Abstract
BACKGROUND: Enteroviruses, including Coxsackie B (CVB) viruses, can cause severe diseases such as myocarditis, pancreatitis, and meningitis. Vaccines can prevent these complications, but conserved non-neutralizing epitopes in the viral capsid may limit their effectiveness. The immunodominant PALXAXETG motif, located in the VP1 N-terminus, is a highly conserved region in enteroviruses that elicits non-neutralizing antibody responses. Virus-like particles (VLPs) offer a safe and effective vaccine platform because of their structural similarity to native viruses but lack viral genetic material. Importantly, VLPs can be structurally modified to exclude specific epitopes. METHODS: Here, we produced a modified CVB1 virus-like particle (VLP) vaccine lacking 15 amino acids from the PALXAXETG motif (designated VLPΔpalxa) using the baculovirus-insect cell expression system. To confirm the structural integrity, we determined the crystal structure of the modified VLP with 3.2 Å resolution. We then conducted comprehensive immunogenicity studies in mice, including dose titration, comparison of two versus three immunizations, and post-vaccination viral challenge. In addition, we evaluated the impact of the AS04 adjuvant on the immunogenicity of unmodified and modified CVB1-VLP vaccines and the formalin-inactivated CVB1 vaccine. RESULTS: The yield of CVB1-VLPΔpalxa was 29.5 mg/L, and the particles were shown to assemble similarly to unmodified CVB1-VLP. CVB1-VLPΔpalxa induced robust antibody responses, with neutralizing antibody titres comparable to or exceeding those elicited by unmodified VLP or inactivated virus vaccines. A 2 µg dose was identified as optimal, providing the highest neutralizing antibody titres. A third immunization significantly increased antibody levels, and all non-adjuvanted vaccines protected the mice from CVB1 challenge after the third dose. The addition of AS04 significantly enhanced the antibody response, particularly in both VLP groups. CONCLUSIONS: We demonstrated that with targeted structural modification of the CVB1-VLP capsid, immunodominant antibody responses against the conserved PALXAXETG motif can be avoided. We demonstrate that structural modification of CVB1-VLP is a viable strategy. Since the deleted epitope is known to be non-neutralizing, its deletion may help focus the immune response on more protective targets and thereby improve vaccine efficacy. The modified VLPs, particularly when adjuvanted, offer a promising approach for developing safe and effective enterovirus vaccines.