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首页> 外文会议>Nanotechnology in medicine II: briding translational in vitro and in vivo interfaces >MODULAR CONTROL OF INNATE IMMUNE SIGNALING USING SELF-ASSEMBLY OF IMMUNE SIGNALS
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MODULAR CONTROL OF INNATE IMMUNE SIGNALING USING SELF-ASSEMBLY OF IMMUNE SIGNALS

机译：使用免疫信号的自组装对初始免疫信号进行模块化控制

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Vaccines play an increasingly important role in preventing and treating diseases ranging from infectious pathogens to cancer because these technologies harness the specificity of the immune system to clear disease without targeting the body's own cells. To realize these goals, new understanding of adjuvants - molecules added to vaccines to enhance function - is needed to support design of next-generation vaccines that elicit responses tailored for specific diseases. We recently reported a simple nanotechnology platform based on self-assembly of peptide antigen and a molecular toll-like receptor agonist (TLRa) to create modular vaccine designs (ACS Nano 2016, ACS Nano 2015). These structures - termed immune polyelectrolyte multilayers (iPEMs) -juxtapose antigen and TLRa at high densities, and offer 100% cargo loading since no carrier component is needed. This modularity also creates the possibility of rationally designing iPEMs that trigger multiple immune pathways with distinct control over the relative activation levels. In cancer, for example, activating multiple innate pathways has been linked to improved patient outcomes in human clinical trials. To exploit iPEMs in this manner, we designed iPEM architectures incorporating a conserved human cancer antigen (Trp2), and a range of molecularly-defined TLRa that spanned different TLRa classes and species (i.e., mouse and human): agonists for TLR3, TLR9, and TLR13. iPEMs were assembled from Trp2 and one, two, or three TLRas, or alternatively, using two different TLRas at varying compositions. To form carrier free capsules using these design schemes, Trp2 was appended with cationic amino acids, then adsorbed onto a sacrificial colloidal template, with alternating adsorption steps employing the specified TLRas (anionic). Centrifugation and wash steps were performed after each adsorption, then the template was dissolved using a chelator (EDTA) to form carrier-free capsules formed entirely from tumor peptide and each TLRa composition. All components were labeled to facilitate measurement of composition by fluorimetry and confocal microscopy. Using this approach, we discovered iPEMs could be assembled from any combination of Trp2 and the TLRas (Fig. 1A). Quantification revealed further confirmed iPEM capsules consisting of the corresponding peptide antigen and TLRa ligands (Fig. 1B). iPEMs incubated with primary dendritic cells isolated from BL6 mice revealed a high degree of colocalization of each iPEM component within cells. For example, iPEMs consisting of Trp2 and all three TLRas revealed that 90% of cells positive for at least one iPEM component were positive for all four components (Fig. 1C). Compared with equivalent free mixtures, iPEMs drove synergistic activation of DCs measured using flow cytometry for common surface activation makers (e.g., CD80, CD40, CD86) (Fig. 1D). Importantly, iPEMs also allowed modular control of TLR signaling, revealed using iPEMs built from Trp2 while varying the input ratio of TLR3a:TLR9a to control the final composition. iPEMs with a high TLR3a:TLR9a ratio triggered a high level of TLR3 signaling (Fig. 1E). As the ratio decreased, TLR3a signaling decreased, while TLR9a signaling increased. This rational control could contribute to more effective vaccines that use molecular adjuvant assembly to direct specific combinations and levels of multiple innate signaling pathways.
机译：疫苗在预防和治疗从传染性病原体到癌症的各种疾病中发挥着越来越重要的作用，因为这些技术利用免疫系统的特异性清除疾病而不针对人体自身的细胞。为了实现这些目标，需要对佐剂有新的认识，即添加到疫苗中以增强功能的分子，以支持设计引发针对特定疾病的反应的下一代疫苗。我们最近报告了一个简单的纳米技术平台，该平台基于肽抗原的自组装和分子收费样受体激动剂（TLRa）来创建模块化疫苗设计（ACS Nano 2016，ACS Nano 2015）。这些结构-称为免疫聚电解质多层（iPEM）-在高密度下并置抗原和TLRa，并提供100％的货物装载量，因为不需要载体成分。这种模块性还提供了合理设计iPEM的可能性，这些iPEM可以触发多个免疫途径，并且可以相对控制相对激活水平。例如，在癌症中，在人类临床试验中，激活多种固有途径与改善患者预后相关。为了以这种方式利用iPEM，我们设计了iPEM架构，其中整合了保守的人类癌症抗原（Trp2）和一系列分子定义的TLRa，跨越了不同的TLRa类和物种（例如，小鼠和人类）：TLR3，TLR9，和TLR13。 iPEM由Trp2和一个，两个或三个TLRas组装而成，或者使用两个不同的TLRas以不同的组成组装而成。为了使用这些设计方案形成无载体的胶囊，将Trp2附加阳离子氨基酸，然后吸附到牺牲胶体模板上，并采用指定的TLRas（阴离子）交替进行吸附。每次吸附后进行离心和洗涤步骤，然后使用螯合剂（EDTA）溶解模板以形成完全由肿瘤肽和每种TLRa组合物形成的无载体胶囊。标记所有成分以利于通过荧光测定法和共聚焦显微镜测量组成。使用这种方法，我们发现iPEM可以由Trp2和TLRas的任何组合组装而成（图1A）。定量显示进一步证实的iPEM胶囊由相应的肽抗原和TLRa配体组成（图1B）。与从BL6小鼠分离的原代树突状细胞孵育的iPEM显示出细胞内每个iPEM组分的高度共定位。例如，由Trp2和所有三个TLRas组成的iPEM揭示，至少一种iPEM组分呈阳性的细胞中有90％对所有四个组分呈阳性（图1C）。与同等的游离混合物相比，iPEM推动了使用流式细胞仪对常见表面活化剂（例如CD80，CD40，CD86）进行DC的协同活化（图1D）。重要的是，iPEM还允许对TLR信号进行模块化控制，这是使用从Trp2构建的iPEM揭示出来的，同时通过改变TLR3a：TLR9a的输入比例来控制最终成分。具有高TLR3a：TLR9a比的iPEM触发了高水平的TLR3信号传导（图1E）。随着比率的降低，TLR3a信号减少，而TLR9a信号增加。这种合理的控制可能有助于使用分子佐剂组装来指导多种先天信号通路的特定组合和水平的更有效的疫苗。

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