Virus World

The COVID-19 pandemic has affected hundreds of millions of individuals and caused more than six million deaths. The prolonged pandemic duration and the continual inter-individual transmissibility have contributed to the emergence of a wide variety of SARS-CoV-2 variants. Genomic surveillance and phylogenetic studies have shown that substantial mutations in crucial supersites of spike glycoprotein modulate the binding affinity of the evolved SARS-COV-2 lineages to ACE2 receptors and modify the binding of spike protein with neutralizing antibodies.

The immunological spike mutations have been associated with differential transmissibility, infectivity, and therapeutic efficacy of the vaccines and the immunological therapies among the new variants. This review highlights the diverse genetic mutations assimilated in various SARS-CoV-2 variants. The implications of the acquired mutations related to viral transmission, infectivity, and COVID-19 severity are discussed. This review also addresses the effectiveness of human neutralizing antibodies induced by SARS-CoV-2 infection or immunization and the therapeutic antibodies against the ascended variants.

Published March 30, 2024:

https://doi.org/10.1007/s15010-024-02223-y 

The glycosylation of viral envelope proteins can play important roles in virus biology and immune evasion. The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) includes 22 N-linked glycosylation sequons and 17 O-linked glycosites. Here, we investigated the effect of individual glycosylation sites on SARS-CoV-2 S function in pseudotyped virus infection assays and on sensitivity to monoclonal and polyclonal neutralizing antibodies. In most cases, removal of individual glycosylation sites decreased the infectiousness of the pseudotyped virus. For glycosylation mutants in the N-terminal domain (NTD) and the receptor binding domain (RBD), reduction in pseudotype infectivity was predicted by a commensurate reduction in the level of virion-incorporated spike protein.

Notably, the presence of a glycan at position N343 within the RBD had diverse effects on neutralization by RBD-specific monoclonal antibodies (mAbs) cloned from convalescent individuals. The N343 glycan reduced overall sensitivity to polyclonal antibodies in plasma from COVID-19 convalescent individuals, suggesting a role for SARS-CoV-2 spike glycosylation in immune evasion. However, vaccination of convalescent individuals produced neutralizing activity that was resilient to the inhibitory effect of the N343 glycan.

Preprint at bioRxiv (June 30, 2023):

 https://doi.org/10.1101/2023.06.30.547241 

Researchers at The University of Queensland have discovered viruses such as SARS-CoV-2 can cause brain cells to fuse, initiating malfunctions that lead to chronic neurological symptoms. Professor Massimo Hilliard and Dr. Ramon Martinez-Marmol from the Queensland Brain Institute have explored how viruses alter the function of the nervous system. Their research is published in Science Advances. SARS-CoV-2, the virus that causes COVID-19, has been detected in the brains of people with "long COVID" months after their initial infection. "We discovered COVID-19 causes neurons to undergo a cell fusion process, which has not been seen before," Professor Hilliard said. "After neuronal infection with SARS-CoV-2, the spike S protein becomes present in neurons, and once neurons fuse, they don't die. They either start firing synchronously, or they stop functioning altogether." As an analogy, Professor Hilliard likened the role of neurons to that of wires connecting switches to the lights in a kitchen and a bathroom.

Professor Massimo Hilliard and Dr. Ramon Martinez-Marmol from the Queensland Brain Institute have explored how viruses alter the function of the nervous system. Their research is published in Science Advances.

SARS-CoV-2, the virus that causes COVID-19, has been detected in the brains of people with "long COVID" months after their initial infection. "We discovered COVID-19 causes neurons to undergo a cell fusion process, which has not been seen before," Professor Hilliard said. "After neuronal infection with SARS-CoV-2, the spike S protein becomes present in neurons, and once neurons fuse, they don't die. They either start firing synchronously, or they stop functioning altogether." As an analogy, Professor Hilliard likened the role of neurons to that of wires connecting switches to the lights in a kitchen and a bathroom. "Once fusion takes place, each switch either turns on both the kitchen and bathroom lights at the same time, or neither of them," he said. "It's bad news for the two independent circuits." The discovery offers a potential explanation for persistent neurological effects after a viral infection.

"In the current understanding of what happens when a virus enters the brain, there are two outcomes—either cell death or inflammation," Dr. Martinez-Marmol said. "But we've shown a third possible outcome, which is neuronal fusion." Dr. Martinez-Marmol said numerous viruses cause cell fusion in other tissues, but also infect the nervous system and could be causing the same problem there. "These viruses include HIV, rabies, Japanese encephalitis, measles, herpes simplex virus and Zika virus," he said. "Our research reveals a new mechanism for the neurological events that happen during a viral infection. This is potentially a major cause of neurological diseases and clinical symptoms that is still unexplored." The researchers acknowledge the collaborative efforts of Professor Lars Ittner and Associate Professor Yazi Ke from Macquarie University, Associate Professor Giuseppe Balistreri from University of Helsinki and Associate Professor Kirsty Short and Professor Frederic Meunier from The University of Queensland.

Original research published (June 7, 2023) in Science Advances:

https://www.science.org/doi/10.1126/sciadv.adg2248 

A new study by researchers from the United States, Spain and Japan investigated the direct effect of the SARS-CoV-2 spike (S) protein on platelet morphology. For the first time, the researchers directly visualized the binding of the S-protein to the platelet plasma membrane. The S-protein is crucial for understanding the molecular mechanisms of SARS-CoV-2 infection. The S-protein is crucial for understanding the molecular mechanisms of SARS-CoV-2 infection. The SARS-CoV-2 S-protein consists of a S1 and a S2 subunits that are separated by host cell proteases. The receptor-binding domain in the S1 subunit is responsible for attachment to host cells. S-protein is suggested to interact not only with the angiotensin-converting enzyme 2 (ACE-2) receptors, but also with several other host receptors including neuropilin-1 and CD147. SARS-CoV-2 is the only beta-coronavirus containing an RGD (Arg-Gly-Asp) tripeptide motif in the receptor-binding domain, which is typically recognized by several members of the integrin membrane receptor family. SARS-CoV-2 infection is associated with abnormalities in coagulation in severe cases. One of the major pathological symptoms in these patients is abnormal platelet behavior. During SARS-CoV-2 infection, several other procoagulant players, such as the formation of neutrophil extracellular traps, the release of tissue factor, elevated fibrinogen levels and dysregulated release of cytokines, create a hypercoagulative environment in the context of COVID-19.

COVID-19 patients have conditions such as a low platelet counts (thrombocytopenia), microvascular thrombosis, and coagulation, suggesting that SARS-CoV-2 can directly cause platelet dysfunction. Platelets isolated from COVID-19 patients exhibit abnormalities such as hyperactivity and increased spreading behaviour. Cytokines, antiphospholipid antibodies, interactions with other immune cells, and direct interaction between SARS-CoV-2 and platelets are potential causes of these abnormalities. The isolated platelets from healthy donors mixed with SARS-CoV-2 or the SARS-CoV-2 S-protein show a faster thrombin-dependent clot retraction, and activate platelets independent of thrombin with upregulation of signaling factors. To assess the direct effect of SARS-CoV-2 S-protein on platelet morphology, the researchers isolated platelets from healthy human blood donors, tested their morphological changes in the presence of S-protein, and visualized the effect of S-protein on platelet morphology using live imaging and cryo-electron tomography. The results showed that the S-protein triggers the dynamic deformation of platelets in elongated morphologies, leading in some cases to their irreversible activation. Platelets incubated with the S-protein showed extensive deformations, consistent with light microscopic observations, while intact platelets showed their typical disc-like morphology. Cryo-electron tomography revealed the formation of actin-rich filopodia at the end of the elongated platelets. The orientation analysis showed that the S-protein binds to the membrane surface through various angular distributions. To test the effects of the S-protein on platelet activation, the researchers performed a solid-phase sandwich ELISA test and a Western blotting. In the ELISA assay, platelet factor 4 was measured to test the secretion of alpha-granules, a marker of platelet activation. The results showed an increase in platelet factor 4 secretion in platelet samples in the presence of S-protein. Moreover, the research team tested the interaction between the S-protein and platelet-expressed integrins. The presence of ACE-2, the main S-protein receptor, on the surface of platelets is still not conclusive. On the contrary, integrin receptors are the main class of receptors expressed in platelets.

Given the possibility that ACE-2 is not widely expressed on platelets, the authors speculated that the S-protein could recognize integrin receptors directly. They tested the binding of S-protein to known platelet integrin receptors αIIbβ3, αvβ3, and α5β1, enriched in the tissue but also expressed on platelets, all recognizing the RGD ligand motif. They found a weak but direct binding of integrin α5β1 and αvβ3 to the S-protein, while integrin αIIbβ3 did not interact with it. According to researchers, the binding of the SARS-CoV-2 is mediated by integrin receptors, based on the following reasons; 1) the activation of platelets is governed by filopodia formation; 2) filopodia formation is initiated by integrin receptors; 3) the major receptors on the platelets are integrin receptors and 4) SARS-CoV-2 S protein contains a “RGD” sequence in the receptor binding domain, which is recognized by a subtype of integrin. The researchers noted that a weak affinity of S-protein to platelet integrin receptors and the reversible binding may reflect the fact that blood clotting defects observed in COVID-19 patients are rare complications and occur in severe cases of COVID-19. The researchers also said that other platelet receptors could also be responsible for the interaction with the S-protein. They concluded that their results shed light on the abnormal platelet behavior leading to coagulopathic events and microthrombosis caused by SARS-CoV-2 infection.

Research published in Nature Communications (Feb. 4, 2023):

https://doi.org/10.1038/s41467-023-36279-5 

The diagnosis and management of post-acute sequelae of COVID-19 (PASC) poses an ongoing medical challenge. Identifying biomarkers associated with PASC would immensely improve the classification of PASC patients and provide the means to evaluate treatment strategies. We analyzed plasma samples collected from a cohort of PASC and COVID-19 patients (n = 63) to quantify circulating viral antigens and inflammatory markers. Strikingly, we detect SARS-CoV-2 spike antigen in a majority of PASC patients up to 12 months post-diagnosis, suggesting the presence of an active persistent SARS-CoV-2 viral reservoir. Furthermore, temporal antigen profiles for many patients show the presence of spike at multiple time points over several months, highlighting the potential utility of the SARS-CoV-2 full spike protein as a biomarker for PASC.

Preprint available in medRxiv (June 16, 2022):

https://doi.org/10.1101/2022.06.14.22276401 

Salk researchers and collaborators show how the protein damages cells, confirming COVID-19 as a primarily vascular disease. 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 they 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. “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.

Original findings published in Circulation Research (March 31, 2021):

https://doi.org/10.1161/CIRCRESAHA.121.318902

SARS-CoV-2 mortality has been extensively studied in relationship to a patient’s predisposition to the disease. However, how sequence variations in the SARS-CoV-2 genome affect mortality is not understood. To address this issue, we used a whole-genome sequencing (WGS) association study to directly link death of SARS-CoV-2 patients with sequence variation in the viral genome. Specifically, we analyzed 3,626 single stranded RNA-genomes of SARS-CoV-2 patients in the GISAID database (Elbe and Buckland-Merrett, 2017; Shu and McCauley, 2017) with reported patient’s health status from COVID-19, i.e. deceased versus non-deceased. In total, evaluating 28,492 loci of the viral genome for association with patient/host mortality, two loci, 12,053bp and 25,088bp, achieved genome-wide significance (p-values of 1.24e-12, and 1.24e-26, respectively).

Mutations at 25,088bp occur in the S2 subunit of the SARS-CoV-2 spike protein, which plays a key role in viral entry of target host cells. Additionally, mutations at 12,053bp are within the ORF1ab gene, in a region encoding for the protein nsp7, which is necessary to form the RNA polymerase complex responsible for viral replication and transcription. Both mutations altered amino acid coding sequences, potentially imposing structural changes that could enhance viral infectivity and symptom severity, and may be important to consider as targets for therapeutic development.

Preprint available at bioRxiv (Nov. 24, 2020):

https://doi.org/10.1101/2020.11.17.386714

The authorization raised immediate questions about who would get access to the antibody treatments, which are in short supply. The Food and Drug Administration has granted emergency authorization of a Covid-19 antibody treatment made by Eli Lilly that is similar to a therapy given to President Trump shortly after he contracted the coronavirus. The decision, announced on Monday by the agency, is likely to be seen as a valuable tool to treat patients with Covid-19 at a time when the pandemic is raging across the United States, hospitals are overwhelmed and doctors have few options to treat the disease. Eli Lilly said that its treatment, called bamlanivimab, should be administered as soon as possible after a positive coronavirus test, and within 10 days of developing symptoms. The authorization applies only to people newly infected with the virus, and the agency said it should not be used in hospitalized patients. It is authorized for people who are 12 and older and at risk for developing a severe form of Covid-19 or being hospitalized for the condition. The F.D.A. said that included people who were over 65 and obese — a key group that early studies have shown can benefit the most from the treatment. “It’s a great day for science and medicine — sort of a feat of what’s possible,” said Dr. Daniel M. Skovronsky, the chief scientific officer of Eli Lilly. The company and its collaborators, including the National Institutes of Health, he said, were able “to create a new drug, manufacture it, test it in clinical trials, and get it authorized for use in just seven months.”

In October, the company announced that it had reached a $375 million deal to sell 300,000 doses of the treatment to the U.S. government. The emergency authorization for Eli Lilly raised immediate questions about who would get access to the treatment at a time when emergency authorizations for coronavirus vaccines might still be weeks or months away. The news came on the same day that Pfizer announced positive early results from its coronavirus vaccine trial. That vaccine might get emergency authorization sometime this year, but even then it would not be available to most Americans until well into 2021.  In a statement on Monday, Alex M. Azar II, the health secretary and a former executive at Eli Lilly, said the F.D.A.’s emergency authorization for bamlanivimab was a “step forward” in “bridging us to the rollout of safe and effective vaccines.” Eli Lilly has said that it expects to have enough doses to treat up to one million people by the end of the year, and that it will be able to significantly increase production thereafter. But that means that even in the best-case scenario, there won’t initially be enough to curb a virus that is now infecting more than 110,000 people a day in the United States. “It’s kind of the best times for these therapies to enter, because they can have an impact,” said Dr. Walid F. Gellad, who leads the Center for Pharmaceutical Policy and Prescribing at the University of Pittsburgh. “It’s also the worst time because we don’t have enough doses, and it’s going to add to the backlog of testing.”...

Lilly's press release (Nov. 9, 2020): 

https://investor.lilly.com/news-releases/news-release-details/lillys-neutralizing-antibody-bamlanivimab-ly-cov555-receives-fda

FDA letter of authorization (Nov. 9, 2020):

https://www.fda.gov/media/143602/download

Like a key, SARS-CoV-2—the virus that causes coronavirus disease 2019 (COVID-19) - attaches to specific molecules on the host cell surface, opening gateways into the cell interior. Viral entry into host cells triggers a prodigious immune response. Much of this battle is waged within the lungs, which explains why many patients hospitalized with COVID-19 have severe respiratory symptoms. Respiratory symptoms, however, are only part of the story. Increasing evidence points toward blood vessel inflammation as having a crucial impact on the severity of COVID-19. In addition, anywhere from 30 to 80 percent of patients experience neurological symptoms, including dizziness, headache, nausea, and loss of concentration. These symptoms suggest that SARS-CoV-2 also affects cells of the central nervous system. While there is no evidence yet that the virus invades the brain, new work by scientists at the Lewis Katz School of Medicine at Temple University shows that the spike proteins that extrude from SARS-CoV-2 promote inflammatory responses on the endothelial cells that form the blood-brain barrier. The study, published in the December print issue of the journal Neurobiology of Disease, is the first to show that SARS-CoV-2 spike proteins can cause this barrier to become "leaky," potentially disrupting the delicate neural networks within the brain.

"Previous studies have shown that SARS-CoV-2 infects host cells by using its spike proteins to bind to the angiotensin converting enzyme 2 (ACE2) on the host cell surface," explained Servio H. Ramirez, Ph.D., Professor of Pathology and Laboratory Medicine at the Lewis Katz School of Medicine at Temple University and principal investigator on the new study. ACE2 is expressed on endothelial cells, which form the inner lining of blood vessels, and serves a central role in mediating different functions of the cardiovascular system. According to Dr. Ramirez, "since ACE2 is a major binding target for SARS-CoV-2 in the lungs and vasculature of other organs in the body, tissues that are behind the vasculature, that receive blood from affected vessels, are at risk of damage from the virus." It has been unclear, however, whether ACE2 is also present in the brain vasculature or whether its expression changes in health conditions that worsen COVID-19, such as high blood pressure (hypertension). To find out, the team began by examining postmortem human brain tissue for vascular ACE2 expression, using tissues from individuals without underlying health conditions and from individuals in whom hypertension and dementia had been established. Analyses showed that ACE2 is in fact expressed throughout blood vessels in the frontal cortex of the brain and is significantly increased in the brain vasculature of persons with a history of hypertension or dementia.

The researchers then investigated the effects of the SARS-CoV-2 spike protein on brain endothelial cells in cell culture models. Introduction of the spike protein, particularly a portion designated subunit 1, produced substantial changes in endothelial barrier function that led to declines in barrier integrity. The researchers also uncovered evidence that subunit 2 of the SARS-CoV-2 spike protein can directly impact blood-brain barrier function. "This is of importance because unlike subunit 1, subunit 2 of the spike protein doesn't bind to ACE2, meaning that a breach to the blood-brain barrier could occur in a manner that is independent of ACE2," explained postdoctoral fellow and first author on the new report Tetyana P. Buzhdygan, Ph.D. Dr. Ramirez's team further investigated the effects of SARS-CoV-2 spike proteins on tissue-engineered microfluidic constructs designed to mimic a human brain capillary. "The tissue-engineered microfluidic models allow recapitulation of the 3-D cyto-architecture and mechanical forces caused by fluid movement, which the vasculature is continuously exposed to," said Allison M. Andrews, Ph.D., Assistant Professor in the Department of Pathology & Laboratory Medicine at LKSOM and a co-author on the report. Experiments showed that binding of spike protein subunit 1 increased barrier permeability in the engineered vessel-like constructs. "Our findings support the implication that SARS-CoV-2, or its shed spike proteins circulating in the blood stream, could cause destabilization of the blood-brain barrier in key brain regions," Dr. Ramirez said. "Altered function of this barrier, which normally keeps harmful agents out of the brain, greatly increases the possibility of neuroinvasion by this pathogen, offering an explanation for the neurological manifestations experienced by COVID-19 patients."...

Original study published in Neurobiology of Disease:

https://doi.org/10.1016/j.nbd.2020.105131

Scientific American is the essential guide to the most awe-inspiring advances in science and technology, explaining how they change our understanding of the world and shape our lives. While the world waits for a COVID-19 vaccine, many researchers are focused on developing effective therapeutics that can be rolled out quickly and cheaply. Monoclonal antibodies—a potentially promising laboratory-manufactured therapy modeled on antibodies extracted from the blood of recovering patients—made headlines recently when President Trump received a not-yet-approved antibody cocktail made by the company Regeneron. And pharmaceutical giant Eli Lilly recently announced that its monoclonal antibody reduced the risk of hospitalization in 300 people who had mild or moderate symptoms of COVID-19, in a small clinical trial. But David Baker, a biochemist at the University of Washington’s Institute for Protein Design, and his colleagues think they can produce an even better therapy. They have designed a synthetic peptide—a short string of amino acids, the building blocks of proteins—20 times smaller than a monoclonal antibody that is designed to bind to the infamous “spike” protein on the surface of the SARS-CoV-2 virus particle. Doing so would directly block the virus from binding to the ACE-2 receptors on human cells, functioning much like an antibody produced by an infected person’s immune system. Baker and his colleagues described these “miniprotein inhibitors” in September in Science. Although the study only tested these synthetic proteins in the lab, mixing viral particles with monkey cells in vitro, he says that unpublished data show they can protect mice and hamsters from SARS-CoV-2 infection. 

“We built these [tiny proteins] from scratch based on ‘first principles,’ using computers to model all the biochemical details of a theoretical protein that could stick to the virus,” explains Baker, who was awarded a $3 million Breakthrough Prize earlier in September for his decades of work pioneering the field of synthetic protein design. His team used computers to digitally design more than two million candidate “miniproteins,” crunched the data using algorithms, sifted out 118,000 candidate genes that encode these proteins, manufactured the proteins from scratch, and tested them directly against the virus in the lab—finding that seven designs could effectively bind to and thus disable the virus. Over the course of 3.5 billion years evolution has produced an incredible array of proteins and peptides. In recent years biochemists have tracked down and used some of these to create new drugs, such as Eptifibatide, an antiplatelet drug administered to prevent heart attacks whose active ingredient is extracted from the venom of the southern pygmy rattlesnake. The Protein Data Bank, an online repository of protein sequences and educational tools, contains the amino acid sequences and full 3-D structures for more than 160,000 peptides and proteins—but the natural world contains hundreds of millions of proteins. 

“It’s very challenging to discover in nature a peptide that does exactly what you want it to do,” explains Gaurav Bhardwaj, also a biochemist at the Institute for Protein Design, but who was not involved in the Science study. He is trying to design a bespoke peptide that would prevent SARS-CoV-2 from replicating within human cells. “Now we can computationally explore the possible design configurations for a peptide in order to perform the exact functions that we want.” Every protein’s function depends on its structure. Interactions between the atoms of the protein’s amino acids cause these chains to self-assemble in less than a second into a complex array of spirals and pleats. As the chain of amino acids grows, these helices and rippled sheets stack on top of and around one another into a dizzyingly complex series of folds, and it is these folds that give proteins their shape and function. Yet figuring out how one amino acid sequence turns into a specific fold has been a torturously difficult task, and it was only in the 1990s—with ever expanding databases of protein information—that scientists could begin to link sequence to form. “We can make up completely new proteins that have never been seen in nature because we now understand the nature of protein folding,” Baker says. “Our ability to use computers to design ‘de novo’ proteins has really only come into its own in the last few years–we might not have been able to apply ourselves to COVID-19 if the pandemic had happened five years ago....

The COVID-19 pandemic is still creating economic and health-related impact over the world. A new study published on the preprint server bioRxiv in August 2020 shows that a gp96-Ig-secreting vaccine candidate based on the expression of full-length spike protein generates a potent T cell response.   The spike or S protein of SARS-CoV-2 is still the best choice for a COVID-19 vaccine as it is both abundantly present and highly immunogenic. Antibodies against this protein neutralize the binding of the virus to the mammalian host cell. However, the presence of glycosylated residues shields the antibody from being able to neutralize the virus properly. Moreover, antibody decay has been shown to occur in recovering individuals, even more rapidly than with the earlier SARS outbreak. T cells are responsible for robust and long-lasting immunity as they induce the formation of memory cells to protect against viral infection, and these cells persist for years rather than the short-lived memory B-cells induced by SARS-CoV-2. Most recovered COVID-19 patients show specific CD8+ T cells. There is a specific subpopulation of tissue-resident memory (TRM) cells, which can respond rapidly to tissue infection without being primed in the lymph nodes. These have the marker molecules CD69 and CXCR620, while interactions between CXCR6-CXCL16 regulate the localization and number of these specific T cells in the lungs. These cells are essential in virus clearance. The researchers, therefore, aimed to generate TRM, since their persistence may lead to improved immunity compared to antibody-only responses.

The Novel Vaccine Candidate

The researchers used a genetically engineered gp96 construct, called the fusion protein gp96-Ig. Here, they replaced the C-terminal KDEL-retention sequence with the Fc portion of immunoglobulin G1 (IgG1). This fusion protein was encoded inside a plasmid vector that is transfected into a cell line for study. The cell line secretes the gp96-Ig. This protein complexes with the antigenic peptides so that the antigens derived from the cell are specifically cross-presented by gp96-Ig in vivo. The most significant benefit of this approach is its ability to allow any viral peptide that is bound to gp96 to produce a durable and potent response.

T Cell Induction

Previous research by the same team has shown, over 20 years, the immunogenic potential of gp96-Ig, whether from an allogeneic or xenogeneic cell primed with the specific antigen, to produce potent and specific T cells against multiple viral antigens. They adapted this technology to show its suitability for vaccine production to induce T cell immunity in lungs, airways, and other relevant epithelial sites Secondly, this technology can work in synchrony with other therapies or vaccines, either as a booster or as a second line of defense, and is amenable to large-scale manufacture. This technology has already been validated in animal and human experiments. This induces protein-specific CD8+ and CD4+ T helper 1 (Th1) responses against tumors, various viruses like HIV and Zika, and malaria, in various models. It not only delays virus infection but also improves the survival of patients with non-small cell lung cancer in clinical trials.

How the gp96-Ig Vaccine Works

The principle of this cell-based vaccine is that gp96-Ig can chaperone protein antigens for efficient endocytosis, allowing them to be cross-presented by activated antigen-presenting dendritic cells to CD8+ T cells, which causes an avid, specific T cell response. This technology was used to create a vaccine that actually brings the spike protein straight to the dendritic cells.

As a result of such a presentation, the primed and activated T cells, specifically targeting the viral S protein can now detect and kill the lung epithelial cells that are infected by SARS-CoV-2.

The gp96-Ig platform is an endoplasmic reticulum chaperone protein that participates in the transfer of antigenic peptides from the cytosol to MHC class I molecules in an orderly fashion. The complexes formed by gp96-antigen binding are mostly internalized by subsets of antigen-presenting cells that have the CD91 receptor on the surface. Once gp96 is internalized, it can present the complex antigens to MHC I and II molecules, helping to activate specific CD4 and CD8 T cells...

Preprint available in bioRxiv (August 24, 2020):

https://doi.org/10.1101/2020.08.24.265090

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy.

The structure and distribution of S on the virion surface, however, has not been characterized. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S present on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.

Published in Nature (August 17, 2020):

https://doi.org/10.1038/s41586-020-2665-2 

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 

The Orthocoronaviridae subfamily is large comprising four highly divergent genera. Four seasonal coronaviruses were circulating in humans prior to the coronavirus disease 2019 (COVID-19) pandemic. Infection with these viruses induced antibody responses that are relatively narrow with little cross-reactivity to spike proteins of other coronaviruses. Here, we report that infection with and vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces broadly crossreactive binding antibodies to spikes from a wide range of coronaviruses including members of the sarbecovirus subgenus, betacoronaviruses including Middle Eastern respiratory syndrome coronavirus (MERS CoV), and extending to alpha-, gamma- and delta-coronavirus spikes. These data show that the coronavirus spike antibody landscape in humans has profoundly been changed and broadened as a result of the SARS-CoV-2 pandemic. While we do not understand the functionality of these crossreactive antibodies, they may lead to enhanced resistance of the population to infection with newly emerging coronaviruses with pandemic potential.

Preprunt in medRxiv (August 6, 2023):

https://doi.org/10.1101/2023.08.01.23293522 

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has been associated mainly with a range of neurological symptoms, including brain fog and brain tissue loss, raising concerns about the virus's acute and potential chronic impact on the central nervous system. In this study, we utilized mouse models and human post-mortem tissues to investigate the presence and distribution of the SARS-CoV-2 spike protein in the skull-meninges-brain axis. Our results revealed the accumulation of the spike protein in the skull marrow, brain meninges, and brain parenchyma. The injection of the spike protein alone caused cell death in the brain, highlighting a direct effect on brain tissue. Furthermore, we observed the presence of spike protein in the skull of deceased long after their COVID-19 infection, suggesting that the spike's persistence may contribute to long-term neurological symptoms. The spike protein was associated with neutrophil-related pathways and dysregulation of the proteins involved in the PI3K-AKT as well as complement and coagulation pathway. Overall, our findings suggest that SARS-CoV-2 spike protein trafficking from CNS borders into the brain parenchyma and identified differentially regulated pathways may present insights into mechanisms underlying immediate and long-term consequences of SARS-CoV-2 and present diagnostic and therapeutic opportunities.

Preprint in bioRxiv (April 5, 2023):

https://doi.org/10.1101/2023.04.04.535604 

Study implicates lack of key hormone, battle-weary immune cells, and reawakened viruses.  An ambitious study of people with Long Covid, the mysterious, disabling symptoms that can trail a SARS-CoV-2 infection, has turned up a host of abnormalities in their blood. The clues add to a body of evidence hinting at drivers of the condition and potential treatments worth testing. They also suggest that, as many scientists and patients have suspected, Long Covid shares certain features with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), another condition thought to follow an infection. The new study, posted as a preprint last week, was modest in size, examining just 99 people with Long Covid. “But it went very deep, it went into granular aspects of the T cells, the antibody response,” says Eric Topol, director of the Scripps Research Translational Institute, who was not involved in the work. “This is exploratory, but it’s the foundation for much bigger studies.” The Long Covid patients, most of them struggling with intense fatigue, brain fog, and other symptoms, had low levels of cortisol, a stress hormone that helps the body control inflammation, glucose, sleep cycles, and more. Features of their T cells indicated their immune system was battling unidentified invaders, perhaps a reservoir of SARS-CoV-2 or a reactivated pathogen such as Epstein-Barr virus. Other groups studying Long Covid patients have reported similar results this year, including in a January Cell paper that documented low cortisol in those with long-lived respiratory symptoms, and reactivation of viruses in patients with neurological issues. Collectively, these data “make me think about what other drugs we can test,” such as virus-directed antibodies or targeted anti-inflammatories to tame the immune system, says Emma Wall at University College London and the Francis Crick Institute, who co-leads a large trial of potential Long Covid therapies.

David Veesler and their colleagues, showed a consequence of this dramatic transformation: only one of eight antibody treatments for COVID used in hospitals—which are based on natural antibodies—still bound effectively to RBDs. Other research has shown that mutations on RBDs and a second site called the N-terminal domain enable the virus to avoid antibodies gained by vaccination or infection. Thanks to Omicron’s convincing disguise, the variant had little to slow it down, and it spread with lightning-fast speed. Vaccines, however, still warded off serious illness, especially with booster shots.  It stabilized. When Omicron drastically altered its spike to hide from the immune system, those changes eliminated some chemical residues the spike needed to attach to ACE2. But other mutations compensated: RBDs formed new chemical bridges to still effectively bind to the protein, according to another study in Science. “It clearly lost some residues important for binding, but it made them up with other interactions,” says Subramaniam, who was the senior author of the paper. The spike protein also became sturdier. In other variants, two subunits within the spike, S1 and S2, are loosely connected. This allows them to split apart quickly so the spike can bury itself in a human cell when the virus encounters one. The downside of this delicate arrangement, however, is that many spikes split prematurely, before getting close to a cell. Once asunder, the spikes can no longer help the virus attach.

Mutations in Omicron led to slim molecular bridges that hold the subunits together better, according to several studies. One was published in the Journal of Medical Virology, and the others were released as preprint papers that have not yet been formally reviewed by other scientists. “This virus has really protected itself from prematurely triggering,” says Shan-Lu Liu, author of one of the papers and director of the Viruses and Emerging Pathogens program at the Ohio State University. “When the virus is in the right place at the right time, it can be triggered and get into the cell, but not prior to that.”  It slipped in the side door. Across previous variants, there was one constant: the virus relies on a protein on the surface of human cells called TMPRSS2 (pronounced “tempress two”) to help it break through the cell membrane. But Omicron did not use TMPRSS2. It took a wholly different route into the cell. Instead of breaking down the front door, it slipped in through the side. While other variants require both the ACE2 and TMPRSS2 proteins to inject their genome into a cell, Omicron bound only to ACE2. Then it was engulfed in a hollow bubble called an endosome. The bubble drifted into the cell, where the virus broke out and began a takeover.....

Antibody treatment for COVID-19 seems to have spurred mutations in the SARS-CoV-2 that infected a man with a compromised immune system.In mid-2020, a man was admitted to hospital with COVID-19. He had been diagnosed with cancer in 2012; the illness and his treatment had probably weakened his immune system. The man’s COVID-19 was treated with two courses of the antiviral drug remdesivir and, later, two courses of convalescent plasma — antibody-laden blood from people who had recovered from COVID-19. He died 102 days after admission. Ravindra Gupta at the University of Cambridge, UK, and his colleagues analysed viral genomes obtained from the man during his illness (S. A. Kemp et al. Nature https://doi.org/10.1038/s41586-021-03291-y; 2021). The viral populations in his blood changed little after remdesivir treatment. But after each course of convalescent plasma, the samples were dominated by viruses with a particular pair of mutations in the SARS-CoV-2 spike protein, the main target of the immune system. Experiments showed that one of the mutations weakened the potency of the antibodies in the convalescent plasma, yet also reduced the virus’s infectivity. The second mutation restored infectivity. The potential for viral evolution means that convalescent plasma should be used cautiously when treating people with compromised immunity, the authors say.

Research published in Nature (Feb. 5, 2021):

https://doi.org/10.1038/s41586-021-03291-y 

The evolution of SARS-CoV-2 humoral response in infected individuals remains poorly characterized. Here, we performed a longitudinal study of sera from 308 RT-qPCR+ individuals with mild disease, collected at two time-points, up to 6 months post-onset of symptoms (POS). We performed two anti-S and one anti-N serology assays and quantified neutralizing antibodies (NAbs). At month 1 (M1), males, individuals > 50 years of age or with a body mass index (BMI) > 25 exhibited higher levels of antibodies. Antibody levels decreased over time. At M3-6, anti-S antibodies persisted in 99% of individuals while anti-N IgG were measurable in only 59% of individuals. The decline in anti-S and NAbs was faster in males than in females, independently of age and BMI. Our results show that some serology tests are less reliable overtime and suggest that the duration of protection after SARS-CoV-2 infection or vaccination will be different in women and men.

Preprint available in medRxiv (Nov. 15, 2020):

https://doi.org/10.1101/2020.11.12.20230466

A study involving more than 5,000 COVID-19 patients in Houston finds that the virus that causes the disease is accumulating genetic mutations, one of which may have made it more contagious. According to the paper published in the peer-reviewed journal mBIO, that mutation, called D614G, is located in the spike protein that pries open our cells for viral entry. It’s the largest peer-reviewed study of SARS-CoV-2 genome sequences in one metropolitan region of the U.S. to date.  The paper shows “the virus is mutating due to a combination of neutral drift — which just means random genetic changes that don’t help or hurt the virus — and pressure from our immune systems,” said Ilya Finkelstein, associate professor of molecular biosciences at The University of Texas at Austin and co-author of the study. The study was carried out by scientists at Houston Methodist Hospital, UT Austin and elsewhere.  During the initial wave of the pandemic, 71% of the novel coronaviruses identified in patients in Houston had this mutation. When the second wave of the outbreak hit Houston during the summer, this variant had leaped to 99.9% prevalence. This mirrors a trend observed around the world. A study published in July based on more than 28,000 genome sequences found that variants carrying the D614G mutation became the globally dominant form of SARS-CoV-2 in about a month. SARS-CoV-2 is the coronavirus that causes COVID-19.

So why did strains containing this mutation outcompete those that didn’t have it? Perhaps they’re more contagious. A study of more than 25,000 genome sequences in the U.K. found that viruses with the mutation tended to transmit slightly faster than those without it and caused larger clusters of infections. Natural selection would favor strains of the virus that transmit more easily. But not all scientists are convinced. Some have suggested another explanation, called “founder’s effects.” In that scenario, the D614G mutation might have been more common in the first viruses to arrive in Europe and North America, essentially giving them a head start on other strains. The spike protein is also continuing to accumulate additional mutations of unknown significance. The Houston Methodist-UT Austin team also showed in lab experiments that at least one such mutation allows spike to evade a neutralizing antibody that humans naturally produce to fight SARS-CoV-2 infections. This may allow that variant of the virus to more easily slip past our immune systems. Although it is not clear yet whether that translates into it also being more easily transmitted between individuals.

The good news is that this mutation is rare and does not appear to make the disease more severe for infected patients. According to Finkelstein, the group did not see viruses that have learned to evade first-generation vaccines and therapeutic antibody formulations. “The virus continues to mutate as it rips through the world,” Finkelstein said. “Real-time surveillance efforts like our study will ensure that global vaccines and therapeutics are always one step ahead.” The scientists noted a total of 285 mutations across thousands of infections, although most don’t appear to have a significant effect on how severe the disease is. Ongoing studies are continuing to surveil the third wave of COVID-19 patients and to characterize how the virus is adapting to neutralizing antibodies that are produced by our immune systems. Each new infection is a roll of the dice, an additional chance to develop more dangerous mutations.

Preprint of  Study available at medRxiv (Sept. 29, 2020):

https://doi.org/10.1101/2020.09.22.20199125

A spike protein mutation D614G became dominant in SARS-CoV-2 during the COVID-19 pandemic.  However, the impact on viral spread and vaccine efficacy remains to be defined. Here, we engineer the D614G mutation in the USA-WA1/2020 strain and characterize its effect. D614G enhances replication on human lung epithelial cells and primary human airway tissues through an improved infectivity of virions. Hamsters infected with the G614 variant produced higher infectious titers in the nasal washes and trachea, but not lungs, confirming clinical evidence that the D614G mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increases transmission. Sera from D614-infected hamsters exhibit modestly higher neutralization titers against G614 virus than against D614 virus, indicating that (i) the mutation may not reduce the ability of vaccines in clinical trials to protect against COVID-19 and (ii) therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this mutation in viral spread, vaccine efficacy, and antibody therapy.

Published in Nature (Oct. 26, 2020):

https://doi.org/10.1038/s41586-020-2895-3

University of Pittsburgh School of Medicine scientists have isolated the smallest biological molecule to date that completely and specifically neutralizes the SARS-CoV-2 virus, which is the cause of COVID-19. This antibody component, which is 10 times smaller than a full-sized antibody, has been used to construct a drug — known as Ab8 — for potential use as a therapeutic and prophylactic against SARS-CoV-2. The researchers report today (September 14, 2020) in the journal Cell that Ab8 is highly effective in preventing and treating SARS-CoV-2 infection in mice and hamsters. Its tiny size not only increases its potential for diffusion in tissues to better neutralize the virus, but also makes it possible to administer the drug by alternative routes, including inhalation. Importantly, it does not bind to human cells — a good sign that it won’t have negative side-effects in people. Ab8 was evaluated in conjunction with scientists from the University of North Carolina at Chapel Hill (UNC) and University of Texas Medical Branch (UTMB) at Galveston, as well as the University of British Columbia and University of Saskatchewan.

“Ab8 not only has potential as therapy for COVID-19, but it also could be used to keep people from getting SARS-CoV-2 infections,” said co-author John Mellors, M.D., chief of the Division of Infectious Diseases at UPMC and Pitt. “Antibodies of larger size have worked against other infectious diseases and have been well tolerated, giving us hope that it could be an effective treatment for patients with COVID-19 and for protection of those who have never had the infection and are not immune.” The tiny antibody component is the variable, heavy chain (VH) domain of an immunoglobulin, which is a type of antibody found in the blood. It was found by “fishing” in a pool of more than 100 billion potential candidates using the SARS-CoV-2 spike protein as bait. Ab8 is created when the VH domain is fused to part of the immunoglobulin tail region, adding the immune functions of a full-size antibody without the bulk. Abound Bio, a newly formed UPMC-backed company, has licensed Ab8 for worldwide development. Dimiter Dimitrov, Ph.D., senior author of the Cell publication and director of Pitt’s Center for Antibody Therapeutics, was one of the first to discover neutralizing antibodies for the original SARS coronavirus in 2003. In the ensuing years, his team discovered potent antibodies against many other infectious diseases, including those caused by MERS-CoV, dengue, Hendra and Nipah viruses. The antibody against Hendra and Nipah viruses has been evaluated in humans and approved for clinical use on a compassionate basis in Australia.

Clinical trials are testing convalescent plasma — which contains antibodies from people who already had COVID-19 — as a treatment for those battling the infection, but there isn’t enough plasma for those who might need it, and it isn’t proven to work. That’s why Dimitrov and his team set out to isolate the gene for one or more antibodies that block the SARS-CoV-2 virus, which would allow for mass production. In February, Wei Li, Ph.D., assistant director of Pitt’s Center for Therapeutic Antibodies and co-lead author of the research, began sifting through large libraries of antibody components made using human blood samples and found multiple therapeutic antibody candidates, including Ab8, in record time. Then a team at UTMB’s Center for Biodefense and Emerging Diseases and Galveston National Laboratory, led by Chien-Te Kent Tseng, Ph.D., tested Ab8 using live SARS-CoV-2 virus. At very low concentrations, Ab8 completely blocked the virus from entering cells. With those results in hand, Ralph Baric, Ph.D., and his UNC colleagues tested Ab8 at varying concentrations in mice using a modified version of SARS-CoV-2. Even at the lowest dose, Ab8 decreased by 10-fold the amount of infectious virus in those mice compared to their untreated counterparts. Ab8 also was effective in treating and preventing SARS-CoV-2 infection in hamsters, as evaluated by Darryl Falzarano, Ph.D., and colleagues at the University of Saskatchewan. Sriram Subramaniam, Ph.D., and his colleagues at the University of British Columbia uncovered the unique way Ab8 neutralizes the virus so effectively by using sophisticated electron microscopic techniques.

Original study published in Cell (August 31, 2020):

https://doi.org/10.1016/j.cell.2020.09.007

At the start of a COVID-19 infection, the coronavirus SARS-CoV-2 docks onto human cells using the spike-like proteins on its surface. The spike protein is at the centre of vaccine development because it triggers an immune response in humans. A group of German scientists, including members of the European Molecular Biology Laboratory (EMBL) in Heidelberg, the Max Planck Institute of Biophysics, the Paul-Ehrlich-Institut, and Goethe University Frankfurt have focused on the surface structure of the virus to gain insights they can use for the development of vaccines and of effective therapeutics to treat infected patients. The team combined cryo-electron tomography, subtomogram averaging, and molecular dynamics simulations to analyze the molecular structure of the spike protein in its natural environment, on intact virions, and with near-atomic resolution. Using EMBL's state-of-the-art cryo-electron microscopy imaging facility, 266 cryotomograms of about 1000 different viruses were generated, each carrying an average of 40 spikes on its surface. Subtomogram averaging and image processing, combined with molecular dynamics simulations, finally provided the important and novel structural information on these spikes. The results were surprising: the data showed that the globular portion of the spike protein, which contains the receptor-binding region and the machinery required for fusion with the target cell, is connected to a flexible stalk. "The upper spherical part of the spike has a structure that is well reproduced by recombinant proteins used for vaccine development," explains Martin Beck, EMBL group leader and a director of the Max Planck Institute (MPI) of Biophysics. "However, our findings about the stalk, which fixes the globular part of the spike protein to the virus surface, were new." 

"The stalk was expected to be quite rigid," adds Gerhard Hummer, from the MPI of Biophysics and the Institute of Biophysics at Goethe University Frankfurt. "But in our computer models and in the actual images, we discovered that the stalks are extremely flexible." By combining molecular dynamics simulations and cryo-electron tomography, the team identified the three joints—hip, knee and ankle—that give the stalk its flexibility. "Like a balloon on a string, the spikes appear to move on the surface of the virus and thus are able to search for the receptor for docking to the target cell," explains Jacomine Krijnse Locker, group leader at the Paul-Ehrlich-Institut. To prevent infection, these spikes are targeted by antibodies. However, the images and models also showed that the entire spike protein, including the stalk, is covered with chains of glycans—sugar-like molecules. These chains provide a kind of protective coat that hides the spikes from neutralizing antibodies: another important finding on the way to effective vaccines and medicines.

Study Published in Science (August 18, 2020):

https://doi.org/10.1126/science.abd5223