A Celigo Whole Well Imaging Cytometer for the Crick Covid neutralisation pipeline

The Crick achieves operational and research efficiencies and economies-of-scale through centralised facilities and functions, known as Science Technology Platforms, that provide all researchers at the Crick, irrespective of affiliation, with access to cutting-edge equipment for research and laboratory enabling functions such as the High Throughput Screening facility (HTS).

The Crick Covid Surveillance unit (CCSU) is an offshoot of the HTS facility and was established to run a pipeline of activity delivering a high throughput live-virus antibody neutralisation assay to measure the ability of patient serum to prevent cell infection by SARS-CoV-2 across thousands of samples simultaneously. This unique neutralisation assay platform (developed in the HTS facility) is essential to many ongoing research studies, for example those assessing the likely impact of Variants of Concern on current vaccinated populations and in clinically vulnerable cohorts. The pipeline is able to generate neutralization data against new VOCs (e.g. Delta and Omicron) within 2-3 weeks upon receipt of a novel virus sample and can process thousands of samples per week at GCP level. Data from this pipeline has measured neutralising antibody titres against Delta and Omicron variants in participants in the Crick-Legacy study (recipients of both the Pfizer and Oxford-AstraZeneca vaccines). These data are shared with UK government scientific advisory boards, contributing to the extension of UK pandemic restrictions to allow more people to receive a second vaccine dose in summer 2021 and the successful booster campaign. Publications reporting these findings have been cited nearly 600 times. This pipeline also enabled the comparison of antibody titres in healthy adults with those of both cancer patients (Crick-CAPTURE study) and kidney dialysis patients (Crick-NAOMI study), providing important data on prioritisation of vulnerable patient groups. In addition, data on the in vitro neutralising efficacy of the synthetic monoclonal antibody, Sotrovimab was shared with NHSE and the Chief Medical Officer and supported the ongoing use of this drug for patients infected with Omicron BA.1 and BA.2. There are no other similar pipelines capable of this scale of live virus assay in the UK (possibly internationally) and with near real time reporting, the data produced remains a vital tool in the UK and international pandemic preparedness and response.

Currently, the neutralisation pipeline is made robust by using multiple HTS liquid handling devices to ensure continuity of delivery. However, the primary imaging (and measurements derived from them) is restricted to one microscope, the Opera Phenix. This reliance on one imaging device is a weak point for this pipeline. This proposal is to deploy the Celigo scanning cytometer in this assay and thereby remove the dependence on one machine for a pipeline that needs to remain operational. Moreover, acquisition on the Celigo is 4-5 times faster meaning that pipeline throughput and capacity can be significantly increased.

The Covid neutralisation pipeline accounted for 50% of HTS usage over 2020/21. Having a high-end microscope occupied with one type of assay necessarily restricts access by other users (currently measured in weeks of delay for access to the Opera Phenix). By moving the primary imaging platform for the CCSU activity to the Celigo, we would greatly increase the availability of high content microscopy to other Crick researchers and thereby improve service delivery for more than 100 Crick researchers who used the HTS facility over 20/21.
The availability of a Celigo imager will also enable improvements to existing activities requiring analysis of cell populations (at scale) in the areas of radiation biology, stem cell biology and general cell viability. In addition, there will be new opportunities for users to explore automation of previously manual assays e.g. Influenza plaque assays

The unique live-virus SARS-CoV-2 neutralisation platform (created by the HTS and now run by the CCSU) produces data for longitudinal cohort studies vital for monitoring the likely impact of emergent VOC's and informs UK and international responses and future pandemic preparedness. We have ensured the robustness and reproducibility of the pipeline by employing strict SOP's and enabling multiple liquid handling devices to prosecute these screens. One vulnerability is that the imaging relies solely on one microscope to generate data; the assay output is the ratio of infected cells (as measured by immunofluorescence of a viral marker) to total cells in a well (estimated from a DAPI stain) in 384-well format. Imaging does not require high resolution but does require multiplate automated acquisition and low resolution, whole well imaging. The proposal is to use the Celigo whole well scanner with a Thermo Orbitor (repurposed by the HTS facility) driven by Momentum scheduling software to take on the imaging of this assay. This would increase pipeline robustness and increase throughput (decreasing acquisition times from 15 to 3 mins per plate).
In addition, this single assay accounts for 50% of HTS output annually, meaning other user users are waiting weeks to access the high content analysis assays (an increasingly common standard format for many) e.g. multiplexed panels of DNA damage markers and cell painting. Migration of the neutralization assay to the Celigo platform would free up HCA capacity to the 100+ HTS users at the Crick and thereby improve service provision. Further, the Celigo will improve the throughput and accessibility of a number of other common assay types in particular clonogenic survival assays as used in radiosensitivity or drug re-purposing screens or viability assays where simple cell counting is required. The Celigo would also create new opportunities to monitor stem cell colony morphology and further automate Influenza viral plaque assays.