• epitope mapping
• Nucleoprotein
• peptides
• Sars-Cov-2

SARS-CoV-2 is an enveloped ssRNA+ virus belonging to the Coronavirus species (strictly correlated to the SARS virus and also related to the MERS virus). Coronaviruses (CoVs) have large, 30-kb non-segmented genomes consisting of several universal open reading frames (ORFs - such as ORF1ab, which code for non structural proteins) and some virus-specific accessory proteins. Among them particularly important in the relationships with the host are the Spike protein (S - decorating the surface of all CoVs and mediating viral entry), Membrane (M), Nucleocapside (or N protein which is involved in genome packaging and is found in the mature virion core) and the Envelope (E). SARS-CoV-2 infection led to a pandemic condition through the acquisition of interhuman transmissibility and a rapid spread among subjects.
Viral infection is characterized by a striking diversity in clinical presentation, ranging from asymptomatic to severe disease (defined as Coronavirus Disease –Covid-19) and potentially leading to death. Disease elapse depends on timely and coordinated innate and adaptive immune response thanks to which the virus can be controlled and eliminated. Thus, the role of the adaptive immune response in SARS-CoV-2 infections is critical, but the repertoire of epitope targets linked to infection is not comprehensively defined; the majority of immune responses to the virus are directed to the spike protein, nucleocapsid and to the ORF1ab. While some epitopes are identified in the majority of samples, others are rare, and have been found variation in the number of epitopes targeted between individuals. Despite mounting evidence that the SARS-CoV-2 N protein may be highly antigenic in the context of COVID-19, there has been limited effort to fully characterize antibody responses during SARS-CoV-2infection. It’s now clear that the Nucleocapside (N) protein, which participates in RNA package and virus particle release, is largely a dimer in solution. It has also been proven that SARS-CoV-2 N protein contains two distinct RNA-binding domains (the N-terminal domain [NTD] and the C-terminal domain [CTD]) characterized by serine/arginine-rich (SR-rich) positively charged domains (SRD). The analysis of immune response to SARS-CoV-2 proteins in infected subjects led to the observation that anti-N proteins antibodies are present in the early stage of infection and persist during time more than other antibodies. However, the high sequence homology with N protein from other CoV(92.52%withSARS-CoV-1) might be the responsible of false positive results and low specificity of antibodies towards the whole protein.
To shed light on the antigenic characteristics of the N protein and to clarify the immunodominant regions targeted by the humoral immune response during SARS-CoV-2 infections, we performed a bioinformatic analysis using B cell epitope prediction databases. We identified 6 different sequences and in collaboration with the Laboratory of Peptide & Protein Chemistry and Biology of the University of Florence, we developed the corresponding panel of synthetic 20-linear peptides. Sera from 148 patients affected by Covid-19 and admitted to Intensive Care Unit, were collected during different waves of the italian pandemia. Immunoenzymatic analysis of anti-N proteins were performed using whole recombinant protein and epitope mapping was obtained with ELISA employing the 6 different N-derived synthetic peptides. Preliminary results suggest that hospitalized CoViD-19 patients develop IgG antibodies against peptides internal to N region and that there is a high heterogeneity in targeted regions among patients. Studies are in progress to analyse more deeply the characteristic of this immune response, with the analysis of the IgA isotype and to correlate specific anti N-peptide antibodies to clinical symptoms.