The ongoing evolution of SARS-CoV-2, driven by mutations and cross-species transmission, necessitates continuous vaccine updates. Since late 2019, the virus has accumulated mutations leading to variants of concern (VOCs) like Alpha, Beta, Gamma, Delta, and Omicron, which exhibit varying degrees of immune escape from pre-existing immunity acquired through vaccination or natural infection. The spike protein's evolution is primarily responsible for this immune evasion, significantly impacting viral transmission and host tropism. First-generation vaccines based on the Wuhan-Hu-1 strain show declining neutralizing activity against subsequent VOCs, particularly Delta and Omicron, highlighting the need for updated vaccines. While bivalent vaccines, incorporating both the original Wuhan-Hu-1 strain and Omicron lineages, were initially introduced, they proved less effective than single-antigen BA.1/BA.5 booster vaccines, likely due to immune imprinting. The rapid evolution of SARS-CoV-2, especially the expansion of the Omicron lineage with diverse subvariants dominating different regions, makes adapting vaccines to a single strain suboptimal. This situation mirrors the annual influenza vaccine challenge, where wild-type strain selection leads to variable vaccine efficacy. This study hypothesizes that vaccine candidates expressing diverse epitopes would be more effective than using single or bivalent wild-type strains. Three computationally designed spike antigens—one pre-Delta (T2_32) and two post-Gamma (T2_35 and T2_36)—were developed and evaluated in small animal models to assess their immunogenicity and breadth of neutralization.

The emergence of SARS-CoV-2 variants with immune evasion capabilities has been extensively documented. Studies have shown the declining efficacy of initial vaccines against newer variants, necessitating the development of updated vaccines. Research on the impact of mutations in the spike protein, particularly in the RBD and NTD regions, on immune escape and viral transmission has been crucial in informing vaccine design. The use of bivalent vaccines and the issue of immune imprinting have also been subjects of recent studies, revealing the limitations of current approaches. Existing literature underscores the need for a proactive strategy to combat the continuous emergence of vaccine-evasive variants, highlighting the importance of computationally designed antigens that incorporate multiple epitopes from various VOCs.

The study employed a computational antigen design approach to create three novel spike-based antigens. For T2_32 (pre-Delta), mutations from Alpha, Beta, and Gamma variants in immunodominant regions (identified using IEDB database and literature) were mapped onto the Wuhan-Hu-1 spike protein scaffold. Stabilizing mutations (K986P, V987P, Q498R) and a 19-amino-acid C-terminal deletion (dER) were introduced to enhance expression and stability. T2_35 and T2_36 (post-Gamma) were designed to address Omicron's emergence, combining Delta and Omicron BA.1 mutations in different spike regions. T2_35 incorporated Delta mutations in NTD/S2 and Omicron BA.1 mutations in RBD/S1-CTD, while T2_36 used the opposite combination. Additional stabilizing mutations, Furin cleavage site modification (682RRAR to GSAS), and mutations Q677H and I834V (observed in circulating variants) were included in T2_35 and T2_36. A Furin cleavage site modified version of T2_32 (T2_32_mFur) was also generated. Immunogenicity was assessed in two animal models: guinea pigs (T2_32 vs. Wuhan-Hu-1, DNA prime-MVA boost) and mice (T2_32_mFur, T2_35, T2_36 vs. Wuhan-Hu-1, Omicron BA.1, mRNA immunization). Neutralizing antibody titers against various VOCs were measured using a pseudotype-based microneutralization assay. Statistical analyses (Mann-Whitney U tests) were performed to compare neutralizing antibody responses between different antigens.

In guinea pigs, T2_32 showed superior neutralizing activity against VOCs compared to Wuhan-Hu-1 after a DNA prime-MVA boost regimen. Heterologous boosting with MVA-T2_32 further enhanced neutralizing responses. In mice, T2_32_mFur, T2_35, and T2_36 exhibited broader neutralization compared to Omicron BA.1, particularly against pre-Omicron and post-Omicron variants. T2_35 showed superior neutralization against post-Omicron variants, while T2_36 demonstrated higher activity against pre-Omicron VOCs. T2_32_mFur exhibited comparable neutralization to BA.1, and superior neutralization to Wuhan-Hu-1 against many Omicron sublineages. Although neutralizing titers were observed against numerous VOCs, titers against more recent variants like BQ.1.1 and XBB.1.5 were lower than those against Delta or BA.2, potentially due to increasing divergence in mutations. Overall, the computationally designed spike antigens demonstrated broader and more robust neutralization than wild-type spike antigens across a range of SARS-CoV-2 variants spanning over two years of viral evolution.

The results support the development of computationally designed, multi-epitope spike antigens as a superior strategy for COVID-19 booster vaccines compared to the current practice of using wild-type spike antigens from past variants. The superior breadth of neutralization observed for T2_32, T2_35, and T2_36, even against variants emerging long after their design, highlights the potential of this proactive approach. The data validate the hypothesis that incorporating multiple epitopes from diverse VOCs leads to broader immune responses, effectively countering immune escape. The study provides strong evidence for using such designed antigens, especially for boosting individuals previously immunized with first-generation vaccines. The observed differences in neutralization profiles between T2_35 and T2_36 against pre- and post-Omicron variants also underscore the role of specific epitopes and mutations in determining immune responses. This approach offers a potential solution to the continuous challenge posed by evolving SARS-CoV-2 variants.

This study demonstrates the superior efficacy of computationally designed, multi-epitope spike antigens for COVID-19 vaccines compared to wild-type strain-based approaches. The designed antigens (T2_32, T2_35, and T2_36) showed broader neutralization against a wide range of SARS-CoV-2 variants, including those emerging long after their initial design. This proactive strategy offers a significant advantage over reactive vaccine updates based on currently circulating strains. Future research should focus on further optimizing these designs, evaluating their efficacy in larger animal models, and conducting clinical trials to assess their performance in human populations. This approach has the potential to provide more durable and comprehensive protection against future SARS-CoV-2 variants.

The study used small animal models (guinea pigs and mice), which may not fully replicate the immune responses in humans. The number of animals in each experimental group was relatively small, limiting the statistical power of the analyses. The study primarily focused on neutralization capacity; other aspects of immune response (e.g., T cell responses) were not extensively evaluated. The observed decrease in neutralization titers against some of the latest VOCs suggests the need for ongoing adaptation and refinement of these vaccine designs to accommodate highly divergent variants. While this study proves the concept, further research is needed to ensure that efficacy will continue to be high against many new variants as well as across diverse populations.