The COVID-19 pandemic underscored the urgent need for rapid and efficient vaccine development. Current vaccine strategies, such as mRNA, DNA plasmid, and protein-based vaccines, have shown varying degrees of efficacy. While mRNA vaccines have demonstrated high efficacy, other approaches are still under development or have limitations. Nanoparticle (NP)-based vaccines have emerged as a promising alternative due to their potential for safe, efficient, and scalable antigen delivery. However, existing NP platforms offer limited control over antigen presentation parameters, hindering rational vaccine design. This study leverages the precise patterning capabilities of DNA origami nanoparticles to address this limitation. The researchers hypothesized that precisely patterning multiple copies of the SARS-CoV-2 RBD trimer on DNA-NPs, along with adjuvants, would lead to a robust and protective immune response. The study's importance lies in its potential to contribute to the development of safer and more effective vaccines against infectious diseases, particularly given the potential for rapid adaptation and emergence of new viral variants. This approach could be pivotal in creating vaccines offering long-lasting immunity and protection against diverse viral strains.

The literature review covers existing SARS-CoV-2 vaccine strategies, highlighting the successes of mRNA vaccines and the ongoing development of other approaches like viral-vector and protein nanoparticle vaccines. It also discusses the importance of the RBD of the spike protein as a potent immunogen and its role in viral entry. The review notes that multivalent display of RBDs is crucial for inducing strong immune responses compared to monomeric RBDs. However, limitations in controlling antigen presentation parameters on existing NP platforms are emphasized, leading to the proposal of DNA origami as a superior platform for precise antigen presentation and modulation of immune cell activation.

The researchers designed a DNA pentagonal bipyramid (PB) nanoparticle using the DAE-DALUS software. The PB was assembled using a long single-stranded DNA scaffold and staple strands. The PB's two surfaces were modified with ssDNA overhangs to facilitate the attachment of the reconstituted RBD trimer (prepared via Protein G-Fc conjugation) and CpG adjuvants. The successful folding and modification of the DNA-NPs were validated using agarose gel electrophoresis, atomic force microscopy (AFM), and dynamic light scattering (DLS). The stoichiometry of PG and RBD on the DNA-NP was quantified via fluorescence measurements. The stability of the DNA-NPs was evaluated using a FRET-based assay. The binding affinity of the RBD-presenting DNA-NPs to the ACE2 receptor was determined using surface plasmon resonance (SPR). BALB/c mice were immunized with different DNA-NP vaccine constructs (including varying concentrations of RBD and the inclusion of CpG adjuvants), and antibody responses were evaluated using ELISA and virus neutralization assays. Finally, in vivo protection against SARS-CoV-2 challenge was assessed in K18-ACE2 mice, monitoring survival rates and weight changes. Detailed protocols were provided for DNA origami scaffold production, nanoparticle folding, protein conjugation, and various characterization techniques including AFM, DLS, fluorescence spectroscopy, FRET, SPR, ELISA, and plaque reduction neutralization assays. Specific reagents, cell lines, and animal models used were also detailed.

The researchers successfully designed and constructed DNA-NPs displaying ten copies of the SARS-CoV-2 RBD trimer and CpG adjuvants. Characterization showed a high folding yield (96%), efficient protein conjugation, and stability of the DNA-NPs in simulated physiological conditions. SPR studies indicated high binding affinity of the RBD-presenting DNA-NPs to the ACE2 receptor. Immunization of mice with the DNA-NPs resulted in high antibody titers. Importantly, mice immunized with the DNA-NP vaccine constructs exhibited significant protection against SARS-CoV-2 challenge, with the 1 µg RBD-PB-CpG group showing no mortality or weight loss. The protective immunity was durable, with high antibody titers observed even two months post-immunization. These findings suggest that the spatial organization and concentration of antigens are crucial for maximizing the cellular response. Specific data points included a 96% folding yield for the DNA-NPs, ~100% PG coverage and ~74% RBD coverage on the NPs, a Kd of 1.35 nM for the RBD-trimer-DNA-NP binding to ACE2 (compared to 4.47 nM for the free RBD monomer), and complete survival in the 1 µg RBD-PB-CpG immunization group after viral challenge.

The findings demonstrate the efficacy of DNA origami as a platform for developing safe and effective SARS-CoV-2 vaccines. The precise control over antigen presentation afforded by DNA origami contributes to the high immunogenicity observed. The inclusion of CpG adjuvants further enhances the immune response. The significant protection against viral challenge highlights the potential of this approach for real-world application. The results also emphasize the importance of optimizing antigen organization and dose for maximizing vaccine efficacy, which could translate to cost-effectiveness in vaccine production. The study's success provides a strong foundation for exploring DNA-NPs as next-generation vaccine platforms.

This study successfully demonstrates the potential of DNA origami nanoparticles as a novel vaccine platform for SARS-CoV-2. The precise control over antigen presentation and the inclusion of CpG adjuvants resulted in a robust protective immune response in mice. This strategy offers advantages in safety, efficacy, and scalability, paving the way for future vaccine development targeting various infectious diseases. Future research could focus on scaling up DNA-NP production and exploring the use of this platform for multivalent vaccines targeting multiple viral strains or other pathogens.

The study was conducted in a mouse model, and the results may not be directly translatable to humans. Further research is needed to evaluate the safety and efficacy of this DNA-NP vaccine in larger animal models and ultimately, in human clinical trials. The current methodology for large-scale production of ssDNA scaffolds requires optimization to reduce costs and improve feasibility.