Investigation of Innate Immune Responses against HIV with Camelid Nanobodies

生殖细胞系编码的受体和衔接子感知感染的迹象，并启动适当的先天免疫反应。虽然这些反应可以减少人类免疫缺陷病毒（HIV）的感染，但炎症的异常激活也与HIV发病机制有关。尽管在疾病进展过程中CD4 T细胞的大量丢失与细胞凋亡有关，但HIV诱导的炎性小体组装的分子细节和对HIV的先天免疫应答的其他基本方面尚不清楚。虽然以前的研究主要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感染的先天免疫应答：我们的目标是直接可视化和量化炎症小体组装和抗病毒反应在人类原代细胞（混合物）与新的炎症小体生物传感器和纳米抗体。纳米抗体是来源于骆驼科动物仅重链抗体的单结构域抗体，我们将通过酶促或遗传修饰定制生成并配备额外的功能。我们将通过流式细胞术和ImageStream分析检测组装炎性小体或安装抗病毒转录反应的单个细胞。HIV病毒株将配备一种新的遗传编码的炎性体生物传感器，可以揭示生产性或流产感染的原代细胞培养物中的炎性体组装，包括PBMC和扁桃体人淋巴细胞聚集体培养物（HLAC）。这将使我们能够对响应细胞进行分类并分析它们的转录组。纳米抗体将用于通过先进的显微镜技术可视化所涉及的分子相互作用，识别新的相互作用，并耗尽原代细胞中的蛋白质进行功能分析。我们将进一步剖析NLRX 1的分子调控，NLRX 1是骨髓细胞中HIV感染所需的关键抗炎宿主因子，在SIV感染的猕猴中早期上调。使用针对NLRX 1的定制生成的纳米抗体，我们将评估其在HIV感染的原代细胞中的功能，分析其通过寡聚化、翻译后修饰或定位的调控，并将通过邻近标记方法鉴定新的相互作用伙伴。我们假设NLRX 1是一个复杂调节的信号中枢，它整合输入以广泛协调抗炎过程。我们将生成，应用和共享新的工具来可视化炎症反应及其调节。因此，我们的工作将有助于揭示HIV感染如何触发和避免相关原代细胞模型中的先天免疫系统。

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.