, M.J., 1989. Schistosoma bovis as an immunological analogue of S. haematobium. Parasite Immunology 11, 329–340. Aronstein, W.S., Dalton, J.P., Weiss, J.B., Strand, M., 1985. IdentiWcation and characterization of a major Schistosoma mansoni glycoprotein antigen cross-reactive with Fasciola hepatica. American Journal of Tropical Medicine and Hygiene 34, 879–888. Balluol, J.M., Sondermeyer, P., Dreyer, D., Capron, M., Grzych, J.M., Pierce, R.J., Carvallo, D., Lecocq, J.P., Capron, A., 1987. Molecular cloning of a protective antigen of schistosomes. Nature 326, 149– 153. Bahghat, M., Francklow, K., DoenhoV, M.J., Li, Y.L., Ramzy, R.M., Kirsten, C., Ruppel, A., 2001. Infection induces antibodies against the cercarial secretions, but not against the cercarial elastases of Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, and Trichobilharzia ocellata. Parasite Immunology 23, 557–565. CaVrey, C.R., Mathieu, M.A., GaVney, A.M., Salter, J.P., Sajid, M., Lucas, K.D., Franklin, C., Bogyo, M., McKerrow, J.H., 2000. IdentiWcation of a cDNA encoding and active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris. FEBS Letters 466, 244–248. Cesari, I.M., Ferrer, A., Kombila, M., Richard, E., Decam, C., Li-Shu, Q., Bout, D., Richard-Lenoble, D., 1998. SpeciWcity of the solid phase alkaline phosphatase immunocapture assay for the diagnosis of human Schistosoma mansoni infection. Transactions of the Royal Society of Tropical Medicine & Hygiene 92, 38–39. Correa-Oliveira, R., Dusse, L.M.S., Viana, I.R.C., Colley, D.G., Carvalho, O.S., Gazzinelli, G., 1988. Human antibody response against schistosomal antigens. Antibodies from patients with Ancylostoma, Ascaris lumbricoides, or Schistosoma mansoni infections react with schistosome antigens. American Journal of Tropical Medicine and Hygiene 38, 348–355. Chlichlia, K., Bahgat, M., Ruppel, A., Schirrmacher, V., 2002. DNA vaccination with asparaginyl endopeptidase (Sm32) from the parasite Schistosoma mansoni: anti-fecundity eVect induced in mice. Vaccine 20, 439–447. Dissous, C., Torpier, G., Duvaux-Miret, O., Capron, A., 1990. Structural homology from the human trematode Schistosoma mansoni and its intermediate host Biomphalaria glabrata. Molecular and Biochemical Parasitology 43, 245–256. Duvall, R.H., Dewitt, W.B., 1967. An improved perfusion technique for recovering adult schistosomes from laboratory animals. American Journal of Tropical Medicine and Hygiene 16, 483–486. Guide for the Care and Use of Laboratory Animals. Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council, National Academy Press, Washington, DC, 1996 (www.nap.edu/openbook/0309053773/html). Harn, D.A., Gu, W., Oligino, L.D., Mitsuyama, M., Gebremichael, A., Richter, D., 1992. A protective monoclonal antibody speciWcally recognizes and alters the catalytic activity of schistosome triosephosphate isomerase. Journal of Immunology 148, 562–567. Hoops, T.P., Woods, K.R., 1981. Prediction of protein antigenic determinants from aminoacid sequences. Proceedings of the Natural Academy of Science, USA 78, 3824–3828.
190
S. Losada et al. / Experimental Parasitology 111 (2005) 182–190
Houghten, R.A., DeGraw, S.T., Bray, M.K., HoVmann, S.R., Frizzel, N.D., 1986. Simultaneous multiple peptide synthesis: the rapid preparation of large number of discrete peptides for biological, inmunological and methodological studies. BioTechniques 4, 522–528. Kane, R.A., Southgate, V.R., Rollinson, D., Littlewood, D.T., Lockyer, A.E., Pagès, J.R., Tchuem Tchuente, L.A., Jourdane, J., 2003. A phylogeny based on three mitochondrial genes supports the division of Schistosoma guineensis into two separate species. Parasitology 127, 131–137. Klinkert, M.Q., Ruppel, A., Beck, E., 1987. Cloning of diagnostic 31/32 kilodalton antigens of Schistosoma mansoni. Molecular and Biochemical Parasitology 25, 247–255. Klinkert, M.Q., Felleisen, R., Link, G., Ruppel, A., Beck, E., 1989. Primary structures of Sm31/32 diagnostic proteins of Schistosoma mansoni and their identiWcation as proteases. Molecular and Biochemical Parasitology 33, 113–122. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lanar, D.E., Pearce, E.J., James, S.L., Sher, A., 1986. IdentiWcation of paramyosin as schistosome antigen recognized by intradermally vaccinated mice. Science 234, 593–596. Lockyer, A.E., Olson, P.D., Ostergaard, P., Rollinson, D., Johnston, D.A., Attwood, S.W., Southgate, V.R., Horak, P., Snyder, S.D., Le, T.H., Agatsuma, T., McManus, D.P., Carmichael, A.C., Naem, S., Littlewood, D.T., 2003. The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland, 1858. Parasitology 126, 203–224. MerriWeld, R.B., 1963. Solid-phase peptide syntesis. The syntesis of a tetra peptide. Journal of American Chemistry Society 85, 2149– 2455. Michel, A., Ghoneim, H., Resto, M., Klinkert, M.Q., Kunz, W., 1995. Sequence, characterization and localization of a cysteine proteinase cathepsin L in Schistosoma mansoni. Molecular and Biochemical Parasitology 73, 7–18. Morgan, J.A.T., DeJong, R.J., Kazibwe, F., Mkoji, G.M., Loker, E.S., 2003a. A newly-identiWed lineage of Schistosoma. International Journal for Parasitology 33, 977–985. Morgan, J.A.T., DeJong, R.J., Lwambo, N.J., Mungai, B.N., Mkoji, G.M., Loker, E.S., 2003b. First report of a natural hybrid between Schistosoma mansoni and S. rodaini. The Journal of Parasitology 89, 416–418. Murare, H.M., Dunne, D.W., Bain, J., DoenhoV, M.J., 1992. Schistosoma mansoni: control of hepatotoxicity and egg excretion by immune serum in infected immunosuppressed mice is schistosome species-speciWc, but not S. mansoni strain-speciWc. Experimental Parasitology 75, 329–339. Newport, G.R., McKerrow, J.H., Hedstrom, R., Petitt, M., McGarrigle, L., Barr, P.J., Agabian, N., 1988. Cloning of the proteinase that facilitates infection by schistosome parasites. Journal of Biological Chemistry 263, 13179–13184. Noya, O., Fermín, Z., Alarcón de Noya, B., Losada, S., Colmenares, C., Hermoso, T., 1995. Humoral immune responses of children with chronic schistosomiasis. Isotype recognition of adult worm antigens. Parasite Immunology 17, 319–328. Noya, O., Alarcón de Noya, B., 1998. The multiple antigen blot assay (MABA): a simple immunoenzymatic technique for simultaneous screening of multiple antigens. Immunology Letters 63, 53–56. Noya, O., Alarcón de Noya, B., Ballén, D., Bermúdez, H., Bout, D., Hoebeke, J., 2001. Immunogenicity of synthetic peptides from the
Sm31 antigen (cathepsin B) of the Schistosoma mansoni adult worms. Parasite Immunology 23, 567–573. Noya, O., Alarcón de Noya, B., Guzmán, F., Bermúdez, H., 2003. Immunogenicity of Sm32 synthetic peptides derived from the Schistosoma mansoni adult worm. Immunology Letters 88, 1–9. Noya, O., Patarroyo, M.E., Guzmán, F., Alarcón de Noya, B., 2003a. Immunodiagnosis of parasitic diseases with synthetic peptides. Current Protein and Peptide Science 4, 299–308. O’Farrell, P.H., 1975. High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250, 4007–4021. Pagès, J.R., Jourdane, J., Southgate, V.R., Tchuem Tchuente, L.A., 2003. Reconnaissance de deux espèces jumelles au sein du taxon Schistosoma intercalatum Fisher, 1934, agent de la schistosomose humaine rectale en Afrique. Description de Schistosoma guineensis n. sp. In Taxonomie, Ecologie et Evolution des Métazoaires Parasites. Tome II. Eds. Combes C. & Jourdane J. Universitaires de Perpignan. pp. 139–146, 380 pp. Rege, A.A., Wang, W., Dresden, M.H., 1992. Cystein proteinases from Schistosoma haematobium adult worms. The Journal of Parasitology 78, 16–23. Riggione, F., Pulido, M., Noya, O., 1996. Plasmodium falciparum: surface modiWcations of infected erythrocytes from clinical isolates. Parasitological Research 82, 490–496. Rollinson, D., Kaukas, A., Johnston, D.A., Simpson, A.J.G., Tanaka, M., 1997. Some molecular insights into schistosome evolution. International Journal for Parasitology 27, 11–28. Salter, J.P., Choe, Y., Albrecht, H., Franklin, C., Lim, K.C., Craik, C.S., McKerrow, J.H., 2002. Cercarial elastase is encoded by a functionally conserved gene family across multiple species of schistosomes. Journal of Biological Chemistry 277, 24618–24624. Smith, M.A., Clegg, J., Webbe, G., 1976. Cross-immunity to Schistosoma mansoni and S. haematobium in the hamster. Parasitology 73, 53–64. Smith, A.M., Dalton, J.P., Clough, K.A., Kilbane, C.L., Harrop, S.A., Hole, N., Brindley, P.J., 1994. Adult Schistosoma mansoni express cathepsin L proteinase activity. Molecular and Biochemical Parasitology 67, 11–19. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the Natural Academy of Science, USA 76, 4350–4354. Valdivieso, E., Bermúdez, H., Hoebeke, J., Noya, O., Cesari, I.M., 2003. Immunological similarity between Schistosoma and bovine cathepsin D. Immunology Letters 89, 81–88. Wasilewski, M., Lim, K., Phillips, J., McKerrow, J., 1996. Cysteine protease inhibitors block schistosome hemoglobin degradation in vitro and decrease worm burdens and egg production in vivo. Molecular and Biochemical Parasitology 81, 179–189. Wolowczuk, I., Auriault, C., Bossus, M., Boulanger, D., Gras-Masse, H., Mazingue, C., Pierce, R., Grezel, D., Ried, G., Tartar, A., Capron, A., 1991. Antigenicity and immunogenicity of a multiple peptidic construction of the Schistosoma mansoni Sm28-GST antigen in rat, mouse and monkey. Journal of Immunology 146, 1987– 1995. Xu, C., Verwaerde, C., Gras-Masse, H., Fontaine, J., Bossus, M., Trottein, F., Wolowczuk, I., Tartar, A., Capron, A., 1993. Schistosoma mansoni 28 kDa Glutation S-transferase and immunity against parasite fecundity and eggs viability. Journal of Immunology 50, 940– 949.