Vaccination with Toxoplasma gondii SAG-1 Protein Is Protective against Congenital Toxoplasmosis in BALB/c Mice but Not in CBA/J Mice

Mice.

Male and female inbred JRJ BALB/c mice, male and female inbred CBA/J mice, and female outbred OF1 Swiss mice were obtained from Centre d'Elevage R. Janvier (Le Genest St. Isle, France).

Parasite.

Cysts of the avirulent Me49 strain of T. gondii were obtained from the brains of orally infected CBA/J mice and prepared as previously described (3). Tachyzoites of the virulent RH strain of T. gondii were harvested from peritoneal fluid samples from Swiss OF1 mice infected with T. gondii intraperitoneally and used to prepare the T. gondii lysate antigen (TLA) as previously described (27).

Mouse immunization.

Female BALB/c and CBA/J mice (15 mice/group) were used at 8 to 10 weeks of age. Recombinant SAG1 protein expressed in Escherichia coli was kindly provided by Roche Diagnostics, Basel, Switzerland. Mice were immunized subcutaneously twice a week with 1 μg of SAG1 in sterile lipopolysaccharide-free saline (cumulative dosage, 4 μg). Control mice were injected with saline.

Congenital infection model.

Mice were allowed to mate 4 weeks after the last immunization. Females were placed in the male's bedding for 48 h to synchronize the estrus and were then caged. Two females were placed in a cage with one male for one night. The following day was designated day 1. The pregnant females were infected perorally on day 12 of pregnancy with 10 cysts of T. gondii Me49 strain. In order to avoid possible T. gondii contamination of the pups through lactation, pregnant females were sacrificed on the last day of gestation, day 19 (day 7 postinfection), and fetuses were aseptically removed for maternofetal transmission studies. For immunological studies, the blood and spleens of the mothers were removed under sterile conditions. Each experiment was repeated three times, and the results shown here are from one representative experiment.

Detection of congenital infection.

Fetuses and placentas were homogenized separately in 1-ml portions of phosphate-buffered saline (PBS), pH 7.2, and inoculated intraperitoneally into Swiss mice. Five weeks later, the Swiss mice were bled and tested for specific T. gondii antibodies using an immunofluorescence assay. Briefly, sera diluted 1/25 in PBS were applied on slides containing formalin-fixed T. gondii tachyzoites (bioMérieux, Marcy l'Etoile, France) for 25 min at 37°C, and fluorescein isothiocyanate-labeled anti-mouse polyvalent immunoglobulin (immunoglobulin G [IgG], IgA, and IgM) conjugates (Sigma, St. Louis, Mo.) diluted 1/125 in PBS were then added for 25 min at 37°C.

Measurement of maternal antibody response.

Specific anti-SAG1 IgG1 and IgG2a antibody titers were measured using an enzyme-linked immunosorbent assay adapted from the assay in reference 17. IgG titers were expressed as the last dilution giving an absorbance value that was twice as much as the mean absorbance value for three negative controls.

Measurement of maternal cytokine production.

Spleen cells were prepared as described previously (7). Cells were stimulated with TLA (1 μg/ml). Positive controls were assayed with concanavalin A (2 μg/ml) in all experiments (data not shown). Culture medium was used for negative controls. The concentrations of gamma interferon (IFN-γ), interleukin-10 (IL-10), and IL-4 were measured in the sera and in spleen cell supernatants using OptEIA mouse sets (BD Biosciences Pharmingen) in 96-well microtiter plates (Nunc).

Statistical analysis.

Differences in congenital transmission were evaluated by Fisher’s exact test, and differences in the maternal immune response were evaluated by Student’s t test. A P of <0.05 was considered significant.

Maternofetal transmission of T. gondii is reduced in BALB/c mice and increased in CBA/J mice vaccinated with SAG1.

Vaccination did not modify the number of fetuses per litter (7.8 ± 0.7 in the vaccinated group versus 8.2 ± 1.0 in the vehicle-treated control group in BALB/c mice; 7.0 ± 2.0 in the vaccinated group versus 8.3 ± 0.7 in the vehicle-treated control group in CBA/J mice). No abortions were observed. The results for maternofetal transmission of T. gondii on day 19 of gestation (day 7 postinfection) are shown in Fig. 1. In BALB/c mice, vaccination with SAG1 significantly reduced transmission of the parasite to the fetuses in comparison with the vehicle-treated control animals (14 of 47 fetuses positive [29.8%] versus 21 of 33 fetuses positive [63.6%]; P = 0.0033). Conversely, in CBA/J mice, transmission of the parasite was increased in the vaccinated group in comparison with the vehicle-treated control group (14 of 28 fetuses positive [50%] versus 8 of 25 fetuses positive [32%]; P = 0.2651). The difference was not significant and may be due to the limited number of fetuses.

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FIG. 1.

Maternofetal transmission of T. gondii in BALB/c and CBA/J mice. The presence or absence of T. gondii infection was assessed in the fetuses before delivery, on the last day (day 19) of gestation (day 7 of infection). Results are from one representative experiment. Vehicle-treated control BALB/c mice (four litters, 33 fetuses) and SAG1-vaccinated BALB/c mice (six litters, 47 fetuses) and vehicle-treated control CBA/J mice (three litters, 25 fetuses) and SAG1-vaccinated CBA/J mice (four litters, 28 fetuses) were studied in this experiment. The numbers of infected fetuses from vehicle-treated (control) versus SAG1-immunized mice were significantly different for the BALB/c mice (P = 0.0033) and are indicated by the two asterisks.

The protective effect of vaccination with T. gondii SAG1 protein has been well demonstrated in acute toxoplasmosis (1, 2, 22, 32). Until now, only one study used SAG1 protein (three 10-μg doses), and that study combined SAG1 with a type 1 adjuvant in a congenital toxoplasmosis model in guinea pigs and showed a reduction in the incidence of infection from 83 to 34% in neonates (13). Our results are the first demonstration that vaccination with a recombinant SAG1 protein without adjuvant reduces maternofetal transmission of T. gondii by more than 50% during primary infection in gestating BALB/c mice. Conversely, in CBA/J mice, passage of the parasite through the placenta is increased by more than 50%.

The influence of the genetic background on the immune response is well-established in a number of infectious models. In mice, the genes from the H-2 regions of the major histocompatibility complex have been related to different immune responses and susceptibilities to acute or chronic toxoplasmosis (28). BALB/c (H-2^d) mice are resistant to acute and chronic toxoplasmosis, whereas CBA/J (H-2^k) mice are resistant in the acute phase but susceptible in the chronic phase of infection. Moreover, primary toxoplasma infection induces a protective immune response in BALB/c mice, which prevents transplacental transmission of the parasite to the fetus during subsequent reinfection, making this a particularly relevant model with respect to human infection (25). Conversely, in CBA/J mice, primary infection does not provide any fetal protection in case of reinfection (unpublished data).

The reduction in vertical transmission of T. gondii in vaccinated BALB/c mice is associated with a mixed type 1- and type 2-specific maternal immune response.

The SAG1-vaccinated group produced high titers of specific anti-SAG1 IgG1 antibodies and low titers of IgG2a antibodies, whereas no anti-SAG1 antibodies were detected in the control group on day 7 postinfection (Table 1). Measurement of serum cytokines revealed significantly higher IFN-γ (P < 0.0001) and IL-10 concentrations (P = 0.0074) in the vaccinated group (Table 2). Low concentrations of IL-4 were measured in both groups. The study of the capacity for ex vivo production of cytokines by the spleen cells restimulated with TLA revealed a comparable profile (Table 3). Only the spleen cells from SAG1-vaccinated BALB/c mice produced IFN-γ and IL-10. IL-4 was not detected.

The increase in vertical transmission of T. gondii in vaccinated CBA/J mice is associated with a type 2 maternal immune response.

In the CBA/J mouse model, vaccination with SAG1 allowed significant production of specific anti-SAG1 IgG1 antibodies without production of IgG2a antibodies on day 7 postinfection (Table 1). No anti-SAG1 antibodies were detected in the vehicle-treated control group on day 7 postinfection. Measurement of serum cytokines revealed a significant increase in IL-10 and IL-4 concentrations (P < 0.0001 and P = 0.0036) without any significant change in IFN-γ concentration in the vaccinated animals compared to the control animals (Table 2). The ex vivo production of the same cytokines in the culture supernatant of spleen cells restimulated with TLA also revealed comparable IFN-γ concentrations in both groups, with a significant higher IL-10 concentration (P = 0.0052) in the SAG1-vaccinated animals (Table 3).

We had previously characterized the immune response to vaccination with SAG1 in noninfected, nonpregnant mice and obtained comparable profiles with a mixed immune response in BALB/c mice (IgG1 and IFN-γ but no IL-10 [unpublished data]) and a predominant type 2 immune response in CBA/J mice (17). The major role of the type 1 immune response has been widely demonstrated in acquired acute toxoplasmosis, during which IFN-γ and IL-2 have a protective effect via activation of cellular immunity (13). On the basis of these facts, numerous vaccine protocols using the SAG1 protein combined with type 1 adjuvants (2, 4, 17, 32) or, more recently, DNA vaccines including the SAG1 gene (1, 9) have demonstrated their efficacy in acute toxoplasmosis. The protection induced by SAG1 was shown to be transferable to naive mice through the CD8 T lymphocytes (16, 32). However, the data obtained in nonpregnant mice must be carefully extrapolated to congenital models, since pregnancy modifies the balance between the type 1 and type 2 immune responses by generating a type 2 environment essential to maintenance of the pregnancy (21). Our results suggest a protective effect of the type 1 response, since the detrimental action of vaccination in CBA/J mice was associated with an isolated stimulation of the type 2 response, whereas the protection obtained in the BALB/c model appears to be related to a stimulation of the type 1 response with an increase in IFN-γ, as also observed by Roberts et al. (26).

The mechanism of action of IFN-γ has not been elucidated in congenital toxoplasmosis. It may reduce the number of circulating parasites by activating the cytotoxic functions of the maternal phagocytes, NK cells, and T lymphocytes or act on the passage of the parasite through the placenta. In contrast with vaccination in nonpregnant mice, we could not detect any detrimental effect of IL-10 which was increased in both vaccinated CBA/J and BALB/c mice. Beside its major function of maintaining pregnancy (8, 21), the role of IL-10 in congenital toxoplasmosis is not clear. It has been shown to stimulate the cytotoxic activity of uterine NK cells (33) and may reduce adhesion of the parasite to the trophoblastic cells by reducing the expression of adhesion molecules induced by proinflammatory cytokines on the surfaces of the trophoblastic cells, as already described for Plasmodium falciparum (18). Although the mechanisms of transplacental passage of T. gondii have not been elucidated, this passage may be under IL-4 control. The increased transplacental passage of T. gondii in vaccinated CBA/J mice was associated with an increase in serum IL-4 levels. This is consistent with the reduction in fetal infection observed in BALB/c IL-4^−/− mice (30).

In our models and as already demonstrated previously (12, 22), vaccination with SAG1 led to a marked production of specific antibodies. Although the roles of the antibodies during toxoplasmosis are controversial, some in vivo and in vitro studies suggest that they may participate in the protective response (14, 20, 31). However, most vaccination studies with SAG1 could not correlate antibody levels and vaccine efficiency (4, 12, 16), perhaps because they measured total IgG levels while IgG properties differ between subclasses. We measured high titers of IgG1 and no or weak production of IgG2a antibodies in the vaccinated groups despite IFN-γ production. This apparent discrepancy could reflect the response to SAG1 vaccination, which is known to induce mainly IgG1 (17, 22), and not the response to infection, since the antibodies were measured very early on day 7 after infection. This IgG1 production could also be stimulated by other cytokines, such as IL-5 or IL-21, that we did not study.

In conclusion, our results show that, in the BALB/c model, vaccination with the SAG1 protein reduces maternofetal transmission of T. gondii by 50% and that this protection is associated with a mixed type 1 and 2 maternal immune response. This balance between the type 1 and type 2 responses is a critical parameter in the event of infection during pregnancy, since it should block the vertical transmission of the parasite while at the same time maintaining the pregnancy. We are currently exploring the roles of humoral and cellular immunity in the protection conferred by vaccination against congenital toxoplasmosis. In addition, experiments are currently under way to provide a better understanding of the mechanisms involved in passage of the parasite through the placenta and those underlying the protection provided by primary infection.