A New Inactivated Coxsackievirus B2 Vaccine: Biological Properties, Immunogenicity, and Protective Effects in Mice

Background: Coxsackievirus B2 (CVB2) causes a range of diseases, including hand, foot, and mouth disease; myocarditis; acute flaccid paralysis; meningitis; and encephalitis. However, no specific antiviral drugs or vaccines are currently available for CVB2. Methods: We used plaque purification, virus titre determination, and serial passaging to screen and identify an inactivated CVB2 vaccine candidate strain, KM31-C05, which exhibited high viral titres and good genetic stability. Comprehensive biological characterization of this candidate strain was performed, including phylogenetic analysis, virulence assessment in BALB/c mice, one-step growth curve analysis, optimization of the multiplicity of infection, as well as determination of viral load, pathological evaluation, and immunohistochemical analysis in tissues of BALB/c suckling mice post-challenge. An experimental inactivated vaccine was prepared using KM31-C05 to evaluate its immunogenicity and protective efficacy. Results: The viral titres of KM31-C05 reached 10⁸ CCID50/mL. After 20 serial passages, only three amino acid mutations were identified (VP3-G165V, VP1-N84K, and VP1-D129N). Although the two VP1 mutations were located in surface-exposed loops, the strain maintained high neutralizing titres across passages, indicating good genetic stability. However, whether these sites affect virulence and replication requires further investigation. Phylogenetic analysis revealed that this strain belonged to genotype C, which is consistent with the strains circulating in mainland China in recent years. The experimental inactivated vaccine prepared from KM31-C05 induced effective neutralizing antibodies (1:128-1:256) in BALB/c mice and provided complete protection to suckling mice against lethal challenge with this CVB2 strain in maternal antibody protection experiments. Conclusions: KM31-C05 demonstrates potential as a CVB2 vaccine candidate in China and provides a theoretical basis for the development of a CVB2 vaccine.