Rapid Benchtop Purification of SARS-CoV-2 for Immunological Studies: Minimizing Cytotoxic Effects on Monocytes

Introduction: Understanding COVID-19 pathogenesis and immune responses relies on robust in vitro systems that utilize purified SARS-CoV-2 virions. This study focused on developing a rapid, benchtop purification method for SARS-CoV-2, designed to eliminate non-viral substances and cytopathic agents, enabling immunological experiments.

Methods: The SARS-CoV-2 delta variant was propagated in Vero E6 cells within a BSL-4 laboratory. Complete virus inactivation was achieved using UV irradiation and was confirmed via standard virological assays. The purification procedure involved filtration through a 0.22 μm membrane, followed by anion exchange chromatography, with desalting performed as an optional step. PBMCs were isolated from healthy donor blood using SepMate™ tubes with slight modifications to the manufacturer’s protocol. These PBMCs were incubated with either purified or unpurified virus, and cells and immunological interactions were analyzed through flow cytometry.

Results: The purification method successfully removed 99% of non-viral proteins, leaving primarily viral proteins. RT-PCR and rapid antigen tests demonstrated an approximately 70% recovery of viral RNA. Flow cytometry confirmed that the unpurified virus caused significant, almost total, CD14+ monocyte destruction. In contrast, monocytes incubated with purified virus maintained viability, indicating reduced cytotoxic effects.

Discussion: This streamlined purification process provides a reliable, rapid tool for preparing SARS-CoV-2 for immunological studies. While anion exchange chromatography was highly effective, observed discrepancies between PCR and rapid antigen test results after desalting merit further exploration. Future studies with active viruses will help determine the infectivity of purified preparations.

Acknowledgment: This research was conducted in collaboration with the National Laboratory of Virology and the Flow Cytometry Core Facility of the University of Pécs. The project was supported by grants 2020-2.1.1-ED-2020-00100 and RRF-2.3.1-21-2022-00010 from the National Research, Development, and Innovation Office of Hungary.