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

自然杀伤（NK）细胞的功能可以受到细胞表面的影响。 其靶细胞的糖基化。CD 56 dim NK细胞的亚群表达唾液酸结合蛋白Siglec， 9.该亚组具有高细胞溶解活性;然而，Siglec-9本身是抑制细胞溶解活性的抑制性受体。 细胞溶解能力，否则这种高细胞毒性群体。利用这个群体的细胞毒性能力 尚未被评估为根除艾滋病毒的方法。在我们的初步研究中， 细胞，我们发现Siglec-9+ CD 56 dim NK细胞的水平与CD 4 + T细胞相关的HIV DNA呈负相关。 抗逆转录病毒治疗（ART）抑制HIV感染。此外，Siglec-9+ CD 56 dim NK细胞表现出明显的细胞毒性。 与Siglec-9- NK细胞相比，对HIV+细胞的细胞毒性更高。这些数据与高度一致 Siglec-9+ NK细胞的细胞毒性性质。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。下一步聚焦目标 细胞，我们发现HIV潜伏感染的CD 4 + T细胞表现出高水平的Siglec-9配体，α2-3唾液酸， 与HIV感染或未感染的细胞相比。我们还开发了一种新的方法来阻止 通过将唾液酸酶（切割唾液酸的酶）与四种 HIV广泛中和抗体（bNAb）。这些共轭物（在手）可以与药物结合使用 重新激活HIV潜伏感染的细胞，以实现功能性HIV治愈。我们对其中一种结合物进行了初步测试 并发现它能够选择性地去唾液酸化HIV+细胞的表面，并增强NK细胞杀死这些感染者的能力。 体外细胞总之，我们的数据支持我们的中心假设，即Siglec/唾液酸聚糖相互作用有助于 HIV感染细胞逃避NK免疫监视的能力，以及通过阻断这些相互作用， HIV感染细胞的选择性去唾液酸化将增强NK细胞清除HIV感染细胞的能力。 在目标1中：我们将检验Siglec-9/唾液酸相互作用有助于HIV感染能力的假设。 潜伏感染的细胞以逃避NK免疫监视。在（1a）中，我们将确定Siglec-9在细胞内的作用。 NK细胞杀死HIV+细胞的能力，在（1b）中，我们将确定α2-3唾液酸在HIV+细胞杀伤能力中的作用。 潜伏感染的CD 4 + T细胞以逃避NK细胞的杀伤。目标2：我们将检验HIV bNAb- 唾液酸酶缀合物在体外（2a）和离体（2b）减小HIV储库的大小，和（2c）延迟病毒感染。 使用脾注射的原发性HIV感染储库（SPHIR-IL 15）的改良版本的体内反弹 具有高NK寿命的非胎儿人源化小鼠模型。我们还将确认bNAb- 唾液酸酶偶联物通过检查Fc介导的功能的作用增强NK细胞抗病毒功能， NK靶向中的信号结合。我们的跨学科方法是利用最新的进展， 糖免疫学的新兴领域，以增强NK细胞杀死ART抑制个体中HIV+细胞的能力。 我们的目标是提供一种新的机制和方法，可以利用它来功能性地治愈HIV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.