Regulation Of Hepatitis B Virus Mutation And Effect Of Immunoprophylaxis In Mother-to-child Transmission

Chronic hepatitis B virus (HBV) infection is an important public health issue globally, es-pecially in Mainland China. HBV virus can be transmitted vertically or horizontally. Perinataltransmission plays a major role in chronicification of HBV infection in high endemic coun-tries. Approximately90%of the HBV infection in infants will initiate the process of chronicinfection, which later may result in liver cirrhosis or hepatocellular carcinoma.In China, hepatitis B vaccine for routine immunization in infants has officially been rec-ommended since1992. Since then, the prevalence of HBV infection in vaccinated childrenhas been dramatically decreased.According to the Chinese national guideline’s HBV recommendation, a timely birth doseprovided within24hours after birth is the essential step of HBV vaccine. Neonates born toHBsAg-negative mother will be administrated5μg or10μg yeast recombinant hepatitis Bvaccines within the first24hours after delivery and followed by another two doses of vac-cines at first and sixth month, respectively. Neonates of HBsAg positive mothers are adminis-trated hepatitis B immuneglobulin (HBIG) within24hours after birth as soon as possible(preferably within12hours), and inoculated with10μg of recombinant yeast hepatitis B vac-cine or20μg recombinant CHO vaccine in different body sites, and followed by two doses ofHBV vaccines at1month and6month, respectively.Due to the implementation of the universal infant HBV vaccination program, HBV infec-tion through mother-to-child transmission has been controlled. Unfortunately, there are stillsome cases who are failed to generate neutralizing antibody or still infected. Very high ma-ternal viremia, maternal HBeAg positive in utero infection, or immune-escaped mutants ofHBV are possible reasons for vaccination failure.To assess the protective effects of current neonatal vaccine program and identify the riskfactors of HBV infection via mother-to-child transmission, the following three systematicstudies were carried out in eastern china:Part IWe enrolled362matched mother-infant pairs with positive maternal HBsAg to assess therate and risk factors of intrauterine transmission of HBV. Peripheral blood from HBsAg posi- tive pregnant women and cord blood of their newborns were obtained, and HBsAg, anti-HBs,HBeAg, anti-HBe, anti-HBc, and HBV-DNA level were detected. Infants with appropriatelyvaccinated were prospectively followed up.Although most (67.4%) of the HBsAg(+) mothers were HBeAg(),36.1(88/244)%had de-tectable HBV DNA in mothers, and their neonatal viremia was detected in5of244neoborns(2.0%).93.2%(110/118) pregnant mothers with HBsAg(+) and HBeAg(+) had detectableHBV DNA, their neonatal HBV viremia was detected in24of118newborns (20.3%). How-ever, the HBV intrauterine infection in newborns with maternal HBsAg positive was8.0%(29/362).When the HBsAg-positive mothers with higher viral loads (106copies/mL), their new-borns were more likely to have intrauterine HBV infection, with the transmission rate of23.3%. In contrast, the prevalence of intrauterine infection in the group whose mother with noor low maternal DNA level was1.9%(2=45.670, P<0.001). The result indicated that themahteral HBeAg positive (Odds Ratio, OR=12.2) and high level DNA viremia (OR=15.4)detected in mother were associated with increased risks for HBV intrauterine infection.Part IIIn this part, the immunogenicity and protective effects of current HBV vaccine programmewere evaluated between the low risk (born to HBsAg negative mother) and high risk (born toHBsAg positive mother) infants. A total of1299low risk cases provided with5μg recombi-nant HBV vaccine at the0,1and6month after birth were taken as group A. A total of1122low risk cases provided with10μg recombinant HBV vaccine at0,1and6month after birthwere taken as group B. A total of339cases of high risk infants were vaccinated with10μgrecombinant hepatitis B vaccine as the0,1,6month schedule with an additional HBIG(out-of-pocket) within24hours after birth and taken as group C. The anti-HBs, HBsAg, andanti-HBc status were compared in these three groups.The geometric mean concentration (GMC) of anti-HBs were714.79mIU/mL,2422.47mIU/mL, and1271.77mIU/mL and the positive rates of anti-HBs (>10mIU/mL) were99.61%,99.73%,97.34%in groups A, B, and C, respectively. Seven children born toHBeAg-positive mothers were chronically infected. Twenty-four months after vaccination, theanti-HBs GMC quickly decreased to25.02mIU/mL in group A,48.01mIU/mL in group B,and35.86mIU/mL in group C. However, the positive rates of anti-HBs were still high, with72.98%,85.90%and79.54%in each group, respectively. About6.7%of children born to HBeAg-positive mothers developed into chronic HBV in-fection, while none of children with maternal HBeAg negative initiated the chronicification.Our data indicated the administration of HBIG reduced the primary response to HBV vaccine.After two years of the primary vaccination, the anti-HBc positive rate in low risk infantswith5μg vaccine was2.85%, which was higher than in those with10μg vaccine (0.98%)(P=0.008). The anti-HBc positive rate of high risk infants was4.54%, which was higher thanthat in the other two groups, suggesting the break-through infection were related to the expo-sure chances and vaccine protective efficiency.In summary, the current neonatal vaccination programmes, especially the high dose vaccineprogram, are effective. Mothers with both HBsAg and HBeAg positive were supposed to betaken more action to reduce the risk of HBV infection by mother-to-child transmission.Part IIISince the viral mutation status will benefit for designing accurate strategies against HBVinfection, full-length HBV genome of matched ASCs mother-child pairs was amplified andthe HBV mutation was analyzed in this part. Peripheral blood of ASCs children and motherswere collected, and HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc and HBV DNA loadswere detected. HBV genome amplified by nested-PCR were sent for direct sequencing, Forthose whose PCR products failed to be sequenced directly, we cloned the amplicons andre-sequenced. The phylogenetic and mutation analyses were performed using MEGA5.0software. HBV was genotyped using the established phylogenetic tree based on the isolatedgeonome sequences and reference sequences from GenBank.Fifty-nine HBV genome pairs isolated from7month-old to15year-old children and theirmothers were subjected for mutation analysis to trace mother-to-child transmission. All thepairs were genotype C (56.0%) or B (44.0%). The consistency in average nucleic acid se-quence of virus genome between the mother and child isolates were99.4%(range from96.1%to100%), indicating that each pairs had the same root.About18.6%(11/59) of ASCs mother-child pairs had inconsistent amino acid sequences in"" antigen determinant epitope (located in AA124-147) of HBV S gene. The mutation rate ofviral “” determinant was33.8%in children and22.0%in their mothers (P>0.05).For G145R mutant in “” determinant,5.1%of children was found while undectable inmothers. The pre-S deletion mutations including the N-terminal (AA1-7) of pre-S1, C-terminal half of pre-S1and the N-terminal half of pre-S2deletions, were observed in13.5%of mothers while undetectable in their matched offsprings (P=0.001).In conclusion, high maternal viremia (HBV DNA loads106copies/mL) and maternalHBeAg positivity were the risk factors of vaccination failure occurred in cases with HBV in-trauterine infection. The immunoprophylaxis program against HBV infection was efficient forneonates. Limited HBV mutations occurred in the mother-to-child transmission. The wildstains and mutant strains of either genotype B or C were able to transmit from mother-to-child.Some special mutation strains, such as preS deletions, were rarely passed via mother-to-childtransmission. The incidence rate of immune escape mutations such as “G145R” was very lowin vaccinated children.