Antigenic properties of the SARS-CoV-2 nucleoprotein are altered by the RNA admixture

Abstract

Determining the presence of antibodies to the SARS-CoV-2 antigens is the best way to identify infected people, regardless of the development of symptoms of COVID-19. The nucleoprotein (NP) of the SARS-CoV-2 is an immunodominant antigen of the virus; anti-NP antibodies are detected in persons previously infected with the virus with the highest titers. Many test systems for detecting antibodies to SARS-CoV-2 contain NP or its fragments as antigen. The sensitivity and specificity of such test systems differ significantly, which can be explained by variations in the antigenic properties of NP caused by differences in the methods of its cultivation, isolation and purification. We investigated this effect for the Escherichia coli-derived SARS-CoV-2 NP, obtained from the cytoplasm in the soluble form. We hypothesized that co-purified nucleic acids that form a strong complex with NP might negatively affect NP’s antigenic properties. Therefore, we have established the NP purification method, which completely eliminates the RNA in the NP preparation. Two stages of RNA removal were used: treatment of the crude lysate of E. coli with RNase A and subsequent selective RNA elution with 2 M NaCl solution. The resulting NP without RNA has a significantly better signal-to-noise ratio when used as an ELISA antigen and tested with a control panel of serum samples with antibodies to SARS-CoV-2; therefore, it is preferable for in vitro diagnostic use. The same increase of the signal-to-noise ratio was detected for the free N-terminal domain of the NP. Complete removal of RNA complexed with NP during purification will significantly improve its antigenic properties, and the absence of RNA in NP preparations should be controlled during the production of this antigen.

Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD

“…RBD-NKY protein was purified from a conditioned medium exactly as described in [ 19 ]. SARS-CoV-2 nucleoprotein (NP), tagged with 10× His was purified in-house from the E. coli cytoplasm as described in [ 21 ], absence of RNA was controlled by the Qubit RNA assay (ThermoFischer Scientific, Waltham, MA, USA).…”

Post-COVID Endocrine Disorders: Putative Role of Molecular Mimicry and Some Pathomorphological Correlates

“…Along with the SARS-CoV-2 SP, several other viral antigens have been proven to be highly significant in the immune response against COVID-19. The SARS-CoV-2 Nucleocapsid protein (NP) is its immunodominant antigen; moreover, anti-NP antibodies have the highest titers among all anti-viral specificities in persons previously infected with this virus [20]. The SARS-CoV-2 Membrane protein (MP) is essential in the disease pathogenesis due to its role in anti-interferonogenic and pro-apoptotic effects and also is highly immunogenic [21,22].…”

Post-COVID Endocrine Disorders: Putative Role of Molecular Mimicry and Some Pathomorphological Correlates

“…several other viral antigens have been proven to be highly significant in the immune response against COVID-19. The SARS-CoV-2 Nucleocapsid protein (NP) is its immunodominant antigen, moreover, anti-NP antibodies have the highest titers among all anti-viral specificities in persons previously infected with this virus [18]. The SARS-CoV-2 Membrane protein (MP) is essential in the disease pathogenesis due to its role in anti-interferonogenic and pro-apoptotic effects and also is highly immunogenic [19][20].…”