Abstract
Bacillus subtilis (B. subtilis) spore can serve as an ideal vehicle for expressing heterologous antigens, and elicit specific immune responses by oral administration. In previous studies, we successfully constructed the recombinant B. subtilis spores expressing cysteine protease of Clonorchis sinensis (C. sinensis, B.s-CsCP), and confirmed that oral administration of B.s-CsCP could elicit good protective immune responses in mice. In this study, Gram staining was used to observe the morphology of B.s-CsCP in different form, and the storage of liquid spores and lyophilized spores at different temperatures was compared. The mice were orally immunized with three different doses of spores (2×108, 1×109, and 5×109 CFU/day) for three times in total at biweekly interval. Then, antibody levels of mice were measured, the safety of spores was evaluated, and the changes of gut microbiota after oral gavage of spores (1×109 dose) were investigated. Results showed that B. subtilis was a typical Gram-positive bacterium, and its spore had good resistance to chemical dye. Liquid B. subtilis spores resuspended in sterile water could be stored for a long time at 4 °C or below, while lyophilized spores could be well stored even at RT and better at lower temperatures. Oral administration of B. subtilis spores to mice could stimulate both local mucosal and systemic immune responses in a dose-dependent manner without toxic side effects. Besides, beneficial bacteria producing butyrate such as Odoribacter were increased, while potential pathogens such as Escherichia-Shigella were decreased in mice intestine. Therefore, our work further confirmed that B. subtilis spores expressing CsCP could be a promising oral vaccine against C. sinensis with the advantages of stability, safety, easy storage, and promotion of intestinal health.
Key Points
• Recombinant CsCP B. subtilis spores could be easily preserved in either liquid or freeze-dried state.
• Oral immunization of recombinant spores in mice could increase both local and system immune levels in a dose-dependent manner.
• Oral administration of recombinant spores increased the number of beneficial bacteria and reduced the number of harmful bacteria in the intestinal tract of mice.
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The authors declare that all data generated or analyzed during this study are included in this published article.
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Funding
This work was supported by the national key research and development program of China (2017YFD0501300), the National Natural Science Foundation of China (31900681), Guangxi Natural Science Foundation (2018GXNSFBA050070), Guangdong marine economy promotion projects fund (GDOE[2019]A29), Natural Science Research Project of Guangxi Medical University (GXMUYSF201729), and Guangxi First-class Discipline Construction Project in Basic Medical Sciences (GXMUBMSTCF-G23).
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ZT, ZW, XY, and YH conceived and designed the experiments. ZT and ZW performed the experiments. ZT, ZW, HS, and LZ analyzed the data. ZT, MS-1, MS-2, HJ, ZL, XZ, and XL contributed new reagents or analytical tools. ZT, XY, and YH wrote the paper. All authors read and approved the manuscript.
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All the mice were raised carefully according to the National Institutes of Health animal care and ethical guidelines. All the animal researches were approved by the Sun Yat-Sen University Institutional Animal Care and Use Committee (Permit Numbers: SYXK (Guangdong) 2010–0107). The sample collection of Gram-negative bacteria from a patient was approved by the Ethical Committee of the First Affiliated Hospital of Sun Yat-sen University as IRB_2018 [054].
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Tang, Z., Wu, Z., Sun, H. et al. The storage stability of Bacillus subtilis spore displaying cysteine protease of Clonorchis sinensis and its effect on improving the gut microbiota of mice. Appl Microbiol Biotechnol 105, 2513–2526 (2021). https://doi.org/10.1007/s00253-021-11126-z
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DOI: https://doi.org/10.1007/s00253-021-11126-z