Single mRNA-based Enterovirus D68 Virus-Like Particle Vaccine Overcomes Manufacturing Hurdles and Prevents Neurological Complications
Background
Enterovirus D68 (EV-D68) is a major pathogen causing severe respiratory disease in children. This virus is closely linked to Acute Flaccid Myelitis (AFM), which involves limb paralysis, threatening global public health. Despite causing permanent neurological paralysis similar to polio, there are currently no approved preventive vaccines or treatments, making urgent countermeasures necessary.
The academic community has focused on prevention strategies based on virus-like particles (VLPs). VLPs are safe because they lack genetic material but induce strong immune responses due to their structural similarity to actual viruses. Previous research developed mRNA technology to express the viral capsid precursor structural protein P1 and the protease 3CD, which induces assembly by cleaving P1 into individual proteins. At the time, the method involved administering a mixture of two types of mRNA-Lipid Nanoparticles (LNPs) containing P1 and 3CD separately.
However, the process of independently synthesizing, encapsulating in LNPs, and mixing two types of mRNA was a major obstacle to mass production. This is because the quality control (QC) process, which was required to maintain a constant mixing ratio of the two preparations and guarantee the stability and encapsulation efficiency of each, was excessively demanding. For clinical development and commercialization, a single mRNA design strategy to reduce manufacturing costs and simplify the process has emerged as an urgent challenge.
Key Findings
Researchers overcame manufacturing difficulties by designing a next-generation mRNA structure that expresses both P1 and 3CD as a single transcript. The core challenge was how to connect the two genes within a single mRNA strand and separate them into independent proteins. The team analyzed four designs: 2A peptide, 3CD protease cleavage site (CS), a combination linker of 2A and CS, and an Internal Ribosome Entry Site (IRES).
Experimental results demonstrated that three single mRNA constructs incorporating the 2A peptide, CS, and the 2A-CS composite linker successfully underwent intended cleavage reactions within cells to form VLPs. This indicates that proteins were appropriately separated during the single ribosomal translation process and self-assembled into complete viral envelope structures. On the other hand, the mRNA construct containing an IRES failed to express VLP. This was due to modified nucleosides added to enhance innate immune evasion and intracellular stability of the mRNA vaccine. The modified bases interfered with the formation of the complex secondary structure of the IRES, leading to a sharp decline in ribosome binding and translation efficiency.
When the mRNA formulation that successfully expressed VLPs was injected into mice, high titers of neutralizing antibodies that neutralize EV-D68 were induced. In challenge experiments involving direct intranasal virus injection, it effectively prevented respiratory infection. The vaccine's efficacy was also proven in suppressing neurological diseases. When serum from vaccinated mice was administered to neonatal mice exposed to the virus, the fatal neurological paralysis symptoms caused by EV-D68 infection were completely suppressed. This demonstrated that preventing infant AFM is possible through maternal immunity or antibody transfer alone.
Significance and Outlook
This achievement provides a turning point for significantly reducing LNP manufacturing complexity and production costs by integrating two mRNAs into one. The single-transcript approach is highly advantageous for scaling up mass production facilities as it unifies production lines and minimizes batch-to-batch quality variance. In particular, it holds high value as a platform technology by demonstrating that 2A peptides or enzyme cleavage linkers, rather than IRES, are the optimal solution for modern mRNA synthesis processes using modified nucleosides.
However, several prerequisites remain for clinical application. It must be verified whether the immunogenicity and infection-protective efficacy observed in mouse models are maintained to the same extent in non-human primates or humans. Close monitoring is also required to determine whether the cleavage efficiency of P1 and 3CD expressed by the single mRNA remains consistent during long-term storage or repeated administration. Toxicity assessments to ensure that residual linker sequences do not induce unintended non-specific immune responses in the human body will also be a critical milestone for entering clinical trials.
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.
This research provides the technological foundation to accelerate the commercialization of vaccines to prevent acute flaccid myelitis (AFM), which causes permanent disabilities in infants and young children. By transitioning from the existing method of using two types of mRNA-LNPs to a single mRNA-LNP formulation, manufacturing costs can be reduced by over 40% and production yields can be dramatically increased. Vaccine manufacturers will be able to mass-produce finished products using a single drug substance production line without the complex mixing process of two different substances.
During infectious disease outbreaks, rapid vaccine supply and distribution to low-income countries are key to health security. A single-formulation vaccine with lower costs due to simplified processes can directly contribute to establishing a global vaccine distribution network. It is considered a strong candidate for a vaccine platform capable of enabling rapid emergency approval and mass distribution during pandemic crises involving the simultaneous spread of seasonal respiratory viruses and pediatric paralysis.