最近舉行的反轉錄病毒和伺機性感染會議 (CROI 2023) 上所發布的海報和演講，降低了人們的預期，亦即使用比幹細胞移植（迄今為止，幹細胞移植已治癒了5 名患者）風險更低、成本更低的方法可能很快就能治癒愛滋病毒。
約翰霍普金斯大學的Natalie McMyn 博士發現，儘管在接受抗反轉錄病毒治療 (ART) 的最初幾年中，能夠產生活HIV 的細胞數量（如果停止ART）會減少，但對於連續較長時間接受ART 的人來說，這個產生病毒的細胞之貯庫的大小不再減少，甚至可能開始緩慢增加。 接受 ART 大約十年後，含有 HIV DNA 的細胞數量，無論其是否具有生產力，也會停止下降，並可能增加。
McMyn 的研究係由幾位愛滋病毒治療研究領域的領導者共同撰寫，其中包括 Steven Deeks 教授、Robert Siliciano 教授和 Janet Siciliano 教授。
西雅圖 Fred Hutch 癌症研究所的 Jared Stern 博士在 CROI 開幕前一天舉行的社區治癒HIV研討會上發表演講，探討了其影響。
史特恩表示，透過針對細胞進行免疫破壞（「休克和殺死」策略）或迫使它們永久靜止（「阻止和鎖定」策略），這些的策略的本身可能並不足以實現達成治癒愛滋病毒。一個各種不同策略之間的微妙、連續的平衡可能還是必要的。
背景
病毒貯庫最終地穩定甚至成長的原因是，在抗愛滋病毒療法的早期，許多細胞仍然能夠活躍地產生愛滋病毒顆粒。 這就是為什麼接受 ART 的人的平均病毒量（至少在最初）為每毫升 2-4 個拷貝而不是零。
當這些細胞確實產生活的愛滋病毒時，它們雖然可以感染新細胞，但也會被免疫系統看到。 這既會針對它們進行免疫破壞，也會使它們走上細胞凋亡（程序性細胞死亡）的道路。
然而後來，「細胞分裂」而不是「病毒複製」變得更加重要。 即使是構成大部分病毒貯庫的長壽 T 記憶細胞也必須不時分裂，以補充體內的 T 細胞，這通常是對感染的反應。 因此，後來活躍性的病毒產生變得越來越少，病毒貯庫主要透過細胞分裂來補充。
當受感染的細胞分裂時，它們就只是分裂——它們不必去表達HIV蛋白或以其他的方式開始製造病毒的過程，就像它們所做的那樣去複製它們的DNA，這也包括它們的HIV原病毒DNA。因為 DNA 在細胞分裂過程中突變的機會要小得多，這些細胞含有完全相同的 HIV「克隆」，這稱為克隆擴增。
研究
McMyn 的研究涉及 31 名接受完全抑制性 ART 治療時間中位數時間近 23 年的人，所有病毒都保持了無法偵測到的狀態。 從 CD4 計數接近零開始（這是 20 世紀 90 年代末），開始 ART 15 年後，他們的 CD4 計數中位數升至約 800，但此後略有下降，穩定在 650 左右。
研究人員從每位參與者的血液中提取純化的靜止 CD4 T 細胞，並進行了兩項測試。 首先，他們進行了病毒生長測定。 這測量了在實驗室培養皿中可以被誘導產生病毒顆粒的細胞的比例。
先前在 2000 年代初羅伯特·西利西亞諾 (Robert Siliciano)的研究發現，具生產性的細胞緩慢減少。 他計算出，在 ART 治療下，HIV 病毒貯庫的半衰期為 44 個月，這意味著病毒產生細胞的數量將在兩年多的時間內減少一半。 這意味著在基線時每百萬分之二的靜止 CD4 細胞能夠產生活病毒，這一數字將在 12 年內下降十倍，在 24 年內下降一百倍。
但這與他們所觀察到的情況不同：在接受 ART 治療的前 3-4 年裡，具生產性的感染細胞之比例下降，但隨後停止下降，甚至可能從那時起略有增加。
另一項測試是對細胞進行 DNA 分析，以檢測所有含有原病毒 DNA 長度的細胞。 由於大多數細胞都是碎片或突變的，因此無法產生病毒，因此含有原病毒 DNA 的 CD4 T 細胞大約是能夠產生活病毒的 100 倍。 如果早期研究顯示的趨勢仍持續下去，則我們預計含有 HIV DNA 的細胞比例也會在 24 年內從每百萬人 200 個下降到每百萬人 2 個。
但原病毒 DNA 貯庫僅在 ART 治療的前 9 年左右有所下降，即便如此，下降幅度也不是很大。 此後保持穩定或略有增長，現在大多數研究參與者都回到了開始的狀態。 無論接受 ART 的時間長短，能夠產生活病毒的含 DNA 細胞的比例都是相同的。 31 個人彼此之間含有 DNA 的細胞和具生產性的感染細胞的數量上相差兩個層級，但在個人內部則是相關的。
透過連續稀釋細胞樣本，研究人員對三個個體的 CD4 靜息 T 細胞中的每個 DNA 序列進行了基因定序。 其中兩個人的一些樣本可以產生活病毒，但第三個人的細胞卻不能產生活病毒。 在這個個體中，幾乎所有病毒分離株（DNA 序列）在基因上都彼此不同。 44 個序列中只有 3 個與其他序列中的一個形成一對，顯示是克隆的 DNA。
在另外兩人身上，59 個原病毒 DNA 序列中的 19% 和 97 個原病毒 DNA 序列中的 36% 產生了完全複製的病毒。 在第一種情況下，產生活病毒的每個序列都是完全相同的，顯然源自於一個已分裂的單一細胞。另還有其他三個克隆序列不產生病毒。
在第二種情況下，產生病毒的 35 個序列中有 18 個是完全相同的，另外 3 個序列則幾乎相同。 然而，其他 14 個產生病毒的序列除了一對之外均彼此不同。並且還有其他 11 個克隆序列不產生病毒。 這些主要是相同序列的成對和三聯體，但7 個細胞有一個具有相同 DNA的克隆則沒有生產力。
影響
娜塔莉·麥克敏(Natalie McMyn) 從她的發現中得出的主要結論是，雖然在某些情況下，愛滋病毒貯庫可能不再能夠產生新病毒，但在大多數情況下，細胞增殖會使含有活病毒的細胞貯庫向上儲值——所以我們不應該指望有太多人可以停止抗反轉錄病毒治療，而病毒量卻不會反彈這種情況的出現。。
賈里德·斯特恩 (Jared Stern) 從這項研究和相關研究中得出了更廣泛的結論。
他們說，隨著時間的推移，貯庫在ART的作用下改變了它的性質。 由於活躍的細胞比不活躍的細胞更快被清除或自毀，因此隨著時間的推移，儲庫的生產力會降低，潛伏期也會增加。
「ART 擅長防止細胞被感染，」他們說。 「但這並不能阻止它們保持傳染性」。
「更潛伏」意味著不太可能被激活，但也不太容易被免疫系統發現——這給希望治癒的研究人員帶來了兩雞困境。 他們是否應該嘗試激活更多細胞並警告免疫系統，冒著新一波細胞感染擴大貯庫的危險，或者他們是否應該嘗試保持潛伏狀態，即使對某些人來說，這意味著維持細胞中含有持續存活的病毒DNA？
從本質上講，愛滋病毒感染者在急性愛滋病毒感染期間面臨著如樂透的狀況。 病毒整合到細胞核中的位置相當隨機，但它傾向於選擇最接近細胞核外部的位點，而這些往往通常是基因表現活躍的區域。 如果 HIV 發現自己處於一段很少或從未表達的 DNA 中（即所謂的「基因沙漠」），那麼即使細胞分裂，也不太可能開始產生病毒。
另一方面，如果它被整合到經常活躍的基因中，那麼當該基因激活時它很可能會被表達。 ART 可以阻止 HIV 的酶深入製造病毒成分的過程，但它不能阻止細胞分裂和克隆那些潛在的具有製造的序列。
這意味著，在我們設計出比起整體大規模替換免疫系統還要更安全、更便宜的愛滋病毒治癒方法之前，我們需要回答一些我們不知道答案的突出研究問題：
是什麼決定了細胞是變得活躍（從而死亡）還是潛伏（從而存活）？ 是什麼決定了細胞從潛伏狀態轉變為生產狀態？ 為什麼有些 T 記憶細胞會進行克隆分裂，而有些則不然？ HIV 本身是否在克隆擴增中發揮作用？
治癒方法是否應該減少或增加愛滋病毒的潛伏狀態？ 長期抗反轉錄病毒治療是否足以（至少對某些人來說）產生一個深層潛伏的病毒貯庫，在停止抗病毒治療後仍無法反彈？ 我們怎麼知道這些人是誰？
參考文獻：
McMyn NF 等人。 長期 ART 後可誘導複製的 HIV-1 持續存在。 第 30 屆反轉錄病毒與伺機性感染會議，西雅圖，摘要 396，2023 年。
Stern J. 長期 ART 對 HIV 病毒庫的影響。2023 年反轉錄病毒與伺機性感染會前之社區愛滋病毒治癒研究治療行動小組工作研討會，西雅圖。
Proliferation, not replication: HIV is lifelong because infected cells divide,
not only because they infect others
Gus Cairns / 11 April 2023 / aidsmap
Kateryna Kon/Shutterstock.com
A poster and a talk both presented at the recent Conference on Retroviruses and Opportunistic Infections (CROI 2023) dampened down expectations that a cure for HIV may soon be possible using less risky and expensive methods than the stem-cell transplants that have so far cured five people.
Dr Natalie McMyn of Johns Hopkins University has found that, although during the first few years on antiretroviral therapy (ART) the number of cells capable of producing viable HIV (if ART is stopped) shrinks, in people taking ART continuously for a longer time, the size of this reservoir of virally-productive cells declines no more and may even slowly start to increase. The amount of cells with any HIV DNA in them, whether productive or not, also stops declining after ten or so years on ART and may increase.
McMyn’s study is co-authored by several leaders in HIV cure research, including Professors Steven Deeks, Robert Siliciano and Janet Siciliano.
Its implications were explored in a talk given by Dr Jared Stern of Seattle’s Fred Hutch Cancer Institute at the Community Cure Workshop held the day before CROI opened.
Stern said that strategies aiming to cure HIV either by targeting cells for immune destruction (the ‘shock and kill’ strategy) or by forcing them into permanent quiescence (the ‘block and lock’ strategy) may not be sufficient in themselves to achieve a cure for HIV. A delicate, sequential balance of different strategies may be necessary.
Background
The reason for the eventual stabilisation and even growth of the reservoir is that in the early years on ART, many cells remain that are capable of actively producing HIV viral particles. This is why the average viral load in people on ART is, at least initially, in the region of 2-4 copies per millilitre instead of zero.
When these cells do produce viable HIV, while they can infect new cells, they also become visible to the immune system. This both targets them for immune destruction, and sets them on the pathway towards apoptosis – programmed cell death.
Later on however, cell division, rather than viral replication, becomes more important. Even the long-lived T-memory cells that form most of the viral reservoir have to divide now and then in order to replenish the body’s T-cells, often in response to infection. Because of this, later on, active viral production becomes rarer and the reservoir is mainly replenished by cell division.
When infected cells divide, they do not have to express HIV proteins or otherwise start the process of making virus – they simply split, duplicating their DNA, including their HIV proviral DNA, as they do. These cells contain identical ‘clones’ of HIV, because DNA has much less chance to mutate during cell division. This is called clonal expansion.
The study
McMyn’s study involved 31 people who had been on fully-suppressive ART for a median time of nearly 23 years. All have maintained viral undetectability. Starting with CD4 counts of near-zero (this was the late 1990s), their median CD4 count rose to about 800 15 years after starting ART, but after that declined a little and plateaued at around 650.
The researchers took purified resting CD4 T-cells from each participant’s blood and did two tests. Firstly,
Previous investigations by Robert Siliciano in the early 2000s found a slow decrease in productive cells. He calculated that the HIV reservoir had a half-life of 44 months on ART, meaning the number of virally productive cells would halve in just over two years. This would mean that two per million resting CD4 cells being able to produce viable virus at baseline, that number would have gone down tenfold in 12 years and a hundredfold in 24.
This was not what they observed: the proportion of cells that were productively infected went down for the first 3-4 years on ART but then stopped decreasing and may even have increased very slightly since then.
The other test was a DNA assay of the cells, to detect all cells that contained lengths of proviral DNA. Because most of these are fragmentary or mutated and so can’t produce virus, about 100 times as many CD4 T-cells contain proviral DNA than can produce viable virus. If the trend indicated by earlier studies continued, we’d expect the proportion of cells containing HIV DNA to decline from 200 per million to two per million in 24 years too.
But the proviral DNA reservoir only declined in about the first nine years on ART and even then by not very much. After that it stayed steady or grew slightly and is now back to where it started in most of the participants studied. The proportion of DNA-containing cells that could produce viable virus was the same regardless of the amount of time on ART. The amount of DNA-containing cells and productively-infected cells differed by two orders of magnitude between the 31 individuals, but were correlated within individuals.
By successively diluting cell samples, the researchers genetically sequenced every DNA sequence in the CD4 resting T-cells they sampled in three individuals. From two of these people, viable virus could be produced from some samples, but not from the third person’s cells. In this individual, virtually all viral isolates (DNA sequences) were genetically distinct from each other. Only three out of 44 sequences formed a pair with one of the other ones, indicating cloned DNA.
In the other two people, 19% of 59, and 36% of 97 proviral DNA sequences gave rise to fully replicating virus. In the first case, every single sequence that produced viable virus was identical, clearly originating from one single cell that had divided. There were three other clonal sequences that did not produce virus.
In the second case, 18 out of the 35 sequences that produced virus were identical, and three others almost identical. The other 14 productive sequences were, however, all different from each other apart from one pair, and there were 11 other clonal sequences that did not produce virus. These were mainly pairs and triads of identical sequences, but there was one clone of seven cells with identical DNA that was not productive.
Implications
Natalie McMyn’s main conclusion from her findings was that, while in some cases the HIV reservoir may stop being capable of producing new virus, in the large majority, cellular proliferation keeps the reservoir of cells containing viable virus topped up – so we should not expect there to be too many people who can stop ART without their viral load bouncing back.
Jared Stern drew wider conclusions from this and related studies.
They said that as time goes by, the reservoir changes its nature under ART. Because active cells are cleared or self-destruct more quickly than inactive ones, over time the reservoir becomes less productive and more latent.
“ART is good at preventing cells from becoming infected,” they said. “But it’s no good at stopping them staying infectious.”
‘More latent’ means less likely to activate, but also less visible to the immune system – which creates a dilemma for cure researchers. Should they attempt to activate more cells and alert the immune system, risking the danger of enlarging the reservoir with a new wave of cellular infections – or should they try to maintain latency, even though in some people that means maintaining cells that will contain persistently viable viral DNA?
Essentially, people with HIV face a lottery during acute HIV infection. The virus’ position of integration into a cell’s nucleus is rather random but it tends to pick sites nearest to the exterior of the nucleus, and these tend to be the areas where commonly active genes are being expressed. If HIV finds itself in a length of DNA that is rarely or never expressed – the so-called ‘gene deserts’ – then even if the cell divides, it is unlikely ever to start producing virus.
On the other hand, if it is integrated into a gene that is often active, then it will likely be expressed when the gene activates. ART stops HIV’s enzymes from getting very far into the process of producing viral components – but it does not stop the cell dividing and cloning those potentially productive sequences.
This implies that before we can devise a safer and cheaper cure for HIV than wholesale replacement of the immune system, we need to answer some outstanding research questions we don’t know the answer to:
What determines whether a cell becomes productive (and so dies) or latent (and therefore survives)? What determines that a cell switches from being latent to being productive? Why do some T-memory cells divide clonally and not others? Does HIV itself have a part to play in clonal expansion?
Should a cure make HIV less, or more, latent? Is long-term ART sufficient, at least in some proportion of people, to produce a deeply latent reservoir that can’t rebound when ART is stopped? And how do we know who those people are?
References
McMyn NF et al. Persistence of inducible replication-competent HIV-1 after long-term ART. 30th Conference on Retroviruses and Opportunistic Infections, Seattle, abstract 396, 2023.
Stern J. Effects of long term ART on the HIV reservoir. Treatment Action Group 2023 Pre-CROI Community HIV Cure Research Workshop, Seattle.