Engineered Outer Membrane Vesicles as DNA Vaccine Delivery Vehicles

描述（由申请人提供）：背景：对DNA疫苗的低免疫应答对其潜在的临床应用仍然是一个挑战，并且新的递送策略的开发仍然是一个重要的努力领域。一种临床上用于增强蛋白质和多糖疫苗效力的策略是通过细菌“外膜囊泡”或OMV递送抗原，所述OMV是由革兰氏阴性细菌组成性产生的囊泡。OMV在自然界中被细菌用作蛋白质和潜在的DNA递送载体。这项R21探索性研究资助的目的是明确确定OMV是否可以被工程化以将基于质粒的DNA疫苗递送到哺乳动物抗原呈递细胞。假设：OMV的特性可以被工程化以增强基于质粒的DNA疫苗向哺乳动物抗原呈递细胞的递送。具体目标：1）工程化并定量评估含质粒的OMV的表面以使其与哺乳动物细胞膜的非特异性结合最大化，和2）工程化并定量评估含质粒的OMV的表面以促进其从酸性亚细胞囊泡中释放。OMV的ζ电位约为-40 mV，这降低了它们与哺乳动物细胞膜的结合亲和力。为了增加OMV与哺乳动物细胞的结合，并增加通过非特异性内吞作用或吞噬作用的内化速率，OMV的表面将被工程化以具有多聚赖氨酸序列以产生具有正ζ电位的OMV。通过内吞作用/吞噬作用的大分子结构的内化导致它们在酸性隔室内的隔离和溶酶体酶的消化。为了促进OMV从溶酶体运输途径逃逸，OMV的表面将被工程化以具有多组氨酸序列以破坏酸性囊泡的膜。该假设的验证将支持未来工程OMV更复杂的特性，如细胞特异性配体，融合蛋白和构象蛋白佐剂。

DESCRIPTION (provided by applicant): BACKGROUND: The low immune response to DNA vaccines remains a challenge to their potential clinical use, and the development of new delivery strategies continues to be an area of significant effort. One clinically utilized strategy to enhance the potency of protein and polysaccharide vaccines is to deliver the antigens via bacterial "outer membrane vesicles", or OMVs, which are vesicles constitutively produced by gram-negative bacteria. OMVs are used by bacteria in nature as protein, and potentially DNA, delivery vehicles. The objective of this R21 exploratory research grant is to definitively determine if OMVs can be engineered to deliver plasmid-based DNA vaccines to mammalian antigen presenting cells. HYPOTHESIS: OMV characteristics can be engineered to enhance the delivery of plasmid-based DNA vaccines to mammalian antigen presenting cells. SPECIFIC AIMS: 1) To engineer, and quantitatively evaluate, the surface of plasmid-containing OMVs to maximize their non-specific binding to mammalian cell membranes, and 2) To engineer, and quantitatively evaluate, the surface of plasmid-containing OMVs to facilitate their release from acidic subcellular vesicles. The zeta potential of OMVs is approximately -40 mV which reduces their binding affinity to mammalian cell membranes. To increase the association of OMVs with mammalian cells, and increase the rate of internalization by non- specific endocytosis or phagocytosis, the surface of the OMVs will be engineered to possess sequences of polylysine to create OMVs with positive zeta potentials. Internalization of macromolecular structures by endocytosis/phagocytosis leads to their sequestration within acidic compartments and digestion by lysosomal enzymes. To facilitate the escape of OMVs from the lysosomal trafficking pathway, the surface of the OMVs will be engineered to possess sequences of polyhistidine to disrupt the membrane of acidic vesicles. Validation of the hypothesis will support the future engineering of OMVs with more intricate characteristics, such as cell specific ligands, fusogenic proteins and conformational protein adjuvants.