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>>> The novel coronavirus’ spike protein plays additional key role in illness
Salk researchers and collaborators show how the protein damages cells, confirming COVID-19 as a primarily vascular disease
SALK NEWS
April 30, 2021
https://www.salk.edu/news-release/the-novel-coronavirus-spike-protein-plays-additional-key-role-in-illness/
The novel coronavirus’ spike protein plays additional key role in illness
Salk researchers and collaborators show how the protein damages cells, confirming COVID-19 as a primarily vascular disease
LA JOLLA—Scientists have known for a while that SARS-CoV-2’s distinctive “spike” proteins help the virus infect its host by latching on to healthy cells. Now, a major new study shows that the virus spike proteins (which behave very differently than those safely encoded by vaccines) also play a key role in the disease itself.
The paper, published on April 30, 2021, in Circulation Research, also shows conclusively that COVID-19 is a vascular disease, demonstrating exactly how the SARS-CoV-2 virus damages and attacks the vascular system on a cellular level. The findings help explain COVID-19’s wide variety of seemingly unconnected complications, and could open the door for new research into more effective therapies.
Representative images of vascular endothelial control cells (left) and cells treated with the SARS-CoV-2 Spike protein (right) show that the spike protein causes increased mitochondrial fragmentation in vascular cells.
“A lot of people think of it as a respiratory disease, but it’s really a vascular disease,” says Assistant Research Professor Uri Manor, who is co-senior author of the study. “That could explain why some people have strokes, and why some people have issues in other parts of the body. The commonality between them is that they all have vascular underpinnings.”
Salk researchers collaborated with scientists at the University of California San Diego on the paper, including co-first author Jiao Zhang and co-senior author John Shyy, among others.
While the findings themselves aren’t entirely a surprise, the paper provides clear confirmation and a detailed explanation of the mechanism through which the protein damages vascular cells for the first time. There’s been a growing consensus that SARS-CoV-2 affects the vascular system, but exactly how it did so was not understood. Similarly, scientists studying other coronaviruses have long suspected that the spike protein contributed to damaging vascular endothelial cells, but this is the first time the process has been documented.
In the new study, the researchers created a “pseudovirus” that was surrounded by SARS-CoV-2 classic crown of spike proteins, but did not contain any actual virus. Exposure to this pseudovirus resulted in damage to the lungs and arteries of an animal model—proving that the spike protein alone was enough to cause disease. Tissue samples showed inflammation in endothelial cells lining the pulmonary artery walls.
The team then replicated this process in the lab, exposing healthy endothelial cells (which line arteries) to the spike protein. They showed that the spike protein damaged the cells by binding ACE2. This binding disrupted ACE2’s molecular signaling to mitochondria (organelles that generate energy for cells), causing the mitochondria to become damaged and fragmented.
Previous studies have shown a similar effect when cells were exposed to the SARS-CoV-2 virus, but this is the first study to show that the damage occurs when cells are exposed to the spike protein on its own.
“If you remove the replicating capabilities of the virus, it still has a major damaging effect on the vascular cells, simply by virtue of its ability to bind to this ACE2 receptor, the S protein receptor, now famous thanks to COVID,” Manor explains. “Further studies with mutant spike proteins will also provide new insight towards the infectivity and severity of mutant SARS CoV-2 viruses.”
The researchers next hope to take a closer look at the mechanism by which the disrupted ACE2 protein damages mitochondria and causes them to change shape.
Other authors on the study are Yuyang Lei and Zu-Yi Yuan of Jiaotong University in Xi’an, China; Cara R. Schiavon, Leonardo Andrade, and Gerald S. Shadel of Salk; Ming He, Hui Shen, Yichi Zhang, Yoshitake Cho, Mark Hepokoski, Jason X.-J. Yuan, Atul Malhotra, Jin Zhang of the University of California San Diego; Lili Chen, Qian Yin, Ting Lei, Hongliang Wang and Shengpeng Wang of Xi’an Jiatong University Health Science Center in Xi’an, China.
The research was supported by the National Institutes of Health, the National Natural Science Foundation of China, the Shaanxi Natural Science Fund, the National Key Research and Development Program, the First Affiliated Hospital of Xi’an Jiaotong University; and Xi’an Jiaotong University.
DOI: 10.1161/CIRCRESAHA.121.318902
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>>> COVID-19 is a vascular disease not a respiratory one, says study
Euronews
By Rafael Cereceda
20/08/2021
https://www.euronews.com/2021/05/06/covid-19-is-a-vascular-disease-not-a-respiratory-one-says-study
Healthy endothelial cells (left) and those treated with coronavirus protein S (right) show mitochondrial fragmentation in the vascular system.
A study at the University of San Diego claims to have proof that COVID-19 is not a respiratory illness, but a vascular one.
This could explain blood clots in some COVID patients and other issues like "COVID feet", which are not classic symptoms of a respiratory illness.
The study, published in the journal Circulation Research, shows with precision how virus damages the cells of the vascular system.
It was already known that besides the various symptoms of COVID-19 that coincide with respiratory problems, there are other cardiovascular issues that affect other parts of the body.
What’s new is the team conducting the study, which included scientists from the SALK Institute, showed the form in which the virus attacks the vascular or circulatory system.
The S protein of the virus, the spike that forms the crown, attacks the receptor ACE2, damaging the mitocondrias that generate the energy of the cells, thus damaging the endothelium, which lines the blood vessel.
This is something that has already been observed, but what wasn’t previously known is the exact mechanism and role of the S protein.
This protein is replicated by all of the currently available vaccines.
The scientists created a pseudovirus for the study, which only had the S protein but not the rest of the virus, to show in the lab that this protein is enough by itself to cause disease.
The effects on the respiratory system are a consequence of the inflammation of the vascular tissue in the lungs.
“A lot of people think of it as a respiratory disease, but it’s really a vascular disease,” says assistant research professor Uri Manor, who is co-senior author of the study.
“That could explain why some people have strokes, and why some people have issues in other parts of the body. The commonality between them is that they all have vascular underpinnings.”
Only an effect in serious cases?
Professor Rafael Máñez Mendiluce, who has been treating COVID-19 patients for a year as head of intensive care at Bellvitge University Hospital, says this is no surprise, given the clinical pictures presented by those who come to his department.
A year ago, he explained to us that the greatest risk of COVID-19 was the inflammatory symptoms presented by the patients. He also reminded us that blood clots are phenomena that occur in other viruses.
Máñez Mendiluce also wonders whether vascular problems do not occur only in the most severe patients, once the infection has already "conquered" the respiratory tract, spreading through the blood.
"Generally in mild patients, the infection is limited to the upper airways only,” he says.
He also believes the vascular problem could be related to the inflammatory response of the patient's immune system.
For Máñez Mendiluce, who has 30 years of experience in intensive care, this discovery does not drastically change the treatment possibilities for the most severe cases. He recalls that antithrombotic drug treatments have not proven to be particularly effective and that for the time being it is recommended to focus on the inflammation caused by the immune response.
This is a long-standing problem in intensive care. "We still don't have any treatment for thrombosis caused by the inflammatory response generated by the infection," he explains.
The discussion in the scientific community is still open, he adds. These problems are recurrent in intensive care units.
Máñez Mendiluce thinks this study does not call into question existing vaccines, although it should be better understood why the AstraZeneca and Johnson & Johnson vaccines have caused some rare cases of blood clotting.
The discovery has caused a stir. Some wonder whether it is dangerous for vaccines to inoculate precisely replicas of the S protein that appears to be capable of causing the disease.
Professor Uri Manor said on his Twitter account that, contrary to anti-vaccine claims, the study only shows that COVID-19 is a very insidious disease.
He explains that the amount of S protein in the vaccines is too small to be problematic.
He also says that the messenger RNA vaccine is much safer than getting the disease. "Everyone should get it, I did and everyone in my family did! Our paper just shows that this disease really sucks.
The Salk Institute researchers now hope to better understand the mechanism by which ACE2 receptors damaged by the S protein cause deformations and damage to mitochondria, which then cause problems in vascular tissue.
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>>> Effect of SARS-CoV-2 proteins on vascular permeability
eLife
https://elifesciences.org/articles/69314
Rossana Rauti, Meishar Shahoha, Yael Leichtmann-Bardoogo, Rami Nasser, Eyal Paz, Rina Tamir, Victoria Miller, Tal Babich, Kfir Shaked see all
Department of Biomedical Engineering, Tel Aviv University, Israel; School of Neurobiology, Biochemistry and Biophysics, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel; Sagol School of Neuroscience, Tel Aviv University, Israel; Blavatnik School of Computer Science, Tel Aviv University, Israel; Grass Center for Bioengineering, The Hebrew University of Jerusalem, Israel; The Center for Nanoscience and Nanotechnology, Tel Aviv University, Israel
Research Article Oct 25, 2021
Cited 0 Views 2,735 Annotations 0
DOI: 10.7554/eLife.69314
Article
Figures and data
Abstract
Severe acute respiratory syndrome (SARS)-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5_c145a (catalytic dead mutant of nsp5), and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein–protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by coronavirus disease (COVID-19). While validating the PPI network model, we found that the tight junction (TJ) proteins cadherin-5, ZO-1, and ß-catenin are affected by nsp2, nsp5_c145a, and nsp7 consistent with the model prediction. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-19.
Introduction
Coronavirus disease (COVID-19) caused by the 2019 novel coronavirus (2019-nCoV/SARS-CoV-2) led to a global pandemic in 2020. By late September 2021, coronavirus had infected more than 220 million people worldwide, causing over 4.5 million deaths. After the initial phase of the viral infection, ~ 30% of patients hospitalized with COVID-19 develop severe disease with progressive lung damage, known as severe acute respiratory syndrome (SARS), and a severe immune response. Interestingly, additional pathologies have been observed, such as hypoxemia and cytokine storm which, in some cases, lead to heart and kidney failure, and neurological symptoms. Recent observations suggest that these pathologies are mainly due to increased coagulation and vascular dysfunction (Lee et al., 2021; Libby and Lüscher, 2020; Siddiqi et al., 2020). It is currently believed that in addition to being a respiratory disease, COVID-19 might also be a ‘vascular disease’ (Lee et al., 2021), as it may result in a leaky vascular barrier and increased expression of von Willebrand factor (VWF) (Siddiqi et al., 2020), responsible for increased coagulation, cytokine release, and inflammation (Siddiqi et al., 2020; Teuwen et al., 2020; Aid et al., 2020; Potus et al., 2020; Wazny et al., 2020; Pum et al., 2021; Barbosa et al., 2021; Lin et al., 2020; Matarese et al., 2020; Xiao et al., 2020). Recent studies suggest that the main mechanism disrupting the endothelial barrier occurs in several stages: First, a direct effect on the endothelial cells that causes an immune response of the vascular endothelium (endotheliitis) and endothelial dysfunction. Second, lysis and death of the endothelial cells Teuwen et al., 2020; Xiao et al., 2020 followed by sequestering of human angiotensin I-converting enzyme 2 (hACE2) by viral spike proteins that activate the kallikrein–bradykinin and renin–angiotensin pathways, increasing vascular permeability (Teuwen et al., 2020; Varga et al., 2020). Last, overreaction of the immune system, during which a combination of neutrophils and immune cells producing reactive oxygen species, inflammatory cytokines (e.g., interleukin [IL]-1ß, IL-6, and tumor necrosis factor), and vasoactive molecules (e.g., thrombin, histamine, thromboxane A2, and vascular endothelial growth factor), and the deposition of hyaluronic acid lead to disruption of endothelial junctions, increased vascular permeability, and leakage and coagulation (Libby and Lüscher, 2020; Teuwen et al., 2020; Varga et al., 2020). Of great interest is the effect on the brain’s vascular system. Cerebrovascular effects have been suggested to be among the long-lasting effects of COVID-19. Indeed, the susceptibility of brain endothelial cells to direct SARS-CoV-2 infection was found to increase due to increased expression of hACE2 in a blood flow-dependent manner, leading to a unique gene expression process that might contribute to the cerebrovascular effects of the virus (Pober and Sessa, 2007).
While many studies point out the importance of the vascular system in COVID-19 (Kaneko et al., 2021; Jung et al., 2020b; Nägele et al., 2020), only a few Pons et al., 2020; Chioh et al., 2020; Nascimento Conde et al., 2020; Buzhdygan et al., 2020 have looked at the direct vascular response to the virus. Most of those reports stem from either clinical observations, or in vitro studies or in vivo studies in which animals/cells were transfected with the SARS-CoV-2 virus and their systemic cellular response assessed, without pinpointing the specific viral protein(s) causing the observed changes. SARS-CoV-2 is an enveloped virus with a positive-sense, single-stranded RNA genome of ~30 kb, encoding 29 proteins (Figure 1). These proteins can be classified as: structural proteins: S (spike proteins), E (envelope proteins), M (membrane proteins), N (nucleocapsid protein and viral RNA); nonstructural proteins: nsp1–16; open reading frame accessory proteins: orf3–10 (Kim et al., 2020; Hu et al., 2021). Table 1 summarizes the known effects of specific SARS-CoV-2 proteins (Gordon et al., 2020; Peng et al., 2020b; Procko, 2020; Cornillez-Ty et al., 2009; Romano et al., 2020; Hillen et al., 2020; Chi et al., 2003). The functionality of some of these is still unknown. Moreover, a considerable knowledge gap still exists regarding molecular mechanisms, especially the protein–protein interaction (PPI) pathways (Cowen et al., 2017), leading to tissue dysfunction.
(See original article for detailed list of (SARS)-CoV-2 proteins)
https://elifesciences.org/articles/69314
To tackle these challenges, we cultured human umbilical vein endothelial cells (HUVECs) and systematically transduced them with lentiviral particles encoding 26 out of the 29 viral proteins, separately. The three remaining genes were not included in this study purely for technical reasons. We then examined their effects on HUVEC monolayer permeability and the expression of factors involved in vascular permeability and coagulation. The results were analyzed in the context of virus–host and host–host PPI networks. By combining the insights from the experimental and computational results, we generated a model that explains how each of the 26 proteins of SARS-CoV-2, including a mutated form of nsp5, the catalytic dead mutant termed nsp5_c145a, affects the protein network regulating vascular functionality. Moreover, once the PPI model was validated with our experimental data, we applied it to more than 250 proteins that have been identified in the literature as affected by the SARS-CoV-2 proteins. This enabled us to pinpoint the more dominant SARS-CoV-2 proteins and chart their effects. Overall, this work shows how each of the SARS-CoV-2 proteins differentially affects vascular functionality; moreover, once the model was validated, we applied it to identify how SARS-CoV-2 proteins interact with proteins that have been significantly correlated with changes in cell functionality.
Results
SARS-CoV-2 proteins impair barrier properties affecting cell-junction proteins
Increasing numbers of studies indicate a significant role for the vasculature in the physiological response to SARS-CoV-2. However, neither the exact molecular mechanism that leads to these effects nor the individual contribution of any of the SARS-CoV-2 proteins is known. Plasmids encoding SARS-CoV-2 proteins were cloned into lentivirus vectors, with eGFP-encoding vector used as a negative control. To shed light on the vascular response to the virus, HUVECs were cultured on different platforms, transduced with these lentiviral particles, and assessed for the effects of the virus proteins on different functionalities. Culturing HUVEC on Transwells or glass coverslips (Figure 2a) allowed us to identify how the specific proteins affect endothelial functionality. To ensure proper infection, the control vector included a GFP label, which enabled us to estimate infection efficiency at around 70 % (Figure 2a). Since the most basic function of the endothelium is to serve as a barrier, we sought to identify the changes in endothelium permeability in response to the SARS-CoV-2 proteins, and to pinpoint which of these proteins have the most significant effect. Barrier functions and properties were measured via trans-epithelial-endothelial electrical resistance (TEER), a standard method that identifies changes in impedance values, reflecting the integrity and permeability of the cell monolayer (Srinivasan et al., 2015). The GFP control and nine SARS-CoV-2 proteins did not show any significant change in TEER values (compared to the untreated condition), whereas 18 of the SARS-CoV-2 proteins caused significant changes in value (see plot in Figure 2b). The most dominant permeability changes were observed with nsp5_c145a, nsp13, nsp7, orf7a, and nsp2, with a 20–28% decrease in TEER values (Figure 2—figure supplement 1, and Figure 2c), in which the different SARS-CoV-2 proteins are listed and the gradual color change from red to violet represents the progressive reduction in TEER values. Figure 2—figure supplement 1 shows the comparison in TEER values before the infection and 3 and 4 days after the infection, showing that the permeability changes in the cells exposed to the viral proteins are maintained.
Next, we analyzed some of the proteins that exhibited the most significant (nsp2, nsp5_c145a, and nsp7) or least significant (S) changes in TEER value for changes in expression of the cell-junction proteins such as CD31 (Figure 2d and e), cadherin 1–5, occludin, and ZO 1–3 (presented later), indicating altered barrier functions. Analysis of the immunocytochemistry (ICC) (Figure 2d and e) showed, as expected, that nsp2, nsp5_c145a, and nsp7 significantly reduce the expression levels of CD31 compared to the untreated, eGFP, and S conditions, suggesting a deterioration in barrier function. Hence, these data show a differential effect of SARS-CoV-2 proteins on endothelial functionality and provide a mechanistic explanation for the reduction in endothelial integrity.
Increased endothelial inflammatory response caused by SARS-CoV-2 proteins
It is known that SARS-CoV-2 can cause a severe cytokine storm (Pum et al., 2021; Wang et al., 2020a) and a significant increase in coagulation-related pathologies. As we were interested in identifying the role of the vasculature in these observations, we stained and analyzed the expression level of VWF (Figure 3a and b), which is highly correlated with coagulation (Rietveld et al., 2019). Similar to the CD31 staining, we characterized only those proteins that resulted in a significant decrease in TEER values (nsp2, nsp5_c145a, and nsp7). As shown in Figure 3a and b, the control samples did not exhibit marked expression of VWF, whereas the cells transfected with nsp2, nsp5_c145a, and nsp7 showed a significant change in VWF expression. Moreover, as VWF is also associated with increased inflammation (Kawecki et al., 2017), we monitored changes in cytokine expression due to the different SARS-CoV-2 proteins (Figure 3c). We were particularly interested in IL-6, which has been identified as one of the most dominant cytokines expressed due to SARS-CoV-2 infection (Wang et al., 2020a; Akbari and Rezaie, 2020; Peruzzi et al., 2020; Liao et al., 2020b; Liao et al., 2020a). We observed that 13 out of the 26 proteins caused an increase in IL-6 secretion, 3 of which had resulted in a decrease in barrier function and increased VWF expression.
Correlation between vascular permeability impairment and viral proteins
We then investigated how SARS-CoV-2 causes the observed changes in HUVECs permeability. We collected sets of proteins responsible for specific functionalities of endothelial cells. We also constructed an integrated viral–host and host–host PPI network. For each viral protein and each prior functional set, we measured the network proximity between the viral protein and the human functional set using a network propagation algorithm. We scored the significance of these propagation calculations by comparing them to those obtained on random PPI networks with the same node degrees. Proteins receiving high and significant scores were most likely to interact with the specific SARS-CoV-2 protein and thus might cause the observed functional changes. When comparing the overall effects of the 26 SARS-CoV-2 proteins on endothelial TJ proteins (e.g., cadherin 1–5, occludin, and ZO 1–3), we found a correlation between the effects of the SARS-CoV-2 proteins and TEER values (Figure 4a). Moreover, some of the proteins that significantly affected the TEER parameters (Figure 2c) were also observed to be significantly proximal to the permeability-related set. These included nsp2, nsp7, and nsp13 (Figure 4a). Our algorithm identified cadherin-2, a-catenin, ß-catenin, d-catenin, and ZO 1 and 2 as the most susceptible proteins to SARS-CoV-2 infection (Figure 4b).
To validate our PPI network model, we performed immunostaining of some TJ proteins (ß-catenin, cadherin-5, ZO-1, and occludin) of HUVEC transfected with viral proteins and to compare it to the model prediction. Similar to the CD31 staining, we characterized only those proteins that significantly decreased TEER values (nsp2, nsp5_c145a, and nsp7) compared to the eGFP and untreated condition (Figure 5). As shown in Figure 5a–c, the cells transfected with nsp2, nsp5_c145a, and nsp7 showed a significant reduction in the ß-catenin, cadherin-5, and ZO-1 intensity, confirming the ability of the SARS-CoV-2 proteins to impair endothelial permeability.
Once the model was validated, we used it to identify how the individual SARS-Cov-2 proteins affect nine other different vascular endothelial cells. As a starting point, we created a table (Table 2) (based on the literature) where we compared the expression of 12 different TJ proteins across nine different types of endothelial cells. We then applied the network-based model to identify which endothelial cells are more susceptible to the different SARS-Cov-2 proteins. As can be seen in Figure 6, there are significant differences in the response of various viral proteins on different types of vascular endothelial cells. For example,, the viral proteins nsp13, nsp11, orf6, and S seem to have a significant effect on the different types of vascular endothelial cells, according to the network score detected. However, the proteins m, E, n, nsp12, and nsp8 are the less interactive with the vascular cells.
(see original article for table) -
https://elifesciences.org/articles/69314
As our network propagation model is highly correlated with our experimental results, we applied it to other physiological systems that are known to be affected by SARS-CoV-2. We created a list of all proteins that are known to be affected by the SARS-CoV-2 proteins according to the literature (Supplementary file 1A, white columns). The table was composed of both proteins identified experimentally via western blot, proteomics, and immunohistochemistry (marked in blue) and those identified clinically as being highly correlated with loss of specific functionality in specific tissues (marked in red). We then applied the network-based model to identify which proteins in Supplementary file 1A are most susceptible to the different SARS-CoV-2 proteins. As can be seen in Figure 7—figure supplements 2–7, Supplementary file 1A and B, specific SARS-CoV-2 proteins were identified as affecting specific proteins in specific tissues. As expected, most of the SARS-CoV-2 proteins affected more than one protein, the most salient being nsp11, nsp4, and nsp7 (Figure 7b), each of which was predicted to affect more than 40 different proteins. An additional parameter that should be considered is the protein’s ‘distance’ from the viral proteins. This value represents the number of hops in the PPI network from a given protein to the viral proteins, where a value of 1 represents a direct viral–host connection. We hypothesized that the closer the distance between the viral proteins and the given protein, the more significant the viral effect. Supplementary file 1A (gray columns) and Figure 7c present the calculated distances. Most of the identified proteins in Supplementary file 1A were classified with a distance of 1 or 2 from the virus, suggesting more severe putative effects. A very clear example, are the T cells, macrophages, lung epithelial and cardiomyocytes which show that the most significant effect was by the viral proteins which present a close connection with the relative cell proteins reported. This suggest a potential effect on the related functional or metabolic pathway (Supplementary file 1A).
Discussion
Due to the impact of SARS-CoV-2, many studies have looked at the physiological responses to the virus (Lee et al., 2021; Libby and Lüscher, 2020; Siddiqi et al., 2020; Teuwen et al., 2020; Chioh et al., 2020). In this work, we sought to identify how specific SARS-CoV-2 proteins affect the vasculature by assessing the effect of individual SARS-CoV-2 proteins on endothelial cells (HUVEC). This approach has significant advantages: it enables pinpointing and isolating how each of the SARS-CoV-2 proteins independently affects the endothelial response, and directly measuring endothelial functionality. The HUVEC model, derived from the umbilical cord, is physiologically representative of the human vascular endothelium, allowing the study of the physiological and pathological conditions as well as the effects of novel drugs on human endothelium (Bouïs et al., 2001; Medina-Leyte et al., 2020). Among technical advantages, cultured HUVECs are a simple in vitro vascular endothelial model, particularly suitable for studying endothelial properties and dynamics as well as the putative role of adhesion molecules, the synthesis of extracellular proteins and blood vessel maturation (Vailhé et al., 2001).
The current study showed that almost 70 % (18 out of 26) of the SARS-CoV-2 proteins affect endothelial barrier integrity; however, the most significant proteins were nsp2, nsp5_c145a, and nsp7, which also induced upregulated expression of the coagulation factor VWF and cytokine release. These critical facts can shed light on the multiple pathologies observed in SARS-CoV-2 infection, including cytokine storm, increased coagulation and related diseases (e.g., heart attack and stroke) (Lee et al., 2021; Aid et al., 2020), increased cardiovascular disease, and increased neurological symptoms. The results presented here showed an effect of in vitro cultured endothelial cells, which may lead to vasculature leakiness, consequently causing exotoxicity (i.e., the penetration of toxic reagents from the blood into the brain). While there are many parameters associated with functional changes, the use of advanced tools, including network-based analysis, enabled us to elucidate the specific proteins and the specific interactions that are predicted to cause these changes. The PPI network enabled us to predict that the changes observed in barrier function are possibly due to interactions between host proteins such as cadherin 2, a-catenin, ß-catenin, d-catenin, and ZO 1 and 2, and at least with the viral proteins nsp2, nsp5_c145a, and nsp7. Moreover, we validated our PPI model performing further immunostaining analysis demonstrating not only the ability of the viral proteins to strongly impair TJ expression, but also to confirm the data predicted by our model in which some TJ proteins can be more affected than others.
PPI analysis revealed a highly correlated effect of nsp7 and nsp13 on ß-catenin in endothelial cells (Figure 4b; Jung et al., 2020a; Lengfeld et al., 2017). Interestingly, neither nsp2 nor nsp5_c145a affected a high number of proteins (Figure 7b), whereas nsp7 did, as identified by the network. Analyzing the repertoire of SARS-CoV-2 proteins, we see almost no effect of the structural proteins; rather, mostly nonstructural and open reading frame proteins affected HUVEC functionality, manifested as decreased barrier function and increased cytokine secretion (Figures 2 and 3). While the nonstructural proteins are mainly responsible for replicating viral RNA, the open reading frame proteins are related to counteraction with the host immune system; some of these are localized to the mitochondria and have been shown to alter the mitochondrial antiviral signaling pathway (Miller et al., 2021). We found that the proteins most affecting barrier function (decreased TEER and decreased CD31, ß-catenin, cadherin-5, and ZO-1 expression) and cytokine response (IL-6 secretion and VWF expression) were nsp2, nsp5_c145a, and nsp7 (Figure 2; Figure 3; Figure 6); nsp7 forms a replication complex with nsp8 and nsp12 that is essential for viral replication and transcription (Cowen et al., 2017; Peng et al., 2020a). Peng et al., 2020b suggested that in the core polymerase complex nsp7–nsp8–nsp12, nsp12 is the catalytic subunit, and nsp7 and nsp8 function as cofactors. They further suggested that the mechanism of activation mainly involves the cofactors rather than the catalytic subunit (Peng et al., 2020b). This might explain why we saw mainly an effect of the cofactor proteins on endothelial cells and almost no effect of the catalytic subunit. Network interactions Díaz, 2020 have shown that nsp7 has the most interactions with the host, suggesting a potential target for the treatment of COVID-19. Moreover, no mutations were found in nsp7 compared to nsp2 or nsp5_c145a (Kaushal et al., 2020), suggesting a conserved protein with a vital function in virus survival. The nsp13 protein has both helicase activity and 5’ triphosphatase activity, which play an important role in mRNA capping. We saw a significant effect of nsp13 on barrier function, but hardly any effect on cytokine secretion. Chen et al., 2020, suggested functional complexation between nsp8 and nsp12, the RdRp (RNA-dependent RNA polymerase) replication complex, and nsp13. Given the fact that we observed a substantial effect of nsp7 – one of the proteins of the replication complex – and an effect of nsp13 on HUVEC barrier function, complexation of nsp13 with the replication complex might indicate an important role for this complex in the impaired functionality of the HUVECs, and therefore in the propagation of the disease, and the known vascular damage seen in COVID-19 patients. As suggested by our model, nsp13 seems to have a strong effect also on other types of vascular endothelial cells (Figure 6) as well as on all cell types (Figure 7a), positioning nsp13 as one of the main targets for disease treatment.
It is important to note that the comparison between the different endothelial cell types revealed exciting differences in the TJ protein expression, which correlate to the different properties of the different cell types (Nakato et al., 2019). One of the major differences was that some endothelial cell lines do not have cadherin at all (e.g., HAoEC), or very limited amount of cadherin (e.g., HPAEC, HUAEC, HGSVEC). Our model suggests that some endothelial types (e.g., HUVEC, HUAEC, HGSVEC, HCAEC) are more susceptible to the SARS-Cov-2 virus. It, therefore, suggests that the treatment of one type of endothelial cell might be different from another type but offers the PPI model as a tool for initial prediction. Overall, the combination of identifying the differences in the TJ protein expression between the different endothelial cells and the use of the PPI model enabled us to pinpoint the differences in susceptibility to the disease and to identify which specific proteins have the most significant effect.
Many studies have looked at the SARS-CoV-2 interaction with nonpulmonary/nonvascular tissues (e.g., neurons, hepatocytes, immune components such as lymphocytes, macrophages, etc.) (Lee et al., 2021), as pathological studies identified a viral effect on these tissues, despite their very limited amount, or lack of ACE2 receptors. To better understand how SARS-CoV-2 interacts with and affects other tissues, we consolidated all of the proteins currently known to be affected by the virus into Supplementary file 1A. It is interesting to note that the most dominant SARS-CoV-2 proteins are nsp4, nsp11, and nsp7. Davies et al., 2020, identified the interaction of nsp2 with nsp4, both involved in endoplasmic reticulum (ER) calcium signaling and mitochondrial biogenesis. This suggests a new functional role in the host ER and mitochondrial organelle contact process and calcium homeostasis.
By now it is clear that vasculature plays a significant role in the physiological response to the virus. However, it is still unclear how the virus affects the vasculature, and if it can be found in the blood. This is a critical question, as it has significant consequences on the extent of the virus’s ability to affect the vasculature. Current studies demonstrate that the pulmonary vasculature is significantly affected and is one of the dominant triggers for the pathologies mentioned above. However, involvement with the rest of the vasculature is still unclear, as is whether the virus can be found in an active form in the blood circulation (Peng et al., 2020a; Chang et al., 2020; Orologas-Stavrou et al., 2020; Andersson et al., 2020; Escribano et al., 2020; Wang et al., 2020b). Some studies suggest that even if there are traces of SARS-CoV-2 in the blood, it is not in an active form and cannot cause disease or a systemic response (Andersson et al., 2020). On the other hand, some studies suggest that SARS-CoV-2 can be found in the blood, and can induce the disease and cause both cellular and systemic dysfunction (Peng et al., 2020a; Chang et al., 2020; Escribano et al., 2020). While this question is beyond the scope of this work, it is important to note that if future studies do identify the active form of SARS-CoV-2 in human blood, then the implications of our findings will apply to this systemic response as well (Ahmed et al., 2020; Park et al., 2020).
As already noted, the pathology is probably a combination of multiple conditions and pathways activated by the different proteins. However, our findings might open new avenues for future therapeutics. Moreover, most of the proteins that were identified as affected by SARS-CoV-2 had a distance factor of at most three to the human and viral proteins. This coincides with the current dogma, whereby proteins that have a shorter distance between them are more likely to be affected.
While beneficial, our approach has two major limitations: (a) our inability to identify the effect of multiple proteins and (b) our neglect of the effect of the coronavirus structure and binding on the cellular response. The former point can be overcome by expressing combinations of different SARS-CoV-2 proteins. However, since the SARS-CoV-2 expresses 29 proteins, there are about ~9 × 1030 possible protein combinations. Therefore, we decided to focus on individual proteins and allow further studies to pursue any combinations of interest. Regarding the latter limitation, we did not include the coronavirus structure (including the ACE2 receptors) in this study, because many studies have already demonstrated the cellular response to this structure (Chioh et al., 2020; Yang et al., 2020; Procko, 2020), and how tissues that do not have significant ACE2 expression (neurons, immune components such as B and T lymphocytes, and macrophages) are affected by the virus remains an open question.
Conclusions
Accumulating clinical evidence suggests that COVID-19 is a disease with vascular aspects. However, only a few studies have identified the specific role of each of the SARS-CoV-2 proteins in the cellular response leading to vascular dysfunctions. In this work, we characterized the endothelial response to each of 26 SARS-CoV-2 proteins and identified those that have the most significant effect on the barrier function. In addition, we used PPI network-based analysis to predict which of the endothelial proteins is most affected by the virus and to identify the specific role of each of the SARS-CoV-2 proteins in the observed changes in systemic protein expression. Overall, this work identified which of the SARS-CoV-2 proteins are most dominant in their effect on the physiological response to the virus. We believe that the data presented in this work will give us better insight into the mechanism by which the vasculature and the system respond to the virus, and will enable us to expedite drug development for the virus by targeting the identified dominant proteins.
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References -
See original article for extensive references -
https://elifesciences.org/articles/69314
Background on Ridgeback Bio - >>> Hedge fund manager stands to profit on ‘flip’ of taxpayer-funded coronavirus drug
The Washington Post
By Christopher Rowland
June 25, 2020
https://www.washingtonpost.com/business/2020/06/11/coronavirus-drug-ridgeback-biotherapeutics/
Emory University’s coronavirus pill EIDD-2801 highlights financial speculation on drugs developed with public investment
Rick Bright, former director of the Biomedical Advanced Research and Development Authority, at a House Energy and Commerce subcommittee hearing on May 14. (Greg Nash/The Hill/Bloomberg News)
Clarifications: A previous version of this story gave inadequate prominence to the role of Wendy Holman, the chief executive of Ridgeback Biotherapeutics; it now introduces her by noting her professional status. In addition, this version places greater emphasis on the company’s prior experience developing an Ebola drug and includes mention of the experience of its employees in developing pharmaceutical products. The company, which declined to answer questions during the reporting of the story, said after publication that it will continue to be closely involved in the development of the coronavirus therapy, as the story now indicates.
Ridgeback Biotherapeutics had no laboratories, no manufacturing facility of its own and a minimal track record when it struck a deal in March with Emory University to license an experimental coronavirus pill invented by university researchers with $16 million in grants from U.S. taxpayers.
But what the tiny Miami company did have was a growing team with experience in pharmaceutical development and research and a willingness from its wealthy owners — chief executive Wendy Holman and her husband, hedge fund manager Wayne Holman — to place a bet on the treatment in the midst of the coronavirus pandemic. That wager paid off with extraordinary speed in May when, just two months after acquiring the antiviral therapy called EIDD-2801 from Emory, Ridgeback sold exclusive worldwide rights to drug giant Merck.
The rapid turnaround of rights to a publicly financed drug highlights the frenzy of financial speculation that has accompanied the spread of the coronavirus around the world. Congress and the Trump administration have authorized more than $7 billion for research and industry subsidies in a desperate hunt for therapies and vaccines.
The perception that companies are profiteering during a global medical crisis — especially in cases where inventions were funded by taxpayers — poses political dangers to the pharmaceutical industry.
Demands are increasing in Congress and around the world that drug companies set affordable prices on coronavirus treatments and vaccines and distribute them equitably. Yet the role of middlemen like Ridgeback puts pressure on companies to increase prices, by adding extra costs. It also raises questions about who is financially benefiting by securing monopoly licensing rights to publicly financed inventions.
Ridgeback, which has one other drug in development for Ebola, was a relatively obscure entity when it snapped up EIDD-2801 from Emory in a deal signed March 19. The university, without disclosing terms, said in a news release that Ridgeback “will be responsible for conducting the necessary trials to bring EIDD-2801 to licensure.”
Within days of securing Emory’s licensing deal, the company mounted a campaign to win hundreds of millions in government funding to develop the drug, according to a whistleblower complaint by Rick Bright, former director of the Biomedical Advanced Research and Development Authority, as well as emails obtained by The Washington Post. After failing to secure the government contracts, Ridgeback launched a human safety trial of the drug in the United Kingdom and transferred rights to Merck in late May.
In addition to an upfront payment of undisclosed size from Merck, Ridgeback will receive unspecified “milestone” payments and a share of net proceeds if the drug is approved, the companies said. Merck will conduct future clinical trials, apply for regulatory approvals and manufacture the drug, which has been shown in lab and animal studies to cripple the virus by interfering with viral RNA. Ridgeback said it will remain involved in developing the drug.
Specialists in drug development called Ridgeback’s turnaround unusually rapid.
“I would think that universities … would not normally transfer products to basically a house-flipper,” said Aaron S. Kesselheim, a physician at Brigham and Women’s Hospital in Boston and professor at Harvard Medical School. “I wouldn’t think they would have to engage with speculators, like it appears that Ridgeback Biotherapeutics is.”
Wayne Holman, who holds a medical degree from New York University, is a hedge-fund manager with a long track record of investing in pharmaceutical stocks. He founded his fund Ridgeback Capital Management in 2006. Wendy Holman, chief executive of Ridgeback Biotherapeutics, is a former investment manager who was named to President Trump’s advisory council on HIV/AIDS in 2019.
The Holmans live on Miami’s exclusive Star Island, where they bought two mansions for a combined $47 million in 2014 and tore one of them down. Ridgeback Capital’s headquarters is in a small office building not far away in Coconut Grove, near a private school where Wendy Holman serves on the board of trustees. Ridgeback has one other drug in development that has been supported with $25 million in government contracts, a potential Ebola treatment invented by a division of the National Institutes of Health. The couple did not respond to requests for comment.
EIDD-2801 is among hundreds of projects underway around the world to develop treatments and vaccines to combat the coronavirus amid signs that covid-19, the disease the virus causes, will become a stubborn, endemic illness that lingers in human populations for many years. If it works and is found to be safe, it likely would become a strong rival to Gilead’s remdesivir, the first antiviral to treat the coronavirus, which must be given intravenously.
“EIDD-2801 has several attributes including oral availability, broad antiviral activity versus multiple coronavirus strains, notably SARS-CoV-2, as shown in preclinical studies, and early clinical results showing that it is well-tolerated from a study conducted by Ridgeback,” Merck said in an email.
But the path to Merck’s portfolio of early-stage drugs has grabbed attention.
“Molecule-flipping is a good characterization of what it is,” said James Love, director of Knowledge Ecology International, a nonprofit watchdog group that tracks public investments and intellectual property.
Hunting through scientific papers and forging alliances with academic laboratories is often done by small firms backed by venture capitalists. Start-ups looking to profit by securing rights to new molecules and nurturing their early-stage development are an ingrained part of the ecosystem of drug development. The goal typically is to push a drug far enough through the approval pipeline — a process that often takes years — until a larger company buys the rights and completes commercialization.
With the coronavirus creating huge demand, that activity is accelerating, say specialists in drug licensing and intellectual property.
“Merck is much better positioned and funded to move the drug through development,” said Joseph A. DiMasi, director of economic analysis and research at the Tufts Center for the Study of Drug Development, which receives drug industry funding. “It is the speed with which this has happened that is extraordinary. That speed in the context of a pandemic is a good thing.”
Stock markets show hunger for a coronavirus treatment. But don’t expect a magic bullet.
The flood of government money is spurring attention to diseases that have been neglected by large drug companies. Vaccines and therapies for viruses do not hold the promise of large, lucrative drug sales because they are not taken as regular treatments for chronic conditions. Many virus outbreaks disappear on their own, making it risky for companies to spend on research. To plug the gap, U.S. government agencies support academic research, or invent and develop drugs directly in government labs.
“When it was limited to things like Ebola and SARS, you didn’t see as much as engagement by the private sectors. These were normally backwater areas in neglected disease,” said Love. “People now are rushing in and scaling up.”
Emory had secured pledges of $30 million in government contracts from science and defense agencies in the past five years to develop EIDD-2801 but tapped just more than half of the available funds, an Emory spokeswoman said. The university did not respond to questions about how it picked Ridgeback.
“Emory is proud that we invented EIDD-2801, and we appreciate the partnerships and government support that makes it possible to provide therapies that will benefit society,” Nancy Seideman, Emory’s vice president for academic communications, said in an email. “Any royalties that we receive — if anything — are channeled directly back to serving our educational and scientific mission.”
In instances where taxpayer-financed drugs make it to market via exclusive licensing deals — which typically have undisclosed terms — debates have sprouted around monopoly pricing.
Douglas Throckmorton, deputy director for regulatory programs at the Food and Drug Administration, speaks via teleconference at a Senate Finance Committee hearing on June 2. Some members of Congress are pushing for drug companies to set affordable prices on coronavirus treatments and vaccines. (Stefani Reynolds/Pool/AP)
Liberal members of Congress and consumer advocates have demanded that taxpayers’ investments be factored into government contracts for development of coronavirus treatments and vaccines, and that terms of licensing deals be disclosed to the public. But Democratic proposals to place constraints on prices for covid-19 therapies did not make it into the $3 billion in emergency subsidies for the drug industry that lawmakers approved in March.
Some companies with experimental medicines backed by public investment have been the subject of intense financial speculation. Investors have gambled that Gilead’s drug remdesivir, which has been proven to modestly improve outcomes, will produce a windfall for the company. The company’s stock has been subject to volatile swings in the past three months.
Remdesivir was developed with at least $70 million in public investment, according to advocates, and a debate has begun about costs and access before Gilead has even set a price.
Even finding a covid-19 vaccine won’t be enough to end the pandemic
Moderna is developing a leading vaccine candidate that is co-owned by the NIH. Government interest in the vaccine has not stopped a boost in the company’s stock of nearly 200 percent since the end of February.
EIDD-2801, which was invented as an influenza drug and has demonstrated effectiveness against multiple viruses in lab dishes, works similarly to Gilead’s remdesivir by interfering with viral RNA. But it has the advantage of being a pill, which means it could be taken by people in their homes, soon after symptoms appear. If the drug proves to be safe and effective, it could prevent countless hospitalizations and deaths and reduce the spread of infection.
Drugs in its class have been known to cause genetic mutations that lead to birth defects, but Merck said when it licensed the drug last week that it was “well-tolerated.” Wayne Holman has said the drug can safely be used for a short course of treatment to fight a viral infection.
As for potential pricing, “Merck and Ridgeback are committed to ensuring that any medicines we develop for SARS-CoV-2 will be accessible to patients globally,” Merck said in an email. It would not discuss specifics.
The deal between Emory and Ridgeback was inked by the university’s Drug Innovation Ventures at Emory (DRIVE), a nonprofit tech-transfer corporation led by Emory scientist George Painter, who holds patents related to the drug. Highly accomplished in both laboratories and boardrooms, Painter is the former chief executive of Chimerix, a North Carolina drug company, and a high-ranking official in antiviral research at the former Glaxo Wellcome. Painter did not respond to interview requests.
On March 20, the day after the Ridgeback licensing contract was completed, Painter and other scientists at the University of North Carolina, Vanderbilt University and Emory posted a preliminary scientific paper on the preprint website bioRxiv.org showing EIDD-2801 thwarted SARS-CoV-2 in human cells and in mice infected with other types of coronaviruses. The peer-reviewed journal Science Translational Medicine published the paper on April 6.
At Vanda Pharmaceuticals, a biotechnology firm headquartered in Washington, D.C., founder and chief executive Mihael Polymeropoulos saw the scientific results in the journal and had his company contact Emory about a potential licensing opportunity. It was too late.
“They came back and they said they had already done a deal with this company, Ridgeback,” Polymeropoulos said. “This deal must have happened in record speed.”
The chief operating officer at Emory’s DRIVE told the Daily Report, an Atlanta trade publication, that the licensing contract, which normally would take four to six months to complete, was negotiated and signed in two weeks after a “mad scramble” by university and Ridgeback lawyers rushing to respond to the coronavirus.
Ridgeback’s involvement burst into the broader public sphere in early May, when Bright, the ousted head of BARDA, filed his explosive whistleblower complaint. Bright alleged that he clashed with Robert Kadlec, the Health and Human Services assistant secretary for preparedness and response, over demands that he award BARDA contracts to well-connected companies. HHS has said it “strongly disagrees” with Bright’s allegations.
In his complaint, Bright cited attempts to secure money for EIDD-2801 — first by Painter in November 2019, and then by Wendy Holman in early April — among episodes of alleged political pressure.
Bright said he rejected requests to fund EIDD-2801 because Emory had already received pledges of $30 million from the National Institute of Allergy and Infectious Diseases and the Department of Defense to cover development of the drug, including human safety testing. Without first seeing safety results, Bright said, it did not make sense to back the drug with new infusions of federal cash.
Wendy Holman’s pleas for government money in early April — less than two weeks after Ridgeback secured rights to the drug from Emory — are contained in emails she wrote to government officials. The full emails, excerpts of which were cited in Bright’s whistleblower complaint, were previously disclosed by the journal Science and were obtained by The Post. Holman said in one email to BARDA that she had been in personal contact with Kadlec, who wanted the project to move forward.
“We need this approval to start the clinical trial for EIDD-2801 as soon as possible,” Wendy Holman wrote to a BARDA contracting official on April 7, just days before Ridgeback planned to launch its first human safety trials. “Lives are literally depending on it, Dr. Kadlec is pushing us to move fast, but we can’t without this authorization.”
“To avoid any delay in executing this clinical trial, Ridgeback must receive approval … as soon as possible” to incur expenses in anticipation of a contract, Holman wrote to a related agency within the Office of the Assistant Secretary for Preparedness and Response on April 3. “We desperately need guidance on this.”
Bright said in his complaint that Ridgeback had been seeking $100 million to further the drug’s development. In an April 13 email, a BARDA official said the proposal from Ridgeback could obligate the government to pay the company more than $300 million. The contract official objected to the outlay because Ridgeback had not followed proper application procedures.
Ridgeback went away empty-handed by the time Bright departed in late April. It started its safety trial on April 10, testing the EIDD-2801 drug against a placebo in 122 healthy volunteers in England, according to its disclosure on clinicaltrials.gov. It hired a contract research organization called Covance to conduct the study. The NIH said in an email that Ridgeback conducted the study at its own expense.
In an interview with The Post on April 15, before the controversy erupted, Wayne Holman talked up the drug and made it clear that Ridgeback would be seeking partnerships and investment from the private sector.
“Treating orally and early can change the course of this pandemic. Not only would it treat the person that is sick, but it should theoretically reduce the infectiousness of that person, and the time period they are infectious to others,” he said. “We have inbound interest from pharma companies.”
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| Moderna Inc | 2.65% | |
| Gilead Sciences Inc | 2.43% | |
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| ACADIA Pharmaceuticals Inc | 2.04% |
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| Gilead Sciences Inc | 4.58% | |
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| Biogen Inc | 4.28% | |
| Seattle Genetics Inc | 4.08% | |
| United Therapeutics Corp | 3.93% | |
| Vertex Pharmaceuticals Inc | 3.89% | |
| ACADIA Pharmaceuticals Inc | 3.75% | |
| Biomarin Pharmaceutical Inc | 3.69% | |
| Alnylam Pharmaceuticals Inc | 3.53% |
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