SARS-CoV-2, the virus behind the COVID-19 global pandemic, has presented scientists with a variety of conundrums. Where did the virus come from? Why is there so much clinical variability across patients? Does the virus mutate – if so, to what extent? How does this impact vaccine design? Such puzzles have brought together some of the top researchers from across the globe working in unity to uncover desperately sought-after answers. Nonetheless, certain aspects of COVID-19 infection remain unsolved. One key elephant in the room is the current lack of effective therapeutics with which to fight the virus.
What if we can trick SARS-CoV-2?
The virus invades human cells when its spike (S) protein binds to a cell surface protein known as ACE2, which is involved in a variety of host physiological processes including blood pressure regulation. ACE2 takes on the role of a cellular doorway, and the SARS-CoV-2 S protein acts as a "key" that unlocks the door. Upon entry, a cascade of events take place that result in infection, proving fatal in some patients and leaving others completely asymptomatic.
Fool you, SARS-CoV-2
If you've ever been to a fun house at a carnival before, you're likely to have stumbled upon a scenario in which you are presented with several doors to choose from in order to progress through the house. At least one of those doors will be a trap door. Make a poor choice and you're lost, unable to reach your final destination.
If ACE2 is a cellular doorway for SARS-CoV-2, through which it must enter to cause infection – can we look to present it with an alternative "trick" doorway, effectively fooling the virus with a decoy? Soluble versions of the ACE2 receptor are being explored for this very purpose, and a recombinant form of the human angiotensin-converting enzyme 2 known as APN01 is currently in a Phase II human clinical trial.
"Viruses must bind a receptor on a host cell to attach and enter, so if the receptor is made in a form that is detached from a cell surface (known as soluble) it will compete with the cell receptor for binding sites on the virus. The virus doesn't know the difference and is rendered non-infectious or neutralized," Erik Procko, assistant professor of biochemistry and professor of biophysics and quantitative biology at the University of Illinois, told Technology Networks.
Benefits and challenges of decoy receptors
The great thing about using soluble receptors as decoys, Procko said, is that the virus cannot easily mutate to become resistant. If the virus was to mutate so that it could no longer bind to the decoy, it would also no longer bind to the natural receptor and lose its virulence. But there is a key challenge associated with this approach to fighting viral infections. "A problem with using virus receptors as soluble decoys is that they often bind weakly, nearly always less well than antibodies that are produced by the immune system to bind and neutralize viruses," Procko added. He is the leader of a research team that sought to overcome this challenge adopting a deep mutagenesis approach.1
What is deep mutagenesis?
"Deep mutagenesis is a Big Data approach to experimentally characterize the effects of mutations. Classically, a small number of mutations are introduced in a protein in a targeted way, which requires having some pre-conceived ideas or hypotheses about what mutations are worth making. In deep mutagenesis, thousands of mutations are made, and so it is possible to saturate the protein with mutations that scan the protein sequence. This is far too many to test individually, so instead, all the mutations are mixed together in a single population." – Erik Procko.
Procko and colleagues decided to explore the mutational landscape of ACE2 to further understand the interaction between ACE2 and the S protein of SARS-CoV-2. In doing so, they were able to scour for mutations that might increase the affinity of ACE2 for the S protein.
The researchers created a library of all the possible single amino acid substitutions that could span the interaction surface of ACE2. The library of genetic mutations was transiently expressed in a human embryonic cell line before being incubated with the receptor binding domain (RBD) of SARS-CoV-2 tagged with green fluorescent protein (GFP). A large number of the variant forms of ACE2 failed to bind to the RBD – as measured by the level of fluorescence – when compared to the wild type. However, there were a small number of the mutated forms of ACE2 that demonstrated higher binding signals.
What does “wild type” mean?
In genetics, the allele (form of a gene) that encodes a phenotype that is most commonly expressed in a population is known as the wild-type allele.
Collecting the cells, Procko and colleagues then applied the deep mutagenesis technique. "Following sequencing, we calculated which mutations were the 'good' ones because they are enriched, or become more frequent in the collected cells, whereas 'bad' mutations were lost. In this way, it is possible to characterize thousands of mutations simultaneously in a single experiment," he said.
Trawling through the data, the scientists pinpointed one soluble variant – nicknamed sACE2.v2.4 – which was singled out for further purification and characterization and found to possess a binding affinity comparable to neutralizing antibodies.
Procko explained that this purification and characterization was divided into three main steps, starting with an affinity binding experiment. "We can directly measure how quickly the decoy receptor associates with or goes on the virus spike and how quickly it dissociates or falls off the virus spike. Tight binders associate quickly and dissociate slowly," he said. sACE2.v2.4 was found to outcompete the wild-type soluble sACE2-IgG1 for binding to S protein-expressing cells. "A second lab at the University of Illinois helped by testing whether the decoy receptors can compete with antibodies in patient serum for binding to the viral spike. They found that the natural host receptor is a poor competitor, but the engineered variant [sACE2.v2.4] is very effective at competing with antibodies for the virus," Procko explained. It is important to note here that the sample cohort of COVID-19-positive patients was arguably small (N = 3).
"Virologists from the military labs at Fort Detrick were able to measure neutralization of the virus in tissue culture. The engineered decoy receptor sACE2.v2.4 is a potent inhibitor of the virus that rivals the effectiveness of the best antibodies," Procko added. The efficacy of monomeric sACE2.v2.4 in its ability to neutralize SARS-CoV-2 in culture was nearly two orders of magnitude greater than the wild-type receptor.
But whilst a soluble ACE2 receptor could be good news for spoiling SARS-CoV-2’s plans, could it also cause unwanted problems for our own systems? ACE2 has an important physiological role in regulating blood pressure and volume by cleaving angiotensin peptides into products that cause vasodilation. The issue of potential unwanted knock on effects is clearly a pertinent question for Procko and his team too, one with which they are all too familiar. “For many virus receptors this is a serious concern, and most of my lab's research is dedicated to engineering out the normal physiological roles of receptors to make them virus specific and safe.” However, in the case of ACE2, early results are looking promising: “Soluble ACE2 has been administered to humans and is safe. We are currently putting the engineered ACE2-based decoy in mice and have yet to observe any toxicity.” He continued, “In this case I am cautiously optimistic.”
Unlike the unwanted side-effects seen with other viral receptors, it would appear that the natural roles of ACE2 are actually working to their advantage. When cells become infected with SARS-CoV-2 they die, resulting in reducing levels of ACE2 in the body and consequently reduced ACE2 activity. This impacts blood pressure and volume and can be attributed to some of the clinical signs of COVID-19. “By administering soluble ACE2 as a decoy, not only is virus replication inhibited, but ACE2 activity in the body is rescued to directly address disease symptoms.” It would appear this could be a win all round. Procko added on the bonus effects of using soluble ACE2 receptors – this is "something that antibodies do not do.“
Hurdles for an academic lab, and preparing for future coronaviruses
The decoy receptor sACE2.v2.4 was only tested in cell lines in this study. Technology Networks explored whether Procko has intentions to take this research further into animal models and, perhaps eventually, humans. He said, "Yes, but reaching humans in a clinical trial is very expensive. To receive FDA approval for an investigational new drug, one needs to show safety in animals and that the drug can be manufactured with consistent quality. Both tasks are very expensive."
Procko and team are "giving it their full effort", as the decoy receptor method could present treatment options beyond COVID-19. "The decoy receptor very closely resembles the natural human receptor, and so any virus that uses ACE2 for cell entry is in principal susceptible to the decoy," he explained.
We know that there are other exotic animal coronaviruses out there that humanity may encounter one day. If there's one thing the COVID-19 global pandemic has emphasized, it's the importance of trying to be prepared for such a situation.
Procko provided Technology Networks a glimpse of what his laboratory are working on to that end, "In unpublished work, we have found that the engineered variant binds with high affinity to a half dozen spike proteins from bat coronaviruses, giving us some confidence that this decoy will also be effective against related viruses in the future. Thus far we have yet to find a single coronavirus that uses ACE2 which the decoy doesn't bind."
"The hurdles for an academic lab to reach the clinic are formidable, which is why drug development is dominated by a small number of large biotech companies with financial muscle. But it's not impossible and we are giving it our full effort." – Erik Procko.
Erik Procko was speaking to Molly Campbell and Dr Karen Steward, Science Writers for Technology Networks.
1. Chan et al. (2020). Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2. Science. DOI: 10.1126/science.abc0870