Trace amounts of cobalt serve as an adjuvant for unglycosylated JEV-ED3 expressed in E. coli, inducing a potent virus-neutralizing immune response.

Small viral protein domains expressed in Escherichia coli could serve as a cost-effective alternative for the development of subunit vaccines. However, due to their inherently low immunogenicity, such E. coli-expressed protein domains are rarely used in practice. In this study, we evaluated the ability of five divalent metal ions-Co2+, Cu2+, Mn2+, Ni2+, and Zn2+-to enhance the immunogenicity of an E. coli-expressed Japanese encephalitis virus envelope protein domain III (JEV-ED3; ∼12 kDa). Biophysical analyses using DLS, SLS, CD, and tryptophan fluorescence spectroscopy revealed that trace amounts of metal ions did not alter the conformational integrity of JEV-ED3, which remained natively folded and monomeric in the presence or absence of the tested ions, except for Zn2+. We then assessed the immunogenicity of JEV-ED3 in the presence and absence of these metal ions in a mouse model. Among them, 5 μM Co2+ produced the strongest enhancement, increasing anti-JEV-ED3 IgG titers by more than 15-fold compared to the no‑cobalt control. Furthermore, Co2+ also significantly boosted the anti-JEV antibody response-by over 7-fold-when co-administered with an inactivated JEV vaccine. The cobalt-induced immune response was Th2-biased (elevated IgG1 and IL-4 levels) and long-lasting, as evidenced by increased CD44+CD62L+ memory T cell populations measured by flow cytometry. Importantly, Co2+-induced antibodies exhibited potent live virus-neutralizing activity in the FRNT₅₀ assay. MTT assays confirmed that the metal concentrations used were well below cytotoxic levels. Collectively, these findings suggest that trace amounts of cobalt can markedly enhance the immunogenicity of otherwise weakly immunogenic protein domains, offering a promising strategy for subunit vaccine development.