Immunized mice naturally process in silico-derived peptides from the nucleocapsid of SARS-CoV-2

Abstract Background The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an excellent immunogen that promotes the production of high-titer antibodies. N protein-derived peptides identified using a bioinformatics approach can potentially be used to develop a new generation of vaccines or diagnostic methods for detecting SARS-CoV-2 and its variants. However, further studies must demonstrate their capacity to be naturally processed by the immune system. Objective We aimed to examine the in vivo processing and recognition of in silico-identified peptides using the serum of immunized animals with the complete protein. Methods Recombinant N (Nrec) protein was subcutaneously administered to six Balb/c mice. Enzyme-linked immunosorbent assay (ELISA), western blotting, dot blotting, and immunoprecipitation were performed to evaluate the recognition of the complete protein and in silico-derived peptides. Results The serum of immunized mice recognized ~ 62.5 ng/µL of Nrec with high specificity to linear and conformational epitopes. Dot blot analysis showed that peptides Npep2 and Npep3 were the most reactive. Conclusion Our data confirm the high immunogenicity of the SARS-CoV-2 N protein and provide evidence on the antigenicity of two peptides located in the N-arm/RNA-binding domain (Npep2) and oligomerization domain/C-tail (Npep3), considered the biologically active site of the N protein.