One of the most ambitious research programs on the Ebola virus began over 14 years ago in the start-up laboratory of a newly minted PhD named Erica Ollman Saphire. Saphire had just solved the structure of one of the first antibodies against HIV, and she was in search of a new challenge.
She began reading about the Ebola virus and quickly became intrigued by the simplicity and complexity of the deadly pathogen. Whereas humans are thought to have around 20,000 genes in their genome, the virus contains just seven. As a result, the virus has had to learn to do more with less. For example, one of its genes codes for two different proteins: a spiky glycoprotein that remains on the surface to help the virus attach and drive itself into the host cell, and a second decoy molecule that gets secreted into an infected person’s blood.
For years, researchers had tried in vain to develop antibodies to specifically target the surface protein without being wasted on the secreted protein. What they needed was a structural biologist to tell them how the same string of 295 amino acids was folded up to give rise to the two different proteins. Saphire had found her challenge.
Since then, she has solved the structures of not just these two molecules, aptly named glycoprotein (GP) and secreted glycoprotein (sGP), but also others such as VP35, a viral protein that enables Ebola to evade the host’s immune response. In a 2013 paper published on the cover of Cell, Saphire showed the virus doesn’t follow the one-direction highway of destiny that is the central dogma of molecular biology. Its proteins are frustratingly dynamic, unfolding and refolding into different configurations at different times for different functions.
Saphire’s findings have generated what she describes as a “roadmap for new treatment,” eventually leading to the development of the experimental drug ZMapp used in the treatment of several Ebola patients.
A two-time Burroughs Wellcome Fund awardee, Saphire says she would not have any of the results that she has today if BWF had not believed in her and been willing to invest in such difficult and risky work.
“No study section would have funded my idea,” says Saphire, whose laboratory is housed at the Scripps Research Institute in La Jolla, California. “I wanted to crystallize the Ebola virus glycoprotein, and I knew that was going to take 5 years and cost over a million dollars. I was going to have to express the protein 200 different ways, bind it with 10 antibodies and then grow 50,000 crystals just to find one or two that would diffract. It was the Burroughs Wellcome Fund that told me to go for it, they had confidence in me, and gave me money so I could hire people and buy equipment to get the project to the point that I could secure larger funding.”
In addition to Saphire, BWF awards have enabled at least four other investigators to pursue research that is helping advance the development of therapies and diagnostics for the recent outbreak of the deadly virus, which according to the Centers for Disease Control has claimed more than eight thousand lives.
Recent BWF awardee Jonathan Abraham began looking for ways to prevent the Ebola virus from entering cells when he was, remarkably enough, just an undergraduate. Like researchers far senior to him, Abraham’s efforts were hampered by the lack of a clear structure for the viral glycoprotein. In graduate school he shifted his focus to the New World hemorrhagic fever viruses, which cause similar, often fatal diseases. Abraham learned that antibody-based therapies, which had hit one stumbling block after another when targeted against Ebola, were working really well to combat these other viruses.
He traveled to South America to harvest antibodies from survivors of New World hemorrhagic fevers, then returned to the lab to examine how these molecules bind to the surface glycoprotein of these infectious agents. Now a clinical fellow at Brigham and Women's Hospital, he is using the structure Saphire solved of the Ebola glycoprotein to figure out how antibodies attack the viruses, and what makes Ebola harder to neutralize than other viruses.
Even if the current combination of antibodies contained within ZMapp are proven effective against Ebola, Abraham says his research can inform the design of complementary antibody cocktails and effective vaccines.
“The drug might work well against some strains, but be ineffective against others. That is something that is true in the field of HIV research, where you have to come up with various combinations of antibodies to address different strains of the virus,” says Abraham. “In theory, hemorrhagic fever viruses should be easier to target because they kill so fast the virus doesn’t have as much of a chance to evolve escape mutations.”
Viruses, especially RNA viruses like HIV and Ebola, have relatively high mutation rates and short generation times that enable them to adapt to changes in their host environment. The pressure to adapt is stronger in viruses that cause chronic disease, as the viral machinery is locked in a long battle with the host’s immune system where only the fittest will survive.
Though Ebola might not be evolving as quickly as viruses like HIV or even influenza, it is still evolving. Funding from the Burroughs Wellcome Fund allowed Pardis Sabeti to devise the hypothesis that emerging pathogens like Lassa and Ebola have been circulating, undetected, for many years, and might be evolving genes that are important for resistance.
The computational geneticist had already developed a breakthrough tool for scanning the genome for such signatures of natural selection. For the last five years, she has been using this technique to look at the evolution of the Ebola virus. In 2014, Sabeti led the international team that sequenced 99 Ebola virus genomes from the blood of patients in Sierra Leone, one of the countries hardest hit by the recent outbreak.
The study has enabled researchers to track the virus as it has spread and gauge how it evolves as it has moved through the human population. The findings, which were published in the journal Science, traced the start of the epidemic to one traditional healer’s funeral, where 12 mourners were infected. It also indicated that the virus is mutating so rapidly that continued progression of the epidemic could create more opportunities for Ebola to adapt. Though Sabeti says the virus doesn’t appear to be mutating at a faster rate than it did in previous epidemics, she warns that the types of mutations it accumulates could still be cause for concern.
“With the viral population expanding rapidly during an outbreak you get a number of new mutations, some of which can change the functionality of the virus,” says Sabeti, who is on the faculty of the Broad Institute and Harvard University. “Most of these will have no effect, but some of them might, which is why this work is important. The diagnostics, treatments, and vaccines are all based on the sequence of the virus. When the viral sequence changes, there is the opportunity that these will become less effective.”
Currently, Sabeti and her colleagues are continuing to look at the evolution of the virus by sequencing a new set of 600 inactivated clinical samples from Sierra Leone. During this outbreak Sabeti has been a strong proponent of open access and data sharing, releasing her results as soon as they become available and encouraging others to do the same. She is also hoping to use the sequencing data her lab has generated to inform and improve diagnostics, and has recently launched a collaboration with biotech company Illumina and USAID to bring sequencing machines to West Africa.
In addition, Sabeti says she is working on a few more experimental approaches, including one where she will be sequencing the immune response of survivors to try to enhance therapeutics and understand how the human immune system responds to Ebola. The first line of defense against a viral infection comes from the innate immune system, which recognizes invading pathogens and sends out chemical signals called cytokines to activate the antiviral response.
Unfortunately, Ebola has evolved a way to disable this part of immunity so that it can enter cells undetected. By the time the immune response is finally alerted, the virus has already grown out of control, killing cells left and right. At that point the immune system has no choice but to throw everything it has at the virus, triggering a “cytokine storm” that ends up damaging healthy tissue and blood vessels even more than the virus does. Many researchers believe that it is this last-ditch effort that eventually kills the patient.
Infectious disease researcher Michael Farzan wants to find a way to stop the virus long before it gets that far. His laboratory investigates the tactics that viruses employ to invade target cells, and searches for strategies to enhance the immune response to this unfortunate event. Thus far, he has identified a number of host factors that can naturally ward off the virus.
Though Farzan has spent most of his career studying these “innate immune factors” in the context of HIV infection, several years ago he used an award from the Burroughs Wellcome Fund to expand his work to flu and the emerging diseases SARS and Ebola. The Ebola virus has to undergo a remarkably complex process – including steps like attachment, internalization, and membrane fusion -- before it can land in the cytoplasm of the host cell and start making copies of itself.
Farzan discovered that a family of factors called interferon-inducible transmembrane proteins or IFITMs disrupt these steps to block viral entry. In a study published in a 2013 issue of the journal Cell Host & Microbe, his group showed that these factors essentially reroute the virus on its way into cells so it hits a dead end.
“We want to continue to explore how these innate immune factors work because then we will have a better handle on how to use them therapeutically,” says Farzan, who conducts his research at the Scripps Research Institute in Florida. “We also want to know if there is a more focused way to drive these molecules besides interferons, which are about as subtle as a sledgehammer, and just as harmful.”
Also stemming on his work in HIV, Farzan and his collaborators are now in the midst of developing an “alternative vaccine” that uses antibodies to neutralize the Ebola virus. The promising ZMapp antibody cocktail was only given to five Ebola patients because supplies of the experimental drug, which is produced through genetic engineering of tobacco plants, ran out. Farzan’s approach would use human muscle instead of tobacco leaves to generate the drug.
Essentially, the researchers would use a harmless carrier called adeno-associated virus, which already exists in most people, to deliver the genetic building blocks of ZMapp into muscle tissue. There, the antiviral components could be produced for many years to offer protection against the virus. Farzan says he recently received approval to test this form of gene therapy in a secure biocontainment safety level 4 facility at the National Institutes of Health.
No doubt, the virus must be handled with care. Numbers from the current outbreak indicate the highly virulent pathogen has killed 70 percent of the people who have contracted the virus. That kill rate, though astounding, also means that a minority manage to survive. Geography certainly plays a role, as individuals who are close to hospitals and receive prompt medical care have better chances of survival. But genetics can stack the deck in a person’s favor even before they see a doctor.
Natural genetic variation can give some people stronger immune systems, which are more adept at fending off a viral attack or producing antibodies to neutralize the threat. Harvard microbiologist Sean Whelan was part of a team of researchers searching for human mutations that could interfere with Ebola infection. Their genome-wide scan repeatedly uncovered mutations in the same gene, called Niemann-Pick C1 or NPC1.
In a 2011 paper in Nature, the researchers showed that NPC1 encodes a host protein that the Ebola virus glycoprotein must attach to in order to infect cells. Mice that were partly deficient in NPC1 got sick but didn’t die. Cells that lacked the protein, including those from patients with Niemann-Pick type C1 disease, remained unharmed after exposure to the virus. Whelan’s colleagues are now testing whether giving small molecule drug therapies to block NPC1 in healthy individuals can make them less susceptible to infection.
Though therapies to block viral entry points or strengthen immune responses are promising, Whelan is more interested in strategies to disable the virus when it has overcome those devices. Once Ebola has infiltrated the cell, it begins making copies of itself that it can send on a mission to infect other cells. The virus is so prolific that nearly half a billion viral particles can be found in a drop of blood from an infected individual.
A single protein, the polymerase, is responsible for making all these copies. For fifteen years Whelan has been studying the polymerase of the vesicular stomatitis virus, a pathogen that causes blisters and sores similar to foot and mouth disease. Like Ebola, the genome of this virus is encoded by RNA. Whelan believes that the protein will make a good target of broadly active antiviral therapeutics, because parts of its structure appear to be similar across this class of viruses but distinct from proteins that have similar jobs in host cells.
Recently, he used a new technique called cryo-electron microscopy – which involves freezing samples at cryogenic temperatures before imaging – to finally get an atomic resolution structure of the RNA polymerase. Like Saphire, Whelan is optimistic that this latest structure will provide a “road map for new treatments,” this time for ones that inhibit the polymerase. He has recently received a small grant from the National Institutes of Health to pursue the same sorts of analyses for the Ebola virus.
“I think this is a tremendously important therapeutic target,” says Whelan. “I am quite certain that in 2015 we will see a number of publications reporting the structure of polymerases of negative-strand RNA viruses and I think that that will spark a resurgence of interest in using those as the basis of therapeutic strategies for blocking the replication of those viruses.”
Though his line of research may be a hot topic in future years, Whelan has been relatively immune to the media attention that has followed fellow BWF awardees Saphire and Sabeti. Prior to the current outbreak, both researchers had traveled to endemic regions to set up laboratories to study Ebola and its more common cousin, Lassa virus. Now, as Ebola has ravaged West Africa and frightened other parts of the world, the researchers have used their knowledge from time in the field to advocate for more resources to combat and contain the virus.
Sabeti, who has been featured in a number of articles in the New York Times, the New Yorker, and the Washington Post, says that she feels a responsibility to inform the public about her research, but also feels compelled to intensify her efforts in the laboratory. It is a difficult balancing act, and Sabeti has had to develop new strategies, such as doing less individual media appearances and more Q&A’s with the whole research team, to free up time to focus on her research.
Some researchers might take all the recent attention on Ebola as validation of their choice to devote their careers to a virus that so rarely touches this continent. But Saphire has never needed external validation to know that her research program was worthwhile.
“I already knew that this was the most important thing that needed to be done, that it was really compelling, that one day a big outbreak would come, and that antibodies would work,” says Saphire. “Part of me wonders, I have been doing the same work for ten years. All of a sudden you think it is important now, are you going to think it is important next year? You can’t just fund research when it is convenient. The only reason that there are any of these candidate therapies now is because people have been studying Ebola virus for 40 years. If we waited until there was a problem to start the basic research, we would have no hope in overcoming an outbreak.”