Track: Discovery and Basic Research - Biology - Immunogenicity
Category: Poster Abstract
Effect of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Immunodominant Peptide Antigens on Human Oral Mucosal, Nasal and Corneal Epithelial Cells.
Purpose: The purpose of the current study is to understand the effects of SARS-Cov2 immunodominant peptide antigen exposure to human oral mucosal, nasal epithelial and corneal epithelial cells by analyzing inflammatory cytokine responses. Methods: Human gingival fibroblast cell line (HGF-1), human nasal epithelial cell line (RPMI 2650), human corneal cells (primary, PCS 70010, and cell line HCE-2), and human monocyte cell line (THP-1) will be purchased from ATCC, and cultured following standard cell culture procedures. Previously published1 immunodominant, and pathogenic peptide sequences derived from spike (S) protein of SARS-CoV-2 will be synthesized (GenScript). The peptide sequencces are: D01. Amino acid positions 200-210; sequence GYQPIDVVRDLG D07. Amino acid positions 927-937; sequence GLGKLQDVVNQNGE D08. Amino acid positions 942-951; sequence ALNTLVKQLSSN The cultured cells (50,000/ well) will be treated with the peptides in varying concentrations in triplicate for 24-72 hours in 200 μl culture medium. At the end of the indicated time point, culture supernatants will be harvested and analyzed for cytokines interleukin 1 beta (IL-1β), IL-6, interferon gamma (IFN-Υ), and macrophage inflammatory protein (MIP) by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (R&D Systems). Vesicular stomatitis virus (VSV) G peptide (YTDIEMNRLGK) will be used as a control. Viability of the cells following peptide treatments will be monitored using Cell Viability Assay (CVA) kit (Promega). ELISA and CVA plates will be read using a multi-mode microplate reader (SpecraMax, Molecular Devices). Results: The proposed study will be initiated after resumption of normal laboratory activities following institutional opening. It is expected that SARS-Cov-2 immunodominant peptide treatment would trigger inflammatory cytokine release from the cells. Statistical analysis will be performed using SPSS version 25.0, and data will be presented as mean+/- SD with one-way ANOVA to determine the statistical significance. Conclusion: Final conclusion from the proposed research could be made only at the completion of the experiments. It is expected that learning the effects of SARS-Cov-2 antigenic peptides on individual cell type may lead to better understanding of the pathophysiology, and possible development of treatment to Covid-19. References: 1. K.-Y. Hwa, W. M. Lin, Y. -I. Hou and T. -M. Yeh. Peptide Mimicrying Between SARS Coronavirus Spike Protein and Human Proteins Reacts with SARS Patient Serum. Journal of Biomedicine and Biotechnology, Vol 2008, 8 pages, Article ID 326464, doi:10.1155/2008/326464.