Generation of Bacillus subtilis displaying alpha-toxin HlaH35LH48L fused with CotB and CotG, and studying the immune response in mice

Abstract

Introduction: The Bacillus subtilis spore is considered to be a powerful vehicle for surface display and antigen delivery. Being safe and widely used for the purpose of oral bacteriotherapy in humans and animals, B. subtilis spore has potential in the domain of needle-free vaccine development. The spores themselves also behave as an adjuvant. This study aims to generate B. subtilis spores expressing mutant staphylococcal alpha-toxin Hla_H35LH48L on the surface and to study their ability to evoke a specific immune response in mice.

Methods: Vectors carrying the hla_H35LH48L gene fused with the coding genes of anchor proteins, cotB and cotG, were cloned in E. coli before being transformed into B. subtilis. The generation of the B. subtilis new strains via chromosomal integration was confirmed by PCR. Sporulation was observed under the microscope. The expression of the target protein on the spore surface was determined by sporeELISA. The level of IgG in the serum and IgA in the feces of Swiss mice were analyzed using ELISA to learn about the immune response against the B. subtilis spore administrated via the oral route.

Results: The PCR products of an expected size on agarose gel electrophoresis showed successful integration, resulting in the construction of new B. subtilis strains BsHT2331 and BsHT2334. sporeELISA analysis detected a significant HlaH35LH48L expression on the spore surface. The BsHT2331 spores (CotB-HlaH35LH48L) triggered a constant increase in the IgG and IgA levels in mice after three doses as 5.5-fold and 2.5-fold higher than the pre-immunization, respectively (p-value < 0.0001). Meanwhile, the group of mice orally administrated with BsHT2334 (CotG-Hla_H35LH48L) showed a notable IgG titer but minor IgA response.

Conclusion: The results concluded that the construction of two new strains BsHT2331 and BsHT2334 that used spore coat proteins CotB and CotG to display mutant alpha-toxin Hla_H35LH48L on the B. subtilis spore surface was successful. It provided an assessment of the ability of B. subtilis spores to stimulate specific antibody production in mice.