Protective effect of recombinant staphylococcal enterotoxin A entrapped in polylactic-co-glycolic acid microspheres against Staphylococcus aureus infection

Abstract

Staphylococcus aureus is an important cause of nosocomial and community-acquired infections in humans and animals, as well as the cause of mastitis in dairy cattle. Vaccines aimed at preventing S. aureus infection in bovine mastitis have been studied for many years, but have so far been unsuccessful due to the complexity of the bacteria, and the lack of suitable vaccine delivery vehicles. The current study developed an Escherichia coli protein expression system that produced a recombinant staphylococcal enterotoxin A (rSEA) encapsulated into biodegradable microparticles generated by polylactic-co-glycolic acid (PLGA) dissolved in methylene chloride and stabilized with polyvinyl acetate. Antigen loading and surface properties of the microparticles were investigated to optimize particle preparation protocols. The prepared PLGA-rSEA microspheres had a diameter of approximately 5 μm with a smooth and regular surface. The immunogenicity of the PLGA-rSEA vaccine was assessed using mice as an animal model and showed that the vaccine induced a strong humoral immune response and increased the percent survival of challenged mice and bacterial clearance. Histological analysis showed moderate impairment caused by the pathogen upon challenge afforded by immunization with PLGA-rSEA microspheres. Antibody titer in the sera of mice immunized with PLGA-rSEA microparticles was higher than in vaccinated mice with rSEA. In conclusion, the PLGA-rSEA microparticle vaccine developed here could potentially be used as a vaccine against enterotoxigenic S. aureus.

Figure 1

SEA expression and toxicity analysis. (A) SDS-PAGE for the rSEA induced by IPTG (1 mM) at different times; (B) Western immunoblot for rSEA protein, using anti-GST as the capture antibody; (C) histological imaging of liver sections from mice injected with PBS, (D) from mice injected with highly purified SEA protein, (E) low dose of rSEA experimental group, and (F) high dose of rSEA. Black arrows in D & F indicate the focal necrosis of hepatocytes and macrophage infiltration.

Figure 2

Characteristics of the PLGA-rSEA. (A) Scanning electron microscopy (SEM) photograph of PLGA-rSEA. Microsphere size, morphology, and surface appearance were examined by SEM. (B) Antigen-release kinetics. Percentage of released antigen was detected by BCA assay from day 10- 50.

Figure 3

Serum antibody analysis. Antibody levels in mice serum. Sera were collected from vaccinated and unvaccinated controls as described in the text. The error bars indicate standard errors of the means for five mice per group. The asterisks indicate P values (*, P < 0.05; **, P < 0.01, compared with the PBS group).

Figure 4

In vivo evaluation of the PLGA-rSEA vaccine protection. (A) Percent survival after infection; (B) In vivo growth of S. aureus in the liver after challenge; (C) In vivo growth of S. aureus in the spleen after challenge. BALB/c mice were vaccinated with PLGA-rSEA and then challenged i.p. with 10⁸ CFU of S. aureus for bacterial clearance analysis (A and B) and 10⁹ CFU lethal doses of S. aureus for percent survival analysis (C) (*, P < 0.05; **, P < 0.01, compared with the PBS group).

Figure 5

Histological analysis of mice after challenge with S. aureus. (A) Liver histopathology from mice challenged with S. aureus after vaccination with PLGA-rSEA. (B) Liver histopathology of unvaccinated mice challenged with S. aureus. The black arrow indicates moderate infiltration of mononuclear cells around the central veins (C) Spleen histopathology of mice immunized with PLGA-rSEA. (D) Spleen histopathology of unvaccinated mice. (E) Kidney histopathology of mice immunized with PLGA-rSEA. The black arrow indicates erythrocytes and macrophages in the white pulp. (F) Kidney histopathology of unvaccinated mice. BALB/c mice were vaccinated and then challenged i.p. with 10⁸ CFU of S. aureus six weeks after immunization. Mice were sacrificed on day 9 after challenge for this analysis. Tissues were stained with hematoxylin and eosin. Black arrows indicate the necrotic and scaled of the tubular structure.