By Jim Oldfield

An undercurrent of worry ran through the panel discussion on COVID-19 at the University of Toronto.

It was mid-February, three weeks before the disease would bring everyday life in Canada to a standstill. More than 30 researchers from across southern Ontario had come together to talk about the new coronavirus and how they could limit its spread.

The researchers were keen to share resources and co-ordinate work. A few asked if the virus would fizzle and spare Canada the kind of outbreak that was gripping China.

There was a sense of looming challenge ahead, says Natasha Christie-Holmes (PhD ’08), who manages U of T’s Combined Containment Level 3 Unit (C-CL3) for the study of high-risk human pathogens.

“Almost everyone agreed this was the one,” says Christie-Holmes. “This was the virus that hit the right balance of transmissibility and impact. There was no question it would be a pandemic.”

After the panel, Samira Mubareka, an assistant professor of laboratory medicine and pathobiology who helped organize the event, greeted Christie-Holmes with a side hug and said, “We’re gearing up fast, right?”

“That is the plan,” said Christie-Holmes.

Urgency and collaboration marked many early responses to COVID-19 in Canada, from federal economic programs and public health directives to selfless work by frontline clinicians, creative efforts by retailers, and volunteer work by students who cared for elders, sewed face masks and helped with contact tracing.

But the collective need for speed was also acute in the less visible work needed to ready the country for COVID-19 research.

Throughout January, Christie-Holmes and many others at U of T drafted regulatory documents with U of T’s central offices, streamlined training in the C-CL3 and secured approval from the Public Health Agency of Canada to work with the virus.

Those efforts enabled the fundamental research that is now starting to improve diagnostics, underpin new public health and clinical guidelines, address shortages in personal protective equipment, and drive drug and vaccine development that will further control and ultimately end the pandemic.

The C-CL3 at U of T was the only lab in Toronto, and one of just a few in Canada, that could anchor that research.

“CL3 labs are difficult and expensive to operate due to containment and regulatory requirements,” says Scott Gray-Owen, the academic director of the C-CL3 and an infectious disease researcher in the Department of Molecular Genetics. “But their importance becomes clear in a devastating pandemic. They allow exceptional scientists with varied expertise to come together and really make a difference.”

Isolation of the Virus

Making a difference with basic research in a viral pandemic requires the ability to sample and reproduce the virus — and the C-CL3 has been key in that work.

“It was obvious we needed to isolate this virus for research in Canada, especially given the difficulties of shipping it across international borders,” says Mubareka, a microbiologist at Sunnybrook Health Sciences Centre and long-time user of the C-CL3.

While Mubareka was urging the pandemic pivot in the C-CL3, she was coordinating a research conference with Arinjay Banerjee, a postdoctoral fellow in Karen Mossman’s lab at McMaster University in Hamilton. Banerjee had studied diseases that leap from animals to humans, known as zoonoses, at the University of Saskatchewan.

McMaster was still re-establishing its CL3 lab to study the virus when Mubareka and Banerjee began to discuss ways to isolate it.

“After we had our first patient case at Sunnybrook in late January, I said, ‘Let’s do it together here.’ I knew his experience with MERS meant he would have the right expertise, and I didn’t want to take chances with failure or safety,” Mubareka says.

The pair connected with Rob Kozak (Fellowship ’19), an assistant professor of laboratory medicine and a clinical microbiologist at Sunnybrook, and Patrick Budylowski, a doctoral student and one of four new staff hired in the C-CL3 for the pandemic.

Banerjee moved to Toronto in February, took bio-safety training in the C-CL3 and by mid-March had produced two isolates. The group would share the live virus or other viral material with more than 20 researchers over the next few months, and Canadian research on SARS-CoV-2 began to take off.

“That early work was incredibly important,” says Gray-Owen. “Almost all SARS-CoV-2 research at U of T is based on it. Most universities in Canada now have that viral stock, and many are exclusively using those two isolates.”

The Vaccine Race

Mubareka’s lab at Sunnybrook also shipped a sample from Canada’s first COVID-19 case to the National Microbiology Lab in Winnipeg, which shared it with the Vaccine and Infectious Disease Organization – International Vaccine Centre (VIDO-InterVac) at the University of Saskatchewan.

Researchers at VIDO-InterVac also isolated the virus and have developed a promising protein-based vaccine, which they’ve shown to be effective in ferrets. They are now testing it in hamsters and start clinical trials this fall.

Kozak is using the Toronto isolates to evaluate a DNA-based vaccine with Gary Kobinger of Laval University in Quebec City, in work based at the C-CL3. Kozak says their vaccine produces a robust immune response pre-clinically, but securing funding for the work is challenging.

He is optimistic about the Phase 1 trials announced last week by Medicago, a Quebec company that plans to build a production facility in that province.

“All vaccine candidates will hopefully work about the same in terms of efficacy,” Kozak says. “But global supply chains are a huge issue with vaccines and therapeutics for this pandemic. We would be naïve to rely solely on one developer in any country, including our own.”

Matthew Oughton (PGME ’03) is an infectious disease physician at Jewish General Hospital and assistant professor at McGill University in Montreal. He says that too many opportunities to better understand and control the virus have been hindered by a lack of co-ordination and co-operation among groups, governments and countries.

“The example set by Dr. Mubareka and her colleagues in Toronto shows what we can achieve by collaboration, and has provided our country with a basis to confront this virus that will continue to benefit research and our population,” he says.

Drugs for Therapy

The quest to repurpose existing drugs as therapeutics is underway in dozens of labs across the country. Staff in the C-CL3 prioritize the many screening requests they receive based on each drug’s potential. They are testing six compounds now.

One promising approach is a team effort led by Jason Moffat (PhD ’02), a molecular genetics professor and a scientist at U of T’s Donnelly Centre for Cellular and Biomolecular Research. More than 20 researchers with expertise in genomics, proteomics, and computational and systems biology are looking for clinically approved drugs that target genes and proteins that the virus needs in order to replicate.

Their search is guided by CRISPR technology developed in Moffat’s lab, which can precisely identify those genetic factors by knocking them out one at a time.

“I love this study,” says Gray-Owen. “It’s fundamental biology but has really exciting translational potential, because if we find a gene or protein essential for replication and an available drug to target it, we could have a cure or therapy right away.”

Stopping Transmission

The need for therapies is urgent, but so is reducing the spread of the virus through respiratory droplets and surfaces. To that end, i3 BioMedical Inc. in Quebec recently asked Gray-Owen to test its patented antimicrobial technology on surgical masks exposed to the virus.

The technology works on wound dressings, gloves and other medical equipment, and last week the company announced that tests in the C-CL3 showed it deactivates the virus on masks, which could help limit transmission in health care settings and the community.

Transmission has also been a concern for human milk banks, which provide donor milk for vulnerable infants whose mothers are unable to produce enough of their own. Most milk banks in North America and Europe use the Holder method (62.5°C for 30 minutes) to pasteurize donor milk, and while that kills known pathogens, there had been no direct evidence the method worked for coronaviruses.

Sharon Unger (PGME ’99) and Deborah O’Connor recently tested the Holder method in milk spiked with SARS-CoV-2 in the C-CL3, and this month showed definitively that it kills the virus. The finding offers further assurance to families that the donor supply is safe, on the heels of initial reports that found traces of the virus in breast milk.

“This is the time you want to protect human milk feeding the most,” says Unger, a pediatrics professor and neonatologist at Sinai Health. “During the HIV/AIDS pandemic in the 1980s, all but one of the 23 milk banks in Canada closed over fear of spreading the virus. We didn’t want a similar situation this time.”

The pair also found that even unpasteurized milk knocked the virus back significantly after 30 minutes. O’Connor, a professor and the chair of nutritional sciences, will now lead research into the anti-viral properties of milk with federal funding announced in July.

The PPE Problem

The global shortage of N95 masks has been a source of intense anxiety and frustration for Canadian health care workers. To help address this problem, groups at U of T are working with local hospitals, the provincial government and Health Canada to validate mask decontamination methods for repeat use.

It’s one of the most comprehensive efforts to re-purpose respirators in the world and could meet the need for thousands of masks.

“We are systematically proving that if you sterilize one of these respirators, it will kill the actual virus while maintaining the mask’s integrity for re-use,” says Ori Rotstein (MD ’77, PGME ’82, MSc ’83), a surgery professor and the vice-president of research and innovation at Unity Health Toronto.

Unity Health will send unused respirators to the C-CL3, where staff will seed them with the virus and return them to Unity for decontamination with vaporized hydrogen peroxide. The C-CL3 staff will then test the masks to ensure the treatment has killed the virus.

James Scott and his lab will assess the masks for filtration efficiency after sterilization.

Scott is a professor in U of T’s Dalla Lana School of Public Health and Faculty of Medicine who has experience in aerosol science. He says the project has encountered many challenges including regulatory requirements, variations in sterilization equipment among hospitals, and a market for N95 respirators that he calls the “Wild West” when it comes to quality and claims about performance and testing.

“Frontline staff and the unions that represent them have legitimate concerns about this process, I completely get that,” Scott says. “I would ask the same questions. That’s another reason why we want to make sure our testing is accurate.”

The project includes other Toronto teaching hospitals and may span the province if the Ontario government follows through on plans to set up standardized sterilization centres. The U of T group is also developing bio-surrogates for the virus that will allow other hospitals to test their own equipment without the live virus.

A Better Future

New facilities and partnerships may be one of the most positive outcomes of COVID-19 in Canada and around the world. For the C-CL3, the legacy of this pandemic will include a city-wide biobank and a core virology facility, along with a much larger budget.

But whether that new funding and the frenzy of interest in prevention of infectious diseases will last is anyone’s guess.

Banerjee, now back at McMaster after isolating the virus in Toronto, isn’t betting on it.

“We see phases of interest in our research on high-impact zoonotic viruses, especially after an outbreak, but then people forget,” he says. “As humans, we move on very quickly. That has to change.”

We need to stop encroaching on wildlife and educate ourselves about pathogen spillover at the animal-human interface, he says. That would mean better regulations on deforestation, habitat loss and mixed farming practices that facilitate viral spread, as well as more effective management of wet markets.

“Look, it’s not like bats moved into our house. There’s plenty of evidence that shows when bats are stressed, they shed more virus,” Banerjee says.

“We need to tackle COVID-19 now. But in the future, instead of spending billions on a potential vaccine or therapeutic after a new virus has emerged, we should invest a fraction of that money to understand where these pathogens are coming from and why.”