Linker-mediated single-transcript mRNA vaccine expressing virus-like particles of enterovirus D68

Enterovirus D68 (EV-D68) causes pediatric respiratory illness and is linked to acute flaccid myelitis (AFM), posing a global health threat. We have previously reported that an mRNA vaccine expressing a virus-like particle (VLP) of EV-D68, which co-administers two mRNA-lipid nanoparticles (LNPs) encoding the viral structural protein P1 and viral protease 3CD, conferred protection against infection. Nonetheless, the requirement for two mRNA-LNPs presents challenges in manufacturing and quality control. Here, we investigated the potential of single mRNA constructs encoding both P1 and 3CD, connected by linker sequences. mRNA constructs encoding both P1 and 3CD in a single transcript were designed with linkers such as 2A peptides, 3CD protease cleavage sites (CS), combinations of 2A peptides and CS, or internal ribosome entry sites (IRES). Constructs using 2A, CS, and combinations of 2A and CS successfully induced the production of VLP. In contrast, mRNA with IRES failed due to modified nucleosides. Mice vaccinated with mRNAs that successfully expressed VLP developed neutralizing antibodies against EV-D68. They also demonstrated a protective effect against intranasal challenge, and their serum prevented neonatal mice from developing neurological symptoms caused by EV-D68 infection. These mRNA designs may reduce manufacturing complexity and cost, facilitating the development of effective EV-D68 mRNA vaccines.