Neutralizing antibodies against the Japanese encephalitis virus are produced by a 12 kDa E. coli- expressed envelope protein domain III (EDIII) tagged with a solubility-controlling peptide

IF 4.5 3区医学 Q2 IMMUNOLOGY

Vaccine Pub Date : 2025-04-22 DOI:10.1016/j.vaccine.2025.127143

Md. Din Islam , M. Monirul Islam , Ayae Inoue , Sanjida Yesmin , Subbaian Brindha , Takahiro Yoshizue , Hiromichi Tsurui , Takeshi Kurosu , Yutaka Kuroda

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引用次数: 0

Abstract

Escherichia coli is a powerful and cost-effective platform for producing recombinant proteins. However, E. coli- produced proteins lack side-chain glycosylation and may be misfolded due to non-native disulfide bonds, often leading to poor immunogenicity. As a result, they are commonly perceived as unsuitable for use as antiviral vaccine antigens. This study addresses this challenge using the small 12 kDa envelope protein domain III of the Japanese encephalitis virus (JEV-EDIII) as a model. We demonstrate that the low immunogenicity of E. coli- produced proteins can be effectively overcome by employing a solubility-controlling peptide tag (SCP-tag) composed of five isoleucines (C5I). E. coli-produced JEV-EDIII oligomerized into 100 nm (Rh) soluble oligomers upon attachment of the C5I-tag, whereas the untagged JEV-EDIII remained monomeric (Rh ∼ 1.9 nm). The C5I-tag significantly enhanced anti-JEV EDIII IgG titers, as evidenced by ELISA, and increased the population of memory T cells in the spleen, as assessed by flow cytometry. Most notably, the C5I-tagged JEV-EDIII elicited neutralizing antibodies, confirmed by the FRNT₅₀ neutralization assay using live JEV. These findings suggest that oligomerization via SCP-tagging offers a promising, adjuvant-free approach for producing neutralizing antibodies with long-term T cell memory, paving the way for developing E. coli- produced, protein domain-based vaccines.

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抗乙型脑炎病毒的中和抗体是由大肠杆菌表达的12 kDa的包膜蛋白结构域III （EDIII）产生的，该结构域III带有可溶性控制肽的标记

大肠杆菌是一种生产重组蛋白的强大而经济的平台。然而，大肠杆菌产生的蛋白质缺乏侧链糖基化，并且可能由于非天然二硫键而错误折叠，通常导致免疫原性差。因此，它们通常被认为不适合用作抗病毒疫苗抗原。本研究利用日本脑炎病毒（JEV-EDIII）的小12 kDa包膜蛋白结构域III作为模型解决了这一挑战。我们证明大肠杆菌产生的蛋白的低免疫原性可以通过使用由五种异亮氨酸（C5I）组成的溶解度控制肽标签（SCP-tag）有效地克服。大肠杆菌产生的JEV-EDIII在附着c5i标签后寡聚成100 nm （Rh）可溶的低聚物，而未标记的JEV-EDIII保持单体（Rh ~ 1.9 nm）。ELISA结果显示，c5i标记显著增强了抗jev EDIII IgG滴度，流式细胞术结果显示，c5i标记增加了脾脏中记忆T细胞的数量。最值得注意的是，c5i标记的JEV- ediii引发了中和抗体，这被使用活JEV的FRNT50中和试验证实。这些发现表明，通过scp标记的寡聚化为产生具有长期T细胞记忆的中和抗体提供了一种有希望的、无佐剂的方法，为开发大肠杆菌生产的基于蛋白质结构域的疫苗铺平了道路。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Vaccine 医学-免疫学

CiteScore

8.70

自引率

5.50%

发文量

992

审稿时长

131 days

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