Screening for regulators of SARS CoV-2 infection and inflammation

National Institute Of Allergy And Infectious Diseases

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Abstract

Cells of the innate immune system, such as dendritic cells and macrophages, constantly patrol host mucosal surfaces and peripheral tissues for signs of infection or injury. Since many people infected with SARS-CoV-2 clear the virus without developing symptoms, aspects of the innate immune response may hold the key to defeating this virus and its variants. The potential for an adverse cytokine storm depends on the interaction of different cell types at the site of infection. This highlights the need to study the immune response to SARS-CoV-2 in a combined environment of both epithelial cells, which are the primary target of infection, and innate immune cells such as macrophages, which are crucial drivers of inflammation. Since this project has been delayed awaiting BSL-3 lab access, we will first summarize our research plan. We propose to establish a co-culture assay which can measure both viral replication in human epithelial cells, an established CoV-2-permissive cell, and IL-6 expression from THP1 human macrophages. As both assay readouts will be FACS-based, this will permit used of a pooled CRISPR screening approach, which will be less labor intensive on a genome-wide scale and more feasible with available equipment in the NIAID BSL-3 lab. Post-infection, we will sort CD32+ THP1 cells from CD324+ (E-cadherin) epithelial cells, and assay the cells for viral levels and IL-6 expression. Viral levels will be assayed using a fluorescent antibody to the spike protein, while IL-6 will be assayed by well-established lab protocols for intracellular cytokine staining. Our expectation is that the epithelial cells will be the primary target for CoV-2 infection, and THP1 the primary IL-6 producers. For this reason, we initially seek to identify regulators of IL-6 in THP1 cells, while limiting the study of host factors of SARS-CoV-2 infectivity to epithelial cells. Once we have established the viral infection and IL-6 expression assays as FACS-based readouts, we will test the reproducibility of the screen assays with positive control genes targeted by specific CRISPR/Cas9 gRNAs. We expect the confirmed human receptor for CoV-2, ACE2, to be an appropriate positive control for viral infection and possibly also for the IL-6 response. We will optimize the viral infection time, multiplicity of infection (MOI), incubation period for golgi block, cell fixation, IL-6 staining conditions and FACS-sorting for both fluorescent virus infection levels and IL-6 expression. Once the primary assay is established, we propose to use an NCATS-provided CRISPR-based lentiviral library in pooled format (Cellecta). THP1 cells and epithelial cells will be separately infected with lentivirus and drug-selected under BSL-2 conditions. To ensure an average of 1 gRNA per cell, we will use an MOI of 0.3 for the lentiviral infection. Control pools of lentivirus-infected THP1 and epithelial cells will be retained for the later sequencing step to identify gRNAs that are lost from the library pool due to effects on cell viability. SARS-CoV-2 preparation and infection will be performed in the BSL-3 lab. As described earlier, we will screen epithelial cells and differentiated THP1 cells in a co-culture system by mixing the CRISPR library-infected cells in a 1:1 ratio. After infection with SARS-CoV-2, cells will be treated with golgi block, fixed with 4% PFA and stained for IL-6, CD32 and CD324 following the previously optimized protocols. We will follow standard operating procedures for BSL-3 CoV-2 work to permit the fixed cells to be FACS-sorted under BSL-2 conditions. Assuming that epithelial cells are the primary viral target and THP1 the primary IL-6 producers, we will sort six populations of cells: Highest and lowest 20% of THP1 cells stained for IL-6 expression and intermediate 60% of IL-6 stained cells as control, and highest and lowest 20% of epithelial cells analyzed for viral fluorescence also with intermediate 60% of cells as control. Genomic DNA will be isolated, gRNA sequences amplified by PCR and deep sequencing will be used to identify gRNAs that are enriched in the high/low 20% sets compared to the intermediate 60%. We will repeat the screen in triplicate to identify gRNAs that appear consistently in the high/low 20% populations. Genes targeted by these gRNAs will be followed up as candidate hits. In FY2021, we have completed pilot assays under BSL-2 conditions with the human coronavirus OC43. We have confirmed we can effectively FACS-separate the epithelial cells and macrophages after co-culture and viral infection, and that this system drives a strong macrophage IL-6 response. We have recently completed the training to gain access to the BSL-3 lab and are now optimizing the epithelial cell/macrophage co-culture conditions for efficient SARS-CoV2 infection.