Ginsenoside Rg1 enhances immune response induced by recombinant Toxoplasma gondii SAG1 antigen

Veterinary Parasitology 179 (2011) 28–34

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Ginsenoside Rg1 enhances immune response induced by recombinant Toxoplasma gondii SAG1 antigen Dao-Feng Qu a,b , Hai-Jie Yu a,b , Zhao Liu a,b , De-Fu Zhang a,b , Qian-Jin Zhou a,b , Hong-Li Zhang a,b , Ai-Fang Du a,b,∗ a b

Key Laboratory of Animal Epidemic Etiology and Immunological Prevention of Ministry of Agriculture, Zhejiang University, Hangzhou 310029, China Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310029, China

a r t i c l e

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Article history: Received 3 September 2010 Received in revised form 10 February 2011 Accepted 14 February 2011 Key words: Toxoplasma gondii rSAG1 Ginsenoside Rg1 Adjuvant

a b s t r a c t Ginsenoside, the most important component isolated from Panax ginseng, exhibits a variety of biological activities. Particularly, ginsenoside Rg1 is known to have immune-modulating activities such as increase of immune activity of T helper (Th) cells. In the present study, we evaluated the immunomodulatory potentials of the Rg1 at three dose levels on the cellular and humoral immune responses of ICR mice against T. gondii recombinant surface antigen 1 (rSAG1). ICR mice were immunized subcutaneously with 50 ␮g Rg1 alone, 100 ␮g rSAG1 alone or with 100 ␮g rSAG1 dissolved in saline containing ginsenoside Rg1 (10 ␮g, 50 ␮g or 100 ␮g). After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that the groups immunized with rSAG1 and Rg1 (50 ␮g, 100 ␮g) developed a high level of specific antibody responses against T. gondii rSAG1, a strong lymphoproliferative response, and significant levels of cytokine production, compared with the other groups. After lethal challenge, the mice immunized with the rSAG1 and Rg1 (50 ␮g, 100 ␮g) showed a significantly increased survival time compared with control mice which died within 6 days of challenge. Our data demonstrate that by addition of ginsenoside Rg1, the rSAG1 triggered a stronger humoral and cellular response against T. gondii, and that Rg1 is a promising vaccine adjuvant against toxoplasmosis, worth further development. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Toxoplasma gondii is an opportunistic intracellular parasite with an extensive range of warm-blooded hosts, including humans (Luder and Gross, 2005). It is found worldwide with a large range of clinical manifestations and high prevalence, which indicates that it is one of the most successful parasites of human (Delibas et al., 2006). Typically humans get infected by ingesting T. gondii oocysts in

∗ Corresponding author at: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310029, China. Tel.: +86 571 86971583. E-mail address: [email protected] (A.-F. Du). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.02.008

water or by eating meat containing the tissue cyst stage of the parasite (Singh, 2003). In the farming industry, congenital toxoplasmosis is also of considerable economic importance, because it is one of the principal causes of abortion, fetal death, and stillbirths in all types of livestock, particularly in pigs, goats and sheep (Ismael et al., 2006; Qu et al., 2009). In recent years, efforts have been made to develop anti-toxoplasma vaccines, the feasibility of which was suggested by the long-term immunity induced by the primary infection, and some T. gondii proteins were investigated as candidates for vaccine (Machado et al., 2010; Tan et al., 2010). Among these vaccine candidates, the main surface antigen 1 (SAG1), highly conserved in T. gondii strains, is the best characterized and was shown to induce both

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humoral and cellular immune responses in mice (Fang et al., 2010; Zhou et al., 2007). Consequently, there were many studies utilizing purified SAG1, recombinant SAG1 or SAG1-derived peptides in animal models (Cardona et al., 2009; Debard et al., 1996; Khan et al., 1991; Petersen et al., 1998; Shang et al., 2009). All of these studies have been encouraging but showed only partial protection against T. gondii. Adjuvants play an important role in the efficacy of vaccines (Sun et al., 2003). In addition to increasing the strength and kinetics of an immune response, adjuvants also play a role in determining the type of immune response generated (El-Malky et al., 2005). Ginseng, the root of Panax ginseng C.A. Meyer, as a safe traditional medicine has been utilized in China for at least 2000 years (Sun et al., 2006). The drug has been believed to stimulate the natural resistance against infections. Recent studies on saponins extracted from the root of P. ginseng (GS) such as Rg1, Re, Rb1, Rc, Rd, have demonstrated that GS has adjuvant activity capable of boosting both cellular (Th1) and humoral (Th2) immune responses. Others have found that a supplement of GS in an alum-adjuvanted bivalent vaccine improved immune responses in pigs to vaccination against porcine parvovirus and Erysipelothrix rhusiopathiae (Rivera et al., 2003a,b). Some researchers observed increased antibody responses and blood lymphocyte proliferation in dairy cattle elicited by vaccination with Staphylococcus aureus mastitis vaccine mixed with an Rb1 fraction of ginsenosides (Hu et al., 2003). Also some experts reported that Rg1 has stronger adjuvant potency than the others after an investigation of ginsenosides Rg3, Rd, Rc, Rb1, Rb2, Re, and Rg2 for their adjuvant effects on the immune responses to ovalbumin (OVA) in mice. Ginsenosides also enhanced the antibody response to viral and bacterial antigens (Rivera et al., 2003b), but the adjuvant effect of ginsenosides in protection from toxoplasmosis has not been examined. In the present study, we investigated the effect of Rg1, the major ginsenoside in the ginseng radix; in assisting the recombinant protein TgSAG1 improve the immune response, promote the cytokine secretion and spleen cell proliferation, and protect the T. gondii-infected mice from death.

2. Materials and methods

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Fig. 1. Chemical structure of ginsenoside Rg1 (C42 H72 O14 ; molecular weight, 801.02).

2.2. Experimental mice Six- to seven-week-old female ICR mice used in this experiment were purchased from Academy of Medical Science, Zhejiang, China, and maintained under pathogen-free conditions in our animal house for use throughout these experiments. 2.3. Parasites and preparation of STAg Tachyzoites of the RH strain of T. gondii were harvested from the peritoneal fluid of ICR mice that had been intraperitoneally infected 3–4 days earlier. Soluble tachyzoite antigen (STAg) was prepared from RH strain tachyzoites as previously described (Qu et al., 2008). Briefly, the obtained tachyzoites were washed with 0.01 mol/l phosphate-buffered saline (PBS, pH 7.4) and sonicated for three 10-min periods at 60 W/s. The toxoplasma sonicate was centrifuged at 2000 × g for 30 min. The protein concentration was determined in the supernatant, which was later used as the source of antigen, by Bradford method. The STAg was stored at −70 ◦ C until use. 2.4. Production and purification of recombinant parasitic SAG1 antigen

2.1. Materials 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), concanavalin A (ConA), RPMI-1640 medium were purchased from Sigma Chemical Co., Saint Louis, MO, USA; goat anti-mouse IgG, IgG1 and IgG2a peroxidase conjugate were from Serotec; fetal calf serum (FCS) was provided by Hangzhou Sijiqing Corp., Hangzhou, Zhejiang, China. Ginsenoside Rg1 extracted from the root of P. ginseng C.A. Meyer was from Hongjiu Ginseng Industry Co. Ltd. (Jilin, China). The Rg1 was a white powder with a purity of 98%; a melting point of 194–196.5 ◦ C; a molecular formula of C42 H72 O14 ; an infrared spectrum (KBr)/cm of 3400, 1620; and a molecular structure as shown in Fig. 1.

Recombinant SAG1 fusion protein was produced in the E. coli system as previously described (Qu et al., 2008). Briefly, the corresponding genes were amplified by PCR which introduce the EcoRI and XhoI restriction sites, respectively. PCR products were digested with EcoRI and XhoI, and cloned into the pET30a system (Novagen Inc., Darmstadt, Germany). The resulting clones were sequenced (Invitrogen Inc., CA, USA). HIS fusion proteins were expressed in E. coli BL21 (DE3). Fusion proteins were purified from supernatant using prepacked HIS-sepharose columns in accordance with the manufacture’s instructions (Sigma Inc., MO, USA). The purified protein was dialysed extensively against PBS to remove imidazole. The purity

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and immunogenicity was confirmed using SDS–PAGE and Western blot. The protein was stored at −70 ◦ C until use. 2.5. Immunization Six- to seven-week-old female ICR mice were divided into six groups, each consisting of twenty mice. Group A, PBS control. Group B was subcutaneously injected with 50 ␮g Rg1. Group C was subcutaneously injected with PBS and 100 ␮g SAG1. Mice in group D–F were subcutaneously injected with 100 ␮g SAG1 and Rg1 of different dosage 10 ␮g, 50 ␮g, and 100 ␮g, respectively. Mice in vaccinated groups were boosted with the same components at the same dose 2 weeks later. 2.6. Measurement of SAG1-specific IgG and subclass Serum SAG1-specific IgG and the subclasses were measured by an indirect ELISA as described previously (Du et al., 2005). In brief, polystyrene 96-well plates (Corning Costar Inc. USA) were coated with 100 ␮l rSAG1 solution (5 ␮g/ml in 50 mM carbonate buffer, pH 9.6) and incubated overnight at 4 ◦ C. After being washed with 0.01 M phosphate buffer solution containing 0.05% of Tween 20 (PBST), the plates were blocked with 5% fetal calf serum in phosphate-buffered saline and incubated for 2 h at room temperature. After washing with PBS containing 0.05% Tween 20 (PBST), individual sera were diluted 1:1000 in 5% FCS/PBS and incubated for 1 h at 37 ◦ C. After the plates were washed, bound antibodies were detected by incubation for 2 h at 37 ◦ C with alkaline phosphatase conjugate goat anti-mouse IgG (ABD Serotec Inc., Kidlington, UK) diluted 1:10,000 in PBS – 5% FCS, and IgG1, IgG2a (ABD Serotec Inc., Kidlington, UK) 1:2000. After being washed again, 100 ␮l of substrate solution (10 mg of ophenylenediamine and 37.5 ␮l of 30% H2 O2 in 25 ml of 0.1 M citrate-phosphate buffer, pH 5.0) was added to each well and further incubated at room temperature for 10 min. The reaction was stopped by adding 50 ␮l of 2 M H2 SO4 to each well. The optical density at 492 nm (OD 492) was read by an automatic ELISA plate reader (Dialab, GmbH, Austria). 2.7. Splenocyte proliferation assay in vitro Splenocyte suspensions were prepared from each group of mice by mincing up the spleens and then pushing through a wire mesh. After the red blood cells were removed using RBC lysis solution (Sigma Inc., MO, USA), splenocytes were re-suspended in DMEM medium supplemented with 10% FCS. Cell numbers were counted with a haemocytometer by trypan blue dye exclusion technique. Cell viability exceeded 95%. Splenocyte proliferation was assayed as previously described (Sun and Pan, 2006). Briefly, Splenocytes were seeded into 4–5 wells of a 96-well flat bottom microtiter plate (Corning Costar Inc., USA) at 5 × 106 cell/ml in 100 ␮l complete medium, thereafter Con A (final concentration 5 ␮g/ml), rSAG1 (final concentration 10 ␮g/ml), or medium were added giving a final volume of 200 ␮l. The plates were incubated at 37 ◦ C in a humid atmosphere with 5% CO2 . After 68 h, 50 ␮l of MTT solu-

tion (2 mg/ml) were added to each well and incubated for 4 h. The plates were centrifuged (1400 × g, 5 min) and the untransformed MTT was removed carefully by pipetting. To each well 200 ␮l of a DMSO working solution (192 ␮l DMSO with 8 ␮l 1 N HCl) was added, and the absorbance was evaluated in an ELISA reader at 570 nm with a 630 nm reference after 15 min. 2.8. Cytokine assays Spleen cell proliferation was assayed as described above, while the cells were stimulated by being incubated with 15 ␮g of rSAG1 per ml or with the medium alone. Cell-free supernatants were harvested and assayed for interleukin-4 (IL-4) activity at 24 h, and gamma interferon (IFN-␥) activity at 96 h. The concentration of IL-4 and IFN-␥ were determined with an ELISA kit (R&D Systems Inc., Minneapolis, USA) as specified by the manufacturer. The sensitivity for the assays was less than 2 pg/ml for IL-4 and IFN-␥. 2.9. Challenge infection ICR mice were challenged intraperitoneally with 50 tachyzoite of T. gondii RH strain 4 weeks after the second immunization. The mice were observed and the time to death was recorded where appropriate. 2.10. Statistical analyses Antibody responses measured by ELISA, lymphocyte responses measured by MTT were expressed as the mean ± SD (net OD values were used to calculate a mean) and evaluated by analysis of variance (ANOVA) and Duncan’s multiple range tests using the SPASS 13.0 software. P < 0.05 was considered significantly different. 3. Results 3.1. Expression of SAG1and Western blot rSAG1 was synthetized in E. coli (Fig. 2). After purification with prepacked HIS-sepharose columns, the purified protein was dialysed extensively against PBS to remove imidazole. The SDS–PAGE showed that there was only one band, and it can be recognized by serum obtained from STAg-inmunized mice by Western blot. 3.2. Enhanced splenocyte proliferation of lymphocytes To measure the splenocyte proliferative response, the splenocytes from the immunized mice were prepared 2 weeks after the second immunization to assess the proliferative immune responses to ConA or rSAG1 (Fig. 3). The splenocytes from mice immunized with rSAG1 and Rg1 showed a signigicant proliferative response to ConA or rSAG1 (P < 0.05). The splenocyte proliferation in the mice immunized with rSAG1 and Rg1 (50 ␮g, 100 ␮g) were significantly higher than the control groups (P < 0.05). In addition, splenocytes from mice immunized with rSAG1

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Fig. 2. SDS–PAGE and Western blot of Toxoplasma gondii rSAG1 expressed in E. coli. Lane 1: lysate of cells with empty plasmid; lane 2: lysate of cells with plasmid pET30a-SAG1; lane 3: purification of rSAG1; lane 4: lysate of cells with plasmid pET30a-SAG1; lane 5: lysate of cells with empty plasmid. Serum obtained from STAg-inmmunized mice was used as antibody to probe the membrane. Lane 6: lysate of cells with plasmid pET30a-SAG1. Serum obtained from Eperythrozoon-infected mice was used as antibody to probe the membrane.

and Rg1 (50 ␮g, 100 ␮g) proliferated higher levels in response to rSAG1 (P < 0.05). 3.3. Serum specific IgG and IgG isotypes

Fig. 3. Splenocyte proliferative responses to Con A and rSAG1. Mice were immunized s.c. twice at a 2-week interval with 50 ␮g Rg1 alone, 100 ␮g rSAG1 alone as controls, or 100 ␮g rSAG1 in combination with Rg1 (10 ␮g, 50 ␮g and 100 ␮g). Splenocytes were prepared 2 weeks after the last immunization and cultured with Con A (final concentration 5 ␮g/ml) or rSAG1 (final concentration 10 ␮g/ml). Splenocyte proliferation was measured by the MTT method as described in the text. The values are presented as means of the OD 570 ± SD (n = 3), and significant differences with control group were designated as *P < 0.05.

After immunization, specific IgG and the IgG subclasses were detected using ELISA to evaluate the adjuvant effect of ginsenoside Rg1 on the humoral immune response which is shown. All the vaccinated mice generated specific IgG antibodies against rSAG1after the first immunization and the levels of antibody increased after the boost immunization. Throughout the testing period, SAG1 antibody levels of the mice immunized with rSAG1 and Rg1 (50 ␮g, 100 ␮g) were significantly higher than that in control groups (P < 0.05). Co-administration of rSAG1 with Rg1 (50 ␮g and 100 ␮g) induced numerically higher specific antibody level than that with 10 ␮g Rg1 (P < 0.05). In contrast, mice in the control groups did not generate antibody response (Fig. 4). Both IgG1 and IgG2a were produced in the sera of mice after immunization (Fig. 5). The mice immunized with rSAG1 and Rg1 (50 ␮g, 100 ␮g) induced higher specific IgG1 (P < 0.05) and IgG2a than the other groups (P < 0.05). 3.4. Production of IFN- and IL-4 The cell-mediated immunity produced in the immunized mice was evaluated by measuring the amount of

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Fig. 4. Serum rSAG1-specific IgG titers. Mice were immunized s.c. twice at a 2-week interval with 50 ␮g Rg1 alone, 100 ␮g rSAG1 alone as controls, or 100 ␮g rSAG1 in combination with Rg1 (10 ␮g, 50 ␮g and 100 ␮g). Serum samples were prepared at weeks 0, 2, 4, 6, and 8 post-primary vaccinations. Results are expressed as means of the OD 492 ± SD (n = 3) and significant differences with control group were designated as *P < 0.05.

cytokines (IFN-␥ and IL-4) released in the supernatants of the cultures of rSAG1-stimulated spleen cells. The spleen cells from mice immunized with rSAG1 formulated with different doses of adjuvants produced significantly higher levels of IL-4 and IFN-␥ than those from the PBS control group (Table 1). Spleen cells from the control groups did not show detectable cytokine expression when stimulated with rSAG1. 3.5. Protection of mice against challenge with T. gondii following vaccination To test whether Rg1 can help recombinant protein SAG1 induce effective protection against T. gondii infection, the immunized mice were intraperitoneally challenged

Table 1 Cytokine production by spleen cells of immunized ICR mice following in vitro antigenic stimulation by rSAG1. Group

Cytokine production (mean ± SD)

Saline rSAG1/PBS rSAG1/10 ␮g Rg1 rSAG1/50 ␮g Rg1 rSAG1/100 ␮g Rg1

25 33 34 49 54

IL-4 (pg/ml) ± ± ± ± ±

7 5 2 11* 14*

IFN-␥ (pg/ml) 62.0 237.1 286.0 412.0 473.0

± ± ± ± ±

9 39* 45* 66* 110*

Mice were immunized s.c. on day 1 and 14 and spleen cells were collected 2 weeks after the last immunization. Cell-free supernatants were harvested and assayed for IL-4 activity at 24 h, and IFN-␥ activity at 96 h. The values are presented as means ± SD (n = 3), and significant differences with control group were designated as *P < 0.05.

with 50 tachyzoite of T. gondii 4 weeks after last immunization (Fig. 6). No difference was observed among the control groups. However, immunization of mice with rSAG1 and Rg1 increased their survival time (±days) com-

Fig. 5. Determination of the specific anti-SAG1 IgG subclass profile in the sera of immunized ICR mice. Sera were collected on day 28 after first immunization and analyzed by ELISA using STAg. Results are expressed as means of the OD 492 ± SD (n = 3) and significant differences with control group were designated as *P < 0.05.

Fig. 6. Survival rate of mice immunized with Rg1, rSAG1 and Rg1 of different dosages after challenging with T. gondii. Mice immunized with saline were included as negative control. The mice were intraperitoneally challenged with 50 tachyzoites of T. gondii 4 weeks after last immunization and observed for mortality (n = 10 mice per group).

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pared with control mice which died within 6 days of challenge. 4. Discussion In the present study, we have compared the effect of immunization with or without ginsenosides (Rg1) to ascertain whether stronger protective immunity against T. gondii infection could be induced by the addition of Rg1. From the results, mice immunized with T. gondii recombinant SAG1 and Rg1 are partially protected against a lethal dose of the RH strain of T. gondii and survived longer than that in the control groups. Recombinant protein antigens may help overcome the toxicity concerns associated with many of the traditional vaccines prepared with live, attenuated or killed pathogens. However, recombinant proteins are often weakly immunogenic or non-immunogenic on their own, and a vaccine adjuvant is usually needed to enhance the resultant immune responses (Sloat et al., 2010). A recent investigation has revealed that ginseng extract Rg1 has adjuvant properties that can improve immune responses (Sun et al., 2007). Chemical analysis has shown that Rg1 is found not only in the root but also in the stems and leaves of P. ginseng (Sun et al., 2008). This discovery has greatly decreased the cost of Rg1 production. Because of its adjuvant property, safety, and relatively low cost, Rg1 deserves further study of its effects on host immunity. The cellular immune response plays an important role in the control of multiplication and spread of T. gondii infection (Rosenberg et al., 2009). In the present study, vaccinating mice with rSAG1 and Rg1 (50 ␮g, 100 ␮g) induced significant T. gondii specific splenocyte proliferation compared with controls and ELISA assay of IgG isolates. Cytokine production showed that Rg1 (50 ␮g, 100 ␮g) can assist rSAG1 induce higher levels of cellular responses. The increased Th1 and Th2 immune responses reported here might be attributed to the immunomodulatory effect of Rg1 by enhancing activity of T helper cells and NK cells responsive to given antigens. To date, the mechanisms that Rg1 mediates its adjuvant effects are not fully understood. Enhanced immune responses by ginseng saponins have also been found previously in other studies. Sun et al. (2008) showed that Rg1 could induce production of Th1 cytokines in the absence of antigen. Some researchers observed that intraperitoneal injection of Rg1 in mice resulted in predominantly IFN-␥ and IL-2 responses which indicates that the stimulation of innate immune responses could be a potential mechanism by which Rg1 mediates its potent adjuvant effects (Lee and Han, 2006). Although cellular immunity is considered the most important part of the immune response to T. gondii, antibodies do play a role in limiting its spread because macrophages kill intracellular parasites coated with antibodies (Sibley et al., 1993). Immunization of mice with rSAG1and Rg1 (10 ␮g, 50 ␮g, 100 ␮g) resulted in the development of higher levels of total specific IgG antibodies as measured by ELISA. The enhanced IgG response persisted at least for 20 weeks (data not shown). Comparing three dif-

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ferent dosages of Rg1 (10 ␮g, 50 ␮g and 100 ␮g), we found that immunogenicity was dependent on the dosage of Rg1. When the vaccination dosage was increased, the antibody level was enhanced. However, this increasing tendency with the higher dosage is limited. A dosage of 50 ␮g Rg1 and 100 ␮g rSAG1 can induce almost the same level of antibody as 100 ␮g and 100 ␮g rSAG1 do. Ginsenoside Rg1 is one of the ginseng saponins identified in P. ginseng (Sun et al., 2006). Enhanced immune responses by ginseng saponins have also been found previously in other studies. For examples, Song et al. (2009) have recently observed an enhanced IgG response in mice injected with inactivated foot-andmouth disease virus antigen in combination with saponins isolated from ginseng stem and leaf; Rivera et al. (2003b) have found an increased specific HI titers in guinea pigs immunized with co-administration of porcine parvovirus antigen with ginseng saponin. During the evaluation of protection potency, highly virulent T. gondii RH strain was used for challenging infection. A partial protection was obtained in immunized mice, the mice imuunized with rSAG1 and Rg1 (50 ␮g, 100 ␮g) survived longer comparing with the control group. No prior vaccine has been shown to completely protect against intraperitoneally challenge with the RH strain of T. gondii (Cong et al., 2008). The RH strain belongs to the type I genotype which is highly virulent in mice, with a lethal dose of a single viable parasite. The ability of type I strains to uniformly cause lethal infections in mice is not due to the direct effect of the parasite, but is attributed to its rapid ability to achieve tissue burden levels with a lethal outcome, which is also associated with excessive serum levels of Th1 cytokines (Liu et al., 2010). In future experiments, it would be helpful to validate the efficacy of DNA immunization by comparison of the brain tissue cyst burden in vaccinated and control groups using T. gondii strains with different virulence (Peng et al., 2009). A possible explanation for the increased survival of RH infected mice after immunization could be that the initial Th2 response induced by the immunization serve to down regulated the Thl response induced by challenge, and thereby prevent tissue inflammation and pathology by a too strong or long lasting Thl response (Petersen et al., 1998). The present study comprehensively evaluated the immunogenicity and protection potency of recombinant protein SAG1 with the help of ginsenoside Rg1. By the addition of Rg1, this vaccine was able to elicit a stronger humoral and cellular immune response as well as increased survival time of ICR mice challenged with the lethal RH strain tachyzoites, compared with controls. Ginsenoside Rg1 should provide a promising effect for vaccine candidate against toxoplasmosis, deserves further evaluation and development in other animal species.

Acknowledgements This work was supported by a grant from the National Special Research Programs for Non-Profit Trades (Agriculture) (no. 200803017), the Science and Technology Department of Zhejiang Province (no. 2009C12069 and no. 2006C22043).

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