Conjunctival epithelial cells resist productive SARS-CoV-2 infection

Abstract

Purpose : The ocular surface is a defined route of entry of several viral pathogens. Conjunctival epithelial cells, which express viral entry receptors ACE2 and TMPRSS2, constitute the largest exposed epithelium of the ocular surface tissue, and may represent a relevant viral entry route. An important unresolved question is whether SARS-CoV-2 can infect the conjunctival epithelial cells.

Methods : Adult human eyes from three female donors of 52, 78, and 80 years old were donated for research following informed consent. All tissue was provided by NHS Blood and Transplant Tissue and Eye Services or the Newcastle NHS Trust following ethical approval (18/YH/04/20). Conjunctival epithelial progenitors were expanded on 3T3 mitotically inactivated feeder cells and differentiated using an air-liquid interface (ALI) method. Single cell RNA-Seq, with complementary imaging and virological assays were used to define the cellular permissiveness of various epithelial cell types in the conjuctiva and determine the cell type specific innate immune response to SARS-CoV-2 infection.

Results : We generated an organotypic ALI model of conjunctival epithelium, composed of progenitor, basal and superficial epithelial cells and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA Seq, with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to SARS-CoV-2 genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-Kβ activity, without activation of antiviral interferon signalling.

Conclusions : The data presented herein show that conjunctival epithelium is permissive to SARS-CoV-2 infection, but without evidence of productive viral replication. This study was performed in organotypic models derived from three different patients, with single cell RNA-Seq data obtained from the peak infection interval. Future work should assess changes in transcriptome of each conjunctival cell type in frequent intervals after infection and in a larger number of donors to get deeper insights into the refractory nature of these cell to viral propagation.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.