| Tuberculosis was an ancient, but still is a chronic consumptive disease that threatens the human lives globally. There are 30 million cases of TB in the world and 3 million TB cases die annually. BCG is the only available vaccine for prevention of tuberculosis. Investigation results showed that the protective efficacy of the yaccine was from -36% to 80%. A well-designed and controlled survey indicated the BCG efficacy was about 50%. The goal of this study is to characterize and compare the immune responses and protective efficacy in mice induced by varied plasmid DNA vaccines that express the surface antigens of Mtb8.4, Ag85B etc of Mtuberculosis and BCGTen plasmid DNA vaccines that express M tuberculosis surface antigens or fusion proteins as well as human interleukin-2 (hIL-2) using pVAXl as a vector have been constructed. Among them, TB plasmid DNA vaccine TB-V1, TB-V2, TB-V3, TB-V4 and TB-V5 expressed Mtb8.4, Ag85B, Mtb8.4-38kDa CTL/Th epitopes-Ag85B, Mtb8.4-GST and Ag85B-GST antigens or fusion proteins respectively; TB-V6 and TB-V7 plasmid expressed M tuberculosis Mtb8.4 and Ag85B protein with ML-2 signal peptide and TB-V8 expressed Mtb8.4, 38kDa CTL/Th epitope and Ag85B fusion protein with hIL-2 signal peptide; TB-V9 and TB-V10 expressed Mtb8.4-GST, Ag85B-GST fusion proteins with hIL-2 signal peptide respectively. The plasmid of p-IL2s was also constructed as a negative control since hIL-2 signal peptide was inserted into the vector only. The DNA fragments containing the sequences encoding the antigens of Mtb8.4, Ag85B, 38 kDa CTL/Th epitope were generated from M tuberculosis H37Rv genomic DNA by PCR and hIL-2 and GST cDNAwere sub-cloned from the plasmid of pGex-2T-hIL-2 respectively. The constructs of the plasmid DNA vaccine were transformed into bacteria DH5a cells and the plasmid DNA were verified by restriction endonucleases mapping and sequence analysis.For the immunization experiments, C57BL/6 mice were used as test animal and they were divided into 12 groups with 10 mice each. The C57BL/6 mice were immunized by injecting into legs muscles with lOOjig plasmid DNA 3 times at 15 days intervals. Groups No 1 to Group No 5 were immunized with TB-V6~TB-V10, and Groups No. 6 to No. 8 were immunized with p-hIL2, p-WL2+TB-V6 and p-hIL2+TB-V7 (SO^ig/each) respectively. Groups No 9 to No 12 were as controls that were immunized with PBS, plasmid DNA of pVAXl, p-IL2s and BCG. BCG was introdermally injected at the dosage of 105 CPUs only once.15 days after the last boost immunization, 5 mice in each tested group were killed and the spleen lymphocyte suspensions were harvested and cultured in 24-well plates for 72hr at 37. The supernatants were collected and assayed for murine interleukin-2(mIL-2) , IL-6 , IL-10 and murine interferon gamma (mIFN-) activities. A quantitative ELISA assay was used to identify the hIL-2 activity for the serum from the eyes of the mice. 21 days after the last boost immunization, the remaining 5 mice were challenged by injection of 0.1 ml of M tuberculosis H37Rv strain suspension (10 CPUs). 15 days post challenges, the mice were killed and their lungs, livers and spleens were homogenized respectively. The immune response and protective efficacy assay were performed with three repeating by couture 0.1 ml of the each suspension onto the Lowenstein-Jensen slants and the CPUs were counted after 20-30 days culture. The bacterial loads were calculated according to the organ's weight and dilutions.The results showed that the average activities of mIL-2, INF- Y in the speenocyte culture supernatants of the immunized group of TB-V6-TB-V10, p-WL2, TB-V6+p-hIL2 and TB-V7+p-hIL2 were significant different with those of PBS, pVAXl and p-IL2s controls, but were almost as high as BCG immunized controls. However, the activity of mIL-6 and mIL-10 was low both in tested or control groups which indicated that Thl immune response can be induced by the 5 plasmid DNA vaccines TB-V6-TB-V10 which is required for immunity against M tuberculosis. High hIL-2 activity in the sera of the p-hIL2, TB-V6+p-hIL2 an...
|
PDF Full Text Request