Formalin treatment increases the stability and immunogenicity of coxsackievirus B1 VLP vaccine

Minna M Hankaniemi,Virginia M Stone,Tanja Andrejeff,Suvi Heinimäki,Amir-Babak Sioofy-Khojine,Varpu Marjomäki,Heikki Hyöty,Vesna Blazevic,Malin Flodström-Tullberg,Vesa P Hytönen,Olli H Laitinen

doi:10.1016/j.antiviral.2019.104595

Abstract

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.

Highlights

The six Coxsackievirus B serotypes (CVB1-6) are common human pathogens belonging to the family of Enteroviruses (EVs)
CVB1-virus-like particles (VLPs) were produced in High Five insect cells, isolated from the clarified insect cell culture supernatants by Tangential Flow Filtration (TFF) and purified with a combination of subsequent anion and cation exchange chromatographic steps
VLPs were eluted from the SO3 cation exchange chromatography column at a NaCl concentration of 200 mM (Fig. 1a)

Summary

Introduction

The six Coxsackievirus B serotypes (CVB1-6) are common human pathogens belonging to the family of Enteroviruses (EVs). Associations between CVB infections (in particular CVB1) and type 1 diabetes (T1D) have been documented (Laitinen et al, 2014; Sioofy-Khojine et al, 2018). The P2 and P3 regions of the polypeptide contain the non-structural proteins, such as viral protease 3CD, that cleave the P1 region into the structural proteins VP0, VP1 and VP3. These structural proteins assemble into the viral capsid, wherein the viral RNA is encapsidated. VP0 is further processed into VP2 and VP4 in a viral RNA-driven autocatalysis reaction yielding the mature virus (Laitinen et al, 2016). The mature capsid exhibits icosahedral symmetry and consists sixty copies of VP1-4 proteins (Muckelbauer et al, 1995)

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