利用次世代定序分析B型肝炎病毒在干擾素治療中的演變

Abstract

B型次肝炎病毒很適合當作模式來觀察宿主的免疫反應。HBV的演化速度跟RNA病毒一樣快， 因此HBV會因爲宿主的因素（包含免疫反應）而改變。再加上，HBV經常造成慢性的感染，所以該病毒有足夠的時間因爲免疫反應而演化。我們的目標是利用這兩個特色來研究HBV演化的衡量方式如何能提供病人免疫反應的更多資訊。 我們主要的假設是不同的免疫反應帶來不同的演化壓力。本論文的研究目的是找到一個方法利用HBV演變的分析來評估病人免疫反應的品質。最後的目標是探討從這個分析得到的資訊是否跟臨床病情有關係，且是否可以利用這些資訊來協助病人的治療。 23位慢性B型肝炎病人（基因型B）經過24周的干擾素治療。治療前，所有病人屬於HBeAg陽性的狀態。在四個時間點，我們利用次世代定序技術來分析病人的樣本：治療前，治療中點（第12周），治療結束，以及治療後第24周。 我們研究HBV的演變與兩個臨床結果的關係：HBeAg seroconversion和病毒量達到90%以上的減少。這兩個臨床結果在HBV的自然歷史成爲跟免疫系統有關的最重要的結果，而且對病人死亡率的減少和對母親傳染嬰兒機率的減少都有重要的貢獻。目前嬰兒HBV疫苗接種越來越普遍，因此，這兩個臨床結果逐漸成爲預防母親傳染嬰兒的重要工具，利於協助解決HBV大流行。 本研究介紹不同計量衡量方式來分析病毒定序檢測對等位基因不利的演化壓力 （多樣化壓力）。這個工具可以辨認多樣化壓力是針對基因裏頭的哪一個位子，以及衡量其多樣化壓力的强度。 這個分析方法可以在不同的病患組顯示多數的趨勢以及建立病患個人的資料. 我們希望未來更多的進步能讓本論文介紹的計量方法提供醫師更多的資訊，協助預後判斷與治療客製化，改善B型肝炎病患的治療績效。

The Hepatitis B Virus is a promising candidate to observe the immune response of its host. Because the evolutionary rate of HBV is as high as that of RNA viruses, the virus has the opportunity to change in response to host factors, including the immune pressure. In addition, HBV causes a chronic infection that lasts long enough for the virus to change in response to the immune pressure. We aim to use this opportunity to investigate how measurements of HBV evolution may provide information about the immune response of patients. Our main hypothesis is that different immune responses cause different evolutionary pressures. As a result, the goal of this research project is to develop a method to monitor the quality of the patient’s immune response through analysis of its impact on the evolution of the HBV virus. The final objective is to correlate this information with clinical outcomes to evaluate the potential of this analytical method as a tool that might provide additional indications to help guide HBV treatment. To conduct this study, we used samples from 23 patients with chronic HBV genotype B infection that underwent 24 weeks of interferon therapy. We analyzed NGS sequences of the virus before treatment, at the halfway point of treatment, at the end of treatment, and 24 weeks after termination of treatment. All patients were HBeAg-positive at the beginning of the treatment. We studied the differences in HBV evolution that correlated with two clinical outcomes: HBeAg seroconversion and viral load reduction by at least 90%. These two outcomes are the most important immune-related events in the natural course of HBV infection. These two outcomes are particularly important because of their association with a decreased probability of mother-to-child transmission. With the now widespread use of vaccination at birth for children born of HBV carriers, these clinical endpoints are likely to become of increased public health relevance as the next best actionable levers to control the HBV pandemic. We developed quantitative measurements to detect signs of selection against a particular allele (diversifying selection) in the evolution of the virus sequences. This allows for the evaluation of the intensity of the diversifying selection as well as the identification of the precise location of the amino acids that cause the allele to be selected against. This method can be used to derive general trends in groups of patients as well as to build personal profiles of individual patients. We hope that further advances in the development of applications from the methods described in this document will provide clinicians with additional information for their prognosis and for guiding targeted treatment to improve the clinical outcomes of patients suffering from chronic Hepatitis B.