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Microbiology

Novel antibodies target human receptors to neutralize SARS-CoV-2 variants and future sarbecoviruses

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Neha MathurBy Neha MathurMay 17 2023Reviewed by Lily Ramsey, LLM

In a recent study published in the Nature Microbiology Journal, researchers generated six human monoclonal antibodies (mAbs) that prevented infection by all human angiotensin-converting enzyme 2 (ACE2) binding sarbecoviruses tested, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, Delta and Omicron.

They targeted the hACE2 epitope that binds to the SARS-CoV-2 spike (S) glycoprotein rather than targeting the S protein, which all previous therapeutic mAbs for SARS-CoV-2 targeted.

Study: Pan-sarbecovirus prophylaxis with human anti-ACE2 monoclonal antibodies. Image Credit: paulista/Shutterstock.comStudy: Pan-sarbecovirus prophylaxis with human anti-ACE2 monoclonal antibodies. Image Credit: paulista/Shutterstock.com

Background

The emergence of new variants of SARS-CoV-2, especially Omicron sublineages, made all therapeutic mAbs targeting SARS-CoV-2 S obsolete.

Any new S-targeting mAb therapy will also probably have limited utility because SARS-CoV-2 will continue to adapt to human antibodies. Ideally, mAbs developed in anticipation of future pandemics caused by sarbecoviruses should be resilient to mutations that arise in them.

About the study

In the present study, researchers developed hACE2-binding mAbs that blocked infection by pseudotypes of all tested sarbecoviruses at potencies matching SARS-CoV-2 S targeting therapeutic mAbs. The binding affinity of these mAbs to hACE2 was in the nanomolar to picomolar range.

To develop these mAbs, researchers used the KP and Av AlivaMab mouse strains that generate a human Kappa (κ) light chain and Kappa (κ) and Lambda (λ) light chains carrying antibodies, respectively.

They immunized these mice with monomeric and dimeric recombinant hACE2 extracellular domains. Fusion to the fraction, crystallizable (Fc) portion of human immunoglobulin G1 (IgG1) rendered them dimeric.

Further, the team generated hybridomas from mice using sera that inhibited SARS-CoV-2 pseudotyped viruses. They used enzyme-linked immunosorbent assay (ELISA) to screen hybridoma supernatants for hACE2-binding mAbs.

Furthermore, the researchers tested the ability of the six most potent mAbs to inhibit Wuhan-hu-1 S pseudotyped infection in Huh-7.5 target cells.

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The team purified chimeric mAbs from the hybridoma culture supernatants and used a SARS-CoV-2 pseudotype assay to reconfirm their antiviral activity. They also sequenced the human Fab variable regions, VH and VL.

The team cloned VH and VL domains from the six most potent chimeric human-mouse mAbs into a human IgG1 expression vector to generate fully human anti-hACE2 mAbs.

They used single-particle cryo-electron microscopy (cryo-EM) to delineate the structural basis for broad neutralization of anti-hACE2 mAbs.

Specifically, they determined the structure of soluble hACE2 bound to the antigen-binding fragment (Fab) of 05B04, one of the most potent mAbs unaffected by naturally occurring human ACE2 variations.

Finally, the researchers tested these hACE2 mAbs in an animal model and determined their pharmacokinetic behavior.

Results

The researchers identified 82 hybridomas expressing hACE2-binding mAbs, of which they selected ten based on their potency in inhibiting pseudotyped virus infection of Huh-7.5 cells.

These ten mAbs were 1C9H1, 4A12A4, 05B04, 2C12H3, 2F6A6, 2G7A1, 05D06, 05E10, 05G01 and 05H02. Four of the five mAbs from the KP AlivaMab mice, viz., 05B04, 05E10, 05G01, and 05D06, shared identical complementarity-determining regions (CDRs). Conversely, AV AlivaMab mice-derived mAbs were diverse.

While allosteric inhibition of hACE2 activity by the mAbs was theoretically feasible, such inhibition did not occur.

Also, the anti-hACE2 mAbs did not affect hACE2 internalization or recycling, suggesting that the anti-hACE2 mAbs would unlikely undergo accelerated target-dependent clearance from the circulation during in vivo use.

These two findings confirmed that these mAbs would not have harmful side effects based on their target specificity.

In addition, the anti-hACE2 mAbs showed favorable pharmacokinetics and no ill effects on the hACE2 knock-in mice. When used prophylactically in hACE2 knock-in mice, these mAbs conferred near-sterilizing protection against lung SARS-CoV-2 infection.

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Moreover, they presented a high genetic barrier to the acquisition of resistance by SARS-CoV-2.

The six anti-hACE2 mAbs also inhibited infection by pseudotyped SARS-CoV-2 variants, Delta, and Omicron, with similar potency, i.e., half maximal inhibitory concentration (IC50) values ranging between 8.2 ng ml−1 and 197 ng ml−1.

A cryo-EM structure of the 05B04-hACE2 complex at 3.3 Å resolution revealed a 05B04 Fab bound to the N-terminal helices of hACE2.

05B04-mediated inhibition of ACE2-binding sarbecoviruses through molecular mimicry of SARS-CoV-2 receptor-binding domain (RBD) interactions, providing high binding affinity to hACE2 despite the smaller binding footprint on hACE2.

None of the four most potent mAbs affected hACE2 enzymatic activity or induced the internalization of hACE2 localized on the host cell surface. Thus, based on their target specificity, these mAbs shall not have deleterious side effects.

Though these anti-ACE2 antibodies could effectively inhibit sarbecovirus infection in humans, the fact that the antibodies target a host receptor molecule rather than the SARS-CoV-2 S protein will necessitate their testing in terms of safety, efficacy, and pharmacological behavior in primate models before human clinical trials.

Conclusions

SARS-CoV-2 might evolve and start using receptors other than ACE2, creating another genetic hurdle to overcome for researchers working on the development of SARS-CoV-2 therapeutics.

However, the human anti-hACE2 mAbs engineered in this study showed exceptional breadth and potency in inhibiting infection by hACE2-utilizing sarbecoviruses.

Thus, they represent a long-term, ‘resistance-proof’ prophylaxis and treatment for SARS-CoV-2, even for future outbreaks of SARS-like coronaviruses.

In addition, these mAbs might prove particularly useful for susceptible patients like those with immunodeficiency and in which vaccine-induced protective immunity is unattainable or difficult to attain.

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Journal reference:
Microbiology

SARS-CoV-2 infection damages the CD8+ T cell response to vaccination

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Reviewed by Emily Henderson, B.Sc.Mar 20 2023

The magnitude and quality of a key immune cell’s response to vaccination with two doses of the Pfizer-BioNTech COVID-19 vaccine were considerably lower in people with prior SARS-CoV-2 infection compared to people without prior infection, a study has found. In addition, the level of this key immune cell that targets the SARS-CoV-2 spike protein was substantially lower in unvaccinated people with COVID-19 than in vaccinated people who had never been infected. Importantly, people who recover from SARS-CoV-2 infection and then get vaccinated are more protected than people who are unvaccinated. These findings, which suggest that the virus damages an important immune-cell response, were published today in the journal Immunity.

The study was co-funded by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, and led by Mark M. Davis, Ph.D. Dr. Davis is the director of the Stanford Institute for Immunity, Transplantation and Infection and a professor of microbiology and immunology at Stanford University School of Medicine in Palo Alto, California. He is also a Howard Hughes Medical Institute Investigator.

Dr. Davis and colleagues designed a very sensitive tool to analyze how immune cells called CD4+ T cells and CD8+ T cells respond to SARS-CoV-2 infection and vaccination. These cells coordinate the immune system’s response to the virus and kill other cells that have been infected, helping prevent COVID-19. The tool was designed to identify T cells that target any of dozens of specific regions on the virus’s spike protein as well as some other viral regions. The Pfizer-BioNTech vaccine uses parts of the SARS-CoV-2 spike protein to elicit an immune response without causing infection.

The investigators studied CD4+ and CD8+ T-cell responses in blood samples from three groups of volunteers. One group had never been infected with SARS-CoV-2 and received two doses of the Pfizer-BioNTech COVID-19 vaccine. The second group had previously been infected with SARS-CoV-2 and received two doses of the vaccine. The third group had COVID-19 and was unvaccinated.

The researchers found that vaccination of people who had never been infected with SARS-CoV-2 induced robust CD4+ and CD8+ T-cell responses to the virus’ spike protein. In addition, these T cells produced multiple types of cell-signaling molecules called cytokines, which recruit other immune cells—including antibody-producing B cells—to fight pathogens. However, people who had been infected with SARS-CoV-2 prior to vaccination produced spike-specific CD8+ T cells at considerably lower levels—and with less functionality—than vaccinated people who had never been infected. Moreover, the researchers observed substantially lower levels of spike-specific CD8+ T cells in unvaccinated people with COVID-19 than in vaccinated people who had never been infected.

Taken together, the investigators write, these findings suggest that SARS-CoV-2 infection damages the CD8+ T cell response, an effect akin to that observed in earlier studies showing long-term damage to the immune system after infection with viruses such as hepatitis C or HIV. The new findings highlight the need to develop vaccination strategies to specifically boost antiviral CD8+ T cell responses in people previously infected with SARS-CoV-2, the researchers conclude.  

Source:

National Institutes of Health

Journal reference:

Gao, F., et al. (2023). Robust T cell responses to Pfizer/BioNTech vaccine compared to infection and evidence of attenuated peripheral CD8+ T cell responses due to COVID-19. Immunity. doi.org/10.1016/j.immuni.2023.03.005.

Microbiology

Clinical trial shows safety and immunogenicity of temperature-stable experimental TB vaccine

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Reviewed by Emily Henderson, B.Sc.Mar 6 2023

A clinical trial testing a freeze-dried, temperature-stable experimental tuberculosis (TB) vaccine in healthy adults found that it was safe and stimulated both antibodies and responses from the cellular arm of the immune system. The Phase 1 trial was supported by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health. A non-temperature stable form of the candidate previously had been tested in several clinical trials. However, this was the first clinical trial of any subunit TB vaccine candidate in a temperature-stable (thermostable) form. Results are published in Nature Communications.

The experimental vaccine, ID93+GLA-SE, was developed by Christopher B. Fox, Ph.D., and scientists at the Access to Advanced Health Institute (formerly the Infectious Disease Research Institute) in Seattle. It is a recombinant subunit vaccine made from four proteins of Mycobacterium tuberculosis bacteria combined with GLA-SE, an immune-stimulating adjuvant. The freeze-dried formulation does not require refrigeration and is mixed with sterile water just prior to injection. Thermostable vaccines are desirable in settings where maintaining cold or frozen vaccines for long periods can be costly and difficult.

The current trial investigated whether administering temperature-stable vaccine containing both ID93 and GLA-SE in a single vial would be as effective at inducing an immune response as a regimen in which non-thermostable ID93 and liquid GLA-SE are held in two vials and combined prior to injection. A single-vial presentation of a thermostable vaccine would have clear advantages in ease of storage, transport and administration, the investigators note.

Daniel F. Hoft, M.D., Ph.D., director of the Saint Louis University Center for Vaccine Development, led the single-site trial at the university’s School of Medicine. Twenty-three participants received the thermostable single-vial regimen, while 22 participants received the two-vial, non-thermostable regimen. Both vaccine presentations were safe and well-tolerated. Recipients of the single-vialled thermostable vaccine had robust T-cell responses and produced higher levels of antibodies in the blood than those receiving the non-thermostable two-vial presentation.

The investigators note some limitations in this small trial. For example, no established correlates of protection define what immune responses are required for vaccine-induced protection from TB disease. Therefore, it is not possible to say whether the enhanced immune responses seen in the thermostable vaccine formulation would translate to improved protective vaccine efficacy. Nevertheless, they conclude, results of this trial demonstrate “a proof-of-concept that adjuvant-containing vaccines can be formulated in a freeze-dried single-vial presentation without detrimentally impacting clinical immunogenicity or safety characteristics.”

Source:

National Institutes of Health

Journal reference:

Sagawa, Z.K., et al. (2023) Safety and immunogenicity of a thermostable formulation of the ID93 + GLA-SE tuberculosis vaccine candidate in healthy adults. Nature Communications. doi.org/10.1038/s41467-023-36789-2.

Microbiology

Using the origami technique to design RNA nanostructures

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Reviewed by Emily Henderson, B.Sc.Feb 28 2023

Researchers from Aarhus University and Berkeley Laboratory have designed RNA molecules, that folds into nanoscale rectangles, cylinders, and satellites, and have studied their 3D structure and dynamics with advanced nanotechnological methods. In an article in the journal Nature Nanotechnology, the researchers describe their work and how it has led to the discovery of rules and mechanisms for RNA folding that will make it possible to build more ideal and functional RNA particles for use in RNA-based medicine.

The RNA molecule is commonly recognized as messenger between DNA and protein, but it can also be folded into intricate molecular machines. An example of a naturally occurring RNA machine is the ribosome, that functions as a protein factory in all cells. Inspired by natural RNA machines, researchers at the Interdisciplinary Nanoscience Center (iNANO) have developed a method called “RNA origami”, which makes it possible to design artificial RNA nanostructures that fold from a single stand of RNA. The method is inspired by the Japanese paper folding art, origami, where a single piece of paper can be folded into a given shape, such as a paper bird.

Frozen folds provide new insight

The research paper in Nature Nanotechnology describes how the RNA origami technique was used to design RNA nanostructures, that were characterized by cryo-electron microscopy (cryo-EM) at the Danish National cryo-EM Facility EMBION. Cryo-EM is a method for determining the 3D structure of biomolecules, which works by freezing the sample so quickly that water does not have time to form ice crystals, which means that frozen biomolecules can be observed more clearly with the electron microscope. Images of many thousands of molecules can be converted on the computer into a 3D map, that is used to build an atomic model of the molecule. The cryo-EM investigations provided valuable insight into the detailed structure of the RNA origamis, which allowed optimization of the design process and resulted in more ideal shapes.

With precise feedback from cryo-EM, we now have the opportunity to fine-tune our molecular designs and construct increasingly intricate nanostructures.”

Ebbe Sloth Andersen, Associate Professor at iNANO, Aarhus University

Discovery of a slow folding trap

Cryo-EM images of an RNA cylinder sample turned out to contain two very different shapes, and by freezing the sample at different times it was evident that a transition between the two shapes was taking place. Using the technique of small-angle X-ray scattering (SAXS), where the samples are not frozen, the researchers were able to observe this transition in real time and found that the folding transition occurred after approx. 10 hours. The researchers had discovered a so-called “folding trap” where the RNA gets trapped during transcription and only later gets released (see video).

“It was quite a surprise to discover an RNA molecule that refolds this slow since folding typically takes place in less than a second” tells Jan Skov Pedersen, Professor at Department of Chemistry and iNANO, Aarhus University.

“We hope to be able to exploit similar mechanisms to activate RNA therapeutics at the right time and place in the patient”, explains Ewan McRae, the first author of the study, who is now starting his own research group at the “Centre for RNA Therapeutics” at the Houston Methodist Research Institute in Texas, USA.

Construction of a nanosatellite from RNA

To demonstrate the formation of complex shapes, the researchers combined RNA rectangles and cylinders to create a multi-domain “nanosatellite” shape, inspired by the Hubble Space Telescope.

“I designed the nanosatellite as a symbol of how RNA design allows us to explore folding space (possibility space of folding) and intracellular space, since the nanosatellite can be expressed in cells”, says Cody Geary, assistant professor at iNANO, who originally developed the RNA-origami method.

However, the satellite proved difficult to characterize by cryo-EM due to its flexible properties, so the sample was sent to a laboratory in the USA, where they specialize in determining the 3D structure of individual particles by electron tomography, the so-called IPET-method.

“The RNA satellite was a big challenge! But by using our IPET method, we were able to characterize the 3D shape of individual particles and thus determine the positions of the dynamic solar panels on the nanosatellite”, says Gary Ren from the Molecular Foundry at Lawrence Berkeley National Laboratory, California, USA.

The future of RNA medicine

The investigation of the RNA origamis contributes to improving the rational design of RNA molecules for use in medicine and synthetic biology. A new interdisciplinary consortium, COFOLD, supported by the Novo Nordisk Foundation, will continue the investigations of RNA folding processes by involving researchers from computer science, chemistry, molecular biology, and microbiology to design, simulate and measure folding at higher time resolution.

“With the RNA design problem partially solved, the road is now open to creating functional RNA nanostructures that can be used for RNA-based medicine, or act as RNA regulatory elements to reprogram cells”, predicts Ebbe Sloth Andersen.

Source:

Aarhus University

Journal reference:

McRae, E.K.S., et al. (2023) Structure, folding and flexibility of co-transcriptional RNA origami. Nature Nanotechnology. doi.org/10.1038/s41565-023-01321-6.

Microbiology

Simple blood tests for telomeric protein could provide a valuable screen for certain cancers

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Reviewed by Emily Henderson, B.Sc.Feb 21 2023

Once thought incapable of encoding proteins due to their simple monotonous repetitions of DNA, tiny telomeres at the tips of our chromosomes seem to hold a potent biological function that’s potentially relevant to our understanding of cancer and aging.

Reporting in the Proceedings of the National Academy of Science, UNC School of Medicine researchers Taghreed Al-Turki, PhD, and Jack Griffith, PhD, made the stunning discovery that telomeres contain genetic information to produce two small proteins, one of which they found is elevated in some human cancer cells, as well as cells from patients suffering from telomere-related defects.

Based on our research, we think simple blood tests for these proteins could provide a valuable screen for certain cancers and other human diseases. These tests also could provide a measure of ‘telomere health,’ because we know telomeres shorten with age.”

Jack Griffith, PhD, the Kenan Distinguished Professor of Microbiology and Immunology and Member of the UNC Lineberger Comprehensive Cancer Center

Telomeres contain a unique DNA sequence consisting of endless repeats of TTAGGG bases that somehow inhibit chromosomes from sticking to each other. Two decades ago, the Griffith laboratory showed that the end of a telomere’s DNA loops back on itself to form a tiny circle, thus hiding the end and blocking chromosome-to-chromosome fusions. When cells divide, telomeres shorten, eventually becoming so short that the cell can no longer divide properly, leading to cell death.

Scientist first identified telomeres about 80 years ago, and because of their monotonous sequence, the established dogma in the field held that telomeres could not encode for any proteins, let alone ones with potent biological function.

In 2011 a group in Florida working on an inherited form of ALS reported that the culprit was an RNA molecule containing a six-base repeat which by a novel mechanism could generate a series of toxic proteins consisting of two amino acids repeating one after the other. Al-Turki and Griffith note in their paper a striking similarity of this RNA to the RNA generated from human telomeres, and they hypothesized that the same novel mechanism might be in play.

They conducted experiments – as described in the PNAS paper – to show how telomeric DNA can instruct the cell to produce signaling proteins they termed VR (valine-arginine) and GL (glycine-leucine). Signaling proteins are essentially chemicals that trigger a chain reaction of other proteins inside cells that then lead to a biological function important for health or disease.

Al-Turki and Griffith then chemically synthesized VR and GL to examine their properties using powerful electron and confocal microscopes along with state-of-the-art biological methods, revealing that the VR protein is present in elevated amounts in some human cancer cells, as well as cells from patients suffering from diseases resulting from defective telomeres.

“We think it’s possible that as we age, the amount of VR and GL in our blood will steadily rise, potentially providing a new biomarker for biological age as contrasted to chronological age,” said Al-Turki, a postdoctoral researcher in the Griffith lab. “We think inflammation may also trigger the production of these proteins.”

Griffith noted, “When you go against current thinking, you are usually wrong because you are bucking many people who’ve worked so diligently in their fields. But occasionally scientists have failed to put observations from two very distant fields together and that’s what we did. Discovering that telomeres encode two novel signaling proteins will change our understanding of cancer, aging, and how cells communicate with other cells.

“Many questions remain to be answered, but our biggest priority now is developing a simple blood test for these proteins. This could inform us of our biological age and also provide warnings of issues, such as cancer or inflammation.”

Source:

University of North Carolina Health Care

Journal reference:

Al-Turki, T., et al. (2023) Mammalian Telomeric RNA (TERRA) can be translated to produce valine-arginine and glycine-leucine dipeptide repeat proteins. PNAS. doi.org/10.1073/pnas.2221529120.

Microbiology

Structure and function of the first FDA-approved treatment for Ebola virus discovered

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Reviewed by Emily Henderson, B.Sc.Jan 30 2023

Scientists at La Jolla Institute for Immunology (LJI) have uncovered the structure and function of the first FDA-approved treatment for Zaire ebolavirus (Ebola virus).

Inmazeb (REGN-EB3), developed by Regeneron, is a three-antibody cocktail designed to target the Ebola virus glycoprotein. The drug was first approved for clinical use in October 2020, but its exact mechanism of action has remained unclear.

In the cover story of the latest issue of Cell Host & Microbe, LJI researchers present a high-resolution, 3D structure of the three antibodies as they bind to the Ebola virus glycoprotein (the viral protein that launches Ebola virus infection). The model reveals new information about both the drug and the virus, and how their interaction fights infection and protects against future viral mutations.

Before this, we had a general idea of what the drug was doing, but we didn’t know exactly how. We now know which specific amino acids the antibodies are latching onto and how their binding affects the viral glycoprotein.”

Ollmann Saphire, Ph.D., LJI President and CEO, Study’s Senior Author

The new research also shows the potential for Inmazeb in treating additional species of Ebolavirus.

The new study shows how three antibodies (light blue, dark blue, and yellow) used in Inmazeb (REGN-EB3) bind to different regions of the Ebola virus glycoprotein (grey) to combat infection. Image credit: Ethan MacKenzie (Phospho Biomedical Animation)

How the antibody cocktail works

At 3.1 angstroms, the 3D structure is the highest-resolution image of the Ebola virus surface protein ever assembled using asymmetric reconstruction. The researchers achieved this detailed view through an imaging technique called cryogenic electron microscopy (cryo-EM).

“It’s like getting mugshots of a protein,” said first author Vamseedhar Rayaprolu, Ph.D., who spearheaded the project as a postdoctoral associate at LJI and now serves at The Pacific Northwest Center for Cryo-EM. “We take photos of the complex that is frozen in all different angles and then stitch them together to get a 3D model.”

Thanks to these images, the LJI team immediately made a discovery not just about the drug, but also about Ebola virus itself. While the overall structure of the Ebola glycoprotein has been known for some time, one region had yet to be modeled effectively-;the β17-β18 loop on the protein’s glycan cap.

“This piece is normally too floppy to be imaged,” said Rayaprolu, “but when the antibodies were bound to the virus, they locked the loop into place and we were able to finally capture its location and structure.”

The team then confirmed that the drug’s three antibodies bind the glycoprotein at distinct, non-overlapping locations, maximizing their effectiveness by minimizing their redundancy.

Atoltivimab (REGN3470) is the specific antibody that binds the β17-β18 loop. When bound, this antibody can serve as a signal to attract the immune system, flagging infected cells to be killed via effector functions.

A second antibody, called odesivimab (REGN3471), binds to amino acids on the glycoprotein’s receptor-binding site, preventing the virus from attaching itself to human cells.

The third antibody, called maftivimab (REGN3479), binds and warps the glycoprotein’s internal fusion loop, which the virus requires to drive itself into a cell. The researchers also found evidence that maftivimab may be valuable in future therapies against other types of Ebolaviruses.

Fighting more than one virus

“Like with SARS-CoV-2, Ebola virus has changed over time and become different than the original virus,” says study collaborator Robert Davey, Ph.D., Professor in the National Emerging Infectious Diseases Laboratories (NEIDL), of the Boston University Chobanian & Avedisian School of Medicine. As Davey points out, Ebola viruses aren’t the only dangerous members of the larger Filovirus family. This family includes closely related Ebolavirus species, such as Sudan ebolavirus (a 2022 outbreak of Sudan ebolavirus killed at least 55 people in Uganda) and the more distantly related Marburg virus.

Through a series of escape studies led by study collaborators in Davey’s lab and at Regeneron, the team found that Inmazeb could potentially protect against several viruses in the Ebolavirus genus of Filoviruses, including Sudan ebolavirus.

The key appears to be the maftivimab antibody. Maftivimab’s target, the viral glycoprotein’s internal fusion loop, is conserved across these Ebolaviruses. This means the loop structure has not changed significantly, even as other parts of the virus have mutated over time.

“We found that, in general, the antibodies in Inmazeb could be effective against the more closely related viruses,” says Davey. “But for the more distantly related species, such as Marburg, more work needs to be done to devise a new antibody cocktail.”

Could Inmazeb also combat new Ebola virus variants? The researchers found that-;in the presence of all three antibodies-;Ebola virus has to undergo ten rounds of replication and multiple mutations to partially escape the effects of the drug. In contrast, using any single antibody alone leads to escape mutations within only one or two passages.

This finding suggests that Inmazeb can provide lasting immunity against variants. The new findings may also guide the development of novel antibody drugs that target the glycoprotein more broadly or effectively.

“We now understand how subtle shifts in the landing site of different antibodies impact function,” says Rayaprolu. “This tells us the differences between more or less effective immune responses.”

“Knowing exactly where a drug contacts the virus helps us predict whether it is likely to still work on a new viral variant,” adds Saphire. “These methods and the insights from our research collaborators will be integral to the development of next-generation vaccines.”

Source:

La Jolla Institute for Immunology

Journal reference:

Rayaprolu, V., et al. (2023) Structure of the Inmazeb cocktail and resistance to Ebola virus escape. Cell Host & Microbe. doi.org/10.1016/j.chom.2023.01.002.

Microbiology

Cryo-electron microscopy reveals atomic structure of Staphylococcus epidermidis bacteriophage

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Reviewed by Emily Henderson, B.Sc.Dec 18 2022

Cryo-electron microscopy by University of Alabama at Birmingham researchers has exposed the structure of a bacterial virus with unprecedented detail. This is the first structure of a virus able to infect Staphylococcus epidermidis, and high-resolution knowledge of structure is a key link between viral biology and potential therapeutic use of the virus to quell bacterial infections.

Bacteriophages or “phages” is the terms used for viruses that infect bacteria. The UAB researchers, led by Terje Dokland, Ph.D., in collaboration with Asma Hatoum-Aslan, Ph.D., at the University of Illinois Urbana-Champaign, have described atomic models for all or part of 11 different structural proteins in phage Andhra. The study is published in Science Advances.

Andhra is a member of the picovirus group. Its host range is limited to S. epidermidis. This skin bacterium is mostly benign but also is a leading cause of infections of indwelling medical devices. “Picoviruses are rarely found in phage collections and remain understudied and underused for therapeutic applications,” said Hatoum-Aslan, a phage biologist at the University of Illinois.

With emergence of antibiotic resistance in S. epidermidis and the related pathogen Staphylococcus aureus, researchers have renewed interest in potentially using bacteriophages to treat bacterial infections. Picoviruses always kill the cells they infect, after binding to the bacterial cell wall, enzymatically breaking through that wall, penetrating the cell membrane and injecting viral DNA into the cell. They also have other traits that make them attractive candidates for therapeutic use, including a small genome and an inability to transfer bacterial genes between bacteria.

Knowledge of protein structure in Andhra and understanding of how those structures allow the virus to infect a bacterium will make it possible to produce custom-made phages tailored to a specific purpose, using genetic manipulation.

The structural basis for host specificity between phages that infect S. aureus and S. epidermidis is still poorly understood. With the present study, we have gained a better understanding of the structures and functions of the Andhra gene products and the determinants of host specificity, paving the way for a more rational design of custom phages for therapeutic applications. Our findings elucidate critical features for virion assembly, host recognition and penetration.”

Terje Dokland, professor of microbiology at UAB and director of the UAB Cryo-Electron Microscopy Core

Staphylococcal phages typically have a narrow range of bacteria they can infect, depending on the variable polymers of wall teichoic acid on the surface of different bacterial strains. “This narrow host range is a double-edged sword: On one hand, it allows the phages to target only the specific pathogen causing the disease; on the other hand, it means that the phage may need to be tailored to the patient in each specific case,” Dokland said.

The general structure of Andhra is a 20-faced, roundish icosahedral capsid head that contains the viral genome. The capsid is attached to a short tail. The tail is largely responsible for binding to S. epidermidis and enzymatically breaking the cell wall. The viral DNA is injected into the bacterium through the tail. Segments of the tail include the portal from the capsid to the tail, and the stem, appendages, knob and tail tip.

The 11 different proteins that make up each virus particle are found in multiple copies that assemble together. For instance, the capsid is made of 235 copies each of two proteins, and the other nine virion proteins have copy numbers from two to 72. In total, the virion is made up of 645 protein pieces that include two copies of a 12th protein, whose structure was predicted using the protein structure prediction program AlphaFold.

The atomic models described by Dokland, Hatoum-Aslan, and co-first authors N’Toia C. Hawkins, Ph.D., and James L. Kizziah, Ph.D., UAB Department of Microbiology, show the structures for each protein -; as described in molecular language like alpha-helix, beta-helix, beta-strand, beta-barrel or beta-prism. The researchers have described how each protein binds to other copies of that same protein type, such as to make up the hexameric and pentameric faces of the capsid, as well as how each protein interacts with adjacent different protein types.

Electron microscopes use a beam of accelerated electrons to illuminate an object, providing much higher resolution than a light microscope. Cryo-electron microscopy adds the element of super-cold temperatures, making it particularly useful for near-atomic structure resolution of larger proteins, membrane proteins or lipid-containing samples like membrane-bound receptors, and complexes of several biomolecules together.

In the past eight years, new electron detectors have created a tremendous jump in resolution for cryo-electron microscopy over normal electron microscopy. Key elements of this so-called “resolution revolution” for cryo-electron microscopy are:

  • Flash-freezing aqueous samples in liquid ethane cooled to below -256 degrees F. Instead of ice crystals that disrupt samples and scatter the electron beam, the water freezes to a window-like “vitreous ice.”
  • The sample is kept at super-cold temperatures in the microscope, and a low dose of electrons is used to avoid damage to the proteins.
  • Extremely fast direct electron detectors are able to count individual atoms at hundreds of frames per second, allowing sample movement to be corrected on the fly.
  • Advanced computing merges thousands of images to generate three-dimensional structures at high resolution. Graphics processing units are used to churn through terabytes of data.
  • The microscope stage that holds the sample can also be tilted as images are taken, allowing construction of a three-dimensional tomographic image, similar to a CT scan at the hospital.

The analysis of Andhra virion structure by the UAB researchers started with 230,714 particle images. Molecular reconstruction of the capsid, tail, distal tail and tail tip started with 186,542, 159,489, 159,489 and 159,489 images, respectively. Resolution ranged from 3.50 to 4.90 angstroms.

Source:

University of Alabama at Birmingham

Journal reference:

Hawkins, N.C., et al. (2022) Structure and host specificity of Staphylococcus epidermidis bacteriophage Andhra. Science Advances. doi.org/10.1126/sciadv.ade0459.

Microbiology

NIAID awards more than $12 million for the development of antiviral therapies

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Reviewed by Emily Henderson, B.Sc.Nov 21 2022

The National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, recently awarded more than $12 million to three institutions for the development of antiviral therapies to treat diseases caused by viruses with pandemic potential. NIAID may award approximately $61.5 million total over five years if all contract options are exercised. The new product development contracts are part of the Antiviral Program for Pandemics (APP), which aims to accelerate the discovery, development and manufacturing of antiviral medicines.

Antivirals are treatments that fight viral infections by acting directly against the virus. Other types of therapies, not the focus of this program, harness the body’s immune system to fight infection. The new contracts will support the development of promising antiviral candidates from late-stage preclinical studies through investigational new drug application-enabling activities and clinical testing.

Alongside the new product development contracts, NIAID already supports nine Antiviral Drug Discovery (AViDD) Centers for Pathogens of Pandemic Concern. The AViDD Centers conduct research on the early-stage identification and validation of novel viral targets and identification and early-stage characterization of antiviral drug candidates.

The new product development contracts include:

Optimization of Broad-Spectrum Filovirus Inhibitors that Target Viral Glycoprotein
Principal investigator: Terry Bowlin, Ph.D.
Institute: Microbiotix, Inc., Worcester, Massachusetts
Base funding amount: $2,069,416
NIAID contract: 75N93023C00001

Development of a Novel 2-Pyrimidone (SRI-42718) as a Potent Inhibitor of Chikungunya Virus Infection and Disease
Principal investigator: Daniel Streblow, Ph.D.
Institute: Oregon Health and Science University, Portland
Base funding amount: $4,696,452
NIAID contract: 75N93023C00002

Development of an Orally Available Antiviral Drug for Yellow Fever
Principal investigator: Jinhong Chang, M.D., Ph.D.
Institute: Baruch S. Blumberg Institute, Doylestown, Pennsylvania
Base funding amount: $5,493,876
NIAID contract: 75N93023C00003

For more information about the APP, please visit: https://www.niaid.nih.gov/research/antivirals.

Source:

National Institutes of Health

Microbiology

Hybrid virus particles formed from coinfections exhibit immune evasion and expanded tropism

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Dr. Chinta SidharthanBy Dr. Chinta SidharthanNov 1 2022Reviewed by Aimee Molineux

In a recent study published in Nature Microbiology, researchers examined virus-virus interactions using human lung cells coinfected with two co-circulating respiratory infection-causing viruses, respiratory syncytial virus (RSV) and influenza A virus (IAV).

Study: Coinfection by influenza A virus and respiratory syncytial virus produces hybrid virus particles. Image Credit: Kateryna Kon/Shutterstock
Study: Coinfection by influenza A virus and respiratory syncytial virus produces hybrid virus particles. Image Credit: Kateryna Kon/Shutterstock

Background

Intracellular pathogens such as viruses generally exhibit tropism, where they show affinity towards selected cell types. Some cells and tissues can be coinfected by taxonomically different viruses since diverse viruses can have an affinity for the same cell type.

Coinfections provide an ecological niche for viruses to interact with each other. These interactions include pseudotyping, where surface proteins from one virus are incorporated into the other virus, or they could result in genomic rearrangements which could give rise to completely new strains with increased infection potential. While some studies claim that disease outcomes are independent of coinfections, others indicate an increase in severe outcomes due to coinfections. However, the mechanisms of viral interactions during coinfections that determine disease outcomes remain unclear.

About the study

In the present study, Madin-Darby canine kidney cells (MDCK) and human epidermoid carcinoma cells (HEp-2) were used to grow virus stocks of hemagglutinin 1 neuraminidase 1 (H1N1) IAV and RSV strain A2, respectively. Cultured human lung adenocarcinoma cells (A549) were infected with IAV and RSV individually and synchronously at high multiplicity of infection (MOI).

Plaque assays were used to determine infectious titers of IAV and RSV in MDCK and HEp-2 cells, respectively. Immunofluorescence microscopy was used to assess single and coinfected cells to determine the effect of coinfections on viral protein localization and infected cell proportions. Virions with IAV haemagglutinin and RSV fusion glycoprotein immunolabels were examined using super-resolution confocal microscopy to determine whether the mixing of the two viral glycoproteins resulted in the budding viral particles incorporating components of IAV and RSV.

Additionally, cryo-electron tomography was performed to investigate the structural characteristics of the RSV and IAV filaments budding from the coinfected cells. Furthermore, since hybrid viral particles would contain surface glycoproteins of both RSV and IAV, antibodies against the IAV haemagglutinin and RSV fusion glycoprotein were used in neutralization assays against IAV and RSV viruses collected from cells infected individually and synchronously.

The cellular receptor for IAV, sialic acid, was removed using neuraminidase, and the cells were inoculated with IAV and RSV viruses from individually infected or coinfected cells to determine whether incorporation of RSV glycoproteins could expand the IAV receptor tropism of hybrid viral particles. Cells were immunostained for the IAV and RSV nucleoproteins and quantified.

Additionally, human bronchial epithelial cells were coinfected with RSV and IAV, and paraffin-embedded infected cultures were immunostained for IAV haemagglutinin and nucleoprotein and the whole RSV virion to determine whether other relevant biological systems formed hybrid viral particles.

Results

The results reported that in coinfected cells, IAV titers were equal to or marginally higher and the RSV titers were lower than cells individually infected with the two viruses. In contrast, in cells coinfected with IAV and rhinovirus, IAV replication was inhibited. This suggested that coinfection outcomes were dependent on the type of viruses involved and the subsequent virus-specific cellular responses.

Furthermore, the study demonstrated the hybrid viral particles generated during coinfections contained structural, functional, and genomic components from both parental viruses, and were infectious. These hybrid viral particles exhibited evasion of IAV-targeted neutralization and the ability to infect neuraminidase-treated IAV receptor-negative cells, indicating modified antigenicity and wide tropism characteristics.

Neutralization assay using anti-RSV glycoprotein antibodies showed that the entry of hybrid viral particles into cells is mediated by the RSV fusion glycoprotein, which suggested that IAV could recruit an unrelated viral glycoprotein as the functional envelope protein.

Although IAV infections are generally restricted to the upper respiratory tracts, hybrid viral particles with structural and functional components of both viruses could enable IAV infections in the lower respiratory tract regions. These results are indicative of the potential increase in pathogenicity and disease severity, and complications such as viral pneumonia.

Additionally, since IAV experiences high mutation rates, hybrid viral particles infecting the lower respiratory tract could result in the selection of viral particles with increased pathogenesis and greater tropism for the lower regions of the respiratory tract. The results also showed that hybrid viral particles were maintained over multiple infection rounds, and aided the spread of IAV into refractory cell populations.  

Coinfection of human bronchial epithelial cells demonstrated the formation of hybrid viral particles in biologically relevant tissues and indicated that since IAV and RSV both circulate during the same season and share tropism for ciliated epithelial cells, the probability of coinfections and in vivo generation of hybrid viral particles is high.

Conclusions

Overall, the study demonstrated that coinfections by RSV and IAV form hybrid viral particles that exhibit modified antigenicity and expanded tropism, and suggested the possibility of other such hybrid viral particle formations from coinfections involving pleomorphic enveloped viruses such as RSV.

The authors believe that while hybrid viral particle formation is dependent on various factors other than structural compatibilities, such as overlaps in circulation season and geography and tropism, coinfections pose the risk of hybrid virus particles with wide tropism and increased immune evasion.

Journal reference:
Microbiology

Clinical trial to evaluate antiviral drug for monkeypox begins in the Democratic Republic of the Congo

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Reviewed by Emily Henderson, B.Sc.Oct 12 2022

A clinical trial to evaluate the antiviral drug tecovirimat, also known as TPOXX, in adults and children with monkeypox has begun in the Democratic Republic of the Congo (DRC). The trial will evaluate the drug’s safety and its ability to mitigate monkeypox symptoms and prevent serious outcomes, including death. The National Institute of Allergy and Infectious Diseases (NIAID), part of the U.S. National Institutes of Health, and the DRC’s National Institute for Biomedical Research (INRB) are co-leading the trial as part of the government-to-government PALM partnership. Collaborating institutions include the U.S. Centers for Disease Control and Prevention (CDC), the Institute of Tropical Medicine Antwerp, the aid organization Alliance for International Medical Action (ALIMA) and the World Health Organization (WHO).

TPOXX, made by the pharmaceutical company SIGA Technologies, Inc. (New York), is approved by the U.S. Food and Drug Administration for the treatment of smallpox. The drug impedes the spread of virus in the body by preventing virus particles from exiting human cells. The drug targets a protein that is found on both the virus that causes smallpox and the monkeypox virus.

“Monkeypox has caused a high burden of disease and death in children and adults in the Democratic Republic of the Congo, and improved treatment options are urgently needed,” said NIAID Director Anthony S. Fauci, M.D. “This clinical trial will yield critical information about the safety and efficacy of tecovirimat for monkeypox. I want to thank our DRC scientific partners as well as the Congolese people for their continued collaboration in advancing this important clinical research.”

Since the 1970s, monkeypox virus has caused sporadic cases and outbreaks, primarily in the rainforest areas of central Africa, and in west Africa. A multi-continent outbreak of monkeypox in areas where the disease is not endemic, including Europe and the United States, has been ongoing since May 2022 with the majority of cases occurring in men who have sex with men. The outbreak has prompted recent public health emergency declarations from the WHO and the U.S. Department of Health and Human Services. From Jan.1, 2022 to Oct. 5, 2022, the WHO has reported 68,900 confirmed cases and 25 deaths from 106 countries, areas and territories.

According to the WHO, cases identified as part of the ongoing global outbreak are largely caused by monkeypox virus Clade IIb. Clade I, which is estimated to cause more severe disease and higher mortality than Clade IIa and Clade IIb, especially in children, is responsible for infections in the DRC. The Africa Centres for Disease Control and Prevention (Africa CDC) has reported 3,326 cases of monkeypox (165 confirmed; 3,161 suspected) and 120 deaths in the DRC from Jan. 1, 2022 to Sept. 21, 2022.

People can become infected with monkeypox through contact with infected animals, such as rodents, or nonhuman primates or humans. The virus can transmit among humans by direct contact with skin lesions, body fluids, and respiratory droplets, including through intimate and sexual contact; and by indirect contact with contaminated clothing or bedding. Monkeypox can cause flu-like symptoms and painful skin lesions. Complications can include dehydration, bacterial infections, pneumonia, brain inflammation, sepsis, eye infections and death.

The trial will enroll up to 450 adults and children with laboratory-confirmed monkeypox infection who weigh at least 3 kilograms (kg). Pregnant women are also eligible to enroll. The volunteer participants will be assigned at random to receive either oral tecovirimat or placebo capsules twice daily for 14 days, with the dose administered dependent on the participant’s weight. The trial is double-blinded, so participants and investigators do not know who will receive tecovirimat or placebo.

All participants will stay at a hospital for at least 14 days where they will receive supportive care. Study clinicians will regularly monitor participants’ clinical status throughout the study, and participants will be asked to provide blood, throat swab, and skin lesion swab samples for laboratory evaluations. The study is primarily designed to compare the average time to healed skin lesions among those receiving tecovirimat versus those receiving placebo. Investigators will also gather data on multiple secondary objectives, including comparisons of how quickly participants test negative for monkeypox virus in the blood, overall severity and duration of disease, and mortality between groups.

Participants will be discharged from the hospital once all lesions have scabbed over or flaked off, and after they test negative for monkeypox virus in the blood for two days in a row. They will be followed for at least 28 days and will be asked to return for an optional study visit after 58 days for additional clinical and laboratory tests. An independent Data and Safety Monitoring Board will monitor participant safety throughout the duration of the study.

The trial is led by co-principal investigators Jean-Jacques Muyembe-Tamfum, M.D., Ph.D., director-general of INRB and professor of microbiology at Kinshasa University Medical School in Gombe, Kinshasa; and Placide Mbala, M.D., Ph.D., operations manager of the PALM project and head of the Epidemiology Department and the Pathogen Genomic Laboratory at INRB.

“I am happy that monkeypox is no longer a neglected disease and that soon, thanks to this study, we will be able to prove that there is an effective treatment for this disease,” said Dr. Muyembe-Tamfum.

For more information, please visit clinicaltrials.gov and search identifier NCT05559099. The trial timeline will depend on the pace of enrollment. A separate NIAID-supported trial of TPOXX is ongoing in the United States. For information about the U.S. trial, visit the AIDS Clinical Trials Group (ACTG) website and search TPOXX or study A5418.

PALM is short for “Pamoja Tulinde Maisha” a Kiswahili phrase that translates to “together save lives.”  NIAID and the DRC Ministry of Health established the PALM clinical research partnership in response to the 2018 Ebola outbreak in Eastern DRC. The collaboration has continued as a multilateral clinical research program composed of NIAID, the DRC Ministry of Health, INRB and INRB’s partners. PALM’s first study was the randomized controlled trial of multiple therapeutics for Ebola virus disease, which supported the regulatory approvals of the NIAID-developed mAb114 (Ebanga) and REGN-EB3 (Inmazeb, developed by Regeneron) treatments.

Source:

National Institutes of Health