【作者】 倪红霞；

【导师】 曹广文； 许国章；

【作者基本信息】 第二军医大学 ， 流行病学， 2013， 博士

【摘要】 乙肝病毒(HBV)慢性感染是我国主要公共卫生问题。在HBV流行区，慢性乙肝主要由于母婴传播引起。婴儿期感染HBV90%发展成慢性感染，最终约25%又进展为肝硬化和肝细胞癌。1992年开始我国将乙肝疫苗接种纳入计划免疫管理。由于疫苗免疫的普及和降低婴幼儿HBV暴露等干预措施的实施，人群HBsAg阳性率明显下降。我国目前在新生儿中推行的乙肝免疫方案为：新生儿出生后24小时内尽早接种乙型肝炎疫苗，按照0、1、6月程序全程需接种3针；对HBsAg阴性母亲的新生儿可用5μg或10μg酵母或10μg中国仓鼠卵母细胞(CHO)乙型肝炎疫苗免疫；对HBsAg阳性母亲的新生儿，在出生后24小时内尽早接种乙型肝炎高效价免疫球蛋白(HBIG)，剂量不小于100IU，同时接种10μg重组酵母或20μg CHO乙型肝炎疫苗，在1个月和6个月时再各接种1剂乙型肝炎疫苗，可显著提高母婴传播阻断的效果。与大多数国家不同，多年来我国只生产5μg(啤酒酵母)重组乙肝疫苗，我国大部分地区还一直使用低剂量的5μg重组酵母乙肝疫苗，国际上对低剂量疫苗的免疫保护效果较为争议。近年来我国有自主研发的高剂量重组乙肝疫苗上市，局部地区已在所有新生儿中推广使用，但对新剂型疫苗的总体免疫保护效果还缺乏科学的现场考核。现有资料表明，即使是接种高剂量乙肝疫苗或联合接种HBIG，仍有5%左右的HBsAg阳性母亲的新生儿感染HBV。HBV母婴传播途径主要为产前传播(即宫内感染)、产时传播和产后传播，一般认为主动和被动免疫措施对HBV产时传播和产后传播的阻断效果较满意，但对宫内已发生感染的婴儿效果较差。宫内传播是当前乙肝病毒母婴传播的最主要途径，但我们对宫内传播及乙肝疫苗免疫阻断失败的原因和内在机制仍不十分清楚。报道较多的危险因素是母亲HBeAg阳性和HBV DNA高滴度。此外，还可能与疫苗的免疫原性、机体对疫苗的反应性、遗传易感性和病毒变异等多种因素相关。在控制乙肝病毒母婴传播方面还有很多科学问题未得到确切答案。本课题从乙肝病毒宫内传播、病毒变异、新生儿接种不同剂量乙肝疫苗的免疫保护效果三个角度进行研究，为进一步控制乙肝病毒母婴传播提供科学依据。第一部分乙肝病毒宫内感染是母婴传播的主要途径，可能是导致高风险新生儿(母亲HBsAg阳性)出生后乙肝疫苗免疫阻断失败，从而发生HBV慢性感染的主要原因。在上海浦东区的医院产科，对入院生产的HBsAg阳性产妇进行问卷调查，同时采集产妇静脉血及新生儿脐静脉血，并预期随访至儿童完成乙肝疫苗基础免疫(7月龄)后，采集的血标本检测乙肝血清学标志物和HBV DNA定量，并检测母子乙肝病毒全基因序列，明确HBV宫内传播发生率及相关危险因素。对362对入院分娩的HBsAg阳性产妇及其新生儿调查显示，新生儿HBV感染率为8.0%。 HBsAg+/HBeAg+双阳性母亲的新生儿， HBV感染率为20.3%；HBsAg+/HBeAg-单阳性母亲的新生儿，HBV感染率为2.0%，差异有统计学意义(2=36.104, P=0.000)。分析显示，母亲HBV DNA水平与新生儿HBV感染率有剂量反应关系，当母亲HBV DNA大于106copies/mL时，新生儿HBV感染率为23.3%，显著高于母亲HBV DNA小于106copies/mL者的1.9%，差异有统计学意义(2=45.670，P=0.000)。母亲HBeAg与HBV DNA106copies/mL的Kappa一致性为0.864，两指标间存在较强相关性。单因素分析显示，母亲HBeAg阳性新生儿的HBV感染风险OR为12.2(4.5-32.9)，母亲HBV DNA高水平(106copies/mL)新生儿的HBV感染风险OR为15.4(5.7-41.7)，不同分娩方式与新生儿HBV感染无相关性(P=0.246)。小样本量随访结果显示，完成乙肝疫苗基础免疫后，3例宫内感染新生儿，1例免疫阻断失败(HBsAg阳性)；1例对疫苗无应答(抗-HBs阴性)。而39例非宫内感染新生儿均免疫成功。提示HBV宫内感染是疫苗免疫失败的重要原因。23对发生HBV宫内感染母婴的乙肝病毒全基因序列分析显示，各母子对病毒全基因序列同源性在99.4%-100%，15对母子(65.2%)病毒序列完全一致。按不同基因型分别与B2和C2野生型乙肝病毒株序列进行比较，母亲感染的B和C基因型突变株与野生株乙肝病毒均可通过宫内传播，未发现有宫内传播优势的特异突变位点。第二部分对我国获批准用于新生儿免疫的两种重组乙型肝炎疫苗，10μg/0.5mL剂型重组(汉逊酵母)乙肝疫苗和5μg/0.5mL剂型重组(啤酒酵母)乙肝疫苗，在2760名新生儿基础免疫中应用的免疫原性及保护效果进行社区为基础的观察研究。HBsAg阴性母亲所生的(低风险)新生儿分别按0-1-6程序全程接种5μg重组乙肝疫苗(A组，共1299例)和10μg重组乙肝疫苗(B组，共1122例)，及HBsAg阳性母亲所生的(高风险)新生儿按0-1-6程序全程接种10μg重组乙肝疫苗，并在出生时自愿联合接种HBIG(C组，339名)，在乙肝疫苗全程免疫后1-2个月，3组的抗-HBs阳转率分别为99.61%，99.73%和97.34%，抗-HBs抗体的几何平均浓度(GMC)分别为714.79mIU/mL，2422.47mIU/mL和1271.77mIU/mL。两种剂量重组乙肝疫苗在低风险新生儿中使用的抗-HBs抗体阳转率相当，但接种10μg重组疫苗较5μg重组疫苗的抗-HBs抗体应答水平高。随访观察至免后2年，3组抗-HBs阳性率出现差异(2=40.470，P=0.000)，分别为72.98%(A组)，85.90%(B组)和79.54%(C组)。3组抗-HBs抗体水平在免疫后1-2月达到高峰，此后均以指数水平快速下降，免后2年3组抗-HBs抗体的几何平均滴度均在50mIU/mL以下。339例母亲HBsAg阳性的新生儿，其中89.4%(303/339)选择10μg乙肝疫苗联合HBIG接种；全程免疫后1-2个月共有7名儿童HBsAg阳性，乙肝疫苗母婴阻断失败率为2.06%；免疫失败儿童均为HBeAg阳性母亲所生，母亲HBsAg+/HBeAg+双阳性新生儿的免疫失败率为6.67%；母亲HBsAg+/HBeAg-单阳新生儿均未发生免疫失败；两组差异有统计学意义(Fisher’s P=0.000)。母亲HBsAg阳性新生儿乙肝疫苗免疫阻断失败危险因素的单因素分析显示，产妇孕期晚期进行HBIG干预是疫苗免疫失败的危险因素(P=0.013)；母亲HBsAg阳性新生儿单独接种10μg乙肝疫苗的免疫失败率是2.79%，10μg乙肝疫苗联合HBIG接种者的免疫失败率是1.98%，差异无统计学意义（Fisher’s P=0.548），而母亲HBsAg阳性新生儿采用10μg乙肝疫苗联合HBIG接种的抗-HBs抗体GMC为1184.09IU/mL，低于单独接种10μg疫苗者的2331.72mIU/mL，差异有统计学意义(2=6.779，P=0.010)。接种5μg疫苗的低风险儿童，在免疫后2年的抗-HBc阳性率为2.85%，高于接种10μg疫苗组的0.98%(P=0.008)；母亲HBsAg阳性的儿童，免后2年抗-HBc阳转率为4.54%，HBeAg阳性母亲的儿童抗-HBc阳转率更高，提示突破感染与暴露机会和疫苗保护能力均有关。分析6名乙肝疫苗免疫失败儿童及其母亲的的乙肝病毒基因全序列，显示基因型B和C乙肝病毒均可由母婴传播，母子全基因序列同源性为99.38-100%。第三部分对15岁以下HBsAg持续阳性的母子进行问卷调查，同时采集母子配对血清，检测乙肝两对半和HBV DNA定量，用nested-PCR方法分段扩增乙肝病毒全基因序列，直接测序失败样本经TA克隆后再测序，MEGA5.0软件进行序列同源性、基因型和突变分析。59对7月龄至15岁经母婴传播的乙肝病毒慢性感染儿童与母亲，其中B基因型26对(44.0%)，C基因型33对(56.0%)，母子病毒全基因序列同源性在96.1～100%之间，平均为99.4%。儿童“”抗原决定簇突变率为33.8%，母亲为22.0%，儿童G145R免疫逃逸突变发生率为5.1%，母亲为0%，差异均无统计学意义(P>0.05)。preS区缺失突变仅在HBV慢性感染母亲中发现，发生率为13.5%(8/59)，儿童中未检测到此类缺失突变(P=0.001)。位于BCP区与肝癌和肝硬化相关的突变在母婴中发生率均较低，未发现母婴传播优势。母亲和儿童均可检测到BCP/PreC/Core区的缺失突变，但突变未出现在配对母子中。综上所述，母亲HBeAg阳性和HBV DNA高水平(106copies/mL)是乙肝病毒母婴传播的危险因素；野生型和突变型乙肝病毒均可通过母婴垂直传播，未观察到突变病毒株有母婴传播优势；我国目前推荐的乙肝免疫方案能有效保护新生儿，阻断乙肝病毒母婴传播；乙肝疫苗普种后出生的HBV慢性感染儿童，以G145R突变为代表的免疫逃逸突变发生率很低，尚未构成公共卫生问题。更多还原

【Abstract】 Chronic hepatitis B virus (HBV) infection is an important public health issue globally, es-pecially in China’s mainland. 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.更多还原