Virus World

The continued evolution of SARS-CoV-2 requires persistent monitoring of its subvariants. Omicron subvariants are responsible for the vast majority of SARS-CoV-2 infections worldwide, with XBB and BA.2.86 sublineages representing more than 90% of circulating strains as of January 2024. In this study, we characterized the functional properties of Spike glycoproteins from BA.2.75, CH.1.1, DV.7.1, BA.4/5, BQ.1.1, XBB, XBB.1, XBB.1.16, XBB.1.5, FD.1.1, EG.5.1, HK.3 BA.2.86 and JN.1. We tested their capacity to evade plasma-mediated recognition and neutralization, ACE2 binding, their susceptibility to cold inactivation, Spike processing, as well as the impact of temperature on Spike-ACE2 interaction.

We found that compared to the early wild-type (D614G) strain, most Omicron subvariants Spike glycoproteins evolved to escape recognition and neutralization by plasma from individuals who received a fifth dose of bivalent (BA.1 or BA.4/5) mRNA vaccine and improve ACE2 binding, particularly at low temperatures. Moreover, BA.2.86 had the best affinity for ACE2 at all temperatures tested. We found that Omicron subvariants Spike processing is associated with their susceptibility to cold inactivation. Intriguingly, we found that Spike-ACE2 binding at low temperature was significantly associated with growth rates of Omicron subvariants in humans. Overall, we report that Spikes from newly emerged Omicron subvariants are relatively more stable and resistant to plasma-mediated neutralization, present improved affinity for ACE2 which is associated, particularly at low temperatures, with their growth rates.

Preprint in bioRxiv (Jan. 23, 2024):

https://doi.org/10.1101/2024.01.20.576353 

In Europe, the pandemic triggered in 2020 by the SARS-CoV-2 coronavirus is now largely under control. But why this virus is able to spread so efficiently remains unclear. A team of researchers led by Dr. Simone Backes, Dr. Gerti Beliu and Prof. Dr. Markus Sauer of the Julius Maximilians University of Würzburg (JMU) has now shown in a publication in "Angewandte Chemie" that some previous assumptions need to be reconsidered. For example, the virus does not bind with several surface proteins simultaneously to several receptors of the cell to be infected. This assumption has previously been an attempt to explain how viruses increase their infectivity. Binding to a single receptor also does not lead to the subsequent docking of further receptors to the virus. The Würzburg research group has now provided evidence that a single virus binds to a single receptor, opening the door for a highly efficient infection.

What could only be speculated about

SARS-CoV-2 carries an average of 20 - 40 spike proteins on its surface. With these, it binds to ACE2 receptors in the membrane of its target cells, for example in the nose and throat of humans. When these receptors are blocked with antibodies, the cell can no longer be infected. Making the ACE2 receptors and their interaction with the viral spike proteins visible microscopically has not been possible so far. Therefore, much was left to speculation - such as whether the viruses bind to multiple receptors with multiple spikes to facilitate entry into the cell. It was also considered that the receptors are present in the membrane in pairs or groups of three rather, so that they can bind more efficiently to the trimeric spike proteins. Or that they are only combined into such groups after binding to a spike protein. Both depend strongly on the density of the ACE2 receptors in the membrane.

Super-resolution microscopy made it clear

The Würzburg researchers wanted to elucidate this mystery: They labeled antibodies with dyes to make the receptors visible and countable. To do this, they used various cell lines that are used as model systems for SARS-CoV infection, and the single-molecule sensitive super-resolution microscopy method dSTORM, developed in Markus Sauer's research group. It turned out that Vero cells, for example, which are often used as a model for SARS-CoV-2 infection, only have one to two ACE2 receptors per square micrometre of cell membrane. This is very few: "In other membrane receptors, this number is often between 30 and 80," Sauer added. "The average distance between neighbouring ACE2 receptors is about 500 nanometres. It is thus much larger than a virus particle, which measures only 100 nanometres," says Backes. The idea that a virus particle with multiple spike proteins can bind to multiple receptors simultaneously is therefore very unlikely, she adds.

ACE2 receptors are always single

The following open question: Are the receptors also present as pairs or groups of three in the membrane? "No. They only occur there singly. And it stays that way even when a viral spike protein has bound to them," says Beliu, group leader at the Rudolf Virchow Center. For an infection, it is sufficient if a single spike binds to a single receptor. With these results, the JMU team was able to disprove many of the original hypotheses about the interaction of viral particles with multiple ACE2 receptors. It also showed that host cells with higher ACE2 expression are more easily to infect, as expected. However, the lipid composition of the membrane and other factors also influence infection efficiency.

What is next?

The JMU team wants to gather as much detailed knowledge as possible about the cell entry mechanism of coronaviruses in order to better understand the infection process. This could ultimately contribute to better prevention and the development of better drugs against COVID-19. Next, the Würzburg researchers want to analyse the entry mechanism with high-resolution light sheet microscopy.

Published March 2023:

https://doi.org/10.1002/anie.202300821 

ACE2 is the indispensable entry receptor for SARS-CoV and SARS-CoV-2. Because of the COVID-19 pandemic, it has become one of the most therapeutically targeted human molecules in biomedicine. ACE2 serves two fundamental physiological roles: as an enzyme, it alters peptide cascade balance; as a chaperone, it controls intestinal amino acid uptake. ACE2’s tissue distribution, affected by co-morbidities and sex, explains the broad tropism of coronaviruses and the clinical manifestations of SARS and COVID-19. ACE2-based therapeutics provide a universal strategy to prevent and treat SARS-CoV-2 infections, applicable to all SARS-CoV-2 variants and other emerging zoonotic coronaviruses exploiting ACE2 as their cellular receptor.

Published in Cell (March 2, 2023):

https://doi.org/10.1016/j.cell.2023.01.039

Patients infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation and thrombosis. This is thought to be associated with an impaired activity of angiotensin-converting enzyme-2 (ACE-2), which is the main entry receptor of SARS-CoV-2 and which also tightly regulates blood pressure by converting the vasoconstrictive peptide angiotensin II (AngII) to a vasopressor peptide. Here, we show that a significant proportion of hospitalized COVID-19 patients developed autoantibodies against AngII, whose presence correlates with lower blood oxygenation, blood pressure dysregulation, and overall higher disease severity. Anti-AngII antibodies can develop upon specific immune reaction to the SARS-CoV-2 proteins Spike or RBD, to which they can cross-bind, suggesting some epitope mimicry between AngII and Spike/RBD. These results provide important insights on how an immune reaction against SARS-CoV-2 can impair blood pressure regulation.

Available as Preprint at medRxiv (Nov. 2, 2021):

 https://doi.org/10.1101/2021.11.02.21265789 

A team of scientists studying the origin of SARS-CoV-2, the virus that has caused the COVID-19 pandemic, found that it was especially well-suited to jump from animals to humans by shapeshifting as it gained the ability to infect human cells.  Conducting a genetic analysis, researchers from Duke University, Los Alamos National Laboratory, the University of Texas at El Paso and New York University confirmed that the closest relative of the virus was a coronavirus that infects bats. But that virus's ability to infect humans was gained through exchanging a critical gene fragment from a coronavirus that infects a scaly mammal called a pangolin, which made it possible for the virus to infect humans.

The researchers report that this jump from species-to-species was the result of the virus's ability to bind to host cells through alterations in its genetic material. By analogy, it is as if the virus retooled the key that enables it to unlock a host cell's door -- in this case a human cell. In the case of SARS-CoV-2, the "key" is a spike protein found on the surface of the virus. Coronaviruses use this protein to attach to cells and infect them. "Very much like the original SARS that jumped from bats to civets, or MERS that went from bats to dromedary camels, and then to humans, the progenitor of this pandemic coronavirus underwent evolutionary changes in its genetic material that enabled it to eventually infect humans," said Feng Gao, M.D., professor of medicine in the Division of Infectious Diseases at Duke University School of Medicine and corresponding author of the study publishing online May 29 in the journal Science Advances.

The researchers found that typical pangolin coronaviruses are too different from SARS-CoV-2 for them to have directly caused the human pandemic. However, they do contain a receptor-binding site -- a part of the spike protein necessary to bind to the cell membrane -- that is important for human infection. This binding site makes it possible to affix to a cell surface protein that is abundant on human respiratory and intestinal epithelial cells, endothelial cell and kidney cells, among others. While the viral ancestor in the bat is the most closely related coronavirus to SARS-CoV-2, its binding site is very different, and on its own cannot efficiently infect human cells. SARS-CoV-2 appears to be a hybrid between bat and pangolin viruses to obtain the "key" necessary receptor-binding site for human infection.

Original study published in Science Advances (May 29, 2020):

https://doi.org/10.1126/sciadv.abb9153

This study compares angiotensin-converting enzyme 2 (ACE2) gene expression, which has been associated with SARS-CoV-2 cell entry, in the nasal epithelium of children vs adults. 

We conducted a retrospective examination of nasal epithelium from individuals aged 4 to 60 years encountered within the Mount Sinai Health System, New York, New York, during 2015-2018. Samples were collected from individuals with and without asthma for research on nasal biomarkers of asthma. The study was approved by the Mount Sinai institutional review board. Written informed consent was obtained from participants (or their parents for minors). Nasal epithelium was collected using a cytology brush that was immediately placed in RNA stabilization fluid and stored at −80 °C. RNA was isolated within 6 months. RNA samples were checked for quality and sequenced as a single batch in 2018. Sequence data processing included sequence alignment and normalization of gene expression counts across genes and samples....

The results from this study show age-dependent expression of ACE2 in nasal epithelium, the first point of contact for SARS-CoV-2 and the human body. Covariate-adjusted models showed that the positive association between ACE2 gene expression and age was independent of sex and asthma. Lower ACE2 expression in children relative to adults may help explain why COVID-19 is less prevalent in children. A limitation of this study is that the sample did not include individuals older than 60 years.

Published in JAMA (May 20, 2020):

https://doi.org/10.1001/jama.2020.8707

The new coronavirus may be using part of our immune system against us.The novel coronavirus is known to use a keyhole called the ACE2 receptor to "unlock" a cell and get inside, where it replicates and wreaks havoc. Now, researchers have found that the gene that encodes the ACE2 receptor may be stimulated by interferons, one of the body's main defenses against viruses. When a foreign pathogen invades, interferons act like "first responders"; the immune system releases these proteins to alert other cells that there's a pathogen in the body. But when interferons rush to alert the body to the coronavirus, they're actually stimulating the gene that ramps up expression of ACE2 receptors, which may lead to more ACE2 production and allow SARS-CoV-2 to infect even more cells.....

This finding is important, given that interferons are being tested as a possible treatment for the new coronavirus. However, it's still unclear whether an interferon treatment would help or harm patients. "It might be that in some patients, because of the timing or the dose, interferon can contain the virus, while in others, interferon promotes more infection," Ordovas-Montanes said in a statement. "We want to better understand where the balance lies and how we can maintain a productive antiviral response without producing more target cells for the virus to infect."...

Original report to be published in Cell:

https://doi.org/10.1016/j.cell.2020.04.035

https://www.cell.com/pb-assets/products/coronavirus/CELL_CELL-D-20-00767.pdf

A new study published in April 2020 on the preprint server bioRxiv reports that the different levels of susceptibility of various organs to viral attack by the SARS-CoV-2 virus causing the huge pandemic of COVID-19 sweeping around the world can be predicted from the level of expression of two molecules, the ACE2 receptor, and the TMPRSS2 protein.

The SARS-CoV-2 is a catastrophic medical disaster, in which over 2.47 million cases have been reported with more than 169,000 people dead, as of April 20, 2020. The most common cause of death is pneumonia with acute lung injury, acute respiratory distress syndrome (ARDS) being the final event However, in some patients, many other organs also suffer severe damage leading to multi-organ failure and even death following infection. This motivated the current attempt to identify the organs that are vulnerable to this virus to understand better how it affects the body. It is already known that the virus accomplishes cell entry via a molecule called angiotensin-converting enzyme 2 (ACE2), which acts as a receptor for both this virus and the earlier SARS virus. Another protease, called TMPRSS2, within the cell, enhance the transmissibility of the virus. Thus the investigators determined to test the potential usefulness of measuring the level of expression of these two proteins to predict the risk that any given organ will be susceptible to the virus.

The investigators used a method called scRNA-seq to detect the clustering of the gene expression of cells, and thus identify cell types. While most studies indicate that the virus causes mainly lung injury, it is known that the ACE2 receptor is chiefly expressed in the lung’s type II alveolar cells (AT2). This means this cell type is vulnerable to the virus. The TMPRSS2 is required to initiate the expression of the spike (S) protein, which is key to the entry of the virus and its spread between hosts.

Preprint Available at medRxiv (April 18, 2020):

https://www.biorxiv.org/content/10.1101/2020.04.16.045690v1

Immunocompromised patients often fail to raise protective vaccine-induced immunity against the global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Although monoclonal antibodies have been authorized for clinical use, most have lost their ability to potently neutralize the evolving Omicron subvariants. Thus, there is an urgent need for treatment strategies that can provide protection against these and emerging SARS-CoV-2 variants to prevent the development of severe coronavirus disease 2019.

Here, we report on the design and characterization of a long-acting viral entry-blocking angiotensin-converting enzyme 2 (ACE2) dimeric fusion molecule. Specifically, a soluble truncated human dimeric ACE2 variant, engineered for improved binding to the receptor-binding domain of SARS-CoV-2, was fused with human albumin tailored for favorable engagement of the neonatal fragment crystallizable receptor (FcRn), which resulted in enhanced plasma half-life and allowed for needle-free transmucosal delivery upon nasal administration in human FcRn-expressing transgenic mice. Importantly, the dimeric ACE2-fused albumin demonstrated potent neutralization of SARS-CoV-2 immune escape variants.

Published in P.N.A.S (December 2023):

https://doi.org/10.1093/pnasnexus/pgad403

Human monoclonal antibodies (mAbs) that target the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein have been isolated from convalescent individuals and developed into therapeutics for SARS-CoV-2 infection. However, therapeutic mAbs for SARS-CoV-2 have been rendered obsolete by the emergence of mAb-resistant virus variants. Here we report the generation of a set of six human mAbs that bind the human angiotensin-converting enzyme-2 (hACE2) receptor, rather than the SARS-CoV-2 spike protein. We show that these antibodies block infection by all hACE2 binding sarbecoviruses tested, including SARS-CoV-2 ancestral, Delta and Omicron variants at concentrations of ~7–100 ng ml−1. These antibodies target an hACE2 epitope that binds to the SARS-CoV-2 spike, but they do not inhibit hACE2 enzymatic activity nor do they induce cell-surface depletion of hACE2. They have favourable pharmacology, protect hACE2 knock-in mice against SARS-CoV-2 infection and should present a high genetic barrier to the acquisition of resistance. These antibodies should be useful prophylactic and treatment agents against any current or future SARS-CoV-2 variants and might be useful to treat infection with any hACE2-binding sarbecoviruses that emerge in the future. A suite of human monoclonal antibodies block infection by all human ACE2 binding sarbecoviruses, including severe acute respiratory syndrome coronavirus 2 variants, at subnanomolar concentrations in cell culture and protect mice against severe acute respiratory syndrome coronavirus 2 infection.

Published in Nature Microbiology (May 15, 2023):

https://doi.org/10.1038/s41564-023-01389-9 

Scientists have created a first-of-its-kind mouse model of the COVID-19 disease, capable of revealing how the SARS-CoV-2 virus wreaks havoc on multiple organ systems in the animal's body. While this experimental model doesn't directly correspond to human coronavirus cases, it's a breakthrough in other ways, giving us a potential test system for exploring how the deadly respiratory pathogen extends way beyond the respiratory system in many cases of infection. "This mouse model is a really powerful tool for studying SARS-CoV-2 in a living system," explains cardiologist Arjun Deb from UCLA. Mouse models of COVID-19 have been engineered before, but none have gotten us to this point, the researchers say, by demonstrating what extra-pulmonary manifestations of COVID-19 look like. That's been a significant limitation for animal-based research into the virus's progression, and it's due to a couple of reasons. While mouse cells contain an analogue of the ACE2 receptor that the SARS-CoV-2 uses to bind to human cells, the virus doesn't seem to attach to the mouse version of the molecule. Genetically engineering mice with the human version of ACE2 provides a workaround of sorts, but before now, scientists hadn't succeeded in inducing multiple organ failure in mice, which could mimic the way human cases of extra-pulmonary infection tend to present. The shortfall may have been because prior studies used nasal inoculation on mice, infecting the animals with SARS-CoV-2 through the nose, which doesn't seem to develop into full-blown, system-wide coronavirus infections in mice.

In the new study, led by first author Shen Li, a cardiologist at UCLA, the team got around this by injecting the virus into the bloodstreams of engineered mice, where it could reach the human version of ACE2 (called hACE2) in the heart and other vital organs. Unfortunately for the subjects, the experimental tweaks worked. And while it wasn't a good outcome for the mice involved, being able to study systemically induced toxicity in SARS-CoV-2-infected mice could significantly expand our knowledge of the virus's branching capabilities in human cases. Such knowledge is sorely needed. "Among COVID-19 patients, those who have organs involved other than the lungs are most at risk of a bad outcome," Deb says. "So we felt it was really important to understand how the virus affects those other organs." In the experiment, systemic administration of the infection provoked rapid results. Within seven days, the infected mice "demonstrated profound morbidity, severely restricted activity, and were found huddled at the corner of the cage", in contrast with a control group of similarly engineered mice who were spared the infection, remaining healthy. In the same amount of time, the infected mice lost up to about 25 percent of their body weight due to sharply reduced food consumption, necessitating euthanisation. The infected mice also had damaged spleens, irregular heart activity and blood pressure, and altered levels of immune cells – all symptoms resembling human cases of COVID-19.

After the animals were euthanised, analysis of their organs revealed changes in gene expression in multiple tissues, disrupting cellular processes that generate energy in the body. "If a virus snuffs out the energy-generating pathways in multiple organs of the body, that's going to really wreak havoc," Deb explains.  Beyond these effects, the infected mice also bore numerous signs of epigenetic changes, which could explain the altered gene expression evident in multiple organs. It's not known for sure, but the impacts of this could potentially be felt long after an infection has been beaten by the immune system – and, hypothetically speaking, could be the basis of the prolonged symptoms experienced by COVID-19 'long haulers'. "Although the physiological significance of SARS-CoV-2 altered DNA methylation patterns is not clear from our study, our model provides proof of concept that such epigenetic changes do occur soon after SARS-CoV-2 infection and can potentially led to persistent transcriptional changes affecting tissue homeostasis and organ function," the authors write in their paper. "Such epigenetic changes potentially occurring in humans with COVID-19 could lead to symptoms from persistent changes in dysregulated gene expression in infected tissues even in the absence of tissue viral burden."...

Cited Research Published in JCI Insight (Dec. 7, 2020):

https://doi.org/10.1172/jci.insight.145027

The nose is the probable starting point for COVID-19 infections. Richard Boucher and Ralph Baric at the University of North Carolina at Chapel Hill and their colleagues tracked the ease with which the new coronavirus infects various cell types in the respiratory tract. The researchers found a gradient of infectivity that decreases from the upper to the lower respiratory tract: the most easily infected cells are in the nasal cavity, and the least easily infected deep in the lungs. (Y. J. Hou et al. Cell http://doi.org/dw2j; 2020). That gradient mapped neatly onto the distribution of cells that express ACE2, a protein that SARS-CoV-2 uses to enter cells.

The authors speculate that the virus gets a foothold in the nose, then sneaks down the respiratory tract when breathed into the airways. They say the results support the use of masks and preventative measures such as nasal cleansing...

Original Study Published in Cell (May 26, 2020):

https://doi.org/10.1016/j.cell.2020.05.042

Men have higher concentrations of ACE2 in their blood than women. As ACE2 enables coronavirus to infect cells, the findings may explain why men are more susceptible to COVID-19 infection than women. The study, published in the European Heart Journal today, also found that heart failure patients taking drugs targeting the renin-angiotensin-aldosterone system (RAAS), such as angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs), did not have higher concentrations of ACE2 in their blood. 

Dr Adriaan Voors (MD-PhD), Professor of Cardiology at the University Medical Center Groningen (The Netherlands), who led the study, said: “Our findings do not support the discontinuation of these drugs in COVID-19 patients as has been suggested by earlier reports.” Some recent research suggested that RAAS inhibitors might increase concentrations of ACE2 in plasma – the liquid part of blood – thereby increasing the risk of COVID-19 for cardiovascular patients taking these drugs. The current study indicates that this is not the case, although it looked only at ACE2 concentrations in plasma, not in tissues such as lung tissue. In addition, the study cannot provide definitive evidence on the effects of RAAS inhibitors in patients with COVID-19. Its conclusions are mainly restricted to heart failure patients, and the patients did not have COVID-19, so the researchers cannot provide a direct link between the course of the disease and ACE2 plasma concentrations. Prof Voors said: “ACE2 is a receptor on the surface of cells. It binds to the coronavirus and allows it to enter and infect healthy cells after it is has been modified by another protein on the surface of the cell, called TMPRSS2. High levels of ACE2 are present in the lungs and, therefore, it is thought to play a crucial role in the progression of lung disorders related to COVID-19.”

The researchers measured ACE2 concentrations in blood samples taken from two groups of heart failure patients from 11 European countries. There were 1485 men and 537 women in the first group, the index cohort, which was designed to test the researchers’ hypotheses and research questions. Then the researchers validated their findings in a second group of 1123 men and 575 women, the validation cohort. The median (average) age of the participants in the index cohort was 69 years for men and 75 years for women, and in the validation cohort it was 74 and 76 years, respectively. When the researchers looked at a number of clinical factors that could play a role in ACE2 concentrations, including the use of ACE inhibitors, ARBs and mineralocorticoid receptor antagonists (MRAs), as well as a history of chronic obstructive pulmonary disease, coronary artery by-pass graft and atrial fibrillation, they found that male sex was the strongest predictor of elevated ACE2 concentrations. In the index cohort, ACE inhibitors, ARBS and MRAs were not associated with greater ACE2 plasma concentrations, and in the validation cohort, ACE inhibitors and ARBs were associated with lower ACE2 concentrations, while MRAs were only weakly associated with higher concentrations.

Published in European Heart J. (May 10, 2020):

https://doi.org/10.1093/eurheartj/ehaa373

Researchers in British Columbia have identified a set of gene variants that are likely to influence disease progression from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection to COVID-19 disease. They have identified a rare set of variants in specific amino acid residues on the protein angiotensin-converting enzyme 2 (ACE2) that affect the viral spike protein’s ability to bind to and enter human cells. A spike protein is a key structural protein on the surface of SARS-CoV and SARS-CoV2 (the causative agent in COVID-19) virions that enables interaction with human cell receptors and fusion with the host cell membrane....

For their study, the team used the gnomAD database – a resource cataloging genetic coding variants for 141,456 adults. They assessed entries on ACE2 in the database and downloaded files predicting missense variants in the protein. The database cataloged 242 coding missense variants, 15 of which were predicted to be located at or close to the ACE2 binding site for SARS-CoV-2 spike protein. On aggregating the frequency of these variants, the team estimated that they occur in approximately 3.9 per 1,000 males and 8.5 per 1,000 females. However, the distribution of these rare alleles is not even across subpopulations, and the only common one was rs41303171 (thought to encode p.Asn720Asp), which occurred in 1.7% of males and 3.2% of females in the database. 

Although the occurrence of this allele was frequent enough to be identified in genome-wide association studies, it does not lie in the ACE2 domain thought to be bound by SARS-CoV-2 and was therefore not considered a worthwhile candidate for this interaction. The second most common variant was rs4646116 (thought to encode p.Lys26Arg), which was predicted to lie next to the ACE2-Spike protein interface.

Preprint of the original study available at bioRxiV (April 14, 2020):

https://www.biorxiv.org/content/10.1101/2020.04.05.026633v1