Spotlight on CoVaRR-Net’s Pillar 4 Functional Genomics and Structure Function of VOCs: Proof that investing in today will lead to a better tomorrow

Since the beginning of the COVID-19 pandemic and the establishment of CoVaRR-Net, the Pillar 4 – Functional Genomics and Structure Function of VOCs – team has made great progress in developing tools and methods to assess the immune response to SARS-CoV-2 variants due to vaccination and infection.

While their achievements are too numerous to list here, the following is a selection that illustrates the legacy of Pillar 4’s work.

Objectives achieved!

Given the rapid rate of SARS-CoV-2 evolution, Pillar 4’s goal of creating and disseminating protein-based and plasmid reagents for SARS-CoV-2 variants represented a huge task[1]. Often, while reagents for a given variant were being developed and tested, new variants were beginning to emerge, and only time tells which variant is designated a Variant of Concern (VOC).

Despite this – and additional logistics challenges of receiving timely shipments during the holiday rush that coincided with Omicron emergence and border closures – Pillar 4 successfully generated over 30 spike variant DNAs for neutralization assays and further study. By the end of 2024, over 100 spike DNA samples had been distributed to at least 16 laboratories across Canada and internationally. Instructions for how to generate virus-like particles (VLPs) and conduct VLP neutralization assays to test serum samples for protective neutralizing antibodies using these DNA constructs were also distributed on request.

Another Pillar 4 objective was to create well-validated assays to test immune responses to various VOCs. Using existing methods, the team modified an assay to test serum for the presence of neutralizing antibodies that can block variants[2]. They were able to get a measure of how well antibodies in the serum can prevent or “neutralize” the VLP’s entry into the cells. Each lot of VLP they develop requires quality control testing to determine the optimal amounts of VLP vs. positive control blocking antibodies to use in the absence of serum so they can make a baseline comparison for each serum sample. Pillar 4 researchers have developed a small library of over 30 VOCs for these assays, most recently JN.1 and KP.2.

Pillars 4 and 6 (a.k.a. Computational Analysis, Modelling and Evolutionary Outcomes or CAMEO) collaborated to develop these pseudovirus neutralization assays. The assays relied on CAMEO’s variant tracking and modelling to identify those VOCs most likely to cause major disruptions in the health of Canadians with each successive wave of SARS-CoV-2 infection. The resulting assays have been used extensively in over 20 COVID-19 immunity studies across Canada, including in immunocompromised populations.

“Having the resources and personnel in place prior to CoVaRR-Net’s establishment had a major positive impact on Pillar 4’s ability to collaborate with Pillar 6 and identify prominent variants, leading to our successes with implementing these VLP assays,” comments Pillar 4 Lead, Dr. Anne-Claude Gingras, Senior Investigator, Lunenfeld-Tanenbaum Research Institute, Sinai Health System. “In turn, these assays have impacted vaccine prioritization for vulnerable groups.”

Indeed, public health’s recommendation that Canadians over 65 and immunocompromised patients (including solid organ transplant recipients, kidney transplant recipients, stem-cell transplant recipients or patients on immuno-suppressants) should be prioritized for COVID-19 vaccination would not have been possible so quickly without Pillar 4. Pillar Lead Anne-Claude Gingras’s lab at the University of Toronto is one of two high-throughput facilities in the country (the other one being CoVaRR-Net Executive Director Marc-André Langlois’ laboratory at the University of Ottawa) that was able to develop assays and test all the blood samples from studies across the country.

In a more big-picture effort to monitor variants and mitigate future pandemic-level threats, Pillar 4 set out to develop technologies to break technical limitations of current neutralization assays using genetic engineering and synthetic biology strategies.

VLP assays have formed the basis of CoVaRR-Net Pillar 4 research activities, however researchers are currently limited to testing VOCs against serum samples one-by-one, which is reactive, laborious, and time consuming. It delays the evaluation of whether neutralizing antibodies form when faced with various variants by several days or even weeks (and in that time, new variants can arise). Pillar 4’s research teams are working off this approach to develop multiplexed versions of these assays using a genetic tagging approach so that they will be able to measure the neutralization of multiple VOCs – perhaps tens (a mid-throughput approach) or hundreds (a high-throughput approach) – at once.

“It’s an ambitious and crazy approach, but I think it is highly worthwhile and can be done,” says Pillar 4 Deputy, Dr. Nozomu Yachie, Associate Professor, University of British Columbia. “This scalable technology aims to produce and screen multiple synthetic viral variant VLPs in fewer pseudotyping and infection steps and could be modified for future pandemic threats. While research is ongoing, proof-of-principle testing of the mid-throughput approach, using six variants at a time, has shown great promise.”

A lasting impact

While the work is still ongoing, Pillar 4 researchers have left a legacy that already puts us in a far better position in the event of a future pandemic threat.

For example, Pillar 4 reagents and experimental platform tools will enable future research to investigate how SARS-CoV-2 variants and other future pandemic threats interact with host proteins, such as antibodies, to look for correlates of protection to infection or disease.

The team has also developed an extensive spike DNA expression plasmid collection and has made these plasmids available to other researchers upon request. These reagents, distributed to numerous researchers throughout Canada and internationally, support equitable and open research to better understand SARS-CoV-2 function and improve health outcomes.

In terms of impact on forming the next generation of researchers in Canada, Pillar 4 trained 26 highly qualified personnel (HPQ) during the pandemic, 20 of those thanks to CoVaRR-Net funding.

Lessons Learned

CoVaRR-Net has taught us that there is benefit in continuing to invest in academic labs that are at the forefront of their fields. In fact, Canada’s rapid scientific response to the COVID-19 pandemic was only possible because of prior academic work.

“For example, Pillar 4’s contributions to the study of SARS-CoV-2 were only possible (as quickly as they were) because of its pre-existing expertise in viral protein production and its previous CIHR-funded research on the coronavirus spike protein,” Dr. Gingras explains. “Similarly, the establishment and scale-up of the Pillar 4 serology and neutralization assays, and the subsequent collaboration with clinical teams, were only possible because of project grants awarded prior to the COVID-19 pandemic.”

Ultimately, leveraging existing funding, personnel and other resources has proven paramount in the speedy response to a pandemic. As such, Canadian academic labs working in these areas should continue to be provided with base funding to support their peacetime efforts on an ongoing basis, so that they are ready to act with modest additional investment at the onset of a future pandemic.