Assessment of Immunogenic and Antigenic Properties of Recombinant Nucleocapsid Proteins of Five SARS-CoV-2 Variants in a Mouse Model

Abstract

COVID-19 cases caused by new variants of highly mutable SARS-CoV-2 continue to be identified worldwide. Effective control of the spread of new variants can be achieved through targeting of conserved viral epitopes. In this regard, the SARS-CoV-2 nucleocapsid (N) protein, which is much more conserved than the evolutionarily influenced spike protein (S), is a suitable antigen. The recombinant N protein can be considered not only as a screening antigen but also as a basis for the development of next-generation COVID-19 vaccines, but little is known about induction of antibodies against the N protein via different SARS-CoV-2 variants. In addition, it is important to understand how antibodies produced against the antigen of one variant can react with the N proteins of other variants. Here, we used recombinant N proteins from five SARS-CoV-2 strains to investigate their immunogenicity and antigenicity in a mouse model and to obtain and characterize a panel of hybridoma-derived monoclonal anti-N antibodies. We also analyzed the variable epitopes of the N protein that are potentially involved in differential recognition of antiviral antibodies. These results will further deepen our knowledge of the cross-reactivity of the humoral immune response in COVID-19.

Overview of Nucleocapsid-Targeting Vaccines against COVID-19

“…For example, it has been demonstrated that anti-N serology tests based on the ancestral antigen are still able to detect antibody responses to recent SARS-CoV-2 variants [ 18 ]. However, studies have revealed the impact of rare mutations on N antigenicity and immunogenicity [ 31 ], which may potentially alter the performance of N-based test systems and vaccine candidates; so, N protein variability should be taken into account as a cause of potential antigenic mismatch between the epitope composition of an N-based vaccine and the circulating SARS-CoV-2 strain. This limitation is fair considering that a single amino acid substitution in the T-cell epitope is sufficient to affect the peptide immunogenicity [ 101 ].…”

“… Reported mutations in the N protein of SARS-CoV-2 variants. ( a ) The most frequent mutations according to [ 16 , 31 , 77 , 78 ] mapped on the structure of a nucleoprotein dimer (PDB ID 8FG2); blue and yellow circles correspond to NTD and CTD, respectively; ( b ) CD4 + (orange) and CD8 + (blue) T-cell epitopes of the N protein of SARS-CoV-2 strain B.1 (Wuhan) deposited in the Immune Epitope Database, which contain variable residues uniquely distinguishing different VOCs (shown as red stripes). The alignment was visualized using Geneious 10.2.5 Software.…”

“… Structure of the SARS-CoV-2 N protein. ( a ) The scheme of N molecule domains involved in the RNA binding and other interactions; adapted from [ 30 , 31 ]. Numbers indicate the position of N amino acid residues; ( b ) side view of the N protein dimer (PDB ID 8FG2), crystal structure of the NTD (PDB ID 7VNU), NTD complexed with ssRNA (PDB ID 7ACT) and a crystal structure of the CTD dimer (PDB ID 7C22).…”

Nucleoprotein as a Promising Antigen for Broadly Protective Influenza Vaccines

“…It is reasonable to suggest that slow evolutionary changes in the NP can also affect the antigenicity of the protein and lead to the reduced binding of anti-NP antibodies raised to immunization with the developed NP-based vaccines with the NP antigen on the cells infected with contemporary influenza viruses. Similar to influenza NP, the SARS-CoV-2 nucleocapsid (N) protein slowly evolves, and although the N-based serologic tests developed on the ancestral strain are still sensitive enough to detect antibody responses to evolved SARS-CoV-2 variants [ 159 ], these mutations may lead to substantial changes in the immunogenicity and antigenicity of the NP protein [ 160 ].…”

The role of SARS-CoV-2 N protein in diagnosis and vaccination in the context of emerging variants: present status and prospects

“…The N protein has been shown to possess non-specific nucleic acid binding capabilities, making it a potential diagnostic tool detection target ( Li and Li, 2021 ). As for vaccine design, despite SARS-CoV-2 being prone to mutation, the high degree of conservation in the nucleocapsid protein gene sequence ensures a certain level of cross-protective efficacy, enabling the vaccine to protect against virus variations to some extent and mitigate the severity of illness ( Premkumar et al, 2020 ; Shaw et al, 2021 ; Li et al, 2022 ; Rak et al, 2023 ). Moreover, the relatively simple structure of the nucleocapsid protein makes it easier for vaccine researchers to grasp as a target antigen and consequently develop highly specific and immunogenic vaccines ( Du et al, 2009 ; Walls et al, 2020 ).…”