Evaluation of Toxoplasma gondii as a live vaccine vector in susceptible and resistant hosts

Abstract

Background: Toxoplasma gondii has been shown to trigger strong cellular immune responses to heterologous antigens expressed by the parasite in the inbred mouse model. We studied the immune response induced by T. gondii as an effective vaccine vector in chickens and rabbits.

Results: T. gondii RH strain was engineered to express the yellow fluorescent protein (YFP) in the cytoplasm. A subcutaneous injection of the transgenic T. gondii YFP in chickens afforded partial protection against the infection of transgenic E. tenella YFP. T. gondii YFP induced low levels of antibodies to YFP in chickens, suggesting that YFP specific cellular immune response was probably responsible for the protective immunity against E. tenella YFP infection. The measurement of T-cell response and IFN-γ production further confirmed that YFP specific Th1 mediated immune response was induced by T. gondii YFP in immunized chickens. The transgenic T. gondii stimulated significantly higher YFP specific IgG titers in rabbits than in chickens, suggesting greater immunogenicity in a T. gondii susceptible species than in a resistant species. Priming with T. gondii YFP and boosting with the recombinant YFP can induce a strong anti-YFP antibody response in both animal species.

Conclusions: Our findings suggest that T. gondii can be used as an effective vaccine vector and future research should focus on exploring avirulent no cyst-forming strains of T. gondii as a live vaccine vector in animals.

Figure 1

Expression of yellow fluorescent protein (YFP) by T. gondii transfected with the pTgmicYFP plasmid. A, Plasmid map of pTgmicYFP. B, Western blot of YFP expressed by the transgenic T. gondii YFP (TgYFP) and wild type T. gondii (WT) using the rabbit anti-GFP antisera and a sheep anti-rabbit IgG HRP-conjugate. C, T. gondii YFP in murine macrophages observed by fluorescence microscopy, confirming the location of YFP by T. gondii YFP. D, Fitness of T. gondii YFP and wild type T. gondii at a ratio of 7:3 in mice.

Figure 2

The partial protection against challenge with Eimeria tenella YFP (EiYFP) in chickens which were immunized with T. gondii. Leghorn chickens immunized s.c. with two doses of T gondii YFP (TgYFP) or wild type RH strain (5 × 10⁶ for the initial immunization and 10⁷ for the booster dose) or 160 μg recombinant YFP emulsified in Freund complete adjuvant (FCA). The immunized chickens were challenged with 10³ transgenic E. tenella YFP 15 days after the booster immunization. A. Fluorescence images of transgenic E. tenella YFP oocysts. B. Fecal oocyst counts in chickens immunized with T. gondii YFP (TgYFP) or wild type T. gondii tachyzoites (Wild-Tg) or recombinant YFP emulsified in FCA (rYFP) or complete cytomix buffer (un-immunized control) and challenged with the transgenic E. tenella (EiYFP). C. Histopathology of the cecum from chickens described above in Figure B. C₁ Shedding of mucosal cells (arrow); C₂ Inflammatory cells infiltration of lamina propria (arrow).

Figure 3

Priming of AA broiler chickens and rabbits with T. gondii YFP (TgYFP) elicited antigen specific antibody responses. A, YFP specific IgG in chicken sera (1: 25) 10 days after immunization by s.c. injection of 5 × 10⁶ transgenic (TgYFP), wild type (WT) T. gondii tachyzoites or complete cytomix buffer (CCB). B, YFP specific IgG (1:25) 10 days after immunization by s.c. or i.m. injection. C, Serum YFP specific antibody titers in chickens 10 days after a s.c. injection of transgenic T. gondii tachyzoites (TgYFP) or i.m. injection of recombinant YFP (rYFP) protein expressed in E. coli. D, Kinetics of serum antibodies to YFP and tachyzoite antigens in rabbits immunized with the transgenic T. gondii. * P < 0.05, ** P < 0.01.

Figure 4

Serum YFP specific antibody titers in chickens and rabbits immunized with T. gondii YFP and/or rYFP protein. Chickens and rabbits were primed s.c. with 5 × 10⁶ and 1 × 10⁷ T. gondii YFP tachyzoites, respectively. The primed chickens were boosted i.m. 20 days later with 160 μg of rYFP emulsified in Freund complete adjuvant, and the primed rabbits were boosted i.m. 35 days later with 500 μg of recombinant YFP emulsified in Freund complete adjuvant. Serum YFP specific IgG titers were measured 10 days after the booster immunization.

Figure 5

IFN-g and IL-4 mRNA transcripts level of spleen lymphocytes. The spleen lymphocytes were isolated on day 12 after immunization, and restimulated in culture for 16 h with rYFP, IFN-g and IL-4 mRNA transcripts level was measured by Real-time RT-PCR.