New ebolavirus vaccine design seeks to drive stronger antibody defense

Scientists at Scripps Research have unveiled a new Ebola virus vaccine design, which they say has several advantages over standard vaccine approaches for Ebola and related viruses that continue to threaten global health.

In the new design, described in a paper in Nature Communications, copies of the Ebola virus outer spike protein, known as the glycoprotein, are tethered to the surface of a spherical carrier particle. The resulting structure resembles the spherical appearance of common RNA viruses that infect humans -- and is starkly different from the snake-like shape of the Ebola virus.

The scientists say the design is intended to stimulate a better protective immune response than standard vaccine approaches, which often expose the immune system to individual glycoproteins rather than realistic-looking virus particles.

In designing the vaccine, the researchers also modified the outer spike protein to be more stable than the normal, "wild-type" version found in actual Ebola virus. In tests in mice and rabbits, they showed that this stabilized version elicited virus-neutralizing antibodies more strongly than the wild-type glycoprotein used in prior Ebola vaccine approaches.

"Here, we did a step-by-step investigation of glycoprotein stability and how that affects the vaccine's ability to elicit antibodies," says Jiang Zhu, PhD, associate professor in the Department of Integrative Structural and Computational Biology at Scripps Research and inventor of the vaccine. "In the end, we were able to develop a really promising vaccine design."

Continued viral threat

Ebola virus is endemic in various African bat species and can jump to humans, causing outbreaks of hemorrhagic fever with high mortality rates. The largest known outbreak of occurred in West Africa during 2013-2016, killing more than 11,000 people.

About two decades ago, Canadian researchers developed a vaccine against Zaire ebolavirus, more commonly known as Ebola virus. The vaccine, which was later licensed to a major pharma company and is called rVSV-ZEBOV, uses a live virus -- vesicular stomatitis virus -- which has been modified to include the gene for the Ebola virus glycoprotein.

When injected, the rVSV-ZEBOV vaccine infects cells and produces copies of the glycoprotein, eliciting an immune response to protect against future exposure to Ebola virus. Tests in Africa amid the aforementioned outbreak suggested it worked well and it was approved by the Food and Drug Administration in late 2019. However, those tests lacked placebo groups and other standard features of typical large-scale phase-III trials. Thus, questions remain on true efficacy.

In developing their new ebolavirus vaccine design, Zhu and his team focused on the relative instability of the glycoprotein structure as a potential factor in vaccine effectiveness. They investigated the molecular sources of this instability in detail, and eventually came up with a set of modifications that greatly stabilize the glycoprotein. In mice and rabbits, their modified glycoprotein elicited a more potent neutralizing antibody response against two different ebolaviruses -- the Makona strain of Ebola virus and the Uganda strain of Bundibugyo ebolavirus -- and compared those with the wild-type glycoprotein.

The team's design also included special protein segments that self-assemble tightly into a ball-shaped "nanoparticle" that support multiple glycoproteins on their surface. This nanoparticle-based structure presents the glycoproteins to the immune system similar to common human viruses, and thus the body has learned to recognize the spherical particles.

"Think of our nanoparticle as your sport vehicle, with a roof rack that carries a mountain bike and a trunk where you stow your clothes, gears and food," Zhu explains. "The only difference here is that the Ebola virus spike is your mountain bike, and the locking domains and T-cell epitopes are your stuff in the trunk. We call that a multilayered design."

A new approach

This nanoparticle design is distinctively different from other nanoparticle platforms. Zhu explains that in his team's design, the genetic codes of the optimized glycoprotein, the nanoparticle-forming unit, the locking domain and the T-cell epitope are all contained in a single piece of DNA. In cells, this DNA generates a single protein chain that can self-assemble, forming the right structure and associating with other identical chains to create a virus-like protein ball with multiple layers.

"The idea is that the all-in-one design simplifies the manufacturing process and drives the vaccine cost lower," Zhu says.

His team already has used the nanoparticle platform to create a COVID-19 vaccine candidate, which has shown in animal models that it can induce a powerful antibody response to both SARS-CoV-1 and SARS-CoV-2. It also has shown to be effective against variants.

For Ebola virus, the nanoparticle-based vaccines showed far better results in mouse and rabbit virus-neutralization tests that tests that used only glycoproteins to stimulate immune response. Inoculating animals with the Ebola wild-type glycoprotein, which tends to fall apart, led to signs suggesting a vaccine phenomenon known as antibody-dependent enhancement -- in which a vaccine elicits not only virus-neutralizing antibodies, but also antibodies that paradoxically increase the virus's ability to infect cells. The researchers found that their best nanoparticle-based designs only minimally elicit these bad antibodies.

"There are a lot of things in the Ebola virus vaccine field that still need to be examined carefully, but in this study, we ended up with two nanoparticle-based designs that seem very suitable for further optimization and testing," Zhu says.

He says the vaccine approach can be extended to other members of the same virus family, such as Marburg virus, which is also a major threat. Ebolaviruses and marburgvirus both belong to a group of viruses, known as filoviruses, that have a bizarre thread-like shape when seen under a microscope.

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Study reveals how some antibodies can broadly neutralize ebolaviruses

Some survivors of ebolavirus outbreaks make antibodies that can broadly neutralize these viruses -- and now, scientists at Scripps Research have illuminated how these antibodies can disable the viruses so effectively. The insights may be helpful for developing effective therapies.

Ebolavirus is a family of often-deadly viruses that includes Ebola virus and many lesser-known viruses such as Bundibugyo virus, Sudan virus and Reston virus.

Structural biologists at Scripps Research used electron microscopy techniques to visualize a set of antibodies that target a key site on these viruses called the "glycan cap." Their research showed that the antibodies work against ebolaviruses using the same three mechanisms to prevent the virus from infecting host cells.

The research, published in Cell Reports, is a step toward the creation of an antibody-based treatment that will be useful against a broad range of ebolaviruses.

"We now understand the molecular basis for these antibodies' abilities to neutralize ebolviruses with broad reactivity against different viral species," says the study's first author Daniel Murin, PhD, a staff scientist in the laboratory of Andrew Ward, PhD.

Ward, a professor in the Department of Integrative Structural and Computational Biology at Scripps Research, says he hopes the work will contribute the development of a "cocktail" of therapeutic antibodies that can save lives by treating many forms of the Ebola virus.

"The goal is to provide doctors in Ebola-prone regions their best weapon yet against these deadly outbreaks," Ward says. "The insights we have gained through our structural studies of the virus show how this may be possible."

Ever-emergent Ebola

The first known ebolavirus, now called Zaire ebolavirus or simply Ebola virus, was identified in 1976, named for the site of an outbreak that year near the Ebola river in what was then Zaire and is now the Democratic Republic of Congo.

Other species have since been added to this family of viruses, including Sudan ebolavirus and Bundibugyo ebolavirus. Ebola viruses colonize African fruit bats, often cause disease in chimpanzees and other non-human primates, and trigger outbreaks in humans every few years, on average. Infected people develop a hemorrhagic syndrome that is fatal in roughly half of untreated cases.

Vaccines against Ebola have been developed recently but have not yet been widely used. And although antibody-based treatments also have been developed, none has been shown effective against a broad range of ebolavirus species.

Nevertheless, studies in recent years have shown that some survivors of Ebola infections carry antibodies that, in lab-dish tests, can neutralize multiple ebolavirus species. A surprisingly high proportion of these broadly neutralizing antibodies target the glycan cap, a sugar-slathered site on a stalk-like protein -- called the glycoprotein -- that enables Ebola viruses to enter host cells.

In the new study, Murin and Ward, along with their colleagues in the James Crowe Lab at Vanderbilt University where the antibodies were isolated, used electron microscopy to analyze a set of glycan cap-targeting antibodies from survivors of various ebolaviruses. Their aim was to understand better how these antibodies target the virus so effectively.

Three ways to defeat the virus

Their analysis suggested that the most broadly effective of these glycan cap-targeting antibodies hit the same vulnerable site on the glycan cap, allowing them to thwart viral infectivity in three ways.

First, the antibody displaces a long viral structure near the glycan cap in a way that destabilizes the entire viral glycoprotein structure, sometimes causing it to fall apart.

Second, the glycan cap antibody -- when it binds to its target site -- can block a key event in the infection process, in which an enzyme called a cathepsin cleaves off the glycan cap. Blocking this cleavage event blocks the glycoprotein's ability to enter host cells.

Finally, the glycan cap antibody, by displacing the loose structure near the glycan cap, enables another type of neutralizing antibody to bind to a separate vulnerable site on the virus. Thus, a glycan cap antibody can "synergize" with another antibody to hit the virus significantly harder than either antibody does alone.

The scientists also determined the key genetic elements that allow glycan-cap antibodies to thwart ebolaviruses in these three ways.

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Targeting the shell of the Ebola virus

 As the world grapples with the coronavirus (COVID-19) pandemic, another virus has been raging again in the Democratic Republic of the Congo in recent months: Ebola. Since the first terrifying outbreak in 2013, the Ebola virus has periodically emerged in Africa, causing horrific bleeding in its victims and, in many cases, death.

How can we battle these infectious agents that reproduce by hijacking cells and reprogramming them into virus-replicating machines? Science at the molecular level is critical to gaining the upper hand -- research you'll find underway in the laboratory of Professor Juan Perilla at the University of Delaware.

Perilla and his team of graduate and undergraduate students in UD's Department of Chemistry and Biochemistry are using supercomputers to simulate the inner workings of Ebola, observing the way molecules move, atom by atom, to carry out their functions. In the team's latest work, they reveal structural features of the virus's coiled protein shell, or nucleocapsid, that may be promising therapeutic targets, more easily destabilized and knocked out by an antiviral treatment.

The research is highlighted in the Tuesday, Oct. 20 issue of the Journal of Chemical Physics, which is published by the American Institute of Physics, a federation of societies in the physical sciences representing more than 120,000 members.

"The Ebola nucleocapsid looks like a Slinky walking spring, whose neighboring rings are connected," Perilla said. "We tried to find what factors control the stability of this spring in our computer simulations."

The life cycle of Ebola is highly dependent on this coiled nucleocapsid, which surrounds the virus's genetic material consisting of a single strand of ribonucleic acid (ssRNA). Nucleoproteins protect this RNA from being recognized by cellular defense mechanisms. Through interactions with different viral proteins, such as VP24 and VP30, these nucleoproteins form a minimal functional unit -- a copy machine -- for viral transcription and replication.

While nucleoproteins are important to the nucleocapsid's stability, the team's most surprising finding, Perilla said, is that in the absence of single-stranded RNA, the nucleocapsid quickly becomes disordered. But RNA alone is not sufficient to stabilize it. The team also observed charged ions binding to the nucleocapsid, which may reveal where other important cellular factors bind and stabilize the structure during the virus's life cycle.

Perilla compared the team's work to a search for molecular "knobs" that control the nucleocapsid's stability like volume control knobs that can be turned up to hinder virus replication.

The UD team built two molecular dynamics systems of the Ebola nucleocapsid for their study. One included single-stranded RNA; the other contained only the nucleoprotein. The systems were then simulated using the Texas Advanced Computing Center's Frontera supercomputer -- the largest academic supercomputer in the world. The simulations took about two months to complete.

Graduate research assistant Chaoyi Xu ran the molecular simulations, while the entire team was involved in developing the analytical framework and conducting the analysis. Writing the manuscript was a learning experience for Xu and undergraduate research assistant Tanya Nesterova, who had not been directly involved in this work before. She also received training as a next-generation computational scientist with support from UD's Undergraduate Research Scholars program and NSF's XSEDE-EMPOWER program. The latter has allowed her to perform the highest-level research using the nation's top supercomputers. Postdoctoral researcher Nidhi Katyal's expertise also was essential to bringing the project to completion, Perilla said.

While a vaccine exists for Ebola, it must be kept extremely cold, which is difficult in remote African regions where outbreaks have occurred. Will the team's work help advance new treatments?

"As basic scientists we are excited to understand the fundamental principles of Ebola," Perilla said. "The nucleocapsid is the most abundant protein in the virus and it's highly immunogenic -- able to produce an immune response. Thus, our new findings may facilitate the development of new antiviral treatments."

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Why doesn't Ebola cause disease in bats, as it does in people?

 A new study by researchers from The University of Texas Medical Branch at Galveston uncovered new information on why the Ebola virus can live within bats without causing them harm, while the same virus wreaks deadly havoc to people. This study is now available in Cell Reports.

The Ebola virus causes a devastating, often fatal, infectious disease in people. Within the past decade, Ebola has caused two large and difficult to control outbreaks, one of which recently ended in the Democratic Republic of the Congo.

When a virus brings serious disease to people, it means that humans are not good hosts for the virus. Viruses depend on a living host for their survival and have natural reservoirs -- a hosting animal species in which a virus naturally lives and reproduces without causing disease. Bats are likely a natural reservoir for the Ebola virus, but little is known about how the virus evolves in bats.

Like most other RNA viruses, Ebola's molecules are structured in a way that makes them more prone to genomic errors and mutations than other types of viruses. Because of this, Ebola and similar viruses have a remarkable ability to adapt to and replicate in new environments.

In the study, the research team, led by Alex Bukreyev, a UTMB virologist in the departments of pathology and microbiology and immunology, working with the team of Raul Andino, University of California, San Francisco, investigated how the Ebola virus adapts to both bat and human cells. They assessed changes in mutation rates and the structure of Ebola virus populations repeatedly in both bat and human cell lines using an ultra-deep genetic sequencing.

"We identified a number of meaningful differences in how the Ebola virus evolves when placed in a human cell line relative to a bat cell line," Bukreyev said. "For instance, the RNA editing enzyme called ADAR within bat cells play a greater role in the replication and evolution of the Ebola virus than do such enzymes in human cells. We found that the envelope protein of Ebola virus undergoes a drastic increase in certain mutations within bat cells, but this was not found in human cells. This study identifies a novel mechanism by which Ebola virus is likely to evolve in bats."

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Scientists find ally in fight against brain tumors: Ebola

Brain scan

Glioblastomas are relentless, hard-to-treat, and often lethal brain tumors. Yale scientists have enlisted a most unlikely ally in efforts to treat this form of cancer -- elements of the Ebola virus.

"The irony is that one of the world's deadliest viruses may be useful in treating one of the deadliest of brain cancers," said Yale's Anthony van den Pol, professor of neurosurgery, who describes the Yale efforts Feb. 12 in the Journal of Virology.

The approach takes advantage of a weakness in most cancer tumors and also of an Ebola defense against the immune system response to pathogens.

Unlike normal cells, a large percentage of cancer cells lack the ability to generate an innate immune response against invaders such as viruses. This has led cancer researchers to explore the use of viruses to combat a variety of cancers.

Using viruses carries an obvious risk -- they can introduce potentially dangerous infections. To get around this problem, scientists, including van den Pol, have experimented with creating or testing chimeric viruses, or a combination of genes from multiple viruses. They have the ability to target cancer cells without harming patients.

One of the seven genes of the Ebola virus that helps it avoid an immune system response also contributes to its lethality. This intrigued van den Pol.

He and the study's first author, Xue Zhang, also of Yale, used a chimeric virus containing one of gene from the Ebola virus -- a glycoprotein with a mucin-line domain (MLD). In wild-type Ebola virus, the MLD plays a role in hiding Ebola from the immune system. They injected this chimeric virus into the brains of mice with glioblastoma -- and found that the MLD helped selectively target and kill deadly glioblastoma brain tumors.

(The team worked with the MLD glycoprotein, not with the full Ebola virus.)

Van den Pol said MLD's beneficial effect appears to be that it protects normal cells from infection -- but not cancer cells, which lack the ability to mount an immune response to pathogens.

A key factor may be that the virus with the glycoprotein MLD replicates less rapidly, potentially making it safer than viruses without the MLD part of the glycoprotein, he said.

In theory, such a virus might be used in conjunction with surgery to eliminate glioblastoma tumors and help prevent a recurrence of cancer, he said.

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Candidate Ebola vaccine still effective when highly diluted, macaque study finds

A single dose of a highly diluted VSV-Ebola virus (EBOV) vaccine -- approximately one-millionth of what is in the vaccine being used to help control the ongoing Ebola outbreak in the Democratic Republic of the Congo (DRC) -- remains fully protective against disease in experimentally infected monkeys, according to National Institutes of Health scientists. The NIH investigators completed the vaccine dosage study using cynomolgus macaques and an updated vaccine component to match the EBOV Makona strain that circulated in West Africa from 2014-16. The study appears in Lancet's EBioMedicine.

Nearly 250,000 people have received the investigational VSV-EBOV vaccine since August 2018 as part of a "ring vaccination" program to help stem the outbreak. The vaccine appears to be safe and highly effective. The manufacturer has announced that it has submitted a biologics license application to the U.S. Food and Drug Administration. VSV-EBOV is based on a live-attenuated vesicular stomatitis virus and delivers an EBOV protein to elicit protective immune responses. With the continued need to vaccinate individuals in the DRC and surrounding countries, a potential shortage of VSV-EBOV vaccine is a concern and further dose adjustment is a possible solution.

Scientists from NIH's Rocky Mountain Laboratories (RML), part of the National Institute of Allergy and Infectious Diseases, tested several dosage strengths, including one with 10 million plaque-forming units (PFU). They determined that a vaccine with 10 PFUs was just as effective as the highest dose tested (a dose which was still lower than the one currently in use in the DRC). They vaccinated macaques 28 days prior to infecting them with a lethal dose of EBOV and then monitored the animals for 42 days after infection. Even the macaques given the lowest dose appeared completely protected from disease due to EBOV.

The scientists say their study findings could help make more vaccine available for more people and may reduce adverse reactions to the vaccine because of the smaller amount of active ingredient. Such reactions can include injection site irritation, headache, fatigue, fever, chills, myalgia, and arthralgia. Demonstrating that the vaccine appears effective with adjusted dosing also could ease the burden on vaccine production.

The authors say that although results from preclinical and clinical studies can differ, these promising findings in macaques of complete protection with a lower-dose VSV-EBOV vaccine help support the possibility of similar clinical trials in people.

From Science Daily