Intranasal Immunization With ESA And STAg Induces Protective Immune Responses Against Toxoplasma Gondii

Objective To determine the optimal condition of taking excreted/secreted antigens(ESA) by co-culture of Toxoplasma gondii tachyzoites and Vero cells.To study the mucosal and systemic immune responses and protection against Toxoplasma gondii after intranasal immunization with ESA taken by co-culture of Toxoplasma gondii tachyzoites and Vero cells, ESA taken from peritoneal fluids of infected mice and soluble tachyzoite antigen(STAg).Methods This study included three parts.The first part was to study the effects of different co-culture condition on ESA protein level.First,we investigate the effects on ESA protein level by different serum density and different culture time in medium containing serum. There were four groups divided by different serum density(0.5%,1%,2%,4%).In each group, there were four different culture time in medium containing serum(3h,6h,12h,24h).RH strain tachyzoites of Toxoplasma gondii and Vero cells were co-cultured in medium containing different serum density.After 3h,6h,12h or 24h,the medium was taken off and replaced by serum-free medium.On day 7 of co-culture,the culture supernate was collected and detected for protein level.Then we investigate the effects on ESA protein level by different co-culture time and different culture time in medium containing serum.There were four groups divided by different co-culture time(7d,9d,11d,13d).In each group,there were four different culture time in medium containing 1%serum(12h,24h,48h,72h).RH strain tachyzoites of Toxoplasma gondii and Vero cells were co-cultured in medium containing 1%serum.After 12h,24h,48h or 72h,the medium was taken off and replaced by serum-free medium.After 7d,9d,11d or 13d of co-culture,the culture supernate was collected and detected for protein level.The second part was to study the mucosal and systemic immune responses after intranasal immunization with ESA of two different origin and STAg.BALB/c mice were randomly divided into four groups and were intranasally immunized with 20μl PBS per mouse or 20μg ESA in vitro,ESA in vivo or STAg per mouse,respectively,twice at an intemal of 2 weeks.Mice were killed on day 14 and 44 after the last immunization,respectively.Spleen lymphocytes and intestinal intraepithelial lymphocytes(IEL)were counted.Serum IgG and slgA in intestinal washes were detected by ELISA.The third part was to observe the effects on protection of mice against Toxoplasma gondii by intranasal immunization with ESA of two different origin and STAg.BALB/c mice were randomly divided into four groups,and were intranasally immunized with 20μl PBS per mouse or 20μg ESA in vitro,ESA in vivo or STAg per mouse,respectively,twice at an internal of 2 weeks.All mice were challenged intragastrically with 1×10~4 tachyzoites per mouse on day 14 after the last immunization.The health condition of mice was observed and the weight of infected mice was recorded every day after challenge.Mice were killed on the 30th day after challenge,the tachyzoites in their spleens and brains were counted.Results RH strain tachyzoites of Toxoplasma gondii and Vero cells were co-cultured in different condition.When serum density was 1%and culture time in medium containing serum was 12h or 24h,ESA protein level was higher than other condition(P<0.05).When co-culture time was 13d and culture time in medium containing serum was 12h or 24h,ESA protein level was significantly higher than other condition(P<0.001).After intranasal immunization,the condition of mice in ESA in vivo group was not good after the last immunization,while mice in other groups were healthy.On day 14 after the last immunization,cell proliferation of spleen lymphocytes(P<0.05)and IEL(P<0.01)were observed in all antigen groups.On day 44 after immunization,spleen lymphocytes and IEL in two ESA groups were still higher than that of PBS group(P<0.05).The level of serum IgG in all antigen groups was higher than that of PBS group on both day 14(P<0.001)and day 44(P<0.05) after immunization.On day 14 after immunization,the level of sIgA in ESA in vitro group (P<0.05),ESA in vivo group(P<0.001)and STAg group(P<0.001)was higher than that of PBS group.On day 44 after immunization,the level of sIgA in two ESA groups was still higher than that of PBS group(P<0.05).After intranasal immunization,mice in ESA in vivo group appeared slight piloerection and lassitude,while mice in other groups were healthy.The weight of mice in PBS group gradually decreased after challenge,and going palliative 15 day later,while that of ESA in vitro group and STAg group(P<0.05)remained increasing,and that of ESA in vivo group did not show significant increase.The tachyzoite load in spleens and brains in all antigen groups was significantly lower than that of PBS group(P<0.05).Conclusion When RH strain tachyzoites of Toxoplasma gondii and Vero cells were cultured in medium containing 1%serum for 12h or 24h,and then in serum-free medium,after 13 days of co-culture,higher yield of ESA protein in the culture supetnate was obtained.Intranasally immunized with ESA in vitro,ESA in vivo or STAg can effectively induce the mucosal and systemic immune responses against Toxoplasma gondii,and the responses induced by two kinds of ESA can persist for a longer time than that of STAg.ESA from peritoneal fluids had toxic side effect on mice,ESA in vitro and STAg may be attractive candidate for Toxoplasma gondii vaccine.