mBiosphere

Staying one step ahead of Ebola virus genomic changes

During the course of the current West African outbreak of Ebola virus disease, the US Army has put troops, medical personnel, building and medical supplies on the ground to fight the epidemic. Now, another critical piece of the medical arsenal has been sent—genomic sequencing capabilities and viral geneticist Captain Jeffrey Kugelman to track genetic changes in the virus as they happen in real-time.

“This virus mutates rapidly and it’s an ongoing concern,” says Kugelman, from Charlesville, Liberia, where he is working at the Liberian Institute for Biomedical Research. He and his collaborators published findings in mBio this week showing that the Ebola virus variant from the current outbreak has acquired genetic changes over the last four decades that might interfere with some therapies currently being tested in humans. Their study highlights the importance of ongoing biosurveillance of the virus’s genome to check for mutations that might disrupt the ability of medical workers to diagnose or treat the disease.

“There is an increasing need to have genomics biosurveillance during outbreaks,” says Gustavo Palacios, senior author on the study and director of the Center for Genome Sciences at the U.S. Army Medical Research Institute of Infectious Diseases in Frederick, Maryland where the analysis was done. “Especially when sequence-based therapies offer promising ways to attack the virus, constant surveillance is needed to see if those therapies are compromised by the natural genetic drift of the pathogen.”

Many of the most promising drugs being developed to fight Ebola are such sequence-based therapies that bind and target a piece of the virus’s genetic sequence or a protein sequence derived from that genetic sequence. These include monoclonal antibody, siRNA (small-interfering RNA) and PMO (phosphorodiamidate morpholino oligomer) drugs currently being used experimentally to treat a handful of patients.  None of these drugs have been approved by the US Food and Drug Administration, but are being used under the emergency containment protocol of the World Health Organization.

“We wanted to highlight where genetic drift, the natural process of evolution on an RNA virus genome, could affect the development of these countermeasures,” says Palacios. The specific binding sites that each therapy targets have already been published. “Our work was to highlight the genetic changes in the current Ebola virus strain that could affect these sequence-based drugs that were designed using previous strains.”

He and Kugelman, along with USAMRIID colleagues and collaborators at Harvard University and the Massachusetts Institute of Technology, both in Cambridge, Massachusetts, compared the current strain, named EBOV/Mak, with two previous strains that caused smaller outbreaks in 1976 and 1995 in Zaire (now the Democratic Republic of the Congo). In each comparison, they found more than 600 genetic mutations in the form of single nucleotide polymorphisms (SNPs).

Next, they zoomed in on just the mutations that occurred in the genetic sequences targeted by the therapeutics. Of those, they found 10 new mutations that might interfere with the actions of the sequence-based drugs currently being tested. Three of these mutations appeared during the current West African outbreak.

“The virus has not only changed since these therapies were designed, but it’s continuing to change,” says Kugelman. All of the therapies were designed in the early 2000’s based on the 1976 or 1995 Ebola virus strains.

He and Palacios say that drug developers need to assess their therapies for efficacy against these mutations to ensure that resources are not wasted further developing a drug that might be less effective or not effective at all against the current virus variant.  Palacios adds that regulatory agencies must also take into account the speed with which virus pathogens can evolve.

“If we agree that sequence-based, targeted therapies are a tool for the future, then we also need to change the way they are approved to allow for more flexibility,” he says.  A more agile regulatory system is needed to respond to genetic changes that might require updated versions of sequence-based therapies.

Two of the therapies, TKM-Ebola, a combination of siRNA components, and ZMapp, a combination of antibodies, have already been used to treat a few patients in the US and Europe in combination with standard medical care.  A clinical trial of TKM-Ebola will start in Sierra Leone in the coming months, which Palacios says is key: “Clinical trials are so important because they have the statistical power to determine if the survival rates are directly related to the drug, beyond supportive care.”

Palacios notes that real-time biosurveillance of the virus’ genome is needed to make sure that current diagnostic tests and therapies remain effective. In the current study, Palacios and Kugelman analyzed patient samples mostly from May to July 2014. However, they would like to monitor genetic changes as the epidemic continues to unfold. That is why Kugelman is now on the ground in Liberia, tracking the genetic changes that might occur in the virus as it is transmitted from human to human in near real-time.

“It’s not fast enough to help the current patients, but within 4-5 days after we get patient samples, we can characterize the outbreak genetically as it happens,” says Kugelman. “We will have virus sequence information within a week, instead of a months-long wait.”

 

--Kendall Powell

Cold Plasma Treatment Cuts Norovirus Germs

When Günter Klein, head of the Institute of Food Quality and Food Safety at the University of Veterinary Medicine Hanover in Germany, and colleagues investigated a norovirus outbreak at a German military facility, they noticed something interesting. Although consumption of norovirus-contaminated salad caused the peak of the illnesses, virus also could be detected on several common surfaces tested, including a computer keyboard, a doorknob and a mail collection box. 

“From this study and others it was obvious that surfaces, with which many people have direct contact, are crucial for the spread of the pathogen,” Klein says.

Normally after such an outbreak, surfaces are disinfected with chemicals and closed down for a certain amount of time. But disinfectants have to be applied for 30 to 60 minutes at least, he says, and can potentially damage the surface material. In addition, cross resistances can develop. His team wanted to see if using a technology like cold atmospheric pressure plasma (CAPP) would be effective against norovirus.

CAPP, a type of gas used to kill bacteria without harming surfaces or human tissues, is being used in medical applications like wound healing. Some scientists also are investigating its potential to remove bacteria from fruits, vegetables and meats. CAPP can be applied for a much shorter time and does not leave residues.

Their study, published this week in mBio, showed that CAPP was able to significantly reduce the number of virus particles in norovirus samples. The finding is exciting because noroviruses typically are very stable in the environment, resisting treatment by detergents or chlorine, freezing or heating, Klein says. Human norovirus is the most frequent cause of epidemic nonbacterial acute gastroenteritis worldwide, causing over 19 million cases of illness in the United States alone each year.

            “CAPP is an environmentally friendly, low energy method that decreases the microbial load on surfaces,” Klein says. “The technology is effective against viruses with a high tenacity, like noroviruses. Its successful application in medical therapy should be transferred to other areas.”

To investigate CAPP’s impact on norovirus, the team prepared on sterile petri dishes three dilutions of a 2011 stool sample from a German soldier infected with norovirus. They treated the samples with CAPP for varying lengths of time, up to 15 minutes, in a plasma chamber.

            After treatment, the scientists observed that samples treated for the longest time had the lowest viral load. CAPP reduced the number of potentially infectious virus particles from 22,000 (similar to what would be found on a surface touched by someone infected with norovirus) to 1,400 after 10 minutes, and to 500 after 15 minutes. Some reductions in viral load were seen in as little as one to two minutes of treatment.

“Cold plasma was able to inactivate the virus on the tested surfaces, suggesting that this method could be used for continuous disinfection of contaminated surfaces,” Klein says. Although plasma could not eliminate the virus completely, he says, “a reduction is still important to lower the infectious dose and exposure for humans.”

            In future studies, Klein’s team will test plasma’s disinfection properties on additional surfaces and types of norovirus, and use electron microscopes to examine the structure of the virus before and after CAPP treatment.

-- Karen Blum

Commensal bacteria are friends, then foes to keep population in check

Martin Blaser, 66, and Glenn Webb, 72, often discuss aging on their long summer hikes up to the Continental Divide near Fraser, Colorado. Not their own, but rather that of the human population. 

No other species has such a long, extended period of aging past the age of reproduction, or senescence. The two long-time collaborators often wondered and debated while they walked, why would evolution have selected such an age structure for the human population? After all, when resources are limited, the more mouths to feed past the age of reproduction, the greater the burden on the young’s survival.

Blaser, a microbiologist at New York University Langone Medical Center who has spent 30 years studying the commensal gut microbe Helicobacter pylori, decided to view the question from the symbiont’s point of view. Although a peaceful co-existor for much of our lives, H. pylori is a major cause of stomach cancer and that risk increases with age. In some parts of the world, the disease accounts for 7-8% of total mortality. 

“That’s a lot for one disease and one microbe,” says Blaser.  He thought that perhaps our microbes, involved in most everything we do, might also be involved in our biological clocks. “I began to think that a real symbiont is an organism that keeps you alive when young and kills you when you are old. That’s not particularly good for you, but it’s good for the species .” It’s also beneficial to the microbe community to keep the human population healthy, because if the human population crashes completely, then no more microbial homes.

Webb, a mathematician at Vanderbilt University, could devise a mathematical model to test this hypothesis. Webb’s forte is coming up with nonlinear differential equations to describe dynamic biological processes.

“Differential equations are all about change,” says Webb. “There’s a kind of magic in them that relates different rates of change to each other. And that relationship between them, the combination of those rates acting together, can reveal something unintuitive or unappreciated about the entire biological process.”

Webb and Blaser incorporated three types of mortality into their model of an early human, hunter-gatherer population: all-cause mortality, the mortality created by the burden of a large senescent population on the juvenile population, and finally the mortality associated with particular microbes. Running a baseline version of the model, with the microbial mortality component set to zero, the team showed in a study published this week that the population stabilizes at around 200 years with about 7,000 individuals and a mean age of 18.

Using this baseline model, they next tweaked the starting parameters, such as doubling the fertility rate. Intuition says that boosting fertility should help more juveniles survive and the population thrive. But surprisingly, it causes wild oscillations in the population over time.

“You get into a boom or bust cycle and the oscillation is so great that the population can easily reach extinction,” if a low point coincides with a crisis such as a food shortage , says Blaser. “This might help explain why modern hunter-gatherers don’t have 20 kids, but rather six on average.”

If a higher birth rate was not the answer, perhaps another solution would be to increase the mortality affecting only senescent individuals. Enter the microbes.

Blaser and Webb ran the model, adding in mortality risks based on particular microbial profiles. In one version they added a Shigella-like risk, which increases mortality only for children, and the population crashed to zero.

Next, they ran a version incorporating risk from a microbe like H. pylori, which affects older adults and increases with age. At the same time, they held juvenile mortality to a constant, low level. In this scenario, the human population reaches a stable equilibrium. 

“If you have an organism that preferentially kills old people, then you get a robust population,” says Blaser. “This is what Nature is doing,” he argues.

Webb says the modeling also revealed an underlying truth about human population growth—that we have the right fertility and mortality rates to maintain our species, even with our very unusual age structure of prolonged juvenile and senescent phases. “That’s what mathematics does for you. It verifies that our age structure is very stable and has been for a very long time.”

Blaser envisions a phase shift in our relationship with our symbiotic microbes over our lifetimes. Early on, natural selection has favored organisms that aid digestion or other metabolic functions and those that help protect against pathogenic invaders. But as we age, selection favors organisms that begin contributing to inflammation and degenerative processes. He points to species like Bacteroides that make vitamins for us, but if they escape the gut, can kill us.

In modern times of increased longevity, the cancer, inflammation, and degeneration these microbes cause place a major burden on the aging population. “Living with diseases caused by various microbes is inherent in our species,” notes Webb. “But the question is, can we improve longevity or manage it better?”

Blaser cautions that simply getting rid of microbes that cause mischief late in life may not be the best solution—especially if their beneficial effects on immunity and metabolism in early life aren’t fully understood.

“Our relationship with microbes is a fact of life in all directions.”

 

Seasonal Flu Vaccines Boost Immunity To Many Strains of Influenza

Paul Thomas was merely trying to develop a more reliable test for exposure to avian influenza viruses when he made a fairly startling finding: Seasonal flu vaccines may protect individuals not only against the strains of flu they contain but also against many additional types.

An immunologist who works with T cell biology, Thomas hypothesized that T cell responses would provide greater specificity than antibody-based tests in terms of determining if an individual had been infected, because for a T cell response to be made, the virus has to have invaded the body. While this hypothesis turned out to be incorrect, his team did find among a group of bird scientists studied that those who reported receiving flu vaccines had a strong immune response not only against the seasonal H3N2 flu strain from 2010, when blood samples were collected for analysis, but also against flu subtypes never included in any vaccine formulation.

The results, published this week in mBio, are exciting “because it suggests that the seasonal flu vaccine boosts antibody responses and may provide some measure of protection against a new pandemic strain that could emerge from the avian population,” said Thomas, an Associate Member in the Department of Immunology at St. Jude Children’s Research Hospital in Memphis, Tenn. “There might be a broader extent of reactions than we expected in the normal human population to some of these rare viral variants.”

Because avian influenza viruses have an important role in emerging infections, Thomas and colleagues tested whether exposure to different types of birds can elicit immune responses to avian influenza viruses in humans. They studied blood samples from 95 attendees of the 2010 annual meeting of the American Ornithologist Union. They exposed plasma from the samples to purified proteins of avian influenza virus H3, H4, H5, H6, H7, H8 and H12 subtypes using ELISA and hemagglutination inhibition (HAI) to see how many different viruses participants reacted to, and how strongly.

In the ELISA tests, 77 percent of participants had detectable antibodies against avian influenza proteins. Most individuals had a strong antibody response to the seasonal H3N2 human virus-derived H3 subtype, part of the 2009-2010 vaccine. However, many also had strong measurable antibody responses to group 1 HA (avian H5, H6, H8, H12) and group 2 HA (avian H4, human H7) subtypes. Sixty-six percent of participants had some level of detectable antibodies against four or more HA proteins that sit on viral surfaces, and a few had responses to all subtypes tested, most of which have not previously been detected in humans.

The team also found that participants who had significant antibody responses did not necessarily also have significant T cell responses to avian viruses, indicating that these two immunity arms can be independently boosted after vaccination or infection; that individuals who reported receiving seasonal influenza vaccination had significantly higher antibodies to the avian H4, H5, H6, and H8 subtypes; and that participants with exposure to poultry had significantly higher antibody responses to the H7 subtype, but to no other subtypes. Exposure to other types of birds did not play a role in immunity.

A person’s immune response on the ELISA test did not necessarily predict response on the HAI test, and vice versa. As HAI antibodies only target the “head” of the HA while ELISA antibodies can be against the head or the relatively conserved “stalk” domain, this result indicated that some individuals were more likely to target the conserved stalk region (i.e. show greater reactivity in ELISA than in HAI).

The work has opened up a lot of questions in figuring out why people mount different types of responses, and potentially how the seasonal vaccine may play a role in boosting these responses, Thomas said. He has started additional studies in other groups of people with varied vaccination and infection histories to tease apart what exposures boost immunity against avian influenza viruses.