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
(2023)
Editorial: Methods and advances in induced pluripotent stem cells-ophthalmology
in Frontiers in Cell and Developmental Biology

Armstrong L
(2021)
In the eye of the storm: SARS-CoV-2 infection and replication at the ocular surface?
in Stem cells translational medicine

Atalay E
(2024)
Animal Models for Limbal Stem Cell Deficiency: A Critical Narrative Literature Review.
in Ophthalmology and therapy

Cartes C
(2021)
Referral Patterns of Patients with Limbal Stem Cell Deficiency to a Specialized Tertiary Center in the United Kingdom.
in Ophthalmology and therapy

Collin J
(2021)
A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells.
in The ocular surface


Djidrovski I
(2021)
Sars-Cov-2 Infects an Upper Airway Model Derived from Induced Pluripotent Stem Cells
in Stem Cells


Figueiredo GS
(2023)
Understanding Ocular Surface Inflammation in Tears Before and After Autologous Cultivated Limbal Epithelial Stem Cell Transplantation.
in Ophthalmology and therapy

Jackson R
(2021)
Conjunctival epithelial cells resist productive SARS-CoV-2 infection
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 | 09/2022 |
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 |
Title | single cell RNA-Seq data of organotypic air-liquid-interface model of human conjunctival epithelium |
Description | An in vitro human conjunctival model devised by our group. Single cell RNA-Seq was carried out to define their cellular composition, data deposited in GEO. The model was also infected with SARS-CoV-2 and single cell RNA-Seq data was also generated from infected model. |
Type Of Material | Database/Collection of data |
Year Produced | 2022 |
Provided To Others? | Yes |
Impact | The data has been widely used by the scientific community especially to assess the response of conjunctival epithelium to SARS-CoV-2 infection. |
URL | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE191232 |
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 | Genetics matters |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Genetics matters is an annual event organised by Newcastle University to celebrate Rare Disease Research day. Research groups organise a table each showcasing their work. Members of the public visit each table, engaging with the researchers and their projects. |
Year(s) Of Engagement Activity | 2023 |
Description | Genetics matters |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Genetics Matters is an annual event organised by Newcastle University focusing on rare diseases and their diagnosis and treatment. My research team organises a table showcasing our work in eye disease and stem cells. |
Year(s) Of Engagement Activity | 2024 |
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 | Prof. Lako key speaker at the Translational Scientist webinar organised by 10x genomics |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Prof. Lako described her group's work on single cell sequencing of human adult and developing cornea. |
Year(s) Of Engagement Activity | 2021 |
Description | Prof. Lako: Invited speaker at the European Single Cell Discovery Symposium 2022 The tale of a single cell Cambridge 26th May 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Research talks focusing on application of single cell technologies to medical and clinical projects. |
Year(s) Of Engagement Activity | 2023 |
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 |