Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in human and animal. To determine the mechanism of a bovine lactoferricin-lactoferrampin (LFCA)-encoding Lactobacillus reuteri CO21 (LR-LFCA) to enhance the intestinal mucosal immunity, we used a newborn piglet intestine model to study the intestinal response to ETEC. Pigs were chosen due to the anatomical similarity between the porcine and the human intestine.4-day-old piglets were orally administered with LR-LFCA, LR-con (L. reuteri CO21 transformed with pPG612 plasmid) or phosphate buffered saline (PBS) for three consecutive days, within 21 days after these treatments, we found that LR-LFCA can colonize the intestines of piglets, improve the growth performance, enhance immune response and is beneficial for intestinal health of piglets by improving intestinal barrier function and modulating the composition of gut microbiota. Twenty-one days after, piglets were infected with ETEC K88 for 5 days, we found that oral administration of LR-LFCA to neonatal piglets attenuated ETEC-induced the weight loss of piglets and diarrhea incidence. LR-LFCA decreased the production of inflammatory factors and oxidative stress in intestinal mucosa of ETEC-infected piglets. Additionally, LR-LFCA increased the expression of tight junction proteins in the ileum of ETEC-infected piglets. Using LPS-induced porcine intestinal epithelial cells (IPEC-J2) in vitro, we demonstrated that LR-LFCA-mediated increases in the tight junction proteins might depend on the MLCK pathway; LR-LFCA might increase the anti-inflammatory ability by inhibiting the NF-κB pathway. We also found that LR-LFCA may enhance the antioxidant capacity of piglets by activating the Nrf2/HO-1 pathway. This study demonstrates that LR-LFCA is effective at maintaining intestinal epithelial integrity and host homeostasis as well as at repairing intestinal damage after ETEC infection and is thus a promising alternative therapeutic method for intestinal inflammation.

Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) has led to serious economic losses to the swine industry worldwide. In this study, an oral recombinant Lactobacillus casei vaccine against PEDV infection targeting the intestinal microfold (M) cells and dendritic cells (DCs) for delivering the core neutralizing epitope (COE) of PEDV spike protein was developed with M cell-targeting peptide (Col) and dendritic cell-targeting peptide (DCpep). The immunogenicity of the orally administered recombinant strains was evaluated. After immunization, significantly higher levels of anti-PEDV specific IgG antibodies with PEDV neutralizing activity in the sera and mucosal sIgA antibodies in the tractus genitalis, intestinal mucus, and stools were detected in mice orally administered with the recombinant strain pPG-COE-Col-DCpep/L393, which expressed DCpep and Col targeting ligands fused with the PEDV COE antigen, compared to mice orally immunized with the recombinant strain pPG-COE/L393 without the DCpep and Col targeting ligands. Moreover, in response to restimulation with the PEDV COE antigen in vitro, a significant difference in splenocyte proliferation response and Th2-associated cytokine IL-4 level was observed in the group of mice orally immunized with pPG-COE-Col-DCpep/L393 (p < 0.05) compared to the groups of mice that received pPG-COE-Col/L393 and pPG-COE-DCpep/L393, respectively. The intestinal M cells- and DCs-targeting oral delivery of genetically engineered Lactobacillus expressing the COE antigen of PEDV can efficiently induce anti-PEDV mucosal, humoral, and cellular immune responses via oral administration, suggesting a promising vaccine strategy against PEDV infection.

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, is the causative agent of porcine epidemic diarrhea (PED) that damages intestinal epithelial cells and results in severe diarrhea and dehydration in neonatal suckling pigs with up to 100% mortality. The oral vaccine route is reported as a promising approach for inducing protective immunity against PEDV invasion. Furthermore, dendritic cells (DCs), professional antigen-presenting cells, link humoral and cellular immune responses for homeostasis of the intestinal immune environment. In this study, in order to explore an efficient oral vaccine against PEDV infection, a mucosal DC-targeting oral vaccine was developed using Lactobacillus casei to deliver the DC-targeting peptide (DCpep) fused with the PEDV core neutralizing epitope (COE) antigen. This probiotic vaccine could efficiently elicit secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in vivo. Significant differences (p < 0.05) in the immune response levels were observed between probiotics expressing the COE-DCpep fusion protein and COE antigen alone, suggesting better immune efficiency of the probiotics vaccine expressing the DC-targeting peptide fused with PEDV COE antigen. This mucosal DC-targeting oral vaccine delivery effectively enhances vaccine antigen delivery efficiency, providing a useful strategy to induce efficient immune responses against PEDV infection.

Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets. In our previous study, a genetically engineered Lactobacillus casei oral vaccine (pPG-COE-DCpep/L393) expressing a dendritic cell (DC)-targeting peptide fused with porcine epidemic diarrhea virus (PEDV) COE antigen was developed. This vaccine induced significant levels of anti-PEDV specific IgG and IgA antibody responses in mice, indicating a potential strategy against PEDV infection. In this study, pPG-COE-DCpep/L393 was used for oral vaccination of newborn piglets against PEDV. We then assessed the immune responses and protection efficacy of pPG-COE-DCpep/L393. An indirect enzyme-linked immunosorbent assay (ELISA) showed that the recombinant Lactobacillus vaccine elicits a specific systemic and mucosal immune response. The T-helper cells mediated by pPG-COE-DCpep/L393 and PEDV infection display a Th1 phenotype. The histopathological results showed that pPG-COE-DCpep/L393 promotes lymphocyte proliferation and effectively protects piglets against PEDV infection. The transforming growth factor-β level indicated that the recombinant Lactobacillus vaccine plays a role in anti-inflammatory responses in mesenteric lymph nodes during PEDV infection. These results show that pPG-COE-DCpep/L393 is a potential vaccine against PEDV infection.

Clostridium perfringens α-toxin is one of the major virulence factors during C. perfringens infection, causing hemolysis of erythrocytes in various species. Here, genetically engineered Lactobacillus casei (pPG-α/L. casei 393) constitutively expressing the toxoid of C. perfringens α-toxin was generated and its immunogenicity in mice for induction of protective immunity against the α-toxin was evaluated via oral immunization. The α-toxoid was constitutively expressed by pPG-α/L. casei 393 without a specific inducer, as confirmed by western blotting, laser confocal microscopy, and flow cytometry. In an experiment on BALB/c mice to evaluate the oral immunogenicity of pPG-α/L. casei 393, significant levels of a specific secretory IgA (sIgA) antibody in the intestinal mucus and feces and an IgG antibody in the serum of the probiotic vaccine group were detected after booster immunization (p < 0.05) as compared with the pPG/L. casei 393 and PBS control groups. These antibodies effectively neutralized C. perfringens natural α-toxin. Moreover, significantly higher levels of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and interferon (IFN) γ in the serum and increased proliferation of spleen lymphocytes obtained from mice orally immunized with pPG-α/L. casei 393 were detected. With a commercial C. perfringens type A inactivated vaccine as a control, immune protection provided by the probiotic vaccine against C. perfringens α-toxin was evaluated, and 90% and 80% protection rates were observed, respectively. Therefore, strain pPG-α/L. casei 393 effectively elicited mucosal, humoral, and cellular immunity, suggesting that pPG-α/L. casei 393 is a promising candidate for development of a vaccine against C. perfringens α-toxin.