Viral ASAPseq definition of CD4+ T cell viral reservoirs

虽然联合抗逆转录病毒疗法（ART）有效地控制了大多数艾滋病毒感染者的病毒血症， （PLWH），由于艾滋病毒宿主，诱导病毒缓解的广泛适用策略仍然难以捉摸 坚持不懈在确定前病毒的病毒学特征方面已经取得了实质性进展， 然而，在我们的理解中仍然存在重大差距， 受感染的CD4+ T细胞构成了细胞HIV库，限制了我们发展有效免疫的能力。 艾滋病治疗策略。我们已经开发了一种新的高通量10X Genomics单细胞测定法， 通过整合的前病毒DNA的存在来鉴定HIV感染的细胞。术语为“病毒ASAPseq”[用于检测 转座酶可降解Chomatin（ATAC）表面抗原谱测序，ASAPseq，使用病毒 比对，病毒ASAPseq]，该测定允许检测常染色质内的HIV前病毒DNA， 用154标记细胞表面抗原标记，产生HIV感染细胞的多模式单细胞分辨。 该测定不需要离体活化来检测整合的病毒DNA的存在，从而 保持受感染细胞的天然状态。我们已经在几个国家成功地试行了这一程序。 条件，包括体外感染的原代CD4+ T细胞，来自病毒血症的原代淋巴结CD4+ T细胞， PLWH和来自ART处理的PLWH的外周血CD4+ T细胞。我们进一步发展了生物信息学 使用自体病毒序列比对识别前病毒DNA的策略，定义表面标记物 HIV感染细胞的表观遗传组成，并表征感染细胞的表观遗传结构。因此，我们 准备第一次直接离体获得对HIV储库组成的直接了解。这里我们 将应用这项新技术来测试HIV感染的CD4+ T细胞具有细胞表面或细胞外基质的假设。 与ART治疗的PLWH中未感染的CD4+ T细胞不同的表观遗传标记。我们将进一步试点新的 策略，scGETseq，能够同时和分别测序常染色质和异染色质 区分和分析ART治疗的PLWH的活性和潜伏库中整合的HIV前病毒。 总之，我们的研究有可能提供第一个完整的了解未经操作的CD4+ T细胞， 直接离体储存HIV。

While combination antiretroviral therapy (ART) effectively controls HIV viremia in most people living with HIV (PLWH), broadly applicable strategies to induce viral remission have remained elusive due to HIV reservoir persistence. Substantial progress has been made in defining the virological characteristics of the proviral reservoir of latently infected cells during ART. However, significant gaps remain in our understanding of the infected CD4+ T cells that constitute the cellular HIV reservoir that limit our ability to develop effective strategies for HIV cure. We have developed a novel high throughput 10X Genomics single cell assay to directly identify HIV infected cells via the presence of integrated proviral DNA. Termed "viral ASAPseq" [Assay for Transposase Accessible Chomatin (ATAC) Surface Antigen Profile sequencing, ASAPseq, with viral alignment, viral ASAPseq], this assay allows detection of HIV proviral DNA within euchromatin in combination with 154-marker cell surface antigen labeling, yielding multimodal single cell resolution of HIV infected cells. This assay does not require ex vivo activation to detect the presence of integrated viral DNA, thereby preserving the native state of the infected cell. We have successfully piloted this procedure in several conditions, including in vitro infected primary CD4+ T cells, primary lymph node CD4+ T cells from viremic PLWH, and peripheral blood CD4+ T cells from ART treated PLWH. We have further developed bioinformatic strategies to identify proviral DNA using autologous viral sequence alignment, define the surface marker composition of HIV infected cells, and characterize the epigenetic structure of infected cells. Thus, we are poised for the first time to gain a direct understanding of HIV reservoir composition directly ex vivo. Here we will apply this new technique to test the hypothesis that HIV infected CD4+ T cells have a cell surface or epigenetic signature distinct from uninfected CD4+ T cells in ART treated PLWH. We will further pilot a new strategy, scGETseq, capable of simultaneously and separately sequencing euchromatin and heterochromatin to distinguish and profile integrated HIV provirus within the active and latent reservoir of ART treated PLWH. Together our studies have the potential to provide the first full understanding of the unmanipulated CD4+ T cell HIV reservoir directly ex vivo.