Immune Efficacy And Immunopotentiation Of Recombinant Marek’s Disease Virus Expressing Chicken Interleukin-2

Marek’s Disease has been controlled by the use of vaccine, but the continued evolution of MDV towards stronger virulence has made the currently available vaccines lose efficacy. In recent years, chicken interleukin-2(ChIL-2) as immunopotentiator has been widely used in aviculture. We expressed ChIL-2by a recombinant MDV (rMDV-IL2) in an attempt to modulate or enhance the immune response against MDV, thereby increasing the efficacy of CVI988vaccine against a virulent MD challenge. Furthermore, we evaluated the immunopotentiation of rMDV-IL2for the Newcastle disease (ND) La Sota vaccine.The ChIL-2gene was directional cloned into pEASY-M1expression vector, then, the expression cassettes for ChIL-2containing the CMV promoter region was amplified by PCR. The product was cloned into the pU/D vector which contained the homologous sequence to the sorfl-sorf2junction region of the CVI988genome. The transfer plasmid, pU/IL2/D, was constructed. The pU/IL2/D plasmid was transfected into chicken embryo fibroblast cells (CEF) along with CVI988-DNA which containing the green fluoresence protein gene (rMDV-GFP). The cells were passaged and a single virus plaque was picked. The recombinant virus named rMDV-IL2were screened and purified with GFP label. According to the results of PCR, Western blot and MTT assay, we confirmed that the rMDV-IL2successfully cloned into MDV genome and the expressing products can promote lymphopoiesis. Analysis of cultured CEF cells infected with the rMDV-IL2showed that there was no difference in growth kinetics compared with the parent MDV.1day-old chickens were vaccinated with rMDV-IL2and CVI988respectively, and tested the immune organ indexes and the peripheral blood T lymphocyte subsets use flow cytometer. The result shows that the immune organ indexes of rMDV-IL2were higher than CVI988. The indexes of thymus were significant difference in21and28days post immune(p<0.05); the indexes of spleen and bursa of Fabricius were significant difference in21,28and35days post immune(p<0.05). The result of CD4+T lymphocyte showed that rMDV-IL2was higher than CVI988, especially in21and28days post immune, there was significant difference(p<0.05). The result of CD8+T lymphocyte showed that rMDV-IL2was higher than CVI988in7days post immune and achieved the highest level in28days post immune. These suggest that rMDV-IL2can improve the cell-mediated immune response.In the efficacy trials of rMDV-IL2,1day-old chickens were vaccinated with rMDV-IL2and CVI988respectively and then challenged with RB1B in7days post vaccination. The birds were observed for90days. Typical results indicating that vaccination with rMDV-IL2can effectively control weight loss caused by MD and have the higher lymphocyte activation reaction. The immune organ indexes showed that rMDV-IL2was higher in comparison with CVI988, and there was significant difference in21,28and35days post infection(p<0.05). Importantly, with rMDV-IL2inducing81.6%protection compared with78.9%induced by CVI988, there were no significant differences in the clinical outcome of the RB1B challenge.To determine whether ChIL-2expression could increase the antibody response against ND, the1day-old chickens were vaccinated with rMDV-IL2and CVI988respectively and then vaccinated with La Sota vaccine in10day-old. The birds were bled at7,14and21days post vaccination. Detecting the antibody titers of ND by HI assay and ELISA, the results indicated that the birds vaccinated with rMDV-IL2have the higher titer than CVI988at7and14days post vaccination, but not at21days. Importantly, the titer of rMDV-IL2was greater than4log2at7days post vaccination.