Cobalt porphyrin phospholipid (CoPoP) can incorporate within bilayers to enable non-covalent surface-display of antigens on liposomes by mixing with proteins bearing a polyhistidine tag (his-tag); however, the mechanisms for how this occurs are poorly understood. These were investigated using the his-tagged model antigen Pfs25, a protein antigen candidate for malaria transmission-blocking vaccines. Pfs25 was found to associate with the small molecule aquocobalamin, a form of vitamin B12 and a cobalt-containing corrin macrocycle, but without particle formation, enabling comparative assessment. Relative to CoPoP liposomes, binding and serum stability studies indicated a weaker association of Pfs25 to aquocobalamin or cobalt nitrilotriacetic acid (Co-NTA) liposomes, which have cobalt displayed in the aqueous phase on lipid headgroups. Antigen internalization by macrophages was enhanced with Pfs25 bound to CoPoP liposomes. Immunization in mice with Pfs25 bound to CoPoP liposomes elicited antibodies that recognized ookinetes and showed transmission-reducing activity. To explore the physical mechanisms involved, we employed molecular dynamics (MD) simulations of bilayers containing phospholipid, cholesterol, as well as either CoPoP or NTA-functionalized lipids. The results show that the CoPoP-containing bilayer creates nanodomains that allow access for a limited but sufficient amount of water molecules that could be replaced by his-tags due to their favorable free energy properties allowing for stabilization. The position of the metal center within the NTA liposomes was much more exposed to the aqueous environment, which could explain its limited capacity for stabilizing Pfs25. This study illustrates the impact of CoPoP-induced antigen particleization in enhancing vaccine efficacy, and provides molecular insights into the CoPoP bilayer properties that enable this.

中文翻译：

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免疫原性钴卟啉脂质双层的实验和计算观察：纳米域增强的抗原结合

钴卟啉磷脂 (CoPoP) 可以结合到双层中，通过与带有多组氨酸标签 (his-tag) 的蛋白质混合，使抗原在脂质体上的非共价表面展示；然而，人们对这种情况发生的机制知之甚少。这些是使用带有his标签的模型抗原Pfs25进行研究的，Pfs25是疟疾传播阻断疫苗的候选蛋白抗原。Pfs25 被发现与小分子水钴胺素、维生素 B12 的一种形式和含钴的可林大环化合物相关，但没有颗粒形成，因此可以进行比较评估。相对于 CoPoP 脂质体，结合和血清稳定性研究表明 Pfs25 与水钴胺素或次氮基三乙酸钴 (Co-NTA) 脂质体的结合较弱，这些脂质体在水相中的脂质头基上具有钴。与 CoPoP 脂质体结合的 Pfs25 增强了巨噬细胞的抗原内化。用与 CoPoP 脂质体结合的 Pfs25 对小鼠进行免疫会引发抗体，该抗体可识别 ookinetes 并显示出降低传播的活性。为了探索所涉及的物理机制，我们对含有磷脂、胆固醇以及 CoPoP 或 NTA 功能化脂质的双层进行了分子动力学 (MD) 模拟。结果表明，含有 CoPoP 的双层产生的纳米域允许进入有限但足够量的水分子，由于其有利的自由能特性允许稳定，这些水分子可以被 his-tags 取代。NTA 脂质体中金属中心的位置更多地暴露于水环境中，这可以解释其稳定 Pfs25 的能力有限。