LMB joins the fight against COVID-19

The SARS-CoV-2 virus is membrane-enclosed but exposes trimeric protein ‘spikes’ on the outside that are immunogenic. These spikes allow the virus to enter cells by binding to ACE2 receptors on cells. We are making several disabled ‘pseudotyped’ versions of the virus in order to study its infection and for use in virus neutralisation tests (functional assays). These viruses just contain the SARS-CoV-2 spikes on the outside (and hence, are not pathogenic).

We are making several disabled ‘pseudotyped’ versions of the virus in order to study its infection and for use in virus neutralisation tests (functional assays). These viruses just contain the SARS-CoV-2 spikes on the outside (and hence, are not pathogenic).

Groups: Leo James, John Briggs, Andrew Carter, Harvey McMahon, Sean Munro

Outputs: Pseudotyped SARS-CoV-2 virus-like particles (mammalian expression system) <2 months

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion. Papa, G., Mallery, DL., Albecka, A., Welch, LG, Cattin-Ortolá, J., Luptak, J., Paul, D. McMahon, HT., Goodfellow, IG., Carter, A., Munro, S., James, LC. PLOS Pathogens https://doi.org/10.1371/journal.ppat.1009246
– Insight on Research: Furin protease is not essential for SARS-CoV-2 infection 

We are developing effective antigens based on viral spike proteins and nucleoprotein. We are making soluble trimeric spikes and also receptor binding domains (RBDs) that are a small part of the spike. RBD binds to ACE2 on the target cell, and is the part that is most different from other coronaviruses. 

The antigens are made for two reasons: they can be injected into animals and humans to produce an immune response as a vaccine candidate or to make antibodies and their libraries. Secondly, these antigens are needed to run serological tests that would determine if patients have been infected with SARS-CoV-2 already. At the moment, these tests are not very good and we are developing ideas on how to make better antigens, for example, by turning them into multimers to exploit avidity.

Groups: Radu Aricescu, John Briggs, Andrew Carter, Leo James, Jan Löwe, Yorgo Modis, Sjors Scheres

Outputs: SARS-CoV-2 RBD protein (bacterial, yeast and mammalian expression systems) <2 months

SARS-CoV-2 multimerised RBD protein (bacterial, yeast and mammalian expression systems) <2 months

SARS-CoV-2 Spike protein (mammalian expression system) <2 months

A thermostable, closed SARS-CoV-2 spike protein trimer. Xiong, X., Qu, K., Ciazynska, KA., Hosmillo, M., Carter, AP., Ebrahimi, S., Ke, Z., Scheres, SHW., Bergamaschi, L., Grice, GL., Zhang, Y., The CITIID-NIHR COVID-19 BioResource Collaboration, Nathan, JA., Baker, S., James, LC., Baxendale, HE., Goodfellow, I., Doffinger, R., Briggs, JAG. Nature Structural & Molecular Biology 27, 934–941. doi:10.1038/s41594-020-0478-5.
– Insight on Research: Studying the Spike protein of SARS-CoV-2

Combined point of care nucleic acid and antibody testing for SARS-CoV-2 following emergence of D614G Spike Variant. Mlcochova, P., Collier, D., Ritchie, A., Assennato, S.M., Hosmillo, M., Goel, N., Meng, B., Chatterjee, K., Mendoza, V., Temperton, N., Kiss, L., James, L.C., Ciazynska, K.A., Xiong, X., Briggs, J.A.G., Nathan, J.A., Mescia, F., Bergamaschi, L., Zhang, H., Barmpounakis, P., Demeris, N., Skells, R., Lyons, P.A., Bradley, J., Baker, S., Allain, J.P., Smith, K.G.C., Bousfield, R., Wilson, M., Sparkes, D., Amoroso, G., Gkrania-Klotsas, E., Hardwick, S., Boyle, A., Goodfellow, I., Gupta, R.K., The CITIID-NIHR COVID BioResource Collaboration. Cell Reports Medicine doi:10.1016/j.xcrm.2020.100099

SARS-CoV-2 following emergence of D614G Spike Variant. Mlcochova, P., Collier, D., Ritchie, A., Assennato, S.M., Hosmillo, M., Goel, N., Meng, B., Chatterjee, K., Mendoza, V., Temperton, N., Kiss, L., James, L.C., Ciazynska, K.A., Xiong, X., Briggs, J.A.G., Nathan, J.A., Mescia, F., Bergamaschi, L., Zhang, H., Barmpounakis, P., Demeris, N., Skells, R., Lyons, P.A., Bradley, J., Baker, S., Allain, J.P., Smith, K.G.C., Bousfield, R., Wilson, M., Sparkes, D., Amoroso, G., Gkrania-Klotsas, E., Hardwick, S., Boyle, A., Goodfellow, I., Gupta, R.K., The CITIID-NIHR COVID BioResource Collaboration. Cell Reports Medicine doi:10.1016/j.xcrm.2020.100099

Critical care workers have lower seroprevalence of SARS-CoV-2 IgG compared with non-patient facing staff in first wave of COVID19. Baxendale, H.E., Wells, D., Gronlund, J., Nadesalingam, A., Paloniemi, M., Carnell, G., Tonks, P., Ceron-Gutierrez, L., Ebrahimi, S., Sayer, A., Briggs, J.A.G., Xiong, X., Nathan, J.A., Grice, G.L., James, L.C., Luptak, J., Pai, S., Heeney, J.L., Doffinger, R. medRxiv (pre-print – not yet peer-reviewed)

SARS-CoV-2 spike protein arrested in the closed state induces potent neutralizing responses. Carnell, GW., Ciazynska, KA., Wells, DA., Xiong, X., Aguinam, ET., McLaughlin, SH., Mallery, D., Ebrahimi, S., Ceron-Gutierrez, L., James, LC., Doffinger, R., Heeney, JL., Briggs, JAG. bioRxiv (pre-print – not yet peer-reviewed)

As an alternative to antibodies – evolving the region of the ACE2 receptor to which SARS-CoV-2 binds to make it bind more effectively to the virus and less well to its natural ligand, Angiotensin II, could produce a soluble protein that reduces the ability of the virus to enter cells without disturbing the Angiotensin system, which plays an important role in blood pressure control.

We will use hypermutating cells lines and in vitro selection to rapidly evolve human ACE2 with the aim of producing these candidate viral decoys. This approach is highly innovative and is directly targeted towards an emergency therapy.

Groups: Nick Brindle (University of Leicester), Julian Sale

Outputs: Evolved ACE2 viral decoys; chicken cell line expression & selection system; mutational scanning of the ACE2-COVID-19 spike protein interaction. 3-6 months

We are using electron tomography to obtain structures of parts of the SARS-CoV-2 virus from virus particles. We are also aiming to study the structure of the S protein on virus-like particles in complex with receptors and neutralising antibodies. Structures are available for isolated proteins, but some interactions with antibodies and the membrane fusion event that the virus triggers can only be understood when structures of these complexes and events are available at high-enough resolutions in the context of the virus.

Structures are available for isolated proteins, but some interactions with antibodies and the membrane fusion event that the virus triggers can only be understood when structures of these complexes and events are available at high-enough resolutions in the context of the virus.

Groups: John Briggs, Andrew Carter, Leo James, Sjors Scheres

Outputs: Images of the virus and of virus-like particles, structures of viral proteins in the context of the virus, and structures with ACE2 domain and antibodies bound. 3-6 months

Structures and distributions of SARS-CoV-2 spike proteins on intact virions. Ke, Z., Oton, J., Qu, K., Cortese, M., Zila, V., McKeane, L., Nakane, T., Zivanov, J., Neufeldt, CJ., Cerikan, B., Lu, JM., Peukes, J., Xiong, X., Kräusslich, H-G., Scheres, SHW., Bartenschlager, R., Briggs, JAG. Nature doi:10.1038/s41586-020-2665-2.
– Insight on Research: Studying the Spike protein of SARS-CoV-2
– Science Museum Group blog featuring interview with John Briggs: Coronavirus: The Spike

Structures and function of locked conformations of SARS-CoV-2 spike. Qu, K., Xiong, X., Ciazynska, KA., Carter, AP., Briggs, JAG. bioRxiv (pre-print – not yet peer-reviewed)

In order to replicate, the SARS-CoV-2 virus needs to transcribe and replicate its RNA into more strands of RNA. For this it uses its own RNA-dependent RNA polymerase (RdRp).

We are making the polymerase (nsp12 + nsp7 and 8) and it is planned to image it by cryo-EM in complex with a primed RNA substrate while it is being transcribed, and then to add known inhibitors such as the antiviral drugs Avigan and Remdesivir. We have access to the highest-resolution cryo-EM instrument in the world and aim to exploit that machine for this study since only true atomic resolution will help with the ongoing efforts to find drugs that are more active against SARS-CoV-2.

Groups: David Barford, Jan Löwe, Chris Russo, Sjors Scheres, John Sutherland

Outputs: SARS-CoV-2 RdRp with RNA substrate and small-molecule inhibitors. Cryo-EM structure at atomic resolution (better than 2.0 Å). 3-6 months

Structure of the SARS-CoV-2 RNA-dependent RNA polymerase in presence of favipiravir-RTP. Naydenova, K., Muir, KW., Wu, L-F., Zhang, Z., Coscia, F., Peet, MJ., Castro-Hartmann, P., Qian, P., Sader, K., Dent, K., Kimanius, D., Sutherland, JD., Löwe, J., Barford, D., Russo, CJ. PNAS 118(7) e2021946118
– Insight on Research: Targeting RNA replication in SARS-CoV-2

One mystery of SARS-CoV-2 is how its genes are translated. All its genes contain a 48 bp stem-loop RNA element in their 3’ UTR. We will help find whether this particular element helps the virus to hijack the cell’s translation machinery (ribosomes to make protein) through these elements, by making reporter strains.

We will also find proteins that bind to this RNA element as that might enable us to find inhibitors that are truly specific for this type of virus. Since it is an RNA element, it is not entirely inconceivable that an RNAi-type approach could be used to develop an emergency therapy very quickly.

Groups: Ben Luisi (Cambridge University), Helena Maier (Pirbright Institute), Chris Oubridge, Felix Randow, Bill Scott (University of California at Santa Cruz)

Outputs: Insights into RNA element s2m’s role in translation of SARS-CoV-2 proteins: a potentially entirely new drug / RNAi target. 3-12 months

Antisense oligonucleotides target a nearly invariant structural element from the SARS-CoV-2 genome and drive RNA degradation. Lulla, V., Wandel, M.P., Bandyra, K.J., Dendooven, T., Yang, X., Doyle, N., Oerum, S., O’Rourke, S., Randow, F., Maier, H.J., Scott, W., Ding, Y., Firth, A.E., Bloznelyte, K.B., Luisi, B.F. bioRxiv (pre-print – not yet peer reviewed).

The membrane proteins that surround the SARS-CoV-2 virus are made in the endoplasmic reticulum (ER) and then trafficked to the Golgi when the virus is assembled. In the Golgi the proteins are processed further by carbohydrate modification and proteolytic cleavage. In addition, some of the Spike protein travels beyond the Golgi to the cell surface where it can cause fusion to adjacent cells.

We are investigating how the viral proteins recruit host cell proteins to direct their traffic, and why it may be advantageous to the virus for some Spike to go beyond the Golgi to enable cell fusion and the formation of multinuclear syncytia.

Groups: Sean Munro, Leo James

Output: Identification of host cell factors required for viral replication and an understanding of the significance of syncytia formation.

Sequences in the cytoplasmic tail of SARS-CoV-2 spike facilitate syncytia formation. Cattin-Ortola, J., Welch, L., Maslen, S.L., Skehel, J.M., Papa, G., James, L.C., Munro, S. bioRxiv (pre-print – not yet peer reviewed).

The efficient generation of SARS-CoV2 Viral Like Particles (VLPs) is a fundamental step for the study of the molecular mechanisms of viral entry and for the identification of drugs that might interfere with it.

We have re-purposed our line of research in order to develop novel methods for the generation of recombinant VLPs and for monitoring their infectivity. Our aim is to generate an efficient screening platform on which to test FDA approved drugs with the potential to interfere with viral uptake.

Group: Marco Tripodi

Output: Generation and distribution of an efficient drug screening platform for inhibitors of SARS-CoV2 cell entry. 3 months.

Will patients (and how many) infected with SARS-CoV-2 develop an antibody response and how widespread will this response be in the population as a whole? These are critical questions for understanding the spread of SARS-CoV-2 and informing policy response to the pandemic.

However, current antibody assays are expensive, unreliable and poorly scalable. As an alternative, we propose a phage-based approach to probe patient sera. Filamentous phage can be viewed as a self-assembling nanoparticle detection reagent, which can be cheaply prepared and propagated simply by growth in a bacterial culture. Our approach is fast, cheap and easily scalable to >10E4 assays per day at a cost <1p per assay.

Groups: Phil Holliger, Willem H. Ouwehand (University of Cambridge)

Output: A phage-based assay for detection and analysis of patient anti-SARS-CoV-2 antibody responses. 3-6 months.

There are increasing reports of some patients with COVID-19 experiencing neurological symptoms. However, it is not yet clear whether the virus actually crosses into the brain, or causes these effects indirectly.

We are using neural organoids to investigate the expression patterns of viral entry factors, such as ACE2, and to test whether pseudotyped viruses with SARS-CoV-2 spike protein can enter neural tissues.

Groups: Madeline Lancaster, Leo James

Outputs: Expression analysis of viral entry factors in CNS tissues. 1-2 months.

Testing viral uptake of pseudotyped virus in neural cell types. 2-3 months.

SARS-CoV-2 infects the brain choroid plexus and disrupts the blood-CSF-barrier. Pellegrini, L., Albecka, A., Mallery, DL., Kellner, MJ., Paul, D., Carter, AP., James, LC., Lancaster, MA. Cell Stem Cell doi:10.1016/j.stem.2020.10.001
– Insight on Research: SARS-CoV-2 disrupts the brain barrier in human organoids
– Article in Vox featuring interview with Madeline Lancaster: The many strange long-term symptoms of Covid-19, explained

We are applying our novel phenotype prediction algorithm to DNA data from direct-to-consumer tests collected via online participation from volunteers from around the world. In addition to a personalised questionnaire selecting a few questions from over 6,000 phenotypes, we are asking every participant a few questions on their COVID-19 status.

This early data, with our unique algorithm applied, has the potential to detect different genetic factors to traditional human genetics approaches, and possibly much sooner.

We are also applying our novel phenotype prediction algorithm to participants from the UK Biobank who have tested positive for the coronavirus and have discovered some preliminary candidates for risk variants which could affect chances of survival (results deposited as a preprint on medRxiv). These preliminary findings are unconfirmed, because the datasets are too small, and so efforts now are focused on securing additional data sources, e.g. from other European biobanks.

Groups: Julian Gough

Outputs: Outside chance of a new discovery. If a COVID-19 discovery is made, results will be published rapidly. <2 months

Genetic risk factors for death with SARS-CoV-2 from the UK Biobank. Lu, C., Gam, R., Pandurangan, A.P., Gough, J. medRxiv (pre-print – not yet peer reviewed).

Positive results have the potential to impact healthcare and may lead to development of a genetic risk test by others.

SARS-CoV-2 enters the cells from endocytic compartments and a pH-raising agent, chloroquine, has shown effectiveness against the virus in vitro. Unfortunately, the results from COVID-19 patients suggest chloroquine might not be protective in vivo.

Better understanding of factors that control the mechanism of viral entry could point us towards more effective compounds that could inhibit infection in patients. We will investigate whether SARS-CoV-2 entry can be prevented by manipulating endolysosomal permeability.

Groups: Patrycja Kozik, Leo James

Outputs: Candidate small molecule tools or genetic targets for manipulating SARS-CoV-2 entry. 6 months

The project is designed to determine whether multimerised protein (subunit) SARS-CoV-2 antigens make better vaccine candidates. The antigens we make have to be highly effective, very stable and easy to produce.

We are investigating whether multimerised S, RBD and N elicit stronger antibody responses than non-multimerised antigens using both pseudoviral models as well as live SARS-CoV-2.

Groups: Jan Löwe, Radu Aricescu, Leo James

Outputs: Multimerised S, RBD and N antigens, assess antibody responses and complete vaccination experiments in mice using multimerised antigens.

The project is to investigate the activity of proteases that are encoded in SARS-CoV-2 to understand their activity and specificity and their consequences for infection. The primary question is whether viral proteases target endogenous proteins in host cells for proteolysis.

We are introducing protease traps into physiologically relevant host cell lines to capture protease substrates, which can then be characterised by mass spectrometry. 

Groups: Jason Chin

Outputs: Viral protease traps expressed and activated in mammalian cells, characterisation of viral protease substrates

In addition to contributing scientific research, numerous members of LMB have volunteered to help the NHS and local communities:

• The LMB assisted with a Cambridge-wide call for volunteers, coordinated by Professor Ian Goodfellow at the Department of Pathology, University of Cambridge.
• 15 LMB-based scientists volunteered at COVID-19 testing centres:
  • Tobias Wauer, Elyse Fischer, Hasan Al-Habib, Nadia Cummins, Antonio Galindo, Ewa Gogola, Samuel Lacey, Andrei Mihut, Jack Munns and James Rhodes volunteered as experienced scientists at the COVID-19 National Testing Centre set up by the National Institute of Health Research in Milton Keynes. The 10 volunteers completed 12-hour shifts to help in the national effort to increase COVID-19 testing capacity. 
  • Tobias Wauer shared his experience of volunteering at the UK Biocentre Lighthouse Lab in The Conversation: ‘My new life as a coronavirus tester’
  • Lorena Boquete Vilarino, Shabih Shakeel, Laura Whitworth, Conny Yu and Eszter Zavodszky volunteered as experienced scientists helping to increase local testing capacity for NHS staff at a COVID-19 testing facility located near the LMB on the Cambridge Biomedical Campus.
  • The testing volunteers shared details of their experience – see: Putting science skills to new use during the pandemic
• Nina Rzechorzek has enrolled as an NHS volunteer and a biomedical researcher on the Cambridge call for volunteers.
• LMB’s Health and Safety department has been busy coordinating donations of personal protective equipment (PPE) to Addenbrookes Hospital in Cambridge. Following a request from the University of Cambridge, LMB staff assembled a large donation of essential PPE including gloves, disposable aprons and coveralls, safety glasses and overshoes. The collection was coordinated by Jillian Deans, Head of LMB Health and Safety. Jillian said: “It was amazing how quickly LMB staff responded to the request and we hope that the PPE goes some way to help front line NHS staff.”
• Lesley Drynan and Matt Coleman of Biological Services Group coordinated another large effort in getting over 1,500 masks to the COVID response team, University of Cambridge, that is working with the NHS. The LMB has lent its PortaCount mask-fit testing machine to Addenbrookes Hospital and Liam Bray is training NHS staff on how to use it.
• Several members of LMB are helping out their communities in their personal capacities, such as Freda Chapman from our Domestic Services department. Freda has signed up to be an NHS volunteer responder and is also assisting her neighbour, who is an essential health worker, by providing hot meals.

We are accompanying our COVID-19 Insight on Research articles with interviews with the scientists who carried out the work, to give some perspective of the experiences of transitioning to COVID-19 research and working at the LMB during the height of the ongoing pandemic.

Xiaoli Xiong

“The institute was dark and quiet probably because I often ended up working until rather late at night. There were very few people working in the institute and I missed finding people to discuss various topics including scientific problems.” Read more…

Zunlong Ke

“As a young researcher, I really hoped that I was able to contribute to the global fight against the pandemic. As it turned out, I was fortunate enough to do so and made important observations of the nature of the virus.” Read more…

Laura Pellegrini

“We decided to take advantage of our new organoid model to study SARS-CoV-2 entry into the brain, whether the virus can directly infect neuronal cells and its potential implications in the brain barrier damage.” Read more…

Guido Papa

“I have been studying viruses since my undergraduate degree and, as a virologist, I felt I had to give my contribution to COVID research and join the global fight against the pandemic by putting in place my expertise and knowledge in virology.” Read more…