Over the course of the pandemic, various COVID-19 variants have arisen with the Delta variant currently dominant in the UK. Understanding how some variants may be able to trigger an effective antibody response against other variants, in addition to itself, could help inform future vaccine design.
In their study, published in eLife(29 July), the scientists analysed blood samples collected from patients who had previously been infected with COVID-19 and who were admitted to UCLH for other reasons, samples from health care workers as well as samples collected from patients at different points earlier in the pandemic. They identified COVID-19 antibodies in the blood, and in the lab ran tests to see if antibodies produced after infection with one variant were able to bind to and neutralise other variants.
The study included: • The original strain first discovered in Wuhan, China • The dominant strain in Europe during the first wave in April 2020 (D614G) • B.1.1.7, the variant first discovered in Kent, UK (Alpha) • B.1.351, the variant first discovered in South Africa (Beta)
While antibodies produced by one variant were able to bind to other variants at a similar rate, there were some differences in whether antibodies could neutralise other variants. If an antibody is able to neutralise a virus, this means it can stop the virus from entering the hosts cells in order to replicate.
The researchers found that antibodies produced by the Alpha variant were not able to neutralise the original or D614G strains as effectively, in comparison to neutralising the Alpha variant itself.
Antibodies produced against infection with the D614G strain were able to neutralise both the Alpha and original strains to a similar level as D614G.
Both the Alpha and D614G strains produced antibodies which were not able to effectively neutralise the Beta strain.
There are many elements of the immune system which impact how protected an individual may be against future disease. This includes memory B cells and T cells which equip the immune system to deal with evolving threats. As a result, these findings do not necessarily mean people who were infected with specific variants are less protected against others.
Kevin Ng, co-first author and PhD student in the Retroviral Immunology Laboratory at the Crick says: “It’s important to note that most people who have been infected with the virus will not know which variant they were infected with and it is critical that everyone eligible for the vaccine takes up the opportunity because we know they are effective in some way against all known variants.”
As the antibodies were able to bind to other variants at a similar level, but had differing ability to neutralise other strains, this suggests that there are only a few regions on the spike of the virus which are important to this neutralisation process. It is the mutations within these key sites which impact the ability of antibodies produced by one variant to neutralise another.
Nikhil Faulkner, co-first author and PhD student in the Retroviral Immunology Laboratory at the Crick says: “As the pandemic continues and more variants may arise, research into how infection with one variant impacts immune responses to other variants is important.
“This could help inform experts who are studying and deciding which variants should be included in future booster vaccines. By creating a multivalent vaccine, like with the annual flu shot, we could factor different variants into a single vaccine. This would help to increase the breadth of immune protection against current and future variants.”
Featured image: Image of the ultrastructural morphology exhibited by the 2019 Novel Coronavirus (2019-nCoV) (CDC)- CDC/ Alissa Eckert, MS; Dan Higgins, MAM / Public domain
Reference: Nikhil Faulkner et al, Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains, eLife (2021). DOI: 10.7554/eLife.69317
A licensed drug normally used to treat abnormal levels of fatty substances in the blood could reduce infection caused by the SARS-CoV-2 virus by up to 70 per cent, reveals a study in the laboratory by an international collaboration of researchers.
The research team, led by the University of Birmingham and Keele University in the UK and the San Raffaele Scientific Institute in Italy, has demonstrated that fenofibrate and its active form (fenofibric acid) can significantly reduce SARS-COV-2 infection in human cells in the laboratory. Importantly, reduction of infection was obtained using concentrations of the drug which are safe and achievable using the standard clinical dose of fenofibrate. Fenofibrate, which is approved for use by most countries in the world including the US Food and Drug Administration (FDA) and the UK’s National Institute for Health and Care Excellence (NICE), is an oral drug currently used to treat conditions such as high levels of cholesterol and lipids (fatty substances) in the blood.
The team is now calling for clinical trials to test the drug in hospitalised COVID-19 patients, to be carried out in addition to two clinical trials also currently underway in such patients in research being led by the Hospital of the University of Pennsylvania in the US and Hebrew University of Jerusalem in Israel.
SARS-CoV-2, the virus that causes COVID-19, infects the host through an interaction between the Spike protein on the surface of the virus and the ACE2 receptor protein on host cells. In this study, responding to the global COVID-19 pandemic, the team tested a panel of already licensed drugs – including fenofibrate – to identify candidates that disrupt ACE2 and Spike interactions. Having identified fenofibrate as a candidate, they then tested the efficacy of the drug in reducing infection in cells in the laboratory using the original strains of the SARS-CoV-2 virus isolated in 2020. They found fenofibrate reduced infection by up to 70%. Additional unpublished data also indicates that fenofibrate is equally effective against the newer variants of SARS-CoV-2 including the alpha and beta variants and research is ongoing into its efficacy in the delta variant.
Corresponding author Dr Farhat Khanim, of the University of Birmingham in the UK, explained: “The development of new more infectious SARS-CoV-2 variants has resulted in a rapid expansion in infection rates and deaths in several countries around the world, especially the UK, US and Europe. Whilst vaccine programmes will hopefully reduce infection rates and virus spread in the longer term, there is still an urgent need to expand our arsenal of drugs to treat SARS-CoV-2-positive patients.”
Co-corresponding author Dr Alan Richardson, of Keele University in the UK, added: “Whilst in some countries vaccination programmes are progressing at speed, vaccine uptake rates are variable and for most low middle income countries, significant proportions of the population are unlikely to be vaccinated until 2022. Furthermore, whilst vaccination has been shown to reduce infection rates and severity of disease, we are as yet unsure of the strength and duration of the response. Therapies are still urgently needed to manage COVID-19 patients who develop symptoms or require hospitalisation.”
Co-author Dr Elisa Vicenzi, of the San Raffaele Scientific Institute in Milan, Italy, said: “Our data indicates that fenofibrate may have the potential to reduce the severity of COVID-19 symptoms and also virus spread. Given that fenofibrate is an oral drug which is very cheap and available worldwide, together with its extensive history of clinical use and its good safety profile, our data has global implications – especially in low-middle income countries and in those individuals for whom vaccines are not recommended or suitable such as children, those with hyper-immune disorders and those using immune-suppressants.”
First author Dr Scott Davies, also of the University of Birmingham, concluded: “We now urgently need further clinical studies to establish whether fenofibrate is a potential therapeutic agent to treat SARS-CoV-2 infection.”
The research, published today in Frontiers in Pharmacology, was also carried out in collaboration with the University of Copenhagen in Denmark and the University of Liverpool in the UK.
Reference: Davies et al. ‘The hyperlipidaemic drug fenofibrate significantly reduces infection by SARS-1 CoV-2 in cell culture models’. Frontiers in Pharmacology. Preprint
Juan Robles and colleagues revealed for the first time that the spike protein of SARS-CoV-2 promotes endothelial inflammation through integrin α5β1 and NF-κB pathway. Their study recently appeared in BioRxiv.
Endothelial cells (ECs) mostly exist in the inner layer of all blood vessels and are normally protected by pericytes. They form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, leukocyte infiltration and coagulation, the hallmarks of the severe disease. However, the mechanisms by which EC are dysregulated in COVID-19 are unclear. Now, Juan Robles and colleagues revealed that the spike activates NF-κB pathway through its interaction with integrin α5β1 in EC to elicit inflammation and leukocyte infiltration.
They also suggested that spike promotes hyperpermeability of EC monolayers and leukocyte adhesion via integrin α5β1 by regulating Rho GTPases and eNOS phosphorylation, and we can prevent or block the leukocyte adhesion and hyperpermeability in response to spike and spike receptor-binding domain by using ‘volociximab’, which is a chimeric anti-integrin α5β1 monoclonal antibody and ‘ATN-161’, which is the integrin α5β1 binding peptide.
“Our findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19.”
— they concluded.
Their work was supported by grants A1-S-9620B and 289568 from “Consejo Nacional de Ciencia y Tecnología” (CONACYT) to C.C. Magdalena Zamora is a doctoral student from ‘Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM)’ and received fellowship 768182 from CONACYT.
Reference: Juan Pablo Robles, Magdalena Zamora, Gonzalo Martinez de la Escalera, Carmen Clapp, “The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB”, bioRxiv 2021.08.01.454605; doi: https://doi.org/10.1101/2021.08.01.454605
Note for editors of other websites: To reuse this article fully or partially kindly give credit either to our author/editor S. Aman or provide a link of our article
Patients with a weakened immune system due to immunosuppressive therapies can develop good immune responses to a corona vaccination. A third dose of vaccine may be necessary for those patients who do not produce antibodies. This is shown by a current study by MedUni Vienna.
People affected by autoimmune diseases often need therapy that weakens the immune system. It is precisely in this group that COVID-19 can develop severely. Up until now it was unclear whether a vaccination against SARS-CoV-2 would guarantee a sufficient response, especially in patients who are receiving so-called B-cell-depleting drugs (e.g. rituximab for rheumatoid arthritis). In a recently published study by a cross-departmental team from the Medical University of Vienna coordinated by the Clinical Department for Rheumatology (Head: Daniel Aletaha) of the University Clinic for Internal Medicine III, this question has now been answered. Senior author Michael Bonelli and his study team were able to show that the majority of these patients are still able to develop a humoral and cellular immune response.
Michael Bonelli says: “B cells represent an important cell population for the development of antibodies. We were able to show that patients under B-cell-depleting therapy with rituximab still develop antibodies against SARS-CoV-2 in more than 50% of cases there is possible additional protection through a cellular immune response. This underlines the importance of vaccinating immunosuppressed patients against SARS-CoV-2. “
Sometimes a third vaccination is needed
Daniel Aletaha, Head of the Clinical Department for Rheumatology, explains further: “The findings of this work formed the basis for a now completed randomized booster vaccination study in which we examined whether that group of patients under therapy with rituximab were after Standard vaccination could not produce antibodies, but a third vaccination with a new mRNA vaccine or a switch to vector vaccine still developed humoral or cellular immunity. The results of the first vaccination study are about to be published and will hopefully contribute to the creation of guidelines for the vaccination strategy against SARS-CoV-2 in immunosuppressed patients.”
A follow-on study of the same design, for which volunteers are currently being recruited, will now extend the rituximab study to all patients with immunosuppression and different indications from the fields of rheumatology, neurology, hematology, transplantation, and others. This project is a collaboration between many researchers from different divisions/institutes of MedUni Vienna.
Service: Annals of the Rheumatic Diseases SARS-CoV-2 vaccination in rituximab-treated patients: B cells promote humoral immune responses in the presence of T-cell-mediated immunity. Mrak D, Tobudic S, Koblischke M, Graninger M, Radner H, Sieghart D, Hofer P, Perkmann T, Haslacher H, Thalhammer R, Winkler S, Blüml S, Stiasny K, Aberle JH, Smolen JS, Heinz LX, Aletaha D , Bonelli M. Ann Rheum Dis. 2021 Jul 20: annrheumdis-2021-220781. doi: 10.1136 / annrheumdis-2021-220781.
Using a bioactivity-guided chromatographic approach, in addition to mass-spectrometry (MS), Guillermo H. Jimenez-Aleman and colleagues identified the antiviral metabolite as Pheophorbide a (PheoA), a porphyrin chlorophyll derivative very similar to animal antiviral metabolite Protoporphyrin IX, in the bryophyte Marchantia polymorpha. Their study recently appeared in BioRxiv.
To confirm the antiviral potential of PheoA, a commercially available PheoA stock solution was serially diluted and mixed with a virus stock to inoculate Vero E6 and Huh7-ACE2 cells (human hepatoma cells expressing ACE2). They found that PheoA has an extraordinary antiviral activity against SARS-CoV-2 preventing infection of cultured monkey and human cells, without noticeable citotoxicity. Additionally, it has been shown that, PheoA prevents coronavirus entry into the cells by directly targeting the viral particle.
They also determined the antiviral spectrum of PheoA on different enveloped and non-enveloped viruses. They showed that, besides SARS-CoV-2, PheoA also displayed a broad-spectrum antiviral activity against enveloped (+)strand RNA viral pathogens such as HCV, West Nile, and other coronaviruses, but not against (-)strand RNA viruses, such as VSV.
Finally, they determined whether the addition of PheoA to remdesivir treatment could result in a synergistic effect on viral infection. They showed that increasing concentrations of PheoA (upto 40nM) improved remdesivir efficacy and viceversa.
“Our results indicate that PheoA displays a remarkable potency and a satisfactory therapeutic index, and suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2.”
Reference: Guillermo H Jimenez-Aleman, Victoria Castro, Addis Longdaitsbehere, Marta Gutierrez-Rodriguez, Urtzi Garaigorta, Pablo Gastaminza, Roberto Solano, “SARS-CoV-2 fears green: the chlorophyll catabolite Pheophorbide a is a potent antiviral”, bioRxiv 2021.07.31.454592; doi: https://doi.org/10.1101/2021.07.31.454592
Note for editors of other websites: To reuse this article fully or partially kindly give credit either to our author/editor S. Aman or provide a link of our article
Why do some people get sick and die from COVID-19 while others seem to be completely unaffected? EPFL’s Blue Brain Project deployed its powerful brain simulation technology and expertise in cellular and molecular biology to try and answer this question.
In response to the COVID-19 pandemic, the COVID-19 Open Research Dataset (CORD-19) of over 400,000 scholarly articles was made open access, including over 150,000 with full text papers related to COVID-19, SARS-CoV-2, and other coronaviruses. The CORD-19 dataset is the most extensive coronavirus literature collection available for data mining to date and the coalition behind it has challenged AI experts to apply their skills in natural language processing and other machine learning techniques in order to generate new insights that may help in the ongoing fight against COVID-19.
“Since early 2020, Blue Brain has been proactively contributing to the fight against COVID-19,” explains Prof. Henry Markram, Founder and Director of the Blue Brain Project. “With this call to action, we realized we could use our Machine Learning technologies and Data and Knowledge Engineering expertise to develop text and data mining tools required to try and help the medical community. Blue Brain set out to answer one of the most puzzling aspects of this pandemic – why some people get very sick, while others are completely unaffected”.
Building and using the text and data mining tools
Accordingly, Blue Brain built and trained machine-learning models to mine these papers and extract structured information from text sources. A simple analysis by this text mining toolbox ‘Blue Brain Search’ of the CORD-19v47 dataset revealed papers that all pointed to glucose metabolism as the most frequently mentioned biological variable.
Using Blue Graph, a unifying Python framework that analyses extracted text concepts to construct knowledge graphs, the group constructed specific knowledge graphs to focus on all the findings that considered glucose in the context of respiratory diseases, coronaviruses, and COVID-19. This allowed for the exploration of the potential role of glucose across many levels, from the most superficial symptomatic associations to the deepest biochemical mechanisms implicated in the disease.
From the facts and findings of thousands of papers mined, multiple lines of evidence emerged that elevated blood glucose levels were either caused by abnormal glucose metabolism, or induced during hospitalization, drug treatments or by IV administration. This approach correlated extremely well with COVID-19 severity across the population and revealed how elevated glucose helps virtually every step of the viral infection, from its onset in the lungs, through to severe complications such as Acute Respiratory Distress Syndrome, multi-organ failure and thrombotic events.
“Subsequently, in the paper, we discuss the potential consequences of this hypothesis and propose areas for further investigation into diagnostics, treatments and interventions that may help to reduce the severity of COVID-19 and help manage the public health impact of the pandemic,” discloses Blue Brain’s Molecular Biologist Dr. Emmanuelle Logette.
The potential of open access scientific papers
“Scientists immediately went to work when the pandemic started and within a year published over a hundred thousand papers. But, can anyone read so many papers? Can anyone see and understand all the patterns across all this research?” asks Prof. Henry Markram. “Fortunately, the coalition behind the CORD-19 dataset convinced all subscription publishers to bring these papers over the subscription paywall and make them openly accessible so that they can be mined with modern machine learning and knowledge engineering technologies”.
“With access to the CORD-19 dataset, Blue Brain quickly assembled an AI tool and targeted it to try and find out why some get sick and others not. Is it enough to just say that older people are more vulnerable? We must find out why. Why do some apparently healthy people die from COVID-19? Why do so many people die in the ICU? To answer these questions, we directed our AI to trace every step of the viral infection from the moment the virus enters the lungs until the time when the virus breaks out of the cells in the lungs and spreads throughout the body to infect the organs,” explains Prof. Markram. “We also built the virus at an atomistic level and developed a computational model of the infection so we could try to test what was coming out of the literature. I think we did find the most likely reason why some people get sicker than others,” he concludes.
An example of this is the team using Blue Brain BioExplorer to visually show the main impacts of high glucose in airway surface liquid on the primary step of infections in the lung and explaining the increased susceptibility to respiratory viruses in at-risk patients.
Blue Brain BioExplorer was built to reconstruct, visualize, explore and describe in detail the structure and function of the coronavirus for this study, and is open source for others to use to answer key scientific questions.
“Pioneering Simulation Neuroscience to better understand the brain has numerous collateral benefits,” states Prof. Markram. “This study shows how Blue Brain’s computing technologies and unique team of multi-disciplinary experts can quickly be redirected to help in a global health crisis.”
A major step forward for science and understanding the brain
“The COVID-19 study also shows why we believe that computational tools are so important to help us understand the brain,” explains Prof. Markram. “The problem is even bigger. There are several million scientific papers that one would need to read and understand to work out what we know about the brain. Does anyone know what we know? But, machines can read so many papers. This is the reason that the Blue Brain has developed some of the most advanced knowledge engineering, mathematical and machine learning accelerator technologies. Actually, this solves only a part of the challenge. With an AI tool that can read all these papers, we would still only know only a small fraction of what the brain contains and how it works. But building model brains using design principles, helps us to try and complete the picture.” he concludes.
Is it right to only open science during a pandemic?
Prof. Markram also expressed his frustration with the all too common practice of locking up of scientific knowledge by subscription publishers. “When the CORD-19 literature dataset was made available to us, we at Blue Brain were able to point our technology at COVID-19 and propose an answer to an important question in the battle against this deadly virus. Therefore, is it right to only make science papers (that are publicly funded) open to the public during a pandemic when the same kind of techniques can be used to help address so many other diseases, accelerate science, and help save the planet from climate change?”
About EPFL’s Blue Brain Project
The aim of the EPFL Blue Brain Project, a Swiss brain research initiative founded and directed by Professor Henry Markram, is to establish simulation neuroscience as a complementary approach alongside experimental, theoretical and clinical neuroscience to understanding the brain, by building the world’s first biologically detailed digital reconstructions and simulations of the mouse brain.
This study was supported by funding to the Blue Brain Project, a research center of the École polytechnique fédérale de Lausanne (EPFL), from the Swiss government ETH Board of the Swiss Federal Institutes of Technology.
Emmanuelle Logette, Charlotte Lorin, Cyrille Favreau, Eugenia Oshurko, Jay S. Coggan, Francesco Casalegno, Mohameth François Sy, Caitlin Monney, Marine Bertschy, Emilie Delattre, Pierre-Alexandre Fonta, Jan Krepl, Stanislav Schmidt, Daniel Keller, Samuel Kerrien, Enrico Scantamburlo, Anna-Kristin Kaufmann, Henry Markram. A machine-generated view of the role of Blood Glucose Levels in the severity of COVID-19. Frontiers in Public Health, 28 July 2021. doi.org/10.3389/fpubh.2021.695139
Featured image: Digital reconstruction of SARS-CoV-2 virus in the lung environment. Credit: Blue Brain Project
Izumi Kimura showed that Lambda spike (S) is highly infectious and T76I and L452Q are responsible for this property.
Lambda S is more susceptible to an infection-enhancing antibody.
RSYLTPGD246-253N, L452Q and F490S confer resistance to antiviral immunity.
The SARS-CoV-2 Lambda BS variant, also known as lineage C.37, is a variant of SARS-CoV-2, the virus that causes COVID-19. It was first detected in Peru in December 2020. On 14 June 2021, the World Health Organization named it Lambda variant and designated it as a variant of interest. Although the vaccination rate in Chile is relatively high (i.e. 60% people received at least one dose), a big Covid-19 surge has occurred in Chile in Spring 2021, which suggested that the Lambda variant is proficient in escaping from the antiviral immunity elicited by vaccination. Besides, that its virological features and evolutionary trait remain unknown.
Now, Izumi Kimura and colleagues revealed the virological features and evolutionary trait of the Lambda variant by performing virological experiments and molecular phylogenetic analysis.
They demonstrated that, there are three mutations which confer resistance to the vaccine-induced antiviral immunity, those are RSYLTPGD246-253N, L452Q and F490S mutations, respectively. Additionally, the T76I and L452Q mutations contributed to enhanced viral infectivity.
“Our data suggest that there are at least two virological features on the Lambda variant: increasing viral infectivity (by the T76I and L452Q mutations) and exhibiting resistance to antiviral immunity (by the RSYLTPGD246-253N, L452Q and F490S mutations).”
— they wrote.
They also demonstrated that the RSYLTPGD246-253N mutation, a unique mutation in the NTD of the Lambda Spike protein, is responsible not only for the resistance to the vaccine-induced neutralization as well as an NTD-targeting NAb, but also for the virological phenotype of the Lambda variant that can associate with the massive infection spread mainly in South American countries.
“Because the Lambda variant is a VOI, it might be considered that this variant is not an ongoing threat compared to the pandemic VOCs. However, because the Lambda variant is relatively resistant to the vaccine-induced antisera, it might be possible that this variant is feasible to cause breakthrough infection.”
— they said.
More studies on the evolutionary traits and virological features of SARS-CoV-2 variants are required in order to explain the possibility of wider spread of epidemic and for assessing the risk of future emerging SARS-CoV-2 variants.
Göttingen researchers have developed mini-antibodies that efficiently block the coronavirus and its new variants
Göttingen researchers have developed mini-antibodies that efficiently block the coronavirus Sars-CoV-2 and its dangerous new variants. These so-called nanobodies bind and neutralize the virus up to 1000 times better than previously developed mini-antibodies. In addition, the scientists optimized their mini-antibodies for stability and resistance to extreme heat. This unique combination makes them promising agents to treat Covid-19. Since nanobodies can be produced at low costs in large quantities, they could meet the global demand for Covid-19 therapeutics. The new nanobodies are currently in preparation for clinical trials.
Antibodies help our immune system to fend off pathogens. For example, the molecules attach to viruses and neutralize them so that they can no longer infect cells. Antibodies can also be produced industrially and administered to acutely ill patients. They then act like drugs, relieving symptoms and shortening recovery from the disease. This is established practice for treating hepatitis B and rabies. Antibodies are also used for treating COVID-19 patients. However, producing these molecules on an industrial scale is too complex and expensive to meet worldwide demand. Nanobodies could solve this problem.
Scientists at the Max Planck Institute for Biophysical Chemistry in Göttingen (Germany) and the University Medical Center Göttingen have now developed mini-antibodies (also known as VHH antibodies or nanobodies) that unite all the properties required for a potent drug against Covid-19. “For the first time, they combine extreme stability and outstanding efficacy against the virus and its Alpha, Beta, Gamma, and Delta mutants,” emphasizes Dirk Görlich, director at the Max Planck Institute for Biophysical Chemistry.
At first glance, the new nanobodies hardly differ from anti-Sars-CoV-2 nanobodies developed by other labs. They are all directed against a crucial part of the coronavirus spikes, the receptor-binding domain that the virus deploys for invading host cells. The nanobodies block this binding domain and thereby prevent the virus from infecting cells.
“Our nanobodies can withstand temperatures of up to 95 °C without losing their function or forming aggregates,” explains Matthias Dobbelstein, professor and director of the University Medical Center Göttingen’s Institute of Molecular Oncology. “For one thing, this tells us that they might remain active in the body long enough to be effective. For another, heat-resistant nanobodies are easier to produce, process, and store.”
Single, double, and triple nanobodies
The simplest mini-antibodies developed by the Göttingen team already bind up to 1000 times more strongly to the spike protein than previously reported nanobodies. They also bind very well to the mutated receptor-binding domains of the Alpha, Beta, Gamma, and Delta strains. “Our single nanobodies are potentially suitable for inhalation and thus for direct virus neutralization in the respiratory tract,” Dobbelstein says. “In addition, because they are very small, they could readily penetrate tissues and prevent the virus from spreading further at the site of infection.”
A ‘nanobody triad’ further improves binding: The researchers bundled three identical nanobodies according to the symmetry of the spike protein, which is comprised of three identical building blocks with three binding domains. “With the nanobody triad, we literally join forces: In an ideal scenario, each of the three nanobodies attaches to one of the three binding domains,” reports Thomas Güttler, a scientist in Görlich’s team. “This creates a virtually irreversible bond. The triple will not let release the spike protein and neutralizes the virus even up to 30,000-fold better than the single nanobodies.” Another advantage: The larger size of the nanobody triad expectedly delays renal excretion. This keeps them in the body for longer and promises a longer-lasting therapeutic effect.
As a third design, the scientists produced tandems. These combine two nanobodies that target different parts of the receptor-binding domain and together can bind the spike protein. “Such tandems are extremely resistant to virus mutations and the resulting ‘immune escape’ because they bind the viral spike so strongly”, explains Metin Aksu, a researcher in Görlich’s team.
For all nanobody variants – monomeric, double as well as triple – the researchers found that very small amounts are sufficient to stop the pathogen. If used as a drug, this would allow for a low dosage and thus for fewer side effects and lower production costs.
Alpacas provide blueprints for mini-antibodies
“Our nanobodies originate from alpacas and are smaller and simpler than conventional antibodies,” Görlich says. To generate the nanobodies against Sars-CoV-2, the researchers immunized three alpacas – Britta, Nora, and Xenia from the herd at the Max Planck Institute for Biophysical Chemistry – with parts of the coronavirus spike protein. The mares then produced antibodies, and the scientists drew a small blood sample from the animals. For the alpacas, the mission was then complete, as all further steps were carried out with the help of enzymes, bacteria, so-called bacteriophages, and yeast. “The overall burden on our animals is very low, comparable to vaccination and blood testing in humans,” Görlich explains.
Görlich’s team extracted around one billion blueprints for nanobodies from the alpacas’ blood. What then followed was a laboratory routine perfected over many years: The biochemists used bacteriophages to select the very best nanobodies from the initially vast pool of candidates. These were then tested for their efficacy against Sars-CoV-2 and further improved in successive rounds of optimization.
Not every antibody is ‘neutralizing’. Researchers of Dobbelstein’s group therefore determined if and how well the nanobodies prevent the viruses from replicating in cultured cells in the lab. “By testing a wide range of nanobody dilutions, we find out which quantity suffices to achieve this effect,” explains Antje Dickmanns from Dobbelstein’s team. Her colleague Kim Stegmann adds: “Some of the nanobodies were really impressive. Less than a millionth of a gram per liter of medium was enough to completely prevent infection. In the case of the nanobody triads, even another twenty-fold dilution was sufficient.“
Also effective against current coronavirus variants
Over the course of the coronavirus pandemic, new virus variants have emerged and rapidly became dominant. These variants are often more infectious than the strain that first appeared in Wuhan (China). Their mutated spike protein can also ‘escape’ neutralization by some originally effective antibodies of infected, recovered, or vaccinated persons. This makes it more difficult even for an already trained immune system to eliminate the virus. This problem also affects previously developed therapeutic antibodies and nanobodies.
This is where the new nanobodies show their full potential, as they are also effective against the major coronavirus variants of concern. The researchers had inoculated their alpacas with part of the spike protein of the first known Sars-CoV-2 virus, but remarkably, the animals’ immune system also produced antibodies that are active against the different virus variants. “Should our nanobodies prove ineffective against a future variant, we can reimmunize the alpacas. Since they have already been vaccinated against the virus, they would very quickly produce antibodies against the new variant,” Güttler asserts confidently.
Therapeutic application in view
The Göttingen team is currently preparing the nanobodies for therapeutic use. Dobbelstein emphasizes: “We want to test the nanobodies as soon as possible for safe use as a drug so that they can be of benefit to those seriously ill with Covid-19 and those who have not been vaccinated or cannot build up an effective immunity.” The team is supported by experts in technology transfer: Dieter Link (Max Planck Innovation), Johannes Bange (Lead Discovery Center, Dortmund, Germany), and Holm Keller (kENUP Foundation). The Max Planck Foundation provides financial support for the project.
The receptor-binding domain of Sars-CoV-2 is known to be a good candidate for a protein vaccine but so far difficult to manufacture economically on a large scale and in a form, which activates the immune system against the virus. Bacteria programmed accordingly produce incorrectly folded material. The Göttingen researchers discovered a solution for this problem: They identified special nanobodies that enforce correct folding in bacterial cells, without obstructing the crucial neutralizing part of the receptor-binding domain. This might allow for vaccines that can be produced inexpensively, can be quickly adapted to new virus variants, and can be distributed with simple logistics even in countries with little infrastructure. “The fact that nanobodies can help with protein folding was previously not known and is extremely interesting for research and pharmaceutical applications,” Görlich says.
Reference: Güttler T, Aksu M, Dickmanns A, Stegmann KM, Gregor K, Rees R, Taxer W, Rymarenko O, Schünemann J, Dienemann C, Gunkel P, Mussil B, Krull J, Teichmann U, Groß U, Cordes VC, Dobbelstein M, Görlich D, “Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies.”, EMBO J (2021). Source
Recep Ahan and colleagues evaluated the activity of griffithsin lectin protein (GRFT) from Griffithsia sp. against the novel human coronavirus, SARS-CoV-2. They demonstrated that this protein can not only block the entry of the Sars-CoV-2 but also inhibit its infection by attaching to spike protein of the Sars-CoV-2, both in vitro VeroE6 cell line and in vivo mouse model. Their study recently appeared in BioRxiv.
Lectin proteins isolated from seaweeds are shown to be potent antiviral agents against enveloped viruses e.g., HIV-1, herpes virus as well as two deadly human coronaviruses, SARS-CoV and MERS-CoV. Antiviral activity of seaweed lectins arises from their affinity to surface glycoproteins on viruses such as gp-120 protein of HIV-1 and spike proteins of SARS-CoV and MERS-CoV. Upon binding to surface proteins, lectins generally block the viral internalization step and thereby prevent the viral infection.
Now, Recep Ahan and colleagues evaluated the activity of griffithsin lectin protein (GRFT) from Griffithsia sp. against the novel human coronavirus, SARS-CoV-2.
For this purpose, they recombinantly expressed GRFT in E. coli with histidine tag and purified. Later, they validated and characterized binding of recombinant GRFT to whole inactivated SARS-CoV-2 as well as purified spike protein from HEK293 with the help of ELISA, ITC and QCM. Finally, they assessed the activity of GRFT in vitro with Vero6 cells, and in vivo with Syrian hamsters.
They demonstrated that griffithsin protein from Griffithsia sp. recombinantly produced in E. coli can bind spike (S) protein of SARS-CoV-2 in vitro and inhibit its infection both in vitro VeroE6 cell line and in vivo mouse model when applied prophylactically. In addition, toxicity assays of rGRFT with mouse models indicated that, it is a tolerable agent even at concentrations higher than its therapeutic concentration window.
“Our results indicate that GRFT is a potent non-mutagenic antiviral agent against SARS-CoV-2, reducing virus transmission through blocking its entry into the cells.”
— they said.
Finally, upon very promising results from in vitro and in vivo assays and experiments, they formulated GRFT as a nasal spray for upcoming human phase trials.
“We believe that GRFT protein-based drugs will have a high impact in preventing the transmission both on Wuhan strain as well as any other emerging variants including delta variant causing high speed spread of COVID-19.”