Studies on the immunity and pathogenesis of important virus infections in Taiwan- Long term immunity induced by hepatitis B vaccine and molecular pathogenesis of enterovirus 71

台灣自1984年開始(1)大規模施打B型肝炎疫苗至今已經超過20年，有效地阻斷了B肝肝炎的母子垂直傳染。10歲以下兒童B型肝炎的帶原率，已經從9.8%下降至1.3%(2)。6至9歲兒童的肝癌發生率，也由每100,000人0.52例降至0.13例(3)。然而，對於B型肝炎疫苗的保護效力可以維持多久，目前沒有定論。現有的研究大部分顯示，不論是第一代血漿製疫苗或是第二代基因重組疫苗， B型肝炎疫苗的保護效力可以維持10到15年以上(4-10)。西元2000年，歐洲的European consensus group on hepatitis B immunity發表一份報告，指出B型肝炎疫苗注射後15年內並不建議追加注射(11)。然而，也有些研究報告卻有不同的結論，它們認為，B型肝炎疫苗注射後10到15年，免疫力便逐漸下降，有失去保護力而發生感染的可能(12-14)。所以，這個問題仍存在相當大的爭議性。本系列研究的目的，在於了解台灣自1984至1992年使用的第一代血漿製(plasma-derived)B型肝炎疫苗，與1992年以後使用的基因工程合成(recombinant)第二代B肝疫苗，是否可以維持15年以上的效果，了解是否有必要追加注射。第一部分的研究中(第二章)，我們與衛生署合作追蹤檢驗78位母親皆為e抗原陽性的B肝帶原者的新生兒個案。這些新生兒在嬰兒時期都確定打過完整四劑的血漿製B肝疫苗，而且打完疫苗以後，都經過抽血確認已經產生具有保護力的B肝抗體。這些個案在七歲的時候也檢驗過一次B肝抗原抗體，確實沒有人的B肝表面抗原(HBsAg)是陽性。我們在這些個案15歲的時候進行這項研究，我們檢驗他們的B肝的表面抗原(HBsAg)、表面抗體(anti-HBs)、以及核心抗體(anti-HBc)。對於測不到保護抗體的個案，給他們一劑追加注射，看看他們對追加注射的反應。研究的結果顯示: 有一位個案的B肝表面抗原呈現陽性，核心抗體也是陽性。顯示其雖然曾經因為注射疫苗產生抗體，可是仍然遭到感染。具有表面抗體的個案有54位(70.1%)。有25(32.5%)位具有B肝病毒核心抗體。表示有相當多的個案曾經受到B肝病毒自然感染。不過B肝疫苗成功地保護了這些個案，使其免於變成B肝的帶原者。23位(29.9%)沒有抗原抗體的個案接受了一劑的B肝疫苗追加注射。追加注射後，有兩位個案沒有產生有保護效力的表面抗體。這是我們第一次證明，曾經接受完整疫苗注射而且確定抗體產生的個案，在15歲左右會遭B肝病毒感染，也有少部分個案(2/77, 2.6%)會對追加注射不產生反應。第三章當中，我們進行了一個大規模的研究，藉由檢驗6,156位15-18歲的高中生的B型肝炎血液標記(HBsAg, anti-HBs, anti-HBc)，來了解國內青少年在接受B型肝炎疫苗15年後，遭B型肝炎感染以及B肝帶原者的比率。並且對不具抗體的個案，進行一劑B肝疫苗追加注射，檢驗其對追加注射的抗體反應。結果顯示，B肝帶原率(HBsAg陽性)有1.7%(107/6156)。4.1%的個案anti-HBc呈現陽性，表示B肝的自然感染仍持續發生中。同時，擁有具保護力的anti-HBs抗體的個案只有37%。換句話說，63%的個案在注射B肝疫苗15-18年以後已經不再具有anti-HBs抗體。接著，有859位HBsAg, anti-HBs, anti-HBc三項皆為陰性的學生，接受一劑B型肝炎疫苗的追加。注射後一個月，有29.3%的學生抗體仍舊小於10 mIU/mL，表示這些個案的免疫記憶已經不復存在。在扣除了可能的未接種完整者，與疫苗不反應者以後，至少有10%的個案已經失去免疫記憶。我們以ELISPOT檢驗細胞免疫記憶的結果，也支持這樣的結果。2004到2007，我們持續追蹤兩群高中生達三年(第四章)。針對於2004年曾給予B型肝炎疫苗追加與未給予追加者，逐年檢驗這些個案的B肝血清標記，比較兩群青少年在3年後遭B型肝炎病毒感染的比率。結果發現，每年大約有9%的個案會失去B肝的表面抗體，而每年新增的B肝自然感染率大約是千分之三。不管有沒有追加注射，都沒有新的帶原者產生。由此看來，雖然部分個案的B肝免疫記憶已經消失，並沒有立即進行大規模追加注射的急迫性。992年以後，台灣所使有的B型肝炎疫苗已經由血漿製(plasma-derived)改為基因工程製(recombinant)。於是我們將研究對象轉為基因工程B肝疫苗。我們針對13-14年前接受基因工程B肝疫苗的國中生，檢驗B肝相關抗原抗體的情形。對於B肝抗原抗體已經呈現陰性的個案，給予追加注射，檢驗抗體反應的情形。以了解基因工程疫苗所提供的保護力，是否可以持續超過13- 14年。對於追加一劑疫苗仍然不會產生抗體的個案，則繼續追加第二劑或第三劑，尋找最佳的追加注射時機與注射方法。研究結果顯示，在我們收集的933位國中生中，B肝抗原的陽性率是是0.2%。再一次證明，B肝疫苗的成效非常顯著，第二代B肝疫苗的效果並不亞於第一代。不過，也有72%的個案既沒有B肝抗原也沒有B肝抗體。我們為543位不具B肝抗原也不具抗體的個案追加注射一劑B肝疫苗。有27.1%的個案在追加一劑B肝疫苗以後仍然沒有產生具保護力的B肝抗體。在追加注射第二劑以以後，有94%的人產生大於10 mIU/mL的抗體，顯示大部份的人並非B肝疫苗的不反應者(non-responder)。大約有21.1%的個案，其體內由B肝疫苗所誘發的B肝免疫記憶已經消失。病毒71型是台灣兒童另外一個重要的病毒性傳染病。自1998年大流行之後，每年都感染不少兒童，並導致重症。今年，又出現了較大規模的流行。在第六章中，我們探討腸病毒71型在台灣的血清流行病學。重點是台灣在1998年腸病毒71型大流行以前是不是就有腸病毒71感染，以及1998年腸病毒71型的發生率與致死率。研究的方法是檢驗腸病毒71型的血清抗體流行病學。我們檢驗1998年前後收集的血清檢體，以micro-neutralization assay檢測中和抗體。另外，利用台灣的疾病管制局在1998年為腸病毒71型病毒感染建立的通報系統，收集腸病毒71型病毒感染有關的重症病例。研究結果顯示，早在1994年，台灣六歲以上的兒童就有大於30%個比例感染過腸病毒71型，成年人的腸病毒71型抗體陽性率更高達50-60%以上。1999年的抗體陽性率較先前的比率都高，反應1998年台灣確實有一次較大規模的腸病毒71型流行。如果將這兩年的陽性率相減，可以估計當年腸病毒71型的發生率是13-22%，年齡較小者發生率較高。每一個年齡大約有35,000到52,000的個案遭腸病毒71型感染。再把死亡病例數放進來分析，可以估計出腸病毒71型在1998年台灣大流行當中，致死率(case fatality rate)大約是89.4/100,000(一歲)到6.6/100,000(四歲)，年齡越大，致死率越低。本研究證實腸病毒71型感染並不是1998年才有的疾病，1998年之前幾年的腸病毒71型感染明顯變少，使人口中可感染宿主數累積，累積了兩三年以後終於爆發大規模的流行。後，在第七章中，我們進到分子生物學的領域，研究腸病毒71型的分子致病機轉。腸病毒為微小RNA病毒科(Picornaviridae)的一員。它包含了一條長約7.5Kb的正股RNA基因體，其中包含了4個形成病毒顆粒的殼蛋白(capsid)以及7個非結構蛋白。腸病毒的感染一方面會引發細胞防衛性的細胞凋亡(apoptosis)，另一方面腸病毒也會抑制宿主細胞的凋亡，使病毒的複製能順利進行(15-18)。然而，目前腸病毒抑制細胞凋亡的機制仍尚未有明確研究。在本研究中，我們首先以yeast two-hybrid方法證明腸病毒71型的2B蛋白與Mcl-1有交互作用，並進一步以GST-pull down assay、共同免疫沈澱法(co-immunoprecipitation)以及共軛焦顯微鏡檢法(confocal imaging)證明Mcl-1與2B的交互作用。目前的研究結果發現，腸病毒71型2B蛋白藉由影響Mcl-1的泛素化(polyubquitinated)而調控Mcl-1的表現量。Mcl-1的增加導致細胞凋亡減少。這些發現，指出了2B蛋白於腸病毒71型調控宿主細胞凋亡反應中扮演重要的角色，已為腸病毒71型調控細胞凋亡的機轉揭開了神秘面紗。Universal neonatal and infant hepatitis B (HB) vaccination program has been launched in Taiwan in 1984 (1) and has resulted in a significant reduction in the prevalence of chronic HBV infections(2), fulminant HB(19), and hepatocellular carcinoma (3). Booster vaccinations are not recommended for immunocompetent subjects 15 years after neonatal vaccination. Nevertheless, there have also been reports of cases of chronic HBV infections that occurred after vaccine-induced protecting antibodies had disappeared [13]. Uncertainties about whether the protection conferred by HB vaccination can truly span 15 years and the need for a booster dose during adolescence or adulthood exist. e aimed to study the duration of HB vaccine-conferred protection and indications for boosters. In different study cohorts, we measured antibody to HB core antigen (anti-HBc), HB surface antigen (HBsAg), and pre- and post-booster titers of HBsAg antibody (anti-HBs) 13-18 years after primary neonatal immunization with plasma-derived or recombinant HB vaccines. In the first study (Chapter 2), we found anti-HBs was undetectable (antibody titer <10 mIU/mL) in 29.5% of 78 children who were born to HB e antigen–positive HBsAg carrier mothers and had developed protective levels of anti-HBs antibodies (>10 mIU/mL) following HB immunization. After a single booster dose of HB vaccine, 2.7% remained anti-HBs-negative. This finding raised the concern about the risk of breakthrough infection. One or more booster immunizations might be needed in seronegative subjects by at least 15 years following neonatal immunization with plasma-derived HB vaccine. n the second part of the study (Chapter 3), we aimed to investigate long-term HB immunity in adolescents who have received plasma-derived HB vaccines in their infancy. In 2004–2005, 6,156 high school students (15–21 years old) who had been vaccinated with plasma-derived HB vaccine as infants were recruited for HB seromarker screening. The immune response to an HB vaccine booster was evaluated in 872 subjects who were seronegative. HB surface antibody (anti-HBs) titers and levels of HB surface antigen (HBsAg)-specific interferon (IFN)-γ- or interleukin (IL)-5-secreting peripheral blood mononuclear cells (PBMCs; measured by enzyme-linked immunospot assay) were determined 4 weeks later. Our results confirmed the vaccine highly efficacious in reducing the HBsAg positivity rate. However, 63.0% of the vaccinees had no protective anti-HBs. After the booster, anti-HBs remained undetectable in 28.7% (158/551) of the subjects who had received complete HB vaccination (4 doses) during infancy. We estimated that at least 10% of the total population had lost their HB vaccine-conferred booster response. A notable proportion of fully vaccinated adolescents had lost immune memory conferred by a plasma-derived HB vaccine 15–18 years later. This decay of immune memory also raised concerns about the need for a booster vaccine for high-risk groups in the long run. e have followed up the study subjects for 3 years (Chapter 4). The incidence of HBV infection (manifested as newly converted anti-HBc) in this age group was about 0.33%. Under such a low incidenc, a 3-year follow-up was not long enough to detect any benefit of booster vaccination in adolescents in this age group. e then shifted our focus to the long-term immunity of recombinant HB vaccine. Similarly, we recruited 933 middle school students and tested their HB seromarkers (Chapter 5). The HBsAg positive rate was as low as 0.3%. 71.9% of study subjects harbored none of HBsAg, anti-HBs, or anti-HBc. A booster vaccination was given to 573 of these subjects. After that, 26.4% of subject remained negative of anti-HBs. A second booster dose raised the positive rate to 93.8%. These results confirmed that immune memory coferred by the recombinant HB vaccine decayed in around 20% (93.8%-73.6%) of vaccinees 13-14 years after the primary vaccination in infancy. n 1998, an epidemic of hand-foot-and-mouth disease and herpangina caused by enterovirus 71 occurred in Taiwan, leaving many fatalities and severely handicapped survivors in its wake. The reasons this rather common pathogen would cause such a large-scale epidemic remain unknown. We performed a seroepidemiological survey to elucidate the epidemiological characteristics of this outbreak, including its incidence and case-fatality rates (Chapter 6). Microneutralization tests for antibodies against enterovirus 71 were used to screen several collections of serum samples. The results showed EV71 was an endemic in Taiwan even before 1998. The EV71 seropositive rates increase with age. Approximately half of all children aged 6 years or older were enterovirus 71 seropositive. In children aged 0.5 to 3 years, seropositive rates were significantly higher in 1999 than in 1997. The incidence of enterovirus 71 infection during the epidemic was estimated to be 13 to 22 percent, with the higher rates in younger children. The case-fatality rate was highest (96.96 per 100,000) in infants aged 6 to 11 months, and declined in older children. Our results showed that enterovirus 71 is endemic in Taiwan. The apparent lack of large-scale enterovirus 71 activity in the three years prior to 1998 might have been the prelude to the epidemic’s appearance in 1998, and suggests that enterovirus 71 infection will reappear every few years. The lack of a protective antibody in younger children may account for the high incidence and case-fatality rate in this age group.nteroviruses have ability to protect host cells from apoptosis induced by various cytotoxic chemicals. The molecular mechanism by which enteroviruses executes its anti-apoptosis function is largely unknown. In Chapter 7, we found a novel molecular mechanism by which enterovirus 71 (EV71) executes its anti-apoptotic activity. By a yeast two-hybrid screen, myeloid cell leukemia-1 (Mcl-1) was identified as an interacting protein of EV71 2B protein. Both in vitro and in vivo binding studies further substantiate the interaction between EV71 2B and Mcl-1. Expression of EV71 2B protein increases stability of Mcl-1, correlating with increased resistance to apoptosis and increment of cell survival. Together, our findings provide a novel mechanism by which EV71 controls survival of its host cell via regulating Mcl-1 stability.口試委員會審定書……………………………………………………………… 1錄……………………………………………………………………………… 2謝……………………………………………………………………………… 7文摘要………………………………………………………………………… 8文摘要…………………………………………………………………………. 11一章 緒論…………………………………………………………………. 14.1 B型肝炎疫苗的長期免疫力.……………………..……………….…… 14.2腸病毒71型的流行病學與分子致病機轉 …………………….……..... 16二章 B型肝炎疫苗保護力消退的證據…………………………….……….. 18. 1 研究背景與目的……………………...………………………………….. 18.2 研究方法………………………………………………………………… 18.3 結果…………………………………………………………………….…19.4 結論與討論……………………………………………………………….20.5 圖表……………………………………………………………………….22三章 血漿製B肝疫苗的長期免疫效力………………………………………263.1 研究目的………………………………………………………………….263.2 研究方法………………………………………………………………….273.3 結果……………………………………………………………………….303.4 結論與討論……………………………………………………………….313.5 圖表……………………………………………………………………….34四章 青少年追加注射B肝疫苗與未追加注射的追蹤比…..………………..404.1 研究目的………………………………………………………………….404.2 研究方法………………………………………………………………….404.3 結果……………………………………………………………………….404.4 結論與討論……………………………………………………………….414.5 圖表……………………………………………………………………….42五章 基因重組B肝疫苗的長期免疫效力……………………………………465.1 研究背景與目的………………………………………………………….465.2 研究方法………………………………………………………………….475.3 研究結果………………………………………………………………….485.4 結論與討論……………………………………………………………….495.5 圖表……………………………………………………………………….50六章 1998年腸病毒71型在台灣大流行前後的血清流行病學………….….52.1 研究背景與目的………………………………………………………….526.2 研究方法………………………………………………………………….526.3 研究結果………………………………………………………………….536.4 結論與討論……………………………………………………………….546.5 圖表……………………………………………………………………….56七章 腸病毒71型與細胞凋亡的調控………………………………………...60.1 研究背景與目的………………………………………………………….607.2 材料與方法……………………………………………………………….617.3 研究結果………………………………………………………………….647.4 結論與討論……………………………………………………………….667.5 圖表……………………………………………………………………….69八章 結論與展望……………………………………………………………….76考文獻……………………………………………………………………...……78錄…………………………………………………………………..…………….9