The COVID-19 pandemic has been prolonged by the emergence of SARS-CoV-2 variants of concern (VOCs), posing a significant threat to disease control. Five VOCs—Alpha (B.1.1.7), Gamma (P.1), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529)—have garnered attention due to their increased pathogenicity, transmissibility, and reduced sensitivity to existing vaccines and monoclonal antibodies (mAbs). This necessitates the development of new vaccine candidates. The SARS-CoV-2 spike protein is crucial for viral entry, making it a prime target for vaccine development. However, mutations in the spike protein of VOCs, such as D614G, N501Y, K417N/T, and E484K, significantly impact its antigenicity, leading to immune escape. While some vaccines remain effective against certain variants like Alpha, others like Beta and Omicron show marked resistance. This highlights the vulnerability of vaccines based on the wild-type spike protein to emerging variants. Recombinant multivalent nanoparticle protein vaccines offer a promising platform, inducing robust and durable humoral immunity compared to monomeric antigens. Previous research has explored the use of the SpyTag/SpyCatcher system and various nanoparticle platforms (Ferritin, E2p, 13-01, and two-component 153-50) to enhance SARS-CoV-2 vaccine immunogenicity. This study focuses on designing a quadrivalent mosaic nanoparticle vaccine incorporating spike proteins from the prototype and three major VOCs to assess its effectiveness against various SARS-CoV-2 variants.

Existing literature highlights the challenges posed by emerging SARS-CoV-2 variants. Studies demonstrate reduced neutralization activity of vaccine-induced or convalescent sera against VOCs, especially Beta and Omicron, underscoring the need for updated vaccine strategies. Several studies explored the use of multivalent nanoparticle vaccines to address this. For example, research using an mRNA vaccine based on the Beta variant spike protein showed increased neutralizing antibody titers compared to the wild-type spike in non-human primates, suggesting the potential of variant-specific vaccines. However, systematic investigation of spike protein variants as a generalized vaccine development strategy remains limited. Studies on recombinant multivalent nanoparticle protein vaccines show their potential to induce robust and long-lasting immunity against SARS-CoV-2 by presenting multiple antigens simultaneously.

This study involved the design, characterization, and evaluation of a quadrivalent mosaic SARS-CoV-2 nanoparticle vaccine. The researchers used computational and structure-guided design to genetically fuse prefusion-stabilized HexaPro spike proteins from the SARS-CoV-2 prototype and three VOCs (Alpha, Beta, and Gamma) to the N-terminus of trimeric 153-50A. The HexaPro construct, known for its increased yield and stability while maintaining native antigenicity, was selected. The resulting HexaPro-153-50A protein was expressed in Expi293FTM cells and purified. A quadrivalent mosaic immunogen (Mosaic NP) was created by in vitro co-assembly of the four HexaPro-153-50A components with pentameric 153-50B.4PT1. Individual HexaPro-based nanoparticles for each variant (WT NP, Alpha NP, Beta NP, Gamma NP) and a cocktail immunogen (Cocktail NP; a mixture of the individual nanoparticles) were also generated. Negative-stain electron microscopy, size exclusion chromatography (SEC), dynamic light scattering, and ELISA were used to characterize the nanoparticles. The binding affinity to the human ACE2 receptor and protein thermostability were assessed. Immunogenicity was evaluated in BALB/c mice and cynomolgus macaques using ELISA, pseudovirus neutralization assays, and authentic virus neutralization assays. Protective efficacy was tested in mice challenged with SARS-CoV-2 B.1.351 and prototype strains. The study also included an analysis of the neutralization breadth against Omicron, Lambda, and other variants with single or combinatorial RBD mutations.

The study demonstrated the successful design and characterization of the quadrivalent mosaic nanoparticle vaccine (Mosaic NP). Negative-stain electron microscopy confirmed the icosahedral architecture of the nanoparticles with surface-displayed HexaPro. Immunoprecipitation analysis using the S2-E12 antibody verified the antigenicity of the nanoparticles. In mice, the Mosaic NP elicited superior binding and neutralizing antibody responses against multiple SARS-CoV-2 variants (including Beta, Gamma, Delta, and Eta) compared to the wild-type HexaPro alone or WT NP. The Mosaic NP also showed equivalent or superior neutralizing activity to Cocktail NP and WT NP. In cynomolgus macaques, the Mosaic NP elicited higher binding antibody titers against multiple variants and comparable or slightly superior neutralizing antibody titers than WT NP. Notably, the Mosaic NP-induced serum showed only a small reduction in neutralization potency against Omicron and Lambda variants compared to the wild type. Importantly, mice immunized with Mosaic NP showed complete protection against SARS-CoV-2 B.1.351 variant-induced body weight loss and lung viral burden reduction. The Mosaic NP also protected mice against the SARS-CoV-2 prototype strain.

The results demonstrate that the quadrivalent mosaic nanoparticle vaccine effectively elicits potent and broad neutralizing antibody responses against multiple SARS-CoV-2 variants, including those not directly included in the vaccine design (Delta and Eta). The slight but consistent advantage of the mosaic NP over the Cocktail NP in eliciting antibodies against multiple variants suggests the benefit of presenting different spike proteins in a mosaic arrangement, potentially enabling more efficient stimulation of B cells. This is in line with the hypothesis that a mosaic arrangement might enhance the breadth and potency of the humoral immune response compared to a simple mixture of monovalent nanoparticles. The study highlights the potential of this approach for developing multivalent vaccines targeting multiple SARS-CoV-2 variants to address the challenge of viral immune escape.

This study provides strong evidence supporting the development of multivalent mosaic nanoparticle vaccines as an effective strategy against SARS-CoV-2 variants. The quadrivalent Mosaic NP demonstrated robust immunogenicity and protection in both mouse and non-human primate models. The data support further investigation of this vaccine candidate in larger animal models and clinical trials to evaluate its efficacy and safety in humans. Future research could explore the inclusion of additional variants and the optimization of the vaccine formulation to further enhance its protective capabilities.

While the study showed promising results, some limitations should be acknowledged. The relatively small sample sizes in the animal studies limit the statistical power of certain comparisons. Further studies with larger animal cohorts are needed to confirm the observed findings. Additionally, long-term efficacy and durability of the immune response need to be assessed in future studies. The study's focus on neutralizing antibodies doesn't fully capture the complexity of the immune response to SARS-CoV-2. Future studies should also analyze the cellular immune response elicited by this vaccine.