Comparison of immune response in sheep immunized with DNA vaccine encoding Toxoplasma gondii GRA7 antigen in different adjuvant formulations

Experimental Parasitology 124 (2010) 365–372

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Comparison of immune response in sheep immunized with DNA vaccine encoding Toxoplasma gondii GRA7 antigen in different adjuvant formulations _ Elzbieta Hiszczyn´ska-Sawicka a,c,*, Hong Li a, Janet Boyu Xu a, Gabriela Ole˛dzka b, Józef Kur c, Roy Bickerstaffe a, Mirosław Stankiewicz a a b c

Faculty of Agriculture and Life Sciences, Lincoln University, P.O. Box 84, Lincoln 7647, Canterbury, New Zealand Department of Medical Biology, Medical University of Warsaw, 73 Nowogrodzka, 02-018 Warsaw, Poland ´ sk University of Technology, Narutowicza 11/12, 80-952 Gdan ´ sk, Poland Department of Microbiology, Chemical Faculty, Gdan

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i n f o

Article history: Received 6 May 2009 Received in revised form 27 October 2009 Accepted 25 November 2009 Available online 3 December 2009 Keywords: Toxoplasma gondii Dense granule antigen GRA7 Sheep Adjuvants Immunization

a b s t r a c t Immunization with plasmid DNA, a relatively novel technique, is a promising vaccination technique. To improve the immune response by DNA vaccination various methods have been used, such as chemical adjuvants or immunomodulatory molecules formulated into microparticles or liposomes. The aim of this research is to evaluate the immune responses of sheep immunized with DNA plasmids encoding Toxoplasma gondii dense granule antigen GRA7 formulated into three different adjuvant formulations. Sixty sheep were injected intramuscularly with the DNA plasmids. Twelve received the liposome-formulated plasmid pVAXIgGRA7, 12 Emulsigen P formulated plasmid pVAXIgGRA7 and 12 Emulsigen D formulated plasmid pVAXIgGRA7. Twelve animals were used as a control and received the vector alone. All the animals were inoculated at week 0, and week 4. Immunization of the sheep with plasmids encoding GRA7, with the different adjuvant formulations, effectively primed the immune response. After the first inoculation, moderate to high antibody responses were observed with the three different adjuvant formulations. A significantly elevated specific IgG2 response was observed in the sheep immunized with liposomes and Emulsigen D as adjuvants. In the group immunized with Emulsigen P as an adjuvant, lower IgG1 and IgG2 antibody levels were developed compared to the other treatment groups. In all the immunized groups, DNA immunization stimulated a IFN-c response. No antibody or IFN-c responses were detected in the control group immunized with an empty plasmid or not immunized. These results indicate that intramuscular immunization of sheep with a DNA vaccine with the adjuvants liposomes and Emulsigen D induce a significant immune response against T. gondii. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Ovine toxoplasmosis, first described by (Hartley et al. (1954), was subsequently recognized as the most common infectious agent involved in abortion, stillbirth and neonatal deaths in sheep (Buxton et al., 2007; McColgan et al., 1988). The existing live vaccine, ToxovaxÒ (Intervet Ltd., New Zealand), based on an attenuated Toxoplasma gondii isolate has been effectively used by sheep producers. However, the live vaccine has a short shelf life, the potential to be hazardous to operatives and there is the risk of the vaccine reversing to the pathogenic phenotype. There is therefore a need for a non-living vaccine with a longer shelf life which is effective in farm animals and safe for humans.

* Corresponding author. Address: Faculty of Agriculture and Life Sciences, Lincoln University, P.O. Box 84, Lincoln 7647, Canterbury, New Zealand. Fax: + 64 3 3253 851. E-mail address: [email protected] (E. Hiszczyn´ska-Sawicka). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.11.015

Immunization with plasmid DNA is a relatively novel technique for the efficient stimulation of a complete immune response against an encoded antigen. It offers several important advantages such as: safety (they do not contain any pathogenic organism that may revert in virulence); promote a specific expression of vaccine coded antigen by host cells; have the ability to deliver multivalent vaccines to a host in a single dose; low quantities (milligrams) of plasmid DNA are sufficient to induce an immune response; manufacturing is cost-effective and storage in the lyophilized form is advantageous for transport and distribution of the product (Beláková et al., 2007). However, the major advantage of DNA vaccines is their ability to generate a cellular immunity with preference for MHC I – restricted CD8+ cytotoxic T cells and MHC II – restricted T helper type 1 (Th1) cells responses (Cohen et al., 1998; De Rose et al., 2002; Bivas-Benita et al., 2005). DNA vaccination can also be effective in inducing long-term antibody responses (Babiuk et al., 2007; Ho et al., 1998). Although the DNA immunization has proven to be very successful in inducing an immune response in mice, it is less effective in inducing immunity in large animals

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and humans (Babiuk et al., 1999). To improve the immune response to DNA vaccination in large animals various methods, such as chemical adjuvants or immunomodulatory molecules formulated into microparticles or liposomes have been evaluated for their ability to augment the potency of DNA vaccination (Babiuk et al., 2003; Greenland and Letvin, 2007; Van Drunen Littel-van den Hurk et al., 2004). A range of promising new adjuvants have been developed which include the EMULSIGENÒ family (MVP Laboratories, Inc.). Conventional adjuvants such as Emulsigen combined with dimethyl dioctadecyl ammonium bromide (DDA/Em) used with subunit vaccines generate a Th1 and Th2-like immune responses (van Rooij et al., 2002; Li et al., 2006). Cationic liposomes are now accepted as effective vectors with low immunogenicity and have been successfully used for gene delivery in human clinical trials (Hyde et al., 2000). Plasmids entrapped within cationic liposomes are not accessible to nucleases, and are, therefore, released over an extended period of time. Synthetic oligodeoxynucleotides (ODN) containing CpG motifs mimic the immune stimulatory effects of bacterial DNA. In vitro studies confirmed that the motif GTCGTT was optimal for several species, including sheep (Kamstrup et al., 2001; Nichani et al., 2004; Pontarollo et al., 2002; Rankin et al., 2001). CpG ODN 2135 has been shown to induce a proliferation in peripheral blood mononuclear cells (PBMC) from sheep and to act as an efficient adjuvant for recombinant protein vaccines in vivo (Rankin et al., 2001). In this study, sheep were chosen as a large animal model to determine the immune responses against T. gondii dense granule antigen 7 (GRA7 antigen) in three different adjuvant formulations: Liposomes, Emulsigen P and Emulsigen D. 2. Materials and methods 2.1. Animals Sixty Coopworth ewes, aged 2 years, were sourced from AgResearch Lincoln, New Zealand, on the basis of being seronegative for T. gondii by an enzyme-linked immunosorbent assay (ELISA) (Institute Pourquier, France), latex agglutination test (Eiken, Japan) and a developed ELISA test for T. gondii (Hiszczyn´ska-Sawicka et al., 2003; Pietkiewicz et al., 2004; Holec et al., 2007) before immunization. The ewes were housed under conventional New Zealand farming conditions and were grazed on pasture for the entire length of the experimental period. All animal manipulations were approved by the Lincoln University Animal Ethics Committee (AEC#130). 2.2. Experiment design The ewes were divided randomly into five groups (n = 12) and treated as follows. Control animals were immunized with 1 mg of empty pVAXIg plasmid (group 1, n = 12) or received no immunisation (group 2, n = 12). There were three treatment groups. Group 3 (n = 12) was immunized with 1 mg of plasmid pVAXIgGRA7 + Liposomes; group 4 (n = 12) with 1 mg of plasmid pVAXIgGRA7 + Emulsigen P and group 5 (n = 12) with 1 mg of plasmid pVAXIgGRA7 + Emulsigen D. All animals were immunized intramuscularly in the dorsal part of the neck. Each animal received two injections 4 weeks apart to a maximum of 2 ml per injection. All ewes were run together for the duration of the trial.

were synthesized by Invitrogen and resuspended in PBS to a final concentration 1 mg/ml. 2.4. Parasites and T. gondii antigens The tachyzoites of the virulent RH strain of T. gondii used for antigen preparation were kindly provided by John Ellis, University of Technology, Sydney, Australia. Tachyzoites were grown in a monolayer of Vero cells in minimal essential medium (MEM) (Invitrogen, CA, USA) supplemented with 2% of fetal bovine serum (FBS) (Invitrogen, CA, USA). Parasites were harvested, washed in phosphate buffered saline (PBS) and used to prepare the T. gondii lysate antigen as previously described (Nielsen et al., 1999). 2.5. Construction of the GRA7 expression plasmid Fragment of the gene encoding the T. gondii GRA7 antigen protein, without the signal sequence, was introduced into pVAXIg vector containing Ig j-chain leader sequence from plasmid pSecTaq2 (Invitrogen, CA, USA) inserted into NheI-HindIII sites of plasmid pVAX1 (Invitrogen, CA, USA). DNA of plasmid pUETGRA7 (Hiszczyn´ska-Sawicka et al., 2003) was used as the template for amplification of the GRA7 gene sequence using a standard PCR amplification protocol and primers: 50 -ATGGATCCGACACATGGACAGCCCAGATC-30 (forward), 50 - GCG AATTCCTAGACGGAGCTCGAATTAATTC-30 (reverse) (introduced BamHI and EcoRI recognition sites, respectively, underlined). The 712 bp PCR product was digested with both BamHI and EcoRI and inserted into the BamHI and EcoRI sites of the eukaryotic expression vector pVAX1. The resulting plasmid was named pVAXGRA7. The 749 bp fragment containing the GRA7 gene was recloned from pVAXGRA7 into pVAXIg using restriction enzymes KpnI/XbaI. The resulting plasmid was named pVAXIgGRA7 (3768bps) and contains a Ig j-chain leader sequence and sequence GRA7 antigen (from 20 to 236 amino acids). The nucleotide sequence of the insert was verified by sequencing. The plasmid was maintained and propagated in Escherichia coli (E. coli) DH5a strain (Invitrogen, CA, USA). The large scale production of endotoxin-free DNA was accomplished using an EndoFree plasmid Giga kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. DNA for vaccination was dissolved in sterile endotoxin-free PBS (Qiagen, Hilden, Germany). 2.6. In vitro expression of construct pVAXIgGRA7 in mammalian cells Chinese Hamster ovary cells (CHO-K1) were transfected with pVAXIgGRA7 or a control plasmid pVAX1 (Invitrogen, CA, USA), using polycationic liposome reagent (Lipofectamine™ 2000, Invitrogen). Cells were cultured in F-12 K Nutrient Mixture, Kaighn’s Modification (Gibco, Invitrogen, USA) as described previously (Patil et al., 2004). Freshly grown CHO-K1 cells were seeded at 2  105 cells/well in 500 ll of growth medium the day prior to transfection. Cells were transfected with plasmids using Lipofectamine™ 2000 according to manufacturer’s instruction. Cells were grown for a further 24 and 48 h before the cell pellets and supernatants were collected and analysed for transgene expression. For immunoblots with transfected cells, SDS–PAGE and immunoblotting were performed. The separated proteins were probed with anti-T. gondii sheep serum. Bound antibodies were detected with peroxidase-labelled rabbit anti-sheep IgG (Pierce, USA). 2.7. Production of recombinant GRA7-His protein

2.3. CpG ODN Unmethylated CpG ODN 2135 – 50 -TCGTCGTTTGTCGTTTTG TCGTT-30 (Pontarollo et al., 2002) with a phosphorotioate backbone

The production of recombinant GRA7 antigen for ELISA and interferon-gamma (IFN-c) test were performed as described in Hiszczynska-Sawicka et al. (2003).

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2.8. DNA-adjuvant complex preparation 2.8.1. DNA-liposomes complex preparation To enhance the efficiency of plasmid DNA uptake in the intramuscular injection, 1 mg of pVAXIgGRA7 plasmid DNA and 500 lg of CpG was complexed with cationic lipid N-[1(2,3-di-oleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) and dioleoyl phosphatidylethanolamine (DOPE) present at ratio 1:1 (w/w) (ESCORTTM Transfection Reagent – Sigma, St. Louis, USA). The DNA/lipid ratio was 1:1 (w/w) and the complex was incubated 15 min at room temperature. 2.8.2. DNA-EmulsigenÒ – D complex preparation 1 mg of pVAXIgGRA7 plasmid DNA and 500 lg of CpG were complexed with EmulsigenÒ – D (MVP Laboratories, Inc., Omaha, USA) to a final concentration of 20%. EmulsigenÒ – D incorporates dimethyldioctadecylammonium bromide (DDA) as an additional immunostimulant for weak antigens. 2.8.3. DNA-EmulsigenÒ – P complex preparation 1 mg of pVAXIgGRA7 plasmid DNA and 500 lg of CpG were complexed with EmulsigenÒ – P (MVP Laboratories, Inc., Omaha, USA) to a final concentration of 20%. EmulsigenÒ – P utilizes HLB (Hydrophile-Lipophile-Lipophile Balance) technology to maximize the stability of the oil-in-water emulsion. 2.9. Serology Serum samples were collected from all the animals prior to the first immunizing injection and thereafter, once per week, until 4 weeks following the second injection. Serum was tested for IgG1 and IgG2 antibodies to recombinant GRA7 antigen produced in E. coli. Antibody levels were measured by ELISA in flat-bottomed 96well ELISA plates (Costar, medium binding Corning Incorporated, USA) coated overnight at 4 °C with 100 ll recombinant GRA7 protein at 10 lg/ml in 0.1 M carbonate buffer pH 9.6. Plates were washed with PBST (PBS pH 7.4 containing 0.05% Tween 20) and then blocked for 2 h at 37 °C in 12% FBS (Invitrogen, CA, USA) in PBST. Sera were diluted 1:400 for IgG1 and 1:80 for IgG2 in PBST buffer and 100 ll added to each well. Plates were incubated 1 h at 37 °C, washed, and monoclonal mouse anti-ovine IgG1 (AgResearch, Upper Hut, New Zealand) or monoclonal mouse anti-ovine IgG2 (AgResearch, Upper Hut, New Zealand) added in dilution 1:500 and 1:100 respectively. Plates were incubated 1 h at 37 °C and then washed six times. Bound antibodies were detected by using horseradish peroxidase (HRP) conjugated rabbit anti-mouse IgG (DacoCytomation, Carpinteria, CA, USA) at 1:2000 dilution for 1 h at 37 °C followed by washing and the addition of o-phenylenediamine dihydrochloride (OPD) (Sigma, St. Luis, USA) as a substrate. For each animal the results were expressed as an optical density (OD) ratio between OD of the sample divided by the OD of the antibody-free control within each ELISA plate (Blanchard et al., 2003). To express the ratio of IgG2/IgG1, calculations were made for each animal followed by the calculation of an average.

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against recombinant bovine IFN-c. These monoclonal antibodies have been shown to cross react with IFN-c of species other then cattle, including sheep (Wood and Jones, 2001). All the procedures were followed according to the manufacturer’s recommendation. The amount of IFN-c was estimated by comparison to a standard curve of recombinant bovine IFN-c. 2.11. Statistical analysis All results were analysed using one-way ANOVA (GenStat v9.1.147). 3. Results 3.1. Expression of T. gondii antigen GRA7 in vitro The ability of recombinant DNA plasmids to express full length GRA7 antigen in vitro was investigated in CHO-K1 cells. A DNA fragment containing the coding sequence of mature T. gondii GRA7 antigen was amplified and cloned into eukaryotic expression vector (pVAXIg). Immunobloting of CHO-K1 cells transfected with pVAXIgGRA7 showed that T. gondii recombinant protein GRA7 was expressed in vitro from this plasmid vector (Fig. 1). The expression vector was designed such that the GRA7 antigen was expected to be at least partially secreted into the culture supernatant. The production of GRA7 was confirmed by immunoblot using polyclonal anti-T. gondii sheep antibodies. Specific immunoreactive protein of approximately 29 Da was detected in supernatants and cells transfected with pVAXIgGRA7 and was absent in cells transfected with control plasmid vector pVAX1 (Fig. 1). These results indicate that recombinant GRA7 was successfully produced and secreted by mammalian cells. 3.2. Interferon gamma production The mean units of IFNc detected in whole blood of sheep stimulated by recombinant GRA7 antigen and soluble tachyzoites antigens are shown in Fig. 2A and B. In both control groups there were no significant differences in IFN-c levels across the experimental period of time (Fig. 2). There were some differences in animal re-

2.10. Interferon gamma test The assay was performed with whole-blood cultures. 1 ml of heparinized blood was dispensed into multidish 48 well plates (Nunc, Denmark) and 20 lg per ml of recombinant GRA7 antigen or soluble toxoplasma antigen added to each well. The culture was incubated for 24 h in 5% CO2 at 37 °C in humidified atmosphere. The supernatants were harvested and stored at -30 °C until assayed for IFN-c by using an ELISA kit (Bovigam™, Prionics AG, Switzerland) that uses specific monoclonal antibodies raised

Fig. 1. Western blot analysis of recombinant GRA7 antigen expressed in CHO-K1 cells using sheep anti-T. gondii antibodies. A. Lane 1: molecular weight proteins marker (Fermentas); Lane 2: cell culture supernatant from pVAXIgGRA7 transfected CHO-K1 cells after 24 h expression; Lane 3: pallet from pVAXIgGRA7 transfected CHO-K1 cells after 24 h expression; Lane 4: cell culture supernatant from pVAXIgGRA7 transfected CHO-K1 cells after 48 h expression; Lane 5: culture supernatant from control CHO-K1 cells transfected with empty plasmid; Lane 6: control CHO-K1 cells transfected with empty plasmid.

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Fig. 2. Kinetics of IFN-c production after whole blood samples restimulation with recombinant GRA7 antigen (A) and soluble toxoplasma antigen (B) in groups 1, 2, 3, 4 and 5 in experimental period of time. Animals were immunized intramuscularly with: pVAXIg (group 1) or not immunized (group 2); pVAXIgGRA7 + Liposomes (group 3), pVAXIgGRA7 + Emulsigen P (group 4), pVAXIgGRA7 + Emulsigen D (group 5). Arrows indicate time of initial immunization (week 0) and booster injection (week 4). Statistical differences inside the groups are shown as  (p < 0.01) and between the groups are shown as  (p < 0.05).

sponses between groups 1 and 2 in the 1st and 2nd week after the first injection but they were not significant. In group 1 (immunized with empty plasmid), the preimmunization level of produced IFNc was 11.41 and 11.43 ng/ml for native antigen and recombinant GRA7 protein respectively. In the non-immunized control group, the preimmunization level of produced IFN-c was 11.4 and 11.41 ng/ml respectively. In group 3 there was a significant difference in IFN-c level after restimulation of blood samples by soluble toxoplasma antigens in the 1st week after the primary injection (p < 0.01) compared to the preinjection level (Fig. 2B). After restimulation of the blood with recombinant GRA7 antigen there was a

significant elevation of IFN-c level in the 2nd week after the booster injection. In group 4, there was a significant elevation of IFN-c level in week 6 only, after restimulation with soluble toxoplasma antigens or recombinant GRA7 antigen. In group 5, significant IFN-c production was detected in the second week after primary injection and from the first week after the booster injection (in weeks 5 and 6). However, IFN-c was only produced in very small quantities in response to the antigens. The average increase in IFN-c concentration was 10.6 ng/ml. Statistical analysis revealed that the group means across time periods are not significantly different, however at certain time points significant differences did

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exist (Fig. 2). The significant difference between both control – and treatment groups (groups 3, 4 and 5) appeared from the 2nd week of the experiment for groups 4 and 5 and after the booster injection for group 3. At certain time points however, there were significant differences (p < 0.05) between treatment groups (groups 3, 4, 5). Production of IFN-c in liposome group (group 3) was significantly higher in week 1 when compared with both Emulsigen groups. For Emulsigen D group the IFN-c expression was higher in weeks 2 and 6 when compared to liposome and Emulsigen P group. The pattern of treatment group differences varied with the restimulation antigen. When recombinant GRA7 was used differences occurred in week 2 whereas with native antigen, differences occurred in weeks 1, 2 and 6 (Fig. 2). 3.3. Antibody response None of the control animals had detectable anti-GRA7 or anti-T. gondii-specific antibodies in their sera. IgG1 and IgG2 antibody levels in the control pVAXIg vaccinated animals (group 1) and notvaccinated animals (group 2) remained at the pre-immunisation levels. The OD ratio was 1.55 and 1.3 for IgG1 and 1.62 and 1.49 for IgG2 respectively (Figs. 3 and 4). The intramuscular immunization of animals with liposome and Emulsigen formulated DNA preparations induced increases in IgG antibody levels against rGRA7 compared to animals non-vaccinated or vaccinated with empty plasmid as shown in Fig. 3. Compared to the Emulsigen P-treated animals (group 4), liposomesand Emulsigen D-treated animals (groups 3 and 5) consistently showed higher IgG antibody responses after the booster injection. The strongest antibody response was observed in group 5 immunized with Emulsigen D as an adjuvant compared to groups 3 and 4 (Fig. 4). Statistical analysis revealed there were significant differences at specific times within each treatment group. In group 5, there was a significant elevation of IgG1 antibodies from the 1st week after the booster injection (p < 0.01). This occurred for group 3 in weeks 6 and 7 and for group 4 in week 6. For IgG2, a significant elevation in the antibody was detected in groups 3 and 5 in the first week after the booster injection and the elevated levels were maintained until week 8. Responses between animals within the same group varied. In group 3 (liposome group) the animals responded evenly (Fig. 3). IgG1 was detected in all the animals from week 6 and IgG2 after the booster immunisation. Of the group 4 animals, only 33% (4 animals from 12) responded with respect to IgG1 and 42% (5 animals from 12) with respect to IgG2. A high antibody level was detected in group 5 animals compared to groups 3 and 4 but there were large variations between animals. In group 5, 2 animals failed to respond to the vaccinations. The isotype nature of the IgG response to the immunization was investigated. Sheep in group 3 exhibited a higher ratio of IgG2 to IgG1 antibody level whilst in group 5 the IgG2/IgG1 ratio was less polarized compared to the animals from the liposomes and Emulsigen P group (Fig. 5). 4. Discussion We have shown here, for the first time, that a DNA vaccine encoding the GRA7 Toxoplasma antigen can induce both humoral and cellular immune responses in sheep characterized by the production of antibodies and IFN-c against T. gondii. Studies on DNA vaccines against T. gondii in mice revealed there were humoral (Angus et al., 2000) and cellular responses (Jongert et al., 2007). In this study we compared the response to DNA immunization using a plasmid carrying the gene of T. gondii GRA7 antigen, formulated by 3 different protocols (Liposomes, Emulsigen D and Emuls-

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igen P). Improving the delivery of a plasmid DNA immunogen may affect a number of the factors that limit immune responses to the vaccine. Most approaches enhance the expression of a plasmid DNA in vivo by protecting the plasmid DNA from degradation and enhancing its uptake by cells. The anti-Toxoplasma DNA vaccine needs to stimulate both humoral and cellular immune responses. Selection of the vaccine delivery/adjuvant system is as important as selection of the immunogen since adjuvants can sometimes lead to the induction of an inappropriate response, e.g. a humoral response instead of a cell mediated response (Altin and Parish, 2006). DNA vaccines are usually not very effective in large animals used to model human infections compared to the responses in mice. In this study we have shown that intramuscular DNA immunization with potentially protective T. gondii antigen GRA7 with 3 different adjuvants induces humoral and cellular immune responses in sheep against T. gondii. We found that although the antigen produced significant immune responses with all the adjuvants used, that statistically higher antibody levels were observed in the liposomes and Emulsigen D groups of animals than in Emulsigen P group. However, after the first injection, the antibody response was only observed in the group immunized with liposomes formulated DNA. This result confirms previous observations that liposomes can improve immune responses to the plasmid encoded vaccines (Perrie et al., 2001; Greenland and Letvin, 2007). In all the DNA immunized groups, the sheep responded and exhibited elevated specific antibody levels after the second injection of DNA. An important observation is that the response in the Emulsigen groups was only generated after the booster immunisation and that some animals failed to respond at all. This observation is similar to that presented by Lauritsen et al. (2009) where pigs vaccinated with Mycoplasma hyosynoviae antigen in different Emulsigen formulation seroconverted after a booster injection. Evaluation of the isotype nature of the IgG responses to immunization revealed that the sheep in the liposomes group exhibited a high ratio of IgG2 to IgG1 antibody level, which is characteristic of a Th1 type response and is typical of chronically infected animals (Denkers, 2003). The production of IgG2 and IFN-c often indicates a shift towards a Th1 immune response, which is a natural outcome of infection with live parasites. Induced immunization in group 3 (liposomes) produced a more balanced response than group 4 (Emulsigen P) or 5 (Emulsigen D). Two different formulations of Emulsigen produced significant differences in their antibody response. Emulsigen D, which incorporates DDA as an additional immunostimulant, induced higher antibody responses after the booster immunisation as demonstrated by the elevated IgG1 and IgG2 levels than Emulsigen P. However, the IgG2/IgG1 ratio was less polarized compared to the liposomes group. Our results confirm those received by Kaur et al. (2009), where DNA vaccine supplemented with Emulsigen induced immune response with predominantly IgG1/IgG2 subclass distribution. Sheep immunized with the three different formulations also showed specific cellular immune responses as characterized by the increased secretion of IFN-c in response to soluble toxoplasma antigen and recombinant GRA7 antigen. In all three treatment groups production of IFN-c was significant when compare to the control groups. In the liposomes group a peak of IFN-c expression was observed in the first week after the 1st injection. Whereas, the production of IFN-c in Emulsigen D group was higher in weeks 2 and 6 when compared to the liposomes and Emulsigen P group. Emulsigen is believed to act, at least in part, by creating an antigen depot at the site of inoculation from which the antigen is slowly released, thus providing prolonged stimulation to the immune system (Ioannou et al., 2002).

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Fig. 3. Individual IgG1 and IgG2 responses of sheep immunized with DNA plasmids. Animals were immunized intramuscularly with: pVAXIg (group 1) or not immunized (group 2) (A); pVAXIgGRA7 + Liposomes (group 3), pVAXIgGRA7 + Emulsigen P (group 4), pVAXIgGRA7 + Emulsigen D (group 5) (B). GRA7 specific antibody levels were measured by ELISA as described in Materials and Methods. Arrows indicate the time of the initial immunization (week 0) and booster injection (week 4).

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week Fig. 4. Specific IgG1 (A) and IgG2 (B) serum antibody ratio in sheep immunized with pVAXIgGRA7 + Liposomes (group 3), pVAXIgGRA7 + Emulsigen P (group 4), pVAXIgGRA7 + Emulsigen D (group 5) following DNA immunization at week 0 and 4. Arrows indicate time of initial immunization (week 0) and booster injection (week 4). Statistical differences inside the groups are shown as  (p < 0.01) and between the groups are shown as  (p < 0.05).

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week Fig. 5. Ratio of IgG2 to IgG1 in groups immunized with: pVAXIgGRA7 + Liposomes (group 3), pVAXIgGRA7 + Emulsigen P (group 4), pVAXIgGRA7 + Emulsigen D (group 5).

GRA7 antigen has been used as a DNA-based immunization procedure using mice as the experimental model. Specific antibody and IFN-c responses against GRA7 protein were detected after the mice were vaccinated with plasmid encoding GRA7 antigen. When a GRA7 encoding plasmid was coinoculated with plasmids encoding GRA1 and ROP2 the immune response was directed mainly against GRA7 suggesting that GRA7 is the main immunodominant character (Jongert et al., 2007). In the research by Jongert et al. (2008), pigs that were vaccinated with pGRA1 plus a pGRA7 cocktail DNA developed significant humoral and Th1 cellular immune responses characterized by the production of IFN-c. Similar results are reported in our research which provides additional evidence that the immune response against T. gondii antigens can be successfully triggered by a DNA vaccine in sheep.

The major purpose of the present work was to investigate whether the vaccination of DNA incorporated into different adjuvant formulations could provide an enhanced immunogenic activity. One way to influence the immune response to an antigen is through the use of an appropriate adjuvant (Greenland and Letvin, 2007; Van Drunen Littel-van den Hurk et al., 2004). We report here that the T. gondii GRA7 antigen, used as a DNA vaccine, was capable of eliciting an immune response. Of the three adjuvant formulations evaluated, liposomes formulated GRA7 vaccine induced a Th1 type of immune response, whilst formulation with Emulsigen D elicited a significant antibody response. It is well known that infection with T. gondii is controlled mainly by cellular immune mechanism (Jongert et al., 2007). However, antibodies also play a role in the regulation of the infection (Angus et al., 2000). Taking this under consideration, liposomes seem to be the best choice among tree adjuvant formulation used in this research. However, the most important outcome of the DNA immunization by the plasmid pVAXIgGRA7 is whether this technology will stimulate protection against Toxoplasma infection. The next stage in the research is to determine the efficacy of the proposed DNA vaccination technique against a Toxoplasma challenge in sheep (Babiuk et al., 1999). Acknowledgments We are grateful to Dr. J. Ellis for providing T. gondii RH strain. The work described herein was funded by Ancare New Zealand Limited. References Altin, J.G., Parish, C.R., 2006. Liposomal vaccines – targeting the delivery of antigen. Methods 40, 39–52. Angus, C.W., Klivington-Evans, D., Dubey, J.P., Kovacs, J.A., 2000. Immunization with a DNA plasmid encoding the SAG1 (P30) protein of Toxoplasma gondii is

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immunogenic and protective in rodents. The Journal of Infectious Diseases 181, 317–324. Babiuk, L.A., van Drunen Littel-van den Hurk, S., Babiuk, S.L., 1999. Immunization of animals: from DNA to the dinner plate. Veterinary Immunology and Immunopathology 72, 189–202. Babiuk, L.A., Gomis, S., Hecker, R., 2003. Molecular approaches to disease control. Poultry Science 82, 870–875. Babiuk, S., Tsang, C., van Drunen Littel-van den Hurk, S., Babiuk, L.A., Griebel, P.J., 2007. A single HBsAg DNA vaccination in combination with electroporation elicits long-term antibody responses in sheep. Bioelectrochemistry 70, 269– 274. Beláková, J., Horynová, M., Krupka, M., Weigl, E., Raska, M., 2007. DNA vaccines: are they still just a powerful tool for the future? Archivum Immunologiae et Therapiae Experimentalis 55, 387–398. Bivas-Benita, M., Ottenhoff, T.H., Junginger, H.E., Borchard, G., 2005. Pulmonary DNA vaccination: concepts, possibilities and perspectives. Journal of Controlled Release 20, 1–29. Blanchard, Ph., Mahé, D., Cariolet, R., Truong, C., Le Dimna, M., Arnauld, C., Rose, N., Eveno, E., Albina, E., Madec, F., Jestin, A., 2003. An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome. Veterinary Microbiology 94, 183–194. Buxton, D., Maley, S.W., Wright, S.E., Rodger, S., Bartley, P., Innes, E.A., 2007. Toxoplasma gondii and ovine toxoplasmosis: new aspects of an old story. Veterinary Parasitology 149, 25–28. Cohen, A.D., Boyer, J.D., Weiner, D.B., 1998. Modulating the immune response to genetic immunization. FASEB Journal 12, 1611–1626. De Rose, R., Tennent, J., McWaters, P., Chaplin, P.J., Wood, P.R., Kimpton, W., Cahill, R., Scheerlinck, J.P., 2002. Efficacy of DNA vaccination by different routes of immunisation in sheep. Veterinary Immunology and Immunopathology 90, 55– 63. Denkers, E.Y., 2003. From cells to signaling cascades: manipulation of innate immunity by Toxoplasma gondii. FEMS Immunology and Medical Microbiology 39, 193–203. Greenland, J.R., Letvin, N.L., 2007. Chemical adjuvants for plasmid DNA vaccines. Vaccine 25, 3731–3741. Hartley, W.J., Jebson, J.L., McFarlane, D., 1954. New Zealand II type abortion in ewes. Australian Veterinary Journal 30, 216–218. Hiszczyn´ska-Sawicka, E., Brillowska-Dabrowska, A., Dabrowski, S., Pietkiewicz, H., Myjak, P., Kur, J., 2003. High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli. Protein Expression and Purification 27, 150–157. Ho, T.Y., Hsiang, C.Y., Hsiang, C.H., Chang, T.J., 1998. DNA vaccination induces a longterm antibody response and protective immunity against pseudorabies virus in mice. Archives of Virology 143, 115–125. Holec, L., Hiszczyn´ska-Sawicka, E., Gasior, A., Brillowska-Dabrowska, A., Kur, J., 2007. Use of MAG1 recombinant antigen for diagnosis of Toxoplasma gondii infection in humans. Clinical and Vaccine Immunology 14, 220–225. Hyde, S.C., Southern, K.W., Gileadi, U., Fitzjohn, E.M., Mofford, K.A., Waddell, B.E., Gooi, H.C., Goddard, C.A., Hannavy, K., Smyth, S.E., Egan, J.J., Sorgi, F.L., Huang, L., Cuthbert, A.W., Evans, M.J., Colledge, W.H., Higgins, C.F., Webb, A.K., Gill, D.R., 2000. Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis. Gene Therapy 7, 1156–1165. Ioannou, X.P., Griebel, P., Hecker, R., Babiuk, L.A., van Drunen Littel-van den Hurk, S., 2002. The immunogenicity and protective efficacy of bovine herpesvirus 1 glycoprotein D plus Emulsigen are increased by formulation with CpG oligodeoxynucleotides. Journal of Virology 76, 9002–9010. Jongert, E., de Craeye, S., Dewit, J., Huygen, K., 2007. GRA7 provides protective immunity in cocktail DNA vaccines against Toxoplasma gondii. Parasite Immunology 29, 445–453.

Jongert, E., Melkebeek, V., De Craeye, S., Dewit, J., Verhelst, D., Cox, E., 2008. An enhanced GRA1-GRA7 cocktail DNA vaccine primes anti-Toxoplasma immune responses in pigs. Vaccine 26, 1025–1031. Kamstrup, S., Verthelyi, D., Klinman, D.M., 2001. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides. Veterinary Microbiology 78, 353–362. Kaur, M., Saxena, A., Rai, A., Bhatnagar, R., 2009. Rabies DNA vaccine encoding lysosome-targeted glycoprotein supplemented with Emulsigen-D confers complete protection in preexposure and postexposure studies in BALB/c mice. FASEB Journal (2009 Sep 25). Lauritsen, K., Riber, U., Nielsen, J., Jakobsen, J., Jungersen, G., 2009. Testing immunogenicity of Mycoplasma hyosynoviae vaccine candidates: induction of antibodies and IFN-response. Abstract. Veterinary Immunology and Immunopathology 128, 329–330. Li, Y.P., Kang, H.N., Babiuk, L.A., Liu, Q., 2006. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models. World Journal of Gastroenterology 12, 7126–7135. McColgan, C., Buxton, D., Blewet, D.A., 1988. Titration of Toxoplasma gondii oocysts in non-pregnant sheep and the effects of subsequent challenge during pregnancy. Veterinary Records 123, 467–470. Nichani, A.K., Mena, A., Popowych, Y., Dent, D., Townsend, H.G., Mutwiri, G.K., Hecker, R., Babiuk, L.A., Griebel, P.J., 2004. In vivo immunostimulatory effects of CpG oligodeoxynucleotide in cattle and sheep. Veterinary Immunology and Immunopathology 98, 17–29. Nielsen, H.V., Lauemoller, S.L., Christiansen, L., Buus, S., Fomsgaard, A., Petersen, E., 1999. Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene. Infection and Immunity 67, 6358–6363. Patil, S.D., Rhodes, D.G., Burgess, D.J., 2004. Anionic liposomal delivery system for DNA transfection. American Association of Pharmaceutical Scientists Journal 15, e29. Perrie, Y., Frederik, P.M., Gregoriadis, G., 2001. Liposome-mediated DNA vaccination: the effect of vesicle composition. Vaccine 19, 3301–3310. Pietkiewicz, H., Hiszczyn´ska-Sawicka, E., Kur, J., Petersen, E., Nielsen, H.V., Stankiewicz, M., Andrzejewska, I., Myjak, P., 2004. Usefulness of Toxoplasma gondii-specific recombinant antigens in serodiagnosis of human toxoplasmosis. Journal of Clinical Microbiology 42, 1779–1781. Pontarollo, R.A., Rankin, R., Babiuk, L.A., Godson, D.L., Griebel, P.J., Hecker, R., Krieg, A.M., van Drunen Littel-van den Hurk, S., 2002. Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs. Veterinary Immunology and Immunopathology 84, 43–59. Rankin, R., Pontarollo, R., Ioannou, X., Krieg, A.M., Hecker, R., Babiuk, L.A., van Drunen Littel-van den Hurk, S., 2001. CpG motif identification for veterinary and laboratory species demonstrates that sequence recognition is highly conserved. Antisense and Nucleic Acid Drug Development 11, 333–340. Van Drunen Littel-van den Hurk, S., Babiuk, S.L., Babiuk, L.A., 2004. Strategies for improved formulation and delivery of DNA vaccines to veterinary target species. Immunological Reviews 199, 113–125. Van Rooij, E.M., Glansbeek, H.L., Hilgers, L.A., te Lintelo, E.G., de Visser, Y.E., Boersma, W.J., Haagmans, B.L., Bianchi, A.T., 2002. Protective antiviral immune responses to pseudorabies virus induced by DNA vaccination using dimethyldioctadecylammonium bromide as an adjuvant. The Journal of Virology 76, 10540–10545. Wood, P.R., Jones, S.L., 2001. BOVIGAM: an in vitro cellular diagnostic test for bovine tuberculosis. Tuberculosis 81, 147–155.