Xu Jian, Sekiguchi Tomofumi, Boonyakida Jirayu, Kato Tatsuya, Park Enoch Y
Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, Shizuoka, Japan.
Department of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, Shizuoka, Japan.
Front Bioeng Biotechnol. 2023 Feb 16;11:1096363. doi: 10.3389/fbioe.2023.1096363. eCollection 2023.
Recent progress has been made dramatically in decorating virus-like particles (VLPs) on the surface or inside with functional molecules, such as antigens or nucleic acids. However, it is still challenging to display multiple antigens on the surface of VLP to meet the requirement as a practical vaccine candidate. Herein this study, we focus on the expression and engineering of the capsid protein VP2 of canine parvovirus for VLP display in the silkworm-expression system. The chemistry of the SpyTag/SpyCatcher (SpT/SpC) and SnoopTag/SnoopCatcher (SnT/SnC) are efficient protein covalent ligation systems to modify VP2 genetically, where SpyTag/SnoopTag are inserted into the N-terminus or two distinct loop regions (Lx and L2) of VP2. The SpC-EGFP and SnC-mCherry are employed as model proteins to evaluate their binding and display on six SnT/SnC-modified VP2 variants. From a series of protein binding assays between indicated protein partners, we showed that the VP2 variant with SpT inserted at the L2 region significantly enhanced VLP display to 80% compared to 5.4% from N-terminal SpT-fused VP2-derived VLPs. In contrast, the VP2 variant with SpT at the Lx region failed to form VLPs. Moreover, the SpT (Lx)/SnT (L2) double-engineered chimeric VP2 variants showed covalent conjugation capacity to both SpC/SnC protein partners. The orthogonal ligations between those binding partners were confirmed by both mixing purified proteins and co-infecting cultured silkworm cells or larvae with desired recombinant viruses. Our results indicate that a convenient VLP display platform was successfully developed for multiple antigen displays on demand. Further verifications can be performed to assess its capacity for displaying desirable antigens and inducing a robust immune response to targeted pathogens.
近年来，在利用功能分子（如抗原或核酸）修饰病毒样颗粒（VLP）的表面或内部方面取得了显著进展。然而，在VLP表面展示多种抗原以满足作为实用疫苗候选物的要求仍然具有挑战性。在本研究中，我们专注于犬细小病毒衣壳蛋白VP2在蚕表达系统中的表达及工程改造，用于VLP展示。SpyTag/SpyCatcher（SpT/SpC）和SnoopTag/SnoopCatcher（SnT/SnC）化学体系是用于对VP2进行基因修饰的高效蛋白质共价连接系统，其中SpyTag/SnoopTag被插入到VP2的N端或两个不同的环区（Lx和L2）。SpC-EGFP和SnC-mCherry被用作模型蛋白，以评估它们在六种SnT/SnC修饰的VP2变体上的结合和展示情况。通过一系列指定蛋白质伴侣之间的蛋白质结合试验，我们发现，与N端融合SpT的VP2衍生的VLP的5.4%相比，在L2区插入SpT的VP2变体显著提高了VLP展示效率，达到80%。相比之下，在Lx区带有SpT的VP2变体未能形成VLP。此外，SpT（Lx）/SnT（L2）双工程嵌合VP2变体对SpC/SnC蛋白质伴侣均显示出共价结合能力。通过混合纯化蛋白以及用所需重组病毒共感染培养的蚕细胞或幼虫，证实了这些结合伴侣之间的正交连接。我们的结果表明，成功开发了一个方便的VLP展示平台，可按需进行多种抗原展示。可以进行进一步验证，以评估其展示所需抗原和诱导针对目标病原体的强大免疫反应的能力。
