Identification and characterization of a full-length cDNA encoding ...

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Biochemical and Biophysical Research Communications 365 (2008) 528–533 www.elsevier.com/locate/ybbrc

Identification and characterization of a full-length cDNA encoding paramyosin of Trichinella spiralis Jing Yang a, Yaping Yang a, Yuan Gu a, Qiang Li b, Junfei Wei a, Shaohua Wang a, P. Boireau c, Xinping Zhu a,* a

Department of Parasitology, School of Basic Medical Sciences, Capital Medical University, 10 Xitoutiao, You An Men, Beijing 100069, PR China b Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101100, PR China c INRA, AFSSA, ENVA, UPVM, JRU BIPAR 956, AFSSA LERPAZ, 94706 Maisons-Alfort, France Received 25 October 2007 Available online 20 November 2007

Abstract A full-length cDNA encoding Trichinella spiralis paramyosin (Ts-Pmy) was cloned by immunoscreening a cDNA library of the adult T. spiralis worm. Ts-Pmy cDNA consists of 2655 bp that encode 885 amino acids. The recombinant protein (rTs-Pmy) was expressed and purified by Ni-affinity chromatography. Western blot analysis showed that rTs-Pmy could be recognized by sera from T. spiralis-infected humans, swine, rabbits, and mice. Immunolocalization demonstrated that Ts-Pmy was abundant on the surface of T. spiralis larvae. BALB/c mice vaccinated with rTs-Pmy demonstrated 36.2% reduction in muscle larvae burden following T. spiralis larvae challenge. Vaccination of the mice with rTs-Pmy resulted in a high level of specific anti-Ts-Pmy IgG antibodies and generated a Th1/Th2 mixed type of immune response, with Th2 predominant. These studies showed that rTs-Pmy induced protective immunity in mice and could be considered as a potential vaccine candidate for trichinellosis. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Trichinella spiralis; Paramyosin; Immune response; Vaccine

The nematode Trichinella spiralis is an intracellular parasite of mammalian skeletal muscle. Trichinellosis is a widespread zoonosis acquired by ingestion of undercooked meat (e.g. pork, game, and horse) containing infective larvae of the Trichinella parasite, which can infect a wide variety of mammalian species, including humans [1]. Human trichinellosis outbreaks occur in many parts of the world, and it has been estimated that as many as 11 million people are infected with this parasite, which is regarded as a reemerging disease in various parts of the world (Eastern Europe, Argentina, etc.) [2]. Trichinellosis is a serious public threat in both developed and developing countries. Outbreaks of human trichinellosis are associated mainly with ethnic groups that prefer raw or lightly processed meat.

*

Corresponding author. Fax: +86 10 8391 1474. E-mail address: [email protected] (X. Zhu).

0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.11.012

For example, out of 548 outbreaks of trichinellosis in China during 1964–1999, 525 outbreaks were caused by eating raw or undercooked pork [3]. Therefore, the development of vaccines capable of preventing swine from becoming infected would make a substantial contribution to disease control. The life-cycle of T. spiralis is different from that of other nematodes, in that all the developmental stages occur in the same host. After ingestion of contaminated meat, the infective larvae are released with the aid of host gastric juice and develop into adult worms in the host intestine in 2–3 days. Five days post-infection, the gravid female begins to release newborn larvae, which penetrate the intestine and migrate to muscular tissue through the blood and lymphatic circulation. The newborn larvae parasitize the muscle cells forming a cyst. Every stage of the life-cycle of T. spiralis can induce a protective host immune response [4]. Trichinella antigens have shown stage specificity, changing qualita-

J. Yang et al. / Biochemical and Biophysical Research Communications 365 (2008) 528–533

tively and quantitatively in different stages of development [5]. A major problem in the development of vaccines against T. spiralis is the complexity of those antigens. It is well known that developing recombinant antigens is a promising strategy for the design of efficient vaccines [6]. Little is known about the T. spiralis genes that are involved in host invasion, parasite survival, and immunity. To investigate the genes and their products with immunological function, cDNA libraries were produced from each stage of T. spiralis. While screening these cDNA libraries with immune sera, some interesting genes were cloned [7–10]. In order to find more immunogenic antigens, the adult cDNA library of T. spiralis was immunoscreened with infected rabbit sera and anti-adult worm rabbit sera. A full-length cDNA encoding paramyosin was identified from the positive clones. Here, we describe the screening, molecular characterization, and protective immunity of T. spiralis paramyosin. Materials and methods Parasites and antigen preparation. T. spiralis (ISS533) was maintained in female ICR mice. Adult worm and muscle larvae were recovered from infected mice by the standard pepsin digestion method as described [11] and infection sera were collected. Crude somatic extracts of adult worms, muscle larvae, and excretory–secretory (ES) products were prepared by conventional methods [12]. Mice. Female BALB/c mice aged 6–8 weeks were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences. Rabbit antisera. Infected rabbit sera were obtained from New Zealand white rabbits by infection with 4000 T. spiralis muscle larvae for 45 days. Antisera against adult extracts were collected from rabbits immunized subcutaneously with 200 lg of crude extract of adult worms emulsified with complete Freund’s adjuvant (FCA, Sigma, USA) followed by two boost injections, each of 200 lg of protein mixed with incomplete Freund’s adjuvant at intervals of 2 weeks. T. spiralis-infected pig sera were kindly provided by Professor Gamble H Ray (National Research Council, USA). Infected human sera. Infected human sera were collected from T. spiralis-infected patients living in the Yunnan Province of China. Immunoscreening adult cDNA library of T. spiralis. The adult worm kZAP||cDNA expression library of T. spiralis [13] was immunoscreened with rabbit anti-T. spiralis adult extract antisera and rescreened with infected rabbit sera (described above) according to the manufacturer’s instructions (Stratagene, USA). DNA sequencing. Phagemid DNA was extracted and sequenced. Nucleotide and deduced amino acid sequences were compared to existing sequences in GenBank by BLAST searching. Expression and purification of recombinant protein. The full-length cDNA of a T. spiralis clone was amplified from phagemid by PCR with primers carrying BamHI and EcoRI restriction sites (forward, 5 0 -CGGGA TCCATGTCTCTGTATCGCAGTCCCAGT-3 0 and the reverse primer 5 0 -CGGAATTCATATTCATGTCCTTCTTCCATCAC-3 0 ). The PCR products were subcloned into expression vector pET-28a(+) with BamHI and EcoRI sites. After transformation into Escherichia coli BL21 (DE3) cells (Novagen, USA), the expression of the recombinant protein was induced by IPTG. The pellet of the bacterial culture was re-suspended, lysozyme was added, and the solution was incubated at 37 °C for 30 min and sonicated. Following centrifugation, soluble and insoluble fractions were collected and analyzed by SDS–PAGE. All of the recombinant proteins were present in inclusion bodies. The proteins were purified by Ni-affinity chromatography (Qiagen, USA) according to the manufacturer’s instructions. The purity of the recombinant protein was determined by SDS–PAGE.

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Raising anti-recombinant protein antibody. Antiserum against recombinant protein was raised in immunized mice. Mice were bled before each immunization. Western blot analysis. Samples including crude somatic extracts of adult worm and muscle larvae, ES products, and the recombinant protein were separated by SDS–PAGE. Then the protein was transferred to PVDF membrane (Millipore, USA). After being blocked with 5% (w/v) skimmed milk powder in TBS-Tween 20, the membrane was incubated with sera from trichinellosis patients, T. spiralis-infected mice, rabbit, and swine, or mouse anti-recombinant protein serum, respectively. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG, goat anti-rabbit IgG, goat anti-pig IgG, or goat anti-mouse IgG (Sigma, USA) were added. Finally, the bands were developed by 3,3 0 -diaminobenzidine tetrahydrochloride (Sigma, USA). Immunohistochemical localization. Muscle larvae of T. spiralis from infected mice were fixed with 3% (v/v) paraformaldehyde, frozen, and cryosectioned [10]. After incubation with the anti-recombinant protein antibody, the section was treated with fluorescein isothiocyanate (FITC)conjugated goat-anti-mouse IgG (Chemicon, USA). The larval section incubated with sera from control mice under the same conditions served as a control. Observations were made with a fluorescence microscope (Lecia DMIL, Germany). Animal immunization and challenge experiments. BALB/c mice were divided into three groups with 10 animals in each. The first group of vaccinated mice was inoculated subcutaneously with the recombinant protein (20 lg/mouse) emulsified with complete FCA, and were boosted twice with the recombinant protein (20 lg/mouse) in incomplete FCA at intervals of 2 weeks. The second and third groups were inoculated with FCA mixed with PBS, or with PBS only as a control under the conditions used for the first group. Two weeks after the final boost, groups of mice were each challenged orally with 400 T. spiralis infective muscle larvae. Worm burden reductions were evaluated by counting the number of muscle larvae recovered from mice sacrificed at 45 days post-infection. Measurement of antibody response. After each immunization, mice sera were collected and titers of anti-recombinant protein IgG and subclasses IgG1 and IgG2a were determined using 96-well microtiter plates (Costar, USA) coated with 100 ll of recombinant protein at a concentration of 1.0 lg/ml in bicarbonate buffer (pH 9.6). After blocking, serum samples diluted with PBS were added. The plates were then washed and incubated with HRP-conjugated goat anti-mouse IgG. For isotype-specific ELISA, after incubation with mouse serum samples, the plates were incubated with goat anti-mouse IgG1 or IgG2a (BD Pharmingen, USA). Then the HRPconjugated rabbit anti-goat IgG-subclass antibodies (BD Pharmingen, USA) were added. The substrate o-phenylendiamine dihydrochloride (OPD, Sigma, USA) was used. Statistical analysis. Data were expressed as means ± standard deviation. Differences among groups were analyzed by Student’s t-test, with P < 0.05 being considered statistically significant.

Results and discussion Molecular characterization of the cDNA encoding paramyosin After immunoscreening of a cDNA expression library (200,000 plaques) with rabbit antisera against T. spiralis adult extracts and T. spiralis-infected rabbit sera, the longest cDNA fragment was sequenced and its amino acid sequence was deduced. The full length of cDNA was 2996 bp. The ORF of the cDNA consisted of 2655 bp encoding 885 amino acids with a predicted molecular mass of 102 kDa and an isoelectric point of 5.4. The cDNA consisted of a 5 0 -untranslated region of 74 bp and a 3 0 -untranslated region of 241 bp followed with a 26 bp poly(A)+ tail.

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The results of a GenBank database search revealed that the predicted amino acid sequence of the cDNA clone has 83% identity with the paramyosin of Caenorhabditis elegans (GenBank Accession No. X08068), 82% with the paramyosin of Onchocerca volvulus (GenBank Accession No. M95813), 81% with the paramyosin of Brugia malayi (GenBank Accession No. U77590), and 34% with the paramyosin of Schistosoma japonicum (GenBank Accession No. AF113971) (Supplementary material, Fig. S1). Therefore, we designated this cDNA fragment Ts-Pmy (T. spiralis paramyosin). Paramyosin, a filamentous, a-helical, and coiled-coil protein of approximately 100 kDa, is present in the muscle of invertebrates including the helminths and forms the core of thick myofilaments that determine their length and stability [14]. It has been reported that paramyosin is present in the tegument and/or on the surface of parasitic worms such as Schistosoma mansoni, S. japonicum, Echinococcus granulosus, and Taenia solium. It is obvious that paramyosin is both a structural component of the contractile apparatus and a good target for vaccine development. Paramyosin of S. mansoni appears to contain strong humoral antigenic epitopes [15]. So far, paramyosin has been defined as a potential vaccine candidate against some helminthiases, i.e. schistosomiasis, cysticercosis, and filariasis [14]. Paramyosin is one of the six schistosome antigens selected by WHO as candidates for vaccine against schistosomiasis [16]. Several vaccination trials have been conducted using either native or recombinant paramyosin protein [17,18]. Studies of human immune responses to paramyosin support the potential of this protein as a vaccine candidate [16]. Therefore, the paramyosin of T. spiralis (Ts-Pmy) could be an important candidate for a vaccine antigen. Expression of recombinant Ts-Pmy (rTs-Pmy) and Western blot analysis The full length of the coding sequence of Ts-Pmy was cloned into a pET28a (+) expression vector and recombinant Ts-Pmy was expressed in E. coli BL21 after induction by IPTG. The rTs-Pmy was analyzed by SDS–PAGE (Fig. 1). The molecular mass of the rTs-Pmy (with the histidine tag) was approximately 110 kDa, which corresponded well to the predicted size of the gene product. The purified rTs-Pmy was used to evaluate its antigenicity by immunoblotting. The results showed that the rTs-Pmy was recognized by sera from trichinellosis patients, T. spiralis-infected sera from rabbits, mice, and swine, as well as by the antibody against rTs-Pmy (Fig. 2A). No recognition was observed with the normal human or corresponding animal sera (data not shown). This result indicates that the paramyosin of T. spiralis is a highly immunogenic antigen and that the antigenicity is preserved in the recombinant molecule. Western blot analysis showed that mouse anti-rTs-Pmy antiserum bound to a band at 110 kDa in the somatic

Fig. 1. Expression and purification of rTs-Pmy. Proteins were analyzed by SDS–PAGE and stained with Coomassie Brilliant blue. Lane 1, protein marker; lane 2, E. coli lysate control (harboring only the pET28a expression vector); lane 3, uninduced E. coli lysate (Ts-Pmy/pET-28a); lane 4, IPTG-induced E. coli lysate (Ts-Pmy/pET-28a); lane 5, rTs-Pmy purified by Ni-affinity chromatography.

extract of T. spiralis muscle larvae and adult worms but not in ES antigens (Fig. 2B). The results indicate that the rTs-Pmy is not a secretory protein but a somatic antigen, and the antibodies elicited by immunized mice recognized the native Ts-Pmy protein. Immunohistochemical localization In order to localize the Ts-Pmy proteins, T. spiralis muscle larvae were reacted with mouse anti-rTs-Pmy serum and then incubated with FITC-labeled goat anti-mouse IgG. Immunofluorescence microscopy revealed strong reactivity of the rTs-Pmy on the surface of T. spiralis larvae. In contrast, very weak reactivity was detected in sections of muscle larvae probed with normal mouse serum (Supplementary material, Fig. S2). These results are consistent with the previous studies of other helminth parasites. Kalinna and McManus [19] reported that paramyosin was located in the tegument of S. japonicum adults, in the post-acetabular glands of cercariae, and on the surface of lung schistosomular. Recent studies on Paragonimus westermani demonstrated that paramyosin was localized in the tegument of the adult parasite [20]. The fact that Ts-Pmy is expressed on the surface of the parasite may provide an opportunity for an anti-rTs-Pmy vaccine, since the tegument of the parasite is constantly in contact with the immune system of the host. Immune responses to rTs-Pmy Mouse serum samples were collected immediately before rTs-Pmy immunization and at 2 weeks after each boost.


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Fig. 2. Western blot analysis of rTs-Pmy. (A) The antigenicity of rTs-Pmy by Western blot. Lane 1, rTs-Pmy recognized by patient sera; lane 2, rTs-Pmy recognized by infected mice sera; lane 3, rTs-Pmy recognized by infected rabbit sera; lane 4, rTs-Pmy recognized by infected swine sera; lane 5, rTs-Pmy recognized by antisera against rTs-Pmy. (B) Western blot analysis of crude somatic extracts from T. spiralis with the antibody against rTs-Pmy. Lane 1, ES antigen; lane 2, purified rTs-Pmy; lane 3, crude somatic extracts of adult worm; lane 4, crude somatic extracts of muscle larvae.

Antibody titers of the serum samples against the rTs-Pmy were measured using ELISA. A high titer of specific antibodies (IgG) was elicited following a boost with rTs-Pmy in all immunized mice (Supplementary material, Fig. S3). IgG subclass antibody levels were measured to further assess the efficacy of rTs-Pmy in induction of the Th1 or Th2 response in vivo. The results demonstrated that the predominant IgG subclass was IgG1, but there was also a significant level of IgG2a. There were significant differences (P < 0.001) in the levels of IgG1 and IgG2a between mice vaccinated with rTs-Pmy and control mice (Fig. 3). Antibody response tests revealed elevated Th2 type IgG1 and Th1 type IgG2a responses, with a predominant IgG1 antibody response in the serum of rTs-Pmy immunized mice. This suggests that FCA-rTs-Pmy vaccination induced a Th1/Th2-mixed type of response, which may be important in host-protective immunity against T. spiralis.

Among the distinct CD4 T helper cell subsets, the Th2 type of immune response is important in host protective immunity to many intestinal nematode infections. Humoral response has a significant influence on Trichinella by participating in entrapment and rapid expulsion of infective larvae, reducing adult worm fecundity and killing newborn larvae [21]. It is known that antibody response is essential in the induction of protection against this parasite, but it is not the only parameter. Indeed, a cellular response has been described in mice infected with T. spiralis [6]. Some experimental results suggest that cellular response as well as humoral immune response to paramyosin may be involved in the mechanisms of protective immunity induced by paramyosin immunization [16]. Vaccination/immunization of paramyosin from another helminth parasite, S. mansoni (Sm97), elicited a protective cell-mediated immunity in mice. Both the native molecule and the recombinant protein of Sm97 had been shown to

Fig. 3. Serum IgG subclass responses in mice vaccinated with rTs-Pmy in Freund’s complete adjuvant (FCA). Antisera from mice immunized with PBS were used as controls. Values are expressed as means ± SD for five mice in each group. **P < 0.001.

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Fig. 4. The muscle larvae counts of vaccinated mice after T. spiralis challenge. BALB/c mice were immunized subcutaneously with FCA-rTsPmy, FCA, or PBS. **A statistically significant difference from the FCA or PBS control group (P < 0.001).

confer significant levels of protection in mice, with the immunity being accompanied by T-cell responses to the antigen and high levels of antibody production [22]. The T. solium paramyosin induced a Th1-like immune response, suggesting the role of a cellular immune response in the establishment of protective status against murine cysticercosis [23]. These results suggest that a combination of humoral and cellular immune response to paramyosin is involved in the protective mechanism against helminth infections. Our results have shown that immunization of rTs-Pmy induced both humoral and cellular responses to T. spiralis infection in mice. Protective immunity elicited by rTs-Pmy The protective response induced by rTs-Pmy against T. spiralis infection was investigated in BALB/c mice. The challenge results showed that vaccination of mice with FCA-rTs-Pmy produced a 36.2% reduction (P < 0.001) in muscle larvae burden compared with the PBS control (Fig. 4). There was no difference in muscle larvae burden between FCA immunized and PBS immunized control mice. These data show that rTs-Pmy possesses a potential vaccine effect for the control of trichinellosis. Our results with rTs-Pmy are similar to those reported from studies of paramyosin vaccines in other parasites. Paramyosin has been subjected to animal trials with different animal models, including mouse, jird (Meriones libycus), pig, sheep, and water buffalo, each resulting in significant and similar levels of protection, ranging from 17% to 87% [14]. For example, B. malayi recombinant paramyosin conferred partial protection against infection in immunized jirds. Therefore, paramyosin is a candidate component for a vaccine to prevent the development of filariasis [17]. Paramyosin is also a strong candidate for a vaccine for S. japonicum and S. mansoni infections, where both native

and recombinant forms of paramyosin have been shown to be immunogenic and to confer protective immunity in mice [19,22]. Several studies have demonstrated the potential value of paramyosin as a vaccine target in T. solium and T. saginata [14]. In addition, nucleic acid vaccination with the T. solium paramyosin-coding sequence elicited the production of anti-paramyosin antibodies in mice and showed 43–48% reductions in the parasite burden [24]. In conclusion, our results demonstrate clearly that paramyosin of T. spiralis induced high levels of specific antibody production. The rTs-Pmy formulated in FCA adjuvant was highly immunogenic in vaccinated mice, resulting in promising protective efficacy in terms of significant reduction of muscle larvae. Collectively, these data suggest that Ts-Pmy is an antigen that triggers host immune responses and is, therefore, a candidate for an antigen vaccine for trichinellosis. The complete Ts-Pmy cDNA sequence has been submitted to GenBank with Accession No. EF429310. Acknowledgments We thank Fengyun Wang and Jin Pan for their technical assistance. This work was supported by grants from the National Natural Science Foundation of China (30571626), the Natural Science Foundation of Beijing (5062005), the Beijing Municipal project for Developing Advanced Human Resources for Education (BAHED), and the PhD Programs Foundation of the Ministry of Education of China (20060025003). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc. 2007.11.012. References [1] M. Ribicich, H.R. Gamble, A. Rosa, I. Sommerfelt, A. Marquez, G. Mira, N. Cardillo, M.L. Cattaneo, E. Falzoni, A. Franco, Clinical, haematological, biochemical and economic impacts of Trichinella spiralis infection in pigs, Vet. Parasitol. 147 (2007) 265–270. [2] Z.Q. Wang, J. Cui, B.L. Xu, The epidemiology of human trichinellosis in China during 2000–2003, Acta Trop. 97 (2006) 247–251. [3] Z.Q. Wang, J. Cui, The epidemiology of human trichinellosis in China during 1964–1999, Parasite 8 (2001) S63–S66. [4] B.Q. Fu, M.Y. Liu, C.M. Kapel, X.P. Meng, Q. Lu, X.P. Wu, Q.J. Chen, P. Boireau, Molecular cloning of a cDNA encoding a putative cuticle collagen of Trichinella spiralis, Vet. Parasitol. 132 (2005) 31–35. [5] P. Boireau, M. Vayssier, J.F. Fabien, C. Perret, M. Calamel, C. Soule´, Characterization of eleven antigenic groups in Trichinella genus and identification of stage and species markers, Parasitology 115 (1997) 641–651. [6] S. Deville, A. Pooter, J. Aucouturier, V. Laine´-Prade, M. Cote, P. Boireau, I. Valleé, Influence of adjuvant formulation on the induced protection of mice immunized with total soluble antigen of Trichinella spiralis, Vet. Parasitol. 132 (2005) 75–80. [7] X.P. Zhu, P. Garcia-Reyna, B.Q. Fu, J. Yang, C.V. Li, Y.P. Yang, M.Y. Liu, G. Ortega-Pierres, P. Boireau, A stage-specific open


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