Parasitol Res (2001) 87: 70±79

Ó Springer-Verlag 2001

ORIGINAL PAPER

Rosario Leyva á Pascal HeÂrion á Rafael Saavedra

Genetic immunization with plasmid DNA coding for the ROP2 protein of Toxoplasma gondii

Received: 18 March 2000 / Accepted: 13 July 2000

Abstract The ROP2 protein of Toxoplasma gondii has previously been proposed as a vaccine candidate against toxoplasmosis. In this work we characterize the immune response induced by injection of plasmid DNA coding for this protein in three strains of mice (BALB/c, C57BL/6, and CBA/J) displaying di€erent levels of susceptibility to toxoplasmosis and compare it with that obtained by vaccination with the live attenuated ts-4 strain of T. gondii. The ROP2 gene was cloned in the eukaryotic expression vector pcDNA3 and the resulting plasmid, named pcDNA3/ROP2, was used to immunize mice. After three immunizations with the plasmid, mice developed antibodies that could be detected by ELISA using a recombinant truncated form of ROP2; and these antibodies also recognized the natural protein by Western blot. Plasmid immunization generated antibodies against the ROP2 of both of the IgG1 and IgG2a isotypes in CBA/J and BALB/c mice and both of the IgG1 and IgG2c isotypes in C57BL/6 mice. However, animals vaccinated with the ts-4 strain generated only IgG2a (in CBA/J and BALB/c mice) or IgG2c (in C57BL/ 6 mice) against ROP2. Kinetic studies of the generation of isotypes indicated that both isotypes were generated at the same time. Mice immunized with the plasmid DNA did not resist a challenge with the virulent RH strain of T. gondii, while mice vaccinated with the ts-4 strain resisted the same challenge. However, in pcDNA3/ROP2-immunized BALB/c mice, death was signi®cantly delayed with respect to the pcDNA3immunized control group. These results suggest that plasmid immunization using the ROP2 gene generates a mixed TH1/TH2 response against ROP2, which is di€erent from that obtained by vaccination with live

R. Leyva á P. HeÂrion á R. Saavedra (&) Departamento de InmunologõÂ a, Instituto de Investigaciones BiomeÂdicas, Universidad Nacional AutoÂnoma de MeÂxico, Ap. Postal 70228, C.U., CP 04510 MeÂxico D.F., MeÂxico e-mail: [email protected] Tel.: +52-5-6223368; Fax: +52-5-6223369

tachyzoites of the ts-4 strain (TH1 response) and is not protective against the highly virulent RH strain of the parasite.

Introduction Toxoplasma gondii is the intracellular protozoan parasite responsible for animal and human toxoplasmosis. In immunocompetent individuals, infection with this parasite usually is clinically asymptomatic, but it may cause severe complications in immunode®cient individuals and in pregnant women (Hughes 1985). In immunode®cient patients, chronic infection with T. gondii can reactivate and produce encephalitis, which is often lethal (McCabe and Remington 1988). In fact, T. gondii is one of the major opportunistic pathogens in HIV-infected patients (McCabe and Remington 1988). Primary T. gondii infection of a mother during pregnancy can lead to abortion, neonatal malformations or other defects which appear during child development (Remington and Krahenbuhl 1982; Wong and Remington 1994). Natural infection with T. gondii generally leads to a state of long-lasting non-sterile protective immunity (Frenkel 1967; Nathan et al. 1984; Suzuki and Remington 1988; Gazzinelli et al. 1991). This protection is Tcell-mediated and involves both CD4+ and CD8+ T cells (Suzuki and Remington 1988; Gazzinelli et al. 1991). Moreover, interferon-c (IFN-c) produced by CD4+, CD8+ and NK cells was shown to be the major mediator of resistance against toxoplasmosis (Nathan et al. 1984; Suzuki et al. 1988; Suzuki and Remington 1990; Gazzinelli et al. 1994). Subunit vaccines against toxoplasmosis should be based on parasite antigens which induce this T-cellmediated protective immunity. We have previously reported the identi®cation and molecular characterization of the ROP2 antigen of T. gondii, a 54-kDa protein component of the rhoptries which is expressed in three stages of the parasite life cycle (Sadak et al. 1988; Saavedra et al. 1991; HeÂrion et al. 1993). The ROP2

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protein was recognized by a human T cell clone (TCC32) isolated from an immune donor, speci®c for the parasite and producing high levels of IFN-c (Saavedra and HeÂrion 1991; Saavedra et al. 1991). The TCC32-de®ned epitope was shown to be conserved among virulent and cyst-forming strains of T. gondii, and was recognized by TCC32 in association with the major histocompatibility complex class II molecule DPw4, the most frequent allele in the caucasian population (Saavedra et al. 1991). It was also found that 68% of T. gondii-immune individuals have T cells speci®c for at least one of three predicted ROP2 epitopes (Saavedra et al. 1996) and that 89% of T. gondii-seropositive individuals possess antibodies against a recombinant truncated form of this antigen (Van Gelder et al. 1993). In the mouse model, it was demonstrated that ROP2 is a target of mucosal immune mechanisms (ChardeÁs et al. 1990). All these properties support the proposal of this protein as a vaccine candidate. Genetic immunization or nucleic acid vaccination is a recently developed method that uses a plasmid vector which express a protein antigen. Injection of animals with the plasmid allows the induction of an immune response speci®c to the protein, which includes antibodies, and both helper and cytotoxic T cells; and it may be protective, as shown in several models (for a review, see Donnelly et al. 1997). Since DNA immunization may induce protective immunity, we were interested to test whether immunization with the gene coding for the ROP2 protein of T. gondii could induce an immune response against the parasite, as an alternative to carrying out immunization studies with puri®ed protein. As a ®rst step to generate a DNA vaccine against toxoplasmosis, in this paper we report the use of the ROP2 gene as a DNA vaccine candidate in the mouse model. In order to test whether the genetic background could in¯uence the immune response against ROP2, we tested the generation of antibodies against the ROP2 protein in the BALB/c (H2d), C57BL/6 (H2b), and CBA/J (H2k) strains of mice, each of which displays a di€erent level of susceptibility to T. gondii (Jones and Erb 1985; Brown and McLeod 1990; Suzuki et al. 1991). We also examined the isotype of the generated antibodies as an indicator of the type of immune response induced. Finally, we compared the immune response induced by DNA immunization with that obtained by vaccination with the live attenuated ts4 strain of T. gondii, which induces a protective immune response against virulent and avirulent strains of the parasite (Waldeland and Frenkel 1983; Waldeland et al. 1983).

Parasites Tachyzoites of the Wiktor strain were obtained by in vitro culture in Vero cells as previously described (Saavedra et al. 1991). Tachyzoites of the RH strain obtained after 15 passages in human foreskin ®broblasts were used for the mouse challenge. The temperature-sensitive mutant ts-4 strain (kindly donated by Dr. E. Pfe€erkorn) was maintained in human skin ®broblasts at 33 °C, as previously described (Pfe€erkorn and Pfe€erkorn 1976; Suzuki and Remington 1988). Bacterial strain Escherichia coli XL1-Blue strain (Stratagene, La Jolla, Calif.) was used as host cell for all plasmid manipulations. Plasmid construction Standard methods were used for DNA manipulation (Sambrook et al. 1989). The full-length ROP2 gene was obtained by PCR using genomic DNA from tachyzoites of the Wiktor strain of T. gondii grown in vitro and AmpliTaq DNA polymerase (Perkin-Elmer, Mexico City, Mexico). The sequences of the 5¢ and 3¢ primers (obtained from Oligotherapeutics, Wilsonville, Ore.) used for the PCR were 5¢ GCTCAAGCTTCCACCATGGAAAACTGTGCGTCGGTC-3¢ and 5¢-CGAGAAGCTTTCATGCCGGTTCTCCATCAGTT-3¢ (HindIII sites underlined). The ampli®ed DNA fragment was puri®ed, digested with HindIII (Lakeside, Mexico City, Mexico), and cloned in the HindIII site of the pBluescript KS(+) vector (Stratagene). The complete sequence of the ROP2 gene was veri®ed by the chain termination method (Amplicycle sequencing kit, Perkin Elmer) and the gene was further subcloned in the HindIII site of the eukaryotic expression vector pcDNA3 (Invitrogen, San Diego, Calif.). A construct containing the ROP2 gene in the correct orientation was identi®ed by restriction enzyme analysis and named pcDNA3/ROP2. Plasmid puri®cation Large-scale preparation of the pcDNA3/ROP2 and pcDNA3 plasmids was carried out using Mega-plasmid columns (Qiagen, Chatsworth, Calif.), following the instructions provided by the manufacturer. The puri®ed plasmids were dissolved in Dulbecco's phosphate-bu€ered solution (DPBS) at a concentration of 1 mg/ml and stored at )20 °C until use. DNA immunization Mice were injected with plasmid DNA dissolved in 100 ll of DPBS as 50-ll doses in each quadriceps. Mice were initially immunized with 50 lg of plasmid DNA and were boosted twice with 100 lg DNA at 3-week intervals. Immunization with ts-4 Mice were intraperitoneally injected with 2 ´ 104 tachyzoites of the ts-4 strain in 100 ll DPBS and were boosted twice with 2 ´ 105 tachyzoites by the same route at 3-week intervals. Mice were bled 2 weeks after each immunization. Production and puri®cation of recombinant ROP2

Materials and methods Mice Six-week-old female BALB/c, C57BL/6 and CBA/J mice were obtained from the Jackson Laboratories (Bar Harbor, Me.) and maintained in pathogen-free conditions in our animal house.

A ROP2 fragment was expressed in E. coli as a fusion protein using the pRSET vector (Invitrogen). The recombinant protein (rROP2) contained the sequence included between residues D-186 and A-561 of ROP2 fused to a 38-residue N-terminal peptide derived from the expression vector. It was puri®ed to >99% homogeneity by a combination of immobilized metal ion anity chromatography and size exclusion chromatography (HeÂrion et al., unpublished data).

72 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis Tachyzoites of the Wiktor strain grown in vitro in Vero cells were washed three times in DPBS, lyzed by boiling in Laemmli sample bu€er, and submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10% polyacrylamide gels, using the discontinuous system described by Laemmli (1970). Proteins were transferred to a nitrocellulose sheet (HybondC, Amersham, Bucks, England) as previously described (Saavedra et al. 1990). After transfer, the nitrocellulose membrane was blocked in washing bu€er (10 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20) containing 1% bovine serum albumin (BSA) for 2 h at room temperature (RT). After washing, 3-mm wide strips were incubated overnight at 4 °C with individual mouse sera, diluted 1:1,000 in washing bu€er containing 1% BSA. After washing, strips were incubated for 2 h at RT with alkaline phosphatase-labeled goat anti-mouse immunoglobulins (Dako, Glostrup, Denmark) diluted 1:10,000 in washing bu€er containing 1% BSA. The phosphatase activity was revealed using the chromogenic substrate nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl-phosphate. Enzyme-linked immunosorbent assay for determination of anti-ROP2 antibodies Flat-bottom, high-binding, styrene enzyme-linked immunosorbent assay (ELISA) plates (Costar, Cambridge, Mass.) were coated by overnight incubation at 4 °C with a 3 lg/ml solution of rROP2 in PBS at pH 7.4 (100 ll/well). Blocking was carried out with 1% BSA in PBS (pH 7.8) with 0.05% Tween 20 (PBS-T20-BSA), for 4 h at RT. After washing with PBS containing 0.05% Tween 20 (PBS-T20), sera diluted in PBS-T20-BSA (100 ll/well) were incubated in the wells overnight at 4 °C. After washing, bound antibodies were detected by incubation at RT for 2 h with horseradish peroxidase-labeled goat anti-mouse immunoglobulins (DAKO) at 1:20,000 dilution in PBS-T20-BSA (100 ll/well). Finally, the peroxidase activity was revealed by adding 100 ll of a solution containing 1.3 mM H2O2 and 0.13 mg/ml 3,3¢,5,5¢-tetramethylbenzidine in 0.1 M sodium acetate/citric acid at pH 6.0 (Bos et al. 1981). The reaction was stopped by adding 100 ll of 1 N H2SO4 and the optical density (OD) was read at 450 nm in an ELISA microplate reader (Bio-Rad, Richmond, Calif.). A serum was considered to be positive when OD > [ODmean + 2 SD] (cut-o€ value), where OD is that of the tested serum, and ODmean and SD respectively are the mean and the standard deviation of the OD of the sera from the DPBS-injected mice. The level of signi®cance of di€erences between the mean OD of each group of mice was determined using the Student's t-test. Determination of antibody isotypes The determination of antibody isotypes was carried out using the ELISA described above, with some modi®cations. Brie¯y, after incubation of the plates with mouse sera and washing, 100 ll of a 1:30,000 dilution of biotinylated rabbit anti-mouse IgG1 or antiIgG2a antibodies (Zymed, San Francisco, Calif.), or a solution containing 0.16 lg/ml of biotinylated mAb 5.7 speci®c for mouse IgG2a of Igh-Cb haplotype (which is expressed by C57BL/6 mice; Pharmingen, San Diego, Calif.) were added and incubated for 2 h at RT. After washing, bound antibodies were revealed by incubation with peroxidase-labeled avidin (Sigma, St. Louis, Mo.), diluted 1:100,000 in PBS-T20-BSA, for 2 h at RT, followed by the chromogenic substrate. A serum was considered to be positive according to the criteria de®ned above. Mouse challenge Mice, immunized according to the di€erent protocols described above, were subcutaneously challenged 3 weeks after the last immunization with 6,000 tachyzoites of the RH strain grown in

human skin ®broblasts. Mortality was assessed and survival curves were compared by the logrank test using the PRISM software (GraphPad, San Diego, Calif.).

Results Cloning of the ROP2 gene The ROP2 gene coding sequence was ampli®ed by PCR using genomic DNA from the Wiktor strain of Toxoplasma gondii as template. HindIII restriction sites were introduced at both extremities to allow further cloning of the gene; and the sequence preceding the initiation codon was modi®ed from the original sequence of the ROP2 gene (Beckers et al. 1994) to ®t the Kozak consensus sequence for optimal protein expression (Kozak 1986). The »1.7-kbp ampli®ed fragment was cloned in the pBKS+ vector to allow veri®cation of the sequence. When compared with the reported sequence of the ROP2-coding cDNA clone Tg34, which was also isolated from the Wiktor strain (Saavedra et al. 1991), we found that a G was missing from position 1,583 of the genomic clone sequence. This G was also missing from the sequence of a genomic clone isolated from the RH strain, as reported by Beckers et al. (1994). Therefore, we conclude that the extra G reported in the cDNA clone Tg34 sequence was probably the result of a sequencing error and that the ROP2 gene sequences of the RH and Wiktor strains are identical. The ROP2-coding sequence was inserted into the HindIII site of the eukaryotic expression vector pcDNA3, under the transcriptional control of the cytomegalovirus early promotor. The resulting plasmid, named pcDNA3/ROP2, was used for immunization of mice. Analysis of the humoral immune response in mice immunized with plasmid DNA Mice were immunized by the intramuscular route with doses of 50±100 lg of pcDNA3 or pcDNA3/ROP2 plasmids. To test whether the genetic background could have an e€ect on the immune response against ROP2 induced by DNA immunization, we used three strains of mice with three di€erent haplotypes (BALB/c [H2d], C57BL/6 [H2b], and CBA/J [H2k]) which are known to display di€erent levels of susceptibility to toxoplasmosis (Jones and Erb 1985; Brown and McLeod 1990; Suzuki et al. 1991). We also immunized mice from each of the three strains with live tachyzoites of the ts-4 strain of T. gondii, which causes infection but does not persist in the host and induces a protective immune response against a challenge with a virulent strain of the parasite (Waldeland et al. 1983). After the third immunization, sera from all animals were tested for the presence of antibodies against ROP2 by ELISA with puri®ed recombinant ROP2 (rROP2).

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A representative result of two experiments is shown in Fig. 1. Four of the ®ve CBA/J mice immunized with the pcDNA3/ROP2 plasmid developed signi®cant levels of antibodies against the ROP2 protein, although there was a high variability in antibody levels among individuals. All BALB/c mice immunized with the pcDNA3/ ROP2 plasmid also produced antibodies against ROP2. C57BL/6 mice developed lower levels of antibodies when compared with the two other strains; and two of the ®ve mice did not seroconvert. Mice immunized with the pcDNA3 plasmid did not generate antibodies against ROP2.

Mice of all three strains vaccinated with ts-4 developed antibodies against ROP2 as well. In the CBA/J strain, only two of the ®ve mice generated signi®cant levels of antibodies. In BALB/c mice, antibodies to ROP2 were detected in four of the ®ve mice, but the levels were statistically lower (P ˆ 0.009) than those observed in mice immunized with the pcDNA3/ROP2 plasmid. All C57BL/6 mice seroconverted and the levels of antibodies were comparable to those observed in plasmid DNA-immunized mice. This experiment shows that mice immunized with plasmid DNA coding for ROP2 develop antibodies against the recombinant protein at similar or higher levels than those generated by vaccination with ts-4. In order to test whether the antibodies generated by plasmid immunization were also able to recognize the natural ROP2 protein, all sera were tested by Western blot using tachyzoites of T. gondii as antigen (Fig. 2). Sera of most mice immunized with pcDNA3/ROP2 plasmid contained antibodies that recognized a 54-kDa antigen. This antigen comigrated with a 54-kDa band de®ned by mAb 4A7, which recognizes the ROP2 protein (Fig. 2, arrow; Sadak et al. 1988). Some sera did not contain antibodies against this 54-kDa antigen when tested by this technique (two sera from CBA/J and two from the C57BL/6 mice); and these same sera were also negative by ELISA. The antibodies recognizing the 54-kDa antigen were absent from the sera of mice injected with DPBS or control plasmid pcDNA3 (Fig. 2). These results show that plasmid immunization induces antibodies able to recognize the natural ROP2 protein. Mice vaccinated with ts-4 also had antibodies which recognized a 54-kDa antigen; however, the intensity of the reaction with this antigen was weaker than that observed in mice immunized with the pcDNA3/ROP2 plasmid (Fig. 2). In addition, the recognition pattern of the other parasite antigens clearly di€ered between the three strains of mice. Analysis of the isotype of the anti-ROP2 antibodies

Fig. 1 Determination of anti-ROP2 antibodies in the sera of mice immunized with plasmid DNA or ts-4. Groups of ®ve mice of strains CBA/J, BALB/c, and C57BL/6 were injected with pcDNA3, pcDNA3/ROP2, live tachyzoites of the attenuated ts-4 strain, or Dulbecco's phosphate-bu€ered saline (DPBS) only, as described in Materials and methods. Sera were obtained 2 weeks after the last immunization and tested for the presence of anti-ROP2 antibodies by enzyme-linked immunosorbent assay (ELISA). Each point represents the mean optical density (OD) at 450 nm of individual sera tested in duplicate at a 1/1,000 dilution. Horizontal bars represent means of the ®ve mice. The cut-o€ values are indicated by dotted lines

We were also interested in determining whether a TH1 and/or TH2 response against ROP2 was generated by DNA immunization and vaccination with ts-4 in the three mouse strains. Therefore, we determined IgG1 and IgG2a against ROP2 in sera as indicators of the type of immune response. As can be observed in Fig. 3, IgG1 against ROP2 was detected in the sera from most mice immunized with the pcDNA3/ROP2 plasmid, although there was heterogeneity in the IgG1 antibody levels among the three strains of mice: CBA/J mice showed the lowest response and BALB/c and C57BL/6 mice showed higher values. IgG1 was not detected in the sera of mice inoculated with the ts-4 strain of T. gondii, nor in control mice immunized with the pcDNA3 plasmid, nor in those injected with DPBS (Fig. 3).

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Fig. 3 Determination of anti-ROP2 IgG1 and IgG2a or IgG2c in the sera of mice immunized with plasmid DNA or ts-4. Sera of mice injected with pcDNA3, pcDNA3/ROP2, ts-4, or DPBS, as described in Fig. 1, were obtained 2 weeks after the last immunization and were assayed for isotype-speci®c anti-ROP2 antibodies by ELISA as described in Materials and methods. Each point represents the mean OD at 450 nm of individual sera tested in duplicate at a 1/1,000 dilution. Horizontal bars represent means of the ®ve mice. The cut-o€ values are indicated by dotted lines

Fig. 2A±C Reactivity of the sera of mice immunized with plasmid DNA or ts-4 by Western blot. Sera from the mice described in Fig. 1 [CBA/J (A), BALB/c (B), and C57BL/6 (C)] were tested by Western blot using Toxoplasma gondii tachyzoites as antigen (3.75 ´ 106 parasites/lane), as described in Materials and methods. The position and molecular weights (in kilodaltons) of standard proteins are shown. The arrow indicates the position of a 54-kDa protein detected with the anti-ROP2 speci®c mAb 4A7 in the same blot

In contrast to the results obtained for IgG1, IgG2a antibodies were detected in CBA/J and BALB/c mice either immunized with the pcDNA3/ROP2 plasmid or infected with the ts-4 strain of T. gondii. BALB/c mice showed a very homogenous IgG2a response, while CBA/ J mice showed an heterogeneous response (Fig. 3). Since C57BL/6 mice express the IgG2c (IgGb2a ) isotype instead of IgG2a (Martin et al. 1998), we used an anti-IgGb2a mAb for detection of this isotype: we found these mice produced IgG2c against ROP2 after DNA immunization and also when infected with ts-4 tachyzoites. In this strain of mice, however, there was a great heterogeneity of response among animals for both IgG1 and IgG2c. All these data indicate that plasmid immunization using the ROP2 gene generates both IgG1 and IgG2a antibodies (IgG2c in C57BL/6 mice) in all three strains of mice, while an infection with the ts-4 strain only induces IgG2a

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or IgG2c antibodies against ROP2. Both ROP2-speci®c IgG1 and IgG2a were also demonstrated by Western blot with parasite lysate (data not shown). To examine whether the IgG1 and IgG2a antibodies induced by DNA immunization appeared simultaneously or sequentially, sera were taken 2 weeks after each immunization and were analyzed by ELISA. As shown in Fig. 4, CBA/J mice immunized with the pcDNA3/ROP2 plasmid developed low levels of antibodies after the second immunization and the levels increased only after the third immunization; IgG1 and IgG2a antibodies appeared according to the same ki-

netics. BALB/c mice immunized with the pcDNA3/ ROP2 plasmid developed antibodies only after the second immunization and the level did not signi®cantly rise after the third immunization; comparable results were obtained for IgG1 and IgG2a antibodies. In C57BL/6 mice, anti-ROP2 antibodies appeared after the ®rst immunization and reached their maximum level after the second immunization. IgG1 antibodies followed the same kinetics, while IgG2c displayed a slight delay. These results show that IgG1 and IgG2a antibodies are generated simultaneously in mice immunized with the pcDNA3/ROP2 plasmid. Mouse challenge In order to test whether DNA immunization generated a protective immunity against T. gondii, immunized mice were challenged with tachyzoites of the virulent RH strain. As can be seen in Fig. 5, all DNA-immunized mice of the three strains died after the challenge. Nevertheless, in the case of the BALB/c strain there was a slight delay (2 days) before death in pcDNA3/ ROP2-immunized mice, when compared to pcDNA3immunized animals; and this di€erence was statistically signi®cant (P ˆ 0.04). As expected, ts-4-vaccinated mice of the BALB/c strain were protected against the challenge, while ts-4-vaccinated C57BL/6 mice only displayed a delay in the time until death.

Discussion

Fig. 4 Kinetics of anti-ROP2 antibody production in mice immunized with plasmid DNA. Groups of ®ve mice of the CBA/J, BALB/c, and C57BL/6 strains were immunized with pcDNA3 or pcDNA3/ ROP2. Sera were obtained before the ®rst immunization and 2 weeks after each immunization, and were tested for the presence of antiROP2 IgG (d), IgG1 (s), and IgG2a (h) or IgG2c (n), by ELISA. For CBA/J and BALB/c strains, each point represents the mean OD at 450 nm ‹ SD of ®ve mice; and for C57BL/6, each point represents the mean OD at 450 nm ‹ SD of the two mice which seroconverted. In all cases, sera were tested in duplicate at a 1/1,000 dilution. Values obtained with the sera of pcDNA3-immunized mice were always <0.12

In the present study, we show that DNA immunization using the ROP2 gene of Toxoplasma gondii generates a humoral immune response against the protein in mice. We observed that mice from the three strains tested (BALB/c, CBA/J, and C57BL/6) seroconverted, although an heterogeneous response was observed among mice of the same strain; and this variability was observed in repeated immunization experiments. Variations in antibody responses were also observed by other authors in inbred strains (Ho€man et al. 1994; Haddad et al. 1997; He et al. 1997; Peet et al. 1997; Moynier et al. 1998) and they have been attributed to di€erential transfection eciencies in individual mice (Haddad et al. 1997). Variability in antibody levels were also observed in ts-4-vaccinated mice. Doses as low as 1 lg plasmid DNA/mouse have been reported to be sucient to induce an immune response, but we required >50 lg of plasmid DNA to induce a response in most animals. The high dose required is probably related to the low expression level of the protein, because we were unable to detect the expression of ROP2 in several eukaryotic cell lines (CHO, P815, L929, and EL4) transfected in vitro with the pcDNA3/ROP2 plasmid (data not shown). However, when a sucient dose of plasmid (100 lg) was injected into the mice, the level of antibodies induced was higher than that induced

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Fig. 5 Survival of mice immunized with plasmid DNA or ts-4 after challenge. Groups of ®ve mice of the CBA/J, BALB/c, and C57BL/6 strains were injected with pcDNA3 (s), pcDNA3/ROP2 (d), ts-4 tachyzoites (j), or DPBS (h) and were challenged with tachyzoites of the RH strain as described in Materials and methods. Results are expressed as percentage of surviving mice. CBA/J mice immunized with pcDNA3 and those immunized with pcDNA3/ROP2 died exactly at the same time; and the curves are superimposed on the graph. CBA/J mice vaccinated with ts-4 and challenged with the RH strain were not included in this experiment

by vaccination with live tachyzoites of the ts-4 strain. Nevertheless, we cannot disregard the fact that the level of expression was too low to induce a T-cell-dependent protective immune response. We were interested in examining whether plasmid immunization generated a TH1 and/or a TH2 immune response. Although cellular immunity could not be studied, we determined the isotype of the anti-ROP2 antibodies as an indicator of the type of immune response, since it has been shown that IFN-c secreted by TH1 cells favors the IgG2a switch, while IgG1 isotypeswitching is regulated by IL-4 produced by TH2 cells (Mosmann and Co€man 1989). In the case of C57BL/6 mice, we measured the IgG2c isotype because, in this strain, the gene for IgG2a is deleted and instead the socalled IgG2c isotype, derived from a separate gene and

not detected by commercial anti-IgG2a antibodies, is expressed (Martin et al. 1998). We found that CBA/J and BALB/c mice developed both IgG1 and IgG2a antibodies both against the recombinant and against the natural ROP2 protein in response to DNA immunization, and C57BL/6 mice developed both IgG1 and IgG2c. Although it was initially reported that DNA vaccination predominantly induces the development of IgG2a (TH1 response) when the DNA is injected in saline (Raz et al. 1996; Feltquate et al. 1997), obtaining a mixed IgG1 and IgG2a response to DNA immunization (as observed in the present work) was also reported recently by other authors for other antigens (Boyle et al. 1997; Leitner et al. 1997; Grifantini et al. 1998; Haddad et al. 1998; Kang et al. 1998; Li et al. 1998; Nass et al. 1998); and it seems to be a common observation. Even while our work was being submitted for publication, DNA immunization against T. gondii was reported for SAG1 (Nielsen et al. 1999; Angus et al. 2000), GRA1, GRA7, and ROP2 (Vercammen et al. 2000). In all cases, the antibodies generated were also of both IgG1 and IgG2a isotypes. We also observed a variability in the response among the animals of the three strains. A few mice produced both isotypes, while others developed predominantly one of the two isotypes. Similar variability between individual mice in the isotype response to intramuscular plasmid vaccination was also reported by other workers (Nass et al. 1998). Multiple parameters can in¯uence the type of immune response obtained by DNA immunization. It has been reported that plasmid DNA contains immunostimulating sequences (CpG) able to induce a TH1-immune response by inducing cells to produce IL-12 (Sato et al. 1996); in fact, the pcDNA3 plasmid used in our work contains two such immunostimulating sequences. It has been shown that both the level of expression of the protein and the di€erential cellular location of the DNA-expressed antigen can in¯uence not only the isotype of the antibodies generated (Boyle et al. 1997; Haddad et al. 1998) but also the strength of the immune response induced (Svanholm et al. 1999; Torres et al. 1999). Moreover, in the case of immunization with a protein antigen, the protein by itself can direct the type of immune response (Zhang and Mohapatra 1993; Milich et al. 1997). In fact, it has been suggested that the identity of antigen and the genetic background of the mice are the more important factors in determining the type of immune response (Pertmer et al. 1996; Haddad et al. 1998). In contrast to the anti-ROP2 immune response induced by DNA immunization, which was characterized by both IgG1 and IgG2a antibodies, the immune response induced by vaccination with ts-4 was characterized by the presence of anti-ROP2 antibodies belonging only to the IgG2a subclass. This type of response is not restricted to the ts-4 strain, since mice of the same strains chronically infected with the Me49 strain of T. gondii also produced antibodies against ROP2 belonging

77

mainly to the IgG2a isotype (data not shown). Thus, the type of immune response induced by DNA immunization against ROP2 di€ers from that obtained by infection with the parasite. Multiple components of the parasite may be responsible for this di€erence. When we studied the kinetics of development of both isotypes in the BALB/c and CBA/J strains, we observed that although the anti-ROP2 antibodies appeared ®rst in BALB/c mice, both isotypes were generated at similar levels at the same time for both strains of mice. In C57BL/6 mice, IgG1 antibodies appeared after the ®rst immunization, while IgG2c appeared after the second immunization. These results suggest that, in our model, both TH1 and TH2 cells were generated simultaneously. One of the main goals of the plasmid immunization is the induction of a protective immune response against the pathogen. In our case, we tested whether the DNAimmunized mice were protected by challenging the animals with the highly virulent RH strain. pcDNA3/ ROP2-immunized CBA/J and C57BL/6 mice died at the same time as the pcDNA3-immunized control mice, while pcDNA3/ROP2-immunized BALB/c mice died 2 days later than the pcDNA3-immunized control mice. The low level of protection observed in this experiment could be due to the high virulence of the RH strain. In fact, Angus et al. (2000) recently demonstrated that mice immunized with plasmid DNA coding for SAG1 were protected against a challenge with an avirulent strain, but not against the RH strain. Vercammen et al. (2000) demonstrated that immunization of C3H mice with a truncated ROP2 gene induced partial protection against an avirulent strain of T. gondii; however, neither the BALB/c nor the C57BL/6 strain was protected. Immunization with truncated GRA2 and GRA7 genes also induced partial protection in C3H mice (Vercammen et al. 2000). Preliminary experiments in our laboratory showed that CBA/J mice immunized with pcDNA3/ ROP2 were partially protected against a challenge with the avirulent cyst-forming Me49 strain of T. gondii, but the C57BL/6 mice were not (Saavedra et al., unpublished data), observations that agree with the results obtained by Vercammen et al. (2000). An important di€erence between our work and that of Vercammen et al. (2000) is that we used the complete ROP2 gene cloned in the pcDNA3 plasmid, in which the ROP2 protein is expressed naturally with its signal peptide, while they deleted the sequence coding for the ROP2 signal peptide and cloned the gene in a plasmid in frame with the tPA signal peptide, which enables the ROP2 protein to be secreted. As mentioned before, manipulation of a gene and cloning in a plasmid that leads to the secretion of the protein can modify the type and strength of the immune response (Boyle et al. 1997; Haddad et al. 1998; Svanholm et al. 1999; Torres et al. 1999). In contrast to animals immunized with plasmid DNA, BALB/c mice vaccinated with the ts-4 strain were protected against challenge with the RH strain, and ts-4vaccinated C57BL/6 mice displayed a delay in time until

death. The protection achieved by vaccination with ts-4 could be due to the induction by the parasite of an immune response to antigens distinct from ROP2 and/or the induction of a di€erent type of immune response. In fact, mice vaccinated with the ts-4 strain developed IgG2a but not IgG1 against ROP2, while mice immunized with plasmid DNA developed both isotypes. This suggests that a TH1 response against ROP2 was generated by vaccination with ts-4, while a mixed TH1/TH2 response was generated by plasmid immunization. In conclusion, we have demonstrated that immunization with the ROP2 gene of T. gondii generates a humoral immune response against ROP2 in three di€erent strains of mice. In BALB/c mice, the induced response was only marginally protective against a challenge with a virulent strain. Cell-mediated immunity and resistance to a challenge with a less virulent strain remains to be evaluated. Acknowledgements We thank Luis Gerardo Molina for helping with the production and puri®cation of recombinant ROP2, Dr. E. Pfe€erkorn for kindly providing the ts-4 strain, and Dr. Jean FrancËois Dubremetz for providing the mAb 4A7. This work was supported by grant 0643P/M from CONACyT. The experiments described comply with the current laws of Mexico.

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