Yonsei Med J.  2007 Jun;48(3):396-404. 10.3349/ymj.2007.48.3.396.

Vaccination against Murine Toxoplasmosis Using Recombinant Toxoplasma gondii SAG3 Antigen Alone or in Combination with Quil A

• Lee YH
• Shin DW
• lee JH
• Nam HW
• Ahn MH

• KMID: 724093
• DOI: http://doi.org/10.3349/ymj.2007.48.3.396

Abstract

PURPOSE
Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.

Keyword

Toxoplasma gondii; recombinant SAG3 antigen; vaccination; Quil A

MeSH Terms

Animals
Antigens, Protozoan/genetics/*immunology/metabolism
Bacterial Proteins/genetics/immunology/metabolism
Blotting, Western
Enzyme-Linked Immunosorbent Assay
Female
Flow Cytometry
Immunoglobulin G/immunology
Interferon-gamma/metabolism
Mice
Mice, Inbred BALB C
Nitric Oxide/metabolism
Protozoan Proteins/genetics/immunology/metabolism
Recombinant Fusion Proteins/genetics/immunology/metabolism
Reverse Transcriptase Polymerase Chain Reaction
Saponins/*immunology
Toxoplasma/growth & development/*immunology
Toxoplasmosis, Animal/*immunology/metabolism/microbiology
Vaccination/*methods

Figure

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  Fig. 1 SDS-PAGE and glutathione-S-transferase (GST) detection of the rSAG3 protein of T. gondii. Lane 1, isopropyl β-D-thiogalactopyranoside (IPTG) induced GST; lane 2, non-induced; lane 3, induced GST fusion SAG3 (rSAG3); lane 4, purified rSAG3.

• 

  Fig. 2 Survival rates of BALB/c mice immunized with rSAG3 protein alone or in combination with Quil A after oral infection with a lethal dose of the T. gondii Me49 strain (1,500 cysts per mouse). Mice were observed daily until 4 weeks after T. gondii infection. Each group consisted of 10 mice.

• 

  Fig. 3 Brain cyst load of mice immunized with rSAG3 alone or in combination with Quil A. Three weeks after the last immunization, mice were orally challenged with a nonlethal dose of the T. gondii Me49 strain (20 cysts per mouse) and evaluated 4 weeks after infection. IC, infection control group; G, GST-immunized group; Q, Quil A-immunized group; G/Q, GST/Quil A-immunized group; rS3, GST fusion SAG3 (rSAG3)-immunized group; rS3/Q, rSAG3/Quil A-immunized group. Data are represented as the mean ± SE of 5 mice.

• 

  Fig. 4 IgG, IgG1 and IgG2a titers of sera from mice immunized with rSAG3 alone or in combination with Quil A. Sera from vaccinated mice were obtained and IgG subclasses measured by ELISA. C, untreated control group. The data are represented as the mean ± SE of 5 mice and are representative one of two separate experiments.

• 

  Fig. 5 Proliferative responses of splenocytes from mice immunized with rSAG3 protein alone or in combination with Quil A. Spleen cells were cultured in the presence of Toxoplasma lysate antigen (TLA) for 72h. Proliferation was assayed by [3H]-thymidine incorporation. The data are represented as the mean ± SE of 5 mice and are representative of two separate experiments.

• 

  Fig. 6 Percentages of T cell subsets in splenocytes from mice immunized with rSAG3 protein alone or in combination with Quil A. Splenocytes were stained with FITC-conjugated CD4, CD8 or γδ mAb, and then analyzed by FACScan. The data are represented as the mean ± SE of 5 mice and are representative of two separate experiments.

• 

  Fig. 7 Relative IFN-γ, TNF-α and IL-10 mRNA expression levels in splenocytes from mice immunized with rSAG3 alone or in combination with Quil A. Differences in the transcriptional level of all genes are expressed relative to the control group (assigned as 1). The data are represented as the mean ± SE of 5 mice and are representative of two separate experiments.

• 

  Fig. 8 Nitric oxide production in peritoneal macrophages from mice immunized with rSAG3 alone or in combination with Quil A. Culture supernatants were obtained from macrophage monolayers, and nitrite levels were measured by Griess assay. The data are represented as the mean ± SE of 5 mice and are representative of two separate experiments.

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