Assessing SARS-CoV-2 entry, replication and prevention in a primary human conjunctival cell model and organ cultured cornea/conjunctiva.
Lead Research Organisation:
Newcastle University
Department Name: Biosciences Institute
Abstract
The SARS-CoV-2 virus, which has caused the COVID-19 pandemic, is highly infectious and predominantly transmitted through respiratory droplets. To enter the host cell SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as a cellular receptor and the transmembrane protease serine type 2 (TMPRSS2) for fusion of viral and cellular membranes. The ocular surface epithelia, conjunctiva and cornea, represent an additional mucosal surface, which can be exposed to respiratory droplets. Several published reports have shown that SARS-CoV-2 can cause conjunctivitis, either as an early sign of infection, or during hospitalization for severe COVID-19 disease. In a recent study of 38 COVID-19 patients, 31.6% had conjunctivitis with 16.7% of these testing positive for SARS-CoV-2 from conjunctival and nasopharyngeal swabs. There is evidence that numerous properties allow the eye to serve as a potential site of virus replication and a gateway for the establishment of respiratory infection, through the nasolacrimal system linking the ocular and respiratory tissues. Our recent data shows co-expression of ACE2 and TMPRSS2 in human superficial conjunctival, limbal and corneal epithelium, suggesting a potential extra-respiratory transmission route of SARS-CoV-2 via the ocular surface. In this proposal we will use human ex vivo differentiated conjunctival and corneal epithelium and organ cultured cornea/conjunctiva as pre-clinical tools to study the entry of SARS-CoV-2 via the ocular surface and to develop effective diagnostic, prophylactics and treatments in the fight against COVID-19. We envisage that proof-of-concept studies developed herein will lead not only to development of eye drops, but also nasal sprays and mouth washes, to provide the much-needed therapies in time of pandemics.
Publications
Armstrong L
(2021)
In the eye of the storm: SARS-CoV-2 infection and replication at the ocular surface?
in Stem cells translational medicine
Djidrovski I
(2021)
SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells.
in Stem cells (Dayton, Ohio)
Jackson R
(2021)
Conjunctival epithelial cells resist productive SARS-CoV-2 infection
Jackson RM
(2022)
Conjunctival epithelial cells resist productive SARS-CoV-2 infection.
in Stem cell reports
Latta L
(2021)
Pathophysiology of aniridia-associated keratopathy: Developmental aspects and unanswered questions.
in The ocular surface
Muus C
(2021)
Single-cell meta-analysis of SARS-CoV-2 entry genes across tissues and demographics.
in Nature medicine
Sungnak W
(2020)
SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes.
in Nature medicine
Tucker NR
(2020)
Myocyte-Specific Upregulation of ACE2 in Cardiovascular Disease: Implications for SARS-CoV-2-Mediated Myocarditis.
in Circulation
| Description | In this study we address an important but unanswered question regarding SARS-CoV-2 tropism via the ocular surface. The conjunctival epithelium comprises the largest exposed part of the ocular surface and expresses the SARS-CoV-2 entry factors, ACE2 and TMPRSS2, which has led to suggestions that the ocular surface can serve as an additional entry portal for the virus. Despite the obvious clinical importance of this question, there is a major lack of data in this area especially in relation to tropism for conjunctival cells. To the best of our knowledge only one study (Singh et al. Ocul Surf. 2021 Sep 25;S1542-0124(21)00106-3) has addressed tropism of SARS-CoV-2 for the conjunctival epithelium, described declining expression of viral proteins from 24 to 72 hours post-infection, suggesting that conjunctival cells may be unable to sustain infection. However the capacity of these cells to support a productive infection, via assembly of nascent viral particles or release of infectious virus, and the responses of individual conjunctival cell subtypes (e.g. superficial or basal conjunctival epithelial cells and epithelial progenitor cells) were not formally assessed. Highlights of our study include: • Development of a new organotypic air-liquid-interface culture model of conjunctival epithelium, which could be maintained successfully up to day 75 of differentiation. • Single-cell RNA-seq analysis, with complementary virological studies, showing that while all conjunctival cell types were permissive to SARS-CoV-2 genome expression, a productive infection did not ensue. • Definition of the early innate immune response, which was characterised by a robust autocrine and paracrine NF-Kß activity, without activation 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 as well as the risks associated with conjunctival transplants. |
| Exploitation Route | These findings will be of use to other researchers and clinicians trying to understand SARS-CoV-2 tropism. By comparing different epithelial surfaces, we will be able to understand why some (for example nasal epithelium) are susceptible to SARS-CoV-2 infection and some (for example ocular surface epithelium) are not. In this informed way, we can then design preventative treatments to stop viral replication in permissive epithelial exposed surfaces. |
| Sectors | Education,Healthcare |
| Description | Generation of B2M deficient limbal stem cells |
| Amount | £250,000 (GBP) |
| Organisation | Fight for Sight |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 10/2022 |
| End | 09/2025 |
| Title | single cell RNA and ATAC-Seq data obtained from adult, embryonic and fetal cornea samples |
| Description | GEO number: GSE155683 http://retinalstemcellresearch.co.uk/CorneaCellAtlas |
| Type Of Material | Biological samples |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | These data are freely available to other researchers. Their utility was exemplified by usage of data for SARS-COv-2 Entry factor studies. |
| URL | http://retinalstemcellresearch.co.uk/CorneaCellAtlas |
| Description | collaboration with dr. Christopher Duncan and his group |
| Organisation | Newcastle University |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | we are optimising an ocular surface model for SARS-COv2 Infection |
| Collaborator Contribution | the collaborating group is providing the virus and sharing us with key techniques for assessing viral replication and cells's immune response |
| Impact | n/a |
| Start Year | 2020 |
| Description | Keynote speaker at Faculty of Medical Sciences, Tirana, Albania |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | The talk focussed on SARS-CoV-2 entry factors, which were derived from our work on single cell RNA-Seq analyses of adult human cornea and conjunctiva. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Prof. Lako Invited speaker at Western Balkan University |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Invited talk at Western Balkan University focusing on single cell RNA-Seq of cornea and applications to SARS-CoV-2 entry into the ocular surface |
| Year(s) Of Engagement Activity | 2022 |
| Description | Speaker at the WT/MRC HCA meeting |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Prof. Lako presented the work performed on the single cell analyses of human adult and developing cornea and application to disease as part of the HCA project funded in her lab. |
| Year(s) Of Engagement Activity | 2020 |