Betacoronaviruses, particularly sarbecoviruses that bind to angiotensin-converting enzyme 2 (ACE2), pose a significant pandemic risk. Two such viruses have already caused major outbreaks: SARS-CoV-1 (2002-2003) and SARS-CoV-2 (ongoing). Bats serve as reservoirs for numerous SARS-CoV-like sarbecoviruses, continually threatening zoonotic spillover. Furthermore, the rapid emergence of SARS-CoV-2 variants, such as those with the N501Y mutation increasing ACE2 binding affinity, poses a challenge to vaccine-induced immunity. Mutations in key RBD epitopes, as seen in the Delta (L452R and T478K) and Omicron variants, further complicate the situation. Therefore, next-generation vaccines providing broader protection against ACE2-binding sarbecoviruses and emerging variants are crucial.

Existing strategies for pan-sarbecovirus and pan-betacoronavirus protection include using synthetic mRNAs expressing chimeric spike proteins or mosaic/cocktail nanoparticles expressing RBDs from different coronaviruses. While effective, these methods require multiple gene constructs, increasing complexity and cost. The study emphasizes the need for a single-antigen vaccine capable of overcoming both zoonotic spillover and immune escape mutations.

This study employed DIOSynVax technology to design antigens. Initially, a phylogenetically informed RBD-based antigen (T2_13) was created by comparing known sarbecovirus sequences. This was further optimized (T2_14-T2_18) by incorporating epitopes recognized by monoclonal antibodies (S309, CR3022, and B38) and introducing a glycosylation site to mask divergent epitopes. These designs were evaluated *in vitro* and *in vivo* in BALB/c mice using DNA immunization. The top performer, T2_17, underwent further testing in guinea pigs and rabbits. To mimic the real-world scenario of a pre-existing immune response, T2_17 was used as a heterologous boost (DNA and MVA) in AZD1222-primed K18-hACE2 transgenic mice. Finally, T2_17 (soluble and membrane-anchored forms, T2_17_TM) was tested as an mRNA immunogen in mice and guinea pigs, using chemically modified mRNA in an LNP formulation. Evaluations included FACS assays, ELISA, pseudovirus neutralization assays, ACE-2 competition assays, and peptide microarrays. Challenge studies using the Victoria and Delta variants of SARS-CoV-2 were conducted in K18-hACE2 mice.

In silico design yielded T2_17, a single RBD-based antigen exhibiting superior binding across various sarbecoviruses in BALB/c mice. T2_17 elicited robust binding antibodies against SARS-CoV and SARS-CoV-2 (ELISA). Guinea pig immunization with T2_17 resulted in significant neutralizing antibodies against both SARS-CoV and SARS-CoV-2; these responses were broader than those induced by a control SARS-CoV-2 RBD antigen (SARS2_RBD_P521N). Rabbit sera demonstrated broad neutralization across various SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, Omicron BA.1). Challenge studies in K18-hACE2 transgenic mice primed with AZD1222 and boosted with T2_17 (DNA or MVA) showed protection against both the Victoria and Delta variants, with improved neutralization of the Delta variant observed in the T2_17 boosted groups. A longitudinal study further indicated sustained antibody levels post-boosting. mRNA delivery of T2_17 (particularly the membrane-anchored T2_17_TM) also showed potent immunogenicity, with neutralization against SARS-CoV, SARS-CoV-2, WIV16, and Omicron BA.1. Higher doses of the T2_17_TM mRNA vaccine were also able to neutralize the XBB.1.5 variant. Peptide microarray analysis revealed that T2_17 elicited a broader RBD-specific antibody response compared to AZD1222.

The study successfully demonstrates a single-antigen RBD-based vaccine, computationally designed using DIOSynVax technology, that elicits broad and potent humoral responses against multiple sarbecoviruses, including SARS-CoV-2 variants. The use of a phylogenetically informed design, combined with epitope optimization and glycosylation site introduction, proved highly effective in generating broadly neutralizing antibodies. The successful use of T2_17 as a booster in a pre-existing immune background highlights its potential in a population already exposed to the virus or vaccination. The robust responses observed across various delivery methods (DNA, MVA, mRNA) underscore the antigen's versatility.

T2_17 demonstrates significant potential as a broadly protective sarbecovirus vaccine. Its single-antigen design, effective across various delivery platforms, and ability to overcome immune escape variants offer a promising solution to the ongoing COVID-19 pandemic and future zoonotic threats. Future research should focus on phase I clinical trials to assess its safety and efficacy in humans and further optimization to account for the continually evolving viral landscape.

While T2_17 showed efficacy against several variants, its neutralization capacity against the latest variants might need further improvement. The study primarily focused on humoral immunity; a comprehensive analysis of cellular immunity would be beneficial. The challenge studies used a relatively small number of animals and a limited set of variants, limiting the generalizability of the results. Further investigation into the optimal dosing strategies for different delivery methods is warranted.