TSOL18/HP6-Tsol, an immunogenic Taenia solium oncospheral adhesion protein and potential protective antigen

Parasitol Res (2008) 102:921–926 DOI 10.1007/s00436-007-0853-8

ORIGINAL PAPER

TSOL18/HP6-Tsol, an immunogenic Taenia solium oncospheral adhesion protein and potential protective antigen R. Michael E. Parkhouse & Pedro Bonay & Luis Miguel González & Elizabeth Ferrer & Teresa Gárate & Cruz M. Aguilar & Milagros M. Cortez A. & Leslie J. S. Harrison

Received: 24 September 2007 / Accepted: 10 December 2007 / Published online: 24 January 2008 # Springer-Verlag 2007

Abstract In this study, we employed Taenia solium mRNA extracted from a tapeworm of Venezuelan origin to clone express and test the recombinant protein of the T. solium homologue of the 18-kDa oncospheral adhesion molecule of Taenia saginata (HP6-Tsag/TSA18). We first confirm the conserved nature of the sequence of the T. solium homologue (TSOL18/HP6-Tsol) and demonstrate that the recombinant protein, which, as with its T. saginata homologue, is characterised by a fibronectin type III homology region, functions as an adhesion molecule. This emphasises the possible importance of TSOL18/HP6-Tsol in tissue invasion, thus providing a rational explanation for its efficacy as a vaccine. As protection against Taenia spp.,

oncospheres is antibody mediated, logically, therefore, TSOL18/HP6-Tsol may also serve as a diagnostic antigen, as is indeed the case for recombinant HP6-Tsag/TSA18.

The nucleotide sequence data for the molecule described in this paper is available in the GenBank, EMBL and DDBJ databases under the accession numbers AM774400, AJ581299, X97000 and X95983.

L. J. S. Harrison (*) University of Edinburgh, Royal (Dick) School of Veterinary Studies, Division of Veterinary Clinical Sciences, Centre for Tropical Veterinary Medicine, Easter Bush Veterinary Centre, Easter Bush, Roslin, Midlothian Scotland, UK EH25 9RG e-mail: [email protected]

C. M. Aguilar Centro de Investigaciones de Enfermedades Tropicales (CIET), Facultad de Ciencias de la Salud, Universidad de Carabobo, San Carlos, Estado Cojedes, Venezuela P. Bonay Centro de Biología Molecular, Universidad Autónoma de Madrid, 28049 Madrid, Spain E. Ferrer : M. M. Cortez A. Departamento de Parasitología and Instituto de Investigaciones Biomédicas (BIOMED), Universidad de Carabobo, Maracay, Venezuela L. M. González : E. Ferrer : T. Gárate Instituto de Salud Carlos III, Centro Nacional de Microbiología, Ctra. Majadahonda Pozuelo Km 2,2, 28220 Majadahonda, Madrid, Spain

Introduction It is a characteristic of several Taenia spp. antigens that they can possess either one or sometimes two fibronectin type III regions (Lightowlers et al. 2003). A good example of this is the Taenia adhesion gene family (TAF; Gonzalez et al. 2007a). These authors considered that taeniids appear to have evolved, along with other adhesion molecules, a

R. M. E. Parkhouse (*) Instituto Gulbenkian de Ciencia, Rua Quinta Grande 6, Apartado 14, P-2780-156 Oeiras Codex, Portugal e-mail: [email protected]

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complex family of genes (TAF) coding for proteins mediating cell adhesion. Such proteins may play an essential role in tissue invasion and in the establishment of concomitant immunity, a strategy that protects the parasite from elimination through death or serious compromise of the host from subsequent superinfection by the same parasite. This current study focused on another Taenia spp. antigen containing a fibronectin type III region with possible utility in diagnosis and protection. TSOL18 (Plancarte et al. 1999) is the T. solium homologue of the 18-kDa oncosphere adhesion protein of Taenia saginata. This is named HP6-Tsag and was cloned from a T. saginata oncosphere cDNA library (Benitez et al. 1996). The sequence was subsequently confirmed, and the protein named TSA18 by Lightowlers et al. (1996). It has been shown to protect cattle from infection with the parasite (Lightowlers et al. 1996; Harrison et al. 2005). In a similar manner, the T. solium homologue of HP6-Tsag, TSOL18 (or HP6-Tsol), protects pigs from experimental challenge with T. solium (Plancarte et al. 1999; Flisser et al. 2004; Gonzalez et al. 2005). Unlike the TAF antigens, the TSOL18 gene appears to be more conserved. It does not form part of a gene family and is reported to vary very little over a wide geographic range (Gauci et al. 2006a). In addition, the protein is considered to be expressed in the oncosphere but not in the metacestode (Gauci et al. 2006b). This is also the case for HP6-Tsag/TSA18, as monoclonal antibody HP6, which is reactive with HP6-Tsag/TSA18, has been shown by immunofluorescence and western blot studies to be reactive with T. saginata hatched and activated oncospheres but not the metacestode (Harrison and Parkhouse 1986). Previous studies demonstrated that T. saginata HP6-Tsag/ TSA18 was a surface/secreted protein of the oncosphere with the structural and functional properties of an adhesion molecule (Bonay et al. 2002), suggesting that the antibodymediated mechanism of protection might be based on neutralization of the adhesion activity, perhaps a prerequisite necessary for tissue invasion by the migrating oncosphere. The objective of this study was, therefore, to determine whether TSOL18/HP6-Tsol also has such a function.

Materials and methods Parasite material An adult T. solium tapeworm was obtained from a consenting adult Venezuelan patient. The collection procedure was given ethical approval from the appropriate body within the University of Carabobo, Venezuela. The patient was treated with Praziquantel (10 mg/kg, Bayer AK) along with a laxative. The expelled worm was collected and then washed several times in sterile 0.01 M phosphate buffered saline,

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0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, (PBS, Sigma Ltd., P4417). The scolex and gravid proglottid samples were retained for morphological examination to verify that the tapeworm was T. solium. The remainder of the tapeworm was divided into three sections consisting of the anterior, medial and the terminal gravid proglottids containing mature eggs. Aliquots of each of the sections were immediately frozen in liquid nitrogen and stored in liquid nitrogen until use. An aliquot of frozen mature proglottids containing tapeworm eggs (0.7 g) was placed on dry ice in a mortar and ground to a fine powder. The frozen powder was suspended in 10 ml Tri Reagent (Sigma Ltd., T-9424) and the total RNA was extracted following the manufacturer's instructions. Polyadenylated RNA (mRNA) was purified from the total RNA by chromatography on oligo (dT) cellulose (Aviv and Leder 1972). The pellet was resuspended in nuclease-free sterile water (Promega Ltd.) and the concentration of mRNA determined by spectrophotometry. Samples were stored at −70°C until required. Isolation and sequencing of the HP6-Tsol (TSOL18) gene The TSOL18 sequence from T. solium (AF017788) was obtained from the EMBL/GenBank to design oligonucleotide primers for the specific amplification of T. solium HP6 by reverse transcriptase-polymerase chain reaction (RT-PCR). Two primers, PTSOL18F 5′-ATGGTGTGTCGGTTTG CTCTCAT-3′ and PTSOL18R 5′- CTACGATCTTCG GACCTTCTTG-3′, were selected using the Primer select Lasergene program (DNASTAR) and obtained from Roche. The RT-PCR reactions were carried out in a Gene Amp 2400 PCR System (Perkin-Elmer) and performed in a single tube. The RT was carried out using 50, 100, 500 and 1,000 ng of mRNA from Venezuelan T. solium proglottids and 50 ng of T. saginata and T. solium gDNA that were combined with 12.5 pmol of reverse primer and with the rest of the components in the reactions according to the manufacturer’s protocols (Retrotools cDNA/DNA polymerase kit, Biotools). The volume of the reaction was adjusted to 20 μl. The RT was run for 30 min at 60°C. The specific components for the PCR were added after RT, including 12.5 pmol of the forward primer. The volume of the reaction was set up to 50 μl using the PCR-specific components according to the manufacturer’s protocol (Retrotools cDNA/DNA polymerase kit, Biotools). The optimised PCR protocol consisted of an initial incubation at 94°C (3 min) followed by 45 cycles: 94°C melting (10 s), 55°C annealing (20 s) and 72°C extension (30 s) then finally, a last extension at 72°C for 7 min. The amplifications were electrophoresed on 1.5% (w/v) agarose gels, with visualization by ethidium bromide staining under UV illumination. DNA fragments (aplicons 393 bp) were cut

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out from agarose gels, purified with the QUIAquick Gel DNA extraction kit according to the manufacturer’s protocols (QUIAGEN) and cloned into pGEM-T (Promega, Madison, WI, USA). The TSOL18/HP6-Tsol DNA inserts from recombinant plasmids were automatically sequenced by fluorescencebase labelling with the Model 377 ABI PRISM system (Perkin-Elmer, Langen, Germany). Homology studies were performed by using Lasergene Software (DNAStar, Madison, WI, USA), TSOL18 (HP6-Tsol) DNA sequence, and predicted amino acid sequence comparisons were carried out using Lasergene Software and conserved domain analysis by CD-search bioinformatics program (NCBI; Marchler-Bauer et al. 2002, 2005) using one RPSBlast algorithm. Expression and purification of recombinant HP6-Tsol (TSO18) protein To express the TSOL18/HP6-Tsol, the full-length TSOL18/ HP6-Tsol cDNA in the pGEM-T vector was used as a template to amplify the truncated HP6-Tsol (TSOL18) fragment by PCR using specific oligonucleotides. The sense TSOL18F7 (5′ GAGAGAGAgaattcTGGCGAG CGGTG 3′) and antisense TSOL18R1 (5′ GAGAGAGActcgagCTACGATCTTCG 3′) primers included EcoRI and XhoI sites. Conditions for the PCRs were 94°C for 1 min (initial denaturation) followed by 30 cycles at 94°C/30 s, 65°C/30 s and 72°C/2 min and 72°C for 7 min (final extension). The truncated TSOL18 PCR product was gel purified and cloned into the expression plasmid vector pGEX-4T-2 (Amersham Biosciences) downstream of glutathione-S-transferase (GST). Escherichia coli strain BL21 was transformed with the construction pGEX-4T-2TSOL18F7R1. The induction of the truncated HP6-Tsol (TSOL18) molecule in pGEX vector was carried out with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG; SigmaAldrich) and 200 μg/ml ampicillin (Sigma-Aldrich) in SOB medium at 37°C for 5 h. For the purification of the recombinant protein, and to improve the efficiency and quality of the purification, a bulk GST purification module (Amersham Biosciences) and a B-PER® bacterial protein extractor reagent (Pierce) were used as described by the manufacturers. The purified proteins were checked by 10.0% SDS-PAGE and Coomassie blue staining. Protein concentrations were determined with a Bradford protein assay kit (Sigma). Preparation and purification of recombinant GST fusion proteins of TEG-Tsol, TEG-Tsag and HP6-Tsag and GST This was carried out exactly as previously described (Gonzalez et al. 2007b).

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Monoclonal antibody HP6 Purified monoclonal antibody HP6 was obtained from a stock (5 mg/ml) solution (Harrison and Parkhouse 1986). Cell adhesion assays Normal rat kidney (NRK) and COS-1 cells were grown in RPMI 1640 medium (Gibco Ltd.) supplemented with 10% v/v (heat inactivated) foetal calf serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). Before use, the cells were detached from culture vessel by EDTA treatment, washed twice with PBS and resuspended at 5×106/ml in RPMI medium with antibiotics but without serum. Calcein-AM (a fluorogenic esterase substrate, Invitrogen Ltd.) was added to a final concentration of 5 μM, mixed and incubated at 37°C for 30 min. The cells were washed twice with pre-warmed (37°C) RPMI and resuspended at 5×106/ml. Ninety-six-well microtitre plates (Nunc Immunoplate II, 4-42404) were pre-coated with the substrates in coating buffer NaHCO2 50 mM, NaCl 50 mM, pH 8.9 (Bovine serum albumin (BSA), GST, TEG-Tsag and TEG-Tsol, fibronectin or recombinant test proteins HP6-Tsol or HP6-Tsag at either 0.5, 1.0 or 2.0 μg/ml) overnight at 4°C. In certain cases, pre-coated wells were then also treated with competing monoclonal antibody HP6 at either 10 or 30 μg/ml at room temperature for 1 h, after which the plates were washed once with the coating buffer and twice with PBS supplemented with 0.01% Tween. The calcein-AM loaded cell suspensions were added to wells of the microtitre plates (100 μl/well) and incubated at 37°C for 20 min before being carefully washed four times with RPMI and twice with PBS to remove nonadherent cells. The fluorescence of the bound cells was measured using a 494-nm excitation filter and 517-nm emission filter in a Fluorstar Optima micoplate fluorimeter (BMG Labtechnologies, Offenburg, Germany). Assays were conkDa

M 1

2

M 3

97 84 55 45 36

Fig. 1 10% (w/v) SDS-PAGE analysis of TSOL18 (HP6-Tsol) recombinant protein expression and purification: lane 1 induced crude cell extract pellet, lane 2 induced crude cell extract supernatant, lane 3 TSOL18 (HP6-Tsol) purified recombinant protein. M molecular mass markers; the numbers indicate the size of the molecular mass markers. The gel was stained using Coomassie blue

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ducted in triplicate and the mean±standard error (SE) of the relative fluorescence units determined.

same fibronectin type III homology is present in both HP6-Tsag and TSOL18 (HP6-Tsol). The truncated recombinant protein TSOL18 (HP6-Tsol) has 14 amino acid less than complete recombinant protein.

Results Sequence data

Expression and purification of recombinant TSOL18 (HP6-Tsol)

The sequence obtained confirmed the previous work (Plancarte et al. 1999) and has been submitted to the GenBank (accession number AM774400). Importantly, the

As can be seen in Fig. 1, purification of the TSOL18 (HP6-Tsol) recombinant protein resulted in a preparation with a unique component of c. 38 kDa.

Fig. 2 Demonstration of the cell adhesion properties of HP6-Tsag and TSOL18 (HP6-Tsol) purified recombinant proteins using either NRK cells (a) or COS cells (b). Microtitre plates were coated with either 0.5 (black), 1.0 (gray) or 2 μg/ml (white) of either fibronectin type III, BSA or GST, TEG-Tsag, TEG-Tsol, HP6-Tsag and TSOL18 (HP6-Tsol), purified recombinant proteins. Where indicated, wells were additionally treated with either 10 or 30 μg/ ml of monoclonal antibody HP6. The results indicate the mean± SE of the relative fluorescence units of three replicate wells as determined using a 494-nm excitation filter and 517 nm emission filter in a Fluostar Optima micoplate fluorimeter (BMG Labtechnologies, Offenburg, Germany)

a Fibronectin GST TEG-Tsag TEG-Tsol HP6-Tsag + Ab 30 HP6-Tsag + Ab 10 HP6-Tsag HP6-Tsol + Ab 30 HP6-Tsol + Ab 10 HP6-Tsol BSA 0

500

1000

1500

2000

Mean +/-se Relative Fluorescence Units

b Fibronectin GST TEG-Tsag TEG-Tsol HP6-Tsag + Ab 30 HP6-Tsag + Ab 10 HP6-Tsag HP6-Tsol + Ab 30 HP6-Tsol + Ab 10 HP6-Tsol BSA 0

500

1000

1500

Mean +/-se Relative Fluorescence Units

2000

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Cell adhesion assays While cell adhesion was generally more pronounced in the COS cell system as opposed to NRK cells, a similar pattern was observed with both these cell types (Fig. 2). It is clear that as with the positive control (fibronectin) and HP6-Tsag, HP6-Tsol also acts as an adhesion protein. The amount of cell adhesion was notably greater in plates coated with 2 μg/ml of test material becoming progressively less when the wells of the microplate were coated at 1.0 μg/ml then 0.5 μg/ml of the various proteins. In addition, cell adhesion was inhibited in a dose dependent manner by MAb HP6. When plates were pre-incubated with monoclonal antibody HP6 (Fig. 2a, b), the inhibition in adhesion was more pronounced in wells treated with 30 μg/ml as opposed to 10 μg/ml of MAb HP6. Readings for the BSA and GST controls were near baseline as were those for purified recombinant proteins TEG-Tsag and TEG-Tsol, which thus served as useful negative controls.

Discussion Neither the recombinant TEG-Tsag nor TEG-Tsol promoted cell adhesion—and so provided useful negative controls for the adhesion assays. It is clear that TSO18 (HP6-Tsol) functions similarly to the previously described HP6-Tsag (Bonay et al. 2002). In this report, we therefore confirm the sequence of TSO18 (HP6-Tsol) of T. solium using, in this case, mRNA and gDNA extracted from a tapeworm of Venezuelan origin. More importantly, we demonstrate for the first time that this molecule, like its T. saginata homologue, HP6Tsag (TSA18), functions as an adhesion molecule, emphasising the possible importance of TSO18 (HP6-Tsol) in tissue invasion, and thus providing a rational explanation for its efficacy as a vaccine. Finally, as protection against Taenia spp. oncospheres is antibody mediated, logically therefore, any vaccine candidate may also serve as a diagnostic antigen, and our preliminary evidence shows that this is indeed the case for recombinant HP6-Tsag (TSA18; Fleury et al. 2003; Ferrer et al. 2003, 2005 2007). Acknowledgements This investigation received financial support from EU/INCO-DC (project CT95-0002), ISCIII-Programa Intramural (MPY 1274/05), ISCIII-RETIC RD06/0021/0019/0016, the Spanish Ministry of Science and Technology SAF2005-00362 and the Consejo de Desarrollo Científico y Humanístico of the Universidad de Carabobo. L. M. Gonzalez was sponsored by fellowships from the ISCIII, RICET-FIS and ISCIII-RETIC RD06/0021/0019 and E. Ferrer by fellowships from RECI, ISCIII and RICET-FIS. We would like to

925 thank nurse C. González of Valle del Rio Hospital Ambulatorio and Dr. Eduardo Borges from the Venezuelan Ministry of Health for their help in the care and treatment of the hospital patients, Lic. Iris Dávila, Glenda Rojas, Yenny Alviares, Maria A. Pieters, Alejandro Aguilar and TSU Maria Lares, Argenis Molina and Jonathan Pérez for their technical support and our students, I. Delgado, N. Depinay, S. Linares and I. Yanez for helping with the field work. The experiments described in this paper comply with the current laws of the countries in which the work was performed.

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