protective immunity against taenia crassiceps murine cysticercosis induced by dna vaccination with a...

Original article

Protective immunity againstTaenia crassiceps murine cysticercosisinduced by DNA vaccination with aTaenia saginata tegument antigen

Gabriela Rosasa, Gladis Fragosob, Teresa Garatec, Beatriz Hernándezd, Patricia Ferrerob,Mildred Foster-Cuevase, R. Michael E. Parkhousef, Leslie J.S. Harrisong,

Sergio López Brionesb, Luis Miguel Gonzálezc, Edda Sciuttob,*a Facultad de Medicina, Unidad de Biomedicina Molecular, Universidad Autónoma del Estado de Morelos, 62210 Cuernavaca, Morelos, Mexico

b Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), A.P. 70228, México D.F., C.P.04510, Mexicoc Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Centro Nacional de Microbiologia, Madrid, Spain

d Facultad de Medicina, Universidad Nacional Autónoma de México, México D.F., Mexicoe Institute for Animal Health, Pirbright Laboratories, Pirbright, Woking, Surrey GU24 0NF, UK

f Instituto Gulbenkian de Ciencia, Rua Quinta Grande 6, 2780–156 Oeiras, Portugalg Department of Tropical Animal Health, Sir Alexander Robertson Centre for Tropical Vetrinary Medicine, Royal (Dick) School of Veterinary Studies,

The University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK

Received 18 March 2002; accepted 22 August 2002

Abstract

This study investigated the protective capacity of the recombinantTaenia saginata Tso18 antigen administered as a DNA vaccine in theTaenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from aT. saginata oncosphere cDNA library, hashomologies withTaenia solium andEchinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice.Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3–81.4%, while lower levels of non-specificprotection were induced in control mice injected with the plasmid pcDNA3.1 (18.8–33.1%) or a plasmid with irrelevant construct,pcDNA3.1/3D15 (33.4–38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid andpcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies.These antibodies were shown to recognize a 66 kDa antigen fraction ofT. crassiceps andT. solium. Splenocytes enriched in both CD4+CD8–and CD4–CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from bothT. solium andT. crassicepscestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres ofT. saginata andT. solium, in the adult tapeworm and in thetegument ofT. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera ofcestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.

© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: DNA immunization; Vaccination; Cysticercosis;Taenia solium; Taenia crassiceps; Taenia saginata

1. Introduction

Echinococcus granulosus, Taenia saginata, Taenia ovisandTaenia solium are the cestodes with the greatest medicaland veterinary importance. Infections with the larval phasecause production losses to the sheep (T. ovis), cattle (T. sagi-

nata) and pig (T. solium) industry. Meanwhile, contact withthe larval phase ofT. solium[1] andE. granulosus[2] causestwo serious diseases, neurocysticercosis and hydatidosis, re-spectively, that greatly affect human health. Neurocysticer-cosis is one of the most frequent neurological disorders inseveral countries of Latin America, Africa and Asia, where itcauses enormous human suffering and great economic loss.Hydatidosis has a worldwide distribution, appearing particu-larly in countries where livestock-raising activities areprominent. In animals and humans, hydatid cysts frequentlyoccur in the liver and lungs, although they can occur at anyanatomical site.

* Corresponding author. Tel.: +52-555-622-3818;fax: +52-555-622-3369.

E-mail address: [email protected] (E. Sciutto)

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© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.PII: S 1 2 8 6 - 4 5 7 9 ( 0 2 ) 0 0 0 2 5 - 4

The extensive similarities in the natural history, pathologyand antigenic compositions among cestodes [2–6] often per-mit the use of a protective or diagnostic antigen from onespecies potentially to be applied to another. Treatment orvaccination of domestic animals as the obligatory intermedi-ate hosts of these parasites that share a two-host life cycleoffers the possibility of controlling transmission. One anti-gen shared by these cestodes is Tso18, which was isolatedfrom a T. saginata oncosphere cDNA library [7,8] and con-tains a full-length cDNA sequence of 1893 bp with an openreading frame of 1680 bp, corresponding to 559 amino acidswith a deduced molecular mass of 65.173 kDa [8]. TheR-Tso18 protein showed 80–84% nucleotide identity withthe major protoscolex surface antigens of E. multilocularis(EM10) and E. granulosus (EG10) [9,10] and was recentlycloned from a T. solium metacestode library (T. Garate,personal communication). Here, we have tested the use of theTso18 antigen of T. saginata as a DNA vaccine in a murinemodel of cysticercosis. This experimental model is obtainedby using Taenia crassiceps, another cestode that naturallyinfects rodents and exhibits extensive antigenic cross-reactivity and cross-immunity with T. solium and developseasily and rapidly in the peritoneal cavity of mice. Thus, ithas been successfully employed to test promising antigensand different vaccine approaches [6,11–14] against T. soliumcysticercosis [15,16]. Our results encourage further investi-gation into the Tso18 antigen as a general vaccine againstdifferent forms of cestodiasis.

2. Materials and methods

2.1. Mice

BALB/cAnN mice, previously characterized as a strainhighly susceptible to cysticercosis [17], were used for vac-cine trials. Mice were bred and kept in our animal facilitiesby single-line breeding system. The experiments reportedherein were conducted according to the principles set forth inthe Guide for the Care and Use of Laboratory Animals,Institute of Laboratory Animal Resources, National Re-search Council, Washington, DC, 1996.

2.2. Parasite and cysticercal antigens

The ORF strain of T. crassiceps[18] has been maintainedby serial intraperitoneal passage in BALB/cAnN femalemice for 16 years in our institute. Parasites for infectionswere harvested from the peritoneal cavity of mice, 1–3months after inoculation of 10 cysticerci per mouse as de-scribed [19].

T. crassiceps and T. solium soluble extracts were preparedby centrifuging the intact cysticerci at 25 000 × g for 60 minat 4 °C [5]. The sedimented material was discarded and thesupernatant containing a mixture of soluble antigens wasrecovered and filter-sterilized.

2.3. Plasmids

Sequences of the experimental Tso18 insert and the irrel-evant control foot and mouth disease virus non-structuralprotein 3D [8,20] were inserted into an expression plasmid(pcDNA3.1, Invitrogen, CA, USA) yieldingpcDNA3.1/Tso18 (pTso18) and pcDNA3.1/3D15 (p3D15),respectively. The Tso18 full sequence was subcloned fromp-Bluescript into pcDNA3.1 vector, according to standardprotocols [21], using EcoRI and XhoI restriction sites.

Plasmids used in this study were isolated in Qiagen max-ipreps (Qiagen Inc, San Diego, CA), according to the manu-facturer’s instructions. DNA was quantified by spectropho-tometry at 260 nm and the final concentration was adjusted to1 µg/µl in sterile saline solution.

2.4. Immunization protocol

Groups of 10 male BALB/cAnN mice were injected oncewith 100 µg of pcDNA3.1, p3D15 or pTso18 recombinantplasmids in 100 µl of saline solution into each quadriceps(200 µg per mouse) through the skin as previously reported[11,12]. From these, seven mice were used for protectionexperiments and three for the study of T-cell reactivity. Con-trol mice were injected only with 100 µl of saline solution.Three independent experiments were conducted.

2.5. Isotype antibody detection by ELISA

Mice were bled from the orbital plexus before and 8 weeksafter immunization, and before infection. Sera were sepa-rated and stored at – 70 °C until individually tested for thepresence of specific antibodies. Antibody titers in the serawere assessed by ELISA using T. solium (TsAg) or T. cras-siceps cysticercal antigens (TcAg) as previously described[22]. Briefly, after incubation of 100 µl per well of TcAg orTsAg (1 µg/ml) overnight at 4 °C, the wells were washed andincubated with 100 µl of mouse sera diluted in PBS–Tween1:100 for 60 min at 37 °C. Antibodies were detected with 100µl of the biotinylated anti-Igs (biotin-conjugated anti-IgG,anti-IgG1, anti-IgG 2a, Sigma, St Louis, MO) diluted tooptimum concentration (1:2000) and incubated for 45 min at37 °C. Bound anti-isotypes were detected using streptavidin-peroxidase (1:2000) over 30 min at 37 °C and 3,5-5,5-tetramethylbenzidine (TMB) as substrate (5 mg/ml, Sigma).The reaction was stopped using 50 µl of 0.2 N H2SO4.Readings of optical density at 450 nm (OD450) were carriedout in a Hum reader ELISA processor (Human Gessellschaftfür Biochemica und Diagnostica, Taunusstein, Germany).

2.6. Immunoelectrotransfer blot

Electrophoresis and immunoblotting using TcAg andTsAg were performed as described elsewhere [5]. Sera fromnon-immunized and DNA immunized mice were diluted

1418 G. Rosas et al. / Microbes and Infection 4 (2002) 1417–1426

1:20 in PBS–Tween 0.05% plus BSA 1% v/v and light milk5% v/v. As positive control, sera from T. crassiceps infectedmice were used. Bound antibodies were detected by incubat-ing each strip in goat anti-mouse IgG (H + L) horseradishperoxidase conjugate (Amersham Laboratories, Bucking-hamshire, UK) diluted 1:1000 for 30 min at room tempera-ture. As substrate, o-chloronaphthol 0.5 mg/ml with 3% v/vH2O2 in PBS, pH 7.3/CH3OH (5:1) was used.

2.7. Infection of mice

Eight weeks after immunization, seven mice from thecontrol and immunized groups were each intraperitoneallyinfected with 10 small (2 mm in diameter), non-buddingcysticerci of T. crassiceps in 0.5 ml of PBS. To determine thelevel of protection, mice were killed 30 days after infectionand the cysts inside their peritoneal cavities were harvestedand counted under a stereoscopic microscope [19]. Organsinside the peritoneal cavity of each mouse were removed andcarefully inspected to detect any remaining T. crassicepslarvae.

2.8. Proliferation assay

Spleen cells from the remaining three control or immu-nized mice were harvested 8 weeks after immunization andcultured in RPMI-1640 medium (Gibco, Gaithersburg, MD).Medium was supplemented with L-glutamine (0.2 mM), non-essential amino acids (0.01 mM), penicillin (100 U/ml),streptomycin (100 µg/ml), 10% fetal bovine serum (FBS)and 2-mercaptoethanol 0.05 mM. Cells were cultured withconcanavalin A (5 µg/ml) as a positive control or with TsAgor TcAg (10 µg/ml) as described elsewhere [14]. Briefly, cellswere incubated at 37 °C in a 5% CO2 humidified atmosphere,in flat-bottomed microtiter plates, at a concentration of 2 ×105 cells per 200 µl final volume. After 72 h, the culturedcells were pulsed (1 µCi per well) for a further 18 h with[methyl-3H]thymidine (Amersham, Life, Science, LittleChalfont England). Then, all cells were harvested and theamount of incorporated label was measured by counting in amodel 1205 b-plate liquid scintillation counter (Wallac Oy,20 101 Turku 10, Finland). All assays were performed intriplicate with three individual mice per group. Data arerepresentative of two independent experiments and are ex-pressed as the mean counts per minute for each experimentalgroup.

2.9. Spleen cell phenotype analysis

After 3 days of culture in vitro with TsAg or TcAg, sple-nocytes were harvested, suspended in cold PBS containing5% of gammaglobulin-depleted FBS plus 0.02% NaN3 andwere stained for 30 min at 4 °C with the following mono-clonal antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (clone RM4-5), phycoerythrin (PE)-conjugated anti-CD8 (clone 53–6.7) FITC-conjugated anti-

CD3 (clone 145-2 C11) and PE-conjugated anti-CD19 (cloneMB19-1) monoclonal antibodies (all from Pharmingen, SanDiego, CA) using 1 µg of each antibody per 100 µl per 106

cells. Non-specific binding of antibodies through Fc recep-tors was blocked with Fc Block (Pharmingen). Stained lym-phocytes were fixed with paraformaldehyde and 10 000 cellswere analyzed with a lymphocyte gate as defined by lightscatter in a FACScan (Becton Dickinson, Palo Alto, CA).Results were expressed as percentage of positive cells.

2.10. Immunolocalization of Tso18 protein

Non-specifically bound host proteins were dissociatedfrom T. crassiceps and T. solium cysticerci, or to T. soliumand T. saginata proglottides using the procedure previouslydescribed [12]. Briefly, tissues were placed on ice cold PBS(vesicular fluid was removed in cysticerci tissues) and incu-bated with 50 mM glycine–HCl, pH 2.5; 0.1% Triton X-100;0.15 mM NaCl for 30 s, pH was restored by adding Tris–HCl,pH 9. After further washing with cold PBS, tissues wereincluded in optimum-cutting-temperature compound(O.C.T.) (Miles, Inc., CA, USA) and frozen at – 70 °C. Sixmicrometer sections were cut with a cryostat, placed onpoly-l-lysine (Sigma)-treated microslides, and fixed in ac-etone for 10 min. Slides were rehydrated with cold PBS, andperoxidase activity was blocked by treatment with 3% H2O2

in methanol for 10 min at room temperature. After washingwith PBS, the slides were blocked with 5% BSA in PBS plus0.1% Triton X-100 (pH 7.4) for 1 h at 4 °C and 30 min at37 °C. Solutions were removed and the slides were incubatedovernight at 4 °C with a pool of sera from immunized mice(pcDNA3.1, p3D15, or pTso18), diluted at 1:5000 in 1%BSA in PBS plus 0.1% Triton X-100 (PBS/A-T). The nega-tive control was done with sera from mice injected withsaline solution, and the positive control with sera from para-sitized mice. After washing three times in PBS/A-T, 5 mineach, the slides were covered with a biotinylated goat anti-mouse IgG (Immunomark Universal Kit, ICN Biomedicals,Costa Mesa, CA) for 30 min at room temperature, rinsed withPBS/A-T, and treated with streptavidin-peroxidase conjugate(HRP conjugated, Immunomark Universal Kit, ICN Bio-medicals) for 30 min at room temperature. Peroxidase activ-ity was visualized by incubating the samples with 3'3-diaminobenzidine (DAB-Plus Kit, Zymed Laboratories,Inc). The slides were counterstained with Mayer’s hematoxy-lin, dehydrated, cleared, and mounted.

2.11. Statistical analysis

The effect of DNA immunization on the parasitic burdenin challenged mice was analyzed using the one-tailed Stu-dent’s t-test, assuming unequal variances. The statistical sig-nificance of the differences between mean values of bindingactivity in ELISA, proliferation assays and flow cytometry

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was performed with the Welch’s unpaired t-test (alternativet-test). The Instat software program (GraphPad, San Diego,CA) performed both statistical analyses.

3. Results

3.1. Induction of protection against T. crassicepscysticercosis by DNA vaccination with pTso18

Fig. 1 shows the results obtained in the three independentexperiments, when the mice were examined 30 days postinfection. Overall, parasite recoveries were higher in experi-ment 3. However, it is evident that, compared to the salinecontrol mice, pTso18 immunized mice exhibited a consistentreduced number of cysticerci (57.3–81.4%) which is alsolower than the non-specific protection observed in controlsimmunized with pcDNA3.1 (18.8–33.1%) or p3D15(33.4–38.8%). Importantly, significant and reproducible lev-els of protection were observed when the parasite burdens ofthe p3D15 and pTso18 immunized mice were compared(34.0%, P = 0.03; 37.2%, P = 0.05; 71.9%, P = 0.001).

3.2. Induction of antibody by DNA immunization withpTso18

Low, but detectable, levels of specific IgG were observedin sera from pTso18-vaccinated mice using both T. crassi-ceps and T. solium as antigen. Significant differences in IgGmainly enriched with IgG1 immunoglobulins were observedin pTso18 mice (P < 0.05) (data not shown).

The repertoire of antigens recognized by sera from immu-nized mice was determined by Western blot. As Fig. 2 shows,all individual sera from pTso18 immunized mice stronglyrecognized a protein of 66 kDa of both T. crassiceps andT. solium cysticerci. Sera from mice immunized with controlplasmid p3D15 and pcDNA3.1 weakly recognized a 66 kDaantigen fraction.

3.3. Sensitization of T cells for subsequent cellproliferation in vitro by DNA immunization with pTso18

To identify the T-cell reactivity to epitope(s) on Tso18, westudied the proliferative response of spleen cells from miceimmunized with DNA. Spleen cells from immunized micewere stimulated in vitro either with TcAg or TsAg (10 µg/ml),or with concanavalineA (5 µg/ml) as a positive control. Fig. 3shows that splenocytes from mice injected with pcDNA3.1and p3D15 responded weakly to parasite antigens in vitro,

Fig. 1. Protective response induced against T. crassiceps cysticercosis byDNA immunization. BALB/cAnN male mice (seven mice per group) wereinjected intramuscularly once with 200 µg of DNA controls (pcDNA3.1 orP3D15), or DNA from pTso18 per mouse or with isotonic saline solution.Eight weeks after immunization, the mice were intraperitoneally infectedwith 10 T. crassiceps cysticerci. Parasitic burden was determined 30 dayspost-infection. As determined by the one-tailed Student’s t-test assumingunequal sample variances, immunization with pTso18 significantly reducedthe parasite burden compared to that observed in p3D15 immunized mice inthe three trials (experiment 1 (P = 0.03); experiment 2 (P = 0.05); andexperiment 3 (P = 0.001)). When compared to the saline controls, non-specific protection was also evident in pcDNA3.1 and p3D15 immunizedmice.

Fig. 2. Identification of the Tso18 native protein in the total extract ofT. crassiceps (Tc) and T. solium (Ts) cysticerci by immunoelectrotransferblot. Electrotransferred strips were developed with pooled sera from control(1, 2) and infected (3) mice and pcDNA3.1 (4–6), pcDNA3.1/3D15 (7–9)and Tso18 (10–12) immunized mice. The arrow shows the 66 kDa protein,which corresponds to Tso18 protein.

Fig. 3. T-cell proliferative responses of splenocytes from control and DNAimmunized mice determined by [3H]thymidine incorporation on day 3 ofculture and incubated with TcAg or TsAg (10 µg/ml) or with concanavalin A(ConA) (5 µg/ml) as positive control. The data show the mean ± S.D. of threeindividual mice from the first experiment and are representative of the twoexperiments.


but higher levels of proliferation were observed in cells frompTso18 immunized mice stimulated with TcAg or TsAg invitro. As seen in Fig. 4, the proportion of CD3 T cellsincreased from 24.5 to 33.7% when splenocytes from pTso18immunized mice were activated in vitro with TcAg and to32.9% with TsAg. A similar increase was observed in bothCD4+CD8– and CD4–CD8+ subpopulations.

3.4. Localization of Tso18 native proteins in differentstages of taeniid parasites

The antibodies induced by DNA immunization withpTso18 were employed to immunolocalize the native proteinin T. crassiceps (data not shown) and T. solium cysticerci(Fig. 5), and in the adult phase and oncospheres of T. solium(Fig. 6) and T. saginata (Fig. 7). Sera from DNA immunizedmice were obtained 8 weeks after the immunization of200 µg of DNA from the orbital sinus. Control sera wereobtained from saline solution injected mice. In T. soliumcysticerci, the Tso18 protein was extensively expressed, be-ing localized in different structures (Fig. 5G, H) such astegument (T), subjacent nuclear layer (N), tegumental spiralcanal (TSC), parenchymal folds (PF) and radical muscles(RM). This protein was also observed in the tegument ofT. crassiceps cysticerci and lightly in the parenchyma (datanot shown). Figs. 6G and 7G show an intense expression ofTso18 protein in T. solium and T. saginata onchospheres,respectively. This protein was also found in adult tissues(Figs. 6H and 7H). Sera from pcDNA3.1 and p3D15 DNAimmunized mice had little or no reactivity when tested ononcosphere, cysticerci and adult tissue.

4. Discussion

This study reports a statistically significant protection inmice against infection with T. crassiceps, when they wereDNA vaccinated with pTso18 antigen sequence originallycloned from T. saginata oncospheres. The plasmid Tso18construct contained the entire Tso18 gene without the poten-tial initial signal peptide [8]. Thus, testing a construct con-taining the signal peptide might yield higher levels of protec-tion, as secretion of the protein often increases the protectiveimmune response stimulated by DNA immunization [12].One dose of pTso18 was enough to reduce the parasiteburden by 57.3–64% versus isotonic saline solution controls,whereas pcDNA3.1 and p3D15 DNA only reduced the para-site burden by 18.8–27.8 and 35.2–38.8%, respectively.These levels of non-specific protection have also been ob-served in previous studies, where up to 30–35% protectionwas shown after three doses of DNA vaccination. This re-sponse has been attributed to stimulation of innate immunityby the CpG repeated sequences of the pcDNA3 plasmid[11,12].

Levels of antibodies generated in this study were highlyvariable and relatively low in most mice, confirming previousexperiences with DNA vaccination [23]. Although there is noobvious correlation between antibody response and protec-tion in the T. crassiceps model [24], protective antibodyresponses may indeed have an as yet undefined role, as theycertainly do confer protection in T. ovis. The antibodiesdetected after pTso18 DNA immunization are largely IgG1,as has been observed after DNA immunization with the genegun [25]. In contrast, in other reports, IgG2 is the predomi-nant isotype elicited by intramuscular immunization [26].

Fig. 4. Lymphocyte populations in antigen-stimulated spleen cells from control and DNA-vaccinated mice (pcDNA3.1, p3D15 and pTso18). Results arepresented as the percentage of positive cells from the first experiment, but are representative of two different experiments. Significant differences where observedin CD3, CD4 and CD8 T cells when compared to pTso18 with all the DNA immunized mice (P < 0.05), as determined with the two-way ANOVA Tukey’s test.


Fig. 5. Immunohistochemical staining of T. solium cysticerci. The sections were incubated with sera from saline solution injected mice (A, B), pcDNA3.1 (C,D), p3D15 (E, F), pTso18 (G, H) immunized mice and T. crassiceps infected mice (I, J) developed with biotinylated goat anti-mouse IgG plus streptavidin-peroxidase conjugate and counterstained with hematoxilin. A, C, E, G, I correspond to the internal view (spiral canal) and B, D, F, H, J to the external sectionof the parasite. G and H show that Tso18 protein is extensively present in different structures of the cisticercy as tegument of the spiral canal (TSC),subtegumental cells (STC), radical muscles (RM), parenchymal folds (PF), subjacent nuclear layer (N), fibrous layer (FL) and muscles (M). Bar = 40 µm.


Fig. 6. Immunohistochemical staining of T. solium oncosphere and adult tissue. The sections were incubated with sera from saline solution injected mice (A, B),pcDNA3.1 (C, D), p3D15 (E, F), pTso18 (G, H) immunized mice and T. crassiceps infected mice (I, J) developed with biotinylated goat anti-mouse IgG plusstreptavidin-peroxidase conjugate and counterstained with hematoxilin. A, C, E, G, I correspond to oncosphere and B, D, F, H, J to adult tissue. G and H showthat Tso18 protein is extensively present in both states of T. solium. G shows the extensively stained oncosphere (O) and embriophore (E), and in H the distalcytoplasm (DC) of the tegument is positive. PC (perinuclear cytoplasm), SC (subcuticular cells). Bar = 40 µm.


Fig. 7. Immunohistochemical staining of T. saginata oncosphere and adult tissue. The sections were incubated with sera from saline solution injected mice (A,B), pcDNA3.1 (C, D), p3D15 (E, F), pTso18 (G, H) immunized mice and T. crassiceps infected mice (I, J) developed with biotinylated goat anti-mouse IgG plusstreptavidin-peroxidase conjugate and counterstained with hematoxilin. A, C, E, G, I correspond to oncosphere and B, D, F, H, J to adult tissue. G and H showthat Tso18 protein is extensively present in both states of T. solium. G shows the extensively stained oncosphere (O) and embriophore (E), and in H the distalcytoplasm (DC) of the tegument is positive. PE (protoplasm extension). Bar = 40 µm.


The increased IgG1 observed after gene gun immunizationsuggests a Th2 response [27], which would be appropriatefor helminth rejection [28]. Interestingly, as shown by West-ern blot analysis, these antibodies clearly recognized a66 kDa antigen fraction in both T. solium and T. crassicepsantigens, in agreement with previous reports [8].

As the Tso18 protein is evidently shared between differentcestodes, it was of interest to examine the stage-specificity ofits expression. In spite of the low level of antibodies deter-mined by ELISA, the detection of the native protein inhistological sections was observed. Immunolocalizationanalysis revealed that the antigen was expressed in all threestages of T. solium and in oncospheres and adult tissue ofT. saginata. The tegumental localization of Tso18 is ofparticular relevance, as it is similar to the homologous EM10Echinococcus[10,29,30], which is localized within the ger-minal layer and membrane structures of developing pro-toscolices [29]. Interestingly, these taeniid genes show sig-nificant similarities with ezrin, radixin, moesin and merlin(ERM) family members [31–34], which are involved in theattachment and the morphology of cells and, in the case ofmerlin, in growth control. Considering that these ERM pro-teins are critical regulators of cytoskeletal–plasma mem-brane interaction, especially in polarized cells, as well asimportant components of signal transduction pathways, po-tential damage induced by the immune system, induced bypTso18 vaccine, would be of great impact on the parasitesurvival.

As shown in Fig. 3, cells from pTso18 immunized miceproliferated more when stimulated with TsAg than TcAg,probably because the T. solium antigens are relatively moreenriched in Tso18. In addition, the proliferating cells wereclearly enriched in both CD4+ and CD8+ T cells (Fig. 4)from pTso18 immunized mice, raising the possible participa-tion of these T cells in the protective immunity induced byvaccination.

In conclusion, the protective capacity exhibited by Tso18,its extensive presence in different cestodes and its abundancein the oncosphere stage as well as other developmental stagesof the parasite encourage further studies towards the use ofTso18 as a multipurpose vaccine in cestodiasis.

Acknowledgements

The authors thank Gerardo Arrellín, Abel Blancas, Al-berto Arzave Suárez, Mercedes Baca, and Marisela Hernán-dez for technical support. The authors also thank Dr. L.Cedillo-Barron for allowing the use of plasmidpcDNA3.1/3D15.

This investigation was partially supported by ConsejoNacional de Ciencia y Tecnología, G25955 m, FundaciónMiguel Alemán, México, British Council, Dirección Generalde Personal Académico IN212401, Universidad NacionalAutónoma de México, The Howard Hughes Medical Insti-tute, the European Union INCO-DC Programme STD4

(Project CD95-0002), and Instituto de Salud Carlos III (Pro-grama Intramural-00/0407).

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