Coxsackie B1 virus-like particle that lacks VP4 protein demonstrates improved vaccine scalability, stability and immunogenicity

Abstract 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. Graphical Abstract