This post was reviewed by Dr. Scott Covey of UBC, one of our subject matter experts.
The paper we’re demystifying today can be found here, if you’d like to follow along.
This is yet another international study that was lead by Dr. Josef Penninger at the University of British Columbia. In addition to Dr. Penninger the team included researchers from Canada, Sweden, Spain and Austria.
SARS-CoV-2, the virus that causes COVID-19, is a coronavirus. SARS-CoV-2 uses a protein called ACE2 that’s found on the surface of some cells to sneak through the cellular membrane and infect the cell. For more information on how nCoV-19 uses ACE2, check out this sidebar, or this paper summary by the amazing M Manolya Sag.
ACE2 is also used by the 2003 SARS virus (SARS-CoV-2) to infect cells. In the years after the 2003 SARS epidemic, The Penninger group was actually the first group to confirm both in cells and in animal studies that ACE2 is the receptor that’s used by the SARS virus to infect cells (the formal name for this virus is SARS-CoV). They also found that having too much ACE2 on cells can lead to more severe disease in mice. Finally, they found that injecting the viral protein used by the SARS virus to bind with cells (also known as the spike protein, see more here) into mice decreased ACE2 expression, which lead to lung injury. With the arrival of the novel coronavirus the Penninger group found themselves uniquely positioned to take that previous knowledge and deploy it against this new enemy. The Penninger group recently published a review where they suggested that ACE2 has two different purposes: one biological, and one more sinister. ACE2 normally works to protect the lungs from injury. However in the case of infection with SARS-CoV or SARS-CoV-2, ACE2 also acts as a the side door that the virus sneaks into the cell through. Pretty sinister, huh?
In a normal human lung, ACE2 is usually found on the surface of a particular type of lung cells called alveolar epithelial type II cells. These cells normally make a sort of slippery mixture of lipids and proteins that helps the lungs and thorax expand during normal breathing and lowers the chance of lung collapse. When these cells are injured- for example by a viral infection- alveoli could collapse and gas exchange through the lungs can be reduced. This means the patient isn’t getting enough oxygen from their breaths, which can cause all sorts of problems. In particular, this could be a major cause of the severe lung injury that has been seen in some COVID-19 patients. ACE2 is also found on a variety of tissues besides the lungs including the heart, kidneys, blood vessels and intestine. This wide distribution of ACE2 might explain the multi-organ trouble that some COVID-19 patients experience.
In this paper the researchers report that clinical-grade human recombinant soluble ACE2 (henceforth known as ‘hrsACE2’) reduces viral growth in cells by a whopping 1000-5000x. Human recombinant soluble ACE2 is a slightly modified (recombinant) human ACE2 protein that is soluble, or floating free in solution, rather than being embedded in a cell membrane. hrsACE2 has already been tested in phase 1 and 2 clinical trials, which makes this particularly exciting. Beyond that, the researchers have shown that the virus can infect blood vessel and kidney organoids (organ-like models grown from stem cells for lab experiments) and that their hrsACE2 treatment significantly reduces this infection at early stages.
Now that we have that overview covered, let’s move to the paper. This particular study is very well laid out and very clearly written, and I’d encourage any readers to also take a look at the original study.
The first thing that the research team had to do was get some virus to be studied. Towards this they pulled out viral particles from a sample taken from a patient in Sweden. They successfully isolated virus particles in February 2020. Once they had virus on hand to study, the researchers shifted to their main research question: can clinical-grade hrsACE2 interfere with SARS-CoV-2 infection?
To test this they used Vero-E6 cells, which were also the cells they used to isolate the virus. They infected the cells with different numbers of SARS-CoV-2 virus particles. The level of virus growth was measured using viral RNA (1 RNA genome = 1 virus). They infected the cells in the presence or absence of hrsACE2 for 1 hour, then washed off the hrsACE2 and incubated the cells for 15 hours. In this experiment they found that 15 hours after the initial infection the cells that were treated with hrsACE2 had a lot less virus in them than the cells that didn’t get the hrsACE2 treatment. From this the researchers concluded that hrsACE2 can prevent the virus from attaching to and sneaking into cells. They found that the level of inhibition was determined by how much virus was present at the time of infection and the dose of hrsACE2- this is called dose-dependency.
(Interestingly, the researchers found that mouse recombinant soluble ACE2 (mrsACE2) does not inhibit infection. This means that the blocking effects of hrsACE2 are very, very specific, which is great news for a potential drug).
As a final experiment in this phase of the study, the researchers infected the cells with SARS-CoV-2 while in the presence of hrsACE2 or mrsACE2 for the full 15 hours. Once again, they found that hrsACE2, but not mrsACE2 (human, but not mouse) lowered the viral infection rates in their cells by a large amount. They also found that neither the human nor the mouse recombinant ACE2 was toxic to the cells.
The next stages of this study were, like all good science, based on some key observations. Firstly, the main spot that SARS-CoV-2 infects seems to be the lung, but the virus has also been found in stool and urine- which indicates a spread to intestines and kidneys. Spread to other organs usually happens via the blood. The virus can get into the blood (viremia) over the course of infection, but viral RNA is very rarely detected in the blood. Finally, the size of the virus (80-100 nm) suggests that the virus has to infect blood vessels first before it can infect other local tissues. This inspired the researchers to test whether SARS-CoV-2 can infect human blood vessels. In addition to this, the researchers knew that ACE2 is strongly expressed in specific parts of the kidney. This, combined with the presence of the virus in urine, meant that they also wanted to test if the virus could infect the kidney.
To test whether the virus could actually infect blood vessels, the researchers had to design another experiment. Now, obviously, there aren’t just spare human blood vessels (or kidneys) lying around for researchers to infect as part of an experiment (to quote our Prime Minister, ‘what a terrible mental image’). To get around this the group used stem cells to grow human capillary organoids. Capillaries are the small blood vessels in the body that form networks through body tissues. Oxygen and other nutrients are delivered to body tissues by the capillaries. Waste and carbon dioxide are picked up and removed by capillaries as well.
As a brief aside: organoids are becoming a big part of scientific research. They are small, simplified models of organs that allow researchers to carry out powerful studies in surroundings that mimic the behaviour of the actual human body.
Once the group had their human capillary organoids ready they infected them with SARS-CoV-2. Following the infection, they detected viral RNA in the blood vessel organoids with an increase in viral RNA (meaning an increase in virus) between days 3-6 after infection. This means that SARS-CoV-2 successfully infected the organoid and successfully made more copies of itself. They found that the viruses being produced by the human capillary organoids were capable of infecting Vero E6 cells- which means that the virus being made in the organoids was still infectious. When hrsACE2 was added to the organoids SARS-CoV-2 infection of the organoid cells was very reduced. hrsACE2 and mrsACE2 weren’t toxic to the blood vessel organoids either- which is great!
To test whether the virus can infect the kidney, the researchers made a kidney organoid from stem cells. When they tested individual cells from their kidney organoids, they found that cells in particular areas of the organoid expressed ACE2 in a way is typically seen in normal kidneys. These kidney organoids were also infected with SARS-CoV-2 and 6 days after infection the researchers found that the virus had successfully replicated and that the kidney organoid cells were producing viral particles that could infect other cells (progeny virus). When they added hrsACE2 to the kidney organoid cultures, they saw a big drop in SARS-CoV-2 infections of the organoid cells, in a dose-dependent way.
From these experiments the researchers concluded that both engineered human blood vessels and engineered human kidney organoids could be infected by SARS-CoV-2, and that this infection could be inhibited by hrsACE2. Their results also suggest that this hrsACE2 might act to protect against lung injury in addition to keeping SARS-CoV-2 from getting into target cells.
Something interesting to point out here- the paper clearly says ‘engineered’ human blood vessels and ‘engineered’ human kidney organoids. Why is this? Why not just say ‘blood vessels’ or ‘kidneys?’ The answer there is that early science- the really groundbreaking, first out of the gate stuff- is often done on model systems or model species. It would be irresponsible to claim that a treatment works all the time or in humans based on data showing it worked on a model system or species. The researchers who wrote this study were very careful to make that distinction, and responsible scientists always are.
As for future directions, the scientists behind this groundbreaking study aren’t willing to sit still. They want to look into whether the infection of vasculature and kidneys has a role in the multi-organ damage seen in some COVID-19 cases. Beyond that, and based on the observation that ACE2 is strongly expressed in cardiac tissue, they are interested in expanding their studies to heart organoids. They also want to study lung organoids. Their hope is that these multiple organoid studies will help them better understand the multi-organ issues that many COVID-19 patients experience.
This study, like all studies, has some limitations and the researchers were not shy about pointing them out. The study is designed to focus on early stages of infection- the first few hours after the virus gets into contact with at-risk cells. While their data shows very nicely that hrsACE2 can block SARS-CoV-2 infection at this early stage, they can’t make any sort of conclusions or predictions as to how effective this treatment would be at later stages of the disease. Additionally, they didn’t look at lung organoids in this study. The lung is the major target of SARS-CoV-2 and would be a good point for future investigation, as stated earlier. Finally, ACE2 in the kidneys has a role in a very complicated biochemical system called the RAS system, which is tied into many external biochemical pathways and can’t be properly simulated by a kidney organoid. This might limit what the researchers can observe using this system. To address these concerns the researchers hope to do further studies into the effect of hrsACE2 in later infection stages, both in cells and in animal models.
Overall this is a groundbreaking piece of research that has made an enormous stride forward in our search for a COVID-19 treatment. The team of scientists responsible for this data were uniquely suited for this investigation given that much of the key information within the field was found out by these very same researchers fifteen years ago! An important takeaway here: science has been in this fight since before we knew we were fighting- and science is going to keep fighting it until the very end.
