Immune response and protective efficacy of recombinant Enterococcus faecalis displaying dendritic cell-targeting peptide fused with Eimeria tenella 3-1E protein

Abstract

Avian coccidiosis causes significant economic losses on the global poultry breeding industry. Exploration of new-concept vaccines against coccidiosis has gradually become a research hotspot. In this study, an Enterococcus faecalis strain (MDXEF-1) showing excellent performance isolated from chicken intestinal tract was used as a vector to deliver Eimeria target protein. The plasmid pTX8048-SP-DCpep-NAΔ3-1E-CWA harboring dendritic cell-targeting peptide (DCpep) fusion with Eimeria tenella NAΔ3-1E gene (3-1E protein-coding gene without start codon ATG and terminator codon TAA) was electrotransformed into MDXEF-1 to generate the recombinant bacteria MDXEF-1/pTX8048-SP-DCpep-NAΔ3-1E-CWA in which NAΔ3-1E protein was covalently anchored to the surface of bacteria cells by cell wall anchor (CWA) sequence. The expression of target fusion protein DCpep-NAΔ3-1E-CWA was detected by Western blot. Each chicken was immunized 3 times at 2-wk intervals with live E. faecalis expressing DCpep-NAΔ3-1E fusion protein (DCpep-NAΔ3-1E group), live E. faecalis expressing NAΔ3-1E protein (NAΔ3-1E group), and live E. faecalis containing empty vector only. The 3 immunized groups were then challenged with homologous E. tenella sporulated oocyst after immunizations, and the immune response and protective efficacy in each group were evaluated. The results showed that serum IgG levels, secretory IgA levels in cecal lavage, proportion of CD4+ and CD8α+ cells in peripheral blood, and mRNA expression levels of IL-2 and IFN-γ in the spleen were significantly higher in chickens in the DCpep-NAΔ3-1E group than in chickens of the NAΔ3-1E group (P < 0.05). Oral immunization to chickens with live E. faecalis expressing DCpep-NAΔ3-1E offered more protective efficacy against homologous challenge including significant improved body weight gain, increased oocyst decrease ratio, and reduced average lesion scores in cecum compared with chickens with live E. faecalis expressing NAΔ3-1E protein. These results suggest that recombinant E. faecalis expressing dendritic cell-targeting peptide fusion with E. tenella 3-1E protein could be a potential approach for prevention of Eimeria infection.

Keywords: DCpep-NAΔ3-1E; Eimeria tenella; Enterococcus faecalis; immune response; protective efficacy.

Figure 1

The schematic illustration of plasmids pTX8048-SP-NAΔ3-1E-CWA harboring synthesized fusion gene fragment SP-DCpep-linker-TrxA-His6. The fusion gene fragment SP-DCpep-linker-TrxA-His6 consists of signal peptide (SP) of Lactococcus lactis secretion protein Usp45, detritic cell-targeting peptides (DCpep) (Curiel et al., 2004), linker sequence (G4S)2, and coding sequence of trxA protein and His6 amino acid and was synthesized by Nanjing GenScript Biotechnology Co., Ltd. (Nanjing, China). The target gene fragment SP-DCpep-linker-TrxA-His6 was digested by Nco Ⅰ/BamH Ⅰ, and then subcloned into Nco Ⅰ/BamH Ⅰ sites of pTX8048-SP-NAΔ3-1E-CWA (A) (Ma et al., 2017) to construct plasmid pTX8048-SP-DCpep-NAΔ3-1E-CWA (B).

Figure 2

Identification of plasmid pTX8048-SP-DCpep-NAΔ3-1E-CWA. The recombinant positive plasmid pTX8048-SP-DCpep-NAΔ3-1E-CWA was identified by digestion with NcoⅠ and Kpn Ⅰ, and an expected 1,071 bp fragment was observed. (M) DNA Marker (DL 5000). Lane (1) Fragment of pTX8048-SP-DCpep-NAΔ3-1E-CWA disgested by Nco Ⅰ and Kpn Ⅰ. Lane (2), (3), and (4) Fragment of pTX8048-SP-DCpep-NAΔ3-1E-CWA digested by Nco Ⅰ, BamH Ⅰ, and Kpn Ⅰ, respectively.

Figure 3

Western blot detection of cell wall–anchored fusion protein SP-TrxA-His6-NAΔ3-1E and SP-DCpep-linker-TrxA-His6-NAΔ3-1E. Target fusion protein SP-TrxA-His6-NAΔ3-1E and SP-DCpep-linker-TrxA-His6-NAΔ3-1E were separated by 12% SDS-PAGE, transferred onto nitrocellulose membranes, and probed with rabbit anti-3-1E polyclonal antibodies (Ma et al., 2011). The expected band corresponding to SP-TrxA-His6-NAΔ3-1E (53 kDa, A) and SP-DCpep-linker-TrxA-His6-NAΔ3-1E (55 kDa, B) was observed. (A) Lane (1) and (3) cell wall–anchored fusion protein SP-TrxA-His6-NAΔ3-1E from nisin-induced MDXEF-1/pTX8048-SP-NAΔ3-1E-CWA. Lane (2) cell wall–anchored fusion protein from nisin-induced MDXEF-1/pTX8048 (negative control). (B) Lane (1) and (3) cell wall–anchored fusion protein SP-DCpep-linker-TrxA-His6-NAΔ3-1E from nisin-induced MDXEF-1/pTX8048-SP-DCpep-NAΔ3-1E-CWA. Lane (2), cell wall-anchored fusion protein from nisin-induced MDXEF-1/pTX8048 (negative control).

Figure 4

(A)The IgG levels in serum and (B) secreted IgA levels in cecal lavage fluid from chickens immunized with 3-1-expressing Enterococcus faecalis MDXEF-1. On day 5 to 7, 21 to 23, and 37 to 39, chickens in each group were orally inoculated with 5 × 10⁹ CFU live bacteria of MDXEF-1/pTX8048-SP-DCpep-3-1E-CWA, MDXEF-1/pTX8048-SP-3-1E-CWA, and MDXEF-1/pTX8048 and PBS (pH7.2), respectively. The sera and cecal lavage fluid were prepared from chickens (n = 5) in each group after 3 immunizations. Specific IgG levels in serum and secreted IgA levels in cecal lavage fluid were determined by indirect ELISA. Data are expressed as mean ± SD (n = 5). ∗P＜0.05, ∗∗P＜0.01.

Figure 5

The percentage of CD4+ and CD8α+ T cells in peripheral blood from chickens immunized with 3-1-expressing Enterococcus faecalis MDXEF-1. After 3 oral immunizations with 5 × 10⁹ CFU live bacteria of MDXEF-1/pTX8048-SP-DCpep-3-1E-CWA, MDXEF-1/pTX8048-SP-3-1E-CWA, and MDXEF-1/pTX8048 and PBS (pH7.2) (control group), lymphocytes in peripheral blood from chickens (n = 5) in each immunized group were isolated by lymphocyte separation medium as per the manufacturer's instruction. The isolated cells were incubated with fluorescein isothiocyanate–conjugated mouse anti-chicken CD4+ antibody (0.5 mg/mL) and phycoerythrin-conjugated mouse anti-chicken CD8α+ antibody (0.5 mg/mL) (Southern Biotech). The relative quantification of T lymphocytes subtypes were measured by flow cytometry (Epics XL MCL， Beckman Coulter). Data are expressed as mean ± SD (n = 5). ∗P＜0.05, ∗∗P＜0.01.

Figure 6

The mRNA expression levels of ChIFN-γ and ChIL-2 in spleen of chickens. On day 5 to 7, 21 to 23, and 37 to 39, chickens in each group were orally immunized with 5 × 109 CFU live bacteria of MDXEF-1/pTX8048-SP-DCpep-3-1E-CWA, MDXEF-1/pTX8048-SP-3-1E-CWA, and MDXEF-1/pTX8048 and PBS (pH7.2), respectively. After 3 immunizations, the mRNA expression levels of ChIFN-γ and ChIL-2 in spleen of chickens (n = 5) was assayed by real-time PCR. The cytokine mRNA levels of individual chicken in each group were divided by mRNA levels of glyceraldehyde-3-phosphate dehydrogenase of the same chicken to normalize the relative mRNA levels of ChIL-2 and ChIFN-γ. Data are expressed as mean ± SD (n = 5). ∗P < 0.05, ∗∗P < 0.01.