探索B型肝炎病毒表面抗原清除相關的病毒和宿主因素及免疫基因甲基化對其病毒–宿主交互作用的影響

Abstract

背景 在慢性B型肝炎感染的治療中，B型肝炎表面抗原(HBsAg)清除目前被認為是功能性治癒(functional cure)。截至目前為止，尚未有長時間的追蹤型研究來探討B型肝炎表面抗原清除的發生率以及相關的影響因子。有研究指出和免疫功能相關的DNA甲基化標記，和B型肝炎病毒量相關以及能夠預測早發性肝癌。 目的 本研究旨在探討和B型肝炎表面抗原清除相關因素以及計算累積發生率；也將找尋DNA甲基化、病毒因子、B型肝炎表面抗原清除之間的關係。 材料與方法 本研究使用縱貫性病毒量資料庫計算B型肝炎表面抗原清除之累積發生率，以及尋找和B型肝炎表面抗原清除相關的影響因子。收案期間為1989至1992年，總共納入1044位男性B型肝炎帶原者並追蹤至2019年5月。DNA甲基化資料庫是源自於縱貫性病毒量資料庫，總共有529位參與者有DNA甲基化資料。我們利用Cox proportional hazard model來計算B型肝炎表面抗原清除的風險和95%信賴區間。DNA甲基化對B型肝炎表面抗原清除免疫反應調控的直接效應及間接效應則利用中介分析來探討。 結果 本研究總共追蹤了15863.42人年，共有290人（27.8%）B型肝炎表面抗原清除；每1000人年發生率為18.28。經過多變項Cox迴歸分析後，年齡、肥胖、脂肪肝、基因型C、核心啟動子突變、低病毒量有較高的B型肝炎表面抗原清除。各基線年齡組間累積B型肝炎表面抗原清除有顯著差異(P<.0001)。早期B型肝炎表面抗原清除(小於50歲)和基線年齡(P<.0001)、麩胺酸丙酮酸轉氨基酵素濃度(P=0.0268)以及B型肝炎病毒量(P=0.0002)之間有顯著相關。在分析的8個甲基化位點中，IFI44L基因上的兩個甲基化位點cg05696877、cg06872964和B型肝炎病毒量及e抗原陽性有顯著負相關；然而無任何甲基化位點和B型肝炎表面抗原清除相關。中介分析結果顯示，IFI44L基因上的兩個甲基化位點(cg05696877,cg06872964)會藉由B型肝炎病毒量和e抗原進而影響B型肝炎表面抗原清除。e抗原在cg05696877位點對B肝表面抗原清除間的相關解釋了相當大的比例(中介百分比=38.4%, P=0.02)。 結論 本研究結果顯示，宿主相關因素及病毒因子皆和B型肝炎表面抗原清除間有顯著相關，且病毒因子可能是與免疫反應相關的DNA甲基化對於B型肝炎表面抗原清除的中介因子。未來需要進行更多研究來釐清DNA甲基化，病毒因子以及B型肝炎表面抗原清除相關的機制。

Background HBsAg seroclearance is currently regarded as the functional cure of hepatitis B virus (HBV) infection. There is paucity of long-term follow-up studies to estimate the incidence and determinants of HBsAg seroclearance. DNA methylation of genes related to immune response have been shown to be associated with HBV DNA levels and predict early-onset of HCC.

Specific Aims This study aimed to calculate the cumulative incidence of HBsAg seroclearance, and investigated viral and host factors related to HBsAg seroclearance. Moreover, the relationships among DNA methylation, viral factors, and HBsAg seroclearance were examined. Materials and Methods We used a longitudinal viral load database to estimate the cumulative incidence of HBsAg seroclearance and identified determinants related to HBsAg seroclearance. Study subjects included 1044 male HBsAg carriers enrolled between 1989 and 1992, and followed up to May, 2019. DNA methylation database was embedded in the longitudinal viral load database, with a total of 529 participants. Cox proportional hazards model was used to calculate the hazard ratios (HRs) and 95% confidence intervals (CIs) for HBsAg seroclearance. Mediation analysis was performed to analyze the direct and indirect effects of DNA methylation in the regulation of immune response on HBsAg seroclearance. Results After 15863.42 person-years of follow-up, HBsAg seroclearance occurred in 290 (27.8%) subjects, with an estimated incidence rate of 18.28 per 1000 person-years. Multivariate analysis by Cox regression revealed that a higher rate of HBsAg seroclearance was associated with older age, obesity, fatty liver, genotype C, BCP double mutations, while a lower rate was associated with higher HBV DNA levels. Age (P<.0001), ALT levels (P=0.0268), and viral load (P=0.0002) were associated with HBsAg seroclearance at an early age (<50 years old). Of the eight methylation CpG sites examined, the two IFI44L sites, cg05696877 and cg06872964 demonstrated an inverse association with viral load and HBeAg positivity, but none of the DNA methylation probes analyzed were associated with HBsAg seroclearance. Mediation analysis showed that methylation of the IFI44L gene (cg05696877, cg06872964) significantly affected HBsAg seroclearance via HBV DNA levels and HBeAg; HBeAg explained a substantial proportion of the association between cg05696877 and HBsAg seroclearance (mediated proportion= 38.40%, P=0.02). Conclusion Both viral and host factors were associated with HBsAg seroclearance, and viral factors might be mediators in the pathway between DNA methylation associated with immune responses and HBsAg seroclearance. Further researches are needed to elucidate the underlying mechanisms among DNA methylation, viral factors and HBsAg seroclearance.