The emergence of SARS-CoV-2 in late 2019 prompted rapid development of vaccines targeting the ancestral Wu-1 strain. This proved effective in reducing severe illness and death. However, the emergence of variants with spike protein mutations significantly reduced the effectiveness of existing vaccines and serum neutralization potency in both infected and vaccinated individuals. For instance, neutralization against the B.1.351 (β) variant was substantially reduced compared to the ancestral strain. The continuous evolution of SARS-CoV-2, particularly the emergence of variants from the B.1.1.529 lineage, necessitates ongoing vaccine updates. While mRNA vaccines have adapted to strain updates, the amenability of other platforms remains less understood. This study focuses on a previously developed receptor-binding domain protein nanoparticle vaccine (RBD-NP), which demonstrated strong protection in preclinical trials and met Phase III clinical trial endpoints. This research investigates variant-updated RBD-NP vaccines based on the ancestral (Wu-1), B.1.351 (β), and P.1 (γ) lineages, benchmarked against prefusion-stabilized (HexaPro) spikes. The study aims to evaluate the stability and immunogenicity of these updated vaccines and their potential to address emerging variants, informing strategies for vaccine updates and pandemic preparedness.

The existing literature highlights the significant challenge posed by SARS-CoV-2 variants, with mutations in the spike protein leading to reduced neutralization efficacy of existing vaccines and therapies. Several studies have demonstrated a significant decrease in neutralizing antibody levels against variants like Beta (B.1.351) compared to the ancestral strain. The rapid development of mRNA vaccines and their adaptability to new strains have been documented, showcasing the effectiveness of this platform. However, other platforms, including protein-based vaccines, require further investigation regarding their ability to adapt to emerging variants. The successful development and licensure of the RBD-NP vaccine provided a strong foundation for this study. Prior research on the RBD-NP platform has shown its effectiveness against the ancestral strain, providing a basis for exploring its potential to address subsequent variants.

The study involved the production of RBD-NPs displaying RBDs from Wu-1, B.1.351 (β), P.1 (γ) lineages, with and without stabilizing Rpk9 mutations (Y365F, F392W, V395I). An E484K point mutant RBD-NP was also created. Prefusion-stabilized (HexaPro) spike proteins with the same VOC mutations (excluding E484K) served as benchmarks. Dynamic light scattering (DLS), negative stain electron microscopy (nsEM), biolayer interferometry (BLI), ELISA, thermal melts, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and size-exclusion chromatography (SEC) were used to characterize the biophysical properties, stability, and antigenicity of the immunogens. Immunogenicity was assessed by immunizing BALB/c and Darwin mice with various vaccine candidates: monovalent RBD-NPs, mosaic RBD-NPs (mRBD-NPs) co-displaying multiple RBDs, cocktails of RBD-NPs (cRBD-NPs), and HexaPro trimers. Neutralizing antibody titers were measured against pseudotyped VSV bearing various SARS-CoV-2 spike proteins. Specific techniques included transient transfection of Expi293F cells for protein production, immobilized metal-ion affinity chromatography (IMAC) for purification, and detailed analysis using various biophysical and immunological assays. Statistical analysis included Kruskal-Wallis tests to compare neutralizing antibody titers.

The study found that RBD-NPs displaying wild-type RBDs from VOCs showed poor solution properties, unlike the stable Wu-1-RBD-NP. Introducing Rpk9 stabilizing mutations improved stability in some cases, but additional mutations might be needed. The RBD-NPs and HexaPro trimers elicited comparable neutralizing antibody levels against various VOCs, with Rpk9 mutations generally enhancing neutralization titers, especially against Omicron BA.1. Cocktail and mosaic nanoparticle formulations, combining different VOC RBDs, also showed robust neutralization breadth. Interestingly, in Darwin mice, Wu-1-RBD-NP elicited the most consistent neutralizing antibody levels against both vaccine-matched and -mismatched variants, while cocktail/mosaic formulations were more effective in BALB/c mice. The study also demonstrated that the shelf-life stability of the RBD-NPs varied depending on the variant RBD included and the temperature. In general, the Wu-1 RBD was more stable than other variants, particularly beta. The addition of the Rpk9 stabilizing mutations improved stability across variants. Notably, only the addition of Rpk9 mutations allowed for the production of OBA.4/5 RBD-NP.

The findings demonstrate that the RBD-NP platform can be adapted to elicit neutralizing antibodies against SARS-CoV-2 variants, highlighting its versatility for vaccine development. The differences in stability and solution properties of the variant RBD-NPs underscore the importance of incorporating stabilizing mutations for reliable strain updates. The comparable neutralizing antibody responses elicited by RBD-NP and HexaPro immunogens suggest that the RBD alone can be sufficient for inducing robust protection against various variants. The superior performance of the Wu-1-RBD-NP in Darwin mice and the effectiveness of cocktail/mosaic formulations in BALB/c mice highlight potential strategies for broad protection. The study's limitations emphasize the need for further research into identifying additional portable stabilizing mutations to improve vaccine efficacy and speed of development for future variants.

This study demonstrates the potential of RBD-NP-based vaccines as a platform for rapidly developing broadly protective coronavirus vaccines. While the RBD-NP platform exhibits strong immunogenicity, the varying stability of different variant-based RBD-NPs highlights a need for further optimization via additional stabilizing mutations. The success of cocktail and mosaic nanoparticle designs suggests a potential strategy for generating vaccines with broader protection against multiple variants. Future research should focus on identifying broadly applicable stabilizing mutations and investigating the combined use of protein nanoparticle vaccines with mRNA technology to leverage the strengths of both platforms.

The study primarily used in vitro and mouse models, which may not perfectly reflect human immune responses. The limited number of variants tested and the focus on specific mutations might not fully capture the complexity of SARS-CoV-2 evolution. Further studies involving a broader panel of variants and human clinical trials are necessary to validate the findings and assess long-term effectiveness.