NEW YORK – In recent years, developments in single cell and single molecule techniques have allowed proteomics researchers to characterize the body's immune response with improved depth and resolution.
Much of this research has been focused on immuno-oncology, but as the SARS-CoV-2 outbreak has turned into a pandemic, a number of companies and researchers in this space have turned their attention to better understanding how patients' immune systems respond to the virus.
"Our interest is in immune architecture and immune content and the populations of the immune system and the tissues they reside in and how they relate to each other and change under the influence of some kind of perturbation," said Stanford University Professor Garry Nolan, whose lab has been at the forefront of several single-cell protein imaging technologies. "So, we’re taking everything that we have been doing with cancer and autoimmune disease and applying it" to COVID-19 research.
Nolan is primarily using two platforms for his COVID-19 work — Akoya Biosciences' CODEX system and IonPath's multiplexed ion beam imaging system, both of which allow for multiplexed protein measurement and imaging at the single-cell level and both of which are based on research and development work done in Nolan's lab. Nolan was also a founder of both companies.
The goal, Nolan said, is to look at the immune response in various tissue types in samples collected from patients who have died from the virus.
"We'll get, I think, a nice view of what is happening to these patients and perhaps begin to correlate why certain forms of tissue damage will be related to, perhaps, preexisting conditions or genetics in these patients," he said. "Because the big question right now is why do some people do just fine while others die from cytokine storms and other seem to die from cardiac issues?"
In immune-oncology work, spatial protein analysis platforms like the CODEX and IonPath systems have allowed researchers to better characterize the tumor microenvironment to understand how interactions between immune cells and tumor cells as well as the spatial organization of these cells influences the course of the disease and patient's response to treatment. Nolan suggested these tools could similarly give researchers a better picture of the immune processes involved in COVID-19 infections.
"You'll see different cell populations," he said. "It's not just the total number of, say, CD4 T cell effectors or one or another of variety of macrophage. These cells work as parts of constantly reassembling machines. Cells come together for a local function and the go off to be parts in different functions. These [cellular] neighborhoods are important because they lead us to think, well, when this cell is next to this cell next to this cell and it always happens at this stage in disease, you can reasonably infer that doesn't happen by chance, that it has a function."
The ability to look at protein expression within these cells then provides further information, Nolan noted. "You can begin to discern what programs are activated within those cells. Are these the cells that are producing the cytokines? Are these the cells that are somehow suppressing locally the immune system?"
Nolan said his team's goal is to analyze mouse models and human tissue samples to generate basic research data on the virus as quickly as possible, putting out their findings as minimally processed data "that people who are working in the field can apply their thoughts, algorithms, and conclusions to."
"The idea is to get raw data out from tissues all over the body," he said. "Once you start to understand the relationship between the cells and how they act, that actually points to mechanism, and mechanism points to intervention."
Fluidigm's CyTOF mass cytometry technology similarly allows for highly multiplexed protein analysis at the single-cell level (in fact, the company has sued IonPath for patent infringement), and the company is supporting COVID-19 research being done by a number of it users, said President and CEO Chris Linthwaite.
Thus far, Linthwaite said, most COVID-19 research using the company's mass cytometry platforms has focused blood-based or cell lysate research, as opposed to the imaging work Nolan's lab is focusing on.
Ruth Montgomery, professor of medicine and epidemiology at Yale University and director of the university's CyTOF facility, is currently using a custom protein panel designed with Fluidigm to analyze cells from the airways of patients collected at 10 sites around the country to investigate how their different immune responses correlate with the course of their infections.
"They are looking at immune cell subsets that can give an indication of the course of COVID-19 progression and recovery and its impact on lung tissue," Linthwaite said.
At Belgium's KU Leuven, researchers have launched a study that is using the CyTOF as well as single-cell RNA sequencing and fluorescence-activated cell sorting to profile COVID-19 patients' immune response in peripheral blood, bronchial alveolar lavage samples, and lung tissue, comparing the immune responses of these patients to the immune profiles of patients suffering other respiratory infections like influenza.
Linthwaite said the company is also seeing uptake of an immune profiling product it launched last year, its MaxPar Direct Immune Profiling Assay, which allows researchers to look at 37 different kinds of immune cells, quantifying 30 proteins across these cells in around five minutes.
Customers "have adopted that panel now to look at immune response or prognostic and mechanistic biomarkers that could potentially guide treatment for COVID-19 patients," he said.
Single-cell proteomics firm IsoPlexis has to date largely marketed its IsoLight platform to researchers doing work on cancer immunotherapy, where it has proved useful for assessing immune cell cytokine production.
Given the apparently important role of overactive cytokine production, the so-called cytokine storm, in COVID-19 deaths, the company is now working with researchers at the Institute for Systems Biology in Seattle to analyze the cytokine production of immune cells taken from COVID-19 patients.
Singapore-based Proteona has likewise targeted its single-cell proteomics efforts at guiding and optimizing cancer immunotherapy but is now using it to address the COVID-19 crisis. The company's technology, called ESCAPE (enhanced single-cell analysis with protein expression), combines single-cell sequencing with DNA-barcoded antibodies targeting proteins of interest to allow researchers to simultaneously assess protein and gene expression.
Proteona hopes to use its platform to analyze B cells from recovered COVID-19 patients to identify antibodies that are highly effective in protecting against the disease.
"We realize that everything we have in place for single-cell proteogenomics would be a pretty good way to identify B cells in healthy, recovered COVID-19 patients, characterize them, and identify B cells that might actually give rise to the best possible antibodies," said CEO Andreas Schmidt.
The company has begun collecting samples in Germany and is now looking to expand to other locations around the world.
Schmidt said the company was particularly interested in pursuing the antibody work given that the blood cancer patients it worked with prior to the pandemic are highly susceptible to the virus.
Treatments for cancers like multiple myeloma typically suppress B cell function, "so you basically end up with no immune response," he said, adding that this means people undergoing such treatments who contract SARS-CoV-2 will very likely die from the virus.
Immunoassay firm Quanterix isn't focused on single cell analysis and has not been as involved in immune-oncology applications, but the company is finding demand for its high-sensitivity assays for studying immune response in COVID-19. Billerica, Massachusetts-based Quanterix's single-molecule array (Simoa) immunoassay technology provides up to 1,000-fold better sensitivity than a conventional ELISA, which makes it useful for looking at low abundance immune system proteins like cytokines and potentially for detecting early signs of seroconversion in infected patients.
"We see this as a moment where our company has been able to pivot a lot of our machinery into actually fighting COVID-19," said Kevin Hrusovsky, the company's president, chairman, and CEO.
The company is currently working with several groups on profiling cytokine and chemokine levels in COVID-19 patients, including a project with researchers at the University Hospital of Lyon to track cytokine and chemokine levels throughout the course of infection with the aim of identifying signatures that could inform clinicians as to how a patient's case might progress.
Quanterix is also developing serology assays for the virus with the idea being that its platform's high sensitivity could allow for testing either earlier in the disease process or perhaps using different, more easily accessible sample types, Hrusovsky said.
"We don't really know yet whether [serology testing] ends up benefitting from sensitivity," he said, but he noted this question was "an intriguing thing to explore," suggesting that a more sensitive assay could be useful if it allowed for testing in samples like saliva or dried blood spots.
"That's the type of thing we want to provide our customers and collaborators the opportunity to explore by getting some [serology] assays built on the Simoa technology," he said.
Reports have suggested SARS-CoV-2 may also have neurological effects, a factor Quanterix is also interested in exploring given the company's traditional focus on neurology. Hrusovsky said the company was working with an Italian team to use its neurofilament light chain (NFl) marker, which detects neuronal death, to look for signs of neurological damage in COVID-19 patients.