Immunoreactivity Analysis of MHC-I Epitopes Derived from the Nucleocapsid Protein of SARS-CoV-2 via Computation and Vaccination

<b>Background</b>: Since 2019, the SARS-CoV-2 virus has been responsible for the global spread of respiratory illness. As of 1 September 2024, the cumulative number of infections worldwide exceeded 776 million. There are many structural proteins of the virus, among which the SARS-CoV-2 nucleocapsid (N) protein plays a pivotal role in the viral life cycle, participating in a multitude of essential activities following viral invasion. An important antiviral immune response is the major histocompatibility complex (MHC)-restricted differentiation cluster 8 (CD8+) T cell cytotoxicity. Therefore, understanding the immunogenicity of SARS-CoV-2 NP-specific MHC-I-restricted epitopes is highly important. <b>Methods</b>: MHC-I molecules from 11 human leukocyte antigen I (HLA-I) superfamilies with 98% population coverage and 6 mouse H2 alleles were selected. The affinity were screened by IEDB, NetMHCpan, SYFPEITHI, SMMPMBEC and Rankpep. Further immunogenicity and conservative analyses were performed using VaxiJen and BLASTp, respectively. EpiDock was used to simulate molecular docking. Cluster analysis was performed. Selective epitopes were validated by enzyme-linked immunospot (ELISpot) assay and flow cytometry in the mice with pVAX-NP_SARS-CoV-2 immunization. Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect whether the preferred epitope induced humoral immunity. <b>Results</b>: There were 64 dominant epitopes for the H-2 haplotype and 238 dominant epitopes for the HLA-I haplotype. Further analysis of immunogenicity and conservation yielded 8 preferred epitopes, and docking simulations were conducted with corresponding MHC-I alleles. The relationships between the NP peptides and MHC-I haplotypes were then determined via two-way hierarchical clustering. ELISA, ELISpot assay, and flow cytometry revealed that the preferred epitope stimulated both humoral and cellular immunity and enhanced cytokine secretion in mice. <b>Conclusions</b>: our study revealed the general patterns among multiple haplotypes within the humans and mice superfamily, providing a comprehensive assessment of the pan-MHC-I immunoreactivity of SARS-CoV-2 NP. Our findings would render prospects for the development and application of epitope-based immunotherapy in lasting viral epidemics.