Targeting Siglec-9/Sialoglycan Interactions to Enhance NK Functions During HIV Infection

项目摘要：自然杀伤(NK)细胞的功能受细胞表面的影响 他们的目标细胞的糖基化。CD56dim NK细胞亚群表达唾液酸结合蛋白Siglec- 9.这个亚群具有很高的细胞溶解活性；然而，Siglec-9本身是一种抑制受体，它抑制 这种高度细胞毒性的种群的细胞溶解能力。利用这一群体的细胞毒能力 尚未被评估为根除艾滋病毒的一种方法。在我们的初步研究中，首先关注NK 细胞，我们发现Siglec-9+CD56dim NK细胞的水平与CD4+T细胞相关的HIV DNA呈负相关 在抗逆转录病毒治疗(ART)期间-抑制艾滋病毒感染。此外，Siglec-9+CD56dim NK细胞表现出 与Siglec-9-NK细胞相比，对HIV+细胞的细胞毒作用更强。这些数据与高度一致的 Siglec-9+NK细胞的细胞毒性质。然而，与Siglec-9的已知抑制功能一致 分子本身，阻断Siglec-9，增强了NK细胞在体外杀死HIV+细胞的能力。下一步关注目标 细胞，我们发现潜伏感染艾滋病毒的α+T细胞表现出高水平的Siglec-9配体，Siglec 2-3唾液酸， 与感染艾滋病毒或未感染艾滋病毒的细胞相比。我们还开发了一种新的方法来阻止 HIV感染过程中唾液酸酶(裂解唾液酸酶)与Siglec/唾液酸的相互作用 艾滋病毒广泛中和抗体(BNAbs)。这些结合物(手头)可与药物联合使用。 重新激活潜伏感染艾滋病毒的细胞，以实现艾滋病毒的功能性治愈。我们在试验中测试了其中一种 并发现它能够选择性地分析HIV+细胞的表面，并增强NK细胞杀死这些感染者的能力 体外培养的细胞。总之，我们的数据支持我们的中心假设，即Siglec/sialoglycan相互作用有助于 HIV感染细胞逃避NK免疫监视的能力以及阻止这些相互作用的能力 选择性去除HIV感染细胞，将增强NK细胞清除HIV感染细胞的能力。 在目标1：我们将测试Siglec-9/唾液酸相互作用有助于HIV能力的假设 潜伏感染细胞以逃避NK免疫监视。在(1a)中，我们将确定Siglec-9在 NK细胞杀伤艾滋病毒+细胞的能力，在(1b)中，我们将确定α2-3唾液酸在艾滋病毒能力中的作用 潜伏感染的CD4+T细胞以逃避NK细胞的杀伤。在目标2中：我们将测试HIV bNAb- 唾液酸酶结合物在体外和体外减小HIV储存库的大小(2a)和(2b)，以及(2c)延迟病毒 使用改良的脾注射HIV感染原发储存库的体内反弹(SPHIR-IL15) 自然杀伤细胞寿命高的无胎人源化小鼠模型。我们还将确认bNAb- 唾液酸酶结合物增强NK细胞抗病毒功能的研究 NK靶向中的Siglec结合。我们的跨学科方法正在利用最近在 糖免疫学的新兴领域，以增强NK细胞在ART抑制的个体中杀死HIV+细胞的能力。 我们的目标是提供一种新的机制和方法，可以用来从功能上治愈艾滋病毒感染。

PROJECT SUMMARY: The functions of Natural Killer (NK) cells can be influenced by the cell-surface glycosylation of their target cells. A subset of CD56dim NK cells expresses the Sialic acid-binding protein Siglec- 9. This subset has a high cytolytic activity; however, Siglec-9 itself is an inhibitory receptor that restrains the cytolytic ability of this otherwise highly cytotoxic population. Harnessing the cytotoxic capacity of this population has not been evaluated as an approach for eradicating HIV. In our preliminary studies and focusing first on NK cells, we found that levels of Siglec-9+ CD56dim NK cells inversely correlate with CD4+ T cell-associated HIV DNA during antiretroviral therapy (ART)-suppressed HIV infection. Furthermore, Siglec-9+ CD56dim NK cells exhibited higher cytotoxicity towards HIV+ cells compared to Siglec-9- NK cells. These data are consistent with the highly cytotoxic nature of the Siglec-9+ NK cells. However, consistent with the known inhibitory function of the Siglec-9 molecule itself, blocking Siglec-9 enhanced NK cells' ability to kill HIV+ cells in vitro. Focusing next on target cells, we found that HIV latently-infected CD4+ T cells exhibit high levels of the Siglec-9 ligand, α2-3 Sialic acid, compared to HIV productively-infected or uninfected cells. We also developed a novel approach to block Siglec/Sialic acid interactions during HIV infection by conjugating Sialidase (enzyme cleaves Sialic acid) to four HIV broadly neutralizing antibodies (bNAbs). These conjugates (in hand) can be used in conjunction with drugs that reactivate HIV latently-infected cells to achieve a functional HIV cure. We pilot tested one of these conjugates and found it able to selectively desialylate the surface of HIV+ cells and enhance NK capacity to kill these infected cells in vitro. Together, our data support our central hypothesis that Siglec/sialoglycan interactions contribute to the ability of HIV-infected cells to evade NK immune surveillance and that blocking these interactions, via selective desialylation of HIV-infected cells, will enhance the capacity of NK cells to clear HIV-infected cells. In Aim 1: we will test the hypothesis that Siglec-9/Sialic acid interactions contribute to the ability of HIV latently-infected cells to evade NK immune surveillance. In (1a), we will determine the role of Siglec-9 in the ability of NK cells to kill HIV+ cells, and in (1b), we will determine the role of α2-3 Sialic acid in the ability of HIV latently-infected CD4+ T cells to evade killing by NK cells. In Aim 2: we will test the hypothesis that HIV bNAb- Sialidase conjugates reduce the size of the HIV reservoir (2a) in vitro and (2b) ex vivo, and (2c) delay viral rebound in vivo using a modified version of the splenic-injected primary HIV-infected reservoir (SPHIR-IL15) non-fetal humanized mouse model with high NK longevity. We also will confirm the mechanism by which bNAb- Sialidase conjugates enhance NK cell antiviral function by examining the role of Fc-mediated functions and Siglec-binding in NK targeting. Our interdisciplinary approach is taking advantage of recent advances in the emerging field of glyco-immunology to enhance NK cell capacity to kill HIV+ cells in ART-suppressed individuals. Our goal is to provide a novel mechanism and approach that can be harnessed to functionally cure HIV infection.