Abstract
Porcine epidemic diarrhea (PED), which is caused by the porcine epidemic diarrhea virus (PEDV), has occurred worldwide and poses a serious threat to the pig industry. Intestine is the main function site of PEDV; therefore, it is important to develop an oral mucosal immunity vaccine against this virus infection. Most traditional plasmid delivery vectors use antibiotic genes as a selective marker, easily leading to antibiotic accumulation and gene contamination. In this study, to explore whether the alanine racemase gene (Alr) could be used as a screening marker and develop an efficient oral vaccine against PEDV infection, a recombinant strain was constructed using Lactobacillus casei with Alr deletion (L. casei ΔAlr W56) to deliver the Alr gene and a core-neutralizing epitope (COE) antigen. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in mice. Compared to the other strains, the recombinant bacteria were able to grow without the addition of D-alanine, revealing that Alr in the plasmid could function normally in defective bacteria. This oral mucosal vaccine would provide a useful strategy to substitute the application of antibiotics in the future and induce efficient immune responses against PEDV infection.
Highlights
Porcine epidemic diarrhea (PED), characterized by watery diarrhea, vomiting, and dehydration, infects pigs at different ages and is caused by the porcine epidemic diarrhea virus (PEDV) [1]
To determine the neutralizing activity of the sera or intestinal mucusal antibodies obtained from the mice immunized with pPG-alanine racemase gene (Alr)-core-neutralizing epitope (COE)/∆Alr W56, 50 μL 2 × serial diluted samples were collected on 40 d post-immunization, mixed with 50 μL of PEDV passages with 200 50% tissue culture infective dose (TCID50) in intestinal epithelial cells (IPECs) and incubated at 37 ◦C for 1 h
All recombinant strains were able to grow on an MRS agar medium in the presence of D-alanine, while only the strain pPG-Alr-COE/∆Alr W56 was able to grow in a medium without D-alanine. These results indicated that the recombinant strains were successfully constructed, and the Alr gene in the plasmid could play a similar role to that in the L. casei W56 genome
Summary
Porcine epidemic diarrhea (PED), characterized by watery diarrhea, vomiting, and dehydration, infects pigs at different ages and is caused by the porcine epidemic diarrhea virus (PEDV) [1]. D-alanine is not a common composition of conventional media, and previous studies have shown that its exogenous addition could recover the normal growth of the Alr gene deletion bacteria [19,21,22]. This suggests that D-alanine selection may be a promising candidate for the substitution of antibiotics in Lactobacillus. Its immunogenicity as an oral vaccine was evaluated through the significant levels of anti-PEDV systemic immunoglobulin G (IgG) and mucosal IgA antibody responses in mice. Our results clearly showed that the recombinant strains were effective at inducing anti-PEDV mucosal immune responses
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