Background: Enteroviruses, including coxsackievirus B1 (CVB1), cause severe diseases such as myocarditis and meningitis, but vaccines are lacking for most enteroviruses. Conserved and immunodominant epitopes, such as VP4 region and VP1 N-terminus may limit vaccine efficacy by inducing non-neutralizing antibody responses. Virus-like particles (VLPs) mimic native viruses without genetic material and can be engineered to exclude epitopes. To address these challenges, we developed a CVB1-VLP lacking VP4.
Methods: Sequence conservation of CVB VP4 protein and the VP1 N-terminal PALXA region was assessed, and BALB/c mice were sequentially immunized with different formalin inactivated CVB vaccines. VLPΔVP4 was produced using baculovirus-insect cell expression system, was purified, and characterized by SDS-PAGE, transmission electron microscopy, dynamic light scattering, cryogenic electron microscopy, three-dimensional image reconstruction and atomic modelling. VLPΔVP4 stability was monitored over five years at 8 °C. Comprehensive preclinical experiments were conducted in mice with VLPΔVP4, VLPΔpalxa and inactivated CVB1. Vaccine immunogenicity was evaluated by neutralization assay, ELISA, ELISpot, and in vitro infection assays.
Results: VP4- and PALXA-regions were conserved among CVB serotypes and sequential mouse vaccinations confirmed the induction of antibodies against these regions, that should be avoided in vaccination. VLPΔVP4 exhibited > 95% purity, expected morphology (~ 30 nm), exceptional stability at 8 °C for five years, and the atomic modelling to 2.7 Å resolution showed that the particles were entirely in expanded form. Excluding VP4 from VLP improved production yield 3.5-fold, enhancing scalability of production. Immunological assays demonstrated that VLPΔVP4 induced slightly Th2-skewed response, but including adjuvant system 04 (AS04) in the vaccine induced balanced humoral and cellular immune response in mice. Sera from all vaccine groups modulated CVB1 infection, but IFN-α induction was lowest in VLP groups, suggesting reduced risk for antibody dependent enhancement of infection. VLPΔVP4 elicited significantly higher IFN-γ responses compared to other vaccines, indicating robust cellular immune response. Antibody responses were comparable across adjuvanted groups, but inclusion of VP4 in the vaccine correlated with weaker systemic T-cell responses.
Conclusions: VLPΔVP4 represents a promising next-generation CVB vaccine candidate with broad applicability against enteroviruses. Removal of VP4 may mitigate the risk for non-beneficial immune imprinting while enabling high purity, long-term stability, and improved manufacturing efficiency.
Keywords: Cellular immunity; Coxsackievirus B1 (CVB1); Cryogenic electron microscopy (cryoEM); Enterovirus; Immune imprinting; Neutralizing antibodies; Structural vaccinology; Virus-like particle (VLP) vaccine.
© 2026. The Author(s).