SARS-CoV-2 is responsible for COVID-19 infection; Structurally, it is made up of four structural proteins called spike (S), membrane (M), envelope (E), and nucleocapsid (N). Recombinant proteins of N and S structural proteins (S1 and RBD) are commonly used as antigens in immunoassays for antibody detection, including lateral flow immunoassays (LFAs), enzyme-linked immunosorbent assays (ELISAs), and chemiluminescent immunoassays (CLIAs). However, some doubts have been expressed about the usefulness of antibody tests due to the antigenic similarity between SARS-CoV-2 and other circulating coronaviruses.21, 22. To avoid a potential cross-reactivity, in silico analysis can be performed to select immunogenic peptides that can be used to standardize novel ELISA for antibody detection or production of monoclonal and polyclonal antibodies for use in standardizing new methods for viral antigen detection, which was the primary objective of this work.
Elevated glycoprotein mediates entry of the coronavirus into host cells by recognizing angiotensin-converting enzyme 2 (ACE2), an attractive antiviral target and a more common antigen for antibody detection. The S protein consists of two functional subunits, S1 and S2, and the S1 subunit consists of an N-terminal domain and a receptor-binding domain (RBD)5, 22. The nucleocapsid protein physically binds the envelope to the RNA+ genome and contains N-terminal RNA-binding domains (N-NTD) and C-terminal dimerization domains.23. 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, vaccines, or multitube diagnostics25,26. In this study, a total of twenty-five peptides were reported, nineteen of them in the S protein and six of them in the N protein. We selected eight peptides, two of which are located in the S1 NTD, five in the RBD and one in the N. Interestingly, previous studies reported Each monomer of Spike protein contains 22 glycosylation sites.27and this post-translational modification plays an important role in immune response evasion and viral pathophysiology and function.28. This approach indicates that the selected peptides, in addition to all properties associated with the induction of the immune response, can be used as antigens in the development of immunoassays. Interestingly, the amino acids in the RBD3 peptide are found almost exclusively in the RBD of SARS-CoV-2 but not present in SARS-CoV according to previous alignments by other authors.29,30.
When we searched for the presence of mutations in SARS-CoV-2 VOCs and their effect on the sequence of 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 alteration in the receptor-binding domain of the S protein, which may contribute to the 40-70% increase in the translocation observed with the Alpha, Beta, Gamma and Omicron variants.6. The delta variant contains amino acid changes in the RBD1 (N440K and G446S) and RBD3 (S477N and T478K) peptides, while Omicron contains 4 amino acid changes in the RBD4 peptide (G496S, Q498R, N501Y, Y505H). These changes are commonly associated with the prevalence, evolution relationship, and neutralization efficacy of each variable, for which future studies should be conducted7,31. The emergence of novel variants of SARS-CoV-2 is a concern for diagnostic testing, however the use of peptides in the MAP8 format could be a new strategy for solving problems associated with the cross-interaction of recombinant proteins. An important consideration is the possibility of obtaining a negative result if only one peptide is used, due to amino acid changes that can affect antibody recognition. Therefore, we suggest a mixture of two or more peptides for serum monitoring.
The eight selected peptides were synthesized into MAP8. This format increases the size and structural complexity of the peptide, allowing exposure of eight epitopes in a single molecule, which favors the use of lower antigen concentrations while using it for immunoassays (0.1 μg/ml in this study). Another interesting property is that this coordination stimulates antibody production32. Using the above-mentioned peptides, their use as antigens to detect antibodies in COVID-19 patients was evaluated using indirect ELISA and induction of the humoral immune response in rabbits. The use of protein N peptides is important because IgG seroconversion appears on average 2 days earlier than protein S33So the use of this peptide can be useful in the early detection of immune response products. To assess the presence of antibodies, in addition to selected peptides, we also obtained recombinant proteins derived from S (trimer and RBD) and N proteins. On both antigens, the results were significantly higher in COVID-19 virus-infected patients than in COVID-19-negative patients. For IgG and total antibodies. In the case of IgM, no significant difference was observed, which may be due to the time that passed from symptom onset to serum collection. Full standardization of this method is still necessary, but the preliminary results deserve further in-depth investigation. Previously, antibodies were tested against N and S proteins, and no significant difference was observed between the two proteins for the detection of total and IgG antibodies34which may mean that all peptides reported during immunoassay development can be mixed to increase the number of available epitopes or used to standardize ELISA methods for each protein individually.
Regarding the use of peptides as antigens to standardize antibody detection methods, several evidence has been published. Wu et al.35Developed an ELISA assay to detect IgM and IgG antibodies against the Spike and Nucleocapsid protein of SARS-CoV. Whereas, during the COVID-19 pandemic, epitopes of antigens derived from SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope) were used as antigens to standardize novel ELISA methods with high sensitivity (91.4%) and specificity (83.7%)36. In another study by Polvere et al., they reported ten predicted peptides derived from the S, M, and N proteins of SARS-CoV-2, seven of which were tested by ELISA for interaction with a pool of serum samples from twenty-four COVID-19 convalescent patients. The results showed differences in interaction with the selected peptides ranging from recognition to only one peptide to all seven peptides.37. The ELISA method used in this study has many similarities with the above examples, thus providing evidence about the potential clinical utility of the reported peptides in standardizing the ELISA method.
Although all peptides can be used as antigens to search for antibodies against SARS-CoV-2, not all of them can be used for induction of the 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 alpha, beta, gamma and omicron VOCs, thus serum derived from vaccinated rabbits can be used to analyze neutralizing activity against these VOCs. The observed result was that the RBD4 peptide containing N501Y (and three additional amino acid changes in the Omicron variant), did not stimulate the humoral immune response in rabbits, but was recognized by serum samples derived from patients.
Stimulation of a positive immune response in rabbits and recognition of peptides by serum samples derived from COVID-19 patients provide evidence for the interaction of selected peptides with MHC molecules, T-cell response, and B-cell activation. The use of synthetic peptides to stimulate the immune response has the advantage that in theory a polyclonal antibody would be as specific as a monoclonal antibody but much easier to produce, and synthetic peptides are cheaper and less time consuming to produce than the synthesis of other antigens, such as as recombinant proteins38. Some trials are still needed to evaluate the clinical application of peptides as antigens (for serosurveillance for VOCs) 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 a full-length protein. In addition, we present a list of seventeen other peptides that can be tested with many applications, such as immunoassay development, antibody production or candidate vaccines.
Experimental procedures to be performed include standardization of ELISA methods using reported peptides for antibody detection in vaccinated or infected patients (with disseminated VOC or novel variants) or recognition of the original protein using serum derived from vaccinated rabbits. In conclusion, this work provides evidence regarding the potential usefulness of selected MAP8 peptides based on silico analysis of the primary amino acid structure of a high glycoprotein and nucleocapsid protein of SARS-CoV-2 to standardize novel immunoassays for the detection of antibodies in serum samples from COVID-19 patients and individuals with They have been vaccinated or to produce antibodies in animal models that can be used to detect antigens in infected individuals.
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