The escalating incidence of emerging infectious diseases, largely due to zoonotic spillover, necessitates the development of effective vaccines. Coronaviruses, such as SARS-CoV, MERS-CoV, and SARS-CoV-2, pose significant global health threats. Neutralizing antibodies (nAbs) targeting the receptor-binding domain (RBD) of the viral spike protein are crucial for protection. While monomeric, dimeric, and trimeric RBD vaccines elicit potent antibody responses, recent variants have demonstrated immune evasion. Multivalent RBD nanoparticles show promise, but existing platforms, relying on stochastic assembly, suffer from drawbacks in antigen distribution uniformity and mass production. This study aims to overcome these limitations using a self-assembling heterotrimeric PCNA scaffold to create a mosaic nanoparticle vaccine with a uniform distribution of six heterologous RBDs, targeting both α- and β-coronaviruses, to elicit broad and potent immune responses.

Existing literature highlights the importance of RBD-specific antibodies in protection against COVID-19 and other coronavirus infections. Multivalent RBD nanoparticles have shown great potential in inducing potent antibody responses. However, challenges remain in creating mosaic nanoparticles with uniform antigen distribution, particularly when incorporating multiple heterologous antigens. Current methods, such as SpyTag/SpyCatcher or sortase-mediated ligation, often result in stochastic assembly, leading to variability in antigen presentation and reduced efficacy. The use of designed protein scaffolds offers an alternative but still faces challenges in scalability and precise antigen placement.

The researchers designed a mosaic nanoparticle vaccine, 6RBD-np, using a heterotrimeric PCNA from *Saccharolobus solfataricus* as a scaffold. Six RBD-SD1s (including the SD1 domain to stabilize the RBD in the down conformation) from SARS-CoV-2 (wild type and variant), SARS-CoV, MERS-CoV, hCoV-HKU1, and hCoV-229E were genetically fused to the N and C termini of the three PCNA subunits. The fusion proteins (S-PCNA1, M-PCNA2, H-PCNA3) were sequentially assembled in vitro to form the 6RBD-np. The nanoparticle's structure and antigen distribution were characterized using SDS-PAGE, Western blot, SEC-MALS, AFM, and TEM. Binding affinity to hACE2 was measured using biolayer interferometry. BALB/c mice were immunized with 6RBD-np, and the resulting antibody responses were evaluated by ELISA and pseudovirus neutralization assays. hACE2 transgenic mice were challenged with SARS-CoV-2 WT and Delta variants to assess protective efficacy. Viral titers, body weight changes, and histopathological analysis were performed to evaluate the effectiveness of the vaccine. Neutralization assays against authentic viruses (WT, Delta, Omicron) and T cell responses (IFN-γ ELISpot assay) were also conducted.

The 6RBD-np was successfully assembled in a defined order, resulting in a uniform distribution of six heterologous RBDs on the PCNA scaffold. SEC-MALS analysis revealed a molecular weight of ~355.7 kDa, and AFM/TEM imaging confirmed a ring-shaped structure with six protruding RBDs (~40 nm in size). The 6RBD-np showed high binding affinity to hACE2. Immunization in BALB/c mice elicited high antibody titers against all six RBD antigens. In hACE2 transgenic mice, 6RBD-np conferred complete protection against both SARS-CoV-2 WT and Delta variant challenges (100% survival rate), unlike other vaccine groups. The 6RBD-np group also demonstrated significantly lower viral titers in lung and brain tissues, reduced histopathological damage, and high neutralizing antibody titers against authentic viruses (WT, Delta, Omicron). Furthermore, 6RBD-np induced robust IFN-γ-producing T cell responses.

The 6RBD-np vaccine demonstrates significant advantages over existing mosaic nanoparticle platforms. Its defined, self-assembling nature ensures uniform antigen presentation, enhancing immunogenicity and broad protection against various coronavirus strains. The complete protection observed in hACE2 transgenic mice against both WT and Delta variants underscores the vaccine's high efficacy. The induction of both strong humoral and cellular immune responses suggests a multi-pronged approach to combating coronavirus infection. The broad cross-reactivity observed, even against α-coronaviruses (hCoV 229E and NL63) not included in the nanoparticle construction, suggests potential for pan-coronavirus immunity. The size of the nanoparticle (~40 nm) falls within the optimal range for efficient immune response elicitation.

This study successfully developed and characterized a novel self-assembling mosaic nanoparticle vaccine, 6RBD-np, for broad coronavirus immunity. The vaccine's uniform antigen presentation, high immunogenicity, and complete protection against SARS-CoV-2 variants in preclinical models highlight its potential as a powerful tool in combating current and future coronavirus threats. Further research should focus on clinical trials to assess its safety and efficacy in humans and explore its adaptability to other emerging viral threats.

The study was conducted in mice, and the findings may not directly translate to human responses. Long-term immunity and the effectiveness against future emerging variants require further investigation. The large-scale production and cost-effectiveness of the 6RBD-np platform also require further optimization for broader applicability.