De novo design of anti-variant COVID-19 vaccine.

Recent studies highlight the effectiveness of hybrid Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) vaccines combining wild-type nucleocapsid and Spike proteins. We have further enhanced this strategy by incorporating delta and omicron variants' spike protein mutations. Both delta and omicron mark the shifts in viral transmissibility and severity in unvaccinated and vaccinated patients. So their mutations are highly crucial for future viral variants also. Omicron is particularly adept at immune evasion by mutating spike epitopes. The rapid adaptations of Omicron and sub-variants to spike-based vaccines and simultaneous transmissibility underline the urgency for new vaccines in the continuous battle against SARS-CoV-2. Therefore, we have added three persistent T-cell-stimulating nucleocapsid peptides similar to homologous sequences from seasonal Human Coronaviruses (HuCoV) and an envelope peptide that elicits a strong T-cell immune response. These peptides are clustered in the hybrid spike's cytoplasmic region with non-immunogenic linkers, enabling systematic arrangement. AlphaFold (Artificial intelligence-based model building) analysis suggests omitting the transmembrane domain enhances these cytoplasmic epitopes' folding efficiency which can ensure persistent immunity for CD4⁺ structural epitopes. Further molecular dynamics simulations validate the compact conformation of the modeled structures and a flexible C-terminus region. Overall, the structures show stability and less conformational fluctuation throughout the simulation. Also, the AlphaFold predicted structural epitopes maintained their folds during simulation to ensure the specificity of CD4⁺ T-cell response after vaccination. Our proposed approach may provide options for incorporating diverse anti-viral T-cell peptides, similar to HuCoV, into linker regions. This versatility can be promising to address outbreaks and challenges posed by various viruses for effective management in this era of innovative vaccines.