Pitt Researchers Discover Llama ‘Nanobodies’ Are Powerful New Coronavirus Treatment

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Pitt Researchers Discover Llama ‘Nanobodies’ Are Powerful New Coronavirus Treatment

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Pitt Researchers Discover Llama ‘Nanobodies’ Are Powerful New Coronavirus Treatment

The researchers used a llama they have dubbed 'Wally'.


11/5/20

https://pittsburgh.cbslocal.com/2020/11 ... treatment/


PITTSBURGH (KDKA) — Llamas could be the new key to helping humans fight the coronavirus.

According to the University of Pittsburgh School of Medicine, medical researchers have discovered a way to extract “tiny but extremely powerful SARS-CoV-2 antibody fragments from llamas.” SARS-CoV-2 is what causes COVID-19 infections.

The researchers used a llama they have dubbed Wally. He is named after the head researcher’s dog and lives on a farm in Massachusetts.

UPMC says, “These special llama antibodies, called ‘nanobodies,’ are much smaller than human antibodies and many times more effective at neutralizing the SARS-CoV-2 virus. They’re also much more stable.”

Dr. Yi Shi, Pitt assistant professor of cell biology, said, “Nature is our best inventor. The technology we developed surveys SARS-CoV-2 neutralizing nanobodies at an unprecedented scale, which allowed us to quickly discover thousands of nanobodies with unrivaled affinity and specificity.”

Researchers say they immunized Wally “with a piece of the SARS-CoV-2 spike protein.” About two months later, they say Wally’s immune system produced these mature nanobodies against the virus.

The researchers then worked to identify the nanobodies in Wally’s blood that most strongly bind to SARS-CoV-2.

The researchers then went to Pitt’s Center for Vaccine Research.

That’s where scientists “exposed the nanobodies to live SARS-CoV-2 virus and found that just a fraction of a nanogram could neutralize enough virus to spare a million cells from being infected.”

UPMC says “these nanobodies can sit at room temperature for six weeks. They can also “tolerate being fashioned into an inhalable mist,” which would deliver antiviral therapy straight to the lungs of the patient.

Doctors say these llama nanobodies could also be a much more affordable treatment for coronavirus.

“Nanobodies could potentially cost much less. They’re ideal for addressing the urgency and magnitude of the current crisis,” Dr. Shi said.

So when could we see this treatment become available?

“It has to be delivered safely. Phase one, two and three trials. Takes time. Can be bulked up rather quickly,” said Director of Vaccine Research Dr. Paul Duprex.

If and when it becomes available, doctors say it would likely be administered as a nasal spray, which would be delivered directly to the lungs for treatment or to prevent the virus from progressing.

“It’s really good to have multiple ways, to have multiple interventions, in development. It just takes time to get things into the pipeline to be used therapeutically,” said Dr. Duprex.

There’s still a full slate of trials for this to go through before it’s approved. Researchers say that while any vaccine can take a long time to develop, this specific drug could move a lot faster.
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Re: Pitt Researchers Discover Llama ‘Nanobodies’ Are Powerful New Coronavirus Treatment

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Llama Nanobodies Could be a Powerful Weapon Against COVID-19

11/5/20


https://www.upmc.com/media/news/110520- ... a-nanobody


PITTSBURGH – Today in Science, researchers at the University of Pittsburgh School of Medicine describe a new method to extract tiny but extremely powerful SARS-CoV-2 antibody fragments from llamas, which could be fashioned into inhalable therapeutics with the potential to prevent and treat COVID-19.

These special llama antibodies, called “nanobodies,” are much smaller than human antibodies and many times more effective at neutralizing the SARS-CoV-2 virus. They’re also much more stable.


“Nature is our best inventor,” said senior author Yi Shi, Ph.D., assistant professor of cell biology at Pitt. “The technology we developed surveys SARS-CoV-2 neutralizing nanobodies at an unprecedented scale, which allowed us to quickly discover thousands of nanobodies with unrivaled affinity and specificity.”


To generate these nanobodies, Shi turned to a black llama named Wally—who resembles and therefore shares his moniker with Shi’s black Labrador.

Shi and colleagues immunized the llama with a piece of the SARS-CoV-2 spike protein and, after about two months, the animal’s immune system produced mature nanobodies against the virus.


Using a mass spectrometry-based technique that Shi has been perfecting for the past three years, lead author Yufei Xiang, a research assistant in Shi’s lab, identified the nanobodies in Wally’s blood that bind to SARS-CoV-2 most strongly.


Then, with the help of Pitt’s Center for Vaccine Research (CVR), the scientists exposed their nanobodies to live SARS-CoV-2 virus and found that just a fraction of a nanogram could neutralize enough virus to spare a million cells from being infected.


These nanobodies represent some of the most effective therapeutic antibody candidates for SARS-CoV-2, hundreds to thousands of times more effective than other llama nanobodies discovered through the same phage display methods used for decades to fish for human monoclonal antibodies.

Shi’s nanobodies can sit at room temperature for six weeks and tolerate being fashioned into an inhalable mist to deliver antiviral therapy directly into the lungs where they’re most needed. Since SARS-CoV-2 is a respiratory virus, the nanobodies could find and latch onto it in the respiratory system, before it even has a chance to do damage.


In contrast, traditional SARS-CoV-2 antibodies require an IV, which dilutes the product throughout the body, necessitating a much larger dose and costing patients and insurers around $100,000 per treatment course.


“Nanobodies could potentially cost much less,” said Shi. “They’re ideal for addressing the urgency and magnitude of the current crisis.”

In collaboration with Cheng Zhang, Ph.D., at Pitt, and Dina Schneidman-Duhovny, Ph.D., at the Hebrew University of Jerusalem, the team found that their nanobodies use a variety of mechanisms to block SARS-CoV-2 infection. This makes nanobodies ripe for bioengineering. For instance, nanobodies that bind to different regions on the SARS-CoV-2 virus can be linked together, like a Swiss army knife, in case one part of the virus mutates and becomes drug-resistant.


“As a virologist, it’s incredible to see how harnessing the quirkiness of llama antibody generation can be translated into the creation of a potent nanoweapon against clinical isolates of SARS-CoV-2,” said study coauthor and CVR Director Paul Duprex, Ph.D.


Additional authors on the study include Sham Nambulli, Ph.D., Zhengyun Xiao, Heng Liu, Ph.D., and Zhe Sang, all of Pitt.
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Re: Pitt Researchers Discover Llama ‘Nanobodies’ Are Powerful New Coronavirus Treatment

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Nanobodies: unharnessed potential in COVID-19 treatment

11/16/20


https://www.news-medical.net/news/20201 ... tment.aspx


As the coronavirus disease 2019 (COVID-19) pandemic continues to cause hundreds of thousands of deaths worldwide every month, scientists continue to work on finding effective therapies to help mitigate its impact on those infected. A new study published in the Journal of Biomolecular Structure and Dynamics in October 2020 discusses the use of nanobodies in diagnosing and treating this deadly disease.

While most COVID-19 cases are mild or asymptomatic, in some cases, the disease causes serious or critical complications, including multi-organ dysfunction and death. The only known way to deal with this virus at present is via non-pharmaceutical interventions (NPIs) such as social distancing, case isolation and contact tracing – and even national or regional lockdowns. However, these are associated with heavy economic disruptions, making the development of effective pharmacological approaches to the pandemic vital.

Human antibody

A human antibody or immunoglobulin molecule is a Y-shaped structure. It comprises four polypeptides, two heavy and two light chains. This allows antibodies to bind to antigens, on the one hand, while mediating their biological activity on the other. Both functions are traceable to different regions of the antibody, the fragment antigen-binding (Fab) and fragment crystallizable (Fc) regions.

The Fc region binds to the cell surface receptors called Fc receptors, and to some complement components. This links antibody binding to immune activation.

Nanobodies


The current study deals with the quest to develop nanobodies (Nbs) or VHHs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which are a novel class of proteins based on antibody fragments with only one domain. They are derived from the heavy chain variable domains of camelids such as camels, llamas and alpacas, lacking a light chain component.

The Nb domains have full antigen-binding potential, with high affinity for their antigens. These have the smallest intact antigen-binding domain (about 15 kDa) and are only about 110 residues in length.

Nb domains have immense potential as therapeutic proteins for several reasons: their small size, stability, high solubility, cost-effective production in a yeast vector and high tissue penetration due to their low molecular weight. However, at present, they cannot be administered systemically because of low gut absorption.

Antiviral advantages of Nbs

Nbs have been explored as antiviral agents in many respiratory illnesses, such as Middle East respiratory syndrome (MERS), influenza type A, and respiratory syncytial virus infections. Recently, a pair of VHHs from a llama immunized with the stabilized prefusion form of the receptor-binding domain (RBD) of the coronavirus spike protein was found to neutralize both MERS and SARS coronaviruses. The VHH directed against the spike RBD bound to the spike with high affinity. This is down to its neutralizing capacity.

Three other VHHs have been found which bind to the RBD of SARS-CoV-2 and inhibit the RBD-ACE2 interaction that is essential for viral entry into the host cell, a finding which is important in view of the current pandemic.

Several researchers have reported the anti-inflammatory capabilities of Nbs too, which could help reduce cytokine production and the risk of a cytokine storm in severe cases of COVID-19.

The ability to deliver Nbs by nebulization allows them to reach the lungs directly to antagonize inflammation and inhibit viral replication. Their fusion to the Fc part of human antibodies of different classes allows them to be easily engineered to counter different viruses.

Approved Nbs


Nbs have already been under study for some time, cutting down on the time needed to bring them to the point of use. For instance, the drug caplacizumab is a bivalent approved Nb, used for thrombotic thrombocytopenic purpura. Another is vobarilizumab, an IL-6R Nb, which is being studied for its use in rheumatoid arthritis. Their safety as antivirals does need to be established, however, since they undergo rapid renal clearance due to the low molecular weight. This could produce renal damage or diminish tissue to below its effective concentration. In short, these issues all require further work.

Diagnostic applications


Nbs are also potentially useful for diagnostic applications. Several kits are now approved for serological diagnosis, targeting the IgM and IgG antibodies against the virus, respectively. However, their lack of specificity and sensitivity make them unacceptable for general screening. Nbs could solve this issue as part of a rapid antigen kit, with high sensitivity and specificity against SARS-CoV-2. In fact, enzyme-linked immunosorbent assays (ELISAs) have already been developed on the basis of Nbs, targeting specific antigens and biomarkers.

Conclusion


The future holds immense promise for Nbs as part of the therapeutic arsenal, as well as for diagnosis, against SARS-CoV-2, and for other new coronaviruses that might emerge in the future.
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Re: Pitt Researchers Discover Llama ‘Nanobodies’ Are Powerful New Coronavirus Treatment

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Enzolytics Inc. Shares Current BioClonetics Immunotherapeutics, Inc. Update

https://finance.yahoo.com/news/enzolyti ... 00939.html


We are making great progress on our plans to further develop additional anti-HIV monoclonal antibodies and to now begin the production of fully human monoclonal antibodies targeting the CoronaVirus. On December 1, we are expanding our lab to the campus of Texas A&M University at its Institute for Preclinical Studies. This expansion will allow us to complete production of monoclonal antibodies against both the HIV virus and the CoronaVirus and collaborate with the biopharma experts on the campus. Although we have NIH grant applications pending for the production of anti-HIV and anti-CoronaVirus monoclonal antibodies, we have secured funding that allows us to proceed without delay.

We welcome the recent news from Eli Lilly regarding its production of monoclonal antibodies for treatment of COVID-19 patients. We note that experts agree that for a monoclonal antibody therapy to be effective, a "combination" (or "cocktail") of such antibodies used in combination will likely be needed. Dr. Anthony Fauci, head of NIAID/NIH, has repeatedly clarified (as recently in his keynote address at the AIDS International Conference) that a success in treatment of such viruses can be expected to be found in the use of multiple broadly neutralizing HIV antibodies - meaning several antibodies that neutralize a broad spectrum of a virus in its numerous mutation forms.

Thus, we recognize that while other pharma companies may produce effective antibodies, there will necessarily be a need for additional monoclonal antibodies to be used in tantum with those initially discovered. Also, and unfortunately, the mutation of viruses, both the HIV and the CoronaVirus, will necessitate the production of numerous effective antibodies as the virus mutates around the therapeutics initially discovered.

Here is why we are confident in our technology.
It will be imperative that produced antibodies target a conserved and immutable site on the virus - otherwise the antibody (over time) will be rendered ineffective due to mutation - known as "virus escape". Our anti-HIV monoclonal antibody targets an immutable virus site on the HIV virus - one that is constant within virtually all 6000 now known different HIV isolates (strains) of the virus. The CoronaVirus has structure correlative to that of the HIV virus. Because our primary anti-HIV monoclonal antibody has been proven to neutralize numerous different strains of the HIV virus in tests in 5 international labs, and knowing the binding site on the HIV virus to which our antibody binds resulting in neutralization, this knowledge provides insight necessary to identifying corresponding structure (amino acid sequences) on the CoronaVirus that should be targeted to effectively neutralize the CoronaVirus. Moreover, we have proprietary methodology needed to produce anti-CoronaVirus monoclonal antibodies targeting such known - to us - sites.

Thus, while the Eli Lilly monoclonal antibody will hopefully have lasting effect, if it is targeting a mutable site on the virus, the virus may "escape" around it. And indeed, a close look at the results of the Eli Lilly initial tests of its antibody show that the antibody reduced the effect of the virus in some but not all of the patients receiving the antibody.

Thus, even these initial Eli Lilly trials demonstrate that additional monoclonal antibodies can be expected to be needed to fully treat COVID-19 patients.

The procedure for producing monoclonal antibodies is also significant and our procedure differs from those used by other pharma companies. In some cases, other pharma companies produce "humanized" rat and mouse monoclonal antibodies where the original antibody affinity and specificity are not maintained, and the chances of immunogenicity are increased. Our methodology also differs significantly from other pharma approaches using the transgenic mouse model [a human immune system which has been "grafted" within a mouse model] having been "vaccinated" with specific and selected purified CoronaVirus Proteins.

In contrast, our model starts with human "immune-B cells", obtained from convalescent individuals who have recovered from the CoronaVirus. The primary distinction of our process for creating fully human monoclonals is the starting point - namely from human "immune-B cells" from humans who have survived successfully from a "natural" CoronaVirus infection. From these, we then produce antibodies that target conserved immutable sites on the virus - to avoid "virus escape".

Additionally, our antibodies retain the original natural antibody affinity and specificity and have lower risk of immunogenicity when used as a therapeutic. They will provide broad-spectrum coverage against viral variants with increased potency, stability as a single-domain molecule, and, in the recombinant form, will have accessibility to the virus epitopes (binding sites) not accessible with a whole antibody.

We are actively moving forward in our production and testing of such antibodies.
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Re: Pitt Researchers Discover Llama ‘Nanobodies’ Are Powerful New Coronavirus Treatment

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Scientists isolate anti-COVID-19 nanobodies produced by a llama

12/22/20


https://www.news-medical.net/news/20201 ... llama.aspx


National Institutes of Health researchers have isolated a set of promising, tiny antibodies, or "nanobodies," against SARS-CoV-2 that were produced by a llama named Cormac. Preliminary results published in Scientific Reports suggest that at least one of these nanobodies, called NIH-CoVnb-112, could prevent infections and detect virus particles by grabbing hold of SARS-CoV-2 spike proteins. In addition, the nanobody appeared to work equally well in either liquid or aerosol form, suggesting it could remain effective after inhalation. SARS-CoV-2 is the virus that causes COVID-19.

The study was led by a pair of neuroscientists, Thomas J. "T.J." Esparza, B.S., and David L. Brody, M.D., Ph.D., who work in a brain imaging lab at the NIH's National Institute of Neurological Disorders and Stroke (NINDS).

" For years TJ and I had been testing out how to use nanobodies to improve brain imaging. When the pandemic broke, we thought this was a once in a lifetime, all-hands-on-deck situation and joined the fight. We hope that these anti-COVID-19 nanobodies may be highly effective and versatile in combating the coronavirus pandemic."

- Dr. Brody, Senior Author, Professor, Uniformed Services University for the Health Sciences

A nanobody is a special type of antibody naturally produced by the immune systems of camelids, a group of animals that includes camels, llamas, and alpacas. On average, these proteins are about a tenth the weight of most human antibodies. This is because nanobodies isolated in the lab are essentially free-floating versions of the tips of the arms of heavy chain proteins, which form the backbone of a typical Y-shaped human IgG antibody. These tips play a critical role in the immune system's defenses by recognizing proteins on viruses, bacteria, and other invaders, also known as antigens.

Because nanobodies are more stable, less expensive to produce, and easier to engineer than typical antibodies, a growing body of researchers, including Mr. Esparza and Dr. Brody, have been using them for medical research. For instance, a few years ago scientists showed that humanized nanobodies may be more effective at treating an autoimmune form of thrombotic thrombocytopenic purpura, a rare blood disorder, than current therapies.

Since the pandemic broke, several researchers have produced llama nanobodies against the SARS-CoV-2 spike protein that may be effective at preventing infections. In the current study, the researchers used a slightly different strategy than others to find nanobodies that may work especially well.

"The SARS-CoV-2 spike protein acts like a key. It does this by opening the door to infections when it binds to a protein called the angiotensin converting enzyme 2 (ACE2) receptor, found on the surface of some cells," said Mr. Esparza, the lead author of the study. "We developed a method that would isolate nanobodies that block infections by covering the teeth of the spike protein that bind to and unlock the ACE2 receptor."

To do this, the researchers immunized Cormac five times over 28 days with a purified version of the SARS-CoV-2 spike protein. After testing hundreds of nanobodies they found that Cormac produced 13 nanobodies that might be strong candidates.

Initial experiments suggested that one candidate, called NIH-CoVnb-112, could work very well. Test tube studies showed that this nanobody bound to the ACE2 receptor 2 to 10 times stronger than nanobodies produced by other labs. Other experiments suggested that the NIH nanobody stuck directly to the ACE2 receptor binding portion of the spike protein.

Then the team showed that the NIH-CoVnB-112 nanobody could be effective at preventing coronavirus infections. To mimic the SARS-CoV-2 virus, the researchers genetically mutated a harmless "pseudovirus" so that it could use the spike protein to infect cells that have human ACE2 receptors. The researchers saw that relatively low levels of the NIH-CoVnb-112 nanobodies prevented the pseudovirus from infecting these cells in petri dishes.

Importantly, the researchers showed that the nanobody was equally effective in preventing the infections in petri dishes when it was sprayed through the kind of nebulizer, or inhaler, often used to help treat patients with asthma.

"One of the exciting things about nanobodies is that, unlike most regular antibodies, they can be aerosolized and inhaled to coat the lungs and airways," said Dr. Brody.

The team has applied for a patent on the NIH-CoVnB-112 nanobody.

"Although we have a lot more work ahead of us, these results represent a promising first step," said Mr. Esparza. "With support from the NIH we are quickly moving forward to test whether these nanobodies could be safe and effective preventative treatments for COVID-19. Collaborators are also working to find out whether they could be used for inexpensive and accurate testing."

Source:

NIH/National Institute of Neurological Disorders and Stroke

Journal reference:


Esparza, T.J., et al. (2020) High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme. Scientific Reports. doi.org/10.1038/s41598-020-79036-0.
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Re: Pitt Researchers Discover Llama ‘Nanobodies’ Are Powerful New Coronavirus Treatment

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Promising nanobodies against COVID-19 produced by llamas

1/25/21


https://www.news-medical.net/news/20210 ... lamas.aspx


In this interview, News-Medical speaks to Dr. David Brody about his latest research that involved discovering nanobodies produced by llamas that could help combat coronavirus.

What provoked your research into the ongoing COVID-19 pandemic?

We are neuroscientists, so it was a bit of a shift in direction! For several years TJ and I had been testing out how to use nanobodies to make brain imaging better. When the pandemic broke, we thought this is a once in a lifetime, all-hands-deck situation and joined the fight.

The labs were all closed down in April, and we thought we would much rather work on COVID-19 rather than staying at home.

What are ‘nanobodies’ and how do these differ from regular antibodies?

Nanobodies are derived from a special type of antibodies naturally produced by the immune systems of camelids, i.e. camels, llamas, and alpacas. Most antibodies are made from 4 proteins bound together: two heavy chains and two light chains.

Camelids make special antibodies that are made from just 2 proteins: 2 heavy chains. Nanobodies are created in the laboratory by isolating just the tips of the heavy chains, where the binding occurs. On average, these nanobody proteins are about a tenth the weight of a typical human antibody.

Because nanobodies are more stable, less expensive to produce, and easier to engineer than typical antibodies a growing body of researchers, including our group, have been using them for medical research. For instance, a few years ago scientists showed that humanized nanobodies may be more effective at treating an autoimmune form of thrombotic thrombocytopenic purpura, a rare blotting clotting disorder, than current therapies.

One of the exciting things about nanobodies is that, unlike most regular antibodies, they can be aerosolized and inhaled to coat the lungs and airways. We think this could be especially beneficial for preventing COVID-19 transmission through the air.

Can you describe how you carried out your research into COVID-19 antibodies?

We immunized a llama, named Cormac, five times over 28 days with a purified version of the SARS-CoV-2 spike protein. Then we isolated the DNA that includes the instructions for making antibodies from Cormac’s blood. (By the way, Cormac lives on a beautiful farm in Eastern Washington state with quite a few other llamas. He has a catheter in one of his veins so that people can draw his blood without sticking him with any needles).

Back in the lab here in Bethesda, we tested out hundreds of nanobodies and found that Cormac produced 13 nanobodies that might be strong candidates. We selected nanobodies that could block the attachment of the SARS-CoV2 spike protein to the human ACE2 receptor.

The virus gets inside of the human body by binding to the ACE2 receptor on the outer surface of human cells, so blocking this attachment was a logical target.

What did you discover?

We discovered that one candidate nanobody, called NIH-CoVnb-112 bound very tightly to the part of the SARS-CoV2 spike protein responsible for interacting with the ACE2 receptor.

We next showed that the NIH-CoVnB-112 nanobody could be effective at preventing infections in a petri dish. To mimic the COVID-19 virus, our collaborators in North Carolina genetically mutated a harmless “pseudovirus’ so that it can use the SARS-CoV-2 spike protein to infect cells that produce the human ACE2 receptor. They found that relatively low levels of the NIH-CoVnb-112 nanobodies prevented the pseudovirus from infecting these cells in Petri dishes.

Importantly, as a team, we showed that the nanobody was equally effective in preventing the infections in Petri dishes after being sprayed through the sort of nebulizer that patients might use.

How effective were these nanobodies at combating coronavirus?

We do not know how effective the nanobodies will be in combating coronavirus. There is still a lot of work to do before we will have the answers.

What are the advantages of using nanobodies compared to typical antibodies?

There are a lot of advantages of nanobodies.

Nanobodies are very stable. We can keep them at room temperature for a long time, they resist extreme heat and cold, and they can be dried down into a powder for storage then reconstituted with water when needed. It is much trickier to handle and store typical antibodies.
Nanobodies are less expensive to produce. They can be made in bacteria or yeast, which is much easier than the mammalian cells used to produce regular antibodies.
Nanobodies are easier to engineer than typical antibodies. They are smaller and made from just 1 protein instead of 2.
Nanobodies can be aerosolized and inhaled to coat the lungs and airways. This does not usually work well for typical antibodies.
Nanobodies do not typically activate the human immune system. This could be an advantage or a disadvantage depending on whether we want to activate the immune system or not.

Do you believe that with further research, these nanobodies could potentially be used to combat the coronavirus pandemic?

We think that with further research, the nanobodies could potentially be used to combat the coronavirus pandemic in several ways:

People who are going into a high-risk situation could use a nebulizer at home to coat their airways and lungs with a protective layer of nanobodies.
People who have been exposed to someone with COVID-19 could start treating themselves at home to reduce the chances that they will get sick
People who have COVID-19 could coat their airways and lungs with nanobodies to reduce the transmission of the active virus to others around them. A virus that has nanobodies stuck to it would be expected to be less infectious than a naked virus.
The nanobodies could be used to spray environmental surfaces, providing a coat that will neutralize the active virus that lands on the surfaces.
The nanobodies could be used for environmental sensors to detect the virus in the atmosphere, in fluids, or on surfaces.
The nanobodies could be used to make very inexpensive test kits that are stable at extreme temperatures.

What are the next steps in your research?

There are several important next steps that are underway:

Testing the nanobodies in animal models of COVID-19 infection.
Scaling up production to make enough high-quality nanobodies for larger studies
Testing to see whether the nanobodies will block the new variants of the virus.

Where can readers find more information?

We strongly encourage everyone to read the original paper. It is important for the public to understand the data and be well-educated on this topic.

For this reason, we have published it in a journal called Scientific Reports that is available to everyone in the world with internet access and we have written the paper in a way that we hope is relatively easy to understand.

Here is the link:

https://www.nature.com/articles/s41598-020-79036-0

Several other groups around the world are also working on nanobodies.

Here is a link to a news story from last fall:
https://www.nature.com/articles/d41586-020-02965-3

One company in China called Shanghai Novamab has already scaled-up production of its nanobody.
https://www.researchgate.net/publicatio ... _potential
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Re: Pitt Researchers Discover Llama ‘Nanobodies’ Are Powerful New Coronavirus Treatment

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Llama-derived nanobodies show neutralizing activity against SARS-CoV-2 variants of concern in vitro

2/19/21


https://www.news-medical.net/news/20210 ... vitro.aspx


With the emergence of new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, the prospect of effective containment of viral transmission has receded. Not only are they more transmissible than the earlier strains, but they also offer partial resistance to neutralization by existing antibodies in some cases. Moreover, they can evade detection by current antigen detection kits because of mutations in the spike protein with each of the new variants.

Better tests needed

This development could cause many more false-negative diagnoses to arise, giving rise to a more devastating wave of community spread. The need for more up-to-date antibody arrays is obvious. A new preprint on the bioRxiv* server reports the development of nanoantibodies (nAbs) – also called or nanobodiesTM or single-domain antibodies – that effectively bind the spike protein from the ancestral Wuhan variant as well as the recently detected UK and South African variants.

SARS-CoV-2 mediates host cell receptor attachment via its spike protein, which has two subunits, S1 and S2. The host cell receptor for the virus, the angiotensin-converting enzyme 2 (ACE2), is bound by the receptor binding domain (RBD) of the viral spike S1 subunit.

Mutational escape of detection

The UK variant, SARS-CoV-2 B.1.1.7 lineage, has approximately 20 mutations. Of these, the N501Y affects the RBD, while several are at the N-terminal domain of the S1 subunit of the spike protein. The South African variant, B.1.351 lineage, has a triple mutation in the RBD, namely, N501Y, K417N, and E484K.

These mutations may affect the epitope binding of the monoclonal antibody pairs that are currently used for antigen detection in rapid detection tests.

The benefits of nanobodies

Nanoantibodies overcome this obstacle by using a different mechanism. Since nAbs have a size ten times less than the classical immunoglobulin G (IgG) antibody, comprising a single domain, they creep into crevices on their cognate antigens by inserting the corresponding epitope-contacting antigens.

In contrast, conventional antibodies use a pair of arms comprising the heavy and light chains, to wrap around the epitope exposed on the surface.

The former mechanism of epitope binding allows greater flexibility in the face of individual amino acid substitutions, and has been exploited in the current study.

Llama nanobodies

The researchers first inoculated the SARS-CoV-2 spike S1 subunit and the receptor-binding domain (RBD) into a llama. They applied their already existing nanoantibody (nAb) development pipeline to obtain many groups of nAbs directed against the RBD. These showed strong binding for the RBD of the Wuhan-Hu-1 strain.

Each of these was then characterized. They found that two of them, nAb1 and nAb2, had strong binding affinity for the wildtype RBD.

Binding to all RBD variants


They created a preliminary version of a rapid antigen test, as a lateral flow assay (LFA), for testing against clinical samples predating the emergence of the newer variants. Using a fluorescent reporter protein, linked to a fusion protein containing two nAb1 domains (nAb1 dimer), they tested for binding affinity against the variant SARS-CoV-2.

They found that both nAb1 and nAb2 bound to RBDs from all three viral variants.

In contrast, the monoclonal antibody CR3022 binds to wildtype RBD only. Surprisingly, it failed to bind to the South African RBD variant, even though computational modeling predicted that the RBD would not be significantly affected by the variant's mutations.

nAb1 dimer shows higher binding

The nAb1 dimer has apparently higher binding affinity relative to the nAb1 monomer, perhaps because of cooperative binding, which may involve adjacent RBDs on the same immunobead. Since the natural form of the spike protein is trimeric, and each virion has about 24 trimeric spikes, cooperative binding is likely to boost the sensitivity of detection of the virus in clinical antigen detection kits.

In fact, earlier research by the same team has shown that linear multimers of nAb1 have a higher neutralizing capacity against SARS-Cov-2 infection of human iPSC-derived lung epithelial cells.

The researchers are continuing to test three or more nAb clusters for their ability to detect these variants and possibly others as well, currently in circulation or likely to emerge soon.

Highly sensitive detection

Already, Allele Biotech has produced a rapid antigen test for COVID-19 diagnosis for use at home and at the point of care. This device uses multiple nAbs to bind to hundreds of such binding pockets on the viral surface. This has added a high level of sensitivity, allowing current and future variants to be detected more confidently.

What are the implications?

The nanoantibodies for rapid antigen detection test bind the receptor binding domain (RBD) of the S1 protein from the original COVID-SARS-2 virus as well as those from the UK and South African variants. This finding validates our antibodies used in our assay for the detection of these major variant strains.”

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:


Yamane, D. et al. (2021). Single-Domain SARS-CoV-2 S1 and RBD Antibodies Isolated from Immunized Llama Effectively Bind Targets of the Wuhan, UK, and South African Strains in vitro. bioRxiv preprint. doi: https://doi.org/10.1101/2021.02.15.431198, https://www.biorxiv.org/content/10.1101 ... 5.431198v1
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