Oral Bacillus subtilis spores-based vaccine for mass vaccination against porcine reproductive and respiratory syndrome

Abstract

Porcine reproductive and respiratory syndrome (PRRS) poses a significant challenge in the global swine industry, leading to substantial economic losses and reproductive and respiratory complications. The causative agent, PRRS virus (PRRSV), with its high mutation rate, complicates the development of universally effective vaccines. Furthermore, current PRRS vaccines are limited by high costs and complex administration methods. Therefore, in this study, we aimed to develop an innovative Bacillus subtilis spore-based oral vaccine targeting PRRS. Their oral administration was evaluated in mice and pigs, and blood, saliva, feces, and bronchoalveolar lavage fluid samples were collected for further analyses. Our vaccine induced IgG and IgA immune responses in both models, with swine demonstrating considerable increase in specific antibody and cytokine levels. These results indicate a high potential for more effective and economically viable control of PRRS in commercial pig farming. The ease of administration and cost-effectiveness of the vaccine also offer a feasible option for widespread application. Our results suggest a new direction in veterinary vaccine development, underscoring the potential of B. subtilis spores in creating effective vaccines for large-scale, real-world applications in animal health management.

Keywords: Bacillus subtilis; Mass vaccination; Porcine reproductive and respiratory syndrome; Spore; Swine; Vaccine.

Fig. 1

Construction and expression of recombinant CotB-GP5 fusion proteins in Bacillus subtilis spores. (A) The plasmids, pHT01-cotB-gp5 containing the self-promoter of cotB and linker, were constructed. The genes encoding the GP5 proteins from PRRSV EU (euGP5) and NA (naGP5) types were synthesized and fused each with the cotB coat protein gene. The linker sequence (GGGEAAAKGGG) was included to ensure proper folding and stability of the fusion protein. CotB-GP5 expression in B. subtilis DB104 spores was identified using SDS-PAGE analysis (B) and western blotting (C) as described previously^, and in the Materials and Method section. These images are displayed cropped. (B) The CotB-GP5 bands are indicated by the arrow.

Fig. 2

Immunogenicity of sCotB-euGP5 and sCotB-naGP5 in mice. Mice were administered sCotB-euGP5 (eu) or sCotB-naGP5 (na) either orally or intramuscularly (IM) as described in the Materials and Methods section. Blood, bronchoalveolar lavages (BAL), fecal and saliva samples were collected on days 21 and 42 post-initial administration. (A) Serum anti-IgG concentration in mice was measured using ELISA as described in the Materials and Methods section. (B– D) IgA concentrations in BAL (B), fecal (C), and oral samples (D) were also measured using ELISA. Statistical significance was determined via the Student’s t-test (***p < 0.001).

Fig. 3

Immune response induced by orally administered sCotB-euGP5 and sCotB-naGP5 mixture. The immune responses to oral administration of a mixture of sCotB-euGP5 and sCotB-naGP5 (sCotB-mixGP5) in pigs was examined as described in the Materials and Methods section. Blood, fecal and saliva samples were collected on days 21 and 42 post-initial administration. (A) Serum anti-IgG concentration in mice was measured using ELISA. (B, C) IgA concentration in fecal (B) and oral samples (C) were also measured using ELISA. Statistical significance was determined via Student’s t-test (***p < 0.001).

Fig. 4

Neutralizing antibody titers against PRRSV in the immunized pigs. The neutralizing antibody titers against both PRRSV EU type (A) and NA type (B) were determined using a virus neutralization assay, as described in the Materials and Methods section. The sera were collected from pigs on day 21 and day 42 post-immunization with the sCotB-mixGP5 spores. Statistical significance was determined via the Student’s t-test (***p < 0.001).

Fig. 5

Expression of IL-4 and IFN-γ in the pigs following oral administration of sCotB-mixGP5. The expression levels of IL-4 (A) and IFN-γ (B) were measured to evaluate the cell-mediated immune response in pigs administered with the sCotB-mixGP5, as described in the Materials and methods section. Statistical significance was determined via the Student’s t-test (***p < 0.001, ** p < 0.01).