【作者】 赵琪；

【导师】 程亮；

【作者基本信息】 武汉大学 ， 基础医学， 2024， 硕士

【摘要】 人免疫缺陷病毒-1(HIV-1)慢性感染导致的艾滋病严重威胁人类健康及社会经济发展。目前临床开展的高效抗逆转录病毒疗法(HAART)虽然可以有效抑制患者体内HIV-1复制,但无法完全恢复患者免疫功能,也不能清除体内HIV-1病毒库,导致停药后病毒迅速反弹。因此,目前亟需开发新型治疗策略,达到治愈艾滋病的目的。HIV-1感染诱导机体抗病毒免疫功能耗竭是导致病毒无法被机体有效清除的重要原因。白细胞介素21(IL-21)是γ链(γc)家族细胞因子,主要由CD4~+T细胞产生,其受体表达于多种免疫细胞表面,如CD8~+T细胞等。研究表明,CD4~+T细胞分泌的IL-21能维持CD8~+T细胞效应功能、促进活化的CD8~+T细胞分化为记忆干细胞样T细胞(TSCM),并防止CD8~+T细胞功能耗竭。然而,在HIV-1慢性感染过程中,CD4~+T细胞数量减少,其分泌IL-21能力下降。因此我们推测外源性IL-21治疗能恢复CD8~+T细胞抗HIV-1感染的效应功能。为了进一步增加IL-21的疗效,我们将IL-21与HIV-1受体分子CD4的胞外区(sCD4)及人免疫球蛋白恒定区Fc段进行融合。我们推测sCD4可将IL-21靶向表达gp120蛋白的HIV~+细胞,从而达到原位活化CD8~+T细胞等效应细胞的作用,进而增强IL-21的治疗效果。Fc功能结构域一方面可增加蛋白的稳定性,另一方面Fc可与巨噬细胞及自然杀伤细胞(NK)上Fc受体结合,介导抗体依赖的细胞毒作用(ADCC)。本研究中我们利用双特异性抗体构建中的“Knobs-into-Holes”技术构建了hIL-21×sCD4-Fc异二聚体融合蛋白:即将hIL-21及sCD4分别与含有突变的Fc(Fc6及Fc9)融合,分别构建hIL-21-Fc6以及sCD4-Fc9,然后将带有hIL-21-Fc6和sCD4-Fc9序列的表达载体同时转染293T细胞,成功获得异二聚体蛋白hIL-21×sCD4-Fc。通过体外实验,我们证明:1)hIL-21×sCD4-Fc可以显著促进记忆干细胞样CD8~+T细胞的产生;2)hIL-21×sCD4-Fc可以靶向被激活的HIV-1潜伏感染细胞以及中和游离的HIV-1病毒;3)hIL-21×sCD4-Fc靶向结合HIV~+细胞后,可以显著促进细胞表面HIV-1包膜蛋白gp120构象的开放,从而促进患者血浆中内源性抗体与细胞表面gp120结合;4)hIL-21×sCD4-Fc可以促进HIV-1感染者外周血中CD8~+T细胞和NK细胞分泌效应分子穿孔素,并且增强特异性CD8~+T细胞的杀伤功能;5)在CD8~+T细胞、NK细胞与被感染的CD4~+T细胞共培养体系中加入hIL-21×sCD4-Fc,可以有效抑制病毒复制。综上所述,我们成功构建了多功能融合蛋白hIL-21×sCD4-Fc,并证明hIL-21×sCD4-Fc可以有效靶向HIV-1感染的细胞,同时增强抗HIV-1效应细胞的免疫功能。我们的研究结果提示多功能融合蛋白hIL-21×sCD4-Fc可以作为一种潜在的药物用于艾滋病的免疫治疗。更多还原

【Abstract】 Human immunodeficiency virus-1(HIV-1)infection leads to the development of AIDS,which is a serious threat to public health.Highly active antiretroviral therapy(HAART)can effectively inhibit HIV-1 replication,but the immune function of patients can not be fully restored.In addition,HAART cannot eradicate HIV-1 latent reservoirs,resulting in the quick rebound of virus upon treatment interruption.Therefore,developing a novel therapeutic strategy is necessary to achieve the purpose of curing AIDS.The exhaustion of antiviral immune function induced by HIV-1 infection is an important reason why the virus can not be effectively cleared by the body.Interleukin21(IL-21)is aγchain(γc)family cytokine,which is mainly produced by CD4~+T cells.The receptor of IL-21 is expressed in many types of immune cells including CD8~+T cells.Studies have shown that IL-21 secreted by CD4~+T cells can maintain the effect function of CD8~+T cells,promote their differentiation into stem cell-like memory T cells(TSCM)and prevent their exhaustion.However,in the process of chronic HIV-1infection,the number of CD4~+T cells decreased and lost their ability to secrete IL-21.We speculate that exogenous IL-21 therapy can restore the anti-HIV-1 function of CD8~+T cells.To further enhance the potential efficacy of IL-21,we fused the extracellular region of HIV-1 receptor molecule CD4(sCD4)and the constant region of human immunoglobulin(Fc)withIL-21.We speculate that sCD4 will enable IL-21 to target HIV~+cells and enhance the in-situ activation of effector cells such asCD8~+T cells,thereby enhancing the therapeutic effect of IL-21.Fc domain would increase protein stability,and mediate antibody-dependent cell-mediated cytotoxicity(ADCC)through binding to Fc receptors on macrophages and natural killer cells(NK).In this study,we take advantage of the"Knobs-into-Holes"technology to construct the heterodimer protein hIL-21×sCD4-Fc by fusing hIL-21 with Fc6 and fusing sCD4with Fc9.We co-transfected the expression vector of hIL-21-Fc6 and sCD4-Fc9 into293T cells,and successfully obtained heterodimer protein hIL-21×sCD4-Fc.We demonstrated that:1)hIL-21×sCD4-Fc significantly promoted the differentiation of activated CD8~+T cells to memory stem cell-like T cells;2)hIL-21×sCD4-Fc can target the reactivated HIV-1 latently infected cells and can neutralize free HIV-1 virus;3)Binding of hIL-21×sCD4-Fc to gp120 promoted the conformational opening of gp120and thus enhanced the binding of endogenous antibodies to gp120;4)hIL-21×sCD4-Fc treatment promoted the secretion of perforin by CD8~+T cells and NK cells from peripheral blood of HIV-1 patients and enhanced the cytotoxic function of HIV-1specific CD8~+T cells;5)In a co-culture system,consisting of HIV-1 infected CD4~+T cells plusCD8~+T cells and NK cells,hIL-21×sCD4-Fc treatment effectively inhibited virus replication.In summary,we have developed a multifunctional fusion protein hIL-21×sCD4-Fc successfully.We proved that hIL-21×sCD4-Fc can effectively target HIV-1 infected cells and enhance the function of anti-HIV-1 effector cells at the same time.Our results suggest that hIL-21×sCD4-Fc may provide a potential agent for immunotherapy of AIDS.更多还原