Study On HBV RNA And HBsAg As Serum Markers For Evaluating The Transcription Of CccDNA In Vivo And In Vitro

Part Ⅰ The establishment of anti-HB V cell model in vitro and the dynamic relationship between HBV DNA,HBV RNA,HBsAg and intrahepatic cccDNAObjective:Covalently closed circular DNA(cccDNA)in hepatocytes is the cause of persistent hepatitis B virus(HBV)infection.HBV DNA,HBV RNA and hepatitis B surface antigen(HB sAg)are all serum expression products of cccDNA in hepatocytes.The purpose of this study is to construct an anti-HBV cell model in vitro to explore the dynamic relationship among HBV DNA,HBV RNA,HBsAg and cccDNA in hepatocytes,and to clarify the correlation among HBV RNA,HB sAg and cccDNA in hepatocytes.Methods:(1)The optimal concentration of tenofovir dipivoxil(TDF)and interferon(IFN)for anti-HBV cell model in vitro was screened by CCK-8 cell proliferation toxicity test;(2)HepG2 cell line was selected as the blank control group.HepG2.2.15 cell line was divided into negative control group,TDF intervention experimental group,and IFN intervention experimental group.The anti-HBV cell model was constructed in vitro for long-term culture.When HBV DNA was detected below the lower limit of detection,TDF intervention experimental group was divided into TDF intervention group and TDF combined with IFN group for further culture;(3)The cells and cell supernatant were subcultured every 3 days,the total DNA(tDNA)in the cells was extracted,and the cccDNA loads were detected by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)after PSAD digestion;the tDNA in the cell supernatant was extracted,and the HB V DNA and HB V RNA loads were detected by reverse transcription PCR(RT-PCR)and qRT-PCR;HBsAg levels were detected by enzyme-linked immunosorbent assay(ELISA);(4)The cccDNA,HBV DNA and HBV DNA loads in the same anti-HBV cell models at different time points,and the same index in different anti HBV cell models at different time points were all compared by one-way repeated measurement analysis of variance.The bivariate linear Pearson correlation analysis method was used to analyze the correlation between each cell supernatant index and cccDNA.Results:(1)CCK8 cell proliferation toxicity test showed that the optimal concentration of TDF was 12 μM and IFN was 100 ng/ml when the cell inhibition rate was ≤10%;(2)After 24 weeks of culture,the cell number and biological index of control group were stable at each time point.TDF and IFN intervention groups were stable from baseline to the 20th week,and the number of cells began to decrease significantly at the 24th weeks(compared with baseline,P<0.05);TDF combined with IFN intervention group began to decrease significantly at the 20th weeks(compared with baseline,P<0.05),so we terminated the experiment after 24 weeks of culture;(3)TDF intervention group:cccDNA load was significantly lower than that of the baseline at the 16th,20th and 24th week(P<0.05),but still in the upper limit of detection(LOD)at the 24th week;HBV DNA load was significantly lower than that of the baseline at the 4th,8th,12th,16th,20th and 24th week(P<0.05),and lower than the limit of detection(LLOD)from the 12th week;HBV RNA load was significantly lower than that of the baseline(P<0.05)at the 12th,16th,20th and 24th week,and LLOD at the 24th week;HBsAg level was significantly lower than that of the baseline(P<0.05)at the 12th,16th,20th and 24th week,but still in the upper LOD at the 24th week;(3)IFN intervention group:cccDNA load was significantly lower than that of the baseline(P<0.05)at the 16th,20th and 24th week,but still in the upper LOD;HBV DNA load was significantly lower than that of the baseline at the 8th,12th,16th,20th and 24th week(P<0.05),and LLOD at the 20th week.HBV RNA load was significantly lower than that of the baseline at the 12th,16th,20th and 24th week(P<0.05),and LLOD at the 24th week;HBsAg level was significantly lower than that of the baseline at the 8th,12th,16th,20th and 24th week(P<0.05),but still in the upper LOD at the 24th week;(4)TDF combined with IFN intervention group:TDF intervention group was divided into TDF intervention group and TDF combined with IFN intervention group after HBV DNA was LLOD at the 12th week.CccDNA load of the combined intervention group was significantly lower than that of the baseline at the 16th,20th and 24th week(P<0.05),but still in the upper LOD at the 24th week.HBV RNA load was significantly lower than that of the baseline at the 12th,16th,20th and 24th week(P<0.05),and LLOD at the 20th week.HB sAg level was significantly lower than that of the baseline at the 8th,12th,16th,20th and 24th week(P<0.05),but still in the upper of LOD at the 24th week;(5)From the baseline to the 24th week,cccDNA decreased in combination group>IFN group>TDF group,negative conversion speed of HBV DNA in TDF group>IFN group,HBV RNA decreased in combination group>TDF group>IFN group,HBsAg decreased in combination group>IFN group>TDF group;(6)There was a highly positive correlation between the levels of HBV DNA,HBV RNA,HB sAg and cccDNA at the baseline(r>0.8,P<0.05).HBV RNA and cccDNA loads were highly positively correlated at the 4th week(r=0.834,P=0.005),HBsAg level and cccDNA load were moderately positively correlate at the 4thweek(r=0.700,P=0.036).HBV RNA load was moderately positively correlated with cccDNA load at the 8th week(r=0.701,P=0.009).After 8 weeks of treatment,no significant correlation was found at the other time points.Conclusion:(1)After drug intervention in vitro,cccDNA,HBV DNA,HBV RNA and HB sAg all decreased,with the most obvious decrease of HBV DNA and the slowest decrease of cccDNA.The negative conversion rate of HBV RNA in TDF intervention group is more obvious than that in IFN intervention group,and the decrease of HBsAg in IFN intervention group is faster than that in TDF intervention group;(3)When HBV DNA is negative,TDF and IFN are administered in combination intervention can improve the HBV RNA negative conversion rate and the decrease of cccDNA and HBsAg,but the coadministration cell culture is more toxic in vitro anti-HBV cell model.Part Ⅱ Study on the reliability of HBV RNA combined with HBsAg as serum markers of cccDNA transcription activity in anti-HBV withdrawal cohort in vivoObjective:The persistence and active transcription of covalently closed circular DNA(cccDNA)in liver cells is the root cause of persistent hepatitis B virus(HBV)infection and relapse after discontinuation of nucleos(t)ide analogues(NAs)treatment in chronic hepatitis B(CHB),but cccDNA detection is limited by invasive operation and is difficult to be widely used in clinical practice.HBV RNA and hepatitis B surface antigen(HBsAg)are currently recommended as serum markers to reflect the content and transcriptional activity of intrahepatic cccDNA for CHB patients after NAs antiviral treatment.The Part Ⅰ has clarified the dynamic relationship between HBV RNA,HBsAg and cccDNA in hepatocytes in the anti-HBV cell models,but in vitro experiments cannot completely simulate the situation in vivo.In this part,we intend to explore the relationship among serum HBV RNA and HB sAg levels and cccDNA content and transcription activity through the NAs discontinuation cohort,to study whether serum HBV RNA and HBsAg can be used as serum surrogate markers for cccDNA content and transcription activity,and to verify the reliability of serum HBV RNA and HB sAg to guide the safe discontinuation of NAs in CHB patients.Methods:This part was divided into two small parts,the first part is the exploration part:CHB patients who underwent liver biopsy and discontinuation after NAs antiviral treatment were selected,their liver tissues at the discontinuation point and the serum tDNA at each follow-up point during the antiviral treatment were extracted respectively,real-time fluorescent quantitative PCR method was used to detect liver tissue cccDNA content and serum HBV DNA and HBV RNA loads at each follow-up point,and chemiluminescence method was used to quantitatively detect serum HBsAg levels at each follow-up point.We analyzed the effect of cccDNA content on the outcome of NAs discontinuation,calculated the cut-off value of cccDNA transcription silent statue,and calculated the cut-off value of serum HBV RNA maintained response time and HBsAg level to predict cccDNA silent statue by ROC curve,and proposed the application of HBV RNA and HBsAg to assess cccDNA content and recommendations for safe discontinuation.The second part is the verification part:the K-M method was used to calculate the cumulative virological relapse rate after discontinuation by the serum and relapse status of the patients with discontinuation,the multi-factor Cox proportional hazard regression model was performed to analyze the factors affecting the relapse after NAs discontinuation;According to the recurrence of drug discontinuation,the reliability of the drug discontinuation criteria recommended by the 2017 EASL guidelines and serum HBV RNA and HBsAg to guide CHB patients treated with NAs was verified.Results:(1)A total of 88 patients who underwent liver biopsy were recruited to study the cccDNA transcriptional activity.The average treatment time was 61.8± 20.1 months.After NAs discontinuation,patients who did not relapse were followed up for an average time of 70.8 ± 43.1 months;(2)The AUC of different index predicted relapse:cccDNA>HBsAg>HBV RNA,were 0.901,0.829 and 0.726,respectively;(3)The cccDNA load of patients without relapse for more than 5 years after NAs discontinuation is the lowest,with an average load of 0.65 copies/cell.The cccDNA load of 0.65 copies/cell was the critical value of silent statue;(4)The cut-off value of serum HBV RNA maintained response duration and the HBsAg level predicting cccDNA silent statue at the NAs discontinuation point was 24 months and 2.6 log10 IU/ml,respectively;(5)The coincidence rates of HBV RNA maintained response duration and HBsAg level for separately evaluating cccDNA silent statue were 58.0%and 66.7%,and the combined evaluation coincidence rate was 75.4%;(6)The serum of 134 patients with NAs discontinuation were used to verify the reliability of HBV RNA and HBsAg in guiding safe discontinuation.The average time of treatment was 48.6 ± 20.6 months,the average follow-up time of patients without relapse after NAs discontinuation was 81.9 ± 34.5 months;(7)At the end of observation,a total of 116 cases occurred relapses.The longest observation time for patients without relapse was 133 months,and the cumulative virological relapse rate of the 1st year,5th years,and 10th years after NAs discontinuation were 67.2%,80.7%,and 91.3%,respectively;(8)Multivariate Cox model analysis showed that HBV RNA and HBsAg level at the discontinuation point were positive risk factors for relapse,and the HBV RNA maintained response duration was a negative risk factor for relapse.(9)The 2017 EASL guideline discontinuation criteria guided NAs discontinuation,95 cases were discontinued when the standard was met,39 cases were discontinued when the standard was not met,and the overall coincidence rate for discontinuation verification was 38.1%;(10)The overall coincidence rate of HBV RNA and HB sAg guiding NAs discontinuation was as high as 90.3%;Conclusion:(1)The load of cccDNA in hepatocytes is the most accurate index to predict the outcome of drug discontinuation;the cut off value of cccDNA transcription silence statue is 0.65 copies/cell;(2)Serum HBV RNA maintained response duration and HBsAg level can predict the content of cccDNA in hepatocytes and guide safe NAs discontinuation.Part Ⅲ A prospective cohort study on the effects of NAs/IFN single and combined treatment on serum HBV RNA and HBsAg levels in patients with CHBObjective:Nucleos(t)ide analogues(NAs)and interferon(IFN)are two major types of anti-hepatitis B virus(HBV)drugs which recommended by the current guidelines,but their anti-HBV mechanism and advantages are different.Different observation indexes of different antiviral treatment will affect the judgment of curative effect.In the Part I,we confirmed that Tenofovir dipivoxil(TDF)and IFN combined administration could improve the negative speed of HBV RNA and decrease hepatitis B surface antigen(HBsAg)levels at anti-HBV cell model in vitro.In the Part II,the in vivo cohort study showed that HBV RNA combined with HBsAg could guide the safe discontinuation of NAs in chronic hepatitis B(CHB)patients.This part of the study aims to dynamically detect the changes of serum HBV RNA and HBsAg levels through the clinical cohort of CHB treated by NAs,and evaluate the efficacy of single use of NAs and NAs combined with IFN antiviral treatment.Methods:(1)CHB patients who had been treated with ETV/TDF for more than 3 years,achieved HBV DNA maintained virological response for more than 1 year,and had positive serum HBsAg(<3000 IU/ml)were recruited and randomly divided into two groups.One group was treated with ETV/TDF monotherapy,and the other group was treated with NAs combined with IFN.The patients were both followed up for 72 weeks at least.(2)Serum HBV RNA load and HBsAg levels were detected by fluorescent quantitative polymerase chain reaction(PCR)every 12 weeks.(3)The differences of serum HBV RNA and HBsAg levels between the two groups were compared by one-way repeated measurement analysis of variance,and the differences of cumulative negative conversion rate of serum HBV RNA and HBsAg between the two groups were compared by log rank test.Results:(1)A total of 54 patients were recruited,of which 27 patients were continued to be treated with NAs alone and the other 27 patients were treated with NAs combined with IFN.(2)At each follow-up point,the serum HBsAg level of NAs combined with IFN group was significantly lower than that of NAs alone group(P<0.05).After 72 weeks of follow-up,there was no patient(0/27)in the NAs group achieved HBsAg negative conversion,and 49.44%(13/27)of the patients in NAs combined with IFN group achieved HBsAg negative conversion.There was significant difference in the cumulative negative conversion rate of HBsAg at each follow-up point between the two groups by log rank test(P<0.0001).(3)At the baseline and the 12th week of follow-up,there was no significant difference in serum HBV RNA loads between the two groups(P>0.05).At the 24th,36th,48th,60th,and 72th week of follow-up,serum HBV RNA loads in NAs combined with IFN group were all significantly lower than those of the NAs group(P<0.05).At the enrollment baseline,66%of the patients in the two groups had achived HBV RNA negative conversion.When patients in the NAs combined with IFN group were treated for 36 weeks,100%of them received HBV RNA negative conversion.The NAs group was followed up for 72 weeks,the HBV RNA negative conversion rate was 81.5%finally.The log-rank test showed significant differences in the cumulative negative conversion rate of HBV RNA at each follow-up point between the two groups(P=0.000).(4)In the NAs combined with IFN group,13 patients with HBsAg negative conversion were followed up for an average of 18.3± 8.6 months after stopping IFN,6 of them stopped NAs together,none of the 13 patients had HBsAg,HBV RNA or HBV DNA positive conversion duration follow-up.Conclusion:(1)HBV RNA and HBsAg are reliable and feasible serological indicators for evaluating antiviral efficacy;(2)For CHB dominant population trated with NAs,NAs combined with IFN treatment can significantly improve the HBV RNA and HBsAg negative conversion rate and achieve clinical functional cure as soon as possible.