Enhancement of protective immune responses by oral vaccination with Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae ApxIA or ApxIIA in mice

Abstract

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1beta and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-alpha increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.

Fig. 1

Schematic of protocols for oral vaccine delivery.

Fig. 2

Specific-IgA antibody responses to Actinobacillus pleuropneumoniea AxpIIA or ApxIA toxin in the lung (A), small intestine (B), and sera (C) of mice orally immunized with S. cerevisiae (□, group A; ■, group B; ░, group C; ▧, group D; ▤, group E). Bars represent the mean O.D. values at 405 nm. Error bars represent the standard deviation from the mean. Significant differences between control groups and vaccinated groups are expressed as ^*p < 0.05 and ^**p < 0.01.

Fig. 3

Systemic specific IgG (A) and specific-IgM antibody responses (B) against Actinobacillus pleuropneumoniea AxpIIA or ApxIA toxin in the sera of mice orally immunized with S. cerevisiae (□, group A; ▪, group B; ░, group C; ▧, group D; ▤, group E). Bars represent the mean O.D. values at 405 nm. Error bars represent the standard deviation from the mean. Significant differences between the control and vaccinated groups are expressed as ^*p < 0.05 and ^**p < 0.01.

Fig. 4

Representative specimens stained by immunohistochemistry for IgA-secreting cells in the lungs of mice after the final immunization. A, group B; B, group D; and C, group E. Arrows indicate positive immunoreactive cells. Counterstaining with hematoxylin. ×400.

Fig. 5

Densitometric analysis of IgA immunoreactivity in the small intestines of mice orally immunized with S. cerevisiae (□, group A; ■, group B; ░, group C; ▧, group D; ▤, group E). Results are expressed as the mean relative density. Asterisks indicate significant differences from the PBS-treated group, ^*p < 0.05 and ^**p < 0.01.

Fig. 6

Comparison of pro-inflammatory cytokines IL-1β (A), IL-6 (B), and TNF-α (C) from the lung and sera of mice following oral immunization with S. cerevisiae (□, group A; ■, group B; ░, group C; ▧, group D; ▤, group E). Bars represent the mean concentration of cytokine proteins. Error bars represent the standard deviation from the mean.

Fig. 7

Survival rates of mice immunized with S. cerevisiae after being challenged with the minimal lethal dose (MLD) of an A. pleuropneumoniae serotype 5 Korean isolate (-♦-, PBS-treated control; -□-, vector control; -▴-, oral immunization with 20 mg of S. cerevisiae expressing ApxIA antigen; -○-, oral immunization with 20 mg of S. cerevisiae expressing ApxIIA antigen; -*-, oral immunization with 10 mg each of S. cerevisiae expressing ApxIA and S. cerevisiae expressing ApxIIA antigen).