mBiosphere

Drug Targeting Ebola Virus Protein VP24 Shows Promise in Monkeys

For nearly 10 years, researchers with the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) have been collaborating with Sarepta Therapeutics, Inc., to develop an effective therapeutic agent against Ebola virus and other related viruses, called filoviruses.

When they started, cases of Ebola virus occurred sporadically and affected at most a few hundred people, usually in Africa. Most research focused on developing a product for a biodefense scenario, like if a terrorist group released Ebola virus in a certain population. “The idea that we would have cases of Ebola virus being diagnosed in the U.S. was on just a few people’s radar and was considered a remote possibility,” says Travis K. Warren, PhD, a principal investigator in the Molecular and Translational Sciences Division at the research institute at Fort Detrick, Md.

Now, with Ebola virus rampant in three West African countries, the work of investigators at USAMRIID has taken on added meaning. Despite many efforts to develop vaccines and antiviral medicines against filoviruses like Ebola, there are currently no licensed medical countermeasures against these viruses. Surprisingly, many products being used by doctors to treat patients infected with Ebola virus in West Africa have not been tested in any animal model or in non-human primates, Warren said.

In mBio this week, Warren and colleagues in the Army and at Sarepta published work showing that the experimental medication AVI-7537, which targets the gene coding for a protein in Ebola virus called VP24, protects 75% of Ebola virus-infected monkeys.

 Their research compared drugs called phosphorodiamidate morpholino oligomers (PMOs) --- synthetic “antisense” molecules that target mRNAs within Ebola virus in a sequence-specific fashion and suppress translation. While previous work by the authors showed that a combination PMO targeting the genes that code for VP24 and another protein, VP35, protected rhesus monkeys from Ebola virus infection, the current study revealed that targeting VP24 alone was sufficient to confer protection from Ebola virus. Treatments consisting of placebo or an agenttargeting VP35 alone resulted in no protection (0% survival).

The majority of monkeys treated with AVI-7537 survived Ebola virus infection and showed substantial reduction of virus in their bloodstreams within eight days of treatment, compared to animals receiving a placebo (saline).

“This study demonstrates that we can protect non-human primates from Ebola virus, using only a single antisense agent,” says Warren, lead study author. “It also suggests that VP24 may be a key virulence factor encoded by Ebola virus, and is a promising target for the development of effective anti-Ebola virus countermeasures.”

 During the study, researchers gave Ebola virus-infected rhesus monkeys one of three medications: AVI-7537; AVI-7539, which targets VP35; or a combination treatment that included both AVI-7537 and AVI-7539, called AVI-6002. A fourth group of monkeys received just saline and served as the control group. The animals received these treatments intravenously, once a day for up to 14 days.

Seventy-five percent of animals treated with AVI-7537 and 62 percent of animals receiving the combination treatment, AVI-6002, survived until the end of the study. By contrast, all animals receiving saline or AVI-7539 developed progressive signs of Ebola disease and succumbed.

Additional tests showed that AVI-6002 and AVI-7537 were similar in their ability to reduce viral load, substantially reducing or eliminating infectious virus and viral RNA in the animals’ bloodstreams. Animals treated with AVI-6002 and AVI-7537 also had less liver and kidney damage, a common complication of filovirus infection, than those treated with placebo or AVI-7539.

“The work demonstrates that impairment of VP24 alone is enough to protect against Ebola virus infection and that targeting VP24 may lead to the development of more effective countermeasures against this important viral pathogen,” said senior study author Sina Bavari, PhD, Science Director for the U.S. Army Medical Research Institute of Infectious Diseases.

Phase Ia clinical trials were successfully completed with AVI-7537; the product was well tolerated in healthy human volunteers. Continued development of this product is dependent on continued funding, Bavari said. Additional studies are pending.

-- Karen Blum

Environmental factors may affect how infants’ gut bacteria mature

A group of international researchers has found that environmental factors, like mode of delivery and duration of gestation, may affect how infants’ gut bacteria mature, and that rate is related to later body fat. The work, published in mBio this week, was conducted by epigenetics researchers from the EpiGen Consortium in collaboration with scientists at Nestlé Research Center in Switzerland. 

            Among a group of 75 infants, those who were vaginally delivered and had a longer gestation before birth tended to more quickly develop a more mature gut microbiota, and had typical body fat at 18 months, the researchers found. By contrast, babies who were delivered via Caesarean section and had shorter gestations took longer to acquire a more mature gut microbiota and had lower body fat at 18 months. All but two babies studied were born at term.

            “It seems like the early environment -- mode of delivery, mode of feeding, duration of gestation and living environment --- may be influencing the rate at which babies acquire their gut microbiota,” said senior study author Joanna Holbrook, PhD, a senior principal investigator at the Singapore Institute for Clinical Sciences, “and that in turn has an association with how babies grow and put on body fat.”

            At birth, human infants start accumulating intestinal microbiota until a relatively stable state is reached, Holbrook said.

            It’s surprising to a lot of people that such short differences in gestation can make such an impact on infants’ health, Dr. Holbrook said. Taking longer to achieve the six-month-like gut profile might be evidence of the benefits of a longer gestation, “or it might suggest we need to intervene in the microbiome of children with slightly suboptimal environments,” she said.

            For the study, Holbrook and colleagues used 16s rRNA sequencing to analyze stool samples that had been collected from infants participating in the GUSTO (Growing Up in Singapore Toward healthy Outcomes) study, which includes members of the three main ethnic groups in Singapore: Chinese, Indian and Malay. The samples were taken when the infants were three days old, three weeks old, three months old and six months old. GUSTO, which aims to evaluate the role of developmental factors in the early pathways to metabolic disease, is Singapore's largest birth cohort study to date.

            Researchers investigated the effect of environmental factors including delivery mode and duration of gestation on trajectories of microbial development, along with the associative relationships with later body fat. Their work found that the samples could be classified into three distinct clusters based on when infants’ gut microbiota matured. Of 17 infants who had a more mature, six month-like microbiota profile high in the bacteria Bifidobacterium and Collinsella by day three, 16 were delivered vaginally. Other babies took up to six months to reach that stage.

Infants that acquired a profile high in Bifidobacterium and Collinsella at an earlier age had typical body fat at age 18 months, while those that acquired this profile later had relatively low body fat.

Follow-up studies will examine other drivers that play a role in the infant microbiome, including the mother’s microbiome.

“What we’d like to do eventually is to be able to intervene in these infants and get the optimal microbiome,” Dr. Holbrook said.

-- Karen Blum

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