2016至2017年台灣禽類工作者血清流行病學世代研究－探究人流感疫苗抗體對H5與H6兩亞型的交叉反應

Abstract

禽流感病毒在合宜情況可經由禽鳥傳到人。在2015-2016年，韓國、中國、歐洲及台灣等地均爆發禽流感病毒在禽類間大流行。由於不斷傳出禽流感病毒感染人的病例，本研究主要試圖評估動物工作者的高危險群感染禽流感病毒的實況。為了確認此感染的準確性與打疫苗的影響，必須探究接種季節性人流感疫苗者所產生的抗體是否對於禽流感H5與H6亞型病毒會產生交叉反應。 本研究自2016年10月至2017年7月間，在台灣的台北與雲林地區分別以三時間點招募198位禽類工作者（暴露組）與176位健康成人（非暴露組）：（1）接種季節性流感疫苗前（第一時間點，T1）、（2）接種季節性流感疫苗後一個月（第二時間點，T2）以及（3）流感季節結束後（第三時間點，T3）。T1的血清可提供上個流感季節所殘留抗體的訊息；T2的血清可提供T1至T2間內，因接種疫苗或自然感染所引起抗體的數據；T3的血清可提供抗體因正常情況衰退或受到自然感染提高的資訊。本研究先以血球凝集抑制試驗［hemagglutination inhibition (HI) assay］來檢測血清中的抗體效價，凡對禽流感H6病毒此抗體呈陽性的血清，再以微中和試驗［micro-neutralization (MN) assay］進行二次確認。使用的病毒抗原包含2016年度季節性人流感H1與H3疫苗株（由國光生物科技公司提供）及台灣禽流感H5與H6病毒株。單變項分析以T檢定和卡方檢定比較暴露組與非暴露組之間及接踵季節性流感疫苗與否的差異；多變項分析是以廣義估計方程式了解影響HI抗體效價之多項因素。 結果發現暴露組在男性、身體質量指數、有2016與2009-2015年間的流感疫苗接種史與沒至他地旅遊，均顯著高於非暴露組。約90%與22.3%的暴露組在T1各暴露於雞及兩種禽類，且31-43%的禽流感相關知識不足，但54-85%工作時有保護行為。整體來看，受試者的H1N1與H3N2的HI抗體血清盛行率在T1時各為63%與55.5%［幾何平均效價(GMT)：46與28.9］；在T2時各為67.3%與54.6% （GMT:52.2與35.1）；於T3時各下降為54.5%與48.4%（GMT: 33.1與28.6）。在評估疫苗效力上，藉由T3與T2的HI抗體比值，估計2016年人流感H1N1與H3N2疫苗效力各為28.5%及41.6%；顯示此2016-17年季節性人流感仍有保護之效。最後，再以廣義估計方程式的模型比較，發現流感疫苗接種史為H1N1與H3N2 HI 抗體陽性的主要影響因素。 在異亞型的測試，發現對台灣低致病性禽流感H5的HI抗體全為陰性(0/374)，但10位受試者對低致病性鴨流感H6的HI抗體呈陽性，其中5位為健檢中心人員，但有1位資料不全。因此血清低致病性鴨流感H6N1的HI抗體盛行率，以健檢中心人員為最高(14.7%，5/34)，其次是禽類工作者(1.52%，3/198)，最低是健康對照組(0.7%，1/142)；且這10位受試者中有4位健檢中心人員（2位有接種流感疫苗）的配對血清之MN抗體均為陽性，推測可能由感染引起或存有交叉反應。此外，H1和H6的HI抗體效價間存有顯著正相關(r=0.21, p <0.0001)。 本研究提供科學實證基礎與方向，以因應未來禽流感的公共衛生決策，包括：（1）針對禽類工作者及其相關高危險群進行規律性的禽流感抗體監測；（2）健檢中心人員可能是潛在的高危險群，需對其推動衛生教育以預防感染；及（3）未來將以分子流行病學與血清流行病學研究，加強監測禽流感病毒變遷，並於高危險區定期追蹤哺乳類動物與人感染動物流感病毒的實況，藉此降低動物流感對大眾的威脅。

Avian influenza viruses (AIVs) have potential to transmit from avian species to humans under appropriate epidemiological conditions leading to zoonotic diseases. The pandemic of avian influenza in avian hosts affected Korea, China, European countries, and Taiwan in 2015 and 2016. Since human infection of AIV has been reported continuously, this study attempts to estimate the prevalence of AIV infection in high risk poultry-related workers. To precisely measure this prevalence, we investigated whether there would be possible presence of cross-reactive antibody (Ab) against H5 and H6 AIV subtypes after receiving human seasonal influenza vaccine. This seroepidemiologic cohort study recruited 198 poultry-related workers (exposure group) and 176 healthy adults without exposure to poultry (non-exposure group) in Taiwan from October, 2016 to July, 2017. Serum samples were collected at the three time points [pre-vaccination (T1) providing information of residual Abs from the last influenza season, 1-month post-vaccination (T2) offering data of elicited Abs from vaccination or natural infection from T1 to T2, and after the ending of flu season (T3) measuring the results of Ab waning or natural infection from T2 to T3]. Seroprevalence rates and serotiters of hemagglutination inhibition (HI) Ab were firstly screened, using viral antigens from the 2016 human seasonal influenza vaccine of A (H1N1/pdm09) and A(H3N2) provided by Adimmune Corp. and Taiwan H5/H6 subtypes. The seropositive ones of anti-H6 HI Ab were then reconfirmed by microneutralization (MN) assays. Univariate analysis was conducted to compare differences between the exposure and non-exposure groups, using Student T test and Chi-square test. To control the important confounders, Generalized Estimating Equation (GEE) was used for multivariate analysis to find the factors that may influence the seroprevalence of HI-Ab. The participants in the exposure group showed statistically higher males, body mass index, vaccination frequencies in 2009-2016 and without going abroad. At T1, about 90% and 22.3% of the exposure subjects had exposed to chickens, and two kinds of poultry, respectively. And 31-43% of them had insufficient knowledge of AIVs, but 54-85% had the protective practices at working time. The overall seroprevalence rates against H1N1/pdm09 and H3N2 viruses were 63% and 55.5% [geometric mean titers (GMTs): 46 and 28.9] at T1; 67.3% and 54.6% [GMTs: 52.2 and 35.1] at T2; and declined to 54.5% and 48.4% [GMTs: 33.1 and 28.6] at T3. The vaccine effectiveness (VE) was estimated by the ratio of HI-serotiters at T3 and T2. The VE against influenza A (H1N1)/pdm09 and A(H3N2) were 28.5% and 41.6%, respectively. It indicates that the 2016 human flu vaccine should have protection for seasonal flu during 2016-2017. The GEE model showed that flu vaccination history was a dominant factor for HI Abs seropositive against both subtypes of H1N1/pdm09 and H3N2. In cross-reactivity test, all of the study subjects were anti-H5 HI-Ab(-) (0/374). However, 10 subjects were anti-H6 HI-Ab(+) [5 were health-care personnel, but one had incomplete information]. Therefore, seroprevalence rate against duck LPAI H6N1 among all study subjects was the highest in health-care personnel (14.7%, 5/34), followed by the poultry-related workers (1.52%, 3/198) and healthy control group (0.7%, 1/142). All of the 4 subjects (2 vaccinated), who showed anti-H6N1 HI&MN-Ab(+) were health-care personnel, implying that their Abs might be induced by natural infection or produced from cross-reactive Abs. In addition, there was a significant positive correlation between HI serotiters of H1 and H6 (r=0.21, p <0.0001). Based on this study, future effort in evidence-based public health policies for preparing pandemics includes: (1) establishing a regular AIV sero-surveillance for poultry-related workers and other high risk groups; (2) implementing health education for health-care personnel to prevent infection of animal flu viruses, and (3) conducting molecular and seroepidemiological studies to monitor viral changes and Abs to minimize infection of animal flu viruses and potential public health threat.