Research undertaken on SARS-CoV2 in the MRC Toxicology Unit
Project 1
Title: To determine the “on and off” target effects of drugs that alter ACE2 expression levels on the infectivity of SARS-CoV2
MRC Investigators involved in project: Luis Miguel Martins, Mathew Van de Pette, Ritwick Sawarkar, Kiran Patill
Objective
Recent studies demonstrate that the ACE2 receptor serves as a binding site for the anchoring spike proteins on the surfaces of SARS-Cov-2 and there is some limited, and in some cases conflicting, evidence that ACE-I treatment and angiotensin II type 1 receptor blockers, in addition to some over the counter medication, leads to increased expression and activity of ACE-2, increasing the potential for viral infection/re-infection. The overarching objective of this study is to determine whether drugs that alter ACE2 expression levels alter the infectivity of this new coronavirus.
Two major aims:
1- To determine whether certain drugs (e.g. Ibuprofen, SARTANS etc) alter infectivity by regulating ACE2 levels.
2- To determine if drugs that modulate ACE2 expression levels have a direct effect on the interaction between the virus spike protein and ACE2.
Outcome and timescales
We will provide information about the potential adverse effects (or not), for these widely-taken medicines. The assays (depending on how quickly reagents can be delivered) can be set up within a month.
Resource for wider community.
The screening system that we are establishing can be used to test for the effects of other drugs and we have already been asked by colleagues in the Hospitals in Leicester to test some of these.
Project 2
Title: Devising new methods to detect and visualize SARSCoV2 directly in infected human tissues
MRC Investigators involved in the project: James Thaventhiran, Marion MacFarlane
Objectives
Recent publications detailing the characteristics of a bad prognosis for COVID-19 have confirmed patients experience a fall in lymphocyte numbers. It remains unknown whether COVID-19/SARS-CoV2 infects lymphocytes and this information has profound implications for our understanding of human immunity to this pathogen. Lymphocytes are the central component of immunity that maintain our ability for protection from pathogen re-encounter. If lymphocytes are infected by this novel pathogen, then assumptions that patients previously infected are now immune may be incorrect. We will devise methods to detect the virus so that we can address:
1. Which immune cell types are infected by Sars-CoV2 in affected patients
2. Which sub-classes of B- and T-cells are affected by Sars-Cov2
3. Which immune cells in the GI tract, if any, are infected by virus
4. These methods will importantly also allow confirmation of viral infection in cases of death of suspected COVID-19 respiratory disease where a diagnosis was not obtained. We have been approached by the Coroner’s office who are keen to make use of this technology for testing of post mortem tissue.
Resource, Outcomes and timescales
We will gain a greater understanding of the disease and will have devised a new testing method. We now have access to some COVID-19 positive tissues in which to quantify the extent of viral infection of immune cells, and in particular to identify immune cell subgroups (eg memory T-cells) which are being affected. Our main methodology will be the use of multiplex in situ assays for the detection of viral genome/protein, with simultaneous immunohistochemical cell type identification. These are methods which are well established in our laboratories, and our main exterior requirement is fixed, embedded, COVID-19/SARS-CoV2 tissues, which we are seeking to obtain from third parties to use on a collaborative basis. Secondarily, if possible, we would also hope to directly visualise coronoavirus particles interacting with the immune system by transmission EM.
Resource for wider community
We hope to become a testing centre for SARSCoV2 for the cases where there is no confirmed diagnosis, we will provide new information about the infection.
Project 3
Title: Identification of host RNA binding proteins that are recruited/required by SARS-CoV-2 during its infection cycle to elucidate therapeutic targets
Senior Staff involved in the project: Anne Willis, Kathryn Lilley (Biochemistry University of Cambridge), James Thaventhiran. We are additionally collaborating with colleagues in Oxford and Munich on this project
Objectives
The need for a robust and wide ranging pharmacological treatment of SARSCoV2 is acute. The virus is expected to become endemic and the disease is most severe in the elderly and other immunocompromised populations in which vaccination and immunity are likely to fail. Targeted anti-viral therapy requires definition of the non-redundant biochemical reactions necessary for the viral RNA life-cycle.
The major aims are:
1. To identify the RNA binding proteins (RBP) required for the efficient intracellular targeting, transcription, modification, processing, translation and encapsidation of viral RNAs.
2. This information will be used to target viral replication with repurposed drugs. The canonical protein synthesis machinery (which the data suggest the virus requires for synthesis of viral proteins) is currently being explored as an anti-cancer target by all major pharmaceutical companies and there are many drugs that can be used in this regard which have been through clinical trial and are readily available.
Outcome and timescales
We will identify the host encoded RBPome required for viral infection and new ways to target the virus. This information can be used to support clinical trials using pre-existing agents. We will obtain the data required in 6-8 weeks. We have already asked colleagues in Germany who have established SARSCoV2 growth conditions to test likely drugs for effects on viral replication.
Resource for wider community
A definition of the RBPome for SARSCoV2 new ways to target the virus with repurposed drugs.
Dissemination
We will publish our findings using pre-print platforms, in the first instance, and discuss our findings with the MRC communication team. We have also published several studies after peer review:
Santos Leal et al.Paracetamol Is Associated with a Lower Risk of COVID-19 Infection and Decreased ACE2 Protein Expression: A Retrospective Analysis. COVID (2021)
Yu, Y. et al. Alzheimer′s and Parkinson′s diseases predict different COVID-19 outcomes, a UK Biobank study, Geriatrics (2021)
Travaglio, M. et al. Links between air pollution and COVID-19 in England, Environmental Pollution (2021)
Buckland, M. et al. Treatment of COVID-19 with remdesivir in the absence of humoral immunity: a case report, Nature Communications (2020)
Project 4
Title: The role of short ACE2 in Sars-Cov-2 infection and patients’ susceptibility to COVID-19
MRC Investigators involved in project: Vito Mennella
Summary
SARS-CoV-2 utilizes Angiotensin Converting Enzyme II (ACE2) as an entry receptor [1]. Analysis of ACE2 expression and function is therefore essential for understanding susceptibility at the cellular and individual level, and to develop therapeutic strategies for COVID-19 [2, 3].
We recently discovered that respiratory epithelia express ACE2 as a second, shorter isoform, in addition to the canonical form. We also showed that shortACE2 is the main isoform regulated by
IFNs [4], cytokines released in response to viral infections.
These discoveries highlight an urgent need to improve our understanding of ACE2 expression in cells and how this varies in patients. Since shortACE2 is IFNs-regulated, and lacks main binding site to SARS-CoV-2, we reason that might be part of a mechanism for protecting airway cells from SARS-CoV-2.
Hypothesis and aims:
Our hypothesis is that cellular and individual susceptibility to infection is linked to the expression ratio of short/longACE-2 and that regulating this will inhibit SARS-CoV-2 entry.
Aim 1. Analyse shortACE-2 expression in patients with characteristics associated with susceptibility to COVID-19 such as ethnicity and chronic disease by bulk RNA sequencing.
Aim 2. Determine shortACE-2 expression patterns in the different airway cell types by scRNA sequencing.
Aim 3. Analyse the role of shortACE-2 in SARS-CoV-2 infection by CRISPR/Cas9 and knock-in strategies in primary epithelial cell models.
Outcome and timescales
This proposal will provide critical information to study precisely how each cell type interacts with the virus, to identify key factors underlying why some people are more susceptible to SARS-CoV-2, and to design therapeutic strategies targeting shortACE2 to inhibit SARS-CoV-2 infection. This project is currently funded by UKRI-BBSRC as part of the rapid response to COVID-19 (18 months from Feb 2021).
Resource for wider community.
All results and data will be shared rapidly upon BioXRiv manuscript submission.
References
1. Hoffmann, M., et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, 2020. 181(2): p. 271-280 e8.
2. Bourgonje, A.R., et al., Angiotensin-converting enzyme 2 (ACE2), SARS-CoV-2 and the pathophysiology of coronavirus disease 2019 (COVID-19). J Pathol, 2020. 251(3): p. 228-248.
3. Devaux, C.A., J.M. Rolain, and D. Raoult, ACE2 receptor polymorphism: Susceptibility to SARSCoV-2, hypertension, multi-organ failure, and COVID-19 disease outcome. J Microbiol Immunol Infect, 2020. 53(3): p. 425-435.
4. Blume, C., A novel isoform of ACE2 is expressed in human nasal and bronchial respiratory epithelia and is upregulated in response to RNA respiratory virus infection. Nature Genetics, 2020. 53 (2), 205-214
Other Unit contributions
Equipment: We have donated a PCR machine to Milton Keynes and 6 tissue culture hoods to University Hospitals of Leicester NHS Trust.