Protective effects of a food-grade recombinant Lactobacillus plantarum with surface displayed AMA1 and EtMIC2 proteins of Eimeria tenella in broiler chickens

Abstract

Background: Avian coccidiosis posts a severe threat to poultry production. In addition to commercial attenuated vaccines, other strategies to combat coccidiosis are urgently needed. Lactobacillus plantarum has been frequently used for expression of foreign proteins as an oral vaccine delivery system using traditional erythromycin resistance gene (erm). However, antibiotic selection markers were often used during protein expression and they pose a risk of transferring antibiotic resistance genes to the environment, and significantly restricting the application in field production. Therefore, a food-grade recombinant L. plantarum vaccine candidate would dramatically improve its application potential in the poultry industry. Results:In this study, we firstly replaced the erythromycin resistance gene (erm) of the pLp_1261Inv-derived expression vector with a non-antibiotic, asd-alr fusion gene, yielding a series of non-antibiotic and reliable, food grade expression vectors. In addition, we designed a dual-expression vector that displayed two foreign proteins on the surface of L. plantarum using the anchoring sequences from either a truncated poly-γ-glutamic acid synthetase A (pgsA′) from Bacillus subtilis or the L. acidophilus surface layer protein (SlpA). EGFP and mCherry were used as marker proteins to evaluate the surface displayed properties of recombinant L. plantarum strains and were inspected by western blot, flow cytometry and fluorescence microscopy. To further determine its application as oral vaccine candidate, the AMA1 and EtMIC2 genes of E. tenella were anchored on the surface of L. plantarum strain. After oral immunization in chickens, the recombinant L. plantarum strain was able to induce antigen specific humoral, mucosal, and T cell-mediated immune responses, providing efficient protection against coccidiosis challenge. Conclusions:The novel constructed food grade recombinant L. plantarum strain with double surface displayed antigens provides a potential efficient oral vaccine candidate for coccidiosis.

A novel whole yeast-based subunit oral vaccine against Eimeria tenella in chickens

“…EtAMA1 and EtIMP1 expressed and delivered in a live recombinant bacterial Lactococcus lactis vaccine resulted in reductions in oocyst output and lesion score (31,32). Similarly, EtAMA1 and EtMIC2 co-expressed in Lactobacillus plantarum and delivered as an oral vaccine reduced oocyst output and lesion score following challenge (33). As noted for most recombinant antigen-based vaccines for Eimeria (reviewed in ( 27)), our yeast-based vaccine reduced parasite replication but did not completely prevent parasite development and shedding.…”

Modulatory Effects of Probiotics During Pathogenic Infections With Emphasis on Immune Regulation

“…L. johnsonii La1 (NCC533) reduces active trophozoite of Giardia intestinalis strain WB and infection duration in Meriones unguiculatus (286). Recombinant L. plantarum NC8 (containing Eimeria tenella protein) induced a higher percentage of a T-cell subset (CD3 + , CD4 + , and CD8 + ) and antibody levels, provided protection against E. tenella infection in chickens, and reduced lesion, cecum damage, and oocyst shedding (270). L. salivarius, L. johnsonii, and S. cerevisiae provided protection against Eimeria infection in chickens; reduced oocyst count, improved weight gain and FCR, and stimulated the immune response with higher antibodies (IgM and IgG) titer and lymphoproliferative response (271).…”

Protective Immunity Induced by an Eimeria tenella Whole Sporozoite Vaccine Elicits Specific B-Cell Antigens

“…In recent years, several studies have shown that recombinant vaccines in combination with novel adjuvants may offer a stronger mucosal immunity that stimulates both innate and acquired (humoral and cell-mediated) immune responses against several Eimeria strains [ 30 , 31 , 32 , 33 , 34 , 35 ]. The delivery system of recombinant or subunit vaccine candidates, using eukaryote or prokaryote vectors for feed or water administration application have shown optimistic and realistic results [ 36 , 37 , 38 , 39 , 40 ]. Some of these novel vaccines not only provide antigenic diversity, but cost of production and welfare issues are significantly reduced or eliminated [ 41 , 42 , 43 ].…”

“…Some of these secreted proteins are trafficked to the parasite surface and beyond via the secretory microneme (MIC) organelles, such as AMA1, IMP1, MIC1, MIC2, MIC3, micronemal protein 4 (MIC4), and micronemal protein 5 (MIC5) [ 3 , 4 , 5 , 16 , 30 , 39 , 50 ]. Several of these proteins were identified in our blots based on their homologous protein mass.…”