ABSTRACT
The tragic COVID-19 pandemic, which has seen a total of 655 million cases worldwide and a death toll of over 6.6 million seems finally tailing off. Even so, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise, the severity of which cannot be predicted in advance. This is concerning for the maintenance and stability of public health, since immune evasion and increased transmissibility may arise. Therefore, it is crucial to continue monitoring antibody responses to SARS-CoV-2 in the general population. As a complement to polymerase chain reaction tests, multiplex immunoassays are elegant tools that use individual protein or peptide antigens simultaneously to provide a high level of sensitivity and specificity. To further improve these aspects of SARS-CoV-2 antibody detection, as well as accuracy, we have developed an advanced serological peptide-based multiplex assay using antigen-fused peptide epitopes derived from both the spike and the nucleocapsid proteins. The significance of the epitopes selected for antibody detection has been verified by in silico molecular docking simulations between the peptide epitopes and reported SARS-CoV-2 antibodies. Peptides can be more easily and quickly modified and synthesized than full length proteins and can, therefore, be used in a more cost-effective manner. Three different fusion-epitope peptides (FEPs) were synthesized and tested by enzyme-linked immunosorbent assay (ELISA). A total of 145 blood serum samples were used, compromising 110 COVID-19 serum samples from COVID-19 patients and 35 negative control serum samples taken from COVID-19-free individuals before the outbreak. Interestingly, our data demonstrate that the sensitivity, specificity, and accuracy of the results for the FEP antigens are higher than for single peptide epitopes or mixtures of single peptide epitopes. Our FEP concept can be applied to different multiplex immunoassays testing not only for SARS-CoV-2 but also for various other pathogens. A significantly improved peptide-based serological assay may support the development of commercial point-of-care tests, such as lateral-flow-assays.
ABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has greatly affected all aspect of life. Although several vaccines and pharmaceuticals have been developed against SARS-CoV-2, the emergence of mutated variants has raised several concerns. The angiotensin-converting enzyme (ACE2) receptor cell entry mechanism of this virus has not changed despite the vast mutation in emerging variants. Inhibiting the spike protein by which the virus identifies the host ACE2 receptor is a promising therapeutic countermeasure to keep pace with rapidly emerging variants. Here, we synthesized two ACE2-derived peptides, P1 and P25, to target and potentially inhibit SARS-CoV-2 cell entry. These peptides were evaluated in vitro using pseudoviruses that contained the SARS-CoV-2 original spike protein, the Delta-mutated spike protein, or the Omicron spike protein. An in silico investigation was also done for these peptides to evaluate the interaction of the synthesized peptides and the SARS-CoV-2 variants. The P25 peptide showed a promising inhibition potency against the tested pseudoviruses and an even higher inhibition against the Omicron variant. The IC50 of the Omicron variant was 60.8 µM, while the IC50s of the SARS-CoV-2 original strain and the Delta variant were 455.2 µM and 546.4 µM, respectively. The in silico experiments also showed that the amino acid composition design and structure of P25 boosted the interaction with the spike protein. These findings suggest that ACE2-derived peptides, such as P25, have the potential to inhibit SARS-CoV-2 cell entry in vitro. However, further in vivo studies are needed to confirm their therapeutic efficacy against emerging variants.
