HBV可以治愈吗？ - “HIV-HBV共感染中的单肝细胞分析显示转录

StephenW

Conference on Retroviruses and Opportunistic Infections Seattle, Washington March 4-7, 2019 Back

Can HBV Be Cured? - "Single hepatocyte analysis in HIV-HBV co-infection shows transcriptional silencing"        Reported by Jules Levin CROI 2019 March 4-7 Seattle "Our reasoning is that if a hepatocyte is not transcribing cccDNA, then that hepatocyte is not making viral antigens to be detected by the immune system. So, even if tolerance is reversed, the immune system would not see that hepatocyte as infected. It would still see the hepatocytes that are transcribing cccDNA as infected and eliminate those. We think of these silently-infected hepatocytes as similar to latently-infected T cells in HIV. The new antivirals would not work either unless they are directly eliminating cccDNA such as a CRISPR-cas9 or an anti-viral that destabilizes cccDNA. An anti-viral that works later in the life cycle would not be active if the cccDNA is not transcribing." WEBCAST: http://www.croiwebcasts.org/console/player/41191?mediaType=slideVideo&&crd_fl=0&ssmsrq=1553181672012&ctms=5000&csmsrq=5051 Program Abstract Hepatitis B virus (HBV) is a leading cause of liver failure and hepatocellular carcinoma worldwide. Due to shared modes of transmission, ~10% of people living with HIV also have chronic HBV infection. HBV cannot be cured because the long-lived covalently closed circular DNA (cccDNA) resides in every infected hepatocyte. However, little is known about HBV replication in human livers. Here, we used single-cell laser capture microdissection (scLCM) and droplet digital PCR (ddPCR) to characterize the HBV replication landscape in situ in humans. We quantified cccDNA, total HBV DNA, and pre-genomic RNA (pgRNA) in each hepatocyte, adjusting for intracellular cytoplasmic RNA 7SL. We examined a median (range; total) 255 (52 – 290; 1100) hepatocytes that were individually isolated from five HIV/HBV co-infected persons with increasing exposure to dually-active antiretroviral therapy (DAART) against HIV and HBV (HB1-5; no exposure to >7 years of exposure). Total HBV DNA, cccDNA, and pgRNA were quantified in each cell. The proportion of infected hepatocytes (cccDNA positive) decreased with longer DAART exposure from 100% (HB1) to 33% (HB5)(Table). The median (range) total HBV DNA per cell was 1 cp/cell (0-112 cp/cell); of cccDNA was 1 cp/cell, (0-31 cp/cell); and of pgRNA was 38 cp/cell, (0-1919 cp/cell). The amounts of cccDNA, total HBV DNA, and pgRNA significantly decreased in infected cells with longer DAART duration (p<0.005 for all targets). HBV transcription (pgRNA) did not correspond with cccDNA levels in the same cells (p>0.05). Additionally, we identified cells that contained cccDNA but not pgRNA, defined here as transcriptionally silent, that accumulated with longer DAART from 0% (HB1) to 46.1% (HB5) of infected hepatocytes (Table). Our results indicate that the HBV viral landscape is highly dynamic, and that there is heterogeneity in transcription of cccDNA including complete silencing. Understanding transcriptional silencing in HBV-infected hepatocytes may be critical to emerging immunotherapy and could be exploited to develop a functional cure.

StephenW

HBV可以治愈吗？ - “HIV-HBV共感染中的单肝细胞分析显示转录沉默”


  Jules Levin报道
CROI 2019年3月4日至7日西雅图

“我们的理由是，如果肝细胞不转录cccDNA，那么肝细胞就不会使免疫系统检测到病毒抗原。因此，即使耐受性被逆转，免疫系统也不会看到肝细胞被感染。仍然可以看到转录cccDNA的肝细胞被感染并消除了这些肝细胞。我们认为这些静默感染的肝细胞与HI​​V中潜伏感染的T细胞相似。

除非他们直接消除cccDNA（例如CRISPR-cas9）或使cccDNA不稳定的抗病毒，否则新的抗病毒药物将无法发挥作用。如果cccDNA没有转录，那么在生命周期后期起作用的抗病毒就不会活跃。“

网络发布：
http://www.croiwebcasts.org/cons ... 000&csmsrq=5051

程序摘要

乙型肝炎病毒（HBV）是全球肝功能衰竭和肝细胞癌的主要原因。由于共享传播方式，约10％的艾滋病病毒感染者也患有慢性HBV感染。 HBV无法治愈，因为长寿命的共价闭合环状DNA（cccDNA）存在于每个感染的肝细胞中。然而，人类对肝脏中HBV复制知之甚少。

在这里，我们使用单细胞激光捕获显微切割（scLCM）和液滴数字PCR（ddPCR）来表征人类原位的HBV复制景观。我们量化了每个肝细胞中的cccDNA，总HBV DNA和前基因组RNA（pgRNA），调整细胞内细胞质RNA 7SL。

我们检测了中位数（范围;总数）255（52  -  290; 1100）肝细胞，这些肝细胞分别从5名HIV / HBV合并感染者中分离出来，并且接触到针对HIV和HBV的双重活性抗逆转录病毒疗法（DAART）（HB1- 5;不接触> 7年的暴露）。在每个细胞中定量总HBV DNA，cccDNA和pgRNA。感染的肝细胞（cccDNA阳性）的比例随着DAART暴露时间的延长而从100％（HB1）降低至33％（HB5）（表）。每个细胞的中位数（范围）总HBV DNA为1cp /细胞（0-112cp /细胞）; cccDNA的浓度为1 cp /细胞，（0-31 cp /细胞）; pgRNA的浓度为38cp /细胞，（0-1919cp /细胞）。在具有较长DAART持续时间的感染细胞中cccDNA，总HBV DNA和pgRNA的量显着降低（对于所有靶标，p <0.005）。 HBV转录（pgRNA）与相同细胞中的cccDNA水平不一致（p> 0.05）。此外，我们鉴定了含有cccDNA而不是pgRNA的细胞，这里定义为转录沉默，从感染的肝细胞的0％（HB1）到46.1％（HB5）累积更长的DAART（表）。

我们的结果表明HBV病毒景观是高度动态的，并且cccDNA的转录存在异质性，包括完全沉默。了解HBV感染的肝细胞中的转录沉默可能对新兴的免疫疗法至关重要，可以用于开发功能性治疗方法。

齐欢畅

感谢