Bacillus Toyonensis BCT-7112T Spores as Parenteral Adjuvant of BoHV-5 Vaccine in a Murine Model


Bacterial spores of the genus Bacillus are being evaluated as adjuvant molecules capable of improving the immune response to vaccines. In this study, we investigate whether subcutaneously administered spores of B. toyonensis BCT-7112T could enhance a vaccine immune response in mice. Three groups of mice were subcutaneously vaccinated on day 0 and received a booster on day 21 of the experiment, with the following vaccine formulations: 40 µg of recombinant glycoprotein D (rgD) from bovine herpesvirus type 5 (BoHV-5) adsorbed in 10% aluminum hydroxide (alum) without B. toyonensis spores (group 1) and B. toyonensis (1 × 106 viable spores) + 40 µg of rgD adsorbed in 10% alum (group 2); and B. toyonensis (1 × 106 viable spores) without rgD (group 3). Group 2 showed significantly higher titers (P < 0.05) of total specific serum IgG, IgG2a, and neutralizing antibodies, when compared with the groups 1 and 3. A significantly higher (P < 0.05) transcription level of cytokines IL-4, IL-12, and IFN-γ was observed in splenocytes from mice that received the B. toyonensis spores in the vaccine formulation. In addition, stimulation of the macrophage-like cell line RAW264.7 with spores of B. toyonensis markedly enhanced the cell proliferation and mRNA transcription levels of IL-4, and IL-12 cytokines in these cells. Our findings indicated that the subcutaneous administration of B. toyonensis BCT-7112T spores enhanced the humoral and cellular immune response against BoHV-5 in mice.

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All data generated or analyzed during this study are included in this published article.


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This study was financed in part by the Coordination for the Improvement of Higher Education Personnel (CAPES)-Brazil-Finance Code 001 and the National Council of Technological and Scientific Development (CNPq) for Leite FPL scholarship.

Author information




Santos FDS contributed to experiment design, performed most of the experiments, and wrote most of the manuscript. Maubrigades LR contributed to the performance of most of the experiments, Gonçalves VS contributed to the ELISA and qPCR reactions, Franz HC contributed to the cell culture, Rodrigues PRC contributed to the virus neutralization test, Cunha RC contributed to the experiment design and analyses of results, and Leite FPL designed the study, analysed the results, and wrote the manuscript. All authors of this paper have approved the submission of this manuscript.

Corresponding author

Correspondence to Fábio Pereira Leivas Leite.

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Mice used in this study were provided by the animal unit at Federal University of Pelotas (UFPel). All protocols and procedures were reviewed and approved by the Ethics Committee on Animal Experimentation (CEEA No. 1981) of the UFPel. The CEEA of UFPel is accredited by the Brazilian National Council for the Control of Animal Experimentation (CONCEA).

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Santos, F.D.S., Maubrigades, L.R., Gonçalves, V.S. et al. Bacillus Toyonensis BCT-7112T Spores as Parenteral Adjuvant of BoHV-5 Vaccine in a Murine Model. Probiotics & Antimicro. Prot. (2021).

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  • Spore
  • Bacillus
  • Vaccine
  • Immunomodulation
  • Cytokines