深入研究 HIV隱藏的 DNA 發現有些人最終能免除藥物
資料來源：aidsmap /格斯.凱恩斯/ 2022 年 1 月 24 日/ 財團法人台灣紅絲帶基金會編譯
期刊文章中的這張圖顯示了在所有六名研究參與者的細胞中發現的所有前病毒 DNA 片段是如何分佈在細胞核中的 23 對染色體周圍的。紅線是在一個或多個時間點產生病毒 RNA 的 DNA，藍線是從未發生過的 DNA。可以看出，位於「基因沙漠」中活性基因位點之外的 DNA 很少產生 RNA。
18 個月前，aidsmap.com 報導了一項研究，該研究提出了一些人在不使用藥物的情況下控制 HIV 的能力背後的機制。發表在科學雜誌《細胞》上的一項新研究使用複雜的探針發現了隱藏在六名患者細胞中的每一點 HIV 遺傳物質。找出這是如何自然發生的還有很長的路要走—這是找出如何永久「阻止和鎖定」HIV持續感染的第一步。
研究小組成員 Mathias Lichterfeld 博士在《科學》雜誌上評論說，他們的發現提高了「愛滋病毒與人類和平共處」的可能性。
背景——儲藏窩和控制者
在 HIV 感染期間，HIV 不會像大多數病毒那樣簡單地顛覆細胞成為新病毒的工廠。作為一種反轉錄病毒，它能夠將其基因拼接到我們細胞核中的人類 DNA 中，從而以更親密的方式成為「我們的一部分」。
其中一些細胞是長壽的中央記憶 T 淋巴細胞——它們的工作是記住舊感染，或者是看起來像舊感染之我們稱之為疫苗的東西，。
其中一些細胞，在接受抗反轉錄病毒治療 (ART) 的人中，因此沒有明顯的進展性疾病，但隱藏著製造免疫系統不可見的新 HIV 的指令。它們確實是沉睡的細胞，擁有所謂的難以接近的 HIV 病毒庫。
什麼是精英控制者的基本信息？
在大多數人中，一旦停止 ART，這些儲存細胞就會再次開始製造病毒。但在少數人中，他們沒有。為什麼？
有些人——大約每 200 名愛滋病毒感染者中就有一名——是所謂的「精英控制者」，他們在根本沒有接受過抗反轉錄病毒治療 (ART) 的情況下仍然無法檢測到病毒。
在兩個人身上——一個叫 Loreen Willenberg 的加利福尼亞人和一個以她的阿根廷家鄉命名的 Esperanza 病人——這個過程似乎已經達到了終點。兩人似乎在沒有醫療介入的情況下基本上治癒了自己的愛滋病毒。
更多來自美國的新聞
更多但仍未確定的人數是所謂的「治療後控制者」。這些人患有進行性 HIV 感染和可檢測到的病毒載量，他們開始接受抗反轉錄病毒治療，但隨後由於選擇或意外停藥 – 此後數月或數年仍無法檢測到病毒。
可能有比我們所知更多的潛在治療後控制者，因為可以理解的是，大多數長期接受抗反轉錄病毒治療的人不會停止藥物。最初對他們進行調查的法國研究顯示，在接受 ART 的人數中可能多達十四分之一。巧合的是，去年報導的另一名阿根廷婦女的案例中，她最初患有嚴重的愛滋病毒疾病，並且已經接受抗反轉錄病毒治療十年。由於副作用，她停止了治療，12 年後，愛滋病毒的跡像很少，並且不必重新開始服藥。
我們在 18 個月前報告的研究很重要，因為它首次提出了控制者中病毒控制的潛在原因，從而提出了治癒之途徑。
它尋找隱藏在 64 名精英控制者免疫細胞內 DNA 中的 HIV 基因序列。正如預期的那樣，研究發現相較於其他 HIV 感染者他們體內有較少的 DNA 長度，但也發現有更高比例的 DNA 是完整的並且能夠產生新的病毒。
但至關重要的是 DNA 的位置。 它似乎停留在所謂的「基因沙漠」中—基因組的一部分由垃圾或結構性 DNA 組成，但不是活躍的基因。 這種 DNA 永遠無法喚醒。
研究人員建議，在精英控制者中從一開始，或在治療後的控制者在 ART 治療的幾年中，最值得注意的是—免疫系統發現了微妙的細胞內病毒複製的跡象，病毒 RNA 片段被「讀取」的DNA 並向細胞機器發出信息以開始製造新病毒。這樣，含有最具轉錄活性的病毒 DNA 的細胞就會被免疫系統的 CD8 細胞選擇性地殺死——只留下永久「阻塞和鎖定」的細胞。
新的研究
該研究團隊由Ragon研究所的徐宇博士（及其配偶）和馬蒂亞斯.利希特菲爾德領導，這是哈佛大學、麻省理工學院和麻省總醫院之間的前沿合作。在 2019 年的報告中，我們注意到 Ragon團隊 「現在正在聯繫那些接受穩定抗反轉錄病毒療法 (ART) 超過 20 年的人，以了解他們是否也設法將病毒流放到基因版本中的西伯利亞去了」。
這項新研究告訴我們，有六名這樣的人長期接受抗反轉錄病毒治療。 Ragon 團隊使用最新技術對隱藏在這六名志願者的 T 淋巴免疫細胞中的 HIV DNA 進行了深入的基因探測。
研究參與者的年齡介於 54 至 74 歲之間。一名是女性，一名沒有記錄性別；其他人是男性。在他們最後一次採集細胞樣本之日前，所有人都已接受 ART 至少九年。之所以選擇他們，是因為他們早在 2001 年就採集了適合深度測序的細胞樣本。三人在開始抗反逆轉錄病毒治療之前採集了樣本，或者在一種情況下，在中斷期間採集了樣本。所有六個人都是在開始 ART 後 9 到 15 年之間採集的樣本，有三個是最近的；亦有四個是曾在開始 ART 一年後採集的樣本。
他們使用的複雜的「平行 RNA、整合和前病毒定序」(PRIP-seq) 測定法在患者血液 (PBMC) 中提取了數百萬個自由漂浮的淋巴細胞，並將它們稀釋到每個樣本中有數千個細胞。由於在細胞庫中，只有萬分之一到百萬分之一甚至更少的細胞含有 HIV DNA，這意味著他們能夠在單個細胞中檢測到 HIV DNA 的單個序列。
他們能夠從 1270 個特定細胞中找到個別長度的 HIV DNA。這些「原病毒」中大多數實際上是有缺陷的，無法產生完整的病毒；他們在 6 名患者中發現了 147 個完整的 HIV 基因組——與數百萬個樣本細胞相比，數量非常少。
他們發現 429 個細胞在一個或多個時間點都在積極地產生可測量數量的 HIV RNA，因此無論這些指令是否完整，它們都在積極地轉錄 HIV 的「指令」。在某些情況下，即使它們的 RNA 指令不完整，病毒也可以重新組裝。
PRIP-seq 探針不僅僅可尋找病毒 DNA。它還可以準確地看到，在人類基因組中，每個長度的 HIV 插入的位置 – 因此它是否靠近宿主基因是經常「清醒」並產生自己的 RNA 信息的活動位點，或者它是否被囚禁於一個「基因沙漠」中。
科學家們發現，愛滋病毒幾乎可以將自身整合到基因組的任何地方。他們在每位患者的 23 對染色體中的每一對、活躍位點和鎖定位點中都發現了前病毒。重要的不僅僅是 HIV DNA 在人類 DNA 中的線性位置。與活性基因的接近度是三度空間的問題，因為人類 DNA 在其鏈中包含大約 30 億個單獨的鹼基（「字母」），並且必須錯綜複雜地折疊起來。因此，功能性接近不僅可能發生在每個染色體內，甚至可能發生在染色體之間。
研究人員在初始樣本中發現了 701 個細胞，他們可以在其中建立確切的整合位點，並發現在 117 個細胞中，前病毒 DNA 位於不含功能性基因的區域。即使在這些細胞中，也可以在大約 22 個細胞中檢測到病毒 RNA，但它不太可能發揮作用。
感染 HIV 的細胞產生 HIV RNA 的最強決定因素是其 HIV DNA 靠近一個活躍的人類基因，或是一個基因開啟的人類 DNA 區域。相反地，它不產生 RNA 的第二強決定因素是它靠近被鎖定為非活性形式的區域（透過稱為甲基化的過程）或靠近導致這種情況發生的人類 DNA 區域。
但最能預測細胞不產生 HIV RNA 的因素是研究對象接受 ART 的時間長度—從而證實了研究人員的假設，即隨著時間的推移，能夠轉錄的功能性 HIV DNA 變得越來越少。
了解更多：尋找治癒愛滋病毒之方法
只有三名患者隨著時間的推移採集了幾個 PBMC 樣本，因此可以測量長期的前病毒 DNA 衰變。在第一次檢測 9 年後對細胞進行取樣的兩名患者中，產生活 HIV RNA 的細胞比例下降了五倍，而在 12 年後取樣的一名受試者中幾乎沒有。第 4 名患者在開始 ART 15 年後只採集了一個細胞樣本，而且該患者也沒有 RNA 產生的證據。
因此，在個人層面，他們在六分之四的患者中發現了病毒 DNA 「阻斷和鎖定」的證據。他們認為，這個過程之所以發生，是因為即使在抗病毒藥劑治療(ART) 下，免疫系統也可以檢測到細胞產生的極低水平的 HIV RNA 並選擇性地殺死最活躍的那些，因此只留下病毒 DNA 被阻斷的細胞。
他們指出，依賴於激活整個細胞儲存庫的「踢就殺」治療策略產生了非常令人失望的結果，因為他們所做的只是計算是否導致含有病毒 DNA 的中央記憶 T 細胞總體量之減少—然而他們並沒有。
然而，如果激活做一些更微妙的事情—選擇性地剔除產生最多 RNA 的細胞—那麼這種策略最終可能會產生一個仍含有 HIV DNA 的細胞庫，但沒有一個細胞可以開始產生病毒成分。即使在實驗室培養皿中用強大的基因激活化學物質刺激四名患者的細胞，它們也不會產生任何 HIV RNA。
在產生的病毒 RNA 水平沒有下降情況下，這讓兩個人的故事完全不同，。
「這是找出如何永久［阻斷和鎖定］HIV持續感染的第一步」
人們發現，在其中一個人中，一段很長的病毒 DNA 被拼接到一個名為 KDM2A 的人類基因的中間。這是一種致癌基因——這種基因正常的作用是刺激細胞分裂，但如果它被卡住，就會導致癌症。另一個人方面，病毒 DNA 並不在任何人類的基因內，而是接近一段 DNA，但它卻是染色體結構的強大破壞者，會迫使 DNA 採用更開放的配置，允許基因激活。此人在接受 ART 期間一直保持較低但可檢測到的病毒載量，約為 20 拷貝/ml。
問題是：為什麼在相同的免疫壓力下卻沒有消除這些病毒 DNA持續活躍的期限？
可能的答案是，HIV 的感染不必透過激活、轉錄 RNA 和產生病毒成分來維持自身。如果它們碰巧是在一個分裂的免疫細胞中，它們也可以在不產生任何 RNA 的情況下做到這一點，透過這樣只是複製 DNA——其中包括病毒 DNA。
由於這種所謂的克隆擴增不會提醒免疫系統，因此無法選擇性地剔除活躍的儲存細胞，如果 HIV DNA 恰好位於人類基因組的頻繁激活部分，它就會留在那裡。
這意味著，與 HIV 感染的其他方面一樣，在免疫系統清除有效感染 HIV 的細胞的能力與相同細胞在清除前自我複制的能力之間存在著一場進化上的軍備競賽。
找到顯示您是否是治療後控制者的遺傳特徵並冒著停止 ART 可能的風險，需要復雜的基因測序工具，如本研究所示。所以這不是為診所準備好的東西。
然而，Ragon 團隊的下一步是對長期接受 ART 的患者進行篩查，看是否有跡象顯示他們所有的前病毒 HIV DNA 都被隔離到非生產區域，然後透過非常仔細的醫學監測將一些 ART 去除，以便 ART可以在病毒重新激活的最早跡象時重新啟動。
《科學》雜誌的報導稱，一名志願者已經同意加入這樣的戰略，而 Yu 和 Lichterfeld 正在篩選其他人。
參考文獻：
Einkauf KB、Yu XG、Lichterfeld M 等人。平行分析單個 HIV-1 前病毒的轉錄、整合和序列。 細胞期刊185 期，P.266-282.e15, 2022（開放取得文章）。
http://doi.org/10.1016/j.cell.2021.12.011。
Deep dive into HIV’s hidden DNA finds that some people could eventually drop their drugs
Gus Cairns 24 January 2022
This figure from the journal article shows how all the pieces of proviral DNA found in all six study participants’ cells were distributed around the 23 pairs of chromosomes in the cells’ nuclei. The red lines are DNA that gave rise, at one or more time points, to viral RNA, and the blue lines are DNA that never did. It can be seen that RNA production was rare from DNA that was located outside active gene sites in the ‘gene deserts’.
Eighteen months ago, aidsmap.com reported on a study that suggested a mechanism behind some people’s ability to control their HIV without drugs. A new study, published in the scientific journal Cell, used sophisticated probes to uncover every bit of HIV genetic material hidden in the cells of six patients. It goes a long way towards finding out how this happens naturally – which is a first step in finding out how to ‘Block and Lock’ ongoing HIV infection permanently.
Dr Mathias Lichterfeld, a member of the research team, commented in Science magazine that their findings raise the possibility of “a peaceful coexistence between HIV and humans.”
Background – reservoirs and controllers
During HIV infection, HIV doesn’t simply subvert cells into becoming factories for new virus, in the way that most viruses do. As a retrovirus, it is able to splice its genes into our human DNA in the nucleus of our cells, thereby becoming ‘part of us’ in a much more intimate way.
Some of these cells are the long-lived Central Memory T-lymphocytes – the ones whose job it is to remember old infections, or the things that look like old infections we call vaccines.
Some of these cells, in people who are on antiretroviral therapy (ART) and therefore don’t have obvious, progressive disease, harbour hidden instructions for making new HIV that are invisible to the immune system. They truly are sleeper cells, harbouring a so-called reservoir of inaccessible HIV.
Basic information on what is an elite controller?
In most people, as soon as ART is stopped, these reservoir cells start making viruses again. But in a few people, they don’t. Why?
Some people – about one in 200 with HIV – are the so-called ‘elite controllers’, who remain virally undetectable without ever taking antiretroviral therapy (ART) at all.
In two people – a Californian called Loreen Willenberg and the Esperanza Patient, named after her Argentinian home town – this process seems to have reached its ultimate point; both seem to have essentially cured themselves of HIV without medical intervention.
A larger but still undetermined number of people are the so-called ‘post-treatment controllers’. These are people with progressive HIV infections and detectable viral loads who start ART but who then, by choice or accident, stop their meds – and remain undetectable for months or years thereafter.
There may be many more potential post-treatment controllers than we know of because, understandably, most people on long-term ART don’t stop it. The original French study investigating them suggested it could be as many as one in 14 people on ART. In one case of, coincidentally, another Argentinian woman reported last year, she had initially had severe HIV disease and had been on ART for a decade. She stopped it due to side effects and, 12 years later, has minimal signs of HIV and has not had to restart it.
The study we reported on 18 months ago was important because, for the first time, it suggested an underlying cause for the viral control seen in controllers – and thereby suggested routes towards a cure.
It looked for HIV gene sequences hidden in the DNA inside the immune cells of 64 elite controllers. It found fewer lengths of DNA in them than in other people with HIV, as expected. But it found a higher proportion of DNA that was intact and capable of giving rise to new viruses.
But what was crucial was where that DNA was located. It seemed to be parked in so-called ‘gene deserts’ – parts of the genome that consist of junk or structural DNA but which aren’t active genes. This DNA could never be woken up.
The researchers suggested that in elite controllers, from the start, or in post-treatment controllers, during years on ART, the immune system spotted subtle, intracellular signs of viral replication – most notably, bits of viral RNA that are ‘read off’ the DNA and message the cellular machinery to start making new viruses. This way, the cells containing the most transcriptionally active viral DNA are selectively killed by the immune system’s CD8 cells – just leaving the permanently ‘blocked and locked’ cells.
The new study
The research team is led by Drs (and spouses) Xu Yu and Mathias Lichterfeld at the Ragon Institute, a cutting-edge collaboration between Harvard University, the Massachusetts Institute of Technology, and Massachusetts General Hospital. In the 2019 report we noted that Ragon “is now contacting people who have been on stable antiretroviral therapy (ART) for more than 20 years to find out if they too, have managed to exile their virus to a genetic version of Siberia.”
The new study tells us about six such people who are on long-term ART. The Ragon team used the latest technology to conduct a deep genetic probe into the HIV DNA hidden within the T-lymphocyte immune cells of these six volunteers.
The study participants were aged 54 to 74. One was a woman and one had no gender recorded: the others were men. All had been on ART by at least nine years at the date of their last cell sample. They were chosen because they had had cell samples taken as early as 2001 that were suitable for deep sequencing. Three had had samples taken before starting on ART or, in one case, during an interruption. Four had samples taken a year after starting ART, and all six had a sample taken between nine and 15 years after starting ART, three very recently.
The sophisticated “Parallel RNA, Integration and Proviral Sequencing” (PRIP-seq) assay they used took millions of free-floating lymphocyte cells in the patients’ blood (PBMCs) and diluted them down till they had several thousand cells in each sample. As within the cellular reservoir only one in ten thousand to one in a million cells or even fewer contains HIV DNA, this meant they were able to detect individual sequences of HIV DNA within single cells.
They were able to find individual lengths of HIV DNA from 1270 specific cells. Most of these ‘proviruses’ were in fact defective and incapable of giving rise to whole viruses; they found 147 lengths of complete, intact HIV genome in the six patients – extraordinarily few compared to the millions of cells sampled.
They found 429 cells that, at one or more time points, were actively producing measurable amounts of HIV RNA, and were therefore actively transcribing HIV ‘instructions’, whether or not those instructions were complete. In some circumstances, even if their RNA instructions are incomplete, viruses can reassemble.
The PRIP-seq probe didn’t just look for viral DNA. It can also see exactly where, within the human genome, each length of HIV is inserted – and therefore whether it is near an active site where host genes are often ‘awake’ and producing their own RNA messages, or whether it is marooned in one of the ‘gene deserts’.
The scientists found out that HIV could integrate itself pretty much anywhere in the genome. They found proviruses in every one of the 23 pairs of chromosomes in each of the patients, in active sites and in locked sites. it wasn’t just where the HIV DNA was linearly within the human DNA that mattered, either. Proximity to active genes was a matter of three dimensions, because human DNA contains about three billion individual bases (‘letters’) in its chain and has to be folded up intricately. Functional proximity could therefore happen not just within each chromosome, but even between chromosomes.
The researchers found 701 cells where, in initial samples, they could establish the exact integration site and found that in 117 cells, the proviral DNA was in an area that did not contain functional genes. Even in these, viral RNA was detectable in some 22 cells, but it was very unlikely to be functional.
The strongest determinant of an HIV-infected cell producing HIV RNA was that its HIV DNA was near an active human gene, or an area of human DNA that switched on genes. Conversely, the second-strongest determinant of it not producing RNA was that it was near an area that was locked up into an inactive form (by a process called methylation) or near an area of human DNA that made that happen.
But the strongest predictor of cells not producing HIV RNA was the length of time the study subjects had been on ART – thus confirming the researcher’s hypothesis that functional HIV DNA capable of being transcribed becomes rarer over time.
Find out more: The search for an HIV cure
There were only three patients that had several PBMC samples taken over time and in whom, therefore, longitudinal proviral DNA decay could be measured. The proportion of cells producing viable HIV RNA fell fivefold in two patients whose cells were sampled nine years af