Study Of Prevalence Rate Status Of Chronic Hepatitis B And PD-1/PD-L1 Signal Passway To Be The Immunotherapy Target For Hepatitis B

Hepatitis B virus infection is the serious public health problem.The number of HBsAg carriers is 93 million in China.HBV infection will develop to recovery;asymptomatic HBsAg carrier;chronic hepatitis B(CHB);acute hepatitis B;cirrhosis and hepatocellular carcinoma(HCC).However,the information and knowledge about mechanistic of HBV infection and natural history are lacking in China recently.Many studies reported that immune tolerance specialy for HBV is the main course of chronic infection.The PD-1/PD-L1 signal pass way is highly relevant with immune tolerance and blocking this pass way could restore the function of exhausted T cells;improve the ability of viral clearance.Hence,in order to know the prevalence status of HBV relative disease and study the possibility and method about immunity therapy by blocking PD-1/PD-L1 pass way,we do two parts work as follows.1.Cohort study to investigate the prevalence rate status of CHBBy means of cross-section and review investigation,we collected 2743 HBsAg positive cases in Longan-Guangxi;Zhengding-Hebei;Zhaodong-Heilongjiang;Tongde-Qinghai and Luohe-Henan five regions.We carried out the follow survey in 2006 for these cases. The results of survey include:(1) Average HBsAg yearly natural seroconversion rate is 10.68%;(2) In 758 HBsAg carriers,prevalence rate of asymptomatic HBsAg carrier is 91.50%,CHB is 6.98%,cirrhosis is 1.09%,HCC is 4.3‰;(3) Highly age group of CHB prevalence is 30～,49～;male's is higher than female's.(4) Prevalence rate of cirrhosis and HCC are higher than normal people.According to those data,we could calculate that the CHB number in China is about 6.5 million.Most of them have been ignoring because of its mild symptoms. 2.The study about PD-1/PD-L1 signal passway to be the immunotherapy target for hepatitis B.(1)Express and purify the PD-L1 recombination proteinAccording to analysis result of the human source PD-L1 mRNA gene sequence,by means of codon optimized and synthesized the whole gene sequence of PD-L1.We constructed the thioredoxin-(PD-L1) recombination expression plasmid successfully and expressed the fusion protein in E.coli.The molecular weight of target protein was 47KD. After purification we obtain the recombination PD-L1 protein with purity is 85%,density is 0.4mg/ml.So we have obtained the PD-L1 recombinant protein.The result proved the basic condition for further study on antibody and mutually action between PD-1/PD-L1 and chronic virus infectious.(2)Analyze the bioactivityon of PD-L1 recombination proteinAccording the principle that PD-L1 could combine with its receptor PD-1 specially,we conjugated the HRP on the PD-1,coated PD-L1 on 96 wells plate and carried out ELISA direct combine assay.The result proved that PD-L1 can combine with PD-1 specially.The best proportion of PD-L1 is 4～6μg/ml,HRP(PD-1) is 1:400.Biomolecular interaction analysis(BIA) assay system also proved the special combination of PD-1/PD-L1.The affinity is 129 resonance units(RU).The result shows us that PD-L1 recombination protein has excellent bioactivity.(3) Prepare the monoclonal antibodies against PD-L1 recombination protein.BALB/c mice was immunized with purified PD-L1 protein and its spleen cells were fused with SP 2/0 cell.After identified by ELISA competitive inhibition assay and western-blot,5 strains monoclonal antibody were selected.The serial number of five strains are 5B12,5C7,5F12,6A12,6F1.We took 5B12,5C7,5F12 as samples to prepare ascites.The cells were injected into BALB/c mice's abdominal cavity.Ascites were collected,identified and purified,we obtain purity anti-PD-L1 antibody with its density is 1.8mg/ml.(4)Block PD-1/PD-L1 pass way by anti-PD-L1 and analyze the effect of HBV clearance.Plasmid named AAV/HBV DNA1.2 was injected into C57BL/C mice by t way of tail vein to construct HBV infection model.Anti-PD-L1 was injected into HBV infection model and observe the HBV DNA copies and HBV infectious markers.The result are(1) Average copies of HBV DNA of control group are not less than 1×10⁶ at 7th days.(2) the whole HBV DNA value of treat group is lower than control group significantly,the 28^th day's HBV DNA clearance rate of treat group is high than control group significantly;(3) whole value of HBsAg of treat group are less than control group,the 7^th's,14^th day's HBsAg of treat group are less than control group;(4) The 28^th day's the anti-HBs of treat group is lower than control group.We could make a conclusion that anti-(PD-L1) antibody can block the PD-1/PD-Llpass way and enhance the effect of HBV clearance.In short,our study investigated and analyzed the prevalence of HBV relative disease such as CHB,et al;established the HBV infection survey cohort.We also expressed recombination PD-L1 protein and prepared the anti-PD-L1 antibody,Used the antibody as immune-medicine on HBV infective mice model.The results show us that anti-PD-L1 can block the signal passway.PD-1/PD-L1 signal passway is the possible therapy target of HBV chronic infection.Anti-PD-L1 should be prospective as being immune-medicine for HBV infection.