Adjuvant Effects Of Chicken IL-1β, IL-2, IFN-γ, MGF Expressed By Recombinant Fowlpox Viruses Used In Combination With Conventional And Recombinant Vaccines Against Newcastle Disease Virus

Advances in understanding the cells and molecular interactions linking the innate immune system to adaptive immune response have revealed the critical role that cytokines serve in antigen presentation, cell activation and proliferation and the establishment of immune memory. Cytokines can enhance vaccine-induced immune responses at three levels: ?recruitment of immune reactive cells to inductive sites and activation of professional antigen presenting cells (APCs), (2) proliferation and differentiation of antigen specific T and B cells and enhancement of effector function and (3) transition of cells into the memory cell pool and lymphocyte homeostasis. As an alternative to the use of cytokine-inducing adjuvants, cytokines may also be used directly. Most cytokines have the ability to modify and re-direct immune responses. Virus vectors offer a very attractive means for the delivery of cytokines. Recombinant cytokines delivered by virus vectors can be expressed continuously and have a good biologic activity in vivo. In the present study, the genes of chicken interleukin 1 B (IL-1 B), interleukin 2 (IL-2) and myelomonocytic growth factor (MGF) were amplified by RT-PCR from spleen cells stimulated by ConA. We constructed recombinant fowlpox viruses (rFPVs) expressing chicken IL-1 3 , IL-2 and MGF. Four chicken cytokines (IL-1 P , IL-2, IFN- Y , MGF) expressed by rFPVs were evaluated for their adjuvant effects on immune responses in SPF chickens and commercial chickens vaccinated with suboptimal dose of La Sota virus or minimal immune dose of rFPV expressing Newcastle disease virus (NDV) HN gene (rFPV-HN). We hope to explore a novel vaccine adjuvant.1. Cloning of genes of chicken IL-1 0 , IL-2 and MGFSpleen cells from 4-wk-old chicken were cultured with ConA (10ulml) in RPMI 1640 containing 5% FCS at a concentration of 107 cells/ml. Cells were cultured for 8h, 24h, 48h or 72h respectively at 41 C in 5% CO2 incubator. Total RNAs were extracted from cells cultured for different times and mRNA were isolated from total RNA. The sequences of the PCR primers were designed on the basis of published cDNA sequences of chicken IL-1 B , IL-2 or MGF. The 5' end of downstream primers contained a sequence (AAAAAAAAA) which is the complement sequence of transcriptional terminal signal of vaccinia virus. The PCR reaction mixture was analyzed by agrose gel electrophoresis after amplification. The results showed that the purposed genes were obtained. At the same time we found that chicken IL-1 B , IL-2 or MGF was expressed by ConA-stimulated spleen cells at various time. The time of expression of IL-1 B was the longest and MGF was the shortest. The cloned sequences of chicken IL-1 3 had a change of four nucleotide sites and one amino acid site and the chicken IL-2 had a variation of four and three sites respectively compared with the published sequences. The change of these sites would be the normal variation in different species and have no effect on their biologic activity.2. Construction of rFPVs expressing chicken IL-1B , IL-2 and MGF and assay of biologic activity of the product in vitroThe sequences encoding chicken IL-1 B , IL-2 and MGF were inserted into FPV (282E4) using the transfer plasmid p1175 under the control of the FPV early/late promoter (PE/L)- The plasmid p1175 contains the E.coli LacZ gene under the control of the PFV late promoter (P11), enabling rapid identification of FPV recombinants. Primary CEF monolayers were infected with rFPVs expressing chicken IL-1 3 , IL-2 and MGF at a M.O.I of 1.0. Following incubation at 37C for 72h, culture supernatants were harvested and filtered through a 0.1 um filter to remove infectious FPV. The levels of chicken IL-1 3 , IL-2 or MGF in cell culture supernatants infected with rFPVs were determined according to the standard procedures of XTT/PMS method. The results showed that rFPVs expressed chicken IL-1 3 , IL-2 and MGF effectively.3. Influence of chicken IL-1 B, IL-2, IFN- Y and MGF expressed by rFPVs on vaccination of La Sota or rFPV-HN against Newcastle disease vir...