Type B Coxsackieviruses (CVBs) are a common cause of acute and chronic myocarditis, dilated cardiomyopathy and aseptic meningitis. However, no CVB-vaccines are available for human use. We have previously produced virus-like particles (VLPs) for CVB3 with a baculovirus-insect cell production system. Here we have explored the potential of a VLP-based vaccine targeting CVB1 and describe the production of CVB1-VLPs with a scalable VLP purification method. The developed purification method consisting of tangential flow filtration and ion exchange chromatography is compatible with industrial scale production. CVB1-VLP vaccine was treated with UV-C or formalin to study whether stability and immunogenicity was affected. Untreated, UV treated and formalin treated VLPs remained morphologically intact for 12  months  at 4 °C. Formalin treatment increased, whereas UV treatment decreased the thermostability of the VLP-vaccine. High neutralising and total IgG antibody levels, the latter predominantly of a Th2 type (IgG1) phenotype, were detected in female BALB/c mice immunised with non-adjuvanted, untreated CVB1-VLP vaccine. The immunogenicity of the differently treated CVB1-VLPs (non-adjuvanted) were compared in C57BL/6 J mice and animals vaccinated with formalin treated CVB1-VLPs mounted the strongest neutralising and, CVB1-specific IgG and IgG1 antibody responses. This study demonstrates that formalin treatment increases the stability and immunogenicity of CVB1-VLP vaccine and may offer a universal tool for the stabilisation of VLPs in the production of more efficient vaccines.

Background and aims: Virus-like particles (VLPs) are promising vaccine candidates, usually eliciting broad and strong immune responses in humans. The aim of this study was to produce VLPs for coxsackievirus B1 (CVB1). CVB viruses cause severe morbidity and mortality worldwide, causing a major impact on the health care system, but there are no treatments or vaccines available against CVBs. The specific aims of this study were to find out the most efficient insect cell-line for CVB1-VLP production, and finding a scalable purification method for CVB1-VLP and finally the characterization of CVB1-VLP. Methods: Baculovirus-insect cell expression system was used in the production of CVB1VLPs. Various steps in the CVB1-VLP production were optimized including the type of insect cell line and the flashBAC DNA variant, the Multiplicity Of Infection (MOI) used for CVB1-VLP amplification, baculovirus cultivation time, type of cell growth medium and culture volumes and the CVB1-VLP purification method. VLPs were concentrated either using tangential flow filtration or PEG-precipitation and purified using multi-step ion exchange chromatography (IEX) purification. The quality and the purity of the CVB1VLPs were estimated with SDS-PAGE, Western Blotting, dynamic light scattering and Transmission Electron Microscopy analyzes. Results: The highest CVB1-VLP production level was obtained in High Five cells and they expressed two times more extracellular VLP than Sf9 cells. FlashBAC ULTRA and flashBAC GOLD expressed considerably more extracellular VLP than flashBAC or flashBAC PRIME in High Five cells. Further, the production levels of flashBAC ULTRA and flashBAC GOLD were almost equal. MOI 1 seemed to give the highest production levels. Highest yield was in 50 ml culture and culture volume could not be efficiently scaled up. 67 % pure VLP was produced with ultracentrifugation, while 100 % pure VLP was produced with IEX. Yield for 100 % pure VLP was 0.6 mg/l. Conclusion: Highest VLP yield was achieved with High Five insect cells using 5 days baculovirus infection, 50 ml culture volume, flashBAC GOLD or ULTRA and MOI 1. 100 % pure CVB1-VLP was generated using three step IEX.