HLA基因型的多型性與B型肝炎病毒感染及B型肝炎疫苗反應性的研究

急性B型肝炎病毒(hepatitis B virus, HBV)感染後，因為個體的免疫反應因素，可能產生自發性的病毒廓清而痊癒；或發展成持續性感染的慢性帶原者。疫苗是預防HBV感染的一種重要方法，但是，在接受B型肝炎病毒疫苗(HB vaccine)後，仍有將近5-10 % 的人對 HB vaccine 的反應很差或沒有反應。人類白血球抗原系統(human leukocyte antigen system, HLA) 是參與及連結免疫反應的重要成份，我們提出假說 : 個體對於B型肝炎病毒廓清或持續性感染及對於B型肝炎病毒疫苗的免疫反應，具有高多型性(polymorphic)的HLA 基因，是重要的決定因子。這個題目分成兩個研究子題: (i) HBV 感染後的研究，我們收集了 434名醫學中心的器官捐贈者或受贈者，時間從 2001年一月至2007年九月。個案必須確認有B型肝炎病毒感染史，然後區分成兩組: 慢性帶原者(Chronic Carrier Group “CC”)和自發性廓清(Spontaneously Cleared Group, “SC”)。慢性帶原者16名，定義是B型肝炎病毒表面抗原(hepatitis B surface antigen, HBsAg) 陽性至少6個月以上。自發性廓清者70名，是指 HBsAg 陰性及 B型肝炎病毒表面抗原的抗體(antibodies to HBsAg, Anti-HBs) 和B型肝炎病毒核心抗原的抗體(antibodies to hepatitis B core antigen, Anti-HBc)皆為陽性者。而且我們也刪除了HBsAg 陰性及Anti-HBs陽性，但是Anti-HBc陰性的個案。(ii) 疫苗反應的研究，包含121 個案(48名醫院健康工作者和73名大學院校學生)，他們接受三劑 20 ug/ml B型病毒肝炎疫苗(Engerix B Hepatitis B Recombinant Vaccine). 每一個個案在疫苗前後皆用穩定的商品化分析試劑(VITROS Eci Immunodiagnostic System)分析 HBs Ag, Anti-HBs 和Anti-HBc。每一個案皆用Viogene Blood and Tissue Genomic DNA Extraction Miniprep System 從200 ul 的血液樣本中萃取DNA，並用商品化的 PCR-reverse SSOP分析試劑(PCR-reverse SSOP HLA typing Kit , Dynal Bioteck Ltd. U.K)執行 HLA-A, -B and -DR 分型。統計分析用Chi-square 和 Fisher exact test 等方法適當的比較分析類別變數，P值小 0.05 視為有統計學上的顯著差異。在HBV感染後的研究, 有16個個案符合慢性帶原者(Chronic Carrier Group “CC”)，符合自發性廓清(Spontaneously Cleared Group, “SC”) 有70個個案。疫苗反應的研究，收集121未曾暴露於B型肝炎病毒(HBV-naïve)的人，執行 3 劑B型肝炎疫苗後，有15人為疫苗不反應者(nonresponders, Anti-HBs <10 mIU/ml)，其餘的106人是疫苗有反應者(responders, Anti-HBs ≥ 10 mIU/ml)。接著分別分析全部樣本的HLA A, B 和 DR基因位點，用具有HLA基因序列不同型別特異性的寡核甘酸探針組(sequence-specific oligonucleotide probe hybridization)進行雜交分型。相較於自發性廓清者，慢性帶原者中HLA-DRB1*09有顯著的頻率增加(28.13% versus 4.86%, , OR=4.58, 95% CI: 1.53-13.68, p=0.003). 相較於疫苗有反應者，在無反應者中，有顯著的頻率增加有 HLA-B*55 (16.67% versus 4.72%, OR=4.04, 95% CI: 1.10-14.32, p=0.033)和HLA DRB1*04(36.37% versus 17.45%, OR=2.74, 95% CI 1.11-6.92, p=0.026)。 HLA-DRB1*09和持續B型肝炎慢性感染相關；而HLA-B*55 and HLA DRB1*04 有較高B型肝炎疫苗的不反應風險。在這個研究中，由於受限於研究的樣本數量，或許仍有些可能相關的HLA基因位點無法釐清。

關鍵字

人類白血球抗原

並列摘要

Following an acute hepatitis B virus (HBV) infection, spontaneously clearance or chronic carrier is determined by the host immune response. Vaccination is important in preventing hepatitis B infection. However, approximately 5-10% of vaccines were poor or no response to hepatitis B (HB) vaccine. Since the human leukocyte antigen system (HLA) is an integral component of the immune response, we hypothesized that the highly polymorphic HLA genes are key determinants of viral clearance and immune response to HB vaccine. Subjects in this study were participants into two studies: (i) the HBV infected study, we enrolled a total of 434 persons who had visited Medical Center for renal or heat transplantation, as either recipients or donors, between January 2001 and September 2007. Subjects were confirmed who have ever infected Hepatitis B virus history, and categorized into 2 different groups: the ‘‘Chronic Carrier Group’’ (CC), and ‘‘Spontaneously Cleared Group’’ (SC) according to the course of HBV infection. The “CC” group who had been hepatitis B surface antigen (HBsAg)-positive for at least 6 months. The “SC” group who was HBsAg-negative with antibodies to HBsAg (anti-HBs) and hepatitis B core antigen (anti-HBc). We excluded subjects who were HBsAg-negative and had anti-HBs, but not anti-HBc. (ii) the vaccination study were obtained from 121 persons (48 health care workers and 73 university students) who and before and after three dose of 20 ug/ml hepatitis B vaccine (Engerix B Hepatitis B Recombinant Vaccine). Each sample was assayed for HBs Ag, Anti-HBs and Anti-HBc using commercial available kits (VITROS ECi Immunodiagnostic System). DNA was extracted from 200 ul sample of blood by Viogene Blood and Tissue Genomic DNA Extraction Miniprep System. HLA-A, -B and -DR typing was performed using commercial PCR-reverse SSOP kits. (Dynal Bioteck Ltd. U.K). Chi-square and Fisher exact test was used to compare categorical variables as appropriate. A p less than 0.05 was considered statistically significant. In HBV infected subject, a total of 16 individuals fulfilled our criteria for a “CC” HBV infection and were matched to 70 individuals with “SC”. In vaccination subject, 121 persons who were HBV-naïve were enrolled After 3 doses of HB vaccination, 15 were nonresponders (Anti-HBs <10 mIU/ml) _with and the remaining 106 were responders (Anti-HBs ≥ 10 mIU/ml). HLA typing to HLA A, B and DR locus were undertaken using sequence-specific oligonucleotide probe hybridization. Compared to “SC”, the “CC” had significant increased frequency of HLA-DRB1*09 (28.13% versus 4.86%, OR=4.58, 95% CI: 1.53-13.68, p=0.003). Compared to responders, the non-responders had significant and marginally-significant increased frequency of HLA-B*55 (16.67% versus 4.72%, OR=4.04, 95% CI: 1.10-14.32, p=0.033), HLA DRB1*04 (36.37% versus 17.45%, OR=2.74, 95% CI 1.11-6.92, p=0.026). HLA-DRB1*09 allele be associated with the chronic HBV infection. The other hand, the HLA-B*55 and HLA DRB1*04 alleles be associated with the no responsiveness to hepatitis B vaccination. The possibility of association to other alleles could not be ruled out due to the limited sample size in this study.

並列關鍵字

Human Leukocyte Antigen ； HLA

參考文獻

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HLA基因型的多型性與B型肝炎病毒感染及B型肝炎疫苗反應性的研究

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