Basic Study On The Quality Control Method Of The Recombinant Modified Vaccine Ankara For HIV-1 Subtype C/B'

Our studying team have constructed DNA vaccine and the recombinant modified vaccine Ankara vaccine successfully for HIV-1 subtype C/B' in the prophase's study. The study was cooperated with ADARC and IAVI.The recombinant modified vaccine Ankara vaccine has carried five exogenous genes(gag-pol,env,nef-tat) which was from HIV-1 subtype C/B' CRF virus strains,the recombinant modified vaccine Ankara vaccine has been named ADMVA.Established the quality control method for virus titer,effect in vitro and immunogenicity of the ADMVA vaccine.Referring to the Pharmacopoeia of the People's Republic of China and the detected methods of the other vaccines,technical guiding principle for prevention of the Living Virus Vector,Combined with the self-characteristics of the recombinant modified vaccine Ankara virus,we made a preliminary research on the quality control method for ADMVA vaccine.Detected the infectious titer of ADMVA virus by imunoenzyme technique and verified the sensibility and stability of this method meanwhile,detected five group samples with two method(Imunoenzyme technique and the method of cytopathic effect)simultaneously.The infectious titers was slightly higher than that detected by the method of cytopathic effect for the same group,linear regression analysis showed that the two methods exist a linear correlation(b = 1.046,t = 45.244,P＜0.001). Linear correlation analysis showed that there was a positive correlation between the two method(r = 0.999,P＜0.001).The result shows that the sensibility of the imunoenzyme technique is good;Select two group samples(MVA-01, MVA-02)randomly,different persons detected the infectious titer for 7 times in different time through the method of imunoenzyme technique,the average titer is 6.08 1g IFU/ml for the group MVA-01,and 6.17 lg IFU/ml for the group MVA-02, repectively.The coefficient of variation between plate is 3.26%for the group MVA-01,and 2.87%for the group MVA-02,repectively.It shows the stability of this method is good.Detected the antigen expression in mammalian cells by Western-Blotting, established the quality control method for effect in vitro.The structural protein Gag,Pol and Env protein expressed highly.It proved the correctness of the inserted gene and the expressed frame.The structural protein can be recognized by specific antiserum of HIV-1 subtype C/B' epidemic strain.It shows consistency antigenicity between the modified gene and the virus strain.Do the preliminary test in mice.First,detected the protein concentration of Gag protein by BCA method,the result shows it exited linear correlation between the concentration and the OD value in the dilution multiple from 0 to 500(R~2=0.9952); Linear regression equation is Y=0.0012X+0.005,the concentration of total protein is 809μg/ml.Second,coating antigen with Gag protein,we detected the titer specific neutralizing antibody by enzyme linked immunosorbent assay,and established the quality control method for detection of humoral immunogenicity.The cellular immune response lever of ADMVA was detected by Enzyme Linked ImmunoSpot method.We detected the lever of the IFN-γof the BALB/c mice which inoculated with ADMVA vaccinia by Enzyme Linked ImmunoSpot method.It can detect T-cell specific response level aimed at the four gene fragments env,gag,pol and nef in HIV-1 and evaluate the cellular immune response lever.Based on the above,we established the quality control method for detection of cellular immunogenicity.