Interruption Of Vertical Transmission Of Hepatitis B And Quantification Of HBV Quasispecies In Mother-infant Pairs With Immunoprophylaxis Failure

Although routine immunoprophylaxis has been known to reduce hepatitis B virus(HBV)transmission,0.75-9.66% immunoprophylaxis failure still occurs.The study aimed to investigate the protective efficacy of an improved immunoprophylaxis protocol to prevent mother-to-infant transmission(MTCT)of HBV and to explore the potential risk factors associated with immunoprophylaxis failure and low antibody response.Little is known about the bottleneck of HBV transmission and the evolution of HBV viral quasispecies between mother and child.It is thus essential to understand the viral factors contributing to HBV MTCT.Methods:(1)A prospective observational cohort study was conducted from July 2012 to April 2015.A total of 863 HBsAg-positive mothers and their 871 infants(8 pairs of twins)were included in the study.Two different hepatitis B vaccine(Hep B)doses(20 μg or 10 μg)were administered to the infants based on the hepatitis B e-antigen(HBeAg)status of their mothers.Simultaneously,hepatitis B immunoglobulin(HBIG)was administered to the infants.Initial injections of HBIG and the hepatitis vaccine were given within 2 hours after birth.Rates of HBV infections among the infants were evaluated at 7 months of age.Factors associated with immunoprophylaxis failure and low responses to vaccination were analyzed by unconditional logistic regression.(2)We adopted a newly developed tag linkage deep sequencing method and analyzed the quasispecies of four MTCT pairs that broke through immunoprophylaxis.By assigning unique tags to individual viral sequences,we accurately reconstructed ~1.25 kb HBV haplotypes with a detection limit of 0.1% in individual patients.Shannon entropy and Sorensen index were calculated using vegan package in R language.Multiple sequence alignment was performed with Muscle.Results:(1)At 7 months of age,no immunoprophylaxis failure was observed in the 565 infants born to HBeAg-negative mothers.Among the 306 infants born to HBeAg-positive mothers,immunoprophylaxis failed in 16 infants(5.2%)of the infants and they were found to be HBsAg-positive.Further analysis showed that HBV DNA levels ≥108 IU/ml(OR=4.53,95% CI: 1.19-17.34),delayed vaccination(OR=4.14 95% CI: 1.00-17.18)and inadequate initial injections(OR=7.69,95% CI: 1.71-34.59)were independently associated with immunoprophylaxis failure.Adequate titers of antibody to HBsAg(anti-HBs,≥100 mIU/mL)were present in 96.5% of immunoprophylaxis-successful infants.For full-term infants,birth weights <3000 g was correlated with low immune responses to vaccination(OR=2.47,95% CI: 1.02-5.99).(2)Four mother-child pairs that failed HBV immunoprophylaxis were included in our study,All 8 patients were infected with subtype C HBV virus.To examine the diversity of intra-host viral quasispecies,we calculated the normalized Shannon entropy(Sn)for each patient sample.Sn ranged from 0.33-0.81,There is no significant difference of Sn between samples from mother and child(p=0.2).Phylogenetic trees were built with 10 major quasispecies for all patients.The maximum likelihood method was used for tree construction using PhyML under GTR model.As expected,viral quasispecies were clustered within each mother-child transmission pair,and separated among different patient pairs.Mother and child viral quasispecies showed higher similarity between patient pair 2 and pair 4,demonstrated as mixed branches in the phylogenetic tree.We examined the newly emergent dominant mutations in child sample for patient pair 1 and pair 3 to test if there is selective sweeping of certain beneficial mutations.Notably,a single mutation G145 A became dominant in the child from patient pair 3,and a double mutation D144G-G145 E occurred in the child from pair 1.G145 A and D144G-G145 A were detected in maternal samples with a slightly higher percentage in patient 1 and 3.However,all of these were minor mutations that occurred at a frequency <1%.Conclusions:This improved immunoprophylaxis protocol is effective in preventing perinatal transmission of HBV.Among infants with HBeAg-positive mothers,high HBV viral loads,inadequate and delayed initial injections were associated with immunoprophylaxis failure.The majority of the infants in our study produced adequate levels of protective anti-HBs titers after immunoprophylaxis.Additional efforts to further reduce perinatal transmission should be considered,especially for HBeAg-positive mothers.Selection sweep of vaccine escape mutations were mild which suggests that controlling HBV viral load during pregnancy might be crucial for further reducing the MTCT rate.