#2 - Why are current antiretroviral therapies not sufficient for curing HIV infection?

It is known that a fraction of human immunodeficiency viruses (HIV) enters a latent stage after viruses infect host cells (1-3). The number of the cells which carry latent proviruses is estimated to be 0.1 to 10 cells in every one thousand unactivated CD4+ T cells (4,5). Such latent proviruses somehow perturb the efficacy of antiretroviral drugs used for curing Acquired Immune Deficiency Syndrome (AIDS) due to the fact that none of our current antiretroviral strategies can precisely target on those cells harboring latent proviruses. Owing to this reason, how to develop better antiretroviral strategies for curing HIV infection has been intensively studied by many laboratories since 1996.

New antiretroviral strategies for curing AIDS

Nowadays there are at least four antiretroviral strategies, including (1) stem cell transplantation, (2) CRISPR/Cas9 gene editing, (3) “shock and kill” strategy and (4) “block and lock” strategy.

Stem cell transplantation

Two receptors that play an important role for HIV entry into the host cells are the CD4 receptor and either CCR5 or CXCR4 coreceptor (Figure 1). The rationale for this strategy is to utilize stem cells from the donors whose CCR5 coreceptors carried a deletion of 32 base pairs (CCR5Δ32/Δ32) (6-7); thus HIV no longer can invade their target cells. Although this strategy has been clinically proven to successfully avoid other healthy CD4+ T cells being attacked by the viruses present in patients; unfortunately, due to the difficulty for performing transplantation as well as transplantation rejection and other side effects, such strategy at present cannot be widely used.

Figure 1. Illustration about HIV infection (sci-fi). The moment while HIV (up left) is attacking the host cells (bottom right) with its tentacles.

CRISPR/Cas9 gene editing

Nowadays one of the most well known gene editing technologies is CRISPR/Cas9 (CRISPR is the abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins) (8-10). The most attractive part involved in this technology is that one can precisely remove a piece of DNA sequence by simply giving a piece of RNA sequence that is complementary to our sequence of interest. Because of this reason, scientists thus think of using the CRISPR/Cas9 technology to directly delete proviral DNA sequences present in the host genome (11,12). Even though it is such a beautiful idea, due to the fact that a high frequency of viral genetic mutations occurs in the process of viral infection (mainly during the process of reverse transcription), which leads a difficulty for designing accurate guide RNAs used in this technology. In other words, because proviral DNA sequences are actually highly diverse among each HIV patient (13), this strategy is unfortunately not feasible, either.

“Shock and kill” strategy

As mentioned in the first paragraph, the undetectable level of gene expression renders latent proviruses invisible from our immune cells. In order to conquer this issue, the principle of the “shock and kill” strategy is to give patients histone deacetylase inhibitors used to reactivate gene expression of latent proviruses (this step is therefore called “shock”) (14,15). When latent proviruses are exposed under our immune system, we then treat patients with antiretroviral drugs (this step is then called “kill”) in order to increase the efficacy of drugs.

The success of this strategy relies on the fact that whether or not histone deacetylase inhibitors can fully reactivate all latent proviruses. Vorinostat is at present one of the most popular histone deacetylase inhibitors used in the “shock and kill” strategy. Although vorinostat has been tested to reactivate latent proviruses in the phase of the clinical trial (16), it remains unknown whether all proviruses can be reactivated.

In order to better investigate the efficacy of vorinostat for reactivation of latent proviruses, in our previously study, we isolated the cells with latent infections and added vorinostat, phytohaemagglutinin (PMA) and DMSO in cell culture, respectively. PMA, which is a global T cell activator is used as a positive control; in contrast, DMSO serves as a negative control. Here we again applied the Barcoded HIV Ensembles (B-HIVE) technology (17) mentioned in the previous blog article to measure the transcriptional magnitude of individual latent proviruses reactivated by vorinostat, PMA and DMSO. We observed that some of the proviruses were prone to be reactivated by vorinostat instead of PHA; in contrast, anothers of the proviruses were prone to be reactivated by PHA instead of vorinostat (Figure 2). Based on this finding, we have claimed that histone deacetylase inhibitors with different functions should be considered to be used at the same time in the clinical trial in order to cover as many latent proviruses as possible; this opinion has also been mentioned in Thomas et al. (2020) (18).

Figure 2. Different histone deacetylase inhibitors reactivate different proviruses. Some proviruses were prone to be reactivated by vorinostat but others were not.

“Block and lock” strategy

Although the term “block and lock” was just announced over the past four to five years, a great progress involved in this strategy has been made. Instead of giving histone deacetylase inhibitors used in the “shock and kill” strategy, here we treat patients with RNA and small molecule inhibitors which can trigger epigenetic silencing in order to suppress the retroviral activity.

In the KU Leuven University (Flanders, Belgium), Dr. Debyser and his team continuously investigate the development of novel antiretroviral strategies for a long time. They first designed a series of small molecule inhibitors which function to interfere with the process of HIV DNA integration in 2010. Such small molecule inhibitors are later named as 2-(quinolin-3-yl)acetic acid derivatives (LEDGINs) (19).

Right now you may be just wondering how do such small molecule inhibitors interfere with HIV infection: we observed that HIV DNA integration sites were retargeted to the chromosomal regions where are not favor for gene expression (Figure 3) when the cells have been treated with LEDGINs before HIV infection (20-22). In the meanwhile, we also observed that the transcription level of proviruses showed a decrease in cells treated with LEDGINs compared with those in cells without the LEDGINs treatment. Taken together, LEDGINs are capable of blocking proviruses in the chromosomal regions in which are not favor for gene expression and subsequently locking gene expression of proviruses.

Figure 3. Schematic representation of the impact of LEDGINs on proviruses. Compared with the cells without the LEDGINs treatment (up panel), the selection of HIV integration sites were retargeted out of transcription unit in cells treated with LEDGINs. d and d' represent the distance between HIV integration site and the ChIP-seq signal from H3K36me3.

Summary

More and more novel antiretroviral strategies will be proposed as long as our knowledge about retroviruses accumulates day by day. However, being a scientist who is attempting to do so every day, the securest strategy for curing HIV infection is still “Prevention is better than cure”.

***How to cite this article: Chen, H.-C. #2 - Why are current antiretroviral therapies not sufficient for curing HIV infection? Blog Silence is a Behaviour. (2020).***

--------------------------------------------------------------------------------------------------------------------------------------

抗反轉錄病毒藥物下的漏網之魚－淺談潛伏原病毒對治療後天免疫缺乏症候群的影響

人類免疫缺陷病毒在感染宿主細胞之後，會有非常少量的病毒進入潛伏期（latency）(1-3)。根據目前的研究估算，每一千個未激活的CD4陽性T細胞（resting CD4+ T cells）中，約有0.1 ～ 10個細胞帶有潛伏中的人類免疫缺陷病毒（latent proviruses）(4,5)。這些帶有潛伏原病毒的細胞正是我們治療後天免疫缺乏症候群（Acquired Immune Deficiency Syndrome）的死角。因為這些潛伏中的原病毒，其病毒的基因表現量極低，使得目前使用於雞尾酒療法中的抗反轉錄病毒藥物（antiretroviral drugs），都無法有效地清除那些帶有潛伏原病毒的細胞。正是因為這個原因，從1996年迄今，如何能夠發展出能更有效地治療後天免疫缺乏症候群的策略，成為許多從事反轉錄病毒研究的實驗室的重點之一。

治療後天免疫缺乏症候群的新策略

目前對於治療後天免疫缺乏症候群的新策略，可以簡單地分成四大類：１）幹細胞移植（stem cell transplantation）技術，２）基因編輯（gene editing）技術，３）「先激活，後清除」（shock and kill）策略以及４）「先封鎖，後抑制」（bock and lock）策略。

幹細胞移植技術

在CD4陽性T細胞的表面上，有兩種受體（receptors），對於反轉錄病毒感染細胞的過程扮演不可或缺的角色：一個是CD4受體（CD4 receptors），另一個則是CCR5或是CXCR4輔助受體（coreceptors）（圖一）。幹細胞移植技術的理論基礎正是利用反轉錄病毒對於受體的專一性，選擇在CCR5輔助受體基因上，帶有32鹼基對的缺失（CCR5Δ32/Δ32）的骨髓捐贈者的幹細胞來進行移植（6,7）。雖然在臨床上，幹細胞移植技術已經被證實是可以有效地防堵其它健康的CD4陽性T細胞再次受到體內反轉錄病毒的攻擊；但由於幹細胞移植技術不易施行，以及術後的排斥反應和副作用，此療法目前仍無法被廣泛地用使用。

圖一：反轉錄病毒感染宿主細胞示意圖。反轉錄病毒（左上）利用其觸手對宿主細胞（右下）進行攻擊。

基因編輯技術

CRISPR［CRISPR為Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins（常間回文重複序列叢集/常間回文重複序列叢集關聯蛋白系統）的縮寫］/Cas9基因編輯是最為大家所知曉的基因編剪輯技術之一（8-10）。這個基因編輯技術最令人驚艷的地方在於，我們只需要提供一段和被移除的DNA序列，有高度互補性的RNA序列（complementary RNA sequence），就可以準確地移除此段DNA序列。科學家因此也想利用這個基因編輯技術，來直接剔除那些插入在人類染色體中原病毒的DNA序列（11,12）。然而，反轉錄病毒在感染宿主細胞的過程中，常伴隨著高度基因序列的突變；造成了在患者體內的原病毒的DNA序列，其實是存有高度的變異性（diversity）（13）。也正是因為這個原因，對於利用 CRISPR/Cas9基因編輯技術來治療後天免疫缺乏症候群的可行性也就大打折扣了。

「先激活，後清除」（shock and kill）策略

我們在第一段曾提到，因為潛伏中原病毒的基因表現量極低，使得我們的免疫細胞無法偵測到這些帶有潛伏中原病毒的細胞。為了克服這個問題，「先激活，後清除」策略（14,15）的原理就是先給予病患可以用來再激活（reactivation）潛伏於細胞中原病毒基因的再次表現（此步驟就是所謂的「先激活」, shock）的組織蛋白去乙醯酶抑制劑（histone deacetylase inhibitors）。當所有潛伏中的原病毒都暴露在我們的免疫系統下時，再給予病患進行抗反轉錄病毒藥的治療（此步驟就是所謂的「後清除」, kill）來增加抗反轉錄病毒藥物清除病毒的效果。

在這個策略中最重要的關鍵是，所使用的組織蛋白去乙醯酶抑制劑是否能完全地再激活所有潛伏中原病毒基因的表現。「伏立諾他」（Vorinostat）是目前於「先激活，後清除」療法中，最被廣泛討論的組織蛋白去乙醯酶抑制劑。雖然「伏立諾他」在臨床試驗階段已經被證實是可以再激活潛伏中原病毒的基因表現（16）；即便如此，我們還是不清楚是否所有潛伏中的原病毒都是可以百分之百地被「伏立諾他」再激活。

為了近一步了解「伏立諾他」對於再激活潛伏原病毒的效能，我們分離出帶有活潛伏原病毒的細胞，並加入「伏立諾他」作為實驗組；另外兩組細胞則分別加入植物血凝素（Phytohaemagglutinin，PHA）作為正向的對照組以及二甲基亞碸（DMSO）作為負向的對照組。利用在前一篇文章裡所提到的「高通量反轉錄病毒條碼化」技術（17），我們發現，當給予相同原病毒不同的藥物（在此即是比較「伏立諾他」和植物血凝素對再刺激原病毒基因表現的影響），原病毒基因表現並沒有存在一致性。也就是說，在這個實驗模型中，有一類的原病毒的基因可以經由「伏立諾他」的刺激而高度被激活，然而對於植物血凝素的刺激則沒有反應；而另一類的原病毒基因表現則是可以經由植物血凝素的刺激來高度被激活，但卻對「伏立諾他」的刺激沒有反應（圖二）。因為這個現象的發現，我們認為，在臨床的治療上，應該需要考慮同時施予多種不同功能的組織蛋白去乙醯酶抑制劑的可行性。這個觀點，同樣也在Thomas et al. (2020) （18）中被提及。

圖二：每隻潛伏原病毒對於組織蛋白去乙醯酶抑制劑的感受性並不相同。有些潛伏原病毒的基因表現較易受「伏立諾他」的刺激而表現；然而另ㄧ些潛伏原病毒的基因表現則較易受植物血凝素的刺激而表現。

「先封鎖，後抑制」（block and lock）策略

雖然「block and lock」（依據此策略的原理，我們將它翻譯為「先封鎖，後抑制」）這個字眼所被提出的時間不過只有四、五年的時間；然而我們對於這個策略的研究，卻是日新月異。不同於在上一段所提到的「先激活，後清除」策略，「先封鎖，後抑制」的基本原理則是施予患者，可以誘發基因表面特徵改變而抑制基因表現（即是所謂的epigenetic silencing）的小分子核糖核酸（RNA）或是小分子抑制劑，來達到抑制反轉錄病毒的活性。

在比利時荷語天主教魯汶大學裡，德比傑－（Zeger Debyser）和他的團隊則是長期致力於研究小分子抑制劑來進行治療後天免疫缺乏症候群的研究。他們首次在2010年設計出一系列可以干擾反轉錄病毒DNA插入宿主染色體過程的小分子抑制劑（2-(quinolin-3-yl)acetic acid derivatives, 簡稱為LEDGINs）（19）。

你或許正在納悶，這個小分子抑制劑到底對反轉錄病毒感染宿主細胞有什麼干擾呢？我們發現到，細胞株如果在被反轉錄病毒感染前，先施予這種小分子抑制劑，病毒DNA插入宿主染色體的位置則被轉移到基因較不表現的區域（Out of Transcription Units）（圖三）（20-22）。同時，我們也觀察到這些受到小分子抑制劑干擾的原病毒，其病毒基因的表現較那些沒有受到小分子抑制劑干擾的原病毒有下降的趨勢。也就是說，利用這個小分子抑制劑，我們將原病毒「先封鎖」在不適合原病毒進行基因表現的宿主染色體區域，來達到「再抑制」原病毒基因表現的效果。

圖三：小分子抑制劑LEDGINs對於原病毒的影響。若在病毒感染前，施予細胞小分子抑制劑LEDGINs，病毒DNA插入宿主染色體的位置則被轉移到基因較不表現的區域。而且病毒基因的表現較那些沒有受到有下降的趨勢。d和d' 代表原病毒DNA插入宿主染色體的位置與在宿主染色體上，具有表H3K36me3訊號的區域。

結語

隨著我們對於反轉錄病毒認識的增加，越來越多對於治療後天免疫缺乏症候群的策略將會被提出。然而，對於一個每天都在嘗試，是否能更佳了解反轉錄病毒的我而言，「預防勝於治療」對於杜絕愛滋病毒傳播，或許仍然是最有效的策略。

***請尊重智慧財產權，切勿隨意下載與轉載。引用煩請註明出處：Chen, H.-C. #2 抗反轉錄病毒藥物下的漏網之魚－淺談潛伏原病毒對治療後天免疫缺乏症候群的影響. Blog Silence is a Behaviour. (2020).***

References | 延伸閱讀

1. Chun, T. W. et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc. Natl. Acad. Sci. U. S. A. 94 , 13193–13197 (1997).