流行性感冒病毒偵測暨安養中心老人接種流感疫苗保護效價之探究，2004-2006

Influenza Virological Surveillance and Evolution on Vaccine Protection in Taiwan Elderly at Nursing Homes, 2004-2006

江百善(Pai-Shan Chiang)

指導教授 ： 金傳春

國立臺灣大學/公共衛生學院/流行病學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.03093

摘要

流感病毒的持續抗原漂變致世衛組織須每年更換疫苗株，過去未曾整合當季流感病毒株、血清抗體及臨床症狀來探討類疫苗株與野生株對於宿主的影響。因此本研究包括:（1）流感病毒偵測:自2005年2月至2006年3月在台灣人口密集的台北縣市與人口少的南投縣，建立人流感病毒偵測，欲探究台灣城鄉的流感病毒之差異及人流感病毒是否含有動物流感病毒的基因;與（2）流感疫苗抗體效益評估，分析老人接種疫苗對當季流感的類疫苗株與野生株之抗體消長，探討其抵禦力與抗體衰退因子。
流感病毒偵測的結果，共分離得2005年的7株A/H3N2亞型(6株台北、1株南投)與3株B型流感病毒，幸此7株A/ H3N2(一株來自家中經營鳥店的12歲女孩)的8段基因均屬於人流感病毒，尚無跨越宿主傳播的任何動物流感病毒基因之片斷。在A/H3N2的抗原漂變上，有71.43% (5/7)的2005年A/H3N2自A/California/7/2004(H3N2) [2005-06年北半球人流感疫苗株]往A/Wisconsin/67/2005(H3N2)[2006-07年北半球人流感疫苗株]漂變，因而在血球凝集素蛋白[hemagglutinin (HA)]226位置發生置換(D225N)後，而與後者屬於同一群聚。一2005年南投株與83.33%(5/6)的台北株在HA基因有三個位置的不同（K173E、S193F、D225N），且在193與225兩處，南投株同加州株（S、D），83.33%的台北株同威斯康辛株（F、N），都會區流感病毒株的分子變異與郊區/鄉村是否有所不同尚值得深入研究。另一名南投縣9歲男童於2004年接種人流感疫苗[2004-05流行季疫苗株:A/Fujian/411/2002(H3N2)]，卻於2005年3月已被A/Nantou/　3003/ 2005(H3N2)病毒[(近似2005-6流行季疫苗株:A/California/7/2004(H3N2) ]感染，且出現發燒、咳嗽，此二證據顯示台灣的流感野生株早於疫苗株施打約1年。此外，2株B型流感病毒（B/Taipei/128/ 2005與B/Nantou/ 2007/2005）的HA(nt. 228-1068)基因屬於維多利亞支系(Victoria-lineage)，但另1株(B/Taipei/9002/ 2005)的HA基因屬於山形支系(Yamagata-lineage)；而此3株病毒的NA（nt. 57-1398）基因均屬於山形支系，顯示2005年台灣本土流行有HA/Victoria與NA/Yamagata兩者重組後的B型流感病毒。
在疫苗評估上，2004年10月在台北縣及2005年9月加入新竹縣、南投縣而有三地的三家安養中心，收集：(1) 2004-2005年疫苗接種後的三週（流行前）與八個月（流行後）安養中心90位老人(男:女=50:40，平均77.42±8.39歲）的配對血清，及(2)2005-2006年再加入疫苗接種前三週內而增採老人與工作者3個時點共178支配對血清，包括163位長者（男:女=97:66，平均75.33±11.94歲，155人於2005年接種流感疫苗，8位未接種）及15位安養中心員工（男:女=2:13，平均41.47±12.83歲，14位於2005年接種流感疫苗，1位未接種）。在擇選不同病毒株上，依台灣野生株的HA1基因序列（nt.113-273）分別相對於2005-06及2006-07疫苗株-A/Fujian/411/ 2003(H3N2)與A/California/7/2004(H3N2)-之差異，選取A/Kinmen/618/2003(H3N2)為2004-05的類疫苗株與A/Taiwan/CDC00702/2004(H3N2)為2005-2006年的類疫苗株，以血球凝集抑制試驗（hemagglutination inhibition test, HI test）針對同一人不同時間的配對血清同時量測其HI抗體效價，進行疫苗接種後的抗體效價評估，並合併流感疫苗接種與否、接種劑數、年齡、性別、慢性病與類流感症狀，分析其對抗體效價之影響;另選A/Taipei/ NTU6/ 2004(H3N2)[在S227P相異]與A/Taipei/　00017/2005(H3N2)[在T131N、K145N相異]為兩野生株病毒，與類疫苗株A/Taiwan/CDC00702/ 2004(H3N2)同時比對HI抗體效價，並擇選不同HI抗體效價的血清，進行中和抗體測試，以明瞭不同Year野生病毒株and vaccine strains在中和抗原之差異。
結果發現: (1)在2004-2005年90位老人接種疫苗3週後，對類疫苗株A/Kinmen/618/ 2003(H3N2)與兩野生株A/Taipei/NTU6/2004(H3N2)、A/Taipei/00017/2005 (H3N2)的HI抗體之幾何平均效價（geometric mean titers, GMT）各為97.74、17.55與16.25，且各有90.2%、21.1%與28.9%的老人具HI抗體力價≧1:40 [血清保護率(seroprotection rate）]，而在施打疫苗半年後，對類疫苗株A/Kinmen/ 618/2003(H3N2)與兩野生株[A/Taipei/NTU6/2004(H3N2)、A/Taipei/00017/2005 (H3N2)]的HI抗體之GMT各為71.27、28.28與28.28，且各有90.0%、44.4%與44.4%的HI抗體力價≧1:40，即接種疫苗半年後對類疫苗株的抗體力價降低，對抗野生株的抗體力價卻升高，此外，高血壓（p=0.008）或糖尿病（p=0.013）患者較常出現抗體的顯著衰退；(2)在2005-2006年，178位老人接種疫苗一個月後對於類疫苗株A/Taiwan/　CDC00702/2005(A/H3N2)[A/California/7/2004-like]之HI抗體GMT顯著高於未接種疫苗者(69.99 vs. 15.87, p=0.001），且HI抗體≧1:40的比例亦明顯較高（83.1% vs.44.1%, p=0.013），產生抗體4倍上升也較高(51.1% vs. 11.1%, p=0.019）。有趣的是接種疫苗前老人對抗此2005-2006年的類疫苗株的抗體GMT在接種疫苗前略低於員工(20.76 vs. 25.62)，但其接種疫苗後一個月反高於員工(70.17 vs. 65.63)；卻又在接種後6個月的抗體GMT再降至甚低(26.37 vs. 40)；然而，同處於安養中心的員工在接種流感疫苗半年後之抗體衰退百分比顯著低於長者（40.5% vs. 13.3%, p=0.03）。此說明老人接種流感疫苗的重要性。然而，同處於安養中心的員工在接種流感疫苗半年後之抗體衰退百分比顯著低於長者（40.5% vs. 13.3%, p=0.03）。
在比較不同流感病毒株上，挑選對抗2005-2006年的類疫苗株A/Taiwan/CDC00702/2004(H3N2)的不同HI抗體效價之7支與22支血清，分別量測對抗兩野生病毒株:(1)2005-2006的台灣主要流行株A/Taiwan/ CDC00284/2004(H3N2)（相較A/California/7/2004於 a.a.1-329的不同處：S138A、N188D、S193F、T196A、S209N、D225N）與(2)同流行季且有明顯HA1胺基酸差異的自一位發病前半年內接種過A/　Fujian/411/2002(H3N2)的9歲男孩之南投株A/Nantou/3003/2005(H3N2)（相較A/California/7/2004於a.a.113-273的不同處：S138A、K173E、N188D、T196A）的HI抗體力價。發現7支血清抗A/Taiwan/　CDC00702/2004(H3N2)與抗A/Taiwan/CDC00284/2004(H3N2)兩者的HI抗體效價均彼此一致(差異<2倍)。而22支血清有5人對抗A/Taiwan/CDC00702/2004(H3N2)與南投株A/Nantou/3003/05　(H3N2)兩病毒株呈現HI抗體效價4倍的差異，兩株病毒的HI抗體力價對數值在線性回歸之標準化斜率=0.811。未來尚待增加血清樣本數以釐清疫苗株與本土流行株的抗原性異同。
世代追蹤研究能釐清血清抗體變遷的全貌，了解其接種流感疫苗後因抗原漂變所產生的保護落差。未來應深入探討決定流感病毒的中和抗原決定基位（epitope）與抗原性機轉，研發廣效性疫苗以克服流感病毒抗原漂變的問題。老人在接種流感疫苗後仍能有效誘發免疫反應，因此應每年定期予以接種以避免流感病毒的威脅，對於抗體衰退較快者，可藉由提高安養中心長者、員工及其常接觸的親屬之疫苗接種率以增群體免疫力，並經由衛生教育減少流感病毒的傳播，確保老人健康。

關鍵字

流感病毒 ； 分子流行病學 ； 抗原漂變 ； 病毒基因重組 ； 疫苗評估 ； 老人健康 ； 台灣

並列摘要

The World Health Organization (WHO) changes vaccine strain of influenza virus annually due to antigenic drift of the virus. Currently, there are few evidences integrated with virological, serological, and clinical information for evaluating vaccine effectiveness. This study involved two parts: (1) virological surveillance in both metropolitan Taipei City/County and suburban Nantou County during Feburary, 2005 – March 2006 to investigate possible viral variation in these two areas and the presence of animal influenza virus genes/ segments in human influenza viruses; and (2) influenza vaccine antibody evaluation to understand the level of vaccine-induced antibody, its effect against different circulating wild-type virus strains, and the factors affecting protection and antibody waning.
Virological surveillance obtained 7 A/H3N2-subype (6 from Taipei and 1 from Nantou) and 3 B-type isolates in 2005. Fortunately, based on phylogenetic analysis, there was no inter- transmission among 8 segments of our 7 A/H3N2 human isolates (including a 12-year old female patient whose parents owned a pet bird store). About 71.43% (5/7) of the 2005 A/H3N2 isolates drifted from A/California/7/2004(H3N2) in HA gene (D225N)[the vaccine strain in northern hemisphere 2005-06] to A/Wisconsin/67/2005(H3N2)[the vaccine strain in northern hemisphere 2006-07] . Three different sites in HA gene (K173E, S193F, and D225N) were found among one 2005 Nantou strain and 83.33% of Taipei strains. Moreover, the 193S and 225D of the Nantou strain were the same as A/California/7/2004 (H3N2), whereas the 193F and 225N of the 83.33% Taipei strains were the same as A/Wisconsin/67/2005(H3N2). This demonstrates that molecular evolution of human influenza A viruses in metropolitan is worthwhile investigating their variation compared to those in rural/suburban areas. In addition, a 9 year-old Nantou boy who received vaccination of (A/Fujian/411/2002(H3N2)) in 2004 was infected with A/Nantou/3003/2005(H3N2) [A/California/7/2004-like was the 2005-06 vaccine strain used in northern hemisphere], and revealed fever and cough in Mar 2005. Based on these data, it is clearly that Taiwan’s wild type virus circulated at least one year earlier than the timing of immunization with the selected vaccine strain. On the other hand, two isolated influenza B viruses (B/Taipei/128/ 2005 and B/Nantou/2007/2005) had Victoria-lineage HA genes (nt. 228- 1068), but another B/Taipei/9002/2005 strain had Yamagata-lineage genes. However, those 3 strains all possessed Yamagata-lineage NA genes (nt. 57-1398). Thus, Taiwan experienced an attack of reassorted influenza B virus (HA/NA=Victoria/Yamagata) in 2005.
For the vaccine evaluation, one nursing home in Taipei County was chosen during 2004-05 flu epidemic season, and two cooperative ones in Hsinchu and Nantou County were added to the study during the following 2005-06 flu season. Two sets of serum samples were collected: (1) 90 paired sera (3 weeks and 8 months after the vaccination) from the elderly in nursing homes during 2004-05 (males: females =50:40, age mean±SD = 75.33±11.94), and (2) 178 tripled sera (within 3 weeks before vaccination, 1 month and 6 months after vaccination) from 163 elderly (males: females =97:66, age mean+SD =75.33±11.94, 155 vaccinated in 2005) and 15 nursing home employees (males: females =2:13, age mean+SD =41.47±12.83, 14 vaccinated in 2005) during 2005-06. To elucidate the relationship among vaccine-induced antibody, with or without vaccination, vaccine doses, age, gender, co-morbidity, and influenza-like illness (ILI) symptoms, four A/H3N2 strains were chosen as antigens in hemagglutination inhibition test (HI test), according to the comparison of HA1 gene (nt. 339-819) with the 2004-06 northern hemisphere vaccine strains [A/Fujian/411/2002 (H3N2) in 2004-05 and A/California/ 7/ 2004(H3N2) in 2005-06]: (1) two vaccine –like strains including A/Kinmen/618/ 2003(H3N2) and A/Taiwan/CDC00702/ 2004(H3N2) were served as the 2004-05 and the 2005-06 vaccine-like strains, respectively; (2) two circulating strains in 2004-05, A/Taipei/NTU6/2004(H3N2) [different in S227P] and A/ Taipei/00017/2005(H3N2) [different in T131N、K145N]. Besides, in order to elucidate the diversity of neutralizing epitopes among strains, several sera with different HI antibody serotiters against A/Taiwan/CDC00702/2005(H3N2) were compared for their neutralization capabilities against both wild-type and vaccine-like strains.
Results showed: (1)3 weeks after vaccination in 2004-05 flu season, the geometric mean titers (GMT) of vaccine-induced HI antibody in nursing home elderly against A/Kinmen/618/2003 (H3N2)[vaccine-like strain], Taipei/NTU6/2004(H3N2) and Taipei/00017/2005(H3N2)[two wild-type strains] were 97.74, 17.55, and 16.25, respectively. Moreover, their seroprotection rates (HI antibody titer≧1:40) against these 3 viruses were 90.2%, 21.1%, and 28.9%. Six months after vaccination, the GMT of HI antibody against these three viruses became 71.27, 28.28, and 28.28, and their seroprotection rates were 90.0%, 44.4%, and 44.4%, respectively. The trends of GMT represented descendent changes against vaccine-like strain but increasing changes against the circulating strains. Furthermore, elderly with diabetes (p=0.008) or hypertension (p=0.013) had earlier HI antibody waning rates. (2) During the following flu epidemic season in 2005-06, the vaccinated elderly had higher HI antibody serotiters, seroprotection rates, and the proportions of HI antibody with 4-fold rise against A/Taiwan/CDC00702/2004(H3N2) at one month post-vaccination than unvaccinated ones (GMT=69.99 vs. 15.87, p=0.001; seroprotection rate=83.1% vs. 44.1%, p=0.013; 4-fold increasing=51.1% s. 11.1%, p=0.013). In age effect on vaccination, GMT of the elderly was much lower than that of nursing home employee before the vaccination (20.76 vs. 25.62). However, GMT increased dramatically at one month after the vaccination with even higher serotiters than that of the employees (70.17 vs. 63.63) and then decreased again to the lower level than that of the employees (26.37 vs. 40). Similarly, elderly had higher antibody waning rate than that of employees at 6 month post-vaccination (40.5% vs. 13.3%, p=0.03). Thus, vaccination is important for elderly to boost immune responses.
In comparing virus strain variation, 7 and 22 sera with different HI antibody serotiters against A/Taiwan/CDC00702/2005(H3N2) [2005-06 vaccine-like strain] were tested against two wild-type viruses: A/Taipei/00284/2005(H3N2) [2005-06 major circulating strain in Taiwan] and A/Nantou/3003/2005(H3N2) by HI. tests. Molecular differences of A/Taipei/00284/2005(H3N2) were S138A, N188D, S193F, T196A, S209N, and D225N, while those of A/Nantou/3003/2005(H3N2), the vaccine-escape variant, were S138A, K173E, N188D, and T196A based on the comparison with A/California/7/2004(H3N2) in HA gene (nt. 339-819). The results found that the differences of HI antibody titers between the two strains - A/Taiwan/ CDC00702/2004(H3N2) and A/Taiwan/CDC00284/2004(H3N2) in 7 chosen sera were all≦2 folds; by contrast, 5 out of 22 sera (22.73%) showed 4-fold differences in HI antibody titers against A/Taiwan/ CDC00702/ 2004(H3N2) and A/Nantou/3003/05(H3N2). The adjusted slope of the log-transferred linear regression was 0.811. The exact antigenic differences in wild-type vs vaccine strains need to be clarified by increasing the sample size of sera.
Follow-up cohort study can elucidate vaccine-induced antibody dynamic changes and reveal the differences in influenza vaccine protection against the new viruses generated after antigenic drifts. To develop a universal influenza vaccine for overcoming this problem of virus variation, future research can focus on understanding the mechanisms of neutralization epitopes of influenza viruses and investigating their trends of antigenic changes. Influenza vaccination in elderly can induce humoral immunity successfully implying that annual immunization should be advocated to minimize their health threats. Moreover, to protect elderly with fast antibody waning, both vaccination of their frequent contacts/relatives to increase the level of herd immunity and health education to minimize possible transmission of the virus will certainly assure health of the elderly.

並列關鍵字

Influenza ； Molecular Epidemiology ； Antigenic Drift ； Reassortment ； Vaccine ； Evaluation ； Health in Aging ； Taiwan

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