【作者】 张维璐；

【导师】 徐德忠； 闫永平； 邵中军；

【作者基本信息】 第四军医大学 ， 流行病与卫生统计学， 2011， 博士

【摘要】 研究背景及目的经典免疫学理论认为新生儿免疫是以母源性抗体提供的被动免疫为主,主动免疫功能尚不成熟,这种状态一直持续到出生后的2-3个月,即为窗口期,这一时期很难建立免疫应答,因此大部分疫苗也就延迟接种[1, 2]。然而,有研究显示这一时期也存在主动免疫,HIV、HSV等胞内病原体感染引起的新生儿急性炎症就是主动免疫存在的直接证据[3]。此外,婴儿出生后就接种了乙肝疫苗,其应答率和成人相近[4, 5],这就使得窗口期主动免疫是否成熟再次成为学术界争议的焦点。动物体内发现的一个有趣的现象为我们提供了线索:接种过疫苗的母体其子代疫苗应答率也高,进一步研究表明母体的主动免疫通过初乳的单核细胞(PBMC)传递给子代[6, 7]。既然母源性PBMC在动物子代发挥了主动免疫功能,从而推测人类母源性PBMC也可能有类似作用。据此,我们提出婴儿乙肝疫苗应答的母婴传递假说,探讨经乙肝疫苗免疫的母体,其主动免疫是否可以传递给子代,并试图解释新生儿主动免疫功能不成熟与乙肝疫苗高应答率这一矛盾,本研究在国内外尚属首次。研究方法本研究通过动物实验和人群研究两方面,探讨经乙肝疫苗免疫的母体,其主动免疫是否可以传递给子代。1.动物实验部分:分别于2009年3月-2009年12月和2010年3月-2010年12月在陕西省西安市及甘肃省武威市进行两批动物实验。实验开始前检测母猪血清抗-HBs水平(ELISA方法,上海科华公司试剂盒)。怀孕母猪随机分为实验组与对照组;第一批实验中每只母猪所产小猪均随机分为免疫组与非免疫组,第二批实验中所有小猪均为免疫组。实验组母猪及免疫组小猪进行乙肝疫苗全程免疫(3次乙肝疫苗颈部皮下分两点注射,20μg/次)。小猪免疫完成后设计10次采血时间点采集血清(免疫完成后1w~10w),定性(ELISA方法,上海科华公司试剂盒)及定量(电化学发光法,德国罗氏公司试剂盒)检测抗-HBs水平。分析实验组与对照组母猪所产小猪抗-HBs滴度及阳性率的差异。2.人群研究部分: 2010年5月-2010年10月,在甘肃省武威市人民医院、凉州区医院、凉州区第三医院及永昌镇共收集母亲及其婴幼儿328对(母亲既往无HBV感染史,婴幼儿月龄为8~48月),其中三所医院74对,永昌镇254对,并进行流行病学调查及采集血液标本。进行血清HBsAg定性(ELISA方法,上海科华公司试剂盒)检测,抗-HBs定性(ELISA方法,上海科华公司试剂盒)及定量(电化学发光法,德国罗氏公司试剂盒)检测。采用回顾性队列研究的方法(以母亲抗-HBs滴度≥10 mIU/mL为暴露,并按婴儿三针乙肝疫苗均及时接种来进行分层),分析抗-HBs阳性组与阴性组的母亲所产婴儿接受乙肝疫苗全程免疫后产生抗-HBs滴度的差异。3.统计学分析:采用SPSS 16.0软件建数据库进行双人录入。采用SPSS 16.0软件和SAS 9.1软件进行统计学分析,Excel软件作图。统计学方法主要应用了t检验(或t’检验),χ2检验(或Fisher确切概率法),两个重复测量因素的方差分析,秩和检验,双变量相关分析等。研究结果1.动物实验部分:纳入研究的母猪为9只,生产小猪79只(存活53只,存活率67.1%)。①所有小猪血清(共400份)抗-HBs定性检测结果显示:免疫组小猪在大多数采血时间点抗-HBs为阳性,非免疫组小猪抗-HBs均为阴性;实验组母猪所产小猪的抗体阳性率高于对照组,并且随观察时间延长,总实验组与总对照组的免疫小猪抗体阳性率差异更加明显。②小猪血清抗-HBs定量检测结果显示:单独的每个采血时间点的小猪血清抗-HBs滴度的对数值进行比较,实验组母猪(接种乙肝疫苗的母猪)所产小猪均显著大于对照组(在免疫后4w、5w,P<0.05;在免疫后1 w、2 w、3 w、6 w、7 w、8 w、9 w、10 w,P<0.01);将所有采血点的定量检测结果进行重复测量资料方差分析,发现实验组母猪所产小猪经乙肝疫苗免疫后产生抗-HBs滴度显著大于对照组母猪所产小猪(P<0.001);根据小猪血清抗-HBs滴度分级进行分析,仍然得到一致结果。2.人群研究部分:共调查研究对象328对母亲及其婴幼儿(男婴191名,女婴137名),母亲平均年龄为30±5岁,婴幼儿平均月龄为27±10月。排除HBsAg+母亲及生产后有乙肝疫苗接种的母亲后,列入统计分析的研究对象为285对,其中,分层后婴儿三针乙肝疫苗均及时接种的研究对象为158对。①母亲与婴幼儿血清抗-HBs水平检测结果:共有56.8%(162/285)的母亲和33.0%(94/285)的婴幼儿抗-HBs滴度<10 mIU/mL;43.2%(123/285)的母亲和67.0%(191/285)的婴幼儿抗-HBs滴度≥10 mIU/mL。②对婴幼儿乙肝疫苗免疫接种史的调查显示:第一针、第二针和第三针乙肝疫苗的及时接种率分别为96.8%(276/285),84.2%(240/285)和60.0%(171//285);3针中有≥1针延迟接种即判定为乙肝疫苗延迟接种,共127名婴幼儿,占44.6%。③回顾性队列研究结果显示:分层前(285对)分析结果显示:母亲抗-HBs阳性组(抗-HBs滴度≥10 mIU/mL)与母亲抗-HBs阴性组(抗-HBs滴度<10 mIU/mL)的婴幼儿抗-HBs滴度无显著性差异。分层后研究队列(158对)分析结果显示:母亲抗-HBs阳性组的婴幼儿抗-HBs滴度显著高于母亲抗-HBs阴性组(P<0.05);两组婴幼儿的抗-HBs滴度分级也有显著性差异(P<0.05)。④母乳喂养的调查结果发现,94.0%的新生儿出生7天内有母乳喂养(其中88.8%的婴儿在出生3天内即开始),并且有82.1%的新生儿母乳喂养量占总奶量的2/3以上。分析出生7天内有母乳喂养与婴幼儿抗-HBs滴度水平的关系,结果显示:分层前研究队列(285对)中,有初乳喂养组和无初乳喂养组婴幼儿抗-HBs滴度水平无显著性差异。在分层后研究队列(158对)中:有初乳喂养的婴儿的乙肝疫苗应答率(69.5%)显著高于无初乳喂养的婴儿(28.6%),P<0.05;两组婴儿抗-HBs滴度分级也有显著性差异(P<0.05)。⑤将母亲生产前有乙肝疫苗接种史的研究对象选出,从定性和定量两方面,分析在这部分人群中,母亲抗-HBs与婴幼儿抗-HBs的关系。结果显示母亲抗-HBs阳性组与阴性组的婴幼儿抗-HBs滴度及滴度的分级数据均无显著性差异(P>0.05)。结论1.国内外首次发现在哺乳动物的母代与子代之间存在着乙肝疫苗应答的母婴传递现象:接种过乙肝疫苗的母猪所产小猪经过乙肝疫苗免疫程序后,其血清抗-HBs滴度显著大于对照组母猪所产小猪。2.通过回顾性队列研究,从人群角度探讨本课题提出的婴儿乙肝疫苗应答的母婴传递假说:母亲抗-HBs阳性组的婴幼儿抗-HBs滴度显著高于母亲抗-HBs阴性组的婴幼儿;有初乳喂养的婴儿的乙肝疫苗应答率显著大于无初乳喂养的婴儿。但是,对母亲生产前有乙肝疫苗接种史的研究对象进行分析时,由于许多母亲对生产前乙肝疫苗接种史记忆不清,导致样本量偏少,还未能排除乙肝疫苗应答的遗传易感性对实验结果的影响。3.本研究的结果初步补充了新生儿乙肝疫苗免疫的理论:经乙肝疫苗免疫的母体,其主动免疫可以传递给子代,从而促进子代对乙肝疫苗的免疫应答。如果能够将孕龄妇女的乙肝疫苗接种及乙肝标志物的检测加以重视,则可将新生儿乙肝疫苗免疫时间提前到怀孕母亲这个层次上,进而为降低婴儿乙肝疫苗无/弱应答率以及制定更有效的免疫计划奠定了理论基础。更多还原

【Abstract】 Background:Neonatal immunity is based on passive immunity provided by the maternal antibodies, whose active immune function is not ripe yet.The immature function of the active immune has lasted for 2 to 3 months after their birth. This period of time is referred as the“window period”. It’s hard to get immune response so that most of the vaccinations are delayed. However, studies show that there are active immune during this period as well. Moreover, vaccination with hepatitis B vaccine at birth, have had almost adult response rate, which makes the question of whether the active immune of infants is mature to become a hot topic again. Animal experiments show that: vaccinated parent, the response rate of vaccine of its offspring is also high. The further research indicates that maternal PBMC in the colostrum can directly transfer to the offspring. Population studies have shown that: maternal PBMC can directly transfer to fetus across the placenta in vivo. As maternal PBMC from animal has active immune function in its offspring, we speculate that maternal PBMC from human should have similar active immune function. Therefore, we put forward the hypothesis: The transfer of maternal immunity promotes the immune response of infants about hepatitis B vaccine. And then we may explain the contradiction of immature active immunity and high response rate of hepatitis B vaccine.Methods:This study explores from two aspects: Animal experiment and population study.1. Animal experiment: All animal experiments were conducted between March 2009 and December 2009 in Shaanxi province,March 2010 and December 2010 in Gansu province. All Sows were free from anti-HBV before the start of the experiment detected by Enzyme-linked immunosorbent assay (ELISA) kits, and were randomly allocated to experiment group (EG) or control group (CG). Sows and piglets were ear notched or punched for identification purposes. After deliveries, the piglets were fed with their mother’s milk in the first month, and then they were raised in routine method. In the first batch of animal experiments, the piglets delivered by the sows of EG were randomly assigned to vaccinated group (VG-EG) or non-vaccinated group (NVG-EG), the piglets delivered by the sows of CG were also randomly assigned to vaccinated group (VG-CG) or non-vaccinated group (NVG-CG). Based on the results of the first batch of experiment, anti-HBs of all the piglets in NVG-EG and NVG-CG were negative, so in the second batch of experiment, all the piglets received three doses of vaccination and divided into VG-EG or VG-CG. The sows of EG and the piglets of VG-EG and VG-CG were vaccinated three times with 20μg of recombinant hepatitis B vaccine intradermally in two lateral sides of the neck behind the ear. Blood samples were collected 2ml for each piglet in evacuated test tubes by venipuncture of ear vein using sterile equipment and procedures, respectively, on weeks 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 post-immunization. Anti-HBs of all the serum specimens were tested by using Enzyme-linked immunosorbent assay (ELISA) kits for anti-HBs from Kehua Company in Shanghai and Electro-Chemiluminescence Immunoassay (ECLIA) reagents for anti-HBs from Roche Diagnostics GmbH.2. Population study: The preparation of the multi-centre population study was started in January 2010 and data analysis was completed in December 2010. All field work was conducted in Wuwei city of Gansu province from May to October 2010. A total of 328 mother-baby pairs were recruited, including 74 pairs from three hospitals (Wuwei City People’s Hospital, Liangzhou Hospital and the third hospital of Liangzhou distinct) and 254 pairs from Yongchang county of Gansu province. The inclusion criteria were that the mothers had no history of hepatitis B virus infection and the ages of infants were 8- 48 months. A standard questionnaire was used to compile the basic information, family history of hepatitis B virus infection, the information of breastfeeding and immunization history of mothers and infants through face to face interview with mothers. Blood samples, collected for each study subject, included 5ml for mothers and children above 2 years, or 3ml for children less than 2 years. HBs Ag of all the serum specimens were tested by using ELISA kits for HBsAg from Kehua Company in Shanghai. Anti-HBs were tested by using ELISA kits for anti-HBs from Kehua Company and ECLIA reagents for anti-HBs from Roche Diagnostics GmbH. Retrospective cohort study was used for analyzing the differences between anti-HBs titers of the infants whose mothers had different anti-HBs titers.3. Statistical Analysis: Data were input in duplicates into the database with SPSS 16.0. All statistical analysis was performed using SPSS version 16.0 and SAS version 9.1. Figures were performed using Microsoft office excel 2007. The t test (or t’test), Correlation, the Chi-square (χ2)-test (or Fisher’s exact test), Repetitive measure analysis of variance, Wilcoxon rank sum test were used in the statistical treatment.Results:1. Animal experiment: There are 9 sows and 79 piglets (53 survived, the survival rate is 67.1%). The qualitative results of ELISA demonstrated that the piglets of VG-EG had a significantly higher anti-HBs positive rates when compared with the piglets of VG-CG (P<0.01). The quantitative results of ECLIA demonstrated that the piglets of VG-EG had a significantly higher anti-HBs titers compared with the piglets of VG-CG on weeks 4 and 5 post-immunization (P<0.05); weeks 1, 2, 3, 6, 7, 8, 9 and 10 post-immunization (P<0.01). Moreover, the results of repetitive measure analysis of variance also showed the anti-HBs titers of the piglets of VG-EG were significantly higher (F=14.89,P<0.001). The anti-HBs positive rates (anti-HBs titers≥10 mIU/mL) and anti-HBs high positive rates (anti-HBs titers≥100 mIU/mL) of the piglets of VG-EG at ten different times after immunization were high than the rates of VG-CG.2. Population study: The quantitative results (n=285 pairs) showed: 56.8% (162/285) mothers and 33.0% (94/285) infants were anti-HBs negative (anti-HBs titers<10 mIU/mL);43.2% (123/285) mothers and 67.0% (191/285) infants were anti-HBs positive (anti-HBs titers≥10 mIU/mL). All infants received hepatitis B vaccine in three doses. However, the rates of the first dose, the second dose and the third dose of hepatitis B vaccine which were vaccinated on time were 96.8%, 84.2% and 60.0% seperately. Totally 44.6% (127/285) of the infants were vaccinated delayed (≥one of the three doses of hepatitis B vaccine were delayed). The results of retrospective cohort study demonstrated that the anti-HBs titers of the infants delivered by mothers who were anti-HBs positive (anti-HBs titers≥10 mIU/mL) were significantly higher compared with the infants delivered by mothers who were anti-HBs negative (anti-HBs titers<10 mIU/mL) (P<0.05). Moreover, anti-HBs positive rate of the infants who were breastfed by their mothers was significantly higher than the infants who were formula-fed (69.5% versus 28.6%, P<0.05). In addition, we chose the mothers who had immunity history of hepatitis B vaccine before their delivering. Then we compare the anti-HBs titer, qualitatively and quantitatively, between the infants whose mothers were anti-HBs positive and those whose are negative. The results show that there were no significant differences in the anti-HBs positive rate and titers (P>0.05).Conclusion:1. We found that the transfer from maternal hepatitis B vaccine-specific immunity to offsprings in pigs for the first time all over the world: The anti-HBs titers of the piglets of VG-EG, delivered by the sows vaccinated with hepatitis B vaccine, were significantly higher compared with the piglets which were delivered by unvaccined sows.2. The study results supported the hypothesis we put forward by using retrospective cohort study: The transfer of maternal immunity promotes the immune response of infants about hepatitis B vaccine. The anti-HBs titers of the infants delivered by mothers who were anti-HBs positive were significantly higher than the infants delivered by mothers who were anti-HBs negative; anti-HBs positive rate of the infants who were breastfed by their mothers was significantly higher compared with the infants who were formula-fed. However, when we analyze the results of mothers who had immunity history of hepatitis B vaccine, for an insufficient sample size, we could not rule out the Genetic susceptibility of hepatitis B which may have influence on our results.3. If mothers were immunited with hepatitis B vaccine, active immunity can be transferred from mothers to their infants and promote the immune response against hepatitis B vaccine of infants. The immunity of hepatitis B vaccine and the detection of HBV markers for women of childbearing age should be paid more attention, which can reduce the incidence of weak or non-response of hepatitis B vaccine of infants and improve the hepatitis B vaccine immunization programs.更多还原