Effects Of Ag85A DNA Vaccine On Immunological Functions Of Th1 In Tumor-bearing Mice

PrefaceAg85A complex, a 30 ~32 -kDa family of three proteins ( Ag85A, Ag85B and Ag85C) , constitute a major portion of the secreted proteins in M. tuberculosis and BCG culture filtrates. They possess enzymatic mycolytransferase enzyme activity required for the biogenesis of trehalose - dimycolate ( cord factor) , a dominant structure necessary for maintaining cell wall integrity. Ag85A may be the most essential component for bacterial survival and virulence. Genetic vaccination with plasmid DNA encoding Ag85 A is found to generate robust Th1 - type helper T - cell responses (higher secretion of interleukin - 2 and gamma interfer-on).It is well known that CD8 ~+ T cells play an essential role in antitumor immunity because most tumors are positive for MHC class I but negative for MHC class Ⅱ. Recent interest has been directed towards the use of Thl cells in generating antitumor immunity since people come to realize that efficient priming of antigen specific CTLs by vaccination requires the simultaneous activation of Th lymphocytes. This study is undertaken to investigate whether Ag85A could generate Thl immune response in tumor - bearing host, a weak cellular immunity body. After the quadriceps muscle is infected with Ag85A DNA vaccine, we detect the effects of it on immunological functions of Thl in mice bearing Meth - A fibrosarcoma, which is the first step in the development of an immunotherapy for tumor based on the use of plasmid DNA vaccines containing the gene for myco-bacterial Ag85A.Materials and methodsWith the method of alkaline lysis, Ag85A - V1Jns. tPA plasmid and Vljns. tPA plasmid were extracted and isolated on a 0. 8% agarose gel. After extracting plasmid from gel according to the protocol of QIAquick Gel Extraction Kit, they were transformed into competent Escherichia coli JM109. Plasmid DNA was digested by Bgl II restriction endonuclease and analyzed by agarose gel electrophoresis. Plasmid was prepared by bacterial fermentation and purified by precipitation with Polyethylene Glycol. Plasmid DNA was adjusted to a final concentration of lg/L in saline and stored at -20t.BALB/c female mice, 6 -8weeks, were randomly divided into 3 groups; Ag85A DNA plasmid group (mice were injected with plasmid containing Ag85A -Vljns. tp A) , empty plasmid control group (mice were injected with plasmid containing Vljns. tpA) and saline control group (mice were injected with saline). Meth - A cells(2 x 105 cells) were inoculated subcutaneously into the back of mice and from the next day the mice were injected with 100 g Ag85A vaccine on quadriceps three times(10 -day intervals). As negative controls, mice were injected with empty plasmid and saline alone. Eight days after the third vaccination mice were sacrificed and spleens were removed asepticly. Spleen cells were cultured and cytotoxicity of NK cells was detected. After 48 hours, the supernatants were harvested for the determination of IFN - 7 by ELJSA. All data are expressed as mean ± SD, significance of differences was determined by t test.ResultsOn the 8 th day after the last immunization the cytotoxicity of NK cells and the production of IFN — -y in Ag85 A DNA plasmid group were higher in the supernatants of spleen cell culture than that in saline control group and empty plasmid control group (p < 0.05) . There is no significant difference between empty plasmid control and saline control group(p > 0.05). It was indicated that in-tramuscular injection of Ag85A DNA vaccine could increase cytotoxicity of NK cells and IFN - 7 production in the spleen cell supernatants of mice bearing Meth - A fibrosarcoma.DiscussionIn this study, we demonstrate that intramuscular injection of Ag85A DNA vaccine could increase cytotoxicity of NK cells and IFN - 7 production in the spleen cell supernatants. This is the first study demonstrating the immunological effects of Ag85A on Meth - A fibrosarcoma and is the first step in the development of an immunotherapy for tumor based on the use of plasmid DNA vaccines containing the gene for mycobacterial Ag85A.DNA vaccines are made of a modified form of an infectious organism' s DNA altered by special techniques to provide what are known as eukaryotic promoters in front of the gene or genes on this DNA. This material, when introduced into the body, usually leads to the expression of the genes with the help of processing within host cells. The gene expression ultimately leads to synthesis of infectious organisms' proteins inside the injected cells and then activated host immune responses. As a result, the host immune system responds in a protective manner to produce antigen molecule frequently almost the same way as would occur if the host were actually infected by the true organism, itself. In addition, target gene fragments can be easily selected and combined according to the research objective. Most importantly, DNA vaccine can stimulate not only humoral immunity but also cellular immunity which ordinary vaccines fail to activate.In this research to detect the effects of Ag85A DNA vaccine on immunological functions of Thl in mice bearing Meth - A fibrosarcoma, we injected the Ag85A DNA vaccine on quadriceps. The result indicated that intramuscular injection of Ag85 A DNA vaccine could increase the activity of NK cells and IFN -7 production in spleen cell cultures. The mechanism might be as follows: (1) The antigen - specific CTL and Thl cells can be generated by Ag85 A DNA vaccine to release IL - 2 and IFN - 7, which can generate NK cells; ( 2 ) On account of borader epitope repertoire of Ag85A DNA vaccine than BCG and M. tu-...