呼吸道融合病毒感染之流行病學及預防

Epidemiology and Prevention of Respiratory Syncytial Virus Infections

紀 鑫(Hsin Chi)

指導教授 ： 黃立民 ； 江伯倫

國立臺灣大學/醫學院/臨床醫學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.02143

摘要

人類呼吸道融合病毒的感染會引起細支氣管炎和肺炎等下呼吸道感染，而且是嬰兒與年幼兒童的首要住院原因。RSV相關住院發生率的高峰出現在1個月大到6個月大之間。在具有明確RSV流行季節的溫帶國家中，約有50%的RSV相關住院事件都是發生在未滿6個月大的嬰兒身上，幾乎所有兒童在2歲時都已經至少感染過RSV一次。發生嚴重RSV疾病的前置因子包括年齡較小、早產、慢性肺病（CLD）、先天性心臟病（CHD）、神經肌肉障礙、免疫缺乏，以及唐氏症等。RSV的流行模式會因地理位置而異：在溫帶氣候中，RSV的流行常出現於秋季並持續流行至春季，而在熱帶地區則通常與雨季一致。RSV呼吸道疾病的治療主要為支持性治療。目前一般建議在冬季期間對高風險兒童每個月施用一次palivizumab以預防RSV感染。對於在台灣針對高風險嬰兒施行的RSV免疫預防療程而言，所謂的「RSV流行季節」可能與RSV感染有季節性高峰的其他國家（如美國）有所不同。台灣的全國性流行病學數據有限，且RSV感染帶來的整體疾病負荷仍未有具體的數據。因此，本研究的目的包括：1. 進行人口群研究以測定台灣RSV相關住院事件的嚴重性、危險因子、季節性以及醫療費用。2. 針對過去連續10季在北台灣分離出之臨床病毒株的RSV F蛋白基因，探討其親緣關係、演化變異性及族群動力學特性。3. 測定連續6個月每月施用palivizumab一次的新型預防療程，在高風險早產兒中預防RSV感染時的臨床有效性及安全性。
首先，我們利用台灣的全民健康保險資料庫，針對台灣未滿5歲兒童自2004年至2007年間因RSV而住院的案例，分析其人口群（population-based）年發生率、共存疾病及各項特性。本研究總共探討11081名曾經歷RSV相關住院事件的兒童。人口群平均年住院率在未滿6個月大和未滿5歲的兒童中，分別為每100,000人年1077和232例。發生率尖峰值出現在1個月和2個月大之間。發生風險的男女比為1.4:1（P<0.001）。依季節區分時有顯著的分布模式：每年一致以春季和秋季為高峰（P<0.001）。共有373例（3.3%）曾再次感染RSV。患有共存疾病的943名兒童（8.5%）年齡較大（P =0.001）、必須在加護病房（ICU）內住較長的時間（P <0.001）、氣管插管的發生率較高（P <0.05），且醫療費用較高（P <0.001）。共有888名病患（8%）需要住進加護病房。年齡較小（P <0.001）和患有共存疾病（P <0.001）是需要住進加護病房的獨立預測因子。結論是在台灣RSV感染每年會有兩波流行（春季和秋季達到高峰）。患有共存疾病者，其住院天數、加護病房住院天數會比較長，醫療費用也比較高。年齡較小、早產、先天性心臟病和腦性麻痺都是入住加護病房的預測因子。
接著，我們研究2000-2010年間於北台灣分離出的臨床RSV病毒株，測定F蛋白基因的分子流行病學特性和種系演變特性。針對於2000年7月和2010年6月間因急性下呼吸道感染就診的兒童身上所分離出之RSV病毒株，首先根據F蛋白基因序列進行分型。接著利用鄰接法（neighbor-joining (NJ) method）和最大概似法（maximum likelihood (ML) method）進行親緣關係的重建和評估。RSV F蛋白基因的種系演變模式係以貝氏馬可夫鏈蒙地卡羅框架（Bayesian Markov Chain Monte Carlo framework）分析而得。F蛋白基因所承受的天擇壓力，乃利用Datamonkey網站的介面進行偵測。總數為325株的臨床RSV病毒株中，親緣分析顯示83種A亞群病毒株（RSV-A）可進一步分成3群，而58種B亞群病毒株（RSV-B）則無顯著的分群現象。在細胞毒性T淋巴細胞（CTL）的HLA-B*57和HLA-A*01限制性表位上，可觀察到RSV-A和RSV-B之間有三個胺基酸不同（111、113和114號位置）。在RSV-B中可觀察到一處發生正向選擇（positive selection）的位置，而RSV-A中則無。病毒的演化速率在2000年以前幾乎無變動，接著於2000年和2005年之間減速，並於2005年之後出現顯著較快的演化現象。在每次流行中最主要的RSV-A亞型都會在下一次的流行中被其他亞型取代。結論是在2004年以前，RSV-A感染參與了數次小規模流行，而且只有極少數的病毒株得以演化而在往後數年中再次興起。2005年以後，流通於人群中的RSV-A病毒株明顯有別於前幾年的病毒株，並持續演化至2010年。種系演變模式顯示RSV的演化歧異性在最近5年內顯著上升。這些觀察結果對於疫苗的設計和流行病學研究可能十分具有價值。
台灣自2010年12月開始，一項每月施用一劑palivizumab、連續施用6劑的新型RSV感染預防療程已獲准用於高風險早產兒。為了研究此項新型療程預防RSV感染時的臨床有效性及安全性。我們從2011年4月開始至2013年3月為止，於馬偕紀念醫院小兒科部進行一項前瞻性觀察研究。我們收錄接受palivizumab預防療法的高風險嬰兒作為研究組，並進行12個月的追蹤。我們以傾向分數配對的方式，找出於2000年7月和2008年6月間於同一所醫院內出生並接受追蹤的歷史對照組。主要評估指標為RSV相關住院事件，而次要評估指標包括住院天數和加護病房（ICU）住院天數。這段時間內，我們收錄了127名嬰兒 (108名於妊娠週數≤28週時出生的嬰兒，加上19名妊娠週數為29-35週時出生且患有慢性肺病〔CLD〕的嬰兒）。他們已按照時程完成6劑的palivizumab療程。在研究組內，RSV相關住院事件在出院後6個月內有2件（1.6%），在12個月內有5件（3.9%）。我們以傾向分數配對的方式，為研究組內127名嬰兒配對出對照組內的 127名嬰兒。RSV相關住院率的降幅在出院後6個月內為86%（10.2%比上1.6%，P =0.002），在出院後12個月內則為78%（15.7%比上3.9%，P =0.004）。相較於對照組，加護病房住院率在出院後6個月內由7.1%顯著降為0.8%（P =0.024），在出院後12個月內則由7.9%顯著降為0.8%（P =0.014）。在6.4%的注射次數中曾記錄到不良事件。結論是在RSV感染案例無單季高峰期的地區（如台灣），連續6個月、每月一次的palivizumab肌肉內注射療法能有效預防高危險群病人RSV相關住院。
為了能讓大多數沒有潛在疾病的健康嬰兒也能預防呼吸道融合病毒感染，建立呼吸道融合病毒DNA疫苗之研究平台。我們選用了C57/BL6的小鼠品系作為實驗對象：DNA疫苗由大腿肌肉注射，每次注射100μg (兩腿各50μg)，間隔10天後再注射一次，合計共免疫三針。每次注射前自小鼠尾端採血並保存血清，第三次免疫的10天後，犧牲部份小鼠取得脾臟細胞進行免疫學分析，剩餘的小鼠以106 PFUs RSV經呼吸道感染後記錄體重變化。我們以小鼠血清做為一級抗體 (primary antibody)進行同樣的分析時，三種DNA疫苗的組合都偵測不到F蛋白，實驗顯示小鼠經過三次F蛋白的DNA疫苗注射後，無法產生能辨認F蛋白的抗體。除了中和抗體的生成外，我們也藉分析細胞性免疫反應 (cellular immune responses)來評估疫苗的效果。實驗結果顯示，同時注射F蛋白及GM-CSF的重組DNA時，樹突細胞和巨噬細胞表面的MHC class II皆有上升的趨勢。顯示CD4 T細胞有一定程度的活化，也呼應了抗原呈獻細胞MHC class II表現量上升的結果。然而，進一步分析T細胞產生干擾素(interferon-γ)的能力時，卻看不到干擾素-γ表現量上升，經過抗CD3抗體刺激後，CD4和CD8 T細胞的增生能力也沒有顯著提高。經由以上的實驗結果，我們可以知道，同時注射F蛋白和GM-CSF的重組DNA時，脾臟中CD4 T細胞和抗原呈獻細胞會部分活化，但其程度可能沒有高到會促進細胞增生，而且由於干擾素-γ表現量沒有增加，表示CD4 T細胞免疫反應可能不是往Th1的方向。至於如何藉由改變與調整DNA的劑量、佐劑的使用、注射疫苗的次數，以及評估時使用的檢驗方法是否適宜，以期能得到效果更顯著的疫苗，則需要參考更多文獻和進行更進一步實驗。

關鍵字

人類呼吸道融合病毒 ； F蛋白 ； DNA疫苗 ； 分子流行病學 ； 預防

並列摘要

Human respiratory syncytial virus (RSV) causes lower respiratory tract infection such as bronchiolitis and pneumonia and is the leading cause of hospitalization of infants and young children. The peak incidence of RSV-related hospitalization is between the second and the sixth month of age. In countries with temperate climates that have well defined RSV seasons, approximately 50% of RSV-related hospitalization occurs in infants younger than 6 months and almost all children are infected by RSV at least once by 2 years of age. Predisposing conditions for the development of serious RSV disease include young age, prematurity, chronic lung disease (CLD), congenital heart disease (CHD), neuromuscular impairment, immunodeficiency, and Down syndrome. RSV epidemics occur depending on geographic location; they cluster during the autumn and last until spring in temperate climate and generally coincide with the rainy season in tropical areas. Treatment of RSV respiratory illness is mainly supportive. Monthly palivizumab is currently recommended to high-risk children as a prophylaxis for RSV infection during winter season. The so-called “RSV season” for RSV immunoprophylaxis in high-risk infants in Taiwan might be different from other country with defined seasonal peaks of RSV infections such as United States. The nationwide epidemiologic data are limited and the total burden of RSV infection remains undefined in Taiwan Therefore, the aims of my study include: 1. Perform a population-based study to determine severity, risk factors, seasonality, and medical cost of RSV-associated hospitalization in Taiwan. 2. To investigate the phylogenetic relationship, evolutionary variability, CTL epitopes and population dynamics of the RSV F protein gene of clinical isolates in northern Taiwan over ten consecutive seasons. 3. To determine the clinical effectiveness and safety of the novel six consecutive monthly doses of palivizumab prophylaxis protocol for the prevention of RSV infection in high-risk preterm infants. 4. To estabilish the RSV DNA vaccine platform.
Firstly, we analyzed the annual population-based incidence, underlying diseases and characteristics of hospitalizations due to RSV in Taiwanese children under 5 years of age from 2004 to 2007 by using Taiwan’s National Health Insurance database. A total of 11081 children with RSV-associated hospitalization were studied. Average annual population-based hospitalization incidences were 1077 and 232 per 100,000 children-year in children under 6 months and under 5 years of age, respectively. The peak incidence was between one and two month of age. The male-to-female incidence risk ratio was 1.4:1 (P<0.001). There was a significant seasonal distribution with consistent peaks in the spring and autumn every year (P <0.001). A total of 373 (3.3%) had repeated RSV infection. The 943 children (8.5%) with underlying diseases were older (P =0.001), required longer intensive care unit (ICU) stays (P <0.001), higher rate of endotracheal intubation (P<0.05), and higher medical cost (P <0.001). A total of 888 (8%) patients required ICU care. The younger age (P < 0.001) and underlying diseases (P < 0.001) were independent predictors of requiring ICU care. RSV infection occurred biennially with peaks in spring and fall in Taiwan. Patients with underlying diseases needed longer hospital stay, ICU stay and higher medical cost. Younger age, prematurity, congenital heart disease and cerebral palsy are predictors of ICU care.
Secondly, we studied the molecular epidemiology and phylodynamics of the F protein gene in clinical RSV strains isolated in northern Taiwan from 2000–2010. RSV isolates from children presenting with acute respiratory symptoms between July 2000 and June 2010 were typed based on F protein gene sequences. Phylogeny construction and evaluation were performed using the neighbor-joining (NJ) and maximum likelihood (ML) methods. Phylodynamic patterns in RSV F protein genes were analyzed using the Bayesian Markov Chain Monte Carlo framework. Selection pressure on the F protein gene was detected using the Datamonkey website interface. From a total of 325 clinical RSV strains, phylogenetic analysis showed that 83 subgroup A strains (RSV-A) could be further divided into three clusters, whereas 58 subgroup B strains (RSV-B) had no significant clustering. Three amino acids were observed to differ between RSV-A and -B (positions 111, 113, and 114) in CTL HLA-B*57- and HLA-A*01-restricted epitopes. One positive selection site was observed in RSV-B, while none was observed in RSV-A. The evolution rate of the virus had very little changed before 2000, then slowed down between 2000 and 2005, and significantly evolved faster after 2005. The dominant subtypes of RSV-A in each epidemic were replaced by different subtypes in the subsequent epidemic. We found that before 2004, RSV-A infections were involved in several small epidemics and only very limited numbers of strains evolved and re-emerged in subsequent years. After 2005, the circulating RSV-A strains were clearly different from those of the previous years and continued evolving through 2010. Phylodynamic pattern showed the evolutionary divergence in RSV has significantly increased in recent 5 years. These observations may be valuable for vaccine design and epidemiological studies.
Thirdly, a novel six consecutive monthly doses of palivizumab for RSV prevention protocol has been approved for high risk preterm infants since December 2010. We conducted an observational prospective study at Department of Pediatrics of Mackay Memorial Hospital from April 2011 to March 2013 to determine the clinical effectiveness and safety of this novel protocol for the prevention of RSV infection. We enrolled high-risk infants who received palivizumab prophylaxis as study group and followed up for 12 months. Historic control, those who were born and followed up in the same hospital between July 2000 and June 2008, were enrolled for propensity score matching. Primary endpoint was RSV-related hospitalization, and secondary endpoints included the length of hospital stay and intensive care unit (ICU) care. During the study period, we enrolled 127 infants (108 infants born at ≤ 28 weeks and 19 infants born at 29–35 weeks with CLD). They completed 6-dose palivizumab as scheduled. Among the study group, the RSV-related hospitalizations were 2 (1.6%) within 6 months and 5 (3.9%) within 12 months after discharge. We matched 127 infants in the control group with 127 infants in the study group by propensity score matching. The reductions of RSV-related hospitalization rates were 86% (10.2% vs 1.6%, P =0.002) within 6 months after discharge and 78% (15.7% vs 3.9%, P =0.004) within 12 months after discharge. Compared to the control group, the rate of ICU care significantly decreased from 7.1% to 0.8% (P =0.024) within 6 months after discharge and from 7.9 % to 0.8% (P =0.014) within 12 months after discharge. Adverse events were recorded in 6.4% injections. This six monthly intramuscular administration of palivizumab is effective for prevention of RSV hospitalization in regions with no single seasonal peak of RSV infection such as Taiwan.
Fourthly, in order to protect most of the healthy baby with no underlying disease from respiratory syncytial virus infection, the establishment of respiratory syncytial virus DNA vaccines research platform is needed. We chose the mouse strain C57 / BL6 as experimental subjects: DNA vaccine by intramuscular vaccination to bilateral thigh with a total 100 μg (each leg 50 μg), for a total of three times of immunizations with an interval of 10 days. To measure HRSV-specific serum IgG, sera will be obtained from blood samples collected from the tail prior to each immunization. Ten days after the third immunization, mice were sacrificed for immunological analysis of spleen cells. Mice was challenged with 106 PFUs RSV and then recorded for weight change daily. We used the mouse serum to assay for antibody response. The experiments showed that mice can not generate F protein antibody after three F protein DNA vaccines. We also analyzed cellular immune response to evaluate the effectiveness of the vaccine. Experimental results showed that when injected with the F protein DNA and recombinant GM-CSF, the dendritic cells and macrophages cell surface MHC class II were upregulated. That CD4 T cells display a certain degree of activation also echoes the increases of MHC class II expression. However, further analysis of interferon-γ by T cells falied to demonstrate the increase of interferon-γ performance and CD4 and CD8 T cell proliferative ability were not significant increased through the anti-CD3 antibody stimulation. From above experimental results, when F protein DNA vaccine and simultaneous injection of recombinant GM-CSF DNA, the spleen CD4 T cells and antigen presenting cells partially activated, but the degree might not be so high as to promote cell proliferation. There were no increase in the amount of interferon-γexpressed by CD4 T cells; this implied there might not be good Th1 immune response. In order to get a more significant effect of the vaccine, we need to adjust the dose by DNA, the adjuvant, or the protocol of vaccination. We also need to search more literature and conducted further experiments.

並列關鍵字

human respiratory syncytial virus ； DNA vaccine ； molecular epidemiology ； Fusion protein ； prevention

參考文獻

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