Utility of peptides identified in silico in spike-S1 domain and SARS-CoV-2 nucleocapsid for antibody detection in COVID-19 patients and antibody production

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SARS-CoV-2 is responsible for COVID-19 infection; structurally, it is composed of four structural proteins called tip (S), membrane (M), envelope (E) and nucleocapsid (N). Recombinant N and S structural proteins (S1 and RBD subunits) are commonly used as antigens in immunoassays for the detection of antibodies, including lateral flow immunoassays (LFA), enzyme immunoassays (ELISA) and chemiluminescent immunoassays (CLIA). However, some doubts regarding the usefulness of antibody tests have been expressed due to the antigenic similarity between SARS-CoV-2 and other circulating coronaviruses.21.22. To avoid potential cross-reactivity, in silico analysis can be performed for the selection of immunogenic peptides that could be used for the standardization of a new ELISA for antibody detection or the production of monoclonal and polyclonal antibodies for its use. in the standardization of new methods for the detection of viral antigens, which was the main objective of this work.

The spike glycoprotein mediates entry of the coronavirus into host cells through recognition of angiotensin-converting enzyme 2 (ACE2), which is an attractive antiviral target and the most common antigen for the antibody detection. The S protein is composed of two functional subunits, S1 and S2, the S1 subunit is composed of an N-terminal domain and a receptor binding domain (RBD)5.22. The nucleocapsid protein physically links the envelope to the genome + RNA and contains N-terminal RNA-binding (N-NTD) and C-terminal dimerization domains23. Both proteins were considered primary targets for epitope identification in this work.

Several in silico approaches have been performed to identify antiviral peptides24epitopes for the development of vaccine-based peptides or multiple epitope-based vaccines or diagnostics25.26. In this study, a total of twenty-five peptides were reported, of which nineteen were in protein S and six were in protein N. We selected eight peptides, two of which were located in the S1 NTD, five in the RBD and one in the N. Interestingly, previous studies have reported that each monomer in the spike protein has twenty-two glycosylation sites27and this post-translational modification plays an important role in immune response evasion, function and viral pathobiology28. This approach indicates that the selected peptides, in addition to all characteristics associated with the induction of the immune response, can be used as antigens in the development of immunoassays. Interestingly, the amino acids present in the RBD3 peptide are almost exclusively located in the RBD of SARS-CoV-2 but are not found in SARS-CoV according to previous alignments performed by other authors.29.30.

When we searched for the presence of mutations in SARS-CoV-2 COVs and their effect on the sequence of the selected peptides, we found that in six of the eight peptides, at least one amino acid change was identified. The RBD4 peptide contains the N501Y amino acid change in the S-protein receptor binding domain, which may contribute to the 40-70% increase in transmission seen with Alpha, Beta, Gamma, and Omicron variants6. The Delta variant contains amino acid changes in the RBD1 (N440K and G446S) and RBD3 (S477N and T478K) peptides, while Omicron has 4 amino acid changes in the RBD4 peptide (G496S, Q498R, N501Y, Y505H ). These changes are generally associated with the prevalence, phylogenetic relationship, and neutralizing potency of each variant, for which future studies should be performed.7.31. The emergence of new SARS-CoV-2 variants is a concern for diagnostic testing, but the use of MAP8-formatted peptides could be a novel strategy to address issues associated with recombinant protein cross-reactivity. An important consideration is the possibility of obtaining a negative result if only one peptide is used, due to amino acid changes which could affect antibody recognition. Therefore, we propose the mixture of two or more peptides for serosurveillance.

The eight selected peptides were synthesized in MAP8. This format increases the size and structural complexity of the peptide, allowing the exposure of eight epitopes in a single molecule, which would favor the use of low concentrations of antigen when used for immunoassays (0.1 µg/mL In this study). Another interesting feature is that this format stimulates the production of antibodies32. Using the aforementioned peptides, their use as antigens for the detection of antibodies in COVID-19 patients by indirect ELISA and the induction of humoral immune response in rabbits was evaluated. The use of peptides present in protein N is important because IgG seroconversion appears on average 2 days earlier than protein S33, so the use of this peptide could be useful in the detection of early immune response products. To assess the presence of antibodies, in addition to the selected peptides, we also obtained recombinant proteins derived from S (trimer and RBD) and N proteins. In both antigens, the results were significantly higher in COVID-19 patients positive than in COVID-19 patients negative for IgG and total antibodies. In the case of IgM, no significant difference was observed, which may be due to the time that elapsed between the onset of symptoms and the collection of serum. A complete standardization of this method is still necessary, but preliminary results deserve further investigation. Previously, antibodies against N and S proteins were tested, and no significant difference between the two proteins was observed for the detection of total antibodies and IgG34which could mean that all reported peptides could be mixed during immunoassay development to increase the number of available epitopes or used for standardization of ELISA methods for each individual protein.

In connection with the use of peptides as antigens for the standardization of antibody detection methods, several evidences have been published. Woo et al.35, have developed an ELISA test for the detection of IgM and IgG antibodies against the Spike and Nucleocapsid protein of SARS-CoV. Considering that, during the COVID-19 pandemic, the use of antigenic epitopes derived from the structural proteins of SARS-CoV-2 (Spike, Nucleocapsid, Membrane and Envelope) have been employed as antigens for the standardization of new ELISA methods to high sensitivity (91.4%) and specificity (83.7%)36. In another study by Polvere et al., reported ten predicted peptides derived from the S, M, and N proteins of SARS-CoV-2, seven of which were tested by ELISA for reactivity to a panel of twenty serum samples. -four COVID-19 convalescent patients. Results showed differences in responsiveness to selected peptides ranging from single peptide recognition to all seven peptides37. The ELISA method used in this study has several similarities to the aforementioned examples, thus providing evidence for the potential clinical utility of the reported peptides in the standardization of an ELISA method.

Although all peptides could be used as antigens for the search for antibodies against SARS-CoV-2, not all could be used for the induction of a humoral immune response. The RBD3 peptide was the only peptide that induced a humoral immune response by itself, and the mixture of peptides could induce an immune response due to the increased complexity and molecular weight of the combined peptides. The RBD3 peptide is highly conserved in VOCs Alpha, Beta, Gamma and Omicron, therefore serum derived from immunized rabbits could be used for analysis of neutralizing activity against these VOCs. A notable result was that the RBD4 peptide, which contains N501Y (and three additional amino acid changes in the Omicron variant), did not induce a humoral immune response in rabbits, but was recognized by serum samples from patients.

Induction of positive immune response in rabbits and recognition of peptides by serum samples from COVID-19 patients provide evidence regarding the interaction of selected peptides with MHC molecules, lymphocyte response T and B cell activation. Using synthetic peptides for induction of the immune response has the advantage that the polyclonal antiserum will in theory be as specific as the monoclonal antibody but much easier to produce, and synthetic peptides are cheaper and take less time to produce than synthesizing other antigens, such as as recombinant proteins38. Some experiments still need to be performed to assess the clinical application of peptides as antigens (for VOC serosurveillance) or in the production of antibodies for antigen detection; however, our preliminary results provide not only in silico approaches (localization in 2D and 3D structure, post-translational modifications and VOC analysis) but also experimental data comparing peptides to full-length protein. Additionally, we provide a list of seventeen other peptides that could be tested with multiple applications, such as immunoassay development, antibody production, or vaccine candidates.

Experimental procedures to be performed include standardization of ELISA methods using reported peptides for detection of antibodies in vaccinated or infected patients (with circulating VOCs or new variants) or recognition of native proteins using serum derived of immunized rabbits. In conclusion, this work provides evidence regarding the potential utility of selected MAP8 peptides based on in silico analysis of the primary amino acid structure of SARS-CoV-2 spike glycoprotein and nucleocapsid protein. for the standardization of new immunoassays for the detection of antibodies in serum samples from COVID-19 patients and vaccinated individuals or for the production of antibodies in animal models that can be used for the detection of antigens in infected individuals .

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