Investigation of Innate Immune Responses against HIV with Camelid Nanobodies

生殖系编码的受体和适配器感知感染的迹象，并启动适当的先天免疫反应。虽然这些反应可以减少对人类免疫缺陷病毒(HIV)的感染，但炎症的异常激活也与HIV的发病有关。尽管疾病进展过程中CD4T细胞的大量丢失与上睑下垂有关，但HIV诱导的炎症小体组装和其他基本方面的先天免疫反应的分子细节尚不清楚。尽管以前的研究大多依赖于细胞因子测量和细胞丢失的间接评估，但我们提出了一种不同的方法来研究对HIV感染的先天免疫反应：我们的目标是直接可视化和量化人类原代细胞(与新型炎性小体生物传感器和纳米体的混合物)中的炎性小体组装和抗病毒反应。纳米抗体是来源于骆驼重链抗体的单域抗体，我们将通过酶或遗传修饰定制产生并配备额外的功能。我们将通过流式细胞术和ImageStream分析来检测组装炎症体或启动抗病毒转录反应的单个细胞。HIV毒株将配备一种新型的基因编码的炎症体生物传感器，该传感器可以在生产性或流产感染的原代细胞培养中显示炎症体组装，包括PBMC和扁桃体人类淋巴细胞聚集培养(HLAC)。这将使我们能够对反应细胞进行分类，并分析它们的转录组。纳米抗体将被用来通过先进的显微镜技术可视化涉及的分子相互作用，识别新的相互作用，并在原代细胞中耗尽蛋白质进行功能分析。我们将进一步剖析NLRX1的分子调控，NLRX1是HIV感染所需的一种关键的抗炎宿主因子，在SIV感染的猕猴中早期上调。利用定制的针对NLRX1的纳米抗体，我们将评估其在HIV感染的原代细胞中的功能，通过寡聚、翻译后修饰或定位来分析其调控，并将通过邻近标记方法寻找新的相互作用伙伴。我们推测NLRX1是一个复杂调控的信号中枢，它整合信息来广泛协调抗炎过程。我们将生成、应用和共享可视化炎症反应及其调控的新工具。因此，我们的工作将有助于揭示HIV感染如何在相关的原代细胞模型中触发和避免先天免疫系统。

Germ line-encoded receptors and adaptors sense signs of infection and mount the appropriate innate immune response. While these responses can curtail infection with human immunedeficiency virus (HIV), aberrant activation of inflammation is also linked to HIV pathogenesis. Although the massive loss of CD4 T cells in the course of disease progression has been linked to pyroptosis, molecular details of HIV-induced inflammasome assembly and other fundamental aspects of the innate immune response to HIV are not understood.While previous studies have mostly relied on cytokine measurements and indirect assessment of cell loss, we propose a different approach to investigate innate immune responses to HIV infection: We aim to directly visualize and quantify inflammasome assembly and antiviral responses in human primary cell (mixtures) with novel inflammasome biosensors and nanobodies. Nanobodies are single domain antibodies derived from camelid heavy chain-only antibodies, which we will custom generate and equip with additional functionalities by enzymatic or genetic modification. We will detect individual cells that assemble inflammasomes or mount antiviral transcriptional responses by flow cytometry and ImageStream analysis. HIV strains will be equipped with a novel genetically-encoded inflammasome biosensor that can reveal inflammasome assembly in productively or abortively infected primary cell cultures, including PBMCs and tonsillar human lymphocyte aggregate cultures (HLAC). This will allow us to sort responding cells and analyze their transcriptome. Nanobodies will be used to visualize the involved molecular interactions by advanced microscopy techniques, identify novel interactions, and deplete proteins in primary cells for functional analysis. We will further dissect the molecular regulation of NLRX1, a critical anti-inflammatory host factor required for HIV infection in myeloid cells that is upregulated early in SIV-infected macaques. Using custom-generated nanobodies against NLRX1, we will evaluate its function in HIV-infected primary cells, analyze its regulation by oligomerization, post-translational modification, or localization, and will identify novel interaction partners by proximity labeling approaches. We postulate that NLRX1 is an intricately regulated signaling hub, which integrates input to broadly coordinate anti-inflammatory processes. We will generate, apply, and share novel tools to visualize inflammatory responses and their regulation. Our work will thus help reveal how HIV infection triggers and avoids the innate immune system in relevant primary cell models.