Research

NPA: Dr. Joanne Lemieux

Coronavirus’ RNA genome encodes for two polyproteins that are autocatalytically processed by viral proteases – PLpro and Mpro into 16 non-structural proteins (nsp1–16). Mpro cleaves two polypeptides at 11 sites to produce 13 mature proteins, and is therefore indispensable for virus replication (ref). Since Mpro is also very well conserved it is considered an attractive drug target. Nimatrelvir (PaxlovidTM), developed by Pfizer, a peptidomimetic inhibitor of Mpro, was approved by FDA and released on the market to treat SARS-CoV-2 in December 2021. However, nirmatrelvir suffers from rapid oxidative metabolism and requires co-dosing with ritonavir as part of PaxlovidTM3. We are developing new generation of oral antivirals which initially demonstrated some broad specificity against main strains of coronaviruses.

We aim to identify broadly specific inhibitors and concurrently develop a commercialized multiplex assay, which will include isolated Mpro enzymes from HCoV-229E, -OC43, -NL63, and -HKU1 strains, and MERS, SARS-CoV and SARS-CoV-2 for compound screening. The ability of screening inhibitors against multiple analytes in high-throughput manner will extremely benefit the program in terms of decreasing the cost and time towards our identification of an antiviral inhibitor with broad specificity

NPA: Dr. John Vederas

The overall goal is to construct more metabolically robust inhibitors of the main protease (3CL) of SARS-CoV-2 that causes COVID-19. These are based on the structures of nirmatrelvir (protease inhibitor in Paxlovid™) and of GC376 that cures feline infectious peritonitis (FIP) coronavirus in cats. Such compounds will also be tested with the main protease (3C) of human rhinovirus (a picornavirus) as its active site, substrate and mechanism are have some similarity. Successful analogs will provide leads for single agent antiviral drugs to treat acute COVID-19 and potentially other respiratory viruses.

GC376 is an injectable drug shown to cure usually fatal FIP coronaviral infections in cats. It is structurally related to nirmatrelvir and potently inhibits the SARS2 3CL protease and viral replication. Currently, it is not orally bioavailable and is administered to animals by injection. Specific objectives of this project include:

1) Chemical synthesis of analogs of nirmatrelvir and GC376 with sites of oxidation by CYP3A4 having hydrogen substituted by deuterium to slow metabolism by isotope effect;

2) Synthesis of SARS2 3CL protease inhibitors with the cyclic glutamine analog having methylene (CH2) next to nitrogen replaced by oxygen to block oxidation;

3) Testing of all analogs for rate of oxidative transformation by CYP3A4 and for inhibition of SARS2 3CL protease, FIP coronaviral 3CL protease and human rhinovirus 3C protease.

NPA: Dr. Troy Baldwin

A key element of a strong public health system is the availability of vaccines that stop people from getting sick or dying. As highlighted by the recent pandemic, vaccination reduced hospitalizations and deaths due to COVID-19 and sped up the return to a more normal way of life. Developing improved ways to generate vaccines and understanding the strengths and limitation of new vaccines is critical to prepare us for a future pandemic. We will study the capability of a new vaccine platform we recently created and determine whether it can be used as a universal vaccine platform.

NPA: Dr. Vanessa Meier-Stephenson

Influenza viruses remain one of the leading causes of worldwide pandemics and one of the several respiratory viruses deemed to have a high likelihood of precipitating the next global pandemic. While vaccination will certainly have a strong role in pandemic preparedness and control, so too will the development of directly targeting therapies that can be taken at the first sign of infection. Given influenza’s diverse host reservoirs (i.e., birds and swine), a therapeutics approach will need to derive from a strategy that is both robust and adaptable. One protein complex that is common to all influenza viruses is its multipurpose polymerase. This protein is the virus’ Swiss army knife and plays many roles in its replication process from beginning to end. Interfering with the ‘knife’s’ functioning at even one of its steps could have an important impact on the virus’ ability to replicate. We propose the use of a diverse molecular probe strategy to screen the nooks and crannies of influenza’s polymerase to determine the best way to jam its pocketknife. This process uses a library of millions of molecular probes and sequentially works to find a shortlist of ones that can bind the strongest. We can then use that shortlist of probes to determine how and where they have bound and whether any modifications will be needed before testing in different culture models. This molecular probe approach is not only a valuable tool against influenza virus, but for other potential emerging pathogens, giving us the ability to pivot to whichever key target is of greatest interest.

Developing this platform on-site will have significant implications for turn-around time to probing function and acquiring first drug options.

NPA: Dr. John Klassen

The goal of this project is to develop new treatments for a wide range of viral infections, including COVID19 and the flu. We will do this by creating molecules that make it harder for viruses to enter our cells. Many human viruses use sugars (glycans) on the surface of our cells to help them gain entry. We will make drug molecules that disrupt the production of the sugars that viruses use for cell entry.  We will use advanced analytical methods to identify the molecules that bind strongly and selectively to specific enzymes that produce a specific class of cell surface sugars. We will then test the ability of these molecules to block cell infection by SARS-CoV-2, the virus responsible for COVID19, to demonstrate proof-of-concept.

NPA: Dr. Sue Tsai

Obesity is a leading risk factor for severe respiratory infections (e.g., H1N1 and SARS-CoV2), yet the same population of individuals is also afflicted with increased risks of vaccine failure. Evidence shows that excessive inflammation leads to disrupted insulin signaling and metabolic homeostasis, and our own work indicates that insulin resistance intrinsic to adaptive immune cells impairs immunological memory. Here, we propose a proof-of-concept study, where we will test the effectiveness of administering anti-diabetes agents that ameliorate systemic metabolic homeostasis and insulin sensitivity, in boosting immunological memory. We will carry out a series of experiments where preclinical models of diet induced obesity will be subjected to insulin sensitizing therapies involving adiponectin receptor agonism, followed by survival and immune function analysis. We will then investigate the specific effects of the therapies on memory B cells and T cells via multi-omics approaches, to gain insights into the metabolic pathways that can be leveraged to perpetuate long lasting protection.

NPA: Dr. Wael Elhenawy

The antimicrobial resistance (AMR) crisis has reached alarming levels worldwide. Our arsenal of antibiotics is failing in the face of bacterial infections due to the rise in AMR and the simultaneous decline in drug discovery. While the COVID-19 pandemic is viral, it was associated with a surge in AMR due to secondary infections with multi-drug resistant bacteria. This poses a threat on vulnerable patients in hospitals and nursing homes in Alberta and the rest of the world. Therefore, there is a pressing need to develop new antimicrobials to face the burgeoning AMR crisis. In the proposed work, we combined computational and experimental approaches to expedite the discovery of antimicrobials at a low cost. We developed a screening pipeline that identified new anti bacterial agents.  We were able to show that these drugs have a broad spectrum against several pathogens like Escherichia coli, Salmonella enterica, and Vibrio  cholera. Future work includes testing the efficacy of these drugs in our established infection models. Together, our program provides a framework for the preclinical development of antimicrobial therapeutics.  

NPA: Dr. Matthew Macauley

Vaccines are one of the greatest feats in human history and have been the solution to many medical crises, including the recent SARS-CoV-2 pandemic. In the past, vaccines using inactive versions of the virus have been widely successful. While this has proven effective, it is challenging to make inactivated viral vaccines on scales large enough for the public during a pandemic. Moreover, vaccine quality control on large scales is another challenge. Modern vaccines use smaller pieces of the virus rather than the entire inactivated virus to generate an immune response. These vaccines that use a piece of the virus, rather than the whole virus, are referred to as subunit vaccines. The major advantage of subunit vaccines is their modular construction, enabling rapid scaling and greater quality control. Indeed, it is these advantages that led to lipid nanoparticles (LNPs) being the vaccine of choice during the COVID-19 pandemic. However, a huge open question is whether these types of vaccine elicit the long-term protection characterized by inactivated viruses. A fundamental difference between whole viruses (inactivated or otherwise) and LNPs, is how they initiate immune responses. In this project, we will develop liposomal nanoparticles that exploit the natural features of how viruses first stimulate immune response, to examine if this can produce a more robust immune response. In summary, will marry the advantages of subunit vaccines (modularity and scalability) with how our immune system captures and responds to viruses naturally.

NPA: Dr. Edan Foley

Our intestines host trillions of microscopic lifeforms that shield us from infection by harmful viruses and bacteria. In this project, we will use zebrafish to simultaneously test a panel of potentially beneficial bacteria for ones with the greatest protective benefits against the bacteria that cause salmonellosis, Salmonella enterica. Fish are excellent animals for these experiments. It is easy to work with large numbers in a very short period, their intestines and immune systems are remarkably like humans, and infected fish develop salmonellosis when we challenge them with Salmonella. By identifying probiotic bacteria that protect from salmonellosis, we hope to identify a novel therapeutic option to protect humans from one of the most common bacterial causes of hospitalization. We are particularly excited about the value of this research, as it is important that we have strategies in place to counter infectious bacteria when new pandemics emerge.

NPA: Dr. Maya Shmulevitz

Reovirus is a virus that doesn’t harm humans or any animals. We are testing if reovirus can be used as a vaccine delivery system in animals to generate immune responses against other viruses that are pathogens and harmful. Specifically, we are currently trying to develop reovirus as a possible vaccine system against a virus called norovirus that is a major stomach-bug. We created reoviruses that display small parts of the norovirus so that immunity can be built against those small parts (called antigens). We
now want to test these possible norovirus vaccines in mice. We will introduce the candidate vaccines to mice by two ways: by mouth and by inhalation. We will then test various specific aspects of the immune system to measure how well the mice can protect themselves against norovirus. Importantly, we will also do many assays to measure the safety of the vaccines and possible side effects. Eventually, candidate vaccines that show good activity and safety in mice will need to be tested in pigs with the actual norovirus infection too. Overall, the project builds capacity in Alberta to generate new vaccines and conduct many assays to test them both for how well they work, and their safety.

NPA: Dr. Kevin Kane

A variety of viruses infect the respiratory tract (lung) including influenza viruses, respiratory syncytial virus (RSV), and SARS CoV-2 (CoV-2). These viruses re-infect their hosts, often despite prior vaccination. A major problem is the general lack of long term “memory” for the virus by the immune system, and changes in the viruses that make them appear different and less recognizable by host immune memory, as the viruses evolve. Almost all vaccinations against respiratory viruses are delivered intramuscularly (usually in the arm). This approach does not result in immune memory cells taking up residence in the lung to be able to act as a first line of defense where viruses enter the lung. We, and others, have shown that vaccination via the airway (intranasally) substantially facilitates long term immune memory, and in our case against influenza virus, the immune cells take up residence in the lung and offer very effective protection against subsequent influenza infection. Secondly, to bolster the immune response in the lung we used a small molecule, an activating peptide (AP) that stimulates a receptor in the lung (PAR2). An augmenting effect on the immune response in vaccination, such as by AP, designates that molecule an “adjuvant”. The adjuvant function of AP is particularly useful, as there are few if any effective adjuvants that act in the lung. Thirdly, in our vaccination approaches intranasally, we include peptide fragments of conserved proteins internal to the virus to induce immune responses against these proteins that do not vary, by a subset of white blood cells called CD8+ T cells. In this way, it is much more difficult after vaccination and infection for the virus to replicate and spread when CD8+ T cells eliminate virus infected cells. We will examine the utility of intranasal delivery, AP as an adjuvant, and inclusion of conserved virus segments in vaccination against RSV, to offer long term immune memory, protection from subsequent infection, detectable immune responses that may mediate protection, and minimal or no pathology. These studies will assess the expanded utility of AP as an adjuvant, intranasal delivery and conserved virus proteins as targets in lung immunity against viruses.

NPA: Dr. Matthias Götte

While nobody can predict the nature of the virus that causes the next pandemic, we do know that certain families of viruses are associated with a high pandemic potential. Coronaviruses and influenza viruses are often named in this context, but there are many other viruses of concern. The so-called paramyxoviruses are also on top of this list. The measles virus is a prominent member of this family. However, the paramyxovirus family is with more than 75 viruses very large and vaccines or treatments are often not available. Here we propose to study four prototypic viruses that represent this family. We specifically propose to study the replication machinery of these viruses with the goal to identify inhibitors that block this machine. Ideally, these inhibitors can be developed into effective treatments that prevent severe disease caused by these viruses.

NPA: Dr. Justin Di Trani

Cryogenic electron microscopy (cryoEM) is a powerful technique that allows us to gain structural insights into biological macromolecules of interest. In order to acquire structural data using cryoEM it is necessary to first isolate the macromolecule of interest then apply the purified sample to a cryoEM grid for imaging. While there have been tremendous advancements in microscope technology as well as the algorithms for analyzing cryoEM data there have been much fewer break-through advances in preparing macromolecules of interest for acquisition on the microscope. The aim of this project is to create grids that bind directly to the macromolecules of interest allowing for one-step purification from a complex sample such as cell lysate or clinical isolates. These grids will be an inexpensive, easy-to-use, and robust way of preforming structural analysis on complex samples.

NPA: Dr. Xiali Lilly Pang

An important lesson we learned from COVID-19 pandemic is that the virus is most likely a causing agent for the next high-epidemic or pandemic, especially those viruses which can transmit between animals and humans. We have developed and applied new technologies to detect SARS-CoV-2, human influenza and RSV in sewages and provided early and comprehensive information of illness burden in various communities during and after the COVID-19 pandemic. Wastewater-Based Surveillance (WBS) has made a great contribution for public health in optimizing countermeasures against the pandemic, and the residents can view trends of COVID-19 in their communities and make informed decisions on their daily activities besides adopting essential hygienic procedures. We propose to use an upgraded detecting WBS platform by combining RT-qPCR/digital PCR with novel and comprehensive whole genomic sequencing (nWGS) to continuously monitor viruses likely to cause the next pandemic for 2 years (twice/week). The viruses we target are human influenza A/B, animal influenza (including high-risk subtype H5N1) and RSV. WBS for measles and poliovirus are also ready to be deployed if the public health authority issues an alert to the public. nWGS can detect more than 60 human viral pathogens in one sewage sample. Continuous WBS monitoring allows us to better understand the minor and major genetic changes of targeted viruses over time, seasonal variations of infection, and early detection of emerging virus. The new findings will be immediately reported to the local public health authority for its informed decision-making. The outcomes will also be shared with SPP-ARC investigators for timely development of new vaccine and treatment. Public Health communications and trends of circulating viruses under the surveillance in 10 communities across Alberta will be shared with Albertans via an upgraded WBS dashboard (covering 80% Alberta population). The knowledge gained at the end of the project improves public health resource planning for disease control and prevention, assists researchers and stakeholders with strategic development and investment, and protects the health of all Albertans.

• Assemble genes, genomes, clones, DNA variant libraries, and mRNA using the same platform
• Generate up to 32 different DNA fragments in <24 hr
• Generate up to 10 μg of DNA from cloned de novo synthesized fragments
• Synthesize up to 10 μg of mRNA per reaction in ~20 hours, option to include modified nucleosides - Clone into off-the-shelf or user-supplied custom vectors
• Low error rates (1:10,000 to 1:30,000)
• Built-in thermocycler and chiller

NPA: Dr. Matthew Macauley

Preparing for the next pandemic means having the infrastructure in place to development, test, and manufacture vaccines. Developing and testing new vaccines requires innovative approaches but also a firm understanding of the materials being used. In most cases, these materials are in the nanometer range. For examples, some of the most successful long-lasting vaccines are attenuated viruses, which members of the SPPARC team are aiming at advancing (e.g. Reovirus and Vaccinia virus). Newer generations of vaccines use nanoparticles loaded with mRNA. In both cases, particles are typically in a 50-200 nm range. Such particles require careful characterization to measure their size and heterogeneity (uniformity). While there are instruments that can measure the average size of particles in solution, there is only one reliable instrument, the Malvern Nanosight Pro, that allows for both parameters to be easily quantified. Quantifying heterogeneity is particularly important because it addresses the quality of the preparation - specifically the lack of clumping. Indeed, aggregates can give adverse effects in vivo and is a parameter that is important to understand. Accordingly, this new infrastructure will enable all members of

NPA: Dr. Howard Young

Cryogenic electron microscopy (CryoEM) is an advanced imaging technique used to determine a three-dimensional picture of biological molecules and complexes at near-atomic resolution. In this technique, the sample is rapidly frozen at very low temperatures (below -180°C), trapping it in vitreous ice (a glass-like, frozen state of water). The sample is then imaged from different angles using an electron microscope, where a beam of electrons passes through the sample. The recorded images of the sample can be used to determine atomic-level pictures of biological molecules and complexes. The requested equipment will aid in the preparation of frozen samples for cryoEM.

NPA: Dr. Maya Shmulevitz

Through this grant, the EVOS M7000 microscope and Biorad ChemiDoc™ MP Imaging System were purchased as a means to analyze immune responses to vaccines. The M7000 microscope allows for both light and fluorescent microscopy capabilities, including live on-stage incubation that controls humidity, CO2/O2 and temperature to permit time-course fluorescence analysis.

The imaging system allows for the quantification of experiments such as (1) Western blot analysis in chemiluminescence, chemi-fluorescence, and fluorescence channels, (2) plaque size measurements, (3) Coomassie and other colorimetric gel electrophoresis imaging, (4) UV imaging, (5) bacterial colony imaging and counting, and others. 

NPA: Dr. Sue Tsai

T and B cells are indispensable for an effective immune response specific to the inciting antigen(s). Detection of antigen-specific immune responses is thus an integral part of the vaccine development workflow. The enzyme-linked immune absorbent spot (ELISPOT) assay determines the frequency of antigen-specific T and B cells by detecting released effector molecules such as cytokines, antibodies and other protein analytes in response to activation with antigens in culture. The method is robust, accurate and sensitive, and enables quantitative single cell analysis of low-frequency T and B cells. The advantage of ELISPOT over methods such as flow cytometry is that it is less labor intensive (e.g., does not require highly skilled operator to run the samples) and provides reliable data with high throughput in a relatively short amount of time (e.g., 1-2 hr run time on a flow cytometer vs 3 min ELISPOT read time to analyze 96 samples on a multi-well plate). The newer technology called FluoroSpot also provides multiplexing options that enable the simultaneous detection of multiple cytokines and effector molecules (stained with fluorescent antibody probes) within the same cells.

NPA: Dr. Wael Elhenawy

Multiple apparatuses were acquired as a means to carry out a program focused on identifying inhibitors for cAMP receptor protein (CRP), a key transcriptional regulator in pathogenic bacteria, including: 

1. Emulsiflex-C3 HP Homogenizer
2. AKTA Go and HiLoad 16/600 Superdex 200 pg liquid chromatography system
3. DeNovix DS-11+ Microvolume Spectrophotometer
4. CellASIC ONIX2 Microfluidic System and bacterial plate reader 

NPA: Dr. Olivier Julien

The Lyophilizer BUCHI L250 is a crucial apparatus to ensure proper purification of protein products for bulk and single-cell proteomics. This equipment allows for efficient solvent evaporation prior to LC-MS/MS.

NPA: Dr. Justin DiTrani

To help the greater cryoEM community at the University of Alberta, this grant supports technologies will be centered around sample preparation, grid fabrication, grid preparation, and methods for advanced computational analysis for cryoEM, including:

1. EM ACE600 High Vacuum Sputter Coater
2. BIOCOMP Gradient Station
3. GPU Developer Workstation

NPA: Dr. Vanessa Meier-Stephenson

The ӒKTA pure 150 L is a liquid chromatography system that facilitates the purification of proteins and nucleic acids, supporting affinity, ion exchange, size exclusion chromatography techniques necessary to study cellular targets of viral infection. 

NPA: Dr. David Marchant

A microscope is a basic and integral tool for any microbiology laboratory. The EVOS M7000 microscope allows the ability to capture white-light images – in order to visualize cells, stained tissues, virus and parasite infection, and any other microscopic objects. With the alternate camera, the microscope can capture images of cells and viruses expressing fluorescent proteins or anything labeled with fluorescent antibodies.