Within weeks of the outbreak of the Covid-19 pandemic, Profs. Ray Owens and James Naismith of the Rosalind Franklin Institute created a collaboration between the team based at the Franklin, Diamond Light Source and the Research Complex at Harwell to develop nanobodies against SARS-CoV-2, the virus responsible for causing Covid-19. As successful agents were created, they brought in partners at the UKHSA and Liverpool University to investigate their potential as a therapeutic agent.
The RSC announced on 7 June 2022 that the collaboration has won one of their prestigious Horizon prizes. These prizes are awarded to teams or collaborations who are opening up new directions and possibilities in their field, through ground-breaking scientific developments.
The large team involves researchers from Diamond, the Franklin, Oxford University where the original team of researchers who initiated the nanobody project were based, the UK Health Security Agency, and Liverpool University. The Covid Nanobody Project included nine specialists from Diamond including the Research Complex at Harwell.
The agents developed by the team have now undergone animal trials, and show powerful therapeutic effects as a nasally delivered drug. This approach, it is hoped, can be applied to other emerging diseases. The unique combination of structural analytical and biological expertise at Harwell, alongside the enabling power of the Research Complex made this collaboration successful in a way which would have been very hard to achieve elsewhere.
The collaboration took a skills-based approach as lockdown meant lab work was built around lone and shift working. Safety was paramount, so they used a strict pipeline approach with each researcher focusing on their key skills. This minimised the movement of researchers through physical areas (only the samples moved) and they worked in a shift pattern to both minimise crossover between researchers and maximise productivity.
Dr Halina Mikolajek, Research Scientist who is also responsible for the Crystallisation Facility at Harwell, based in the Research Complex adjacent to Diamond, explained:
This was pretty cool because it meant we all worked only to our strengths and dropped the aspects we are less good at.
Jim Naismith led the computing and analysis and I focussed on producing crystals and collecting the data (both of which I love) and we were fast! To give a sense of speed, I went from protein delivery at 17:00, crystal plate set up at 18:00 to crystals the following morning at 9:00, cryocooling at 12:00 and data collection the following morning. Jim would then convert the data to structure at the end of that day.
Halina kept the crystallisation facility at Harwell running and supported a number of other Covid-19 projects. She led on the crystallisation of the nanobody complexes and has been a key part of the biophysics work. Although she was often working on her own through the night during the pandemic, she says she never felt lonely because she and the others were just so busy.
We were driven by adrenaline to just move forward as fast as we could. We would often not see anybody physically, but all the different researchers from different areas were in touch all the time, which really helped. Everything clicked into place and we were able to move really fast - faster than I think any of us expected.
Summing up their contribution; Halina said:
I am super proud of what was achieved in those first 4 months of Lockdown and take huge satisfaction that what we have jointly designed might be one of the treatments that will be offered to patients with Covid-19 in the near future. The working pattern of nights was tiring and I was pushing my limits in every way but I had the best and most productive time as a researcher ever. Not only in my 4.5 years at Diamond but across my entire career so far.
When I look back, this kind of work is what got me into STEM. It reminded me of how exciting the first year of my PhD was and I still get excited to find crystals and see molecules the human eye cannot see.
Other researchers from Diamond who participated in the Horizon collaboration with The Franklin were: Dan Clare - (Principal EM Scientist, eBIC), Peter Harrison - (Postdoctoral Research Associate in CryoEM, Membrane Protein Lab & eBIC, Julika Radecke - (EM Scientist, eBIC ), Prof. Sir David Stuart and Vinod Vogirala - EM Scientist, eBIC.
Peter Harrison worked with colleagues in the Franklin in the protein production pipeline. This involved the expression and purification of nanobodies, RBD and Spike protein, and preparing samples which were then used for biophysical and structural studies. Peter was also responsible for removing and quantifying LPS levels in protein samples before animal studies, and also assisting in biophysical and EM collections.
Following protein purification, once the samples have been optimised for vitrification, EM grids were made by staff in The Franklin using the Chameleon, a new plunge freezing device shared between Diamond and The Franklin. Grids were then screened on a transmission electron microscope (TEM) to establish optimal freezing conditions. A good EM grid will have thin enough ice to accommodate the entire protein of interest in various orientations. At this point the grids were loaded onto a Titan Krios TEM for high resolution single particle data collections. This has been done by Julika Radecke, followed by microscope alignments, camera tuning and gain reference acquisition and data collection setups.
Vinod Vogirala was involved in initial cryo-EM screenings of several nanobody/SARS-CoV-2 Spike protein complexes and several cryo-EM data collections. He processed the H11 nanobody/SARS-CoV-2 Spike protein complex to atomic resolution. This facilitated better understanding of how the higher affinity mutants, H11-H4 and H11-D4 nanobodies bind to the Spike protein and block it from interacting with the ACE2 receptor.
Dan Clare was involved in cryo-EM data collection of several nanobody/SARS-CoV-2 Spike protein complexes. He processed one of the H11 nanobody/SARS-CoV-2 spike protein complex to near atomic resolution and built an atomic model from the cryoEM density. Analysis of the structure facilitated a better understanding of how the high affinity nanobodies, H11-H4 and H11-D4, bind to the spike protein and stop it interacting with its cellular ACE2 receptor.
Professor Sir David Stuart commented:
The virus behind the pandemic has numerous physio-chemical interactions. Likewise, the treatments for the disease involve more interactions with the human body in order to block the virus. Investigating and understanding these interactions at the molecular level using facilities like Diamond is essential to providing new vaccines and drugs to combat the global pandemic and play a key part in our preparedness to face future challenges.
Find out more about the prize on the RSC website: https://www.rsc.org/prizes-funding/prizes/2022-winners/team-nanobodies
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