• Open Access

A chloroplast-derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice

Authors


  • These two authors contributed equally to the work.

Correspondence (Tel +54 02241 430323; email mclemente@intech.gov.ar)

Summary

The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti-Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34-kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 μg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN-γ, IL-4 and IL-10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4-specific serum antibodies and secretion of IFN-γ, IL-4 and IL-10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads.

Introduction

The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds (Tenter et al., 2000). Toxoplasma gondii is recognized as a category B priority pathogen by the National Institute of Health, Bethesda, USA. In humans, T. gondii infection is widespread, and for most people, the parasite causes an asymptomatic infection. However, T. gondii can cause complications in pregnant women and immunodeficient individuals (Mitchell et al., 1990; Luft and Remington, 1992; Rorman et al., 2006). Also, recent studies have shown that chronic T. gondii infections can induce neuropsychiatric disorders such as schizophrenia (Flegr, 2007; Pedersen et al., 2011). Transmission to humans is either through consumption of food contaminated with tissue cysts and meat products from infected animals or by ingestion of oocysts released in the faeces of infected cats (Hill and Dubey, 2002). In veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock (Dubey, 2004, 2009). Indeed, millions of productive lambs are lost through this infectious disease (Dubey, 2009). On the other hand, treatment of toxoplasmosis is difficult due to the toxic effects of available drugs, and re-infection also occurs rapidly. Thus, the development of either an effective vaccine or new anti-Toxoplasma drugs would be of great value to human and veterinary medicine (Henriquez et al., 2010).

In recent years, significant advances have been made in identifying possible new vaccines that can induce a protective immune response against this parasite. For example, excretory/secretory antigens of T. gondii have been shown to play an important role in stimulating immune responses to T. gondii (Cesbron-Delauw, 1994). The main components of the excretory/secretory fraction are the dense granule (GRA) proteins (Cesbron-Delauw, 1994). In particular, GRA4 appears as an interesting component for the development of an anti-Toxoplasma multiantigenic vaccine, which could be based either on DNA or on recombinant proteins (Martin et al., 2004; Mevelec et al., 2005; Zhang et al., 2007; Sánchez et al., 2011). Its sequence contains a linear epitope that is highly antigenic (Mevelec et al., 1998). Indeed, GRA4 is an effective promoter of the expression of IgA and IgG antibodies (Mevelec et al., 1998; Nigro et al., 2003; Altcheh et al., 2006). Also, immunization with DNA or recombinant GRA4 is able to induce a cellular response with accumulation of IFN-γ (Desolme et al., 2000; Mevelec et al., 2005; Sánchez et al., 2011). On the other hand, oral immunization of C57BL/6(H-2d) mice with truncated soluble forms of GRA4 in association with cholera toxin induces a significant Th2-like mucosal response and partial resistance to oral infection with T. gondii (Mevelec et al., 1998). These results indicate that GRA4 is a potential candidate for oral vaccination against T. gondii, able to induce both mucosal and systemic immune responses.

The production of plant-derived vaccines has been widely assayed in the last 20 years. Several antigens from human and animal pathogens have been correctly expressed and shown to produce a specific humoral response, and in some cases, a protective response against infection in murine models (Kong et al., 2001; Clemente et al., 2005; Gómez et al., 2008; Santi et al., 2008; Kostrzak et al., 2009; Zhang et al., 2009; Laguía Becher et al., 2010; Gonzalez-Rabade et al., 2011). One of the main advantages of plants is that their tissues provide a natural environment for antigen encapsulation, which protects the antigen from degradation (Limaye et al., 2006; Hayden et al., 2012). In this way, when the plant tissue is digested, a sufficient quantity of antigen can be captured from the mucous membranes and stimulate an immune response (Berinstein et al., 2005; Kapusta et al., 2010). For this reason, plants are an ideal vehicle for oral vaccine administration.

Previously, a His-tagged truncated version of the T. gondii GRA4 antigen (residues 163–345), transiently expressed in tobacco leaves using vacuum agroinfiltration, has been found to be able to react with seropositive human sera (Ferraro et al., 2008). Compared to nuclear transformation, chloroplast transformation offers many advantages, including high levels of transgene expression likely due to a high copy number, absence of epigenetic effects, transgene containment via maternal inheritance and multigene expression in a single transformation event (Lössl and Waheed, 2011). In addition, tobacco chloroplast–derived vaccines are able to induce neutralizing antibodies against human or animal infections (Molina et al., 2005; Fernández-San Millán et al., 2008). More importantly, proteins expressed in plastids have been found to be stable in the gut and efficiently transported to the circulatory system (Limaye et al., 2006). For these reasons, the main goal of this study was to explore the expression of T. gondii GRA4 antigen by chloroplast transformation in tobacco plants, and to determine the ability of this antigen to induce protection in a T. gondii murine infection model when administered orally without any adjuvant.

Results

Integration of the GRA4 sequence into the tobacco plastid genome

The GRA4 sequence (corresponding to amino acids 183–345; Mevelec et al., 1998) was expressed from the plastid constitutive promoter and 5′UTR of the psbA gene and translationally fused to the 3′-end of the first 45 nucleotides of the GFP sequence, which has been shown to be a desirable downstream box (DB) to improve protein accumulation in tobacco chloroplasts (Farran et al., 2010). The chimeric gene was cloned into the chloroplast transformation vector pAF-TDHIS for insertion between the trnI and trnA genes in the tobacco plastome (Figure 1a). This vector contains a chimeric aadA gene as the selectable marker, which provides spectinomycin resistance for selecting stable transformants. Additionally, the pAF-TDHIS vector allows the translational fusion of the transgene to the 42-amino acid tetramerization domain (TD) from the human transcription factor p53 (Ortigosa et al., 2010) and introduces a C-terminal 7xHis tag.

Figure 1.

Integration of chimeric GRA4 gene into the plastid genome and homoplasmy verification. (a) Map of the wild-type and GRA4-transformed plastid genomes. The transgenes are targeted to the intergenic region between trnI and trnA. The Gra4 expression cassette includes the psbA promoter and 5′-untranslated region (PpsbA) and the psbA terminator (TpsbA) from tobacco. A downstream box (DB) with the first 15 amino acids of the soluble modified GFP protein was used. The GRA4 sequence was translationally fused to the 42 amino acid tetramerization domain (TD) from the human transcription factor p53. The selectable marker gene aadA is driven by the ribosomal RNA operon promoter (Prrn). The expected sizes of DNA fragments in Southern blot analysis with the restriction enzyme BglII are indicated. The 0.8-kb fragment (P1) of the targeting region for homologous recombination was used as probe. Arrows indicate the direction of transcription. (b) Southern blot analysis of three independent transplastomic tobacco lines is shown. Total DNA was digested with BglII and probed with P1.

The pAF-TDHIS-GRA4 construct was introduced into tobacco (cv. Petite Havana) plastids by biolistic transformation (Daniell, 1997). The stable integration of the foreign gene into the chloroplast genome and the homoplasmy of the transformed plants were confirmed by Southern blot analysis. After two rounds of selection, total leaf DNA from the regenerated plants was digested with the BglII restriction enzyme, which cuts in two positions flanking the insertion site and in one position in the psbA promoter (Figure 1a). The 0.8-kb probe P1, which is homologous to the flanking regions trnI and trnA (Figure 1a), was used. Two of the three transplastomic lines analysed showed the expected hybridization pattern and revealed the absence of residual copies of the wild-type plastome (Figure 1b, lanes 1 and 3). To ultimately confirm homoplasmy, seeds from the T0 generation were germinated in vitro on spectinomycin-selective medium. Lack of segregation for spectinomycin resistance in the T1 generation demonstrated homoplasmy (data not shown). Line 1 was selected for further studies.

Expression and quantification of the GRA4183-345 protein

The recombinant protein GRA4 in transformed chloroplasts (chlGRA4) was detected by Western blot with an anti-GRA4 polyclonal antibody. Leaves from four plants were analysed (Figure 2a). Each sample showed a specific 34-kDa band (the GRA4 antigen fused to the DB and the TD) (Figure 2a). This band showed an electrophoretic mobility higher than the E. coli-derived and purified GRA4 (rGRA4) as a consequence of the translational fusions described above (Figure 2a). ChlGRA4 accumulation in these plants was approximately 6 μg/g of fresh weight (FW) (equivalent to 0.2% of total protein) (Figure 2a), as estimated by Western blot analysis using rGRA4 (Martin et al., 2004), previously quantified. No protein was detected in samples extracted from wild-type control plants.

Figure 2.

Chloroplast-derived GRA4 protein expression. (a) Western blot indicating the expression of chlGRA4 from four independent transplastomic plants. GRA4 polyclonal antibody specific for rGRA4 protein was used. Wild-type (WT) protein served as the control. Lanes 1, 2, 3 and 4: Protein samples from four independent transplastomic plants (transgenic plants 1, 2, 3 and 4, respectively) extracted from 100 mg of leaf fresh weight. 20 and 40 : 20 and 40 ng rGRA4, respectively. (b) Western blot analysis of chlGRA4 protein expression in soluble (S) and insoluble (IN) fractions. Proteins extracted from 100 mg of leaf fresh weight of homogenous stocks of five transplastomic (chlGRA4) plants and wild-type (WT) plants 45 and 90 days after transplanting were loaded in lanes. 100 : 100 μg of rGRA4. Coomassie blue-stained gels were used as controls of the protein loading. M: prestain molecular weight protein marker (Fermentas). The arrow indicates the chlGRA4 protein.

The effect of plant age on chlGRA4 protein expression was analysed in plants growing in the glasshouse at 45 and 90 days. Soluble and insoluble proteins were extracted from fully developed leaves, and chlGRA4 expression was analysed by Western blot (Figure 2b). The specific 34-kDa band was observed in 45 and 90-day-old plants mainly in the insoluble fraction, and no degradation products were detected (Figure 2b). The higher expression level after 90 days of culture in the glasshouse in relation to that after 45 days indicates that chlGRA4 is stable in the stroma of the chloroplast.

Leaves from chlGRA4 over-expressing tobacco plants were excised 120 days after transplanting, lyophilized, ground to powder and stored at 4 °C. ChlGRA4 accumulation in lyophilized plant leaf stocks was estimated by Western blot analysis using a dilution series (10, 20, 40, 60 and 80 ng) of rGRA4 (Martin et al., 2004), previously quantified as reference (Figure S1). The amount of chlGRA4 protein accumulated in the chloroplast was estimated to be approximately 12 μg/g of lyophilized weight. Lyophilized material was chosen for oral administration to mice.

Immunogenicity of chloroplast-derived GRA4

Considering that T. gondii infects the host through the gut mucosa, the efficacy of oral immunization with transplastomic plants expressing chlGRA4 was analysed. C57BL/6(H-2d) mice were immunized orally with leaf extracts from chlGRA4 plants (GRA4 group), leaf extracts from wild-type plants (control group) or PBS buffer (PBS group). To determine whether this vaccination strategy was able to promote protection against T. gondii infection, 2 weeks after the last boost, mice were challenged by intragastric gavage with a nonlethal dose of Me49 tissue cysts and individual parasitemia levels were assessed 1 month later. An important decrease in the brain cyst burden (59%) was observed as compared to the control and PBS groups (Figure 3). No differences in the parasitemia levels were observed between these two control groups (Figure 3).

Figure 3.

Protection of C57BL/6(H-2d) mice against Toxoplasma infection. Eight- to ten-week-old mice (eight mice per group) were immunized on days 0, 7, 14, 21 and 28 by oral administration. Two weeks after the last boost, mice were challenged by gavages with 20 cysts of the Me49 strain (LD50). Thirty days after the challenge, the number of brain cysts in mice was determined. Control: mice vaccinated with leaf extracts from wild-type plants; GRA4: mice vaccinated with leaf extracts from chlGRA4 plants; PBS: mice vaccinated with buffer PBS. Statistical analysis was performed by one-way analysis of variance (anova) using the Bonferroni's multiple comparison test. Results are expressed as the means ± SEM and represent one of two similar experiments. Different letters indicate statistically significant differences (P < 0.001 GRA4 vs. control and PBS).

Systemic immune response generated by chlGRA4

To examine whether leaf extracts from chlGRA4 plants administered orally were able to elicit a systemic immune response, blood samples were obtained 14 days after the immunization schedule was completed and the specific IgG antibodies against rGRA4 were determined by direct ELISA (Figure 4). As shown in Figure 4, GRA4-specific serum IgG antibodies were detected in all mice receiving chlGRA4 with titres of 1000 (Figure S2), while mice vaccinated with leaf extracts from wild-type plants or PBS did not display significant titres. To investigate the dominant Th pattern stimulated following immunization, GRA4-specific IgG isotypes were determined by direct ELISA. Significant levels of IgG2b and IgG1 were observed only in chlGRA4-vaccinated mice with titres of 500 for IgG2b and 250 for IgG1 (Figure 4b, Figure S2). Then, the ability of the vaccination protocol to promote antigen-specific cellular response was assessed in the spleen. The concentrations of Th1- or Th2-associated cytokines were quantified in supernatants of spleen cells restimulated in vitro with excretory-secretory antigens (ESA). Spleen cells of mice immunized with chlGRA4 produced significant levels of IFN-γ, IL10 and IL-4 when compared to the control and PBS groups (Figure 5). These results show that oral immunization with leaf extracts from chlGRA4 plants induces a mixed Th1/Th2 response at the systemic level.

Figure 4.

Determination of specific anti-chlGRA4 humoral response in orally vaccinated C57BL/6(H-2d) mice. Two weeks after the immunization schedule was completed, blood samples (eight mice per group) were obtained and anti-GRA4-specific antibodies were determined by ELISA. (a) Specific total IgG and (b) IgG isotype in sera from mice vaccinated with leaf extracts from wild-type plants (control), leaf extracts from chlGRA4 plants (GRA4) or with buffer PBS (PBS) were determined in duplicate. Mouse sera were diluted 1 : 100 for GRA4-specific antibodies determination (nonsaturating dilutions). Results are expressed as the mean of the OD630 ± SEM and are representative of two experiments. Statistical analysis was performed by one-way analysis of variance (anova) using the Bonferroni's multiple comparison test. Different letters indicate statistically significant differences (P < 0.001 GRA4 vs. control and PBS). Capital letters correspond to statistical analysis of IgG2b isotype. Lower case letters correspond to statistical analysis of IgG and IgG1 isotype.

Figure 5.

Cytokine productions by spleen cells in orally vaccinated C57BL/6(H-2d) mice. Forty-five days after the immunization schedule was completed, spleen and lymph node cells were isolated from mice (five mice per group) and stimulated in vitro with excretory-secretory antigens (ESA) (10 μg/mL). Values for IFN-γ, IL-4 and IL-10 were measured at 72 h of culture. Results are mean cytokine concentrations in spleen cells from the five mice of each experimental group. Control: mice vaccinated with leaf extracts from wild-type plants; GRA4: mice vaccinated with leaf extracts from chlGRA4 plants; PBS: mice vaccinated with buffer PBS. Results from one of two similar experiments are shown and are expressed as the mean ± SEM Statistical analysis was performed by one-way analysis of variance (anova) using the Bonferroni's multiple comparison test. Different letters indicate statistically significant differences (P < 0.001 GRA4 vs. control and PBS).

Mucosal immune response generated by chlGRA4

To examine whether oral administration of leaf extracts from chlGRA4 plants is able to induce antibody-specific response at mucosal levels, intestinal lavages were collected 45 days after the last immunization, and the presence of GRA4-specific secretory immunoglobulin A (sIgA) was evaluated by ELISA. ChlGRA4-vaccinated mice elicited significant levels of GRA4-specific sIgA compared to the control and PBS groups (Figure 6). Then, the ability of this oral vaccination protocol to promote antigen-specific cellular response was assessed in gut-associated lymphoid tissue (GALT). Forty-five days after the immunization schedule was completed, we estimated the concentration of Th1- and Th2-associated cytokines by capture ELISA in supernatants of mesenteric lymph node cells stimulated in vitro with ESA. A significant increase in the amount of secreted IFN-γ, IL10 and IL-4 was observed in mice immunized with chlGRA4 when compared to the control and PBS groups (Figure 7). These results show that a vaccination protocol based on oral administration of leaf extracts from chlGRA4 plants triggers a strong mucosal response characterized by the presence of GRA4-specific sIgA and secretion of Th1- and Th2-associated cytokines in the GALT.

Figure 6.

Determination of specific anti-chlGRA4 sIgA in intestinal lavages of orally vaccinated mice. Forty-five days after the immunization schedule was completed, intestinal lavage samples (five mice per group) were obtained and antibody concentration was determined by ELISA. Results are expressed as the percentage of GRA4-specific IgA with respect to the total IgA present in the sample ± SEM and are representative of two experiments. The total mean IgA values ± SEM for the different groups were as follows: GRA4, 32.58 ± 2.52 ng/mL; control group, 11.22 ± 1.60 ng/mL; and PBS group, 19.54 ± 1.58 ng/mL. Statistical analysis was performed by one-way analysis of variance (anova) using the Bonferroni's multiple comparison test. Different letters indicate statistically significant differences (P < 0.001 GRA4 vs. control; P < 0.01 GRA4 vs. PBS).

Figure 7.

Cytokine productions by lymph node cells in orally vaccinated C57BL/6(H-2d) mice. Forty-five days after the immunization schedule was completed, spleen and lymph node cells were isolated and stimulated in vitro with excretory-secretory antigens (ESA) (10 μg/mL). Values for IFN-γ, IL-4 and IL-10 were measured at 72 h of culture. Results are mean cytokine concentrations in lymph node cells from five mice per experimental group. Control: mice vaccinated with leaf extracts from wild-type plants; GRA4: mice vaccinated with leaf extracts from chlGRA4 plants; PBS: mice vaccinated with buffer PBS. Results from one of two similar experiments are shown and are expressed as the mean ± SEM Statistical analysis was performed by one-way analysis of variance (anova) using the Bonferroni's multiple comparison test. Different letters indicate statistically significant differences (P < 0.001 GRA4 vs. control and PBS).

Discussion

A live toxoplasma vaccine based on the attenuated S48 strain of the parasite has been registered and is currently used in veterinary medicine (Hiszczynska-Sawicka et al., 2011). However, it is not suitable for humans due to the risks for breakthrough infection (Hiszczynska-Sawicka et al., 2011). Therefore, new approaches to vaccine development using subunit vaccines based on T. gondii antigens have been widely initiated (Jongert et al., 2009).

Plant expression systems have been intensively studied to provide an attractive alternative for oral vaccine production (Paul and Ma, 2010). In the present work, the GRA4 antigen of T. gondii was expressed in tobacco plants. Chloroplast transformation was carried out to improve the expression level of the heterologous protein. Previously, the yields of GRA4 in tobacco infiltrated leaves were in the order of 0.01% of total soluble protein, which represents around 0.2 μg/g of fresh weight (Ferraro et al., 2008). Here, the chlGRA4 expression levels in the transplastomic plants were up to 6 μg/g of fresh weight (or 0.2% of total protein). As expected, chloroplast transformation allowed a significant 30-fold increase in GRA4 protein accumulation in the plant. In addition, recombinant protein levels were stable over the whole life cycle of the plants. However, chlGRA4 levels were not as high as the usual figures in plastid transformation. Given that chlGRA4 seems to be stable in the chloroplast, low expression levels could be due to an inefficient translation initiation, despite the use of a DB that has been proven to be useful for protein accumulation (Farran et al., 2010). The combination of a DB with a particular ORF may alter mRNA folding and thus affect translation mediated by the DB, suggesting that empirical optimization is required for each ORF. Gray et al. (2011) demonstrated the specific effects of three DBs in the expression level of beta-glucuronidase in tobacco chloroplasts.

GRA4 is a target antigen for both mucosal and systemic immune responses, and therefore, it is a valuable candidate to be used in oral vaccination strategies (Bout et al., 1995; Mevelec et al., 1998). To our knowledge, this is the first report that tested the immunogenicity of GRA4 antigen in an oral vaccination approach without the use of any adjuvant. It was previously shown that oral administration of chloroplast-derived vaccine antigens produced in tobacco and lettuce conferred combined protection against malaria and cholera (Davoodi-Semiromi et al., 2010).

Oral administration of adjuvant-free chlGRA4 was enough to elicit an immune response capable of inducing partial protection against T. gondii infection measured as a marked reduction in brain cyst loads. Similar results were obtained with the rGRA4 protein administrated intramuscularly in combination with adjuvant-like alum (Martin et al., 2004) or CpG (Sánchez et al., 2011). On the other hand, a protocol based on priming with plant-derived T. gondii SAG1 protein by oral administration and intradermal (i.d.) boosting with rSAG1 expressed in bacteria (Laguía Becher et al., 2010) elicited a protection (50%) similar to that obtained with chlGRA4 (59%). Therefore, the inclusion of exogenous adjuvants or a rGRA4 i.d. boost or GRA4-SAG1 mixed plant materials could be implemented to achieve a higher level of protection against Toxoplasma infection.

Our results also showed that the partial protection against T. gondii infection was a consequence of the development of both an effective mucosal and systemic immune response. Specific mucosal sIgA and serum IgG were produced in mice immunized with chlGRA4. The high levels of serum IgG would be indicating a mucosal origin of these antibodies, which was confirmed by an effective activation of mucosal sIgA. Particularly, sIgA provide immune protection against Toxoplasma in mucosal tissues. However, mucosal defences can also be mediated by cytokines (Dimier-Poisson et al., 2003). Given that the initial activation of T cells probably occurs in Peyer's patches and mesenteric lymph nodes, we investigated the induction of Th1- and Th2 cytokines in the local lymph nodes. The Th1/Th2 cytokine levels in chlGRA4-vaccinated mice were higher than in the other two control groups. These results suggest that the antigen expressed in the chloroplasts would have been recognized by the antigen-presenting cells and presented in the gut-associated lymphoid tissues, resulting in the activation of T helper cells.

chlGRA4 vaccination showed to elicit a mixed IgG2b/IgG1 systemic response with predominance of IgG2b. However, protection against T. gondii infection is mainly attributed to a cell-mediated systemic immunity. IFN-γ has been shown to be a major mediator of resistance against toxoplasmosis (Suzuki et al., 1988; Gazzinelli et al., 1991). Nevertheless, the absence of some type 2 cytokines such as IL-4 or IL-10 can result in an increase in the immunopathology or mortality in mice (Gazzinelli et al., 1996; Henriquez et al., 2010). In this context, restimulation of spleen cells from the chlGRA4-vaccinated group led to the production of IFN-γ, IL-4 and IL-10. The higher levels of production of IFN-γ compared with those of IL-4 and the higher levels of serum IgG2b over IgG1 indicate that a Th1-mediated predominant T-cell response was generated. The presence of large amounts of IL-10 in restimulated splenocytes from chlGRA4-immunized mice suggests that this vaccination protocol also elicits a feedback modulation of this predominant Th1 response. Finally, oral chlGRA4 vaccination also induced the production of IL-4, a Th2 cytokine, suggesting a B-cell contribution to the protection obtained after T. gondii infection in vaccinated mice. These results are supported with the T and B epitopes mapped in the GRA4 sequence (Mevelec et al., 1998). However, additional adjuvants present in the plant material could be contributing with the modulation of the immune response observed in mice vaccinated with chlGRA4 (Gonzalez-Rabade et al., 2011; Corigliano et al., 2011).

In conclusion, our findings suggest that chloroplast-derived GRA4 is able to induce a protective immune response against Toxoplasma infection in mice that correlates with a mucosal and systemic balanced Th1/Th2 response. This indicates that chlGRA4 displays a good potential for toxoplasmosis control by oral vaccination. Moreover, plant expression of GRA4 as well as SAG1 (Clemente et al., 2005; Laguía Becher et al., 2010) provides an excellent possibility for the development of a multicomponent vaccine against Toxoplasma.

Experimental procedures

Genetic engineering of the chloroplast expression vectors

The GRA4 sequence was amplified by PCR from the pQ-GRA4 vector (Martin et al., 2004) using the primers 5′-cccgggCTGCAGTTGAGACAACAGTC-3′ (upstream) and 5′-ctcgagCTCTTTGCGCATTCTTTCC-3′ (downstream), which introduces the SmaI and XhoI sites, respectively. The SmaI-XhoI GRA4 fragment was cloned into the pSK-GFP:EDA plasmid (Farran et al., 2010), which allows the translational fusion of GRA4 to the first 15 amino acids of the soluble modified GFP protein (GenBank accession no. U_70495). The resulting pSK-GFP:GRA4 vector was digested by SalI and XhoI, and the generated fragment, which also contains the promoter and 5′UTR of the tobacco psbA gene, was introduced into the tobacco chloroplast transformation vector pAF-TDHIS (Ortigosa et al., 2010) previously digested by SalI, to produce pAF-GRA4.

Bombardment and regeneration of chloroplast transgenic plants

Gold microprojectiles coated with pAF-GRA4 plasmid DNA were bombarded into in vitro grown tobacco (Nicotiana tabacum var. Petite Havana) leaves using the PDS1000/He (Bio-Rad, Hercules,California) biolistic device, as previously described (Daniell, 1997). After bombardment, leaves were incubated in the dark for 2 days at 28 °C. Leaves were then cut into small pieces (approximately 5 × 5 mm) and placed adaxial side up on regeneration medium (RMOP) in Magenta vessels (Sigma, St. Louis, MO) containing 500 mg/L spectinomycin dihydrochloride as the selecting agent. The regenerated spectinomycin-resistant shoots were subjected to a second round of selection under the same conditions. These regenerated plants were then transplanted and grown in a greenhouse for homoplasmy confirmation and seed production.

Southern blot analysis

Total plant DNA (10 μg) was digested with BglII, separated on a 0.8% (w/v) agarose gel and transferred to a nylon membrane. The digestion by BglII and BamHI of the chloroplast vector generated a 0.8-kb probe (P1) homologous to the flanking sequences (Figure 1a). Probe labelling and hybridization were performed using the chemiluminescent AlkPhos direct labelling and detection system (GE Healthcare, Buckinghamshire, UK). After homoplasmy confirmation by Southern analysis, seeds were harvested and germinated in vitro on spectinomycin-selective medium. The T1 seedlings were isolated and cultured for 4 weeks in MS medium (Sigma). Finally, plants were transferred to pots and grown in a greenhouse for further analysis.

Protein extraction and Western blot analysis

Leaf samples (100 mg) were ground in liquid nitrogen, homogenized in Laemmli buffer 2× (Laemmli, 1970) and the extract heated at 100 °C for 5 min. To separate soluble and insoluble protein fractions, the pulverized samples (100 mg) were homogenized in a soluble protein extraction buffer (20 mm NaPO4, 250 mm NaCl, 0.1% Triton X-100, Fernández-San Millán et al., 2007). After centrifugation at 10 000 g, the soluble fraction (supernatant) and insoluble fraction (pellet) were homogenized in Laemmli buffer 2×. Proteins were separated by SDS-PAGE (15% gel) and transferred onto polyvinylidene fluoride membranes (PVDF; GE Healthcare) using an Electro-transfer unit (Bio-Rad). The membranes were sequentially incubated with rabbit polyclonal anti-GRA4 antibody (1 : 500) and alkaline phosphatase conjugated goat anti-rabbit IgG (1 : 5000; Sigma). After washing, the reaction was developed by the addition of nitroblue tetrazolium/5-bromo- 4-chloro-3-indolyl phosphate (NBT/BCIP) substrate (Promega, Fitchburg, Wisconisn). Prestained protein (Fermentas, Burlington, ON, Canada) was included in Western blots as molecular weight markers. To estimate the GRA4 accumulation in tobacco leaves, E. coli-derived and purified GRA4 (rGRA4) of known concentration was also separated by SDS-PAGE (15% gel), detected by Western blot and used as a standard to build a calibration curve. Amounts of GRA4 in leaf extracts were determined by comparison with the rGRA4 calibration curve using the ‘Gel-Pro analyzer’ software (Media Cybernetics, Rockville, MD).

Animals

Female C57BL/6(H-2d) mice were bred and housed at the animal facilities of the Biotechnology Research Institute (IIB), National University of General San Martin (UNSAM), Buenos Aires, Argentina, and used at 8–10 weeks of age. All procedures requiring animals were performed in agreement with institutional guidelines and approved by the Independent Ethics Committee for the Care and Use of Experimental Animals of the National University of General San Martin (C.I.C.U.A.E., IIB-UNSAM, 09/2011).

Oral immunization of mice

In all cases, mice were deprived of food and water for 2 h prior to each immunization and were gavaged with 83 mg of lyophilized powder resuspended in a final volume of 200 μL of PBS 1× buffer. Mice from GRA4 group were orally immunized with five doses of transplastomic leaf extracts homogenized in PBS 1× buffer, without enrichment or purification, containing the equivalent of approximately 1 μg of chlGRA4 at 1-week intervals. The control group received five doses of wild-type leaf extracts homogenized in PBS 1× buffer, while the PBS group was immunized with five doses of PBS 1× buffer. Eight and five mice per experimental group were used in the challenge infection experiments and cytokine analysis, respectively.

Measurement of antibody responses

Two weeks after the last immunization, blood was collected from the tail vein and sera were stored at −20 °C until analysed for the presence of specific antibodies. Pre-immune serum samples were used as negative controls.

Antigen-specific antibodies were analysed by enzyme-linked immunosorbent assay (ELISA) as previously described (Clemente et al., 2005; Laguía Becher et al., 2010). Briefly, each well of 96-well microtitre plates (Immuno Plate Maxisorp; Nunc, Rochester, NY) was coated overnight at 4 °C with 5 μg/mL of rGRA4 diluted in 0.05 m carbonate buffer (pH 9.6). Rat anti-mouse immunoglobulin G (IgG)–horseradish peroxidase conjugate (1 : 5000) was used as a secondary antibody (Sigma), and rat anti-mouse IgG1- or IgG2a-horseradish peroxidase conjugates (1 : 3000) (Pharmingen) were used for isotype analysis. Immune complexes were revealed with trimethylbenzidine substrate (TMB, One-Step; Invitrogen, Carlsbad, CA), and optical density was read at 630 nm with an automatic ELISA reader (Dynatech MR4000). Sera were used at 1 : 50–1 : 8000 dilutions. To attain a comparative analysis of IgG profiles, serum samples were used at a 1 : 100 dilution. The negative control serum samples included in the assay displayed values lower than the cut-off.

On the other hand, 45 days after the last immunization, five mice were sacrificed and intestinal lavages were obtained by flushing the organs with several aliquots of PBS supplemented with 50 mm EDTA, 5% fetal bovine serum (FBS, GIBCO, Carlsbad, CA) and 10mm PMSF with a final volume of 1 mL. Lavages were then centrifuged to remove debris (10 min at 3000 g), and supernatant fluids were stored at −80 °C. The amount of total and GRA4-specific IgA present in intestinal lavages was determined by ELISA as previously described by Rharbaoui et al. (2002). To establish the IgA standard curve, plates coated with goat anti-mouse IgA (Pharmingen) as capture antibody were further incubated with serial dilutions of purified mouse IgA (Pharmingen). As secondary antibody, biotinylated goat anti-mouse IgA (Pharmingen) was used and plates were developed with trimethylbenzidine substrate (TMB, One-Step; Invitrogen) and optical density was read at 630 nm with an automatic ELISA reader (Dynatech MR4000). GRA4-specific IgA was analysed by a direct ELISA with GRA4 as the coating antigen, as described above, and specific amounts were calculated using the IgA standard curve described before. To compensate for variations in the efficiency of recovery of secretory antibodies between animals, the results were normalized and expressed as percentage of GRA4-specific IgA with respect to the total amount of IgA present in the sample.

Cytokine analysis

Spleens and mesenteric lymph nodes were aseptically removed from five mice per group 45 days after the last immunization.

Single-cell preparations were obtained by crushing spleens through stainless steel meshes followed by suspension in erythrocyte lyses buffer (0.15 m NH4Cl, 1.0 m KHCO3, 0.1 mm EDTA, pH 7.2). The viability of the cells used in the experiments was always higher than 80%, as measured by trypan blue exclusion (Sigma). The cells were then suspended in RPMI 1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% FBS (GIBCO), 2β-mercaptoethanol (final concentration, 5 × 10−5 m), penicillin (100 U/mL) and streptomycin (100 μg/mL). Supernatants from cultured splenocytes and lymph cells (10 × 106/mL) were collected after 72 h of stimulation with excretory-secretory antigens (ESA) of T. gondii (10 μg/mL) and stored at −70 °C. Cytokine production was measured in supernatants at 72 h (IFN-γ, IL-4 and IL-10) by capture ELISA commercial kits (Pharmingen; BD Biosciences, Sandiego, CA). The sensitivity limits for the assays were 20 pg/mL for IFN-γ, 50 pg/mL for IL-10 and 10 pg/mL for IL-4. At least two independent ELISAs were performed for each sample.

Challenge infection

Mice (eight per experimental group) were orally infected with 20 Me49 tissue cysts (sublethal dose) 2 weeks after the last immunization. Mice were observed daily for mortality. One month after the challenge, mice were sacrificed and their brains removed. Each brain was homogenized in 2 mL of PBS with a Dounce tissue grinder. The mean number of cysts per brain was determined by observation under an optical microscope, by counting four samples of 20 μL aliquots of each homogenized brain.

Statistical analysis

Statistical analysis was carried out with the Prism 5.0 Software (GraphPad, San Diego, CA) using one-way analysis of variance (anova). A Bonferroni's test was carried out to compare means. Values of P < 0.05 were considered significant.

Acknowledgements

This work was supported by PIP 168/2010 of the National Research Council (CONICET, Argentina), PICT 691/2007 of the National Agency for Promotion of Science and Technology (ANPCyT, Argentina) and by Grant BIO2005-00155 (Ministerio de Educación y Ciencia, España).

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