2011-2012年台北市學童影響A型新流感H1N1/09抗體反應的因子及探究疫苗抗體對異演化支系B型流感病毒的交叉反應

Factors Associated with Antibody Responses to Influenza A(H1N1)pdm09 Virus and Antibody Cross-reactivity to Influenza B Heterologous Lineage Virus in Taipei Schoolchildren, 2011-2012

劉威(Wei Liu)

指導教授 ： 金傳春

國立臺灣大學/公共衛生學院/流行病學與預防醫學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10170

摘要

2009年新型流感[A(H1N1)pdm09]病毒於全球肆虐，並持續在往後的流感季，取代原季節流感H1N1病毒，在世界各地造成疫情，致使世衛組織於2009年後納入此新病毒為三價疫苗之一。臺灣政府為減少流感疫情，自2009年接種1-6年級小學學童，其後每年均宣導學童接種三價的季節流感疫苗，然學童年年重複接種對新型流感病毒抗體之影響尚缺完整數據；再加上2011-2012年主為B型流感病毒流行，卻又與疫苗株不吻合，也需科學基礎作未來改進疫苗政策之參考。本研究目的有五:(1)探討學童四種接種史(2009和2010年打兩劑、2010年打一劑、2009年打一劑及此兩年均沒打)，對第三年新流感H1N1病毒抗體反應與維持的關係，(2)評估2011-2012年季節流感疫苗引發學童的新流感H1N1病毒免疫力(immunogenicity)和效力(effectiveness)，(3)探尋與此新病毒抗體反應、維持及衰退的相關因子，(4)學童接種疫苗之安全性及(5)檢視B型流感病毒與疫苗株不吻合的異演化支系(lineage)流行株之學童抗體交叉反應。

做法上，自2009年11月至2010年3月間收入台北市兩國小1-6年級學童共935名，進行血清流行病學世代追蹤研究。於家長同意後，始在學校於疫苗接種前(T1)、疫苗接種後一個月(T2)及疫苗接種後四個月(流行季結束T3)三時間點採集學童血液；並以血球凝集抑制試驗(hemagglutination Inhibition assay, HI)測量所有參與學童對新流感病毒H1N1疫苗株(A/California/07/2009)裂解抗原之血球凝集抑制(HI)抗體，並分層抽樣88成三對血清同時測得B型流感病毒疫苗株[B/Brisbane/60/ 2008(Victoria lineage)]與異演化支系流行株[B/Massachussets/02/2012(Yamagata lineage)]之HI抗體以評估交叉反應。統計分析上，以t檢定和卡方檢定先進行單變項分析找出兩組差異的因子；再以變異數分析(analysis of variance, ANOVA)檢定組差異(如過去兩年流感疫苗接種史)；最後以複線性回歸(multiple linear regression)或複邏輯斯回歸(multiple logistic regression)進行多變項分析，調整重要與(邊際)顯著變項，分析T1的抗體維持、T2的抗體反應及T3的抗體衰退之相關因子。

結果顯示，64.0%的學童血清於2011-12流感季打疫苗前(T1)已對A(H1N1)pdm09具保護力之HI抗體(效價≥1:40)；探討過去疫苗史，發現於此T1昔僅2010年接種流感疫苗較僅2009年接種、過去兩年連續接種及均沒施打的學童，有具統計意義較高的新流感H1N1病毒HI抗體幾何平均效價[Geometric Mean Titer, GMT(95%CI)：71.6 (41.7-123.0), 42.0 (25.3-69.6), 34.8 (28.5-42.6), 28.9 (13.1-63.6), p=0.0509]和血清保護率(Seroprotection rate：71.88%, 66.67%, 62.5%, 28.99%, p=0.627)。於當年接種季節流感疫苗一月後(T2)的此病毒HI抗體，雖疫苗接種史四組血清保護率無差異；但其GMT均升高，仍以15位過去均沒接種者最高，其次是32位2010年僅打一劑者，27位在2009年僅打一劑者，最低是185位前兩年連續接種者[GMT(95%CI):557.2(252.8-127.9), 434.1(340.6-555.3), 394.0 (258.1- 601.3), 282.5(242.5-329.0), p=0.0025]；且此T2的抗體反應與T1已具H1N1pdm09保護力的HI抗體維持相關，T1效價≥1:40四組的GMT排序全相一致[9位過去均沒接種者最高，其次是23位於2010年僅打一劑者，18位在2009年僅打一劑者，最低是112位前兩年連續接種者(GMT=940.3，488.1，419.1，366.6, P<0.0001)]，即連續接種者之HI抗體效價未增；且T1流感HI抗體效價低(<1:40)時，此四組T2的HI抗體效價均低致GMT無顯著差異。

評估2011-2012年季節流感疫苗上，探討安全性發現有32.4%(551/1701)的學童在接種疫苗後7天內有不良反應，但均無嚴重副作用；且接種前已有>1:40新流感H1N1血清HI抗體效價的學童在接種後較易發生腫脹[18.5%(58/314) vs 10.7%(18/168) , p=0.0260]。在疫苗效力，464位打完疫苗後一個月者此HI抗體保護力高達97.6%，明顯高於117位未打疫苗組的65.8%(P<0.05)，另打疫苗組有超過75%抗體上升4倍以上；且其抗體保護率並未明顯下降[T2:97.6%, T3:94.6%]，但此抗體幾何平均效價自T2到T3從357.0降至186.8；而其抗體四倍下降者達29.3%。接種後一個月(2011年12月)至流感季末(2012年3月)，打疫苗學童上確實減少69.2%(95%CI = 49.4%-81.2%)的新流感H1N1病毒的自然感染(抗體效校價T2到T3≥2倍上升)。

進一步分析學童於接種前抗體維持、接種後一個月抗體反應和後四個月抗體衰退之相關因子，發現控制年級、性別、學校及洗手習慣後，學童家有老人在接種前較少具新流感H1N1之血清保護力(OR=0.69, 95%CI=0.48-0.99, p=0.0488)；另控制性別、年級後，每週運動三小時以上[log value of reduced serotiter (mean±SD) = -1.39±1.11 vs. -0.98±0.84, p=0.0036]和T2抗體效價較高者為主要影響學童新流感H1N1抗體衰退因子[-1.34±0.98 vs. -1.05±1.02, p=0.0033]。

在B型流感病毒上，從88位學童的血清抗體初步分析，顯示接種疫苗[B/Brisbane(B/Bris)]的學童較未接種者有顯著較高的比例對異演化支系的流行株[B/Massachusetts (B/Mass)]呈HI抗體顯著(≥4倍)上升[28%(19/67) v.s. 14%(3/21)]。細分析知接種疫苗後有54%(31/67)學童從T1到T2對疫苗株(B/Bris)HI抗體顯著上升中，有45％(14/31)學童伴隨對流行株(B/Mass)抗體效價顯著上升，但在疫苗株抗體未顯著上升者，僅14%(5/36)對流行株顯著上升HI抗體。相反地，對流行株的抗體效價顯著上升在未接種疫苗的兒童，僅伴有15-33％對疫苗株抗體的效價顯著上升[33%(1/3)T1~T2, 15% (33/20)T2~T3]。因此接種學童的B/Bris疫苗抗原比未接種疫苗但遇B/Mass感染的兒童更易誘發交叉反應的抗體，反之不然；但此結果仍需更多樣本以確切評估此兩病毒之間的交叉反應和保護力。

由於新流感H1N1病毒於2009入台至2011-12流感季仍持續流行且無明顯抗原變化，加上學童對此病毒完整的接種記錄，此為本研究較過去每年替換季節流感病毒株之優勢。綜言之，2011-2012年季節流感疫苗接種顯著提昇學童對新流感H1N1病毒的保護力;而此新病毒流行後隔年施打裂解流感疫苗的學童較每年施打者誘發較高的幾何平均抗體效價，然未來對感染後再接種或重複接種裂解疫苗是否提昇免疫效益，仍須探研T細胞或B記憶型細胞的影響，以進一步探究最佳的疫苗策略。而常運動者在接種後四月抗體更易衰退，此仍須進一步研究此與流感致病的關係。另初步分析發現該季B流感病毒疫苗株仍可刺激學童對異演化支系之B流感病毒流行株產生交叉反應抗體，未來可進一步測血清中細胞激素(cytokines)[如interleukin(IL)-6、soluble tumor necrosis factor(sTNF)α、IL-4]，以藉由學童世代評估此兩交叉反應抗體對異演化支系流行株彼此的交叉保護情形，期能提供決策者制定更具效益之學童疫苗政策。

關鍵字

學童 ； 2009新型流感病毒 ； 流感疫苗接種史 ； 運動 ； 抗體衰退 ； 血清流行病學 ； B型流感病毒 ； 抗體交叉反應 ； 臺灣 ； 公共衛生防疫政策

並列摘要

The pandemic H1N1 virus [A(H1N1)pdm09] spread globally since 2009, resulting in epidemics around the world. World Health Organization had recommended the A(H1N1)pdm09 as one of the three components involved in the trivalent influenza vaccine in influenza seasons since 2009. Taiwan's government launched mass- vaccination of this novel virus on grade 1-6 elementary schoolchildren in 2009 and then implemented annual vaccination of trivalent flu vaccine. However, the antibody (Ab) responses after repeated annual vaccination have not been available. In addition, the dominant circulating virus during the 2011-2012 was influenza B/Yamagata-lineage with mismatched B/Victoria-lineage vaccine strain. Therefore, this study had the following five aims: (1) to explore the relationship between Ab respones of H1N1pdm09 and their past vaccination histories (2009 and 2010, 2010 alone, 2009 alone, and none in both years). (2) to assess the immunogenicity to this virus and vaccine effectiveness during the 2011-12 flu season, (3) to investigate the factors associated with Ab maintenance in next year and Ab waning post-vaccination in the 2011-12 season, (4) to evaluate the safety of the 2011-12 flu vaccine, and (5) to examine the cross-reactivity Abs between the B/Victoria-lineage from the vaccine strain and the vaccine-mismatched circulating heterologous influenza B/Yamagata-lineage strain.
A seroepidemiological cohort study on 935 grade 1-6 schoolchildren from the two elementary schools in Taipei was conducted from Nov. of 2011 to March of 2012. Blood samples were collected at the three time points: pre-vaccination (T1), 1-month post-vaccination (T2, Dec. 2011), and ending of the flu season (e.g.4-month post-vacci- nation, Mar. 2012, T3). The hemagglutination inhibition (HI) assay was used to measure the HI Abs against A/California/07/2009 (pdmH1N1) for all participants. The selected 88 triplet serum were tested for HI Abs of the two influenza B virus strains [B/Brisbane/ 60/ 2008(Victoria lineage)] and B/Massachussets/ 02/2012(Yamagata lineage)]. In univariate analyses, student t-tests and chi-square tests were applied for finding out the differentiable variables between the two comparison groups; ANOVA was used for testing differences in multiple groups (e.g. the four categories of vaccination history in the past two years). Finally, multiple linear and logistic regression analyses were applied to investigate factors associated with maintaining of anti-A(H1N1)pdm09 protective Ab at T1 and Ab waning at T3 after controlling important variables.
The results showed that 64% of the participants before the vaccination (T1) of 2011-12 flu season already had seroprotective titer (≥1:40) of HI Ab to H1N1pdm09. In vaccination histories, schoolchildren who had received the 2010 vaccine alone showed the highest geometric mean titer (GMT) and seroprotection rate (SR) at T1 than those had vaccinated only in 2009, in both 2009 and 2010-11 flu seasons, and those unvaccinated in 2009/2010 [GMT(95%CI)：71.6(41.7-123.0), 42.0(25.3-69.6), 34.8 (28.5 -42.6), 28.9(13.1-63.6), p=0.0509; SR%：71.9, 66.7, 62.5, 29.0%, p=0.627]. However, the GMTs at T2 (1-month post-vaccination in 2011-12) was significantly the highest in those without any previous vaccination of (H1N1)pdm09, followed by those vaccinated only once either in 2010 or in 2009, and those with repeated vaccination in the past two years [GMT(95%CI):557.2(252.8-127.9), 434.1(340.6-555.3), 394.0 (258.1-601.3), 282.5 (242.5-329.0), respectively, p=0.0025]. This pattern was not only highly correlated with but also consistent with maintaining seroprotective Ab at T1 (e.g. serotiter of T1≥1:40) [GMT=940.3(n=9)，488.1(n=23)，419.1 (n=18)，366.6 (n=112), P<0.0001)]. In other words, repeated flu vaccine did not enhance HI titer too much for children with HI Ab sero- protective at T1 whereas influenza-naive individuals (<1:40 at T1) nor produced higher serotiters of HI Ab to this virus at T2 leading to no difference in past vaccine histories.
In vaccine safety, 32.4% (551/1701) of children occurred at least one adverse event but without any serious side effects within 7 days after the vaccination; swelling occurred more frequently in children who had prevaccination anti-H1N1pdm09 HI Ab 1:40 or greater than those unseroprotective ones [18.5%(58/314) vs 10.7%(18/168) , p = 0.026]. In vaccine effectiveness, 97.6% of the 464 vaccinated children at T2 were seroprotective (significantly higher than 65.8% of the 117 unvaccinated group, P<0.05). In addition, more than 75% of the vaccinated children had ≥4-fold rise in HI serotiter from T1 to T2 and this seroprotection percentage did not decrease significantly from T2 to T3 [T2:97.6%, T3:94.6%], but the GMT declined from 357.0 at T2 to 186.8 at T3, with 29.3% of them declining ≥4-fold. In fact, the influenza vaccination in schoolchildren reduced 69.2% (95% CI=49.4%-81.2%) of the H1N1pdm09 infection[e.g.≥2-fold serotiter rise] between 1-month and 4-month post-vaccination.
Further analysis on factors contributing to anti-H1N1pdm09 HI Ab maintenance at T1 and waning at T3 demonstrated that children living with elderlies were less likely to have seroprotective Hi titer of anti-H1N1pdm09 at T1, after controlling gender, grade, and hand-washing habits after playing[Adjusted Odds Ratio(ORadj.)=0.69, 95% CI= 0.48- 0.99, p=0.0488]. Furthermore, weekly exercise 3 hours or greater and the higher levels of T2’s GMT were the main factors associated with anti-H1N1pdm09 HI Ab waning in T2~T3, after controlling gender and grade [log value of reduced serotiter(Mean±SD)= -1.39±1.11 vs. -0.98±0.84, p=0.0036 ; -1.34±0.98 vs. -1.05±1.02, p=0.0033].
Influenza B virus results showed that the B/Brisbane (B/Bris) vaccinated children had significantly higher percentage to acquire ≥4-fold rise in HI Ab to the Flu B Massachusetts (B/Mass) strain with heterologous lineage] than the unvaccinated group from T1 to T2 [28%(19/67) vs 14%(3/21)]. In addition, 54%(31/67) of the vaccinated students, who had ≥4-fold rises in anti-B/Bris serotiter from T1 to T2, 45%(14/31) of them accompanied with ≥4-fold rises in anti-B/Mass serotiter, whereas only 14% (5/36) of the unvaccinated children with such serotiter increases for wild-type B virus strain. On the contrary, children with ≥4-fold rises in anti-B/Mass HI Ab from T1 to T3, accompanied with 15-33% ≥4-fold rises in anti-B/Bris serotiter [33%(1/3) T1~ T2; 15% (33/20) T2~T3]. Therefore, immunization of B/Bris antigen more easily induced the cross-reactive anti-B/Mass Ab than the vice versa. However, more sample size will be needed for better evaluation between cross-reactivity and cross-protection.
In conclusion, the 2011-12 influenza vaccination in children significantly enhanced the protection from the H1N1pdm09 infection. Vaccination with 2-year interval induced higher HI Ab GMT to this virus than annual vaccination. However, the effect of increasing immunogenicity in either natural infection followed by vaccination or repeated vaccination of the split flu vaccine needs further investigation in both T-cell immunity and B-cell memory. Moreover, exercised children had higher rate of Ab waning, however, more studies will be needed to investigate such a waning would affect the symptoms. The cross-reactivity in anti-B-HI needs to measure serum cytokines [IL-6、sTNFα、IL-4] to understand better the correlation between immune-protection and cross-reactivity for establishing the best vaccination policy.

並列關鍵字

Schoolchildren ； 2009 Pandemic Influenza A(H1N1) Virus ； Influenza Vaccination History ； Exercise ； Antibody Waning ； Seroepidemiology ； Influneza B Virus ； Antibody Cross-reactivity ； Taiwan ； Public Health Policies

參考文獻

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