Interdisciplinary Graduate Program in Genetic Engineering, the Graduate School, Kasetsart University, Bangkok 10900, Thailand.
Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.
Vet Microbiol. 2022 Dec;275:109583. doi: 10.1016/j.vetmic.2022.109583. Epub 2022 Oct 18.
A DNA fragment containing CpG motifs (CpG ODN) is one of the potent immunopotentiators used to improve vaccine efficacy. It can enhance a protective immunity by stimulating both innate and adaptive immune responses. In this study, we designed and constructed a recombinant plasmid carrying the combined CpG ODN to generate an immunopotentiator for boosting the immunogenicity of porcine circovirus type 2 (PCV2) virus-like particles (VLPs). The capsid protein of PCV2b was expressed in insect cells and purified by affinity chromatography. The purified capsid protein was incubated with the CpG ODN in the reaction that allowed VLPs formation and encapsidation of the CpG ODN to occur simultaneously. Morphology of the reassembled VLPs was similar to the PCV2 virions as observed using an electron microscope. When the CpG ODN-encapcidated VLPs was treated with DNase I, the VLPs could protect the packaged CpG ODN from the enzyme digestion. Moreover, we immunized mice subcutaneously with VLPs, CpG ODN-loaded VLPs, or phosphate buffer saline for three times at two-week intervals. The results showed that the CpG ODN-loaded VLPs could elicit significantly higher levels of PCV2-specific neutralizing antibodies and interferon gamma (IFN-γ) expression in the immunized mice compared to those conferred by the VLPs alone. Conclusively, we have proved that the CpG ODN incorporated in VLPs can serve as a potent immunopotentiator for PCV2 vaccine development.
一段含有 CpG 基序的 DNA 片段（CpG ODN）是一种有效的免疫增强剂，可用于提高疫苗的效力。它可以通过刺激先天和适应性免疫反应来增强保护性免疫。在本研究中，我们设计并构建了一种携带 CpG ODN 的重组质粒，以产生一种免疫增强剂，用于增强猪圆环病毒 2 型（PCV2）病毒样颗粒（VLPs）的免疫原性。通过亲和层析从昆虫细胞中表达和纯化 PCV2b 衣壳蛋白。将纯化的衣壳蛋白与 CpG ODN 孵育，允许同时形成 VLPs 并将 CpG ODN 包封。用电子显微镜观察到，重新组装的 VLPs 的形态与 PCV2 病毒粒子相似。当用 DNA 酶 I 处理包封 CpG ODN 的 VLPs 时，VLPs 可以保护包封的 CpG ODN 免受酶消化。此外，我们通过皮下免疫小鼠三次，间隔两周，每次使用 VLPs、CpG ODN 加载的 VLPs 或磷酸盐缓冲盐水。结果表明，与单独使用 VLPs 相比，CpG ODN 加载的 VLPs 可以在免疫小鼠中诱导更高水平的 PCV2 特异性中和抗体和干扰素γ（IFN-γ）表达。总之，我们已经证明，VLPs 中掺入的 CpG ODN 可以作为 PCV2 疫苗开发的有效免疫增强剂。
