SARS-CoV-2 and the immune response; the establishment of a SARS-CoV-2 neutralization assay and subsequent immune utilization

Gormley, Delania (2023) SARS-CoV-2 and the immune response; the establishment of a SARS-CoV-2 neutralization assay and subsequent immune utilization. Masters thesis, Memorial University of Newfoundland.

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Abstract

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has become increasingly important since the onset of the coronavirus disease 2019 (COVID-19) pandemic. In less than a year, there were numerous vaccination guidelines. After the vaccination program, the scientific field raced to answer questions regarding the immune response at the cellular and molecular levels. We aimed to bridge innate and adaptive immune responses to uncover the pathophysiological factors that mediate immune pathogenicity. The introductory part of the project was focused on the adaptive immune response to measure how antibodies and sera affect spike-mediated viral infection. To monitor disease outcomes and determine vaccine efficacy, we evaluated the neutralizing antibody potency using an HIV-1 pseudovirus in a cohort of SARS-CoV-2 infection and/or vaccinated individuals. We hypothesized that high neutralization titer would positively correlate to immune protection and decreased disease severity. The SARS-CoV-2 pseudovirus system and neutralization assay are established and future work could gain neutralization data. These results highlight a valuable complement to ELISA-based methods and the importance of studying spike-mediated viral infection on the immune response. The accompanying part of this research concentrated on the innate immune responses to SARS-CoV-2 ORF3a viral infection. In this regard, it was apparent early on that COVID-19 involved extensive inflammation and immune dysregulation. Therefore, we decided to evaluate the key regulatory factors and mediators involved in inflammasome formation/activation and programmed cell death by an inflammasome array. We hypothesized that the SARS-CoV-2 ORF3a protein would induce pyroptosis through the upregulation of the NLRP3 inflammasome and its components. We found that the CCL5, MEFV, NLRC5, NLRP3, MOK, and TNF genes were all statistically significantly upregulated. These genes have implications in both apoptosis and pyroptosis, and both cell death pathways could be activated simultaneously in response to the SARS-CoV-2 ORF3a protein. These results highlight a future application for therapeutic development for SARS-CoV-2 and other inflammatory diseases.

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