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.

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.