Construction Of Recombinant Lactobacillus Constitutive Expressing IBDV Vp2 Protein And Analysis Of Its Immunogenicity In Chickens

Infectious bursal disease(IBD) caused by infectious bursal disease virus(IBDV), is an immuno suppressive viral disease in chickens that results in enormous economic losses in poultry industry. In this study, the vp2 protein of IBDV as immunogen was expressed by lactobacillus strains i.e. L. plantarum, L. pentoses, and L.casei 393 using p PG612, a surface display vector containing HCE constitutive promoter and Pgs A anchor protein, and in order to improve the protein expression level, T7g10 translational enhancer was introduced into the p PG612. In the present study, we evaluated an ameliorate maneuver for the preparation of efficient competent cells of lactobacillus, and the parameters including washing buffers, optical density 600nm(OD 0.4-0.5), plasmid concentration i.e. 2μ l-6μ l(100ng/μ l), and voltage 2.3-2.4 kv/cm were mainly focused. The high transformation efficiency was recorded in L.casei 393(9.9×102 and 2.4×102), L.pentoses(1.1×103and 3.1×102), L. plantarum(1.2×103 and 3.7×102), with genetically engineered IBDV-vp2 expression plasmids viz.,(i) p PG612-HCE-Pgs A-vp2-rrn BT1T2,(ii) p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2 by electroporation, respectively, followed by further confirmation of electro-transformation by isolation, enzyme digestion and PCR. This optimized method was simpler and more efficient compared to previously employed methods for the preparation of lactobacillus competent cells.The immunogenicity of genetically engineered lactobacillus strains i.e. L.plantarum, L.pentoses, L.casei 393 containing p PG612-vp2(p PG612-HCE-Pgs A-vp2-rrn BT1T2) and p PG612-T7g10-vp2(p PG612-HCET7g10-Pgs A-vp2-rrn BT1T2) used as oral vaccine to induce immunity against IBDV in chickens was evaluated, respectively. Our results showed that the vp2 protein was successfully expressed and displayed on the cell surface of the LPL/p PG612-vp2(PL), LPL/p PG612-T7g10-vp2(TL),LPE/p PG612-vp2(PE), LPE/p PG612-T7g10-vp2(TE), LC/p PG612-vp2(PC) and LC/p PG612-T7g10-vp2(TC), while high level of protein expression was obtained in p PG612-T7g10-vp2, indicating that the T7g10 enhancer can effectively improve vp2 expression. Consequently, significant levels(P<0.05) of immune responses in chickens and effective immune protection(87.5%) for chickens against IBDV were obtained orally administrated LPL/p PG612-T7g10-vp2, suggesting a better immunogenicity. In this study, we demonstrated that recombinant lactobacillus delivery system in vaccinated groups induced strong antibody responses, that antibody responses in groups immunized with p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2 were significantly higher than the groups vaccinated with p PG612-HCE-Pgs A-vp2-rrn BT1T2, we also observed that the group immunized with recombinant L. plantarum showed a higher survival and protection than recombinant L. casei group and recombinant L. pentoses group. Meanwhile, recombinant L. casei group showed higher survival and protection than L. pentoses.We observed large portion of T cells in vp2 vaccinated groups, chickens challenged with IBDV showed increased number of CD4+ and CD8+ T cells. There was a significance(p<0.05) difference of CD4+ and CD8+T cells in all groups, but groups vaccinated with p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2 showed high percentage of CD4+ and CD8+T cells than groups vaccinated with p PG612-HCE-Pgs A-vp2-rrn BT1T2. The vaccinated groups stimulated T cells statistically significant compared to control groups(P<0.05). CD8+(13.3%) and CD4+(21.0%) proliferation showed higher stimulation in group TL in contrast to group PL: CD8+(10.4%) and CD4+(14.0%); same while CD8+(11.4%) and CD4+(20.9.0%) proliferation showed higher stimulation in group TC in contrast to group PC:CD8+(7.2%) and CD4+(11.5%) and in TE group CD8+(8.3%) and CD4+(16.7%) proliferation showed higher stimulation in contrast to group PE:CD8+(7.5%) and CD4+(13.5%). All groups showed higher percentage of CD8+ T cells than CD4+.CD8+ belongs to cytotoxic T cells which involves in lysis of virally infected cells, tumor cells and allografts, and plays a crucial role in immune response. In cellular immune response, same trend was also observed in recombinant L. plantarum which showed more percentage of CD8+ T and CD4+ then L. casei and L. pentoses. High level of IFN-γ, IL-2 and IL-4 cytokines were detected in groups vaccinated with p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2 compared to groups vaccinated with p PG612-HCE-Pgs A-vp2-rrn BT1T2. Instead of this, vaccinated groups showed that the level of IFN-γ was higher than IL-2 and the level of IL-2 was higher than IL-4. Same trend was also observed as L. plantarum shows more Th1 and Th2 cytokines production then L. casei and L. pentoses, and L. casei showed more Th1 and Th2 cytokines production then L. pentoses. The high Th1 cytokines production such as IFN-γ and IL-2 are linked with cellular immune responses, while humoral responses are related to the production of Th2 cytokines such as IL-4. In conclusion, the recombinant L. plantarum containing the p PG612-T7g10-vp2 provides a promising strategy for vaccine development against IBDV via oral administration.