Skip to main content
Infection and Immunity logoLink to Infection and Immunity
. 2000 Feb;68(2):969–972. doi: 10.1128/iai.68.2.969-972.2000

Intranasal Immunization with Toxoplasma gondii SAG1 Induces Protective Cells into Both NALT and GALT Compartments

F Velge-Roussel 1,*, P Marcelo 1, A C Lepage 1, D Buzoni-Gatel 1, D T Bout 1
Editor: J T Barbieri1
PMCID: PMC97233  PMID: 10639474

Abstract

Intranasal (i.n.) immunization with the SAG1 protein of Toxoplasma gondii plus cholera toxin (CT) provides protective immunity. The aim of this study was to analyze the cellular activation of several mucosal compartments after i.n. immunization. Cervical and mesenteric lymph node (CLN and MLN, respectively) lymphoid cell and intraepithelial lymphocyte (IEL) passive transfer experiments were performed with CBA/J mice immunized i.n. with SAG1 plus CT. CLN and MLN cells and IEL isolated 42 days after immunization conferred protective immunity on naive recipient mice challenged with strain 76K T. gondii, as assessed by the reduction in the number of brain cysts. There were proliferative specific responses in nose-associated lymphoid tissue and the CLN and MLN cells from mice immunized with SAG1 plus CT, but no cytokine was detectable. Thus, protective immunity is associated with a specific cellular response in the nasal and mesenteric compartments after i.n. immunization.


Infection with Toxoplasma gondii confers on the host lifelong protective immunity against reinfection. This suggests that prevention of toxoplasmosis is a realistic goal (22).

Studies showing the importance of systemic cell-mediated immunity in protection against toxoplasmosis are unequivocal (9, 11). Infection with T. gondii generally occurs via the oral route and triggers a cellular response in the gut (5, 6). Anti-T. gondii cellular mucosal immunity is strongly mediated by lymphocytes, mainly intraepithelial lymphocytes (IEL) (2, 19).

Some attempts to generate protective immunity against toxoplasmosis have used the peroral route (1). Since all mucosal surfaces have the same mucosal immune system, the intranasal (i.n.) route is a possible alternative for immunization (18). In a previous study, the use of SAG1, the major surface antigen of T. gondii, plus cholera toxin (CT) for i.n. immunization provided potent protection, reducing the cyst burden by 80% (8). Thus, protective immunity can be stimulated by i.n. immunization of the intestine, where the pathogen enters the host. This study was carried out to investigate the mucosal immunity induced by i.n. immunization with SAG1 plus CT.

Parasites.

Cysts of the 76K T. gondii strain were obtained from the brains of orally infected CBA/J mice (Janvier, St Genest/Lisle, France). Tachyzoites of the virulent strain RH of T. gondii were harvested from peritoneal fluid of Swiss OF1 mice infected intraperitoneally and were used to prepare the sonicated antigen (toxoplasmic extract) (6).

Mouse immunizations.

Eight CBA/J mice were immunized i.n. with 20 μg of T. gondii SAG1 plus 0.5 μg of CT (Sigma) at 10 μl per nostril under light ether anesthesia and boosted 4 weeks later in the same way. SAG1 was purified using an anti-SAG1 monoclonal antibody (MAb) affinity column (16), and the purity of the protein was demonstrated by sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis analysis in a PHAST system (Pharmacia), followed by silver staining of the gel (16).

Adoptive transfer of cells.

Immunized CBA/J mice were killed on day 15 after the boost, and cell suspensions were prepared from cervical and mesenteric lymph nodes (CLN and MLN, respectively) and the small intestine. IEL were prepared as previously described (4). Cells (5 × 105) were analyzed with an anti-CD8β MAb (53-5.8 clone; PharMingen) and an anti-Thy 1.2 MAb (53-2.1 clone; PharMingen) on a FACScan (Becton Dickinson, Mountain View, Calif.). Around 3 × 107 and 2 × 106 lymph node cells and IEL, respectively, were intravenously injected into CBA/J recipient mice (six per group).

The day after the adoptive transfer, recipient mice were infected orally with 80 cysts of T. gondii strain 76K. Mice that received IEL were challenged 4 days after cell transfer. Intracerebral cysts were counted 1 month later. Brains were removed and homogenized in 5 ml of phosphate-buffered saline. The average number of cysts per brain was determined by examining eight samples of each homogenate (10 μl each). Results were collected from at least six mice per group and were expressed as the average ± the standard deviation for each group. The analysis of variance (ANOVA) test was used to determine the statistical significance level of differences.

Proliferative T-cell response.

Single-cell suspensions were prepared 15 days after the boost by purifying the cells from CLN, MLN, and nasal tissue (NALT) from four immunized mice. To isolate NALT cells (27), mice were killed by ether asphyxia, the lower jaws were removed, and the palates were cut parallel to the septum and removed. The NALT from each side of the septum was gently removed with fine dissection forceps. Cell suspensions were prepared by passing the tissue suspended in RPMI 1640 medium through nylon net (Blutex; Tripett et Renaud) using a syringe. All cells (5 × 105) were incubated for 5 days with different antigen (toxoplasmic extract) concentrations in RPMI 1640 medium (Gibco) containing 2 mM l-glutamine, 1 mM sodium pyruvate, 50 μg of gentamicin per ml, 5 × 10−5 M 2β-mercaptoethanol, and 5% fetal calf serum. Proliferative responses were assessed over 18 h by measuring [3H]thymidine (18.5 kBq per well [Dositek]) incorporation. Incorporated radioactivity was measured in a liquid scintillation counter (LKB) following filtration on fiberglass filters. Results were expressed as the mean of triplicates ± the standard deviation.

CLN and MLN cells and IEL of i.n. immunized mice transfer protective immunity.

CBA/J mice are resistant to acute T. gondii infection but develop a large number of brain cysts. The protection conferred by adoptive transfer of lymphocytes collected on day 42 from organs of i.n. immunized mice was evaluated. One month after infection, CBA/J mice given MLN or CLN cells from mice immunized with SAG1 plus CT exhibited significantly fewer cysts (50 and 60% reduction; ANOVA test, P < 0.0003 and P < 0.01, respectively) than naive mice or mice given lymph node cells stimulated with CT alone in vivo (Fig. 1). Thus, lymphocytes from CLN and MLN were stimulated by i.n. immunization with SAG1 plus CT and participated in the protection of recipient mice against T. gondii challenge. IEL from mice immunized with SAG1 plus CT were also transferred to recipient mice (Fig. 2). One month after the challenge, the protection conferred by IEL was similar to that obtained with MLN and CLN cells (about 50%) compared to controls.

FIG. 1.

FIG. 1

Adoptive transfer of CLN and MLN primed with SAG1 plus CT to CBA/J mice. For MLN and CLN adoptive transfer, six naive mice were given around 3 × 107 cells from donor mice. They were challenged by the oral route with 80 cysts of strain 76K T. gondii 1 day after the transfer. Protection was assessed by counting the cysts in the brain 1 month later. Statistical significance was calculated by the ANOVA test as indicated by asterisks. For the SAG1-plus-CT group compared to the phosphate-buffered saline (PBS) or CT group: ∗∗, P < 0.0003; ∗, P < 0.01. The results are representative of two independent experiments.

FIG. 2.

FIG. 2

Adoptive transfer of SAG1-plus-CT-primed IEL to CBA/J mice. Six naive CBA/J mice were given 2 × 106 primed or unprimed IEL from donors and challenged by the oral route with 80 cysts of strain 76K T. gondii 4 days after the transfer. These results are representative of two independent experiments. ∗∗, P < 0.01. PBS, phosphate-buffered saline.

Proliferative response of NALT, CLN, and MLN cells.

In order to study activation of the mucosal compartments further, proliferative T-cell responses of NALT, CLN, and MLN were tested. Table 1 shows that a stronger specific proliferative response of NALT cells from the group immunized with SAG1 plus CT was observed when they were incubated with toxoplasmic extract compared to those from the group immunized with CT. Also, cells from the MLN and CLN of these mice also produced a cellular response to toxoplasmic extract incubation (Table 1). Thus, i.n. immunization seemed to induce a specific T-cell response in NALT, CLN, and MLN.

TABLE 1.

Proliferation of lymphocytes from the NALT, CLN, and MLN of mice immunized i.n.a

Cell Immunization group Δcpmb of toxoplasmic extract (10 μg/ml)
NALT SAG1 + CT 12,223 ± 320c
CT  0
CLN SAG1 + CT 3847 ± 481d
CT 77 ± 65
MLN SAG1 + CT 10,013 ± 1276e
CT 797 ± 261
a

Proliferative responses of cells from NALT, CLN, and MLN of mice immunized with SAG1 plus CT cultured in vitro with T. gondii total extract for 4 days were measured. Cells were taken from organs on day 42 after immunization, and proliferation was evaluated by measuring [3H]thymidine incorporation. Welsh's test of Instat gave the levels of statistical significance. 

b

Δcpm was calculated as the counts per minute obtained in the presence of stimulating antigen minus the counts per minute in medium alone. 

c

P = 0.0002. 

d

P = 0.0055. 

e

P = 0.0066. 

The cells from mice immunized with SAG1 plus CT were purified from NALT, CLN, and MLN and stimulated with toxoplasmic extract to test their capacity to produce interleukin-10 or gamma interferon. A small amount of gamma interferon (20 to 67 pg/ml) was produced, but no interleukin-10 could be detected (data not shown).

SAG1-primed IEL have mainly the CD8αβ+ phenotype.

IEL are mainly CD8α+ cells, which comprise CD8αα+ and CD8αβ+ cells (12, 15). A fluorescence-activated cell sorter analysis was performed to explore the change within the IEL population after i.n. immunization (Fig. 3). There was an increase in the Thy-1.2+ and CD8β+ IEL subsets on day 11 following the boost in the groups immunized with SAG1 plus CT and CT. The phenotypic change that occurs within the IEL population after SAG1 immunization might indicate that intestinal IEL are sensitized by immunization by the i.n. route.

FIG. 3.

FIG. 3

Phenotypic analysis of gut IEL subsets from CBA/J mice after immunization with SAG1 plus CT. Lymphocyte suspensions were prepared from four mice, and the relative percentages of Thy1.2+ or CD8β+ fluorescent IEL were determined by cytofluorimetric analysis of 5 × 105 cells.

Although NALT contains more naive than memory T helper cells (28), there is specific local proliferation after i.n. immunization. Moreover, proliferation of CLN may indicate that this specific response was extended to the systemic compartment (20, 28). MLNs also showed specific proliferation. The MLN response might indicate that the stimulation was extended to the common mucosal compartment, and thus, NALT appears to be the origin of a specific mucosal response (7, 8, 21, 24).

IEL are important in the gut mucosal immune system, as they are the first functional barrier to toxoplasmosis, having cytotoxic activity and secreting cytokines (2, 4, 19). Our previous studies demonstrated that the CD8αβ+ subset of IEL from mice infected with T. gondii 11 days before can protect naive mice from a T. gondii challenge (2, 19). The increase in the number of Thy 1.2+ CD8β+ IEL following SAG1 immunization indicates IEL activation. As expected, IEL primed with SAG1 plus TC conferred less protective immunity on recipient mice than did IEL from infected mice (2). Nevertheless, the capacity of primed IEL to protect could be crucial because of their gut location and furthermore because they might be functional early following infection (3).

Since MLN are the first lymphatic organization draining the gut, activation of the lymphocytes at this site might account for protection. Like IEL, MLN lymphocytes might migrate to the gut after their transfer and thus help in blocking parasite invasion (3, 13, 17, 25). Immunization by the i.n. route induces a subset of T helper cells in the CLN and MLN that are implicated in protection (50 and 60%, respectively). Passive transfer of SAG1-primed splenocytes conferred only 30% cyst reduction, whereas 80% cyst reduction was obtained by i.n. immunization with SAG1 plus CT (8). Protective status conferred by i.n. immunization with SAG1 plus CT might be due to the combination of the mucosal immunity represented by the primed IEL and the MLN cells, while the systemic immunity was represented by the spleen (8).

Many studies have used the i.n. route for immunization with potential vaccines with or without CT (10, 14, 23, 26). Vaccinations with aeropharyngeal pathogens have been shown to be successful. They were shown to trigger both mucosal and systemic T- and B-cell responses (20, 28) and thus can be used to target pathogens that invade far from the immunization site, such as the gut or vagina (7, 8, 21, 24). Indeed T. gondii naturally invades the intestine of its host and can be partially controlled by i.n. immunization with the protein SAG1 plus CT (8). Our work has demonstrated that this route of immunization induces mucosal cell populations able to participate in protective immunity. Thus, mucosal immunization, particularly via the i.n. route, has considerable potential for triggering immunity in all mucosal and systemic compartments.

Acknowledgments

This work was supported by a grant from La Ligue (Comité d'Indre et Loire).

We thank D. Tabareau for typing assistance and R. Magné for assistance in purifying SAG1. The English text was edited by O. Parkes and K. Day-Wilson.

REFERENCES

  • 1.Bourguin I, Chardès T, Bout D T. Oral immunization with Toxoplasma gondii antigens in association with cholera toxin induces enhanced protective and cell-mediated immunity in C57BL/6 mice. Infect Immun. 1993;61:2082–2088. doi: 10.1128/iai.61.5.2082-2088.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Buzoni-Gatel D, Lepage A C, Dimier-Poisson I H, Bout D T, Kasper L H. Adoptive transfer of gut epithelial lymphocytes protects against murine infection with Toxoplasma gondii. J Immunol. 1996;158:5883–5889. [PubMed] [Google Scholar]
  • 3.Buzoni-Gatel D, Debabi H, Moretto M, Dimier-Poisson I H, Lepage A C, Bout D T, Kasper L H. Intraepithelial lymphocytes traffic to the intestine and enhance resistance to Toxoplasma gondii infection. J Immunol. 1999;162:5846–5852. [PubMed] [Google Scholar]
  • 4.Chardès T, Buzoni-Gatel D, Lepage A C, Bernard F, Bout D T. Toxoplasma gondii oral infection induces specific cytotoxic CD8α/β+ Thy-1+ gut intraepithelial lymphocytes, lytic for parasite-infected enterocytes. J Immunol. 1994;153:4596–4603. [PubMed] [Google Scholar]
  • 5.Chardès T, Velge-Roussel F, Mévélec P, Mévélec M-N, Buzoni D, Bout D. Mucosal and systemic cellular immune responses induced by Toxoplasma gondii in cyst orally infected mice. Immunology. 1993;78:421–429. [PMC free article] [PubMed] [Google Scholar]
  • 6.Chardès T, Bourguin I, Mévélec M-N, Dubremetz J-F, Bout D. Antibody response to Toxoplasma gondii in sera, intestinal secretions, and milk from orally infected mice and characterization of target antigens. Infect Immun. 1990;58:1240–1246. doi: 10.1128/iai.58.5.1240-1246.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Corthésy-Theulaz I E, Hopkins S, Bachmann D, Saldinger P F, Porta N, Haas R, Zheng-Xin Y, Meyer T, Bouzourène H, Blum A L, Kraehenbuhl J-P. Mice are protected from Helicobacter pylori infection by nasal immunization with attenuated Salmonella typhimurium phoPc expressing urease A and B subunits. Infect Immun. 1998;66:581–586. doi: 10.1128/iai.66.2.581-586.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Debard N, Buzoni-Gatel D, Bout D. Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection. Infect Immun. 1996;64:2158–2166. doi: 10.1128/iai.64.6.2158-2166.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Denkers E Y, Gazzinelli R T. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin Microbiol Rev. 1998;11:569–588. doi: 10.1128/cmr.11.4.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Gallichan W S, Rosenthal K L. Specific secretory immune responses in the female genital tract following intranasal immunization with a recombinant adenovirus expressing glycoprotein B of herpes simplex virus. Vaccine. 1995;13:1589–1595. doi: 10.1016/0264-410x(95)00100-f. [DOI] [PubMed] [Google Scholar]
  • 11.Gazzinelli R T, Hakim F T, Hieny S, Shearer G M, Sher A. Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-γ production and protective immunity induced by an attenuated Toxoplasma gondii vaccine. J Immunol. 1991;146:286–292. [PubMed] [Google Scholar]
  • 12.Guy-Grand D, Cerf-Bensussan N, Malissen B, Malissen-Seris M, Briottet C, Vassili P. Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J Exp Med. 1991;173:471–476. doi: 10.1084/jem.173.2.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Hamann A, Andrew D P, Jablonski-Westrich D, Holzmann B, Butcher E C. Role of α4-integrins in lymphocyte homing to mucosal tissue in vivo. J Immunol. 1994;152:3282–3289. [PubMed] [Google Scholar]
  • 14.Holmgren J, Lycke N, Czerkinsky C. Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems. Vaccine. 1993;11:1179–1184. doi: 10.1016/0264-410x(93)90039-z. [DOI] [PubMed] [Google Scholar]
  • 15.Kaiserlian D. The intestinal epithelial cell: a nonconventional type of antigen-presenting cell. Dyn Nutr Res. 1995;4:32–39. [Google Scholar]
  • 16.Kasper L H, Crabb J H, Pfefferkorn E R. Purification of major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody. J Immunol. 1983;130:2407–2412. [PubMed] [Google Scholar]
  • 17.Kim S-K, Reed D S, Heath R W, Carbone F, Lefrancois L. Activation and migration of CD8 T cells in the intestinal mucosa. J Immunol. 1997;159:4295–4306. [PubMed] [Google Scholar]
  • 18.Kraehenbuhl J P, Neutra M R. Molecular and cellular basis of immune protection of mucosal surfaces. Physiol Rev. 1992;72:221–242. doi: 10.1152/physrev.1992.72.4.853. [DOI] [PubMed] [Google Scholar]
  • 19.Lepage A, Buzoni-Gatel D, Bout D, Kasper L H. Gut-derived intraepithelial lymphocytes induce longterm immunity against Toxoplasma gondii. J Immunol. 1998;161:4902–4908. [PubMed] [Google Scholar]
  • 20.McGhee J R, Mestecky J, Dertzbaugh M T, Eldridge J H, Hirasawa M, Kiyono H. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine. 1992;10:75–88. doi: 10.1016/0264-410x(92)90021-b. [DOI] [PubMed] [Google Scholar]
  • 21.Nardelli-Haefliger D, Roden R B S, Benyacoub J, Sahli R, Kraehenbuhl J-P, Schiller J T, Lachat P, Potts A, de Grandi P. Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice. Infect Immun. 1997;65:3328–3336. doi: 10.1128/iai.65.8.3328-3336.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Remington J S, Desmonts G. Toxoplasmosis. In: Remington J S, Klein J O, editors. Infectious diseases of the fetus and the newborn infant. 3rd ed. Philadelphia, Pa: The W. B. Saunders Co.; 1995. pp. 140–267. [Google Scholar]
  • 23.Tamura S I, Funato H, Nagamine T, Aizawa C, Kurata T. Effectiveness of cholera toxin B subunit as an adjuvant for nasal influenza vaccination despite pre-existing immunity to CTB. Vaccine. 1989;7:503–505. doi: 10.1016/0264-410x(89)90273-9. [DOI] [PubMed] [Google Scholar]
  • 24.Ugozzoli M, O'Hagan D T, Ott G S. Intranasal immunization of mice with simplex herpes virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody response. Immunology. 1998;93:563–571. doi: 10.1046/j.1365-2567.1998.00441.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Williams M B, Butcher E C. Homing of naive and memory T lymphocyte subsets to Peyer's patches, lymph nodes, and spleen. J Immunol. 1997;159:1746–1752. [PubMed] [Google Scholar]
  • 26.Wu H-Y, Russell M W. Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit. Infect Immun. 1993;61:314–322. doi: 10.1128/iai.61.1.314-322.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Wu H-Y, Nikolova E B, Beagley K W, Russell M W. Induction of antibody-secreting cells and T-helper and memory cells in murine nasal lymphoid tissue. Immunology. 1996;88:493–500. doi: 10.1046/j.1365-2567.1996.d01-690.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Wu H-Y, Russell M W. Nasal lymphoid tissue, intranasal immunization and compartmentalization of the common mucosal immune system. Immunol Res. 1997;16:187–201. doi: 10.1007/BF02786362. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES