Recombinant antigens for serodiagnosis of cysticercosis and echinococcosis

Parasitology International 55 (2006) S69 – S73 www.elsevier.com/locate/parint

Recombinant antigens for serodiagnosis of cysticercosis and echinococcosis Yasuhito Sako a,*, Minoru Nakao a, Kazuhiro Nakaya b, Hiroshi Yamasaki a, Akira Ito a a

b

Department of Parasitology, Asahikawa Medical College, Asahikawa, 078-8510, Japan Animal Laboratory for Medical Research, Asahikawa Medical College, Asahikawa, 078-8510, Japan Available online 13 December 2005

Abstract Diagnosis of cysticercosis/echinococcosis is primarily based on imaging techniques. These imaging techniques are sometimes limited by the small size of visualized lesions and atypical images, which are difficult to be distinguished from abscesses or neoplasms. Therefore, efforts have been directed toward identification and characterization of specific antigens of parasites for development of serodiagnostic method that can detect specific antibody. For cysticercosis, glycoproteins of 10 – 26 kDa in cyst fluid of Taenia solium have been widely accepted for serodiagnosis purpose. The glycoproteins consist of a very closely related family of 8-kDa proteins. We identified four genes (designated Ag1, Ag1V1, Ag2 and Ag2V1) encoding the 7- and 10-kDa polypeptides. Based on the antigenicities of these clones, Ag1V1 and Ag2 were chosen as ELISA antigens and the Ag1V1/Ag2 chimeric protein was expressed. The Ag1V1/Ag2 chimeric protein showed the similar sensitivity and specificity as the native glycoproteins. For alveolar echinococcosis, the 65-kDa protein of Echinococcus multilocularis protoscolices and Em18 has been considered as serodiagnostic antigens. The sensitivity and specificity of Em18 are very compatible to those of the recombinant 65-kDa protein. Recently, we demonstrated that Em18 was the proteolytic product of the 65-kDa protein following the action by cysteine proteinases. From the information of N-terminal amino acid sequences, molecular size and isoelectric point of Em18, recombinant Em18 (349K to 508K of the 65-kDa protein, RecEm18) was expressed and evaluated for serodiagnostic value. RecEm18 has the potential for use in the differential serodiagnosis of alveolar echinococcosis. D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Cysticercosis; Alveolar echinococcosis; Cystic echinococcosis; Serodiagnosis; Recombinant protein

1. Introduction The larval stage of the pork tapeworm Taenia solium is responsible for cysticercosis (CC). Humans are accidentally infected by ingestion of T. solium eggs excreted with the feces of individuals harboring the adult tapeworm in the intestinal tract. The oncospheres hatched in the small intestine migrate throughout the body, invade skeletal and other muscle, subcutaneous tissue, or the central nervous system and develop into cysticerci. This disease is crucial as an emerging disease in developing countries [1– 5]. Alveolar echinococcosis (AE), caused by the larval stage of Echinococcus multilocularis, is a serious parasitic disease of humans in Northern Hemisphere countries in the higher latitudes. Humans are infected with E. multilocularis by accidental ingestion of eggs excreted with the feces of carnivores harboring the adult tapeworm of this species. The * Corresponding author. Tel.: +81 166 68 2422; fax: +81 166 68 2429. E-mail address: [email protected] (Y. Sako). 1383-5769/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2005.11.011

eggs hatch in the small intestine of the human host releasing the oncosphere which migrates via the portal system into various organs, mainly the liver, and differentiates into the metacestode stage. The metacestodes propagate asexually like a tumor leading to organ dysfunction. As clinical symptoms usually do not become evident until 10 or more years after initial parasite infection, early diagnosis and treatment are important for reduction of morbidity and mortality [6,7]. At present, diagnosis of CC, AE and cystic echinococcosis (CE) is primarily based on imaging techniques including echography, computed tomography (CT), and magnetic resonance imaging (MRI) in addition to clinical criteria [8,9]. These imaging techniques are useful and reasonably accurate but have sometimes limitations by the small size of visualized lesions and/or atypical images, which are difficult to be distinguished from abscesses or neoplasms. Moreover, these techniques are too expensive and inaccessible in most areas where CC, AE or CE is endemic. Therefore, the development of immunodiagnosis tests that detects species-specific antibodies or antigens in patient specimens is urgently required because of its simplicity

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and reliance. Efforts have been directed toward identification and characterization of specific antigens of T. solium and E. multilocularis metacestodes and several excellent antigens have been identified for serodiagnosis [10 –19]. However, the preparation of native serodiagnostic antigens is under many restrictions, as we need to find and/or maintain infected animals, which are not always practicable. In order to overcome the problems mentioned above, molecular techniques which make possible to produce the immunodiagnostic antigens under stable conditions are very useful. In this short review, we describe our current work on molecular cloning of immunodiagnostic antigen genes and serological evaluation using recombinant proteins for CC and echinococcosis, either AE or CE [20,21]. 2. Cysticercosis 2.1. Immunodiagnostic antigens Gottstein and others [10] reported the species-specific antigens (8 and 26 kDa proteins) in crude extract of T. solium metacestodes. Parkhouse and Harrison [11] described the glycoproteins (GPs) in cyst fluids of T. solium and Taenia saginata using lentil –lectin affinity chromatography purification. Tsang and others [14] characterized the GPs in crude extract of metacestode using lentil – lectin affinity chromatography purification and described the usefulness of GPs (seven GPs ranged from 13 to 50 kDa) for differential serodiagnosis based on immunoblot analysis but not ELISA. These immunodiagnostic antigens have been widely accepted for serodiagnostic purpose. However, these antigens were inapplicable to ELISA system due to the existence of cross-reactive components. Ito et al. [19] developed a simple method to purify diagnostic antigens (10 – 26 kDa antigens under reducing condition) from cyst fluid by preparative isoelectric-focusing electrophoresis (PIEFE) and demonstrated the sensitivity and specificity for differential serodiagnosis of CC in both ELISA and immunoblot analysis in humans [22] and in pigs [23 –25] and dogs [26]. As mentioned above, the 10- to 26-kDa GPs in cyst fluid of T. solium metacestodes have been widely accepted for serodiagnosis purpose [9,14,19]. 2.2. Cloning and characterization of diagnostic antigen candidate genes In order to characterize biochemical properties of these GPs and produce recombinant antigens for serodiagnosis, we carried out cDNA cloning of genes encoding these GPs [20]. As a result of screening of T. solium metacestode cDNA library, fourantigen candidate clones, named Ag1, Ag1V1, Ag2 and Ag2V1 were isolated. These clones, except Ag2V1, encoded a 7-kDa polypeptide, and Ag2 encoded a 10-kDa polypeptide and showed 53 – 94% similarity at the amino acid level. The difference between the polypeptide size predicted from a cDNA sequence and the native antigen size detected by immunoblot analysis suggested the occurrence of N-linked glycosylation since putative N-linked glycosylation sites existed in Ag1 and

Table 1 Results of ELISA using 70 cysticercosis patient sera a

Native glycoproteins Ag1V1/Ag2 chimeric protein a

No. of positive

%

65 66

92.9 94.3

Native glycoproteins were prepared by the method of Ito et al. [19].

Ag1V1. Obregon-Henao et al. [27] demonstrated that purified 12-, 16- and 28-kDa native GPs migrated as 7-kDa proteins after treatment of enzymatic deglycosylation, which indicated that the protein backbone of GPs was similar in size (7 kDa), but extent of glycosylation was different. A sequence homology research revealed that four-antigen candidate clones showed high identities to the cysticercosisspecific antigen of T. solium [28], the immunodiagnostic antigen of Taenia crassiceps [29] and antigen B of Echinococcus granulosus [30], those closely related to the cestode hydrophobic ligand-binding protein characterized in Moniezia expansa [31,32] and Hymenolepis diminuta [33] as briefly reviewed by Ito [34]. 2.3. Expression of Ag1V1/Ag2 chimeric protein and evaluation for diagnostic value using ELISA Based on antigenicities of these clones, Ag1V1 and Ag2 were chosen as immunodiagnostic antigens and the Ag1V1/ Ag2 chimeric protein was expressed [20]. Ag1V1/Ag2 chimeric protein consisted of Ag1V1, Ag2 and five glycines as a spacer and expressed as chitin binding domain/mini-inteins fusion protein, which is used for affinity purification and automatic cleavage of fusion protein. This Ag1V1/Ag2 chimeric protein was slightly modified form of previously reported one (unpublished). In order to assess the diagnostic value of Ag1V1/Ag2 chimeric protein, we further tested its immunoreactivity by ELISA using sera from CC patients (Table 1). A positive reaction to Ag1V1/Ag2 chimeric protein was observed in 94.3% (66/70 cases) of sera from CC patients confirmed to be seropositive by immunoblot analysis [19]. The Ag1V1/Ag2 chimeric protein showed similar sensitivity to native GPs. Since Ag1V1/Ag2 chimeric protein used in this study was expressed using an Escherichia coli system, this protein was not glycosylated. Native antigens might be highly glycosylated, and the carbohydrates were thought to be key antigenic parts for immunodiagnostic sensitivity [27]. However, our data using chimeric protein and the data of Hancock et al. [35] using synthetic peptide of the same molecule indicated that antibodies to peptide, not carbohydrates, sufficiently existed in patient serum to detect T. solium infection, indicating that Ag1V1/Ag2 chimeric protein is a valuable target antigen for differential diagnosis on the serological evaluation. 3. Alveolar echinococcosis 3.1. Immunodiagnostic antigens Using molecular and immunological techniques, many researchers have attempted to identify E. multilocularis-

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A) (kDa) 113 92

1 2 3 4 5 6

B) 1 2 3 4 5 6

50 35 28.9 21

Fig. 1. Results of proteinase inhibition assays. E. multilocularis metacestodes crude extracts were incubated in the absence or presence of proteinase inhibitors for 60 min. (A) Incubation at pH 7.4; (B) incubation at pH 5.0. Lane 1, pre-incubation; lane 2, no proteinase inhibitors; lane 3, serine proteinase inhibitor (AEBSF, 10 mM); lane 4, metalloproteinase inhibitor (EDTA, 5 mM); lane 5, cysteine proteinase and trypsin-like proteinase inhibitor (leupeptin, 50 AM); lane 6, cysteine proteinase inhibitor (E64, 5 AM). After incubation, each extract was characterized by immunoblot analysis using recombinant EM10-immunized rabbit serum. Molecular size markers are indicated on the left. 65-kDa and 52-kDa bands (EM10) are indicated by arrows and Em18 is indicated by an arrowhead.

specific antigens and showed the usefulness of recombinant antigens for serodiagnosis. Vogel and others [12] identified a partial cDNA clone, designated II/3, from an E. multilocularis protoscolex library by screening with a pool of sera from AE patients and the expressed protein was shown to have potential for use in the serodiagnosis of AE. Muller et al. [13] subcloned a fragment of the cDNA II/3, referred to as II/3 – 10, which retained the diagnostic epitopes but was more suited to use in immunoassays. Subsequently, Frosch and others [15] characterized a full-length mRNA of 65-kDa protein from E. multilocularis protoscolices, and showed that the expressed antigen, designated EM10, had potential for use in diagnosis of AE. Contemporaneously, Hemmings and McManus [16] characterized a partial cDNA, designated EM4, encoding an antigen that was also potentially useful for serodiagnosis of AE. The II/3 and EM4 proteins were subsequently confirmed as being fragments of EM10. Recently, we reported another novel antigen, termed Em18 (18-kDa protein under reducing condition), partially purified by PIEFE from E. multilocularis protoscolices and demonstrated its usefulness for highly sensitive and specific diagnosis of AE by immunoblot [18]. Unlike 65-kDa protein described above, the Em18 used routinely for serodiagnosis was prepared from E. multilocularis protoscolices and its recombinant form was not been produced, since Em18 has not been sufficiently characterized using molecular techniques. Therefore, we performed molecular dissection of Em18 to produce recombinant Em18 [21].

as to whether EM10 and Em18 were antigenically same or otherwise related each other. Em18, which had been partially purified by PIEFE, was recognized with patients’ mono-specific antibodies to recombinant EM10 and a rabbit antiserum raised against recombinant EM10. These results indicated a clear antigenic relationship between Em18 and EM10. N-terminal amino acid sequence of Em18 was identical to that of EM10 (349K to 355D), indicating the likelihood that Em18 was a breakdown product of EM10. It has demonstrated that Em18 was a cleavage product of EM10 by proteolysis involved in at least a cysteine proteinase as shown in Fig. 1 (modified from Sako and others [21]). 3.3. Expression of recombinant Em18 (RecEm18) and its evaluation for serodiagnostic value Recombinant Em18 (RecEm18) was prepared by subcloning of the appropriate segment of a cDNA encoding this fragment of the EM10 antigen and expression in an Escherichia coli-based expression system [21]. Based on molecular size and isoelectric point information obtained from the primary structure of EM10 and native Em18, a polypeptide from 349K to 508K of EM10 was chosen for expression as RecEm18. The region selected for expression as RecEm18 excluded those parts of EM10, which showed homology to the human ezrin, radixin and moesin (ERM) family molecules and hence might be more likely to contain epitopes recognized by AE patients. The diagnostic value of RecEm18 was tested in both ELISA and immunoblot using individual sera from patients with AE, CE and CC (Table 2). By ELISA, a positive reaction to RecEm18 was observed in 87.1% (27/31 cases) of sera from clinically confirmed AE patients. Of the 33 CE patient sera, one patient serum exclusively showed reaction to RecEm18 by both methods. This was an Australian CE case with multiple cysts showing a strong positive response against E. granulosus cyst fluid antigens by ELISA and against Arc 5 by immunoelectrophoresis [36]. Five other CE cases with single cysts showing similar high titers were negative against RecEm18. No positive results were observed with sera from patients with CC (n = 10) or healthy persons (n = 15). In order to further assess the diagnostic value of RecEm18, the RecEm18 and affinity-purified native Em18 were compared by ELISA and immunoblot and showed the usefulness of RecEm18 for differential serodiagnosis [37,38]. In addition, Xiao and others investigated the genetic polymorphism of Em18-encoding regions of EM10 gene among E. multilocularis isolates worldwide (Europe, North America and Asia) and showed that there were no differences (in prepara-

Table 2 Results of ELISA and immunoblot using RecEm18 [21]

3.2. Biochemical characterization of Em18 The sensitivity and specificity of Em18 for AE were very compatible to those of recombinant EM10, raising the question

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Alveolar echinococcosis Cystic echinococcosis Cysticercosis

No. of sera examined

Positive (%) ELISA

Immunoblot

31 33 10

27 (87.1) 1 (3.0) 0 (0)

28 (90.3) 1 (3.0) 0 (0)

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tion). These results demonstrated the potentiality of Em18 for use in the serodiagnosis of AE. Despite there being a protein expressed by E. granulosus metacestodes which has a high level of homology to EM10 (98.6% identity at amino acid level) [39,40], this protein does not induce significant levels of cross-reacting antibodies to RecEm18 in CE patients except in a small minority of patients with multiple cysts. It is interesting to speculate the reasons why these similar proteins should seem to have distinctly different antigenicities in AE and CE patients. The two parasites have quite different pathologies, with E. multilocularis being invasive and having intimate contact with host tissues [41] in comparison to E. granulosus which has relatively less direct contact with the host except following cyst rupture [36]. For this reason, B cell responses in CE patients to the EM10 homologue of E. granulosus might be low. Further investigations will be needed to clarify the relationship between different pathogenesis of parasites and the B-cell response to Em18. Acknowledgments This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, Japan and the Japan Society for Promotion of Science to A.I. (10557029, 11694259, 12557024, 17256002) and to Y.S. (12770122). References [1] Schantz PM, Moore AC, Munoz JL, Hartman BJ, Schaefer JA, Aron AM, et al. Neurocysticercosis in an Orthodox Jewish community in New York City. N Engl J Med 1992;327:692 – 5. [2] Craig PS, Rogan MT, Allan JC. Detection, screening and community epidemiology of taeniid cestode zoonoses: cystic echinococcosis, alveolar echinococcosis and neurocysticercosis. Adv Parasitol 1996;38:169 – 250. [3] Simanjuntak GM, Margono SS, Okamoto M, Ito A. Taeniasis/Cysticercosis in Indonesia as an emerging disease. Parasitol Today 1997; 13:321 – 3. [4] White Jr AC. Neurocysticercosis: a major cause of neurological disease worldwide. Clin Infect Dis 1997;24:101 – 13. [5] Wandra T, Subahar R, Simanjuntak GM, Margono SS, Suroso T, Okamoto M, et al. Resurgence of cases of epileptic seizures and burns associated with cysticercosis in Assologaima, Jayawijaya, Irian Jaya, Indonesia, 1991 – 95. Trans R Soc Trop Med Hyg 2000;94:46 – 50. [6] Ammann RW, Hirsbrunner R, Cotting J, Steiger U, Jacquier P, Eckert J. Recurrence rate after discontinuation of long-term mebendazole therapy in alveolar echinococcosis (preliminary results). Am J Trop Med Hyg 1990; 43:506 – 15. [7] Gottstein B, Lengeler C, Bachmann P, Hagemann P, Kocher P, Brossard M, et al. Sero-epidemiological survey for alveolar echinococcosis (by Em2-ELISA) of blood donors in an endemic area of Switzerland. Trans R Soc Trop Med Hyg 1987;81:960 – 4. [8] Pawlowski ZS, Eckert J, Vuitton DA, Amman RW, Kern P, Craig PS, et al. Echinococcosis in humans: clinical aspects, diagnosis and treatment. In: Eckert J, Gemmell MA, Meslin FX, Pawlowski ZS, editors. WHO/OIE manual on echinococcosis in humans and animals: a public health problem of global concern. Paris’ OIE; 2001. p. 20 – 66. [9] Ito A, Craig PS. Immunodiagnostic and molecular approaches for the detection of taeniid cestode infections. Trends Parasitol 2003;19:377 – 81. [10] Gottstein B, Tsang VC, Schantz PM. Demonstration of species-specific and cross-reactive components of Taenia solium metacestode antigens. Am J Trop Med Hyg 1986;35:308 – 13.

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