The Oral Inactivated Porcine Epidemic Diarrhea Virus Presenting in the Intestine Induces Mucosal Immunity in Mice with Alginate-Chitosan Microcapsules

Abstract

The porcine epidemic diarrhea virus, PEDV, which causes diarrhea, vomiting and death in piglets, causes huge economic losses. Therefore, understanding how to induce mucosal immune responses in piglets is essential in the mechanism and application against PEDV infection with mucosal immunity. A method of treatment in our research was used to make an oral vaccine that packaged the inactive PEDV with microencapsulation, which consisted of sodium alginate and chitosan, and adapted the condition of the gut in mice. The in vitro release experiment of microcapsules showed that inactive PEDV was not only easily released in saline and acid solutions but also had an excellent storage tolerance, and was suitable for use as an oral vaccine. Interestingly, both experimental groups with different doses of inactive virus enhanced the secretion of specific antibodies in the serum and intestinal mucus, which caused the effective neutralization against PEDV in the Vero cell by both IgG and IgA, respectively. Moreover, the microencapsulation could stimulate the differentiation of CD11b+ and CD11c+ dendritic cells, which means that the microencapsulation was also identified as an oral adjuvant to help phagocytosis of dendritic cells in mice. Flow cytometry revealed that the B220⁺ and CD23⁺ of the B cells could significantly increase antibody production with the stimulation from the antigens' PEDV groups, and the microencapsulation could also increase the cell viability of B cells, stimulating the secretion of antibodies such as IgG and IgA in mice. In addition, the microencapsulation promoted the expression of anti-inflammatory cytokines, such as IL-10 and TGF-β. Moreover, proinflammatory cytokines, such as IL-1, TNF-α, and IL-17, were inhibited by alginate and chitosan in the microencapsulation groups compared with the inactivated PEDV group. Taken together, our results demonstrate that the microparticle could play the role of mucosal adjuvant, and release inactivated PEDV in the gut, which can effectively stimulate mucosal and systemic immune responses in mice.

Keywords: PEDV; alginate; chitosan; microcapsules; mucosal immunity.

Figure 1

The stability of microcapsules shown by the release rate in (A) PBS, normal saline, hydrochloric acid, and release ability in different months (B). The differences between means were considered significant at * p < 0.05 and very significant at ** p < 0.01.

Figure 2

Enzyme-linked immunosorbent assay measures of antibodies IgA and IgG in feces (A) and serum (B) after the final immunity. The differences between means were considered significant at * p < 0.05 and very significant at ** p < 0.01.

Figure 3

The results of antibody neutralization activity of IgG (A) and IgA (B). Both neutralization percentages of IgA and IgG in the intestinal mucosa and serum.

Figure 4

The flow cytometric analysis results for the percentage of CD11b+ and CD11c+ cell for immunization (A). (B) indicates the percentage of B220+ and CD23+ cell with the flowcytometric analysis.

Figure 5

The expression analysis of genes by qRT-PCR analysis. For the expression analysis of genes, blue and red indicate decreased and increased expression, respectively. For qRT-PCR analysis of the expression of randomly selected novel genes from the immune and tight junction, data are presented as mean ± S.D. (n = 3).