Ongoing evolution of SARS-CoV-2 has generated antigenically distinct variants that challenge the breadth of protection conferred by current vaccines, highlighting the need for innovative, versatile antigen display platforms. The receptor-binding domain (RBD) is a key target of neutralizing antibodies but exhibits limited immunogenicity as a soluble antigen. To overcome this limitation, we developed a nodavirus capsid-derived virus-like particle (VLP) platform for multivariant RBD display.
Chimeric CΔ116-MrNVCP-RBD constructs were generated by genetically fusing the SARS-CoV-2 RBD to a protruding domain–truncated Macrobrachium rosenbergii nodavirus capsid protein (CΔ116-MrNVCP). Constructs were expressed in Escherichia coli and self-assembled into uniform spherical VLPs (13–18 nm) as visualized by TEM. ELISA confirmed surface exposure of the RBD, demonstrating preserved antigenicity.
To assess immunogenicity, BALB/c mice (n=112) were immunized intramuscularly on a four-dose regimen with single, bi- or quadri-mixed VLP formulations, with or without the adjuvant AddaVax. RBD-specific IgG titers were detected in all VLP-RBD groups, while flow cytometric profiling of splenocytes revealed activation of CD4+, CD8+ T cells and F4/80+ macrophages, with response magnitudes varying across formulations. Splenocyte restimulation assay confirmed antigen-specific recall responses across variant combinations, with heightened CD69 and CD44 expression. Multiplex cytokine analysis of splenocytes and sera demonstrated balanced Th1/Th2 profiles, indicating strong yet regulated immune activation, particularly in variant-mixed and adjuvanted groups. Multivariant formulations induced broader, more coordinated immune responses than monovalent VLPs, with adjuvanted formulations consistently outperforming their unadjuvanted counterparts, suggesting adjuvant-enhanced immunity. No adverse histopathological effects were detected, confirming the safety and tolerability of all formulations.
Collectively, these results establish CΔ116-MrNVCP VLPs as a novel platform capable of inducing robust humoral and cellular immune responses, supporting their development as next-generation VLP-based COVID-19 vaccines.