Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Tokyo, 108-8477, Japan.
Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Tokyo, 108-8477, Japan.
Fish Shellfish Immunol. 2024 Sep;152:109803. doi: 10.1016/j.fsi.2024.109803. Epub 2024 Aug 2.
Nervous necrosis virus (NNV) capsid protein plays an important role in producing viral particles without any genetic elements. Thus, NNV is a promising candidate for vaccine development and is widely used for constructing vaccines, including DNA, recombinant proteins, and virus-like particles (VLPs). Our study aimed to investigate the potential of NNV capsid protein (NNV) and NNV capsid protein fused to enhanced green fluorescent protein (NNV-EGFP) through VLP formation and whether their application can induce specific antibody responses against certain antigens. We focused on producing DNA and recombinant protein vaccines consisting of the genes for NNV, EGFP, and NNV-EGFP. The approach using NNV-EGFP allowed NNV to act as a carrier or inducer while EGFP was incorporated as part of the capsid protein, thereby enhancing the immune response. In vitro studies demonstrated that all DNA vaccines expressed in HINAE cells resulted in varying protein expression levels, with particularly low levels observed for pNNV and pNNV-EGFP. Consequently, structural proteins derived from HINAE cells could not be observed using transmission electron microscopy (TEM). In contrast, recombinant proteins of NNV and NNV-EGFP were expressed through the Escherichia coli expression system. TEM revealed that rNNV was assembled into VLPs with an approximate size of 30 nm, whereas rNNV-EGFP presented particles ranging from 10 nm to 50 nm in size. For the vaccination test, DNA vaccination marginally induced specific antibody responses in Japanese flounder compared to unvaccinated fish. Meanwhile, NNV and NNV-EGFP recombinant vaccines enhanced a greater anti-NNV antibody response than the others, whereas antibody responses against EGFP were also marginal. These results indicate that NNV capsid protein-based antigens, presenting as particles, play an important role in eliciting a specific anti-NNV antibody response and have the potential to improve fish immune responses.
神经坏死病毒（NNV）衣壳蛋白在产生无任何遗传元件的病毒颗粒方面发挥着重要作用。因此，NNV 是疫苗开发的有前途的候选物，并广泛用于构建疫苗，包括 DNA、重组蛋白和病毒样颗粒（VLPs）。我们的研究旨在研究 NNV 衣壳蛋白（NNV）和与增强型绿色荧光蛋白（NNV-EGFP）融合的 NNV 衣壳蛋白通过 VLP 形成的潜力，以及它们的应用是否可以诱导针对某些抗原的特异性抗体反应。我们专注于生产由 NNV、EGFP 和 NNV-EGFP 基因组成的 DNA 和重组蛋白疫苗。使用 NNV-EGFP 的方法使 NNV 能够充当载体或诱导物，而 EGFP 则作为衣壳蛋白的一部分被掺入，从而增强免疫反应。体外研究表明，在 HINAE 细胞中表达的所有 DNA 疫苗都导致了不同的蛋白表达水平，其中 pNNV 和 pNNV-EGFP 的表达水平特别低。因此，无法使用透射电子显微镜（TEM）观察源自 HINAE 细胞的结构蛋白。相比之下，通过大肠杆菌表达系统表达了 NNV 和 NNV-EGFP 的重组蛋白。TEM 显示 rNNV 被组装成大约 30nm 的 VLPs，而 rNNV-EGFP 呈现出 10nm 至 50nm 大小的颗粒。在疫苗接种测试中，与未接种疫苗的鱼类相比，DNA 疫苗仅在日本牙鲆中诱导了特异性抗体反应。同时，NNV 和 NNV-EGFP 重组疫苗比其他疫苗增强了更大的抗 NNV 抗体反应，而针对 EGFP 的抗体反应也很微弱。这些结果表明，基于 NNV 衣壳蛋白的抗原呈颗粒状，在引发特异性抗 NNV 抗体反应方面发挥着重要作用，并有可能提高鱼类的免疫反应。
