Oral administration of recombinant Lactobacillus plantarum co expressing FimB, CnaA, NetB and FBA antigens targeting chicken dendritic cells provides effective protection against necrotizing enteritis in broilers

Necrotic enteritis (NE), an acute intestinal disease of broilers caused by Clostridium perfringens, poses a significant threat to global poultry production. The ban on antibiotic growth promoters in feed, particularly in the EU since 2006, has exacerbated this problem, leading to a resurgence of NE and substantial economic losses. To address this challenge, we developed a novel probiotic-based oral vaccine strategy. We engineered three recombinant Lactobacillus plantarum NC8 strains, NC8(pYL560) expressing three secreted C. perfringens antigens (FimB, NetB, CnaA), NC8(pYL594) expressing a fourth surface-displayed antigen (FBA), and NC8(pYL595), which co-delivers these four antigens with a dendritic cell (DC)-targeting nanobody (phi74) fused with FBA. In vitro, the DC-targeting strain NC8(pYL595) showed superior uptake by chicken bone marrow-derived DCs compared to the non-targeting strain (78.4% vs. 46.9%, p < 0.01), along with enhanced adhesion, invasion, and the ability to promote DC maturation (upregulating CD83, CD86, CCL5, CCR7). After a total of six dose immunization, both NC8(pYL594) and NC8(pYL595) could moderately elevate the antigen specific serum IgG, intestinal IgG, IgA and IgM antibodies compared with NC8(pYL560), especially the intestinal IgA and IgG. In addition, the splenic cellular proliferation and productions of intracellular IL-4 and IFN-γ were also moderately increased in both NC8(pYL594) and NC8(pYL595) groups. Most importantly, in a broiler challenge model, oral immunization with either NC8(pYL560), NC8(pYL594) or NC8(pYL595), especially NC8(pYL595), significantly reduced averate intestinal lesions to 1.6, 1.5 and 0.8, respectively, compared with the mock control (lesion score=3.67) and empty vector control (lesion score=3.125). In addition, immunization with NC8(pYL595) resulted in dramatically decreased disease positive rates to 60%, compared with 77.7% in NC8(pYL560) group and 87.5% in NC8(pYL594) group. In conclusion, our findings demonstrate that the integration of multi-antigen co-expression and DC-targeting synergistically enhances the immunogenicity and protective effects of an oral lactobacilli vaccine, offering a promising antibiotic-free strategy for NE control.