Study On Hepatitis B Virus Mother-to-Child-Transmission Blocking Strategy And Correlation Between Virus Mutation And Blocking Failure

Background:Mother-to-child transmission?MTCT?of hepatitis B virus?HBV?is the main cause of chronic hepatitis B?CHB?infection in china.Perinatal transmission still happens in about 10%infants born to HBV infected mothers despite the active-passive immune prophylaxis.Scholars are most interested on problems of HBV MTCT blocking mechanisms,reasons for MTCT blocking failure,and exploring and updating new blocking strategies.High maternal HBV DNA level is the most important risk factor to induce MTCT.Therefore,antiviral therapy of nucleos?t?ide analogue?NA?during pregnancy is recommended.Antiviral therapy during pregnancy can be divided into two situations,one is for chronic hepatitis B patients who has elevated alanine aminotransferase?2×upper limit of normal?ULN?and need to be treated at any time,and the other is for HBV carriers at the third trimester with normal ALT and high viremia to block HBV MTCT.Whether antiviral therapy during pregnancy can block HBV MTCT or not,and how about the safety of fetus need more evidences.Meanwhile,for pregnant women in HBV infected immune-tolerant phase,can antiviral therapy be efficacious or not,can NA treatment lead hepatitis flare or not are unclear.Most studies recommended that drug discontinuation at1-3 months after delivery.However,can mothers give breast feeding to babies?When to stop drug is more suitable and safer?Can treatment in immune-tolerant-phase increase virus mutation to NA resistance?There are no definite answers and need more evidences.Due to correction function insufficiency of reverse transciptase?RT?during HBV replication course,HBV gene heterogeneity is exist widely.Many studies are about influences of S gene mutations on MTCT.However,on the selective pressure of NA drugs widely use in clinic,P gene especially RT gene variation strains are probably selected to be popular.HBV P gene contain S gene.Therefore,whether P gene variation strains can influence hepatitis B surface antigen?HBsAg?characteristic and function,produce new immune-escape strains,and lead to HBV MTCT blocking failure are unclear.Objective:1.Telbivudine?LdT?use in pregnant women,including chronic hepatitis B?CHB?patients and high viremia patients in immune-tolerant phase.Evaluate the efficacy of LdT reducing HBV MTCT,and observe the safety of fetus and infants.2.HBV infected immune-tolerant phase pregnant women with high viremia accepted LdT therapy in the third trimester.Observe the viral response and proportion of ALT elevation to evaluate its efficacy and safety,and to discuss on the time point of drug discontinuation.3.Compare the HBV P/S gene variation characteristics of HBV immune prophylaxis failure mothers-children and immune prophylaxis successful mothers,and calculate the influences of protein secondary structure changes which are produced by these variation on protein functions.Materials and Methods:Part?:Study on efficacy of LdT reducing HBV MTCT and safety of infants.It was a prospective,open-label,multicenter study.Pregnant women of 19 to 45 years old enrolled were from Shengjing Hospital of China Medical University and The Sixth People's Hospital of Shenyang between January 2011 and April 2014.If ALT was normal??40U/L?,she should be in gestation week 24-32,with HBV DNA>1×10⁵IU/ml.If ALT was abnormal,she should be with ALT?2×ULN,and for HBeAg positive,HBV DNA?20000IU/ml or for HBeAg negative,HBV DNA?2000IU/ml.Patients fulfilling the inclusion criteria and agreeing for antiviral therapy were given oral LdT 600mg daily.All infants received standard active-passive immune prophylaxis.MTCT was defined as detectable HBV DNA or HBsAg in peripheral serum samples of infants at 7 months age.Observe the MTCT rate,safety of the agent in fetus and infants.Part?:Efficacy and safety of LdT use and discontinuation in HBV-infected immune tolerant phase pregnant women.HBV infected immune-tolerant phase pregnant women with high viremia(HBV DNA?5 log₁₀IU/ml),normal ALT and without cirrhosis.LdT therapy was accepted from the gestation 24 to 32 weeks until delivery.Serum HBV DNA loads were detected before delivery.If detectable HBV DNA?HBV DNA>20 IU/ml?occurred,antiviral therapy was stopped at delivery immediately,and breast feed was allowed one week after drug discontinuation.Otherwise,the treatment was continued after delivery.Observe the viral response and proportion of ALT elevation to evaluate its efficacy and safety.Part?:Study on gene heterogeneity of HBV immune prophylaxis failure mothers and children.10 pairs of HBV immune prophylaxis failure mothers-children and 18 HBV immune prophylaxis successful mothers with detectable HBV DNA enrolled were from Shengjing Hospital of China Medical University between November 2011 and February 2014.Children'fathers were without HBV infection.Mothers'ages were from 20 to 45 years old,and children were between 1 to 9 years old.Children all completed the full regimen of HBV vaccine.All patients were with normal liver function,and never accepted NA therapy.Detect HBV DNA loads,extract DNA and sequence the whole P gene.Collect 8 HBV standard strains from GenBank,select the same base alignment with our sequence,and analyze with DNASTAR software.Megalign program was for comparing the differences among multi-alignments and analyzing the homology.Phylogenetic tree program was for classifying HBV genotypes.EditSeq program was for amino acid translation and classifying HBV serotypes.Protean program was for analyzing the protein secondary structures.Results:Part?:Study on efficacy of LdT reducing HBV MTCT and safety of infants.?1?There were 135 women in treatment group and 30 patients in observation group.In treatment group,patients with normal ALT were 93?93/135,68.89%?,and CHB patients with ALT?2×ULN were 42?42/135,31.11%?.The average age of 135 pregnancy women in treatment group was 27.81±4.33 years?range:20-42?.The baseline HBV DNA load was7.82±1.04 log₁₀IU/ml?range:4.67-9.53?,the average ALT was 89.20±134.2U/L?range:6-839?.Treatment started at mean gestation weeks of 24.51±5.89?range:4-38?.Of those 30patients without treatment,the average age was 27.43±4.88 years?range:19-39?,the baseline HBV DNA load was 7.61±1.29 log₁₀IU/ml?range:5.11-9.72?,the average ALT was 60.60±120.01U/L?range:9-582?.There were no differences of baseline values between the treatment group and observation group.?2?In the treatment group,there were 2 patients?2/135,1.48%?with fetus growth abnormal after 5 days and 13 days of LdT therapy,separately.The former was with cleft lip,and the latter was with death fetus.These two both were CHB patients.Other 133patients were with fetus growing and developing normal.There were 134 infants without congenital malformations born to these mothers.All the neonates had normal Apgar scores when birth,and developed well.?3?At 7 months after birth,no infant infected with HBV in treatment group,and MTCT rate was 0.In contrast,2 infants out of 21?2/21,9.52%?mothers in observation group followed up at 7 months were HBV-infected?P=0.022<0.05?.Part ?:Efficacy and safety of LdT use and discontinuation in HBV-infected immune tolerant phase pregnant women.?1?91 women?treatment group?were treated with oral LdT 600mg/d,and 26 patients?observation group?did not accept antiviral therapy.The mean HBV DNA of treatment group at baseline was 8.15±0.82log₁₀IU/ml?range:5.54-9.53?.Two women?2/91,2.20%?with HBV DNA declined less than 2log₁₀IU/ml after 1 month of therapy compared to baseline switched LdT to Tenofovir disoproxil fumarate?TDF?.At delivery,the serum HBV DNA levels declined by 4.20 log₁₀IU/ml with mean level of 3.95±0.94log₁₀IU/ml?range:0-5.34??P<0.0001?.Eighty seven patients who discontinued therapy were with HBV DNA rebounded to 8.06±0.83 log₁₀IU/ml?range:5.22-8.95?at 1 month after delivery.?2?Four?4/91,4.40%?patients continued to take LdT after delivery,two?2/91,2.20%?were with undetectable serum HBV DNA before delivery,other two?2/91,2.20%?were with on-treatment hepatitis flare.Other eighty seven?87/91,95.6%?patients stopped the drug at delivery immediately.Only three?3/87,3.45%?patients had off-treatment hepatitis flare,in which two with ALT rising to 4×ULN and 6×ULN separately at 1 month after delivery,and the other 1 with ALT rising to 16×ULN at 3 months after delivery.They all without liver function decompensate and with normal ALT after 1 month of antiviral retreatment.Part?:Study on gene heterogeneity of HBV immune prophylaxis failure mothers and children.?1?In mothers-children of immune prophylaxis failure group,the mean HBV DNA of mothers?failure mothers group,FM group?was 7.44±1.44 log₁₀IU/ml?range:5.82-8.86?,and the mean HBV DNA of children?children group,C group?was 7.56±0.96 log₁₀IU/ml?range:4.44-9.35?.There were no differences between the two groups?P=0.359>0.05?.The mean HBV DNA of mothers in HBV immune prophylaxis successful group?successful mothers group,SM group?was 7.95±0.66 log₁₀IU/ml?range:6.41-8.92?.There were no differences between the two group mothers?P=0.453>0.05?.?2?Nine pairs of mother-child had the same genotype separately.Three pairs had genotype B?M2-C2,M5-C5 and M6-C6?,and six pairs with genotype C?M1-C1,M3-C3,M4-C4,M7-C7,M8-C8 and M9-C9?.Only one pair had different genotype between mother and child,which was genotype C?M10?for mother and genotype B?C10?for child.All the ten pairs of mothers-children were with the same serotype adr.Meanwhile,in the HBV immune prophylaxis successful mothers,four were genotype B?4/18,22.22%?and fourteen were genotype C?14/18,77.78%?.Serotype of them were 13 adr?13/18,72.22%?and 5 adw?5/18,27.78%?,separately.Genotypes and serotypes were with no differences between the two group mothers?P>0.05?.There were four pairs of FM-C with homology of 100%?M1-C1,M7-C7,M8-C8 and M9-C9?,and they were all genotype C.Other two genotype C pairs of FM-C?M3-C3 and M4-C4?with homology of 99.7%and 99.1%,separately.Three pairs of genotype B?M2-C2,M5-C5 and M6-C6?with P gene homology of 99.3%,99%and 98.8%,separately.one pair with different genotype?M10-C10?only had homology of 94%.?3?There were no mutations happened on the P gene RT sites which were usually occurred in strains resistant to NA therapy?80,169,173,180,181,184,202,204,236 and250?,not only in 10 pairs of HBV immune prophylaxis failure mothers-children,but also in 18 HBV immune prophylaxis successful mothers.However,there were 3 pairs of FM-C had 14?14/1032,1.36%?nucleotide point mutations on other sites of RT gene.Among them,four nucleotide point mutations were sense mutation,including ntA289T?C3?,ntC446G?M1/C1?,ntA895T?M3/C3 and M9/C9?and ntG1114A?M9/C9?,and leading to amino acid changes of rtT54S,rtS106C,rtS256C and rtA329T.We found that?-helix,?-pleated sheet and?-turn of protein secondary structure were different from the same genotype reference strains,leading to diferences with molecular weight,length,isoelectric point and antigenic index.S gene had N3S?M10?,A45T?C3?and L98V?M1/C1?mutations.Protein secondary structures analysis showed their antigenic indexes were different from the same genotype standard strains.However,there were no mutations found in the HBsAg“a”determinant?AA124-147?.Conclusions:1.LdT treatment in pregnant women effectively reduced HBV MTCT and it was safe for fetus and infants.2.HBV immune tolerant phase mothers with high viremia who accepted LdT treatment at gestation weeks 24-32 displayed a significant reduction on maternal HBV DNA levels.If with detectable serum HBV DNA at delivery,patients stop the drug at delivery immediately was safe and seldomly?3.45%?with off-treatment hepatitis flare.3.HBV immune prophylaxis failure mothers-children had different mutations in S gene outside of the“a”determinant from HBV immune prophylaxis successful mothers.They were N3S,A45T and L98V.These mutations can lead to protein secondary structures changes.