Conjunctival epithelial cells resist productive SARS-CoV-2 infection

Although tropism of SARS-CoV-2 for respiratory tract epithelial cells is well established, an open question is whether the conjunctival epithelium is also a target for SARS-CoV-2. 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. To address this question, we generated an organotypic air-liquid-interface 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 ensure. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust NF-Kß activity, alongside evidence of suppression of antiviral interferon signalling. Collectively these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies such as personal protective equipment. Overall design: Problem: Respiratory viruses including SARS-CoV-2, responsible for the COVID-19 pandemic, enter via the respiratory tract epithelium, however they can also use the eye surface as an additional en-try point. The conjunctiva has a large unexposed surface area with mucus producing cells. Clinical reports have shown that some of COVID-19 patients suffer from conjunctivitis in the early stages of infection. To date it is not fully established if the conjunctiva can be used as an additional entry and propagation portal for SARS-CoV-2. Results: To assess whether conjunctiva can be infected by SARS-CoV-2, we generated an in vitro model by culturing human conjunctival cells under special conditions, which promote cell differen-tiation and layer stratification through air-liquid contact. In this manuscript, we show that the ex vivo model contains all cells found in the native conjunctiva and moreover expresses the two key viral entry factors, ACE2 and TMPRSS2. Infection of the model with SARS-CoV-2 shows that while all conjunctival cells can be infected with the virus, new viral particles that are necessary for propaga-tion cannot be generated. Importantly, the conjunctival cells responded to viral infection by up-regulating a key signalling pathway, NF-?B, which is often the first line of defence upon viral infec-tion of eye cells. Impact: Our data indicate that the risk of SARS-CoV-2 transmission from a conjunctival organ trans-plant is low. The lack of viral propagation in conjunctiva should also be informative on provision of protective personal equipment used by medical staff and carers of COVID-19 patients.