Bacillus subtilis Spores Displaying Toxoplasma gondii GRA12 Induce Immunity Against Acute Toxoplasmosis

Hongchao Sun ¹ Xiufang Yuan ¹ Wei Zhou ^2* Zhijin Zhou ^2* Fei Su ^1* Yuan Fu ^1* Lili Hao ^3* Xin Liu ^3* Xin Zhou ^1* Shiyi Ye ^1* Lihua Xu ^1* Bin Yu ^1* Junxing Li ^1* Tuan-yuan Shi ^1*

• ¹ Zhejiang Academy of Agricultural Sciences, Hangzhou, China
• ² Zhejiang Provincial Animal Disease Prevention and Control Center, Hangzhou, Jiangsu Province, China
• ³ Southwest Minzu University, Chengdu, Sichuan Province, China

Background: Toxoplasma gondii can infect almost all warm-blooded animals. The drugs used to treat toxoplasmosis have the disadvantage of being toxic and resistance, and the only licensed vaccine entails a risk of virulence restoration. The development of a safe and effective vaccine is urgently needed. Bacillus subtilis has been used as a potential vaccine expression vector. T. gondii GRA12 is a key virulence factor that resists host innate immunity. Methods: A recombinant spore named rBS-GRA12 was constructed by fusing the T. gondii GRA12 protein to the B. subtilis coat protein B. rBS-GRA12 spores were identified by PCR, WB, IFA, amylase activity, and ultrastructural analysis. Immunological experiments were then conducted to assess the immunoprotective effects. Groups of mice immunized with rBS-GRA12 (10 6 , 10 8 , or 10 10 colony-forming units), GRA12 protein emulsified with Freund's adjuvant (FA+GRA12), Freund's adjuvant alone (FA), phosphate buffered saline (PBS), or wild-type B. subtilis spores (WT). Splenocyte proliferation, antibodies, and cytokine expression levels were used to assess immune responses. All groups were inoculated with T. gondii, and survival times and parasite loads in tissues were used to assess protective effects . Results: Amylase activity assays confirmed the generation of recombinant B. subtilis. PCR, WB and IFA confirmed that the rBS-GRA12 spores expressed GRA12. Observation of rBS-GRA12 spores via TEM and SEM indicated that GRA12 expression had no effect on spore morphology or structure. Splenocyte proliferation was significantly greater in all three rBS-GRA12 groups than in the FA+GRA12 group, and IgG and IgG2a subclass titers were higher. Substantial production of IFN-γ, IL-12, and an increase in IL-4 production were evident in the rBS-GRA12-10 8 group. sIgA levels were significantly elevated in all three rBS-GRA12 groups than in the FA+GRA12 group and the control groups. Brain and liver tissues parasite loads were significantly lower in the rBS-GRA12 groups than in any other group. Compared to all other groups, mice in the rBS-GRA12 groups exhibited longer survival times when challenged with T. gondii. Conclusion: Mice immunized with rBS-GRA12 exhibited higher levels of immune responses. These results provide a new perspective for the development of T. gondii vaccines.