A team of scientists has discovered how the deadly Ebola virus hijacks human cells, opening potential avenues to new drugs and a vaccine.

Ebola kills roughly 80% of those who contract it, usually causing them to bleed to death in a few weeks. Since its discovery in 1976, the virus has killed 1,000 people, according to the World Health Organization. Recent Ebola outbreaks in the Republic of Congo and neighboring Gabon in central Africa have killed several dozen people. The virus's mysterious appearances, rapid course and lack of treatment have made it a daunting challenge for public health -- and a potential weapon for terrorists.

Now, a research team says it has answered important basic questions about how Ebola, and a related virus, Marburg, commandeer human cells. Their findings shed light on possible ways to design drug therapies. The Ebola virus, shaped like a shepherd's crook, targets tiny fat platforms called "lipid rafts" that float atop the membrane of human cells. These cholesterol-rich rafts are the viruses' gateway into cells, the assembly platform for making new virus particles, and the exit point where new virus particles bud.

This virus-like particle, the harmless hollow shell of the Ebola virus, may one day be useful in creating an Ebola vaccine. The particle has been disarmed of its genetic material and is unable to replicate.

The team's report, set to be published Monday in the Journal of Experimental Medicine, "is highly significant," says Eric Freed, principal investigator in the laboratory of molecular microbiology at the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health in Bethesda, Md. "It adds another human pathogen to the growing list of viruses that use lipid rafts."

The findings add new insight into the life cycle of viruses and how they subvert human cellular mechanisms. It is a critical early step toward one day creating drugs that would stop viruses from replicating. Ebola and Marburg, both members of a family of hemorrhagic-fever viruses called filoviruses, share the reproduction strategy of viruses ranging from measles and influenza to HIV, which causes AIDS. Their ability to traffic aboard lipid rafts may help them evade the human immune system, researchers speculate.

One of the researchers, [Sina A Bavari (born 1959)], at the U.S. Army Medical Research Institute of Infectious Diseases in Fort Detrick, Md., says, "By understanding how Ebola and Marburg are entering into and budding from the cells, it gives us an avenue to come up with new therapeutics that would alter these pathways." Dr. Bavari's co-author is M. Javad Aman, of Clinical Research Management Inc. in Frederick, Md.

The push to probe Ebola has assumed greater urgency since the Sept. 11 terror attacks, as fears have grown about the existence of weaponized forms of Ebola or Marburg. Such bioterror weapons were in development within the former Soviet Union, according to Kenneth Alibek, a Soviet bioweapons scientist who defected to the U.S. and wrote the 1999 book "Biohazard."

Says [Sina A Bavari (born 1959)], "It doesn't take a Nobel laureate to figure out that something so deadly could be transformed into a bioterror agent." Yet-to-be-developed vaccines and antiviral drugs could be critical elements of bioterror defense against the viruses.

Because their targets, the lipid rafts, are made of fat, known agents such as the popular cholesterol-lowering statin drugs may offer one possible model for new drug therapies. Antifungal drugs, such as nystatin and filipin, that break up fat could be other possible models.

In their research, [Sina A Bavari (born 1959)] and [Drs.] Aman produced harmless copies of the Ebola virus that, it turns out, may be a possible vaccine candidate. The virus-like particles, known as VLPs, are hollow protein shells, gutted of their virulent genome. The researchers say the hollow proteins could elicit an immune defense, because they signal the body that an Ebola invasion is under way without actually causing disease.

"You're basically fooling the body," [Sina A Bavari (born 1959)] says, "but the virus cannot replicate itself." The hollow-protein model is one approach being used in the search for a vaccine for HIV. Future studies will examine whether such a strategy is safe and effective against Ebola and Marburg, he adds.

The researchers say their creation of the hollow VLPs could allow Ebola research to take place more freely in laboratories across the country, on regular lab benches outfitted with suction hoods, to prevent the escape of particles. At present, scientists have to wear space suits and work behind air-locked doors in high-containment Biosafety Level 4 labs when handling live Ebola virus.

Another National Institutes of Health researcher working on Ebola, [Dr. Gary Jan Nabel (born 1953)], of the Vaccine Research Center at NIAID, applauds the report. Dr. Nabel has published studies showing that Ebola vaccine created using experimental "naked DNA" -- the opposite of the hollow VLPs, in a way -- protected monkeys against lethal Ebola infection when given with a booster shot of the vaccine. His team is partnering with [Vical Incorporated], a San Diego biotech company, to produce the vaccine prior to launching studies of its safety in humans.

"People are waiting with bated breath for a drug or vaccine," [Dr. Gary Jan Nabel (born 1953)] said. "These [studies] show that we're on the road that will get us there."

PORTLAND, Ore. [AVI BioPharma], Inc. (Nasdaq:AVII) announced today the presentation of data from two studies evaluating the company's NEUGENE(R) PLUS therapeutic antisense compounds in the treatment of nonhuman primates (NHP) exposed to the Ebola virus. Results of the studies were presented at the National Institutes of Health's Filovirus Animal Workshop by [Sina A Bavari (born 1959)], principal investigator, U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID).

The studies were conducted in collaboration with USAMRIID and funded as part of AVI's two-year, $28 million research contract with the [Defense Threat Reduction Agency] (DTRA) of Fort Belvoir, Va., an agency of the Department of Defense.

The two Ebola studies involved 10 NHP subjects, including two controls. In the first study, three of four treated NHPs survived and the Ebola viral infection was completely eliminated. In the second study, all four treated NHPs survived substantially beyond untreated subjects and all completely eliminated the Ebola virus. Subjects were initially challenged with a 1000pfu of Ebola Zaire and then treated one hour following exposure with a 20 mg/kg dose of two NEUGENE PLUS antisense drugs via subcutaneous (SC) and intraperitoneal (IP) injection. Researchers continued treatment daily for 10 to 14 days via SC and IP injection at 20mg/kg.

[Sina A Bavari (born 1959)] also presented data for two studies evaluating NEUGENE PLUS therapeutics in the treatment of mice and guinea pigs exposed to different strains of the Marburg virus. One hundred percent survival was observed in mice challenged with Marburg, Ravn strain and 100 percent survival was observed in guinea pigs challenged with Marburg, Musoke strain. The NEUGENE PLUS therapeutic is expected to be effective against all known strains of Marburg. These studies were also supported by the $28 million DTRA contract.

"These studies clearly demonstrate the ability of a NEUGENE PLUS treatment to protect against viremia and death associated with Ebola or Marburg exposure," said K. Michael Forrest, AVI's interim CEO. "This research provides the basis for a viable therapeutic response as part of our nation's biodefense preparedness. It also establishes much-needed scientific evidence of therapeutic benefit against two currently untreatable hemorrhagic viruses that trigger devastating outbreaks."

Associated mouse studies demonstrated that AVI's Ebola NEUGENE PLUS therapies are safe and well-tolerated in mice at 50 times the dose used in the NHP studies.

The NEUGENE PLUS molecules used in the study represent a small but significant chemical modification to AVI's antisense "backbone." This change, which creates a positively charged therapeutic molecule that binds more readily with negatively charged RNA virus particles, is one result of an ongoing initiative at AVI to innovate the antisense platform for improved pharmacokinetics and bioavailability in certain therapeutic areas, including the treatment of infectious diseases.

AVI researchers are in the process of conducting additional GMP and GLP toxicology and safety studies using the NEUGENE PLUS compounds as part of the ongoing collaboration between USAMRIID and AVI. The next step in evaluating clinical efficacy of the NEUGENE PLUS drugs against Ebola exposure will measure the impact of delayed treatment of Ebola Zaire threats in a NHP population.

About Ebola Zaire and Marburg Viruses

• Ebola hemorrhagic fever is a severe, often-fatal disease in humans and nonhuman primates (monkeys, gorillas and chimpanzees) that has appeared sporadically since its initial recognition in 1976. The disease is caused by infection with Ebola virus, named after a river in the Democratic Republic of Congo (formerly Zaire) in Africa, where it was first recognized. Ebola virus and Marburg virus are the only two members of a family of RNA viruses called the Filoviridae.
• Researchers have hypothesized that the first patient becomes infected through contact with an infected animal. After the first patient in an outbreak setting is infected, the virus can be transmitted in several ways. People can be exposed to Ebola virus from direct contact with the blood and/or secretions of an infected person.
• The disease is a National Institute of Allergy and Infectious Disease (NIAID) priority A pathogen and a bioterrorism suspect agent of interest to the Department of Defense and Project BioShield. There are currently no approved treatments for Ebola.
• Marburg virus was first recognized in 1967, when outbreaks of hemorrhagic fever occurred simultaneously in laboratories in Marburg and Frankfurt, Germany, and in what is now Serbia.
• Marburg hemorrhagic fever is a rare, severe type of hemorrhagic fever that affects both humans and nonhuman primates. It is caused by a genetically unique animal-borne RNA virus, whose recognition led to the creation of this virus family.

About AVI BioPharma

• AVI BioPharma develops therapeutic products for the treatment of life-threatening diseases using third-generation NEUGENE antisense drugs and ESPRIT exon skipping technology. AVI's lead NEUGENE antisense compound is designed to target cell proliferation disorders, including cardiovascular restenosis. In addition to targeting specific genes in the body, AVI's antiviral program uses NEUGENE antisense compounds to combat disease by targeting single-stranded RNA viruses, including dengue virus, Ebola virus and H5N1 avian influenza virus. AVI's NEUGENE-based ESPRIT technology is initially being applied to potential treatments for Duchenne muscular dystrophy. More information about AVI is available on the company's Web site at http://www.avibio.com.

[...]

• CONTACT: AVI Contact:
• AVI BioPharma, Inc.   /   Michael Hubbars  [...]
•   or  AVI Investor Contacts:
•              Lippert/Heilshorn & Associates Inc.   /    Jody Cain or Brandi Floberg [...]
•  or   AVI Press Contact:
•              Waggener Edstrom Worldwide Healthcare   /    Jenny Moede   [...]

New studies show that treatments targeting specific viral genes protected monkeys infected with deadly Ebola or Marburg viruses. Furthermore, the animals were protected even when therapeutics were administered one hour after exposure—suggesting the approach holds promise for treating accidental infections in laboratory or hospital settings.

The research, which appears in today's online edition of the journal Nature Medicine, was conducted by the U.S. Army Medical Research Institute of Infectious Diseases in collaboration with [AVI BioPharma], a Washington-based biotechnology firm.

Working with a class of compounds known as antisense phosphorodiamidate morpholino oligomers, or PMOs, scientists first performed a series of studies with mouse and guinea pig models of Ebola to screen various chemical variations. They arrived at a therapy known as AVI-6002, which demonstrated a survival rate of better than 90 percent in animals treated either pre- or post-exposure.

Encouraged by these results, the team conducted "proof of concept" studies in which 9 rhesus monkeys were challenged with lethal Ebola virus. Treatment was initiated 30-60 minutes after exposure to the virus. In these studies, 5 of 8 monkeys survived, while the remaining animal was untreated. Further experiments, including a multiple-dose evaluation, also yielded promising results, with 3 of 5 monkeys surviving in each of the AVI-6002 treatment groups when they received a dose of 40 mg per kg of body weight.

According to first author Travis K. Warren of USAMRIID, antisense drugs are useful against viral diseases because they are designed to enter cells and eliminate viruses by preventing their replication. The drugs act by blocking critical viral genetic sequences, essentially giving the infected host time to mount an immune response and clear the virus.

Ebola and Marburg cause hemorrhagic fever with case fatality rates as high as 90 percent in humans. The viruses, which are infectious by aerosol (although more commonly spread through blood and bodily fluids of infected patients), are of concern both as global health threats and as potential agents of biological warfare or terrorism. Currently there are no available vaccines or therapies. Research on both viruses is conducted in Biosafety Level 4, or maximum containment, laboratories, where investigators wear positive-pressure "space suits" and breathe filtered air as they work.

The USAMRIID team next turned its attention to Marburg virus, again screening various compounds in mice and guinea pigs to select a candidate for further testing. They settled upon AVI-6003, a drug that consistently conferred a high degree of efficacy (better than 90 percent survival) in both models.

Investigators conducted two pilot studies in cynomolgus monkeys to assess the efficacy of AVI-6003 against lethal challenge with Marburg virus. As with the Ebola studies, treatments were initiated 30-60 minutes after infection. All 13 animals receiving AVI-6003 survived. Additional research provided important information about the optimal therapeutic dose range of the compound, with a 40 mg per kg body weight dose protecting 100 percent of the monkeys following challenge.

"This report of successful early post-exposure treatment of filovirus hemorrhagic fever is significant on its own," said Colonel John P. Skvorak, USAMRIID commander, "but the drug characteristics of these PMOs also support investigation of potentially broader therapeutic applications."

Senior author [ Sina A Bavari (born 1959)] said USAMRIID has been collaborating with AVI BioPharma since 2004. In February of that year, an Institute scientist working in a Biosafety Level 4 laboratory stuck her thumb with a needle while treating Ebola-infected mice with antibodies. As a precaution, USAMRIID medical experts recommended the investigator be isolated for 21 days to ensure that she had not been infected.

Coincidentally, earlier that very day, [Dr. Patrick Lynn Iversen (born 1955)] from AVI BioPharma had presented a seminar at USAMRIID concerning the efficacy of novel antisense drugs against a range of viruses. When he found out that a USAMRIID scientist had potentially been exposed to Ebola virus, the company volunteered to design and synthesize compounds against the virus to treat her if the need arose.

The team at AVI worked for four straight days to generate human-grade anti-Ebola compounds. In the meantime, their regulatory staff worked with USAMRIID physicians to gain emergency approval from the U.S. Food and Drug Administration to use the compounds if necessary. Five days after the exposure, AVI delivered the compounds to USAMRIID's medical team. 

Fortunately, the scientist had escaped infection with Ebola virus, so the compounds were never used. However, USAMRIID went on to test them in animal models, and has been collaborating with AVI ever since. 

According to the authors, the investigational new drug applications (IND) for AVI-6002 and AVI-6003 have been submitted to the U.S. Food and Drug Administration, and they are now open to proceed with clinical trials.

NOTES: 

• Collaborating on the study were Travis K. Warren, Jay Wells, Kelly S. Donner, Sean A. Van Tongeren, Nicole L. Garza, Donald K. Nichols, Lian Dong, and Sina Bavari of USAMRIID; Kelly L. Warfield and Dana L. Swenson, formerly of USAMRIID; and Dan V. Mourich, Stacy Crumley, and Patrick L. Iversen of AVI BioPharma.
• USAMRIID, located at Fort Detrick, Maryland, is the lead medical research laboratory for the U.S. Department of Defense Biological Defense Research Program, and plays a key role in national defense and in infectious disease research. The Institute's mission is to conduct basic and applied research on biological threats resulting in medical solutions (such as vaccines, drugs and diagnostics) to protect the warfighter. USAMRIID is a subordinate laboratory of the U.S. Army Medical Research and Materiel Command. 
• Reference:    Travis K. Warren, Kelly L. Warfield, Jay Wells, Dana L. Swenson, Kelly S. Donner, Sean A. Van Tongeren, Nicole L. Garza, Lian Dong, Dan V. Mourich, Stacy Crumley, Donald K. Nichols, Patrick L. Iversen and Sina Bavari; "Advanced antisense therapies for postexposure protection against lethal filovirus infections," Nature Medicine (22 August 2010).

The treatment of the two Ebola-stricken, American medical missionaries with the well-documented, ZMapp "plantibodies" drug developed by U.S. and Canadian companies and federal agencies has raised questions of bioethics and process.

But how can Ebola drugs be tested for efficacy if their use is restricted during the most widespread Ebola outbreak since 1976?

As a result, the World Health Organization has scheduled a meeting of medical ethicists for next week how drugs not previously tested in humans might be ethically mobilized during such a crisis. From the WHO announcement:

• “We are in an unusual situation in this outbreak. We have a disease with a high fatality rate without any proven treatment or vaccine,” says Dr Marie-Paule Kieny, Assistant Director-General at the World Health Organization. "We need to ask the medical ethicists to give us guidance on what the responsible thing to do is.”

To recap, the ZMapp combination product of three, humanized antibodies against different parts of the Ebolavirus outer glycoprotein – which continues to be called a "secret serum" – was given to Dr. Kent Brantly and Nancy Writebol prior to their medivac journey from a hospital in Paynesville, Liberia, to the biosafety level-4 isolation ward in Atlanta's Emory University Hospital.

In her deeply-considered article yesterday, Laura Seay at The Washington Post wrote,

• "The optics of the situation were already bad enough: at great expense, two white Americans were whisked away to safety and a level of health care that simply cannot be provided on the fly in a Liberian hospital. The black Liberians they had been treating were left to see whether fate would save or kill them under the same substandard health care system they have always lived under."

• "That what now looks like a miracle cure was only given to the white Americans looks even worse. Why hadn’t anyone reached out to try the serum on Ebola patients sooner, especially if its potential to heal is so promising?"

However, this antibody combination had never been given to humans, not even healthy volunteers.

Other drugs already in Phase 1 safety trials

In contrast, at least two other companies have products that have already been subject to Phase 1 safety trials. Most widely publicized has been TKM-Ebola, a lipid nanoparticle, RNA-interference drug being developed by Vancouver-based, Tekmira Pharmaceuticals, together with a division of the Department of Defense.

Single-dose and multiple ascending dose studies have been progressing and the company announced on July 21 that the FDA was placing a hold on the trials as they conducted an interim analysis of the drug's safety profile.

However, Tekmira just announced today that the FDA verbally confirmed the modification of the trials' status to a "partial hold," thereby allowing the drug to be used in people infected with Ebola.

• "We are pleased that the FDA has considered the risk-reward of TKM-Ebola for infected patients. We have been closely watching the Ebola virus outbreak and its consequences, and we are willing to assist with any responsible use of TKM-Ebola. The foresight shown by the FDA removes one potential roadblock to doing so," said Dr. Mark Murray, CEO and President, Tekmira Pharmaceuticals. "This current outbreak underscores the critical need for effective therapeutic agents to treat the Ebola virus. We recognize the heightened urgency of this situation, and are carefully evaluating options for use of our investigational drug within accepted clinical and regulatory protocols."

The Tekmira drug targets three of the seven genes of the Ebola virus. (Yes, the deadly Ebolavirus wreaks its havoc with only seven proteins. In comparison, we humans have just under 20,000.)

The second drug is a different type modified RNA molecule, AVI-7537, from Sarepta Therapeutics. AVI-7537 is directed against one of the three Ebolavirus genes (VP24) targeted by Tekmira's drug. But its chemistry platform, called PMOplus, is distinctly different from that of Tekmira's TKM-Ebola. AVI-7537 also works via a different mechanism to block the viral protein from being made.

For those interested in the technical aspects of the chemistry and mechanism, this open-access 2012 paper in the journal, Viruses, is invaluable.

AVI-7537 has already shown effectiveness in non-human primates against the Zaire Ebolavirus, the species implicated in the current outbreak. Moreover, Sarepta had been conducting Phase 1 safety trials with the drug alone and together another Ebola-directed PMOplus molecule (AVI-7539, with the combination called AVI-6002).

The work, done with the support of the Department of Defense, was part of a project put on hold in 2012 during the fiscal cliff fiasco.

The Ebola drug proved to have an excellent safety profile in a single dose escalation study and a two-week, daily dosing regimen. So the discontinuation was more likely due to economics than medical concerns. In support of that speculation, the DoD continued to support Sarepta's Marburg virus program.

Sarepta's drug remains available

Sarepta's president and CEO, Chris Garabedian, told Barron's earlier this week that the company had a drug that could be deployed and shipped if a request was made of the company and all permits and authorizations were cleared.

Garabedian told me on Tuesday that company does indeed have clinical trial-quality drug on hand. The Marburg program that has continued "uses the same backbone chemistry," so the safety studies that are continuing with that drug at higher human doses could be applied to AVI-7537.

"We're here to raise awareness that we do have a technology that might be helpful, and that we do have drug substance on hand if we received a request from a government agency," said Garabedian. "We, of course, would have to get the appropriate waivers and approvals from the Department of Defense who supported the development of this compound as well as the FDA in terms of an emergency use authorization."

The U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID) is a co-assignee on the two patents, not expiring until 2025, that cover Sarepta's Ebolavirus drugs.

Beyond the fact that the U.S. government has already made a significant investment in Sarepta's drug, the company already holds an open IND for human clinical trials.

"We're just highlighting that we have drug substance available and we can go to convert that into finished product in vials. Assuming all approvals are there from the various agencies, we could have this ready in a week for compounding in a pharmacy for dosing into a patient," said Garabedian.

Should any agency or institution wish to make such a request, this wouldn't be the first time Sarepta had made a drug available on an emergency basis. In fact, the genesis of Sarepta's Ebola program was the government's acute need for a drug.

"This started with a real-world situation where a researcher at USAMRIID had a needle-stick injury with Ebola and the company was called to see if we could turn around a drug candidate over a weekend," said Garabedian. "Within five or six days, we had drug on a plane to dose this patient."

"Fortunately for this woman, she didn't test seropositive (indicating exposure to the virus) so she didn't receive the drug. But that started our animal work and led to a lot of other collaborations with the government."

We contacted USAMRIID's Dr. Sina Bavari, for more information on the potential clinical utility of AVI-7537. Bavari is the lead investigator in the government partnership and senior co-author on peer-reviewed publications with the Sarepta drugs. While he promptly acknowledged our request, press officer Cmdr. Amy Derrick-Frost, USN, from the Office of the Assistant Secretary of Defense for Public Affairs responded, "At this time, we are not doing interviews."

FORT DETRICK, Md. (Oct. 22, 2014) -- From on-site laboratory support in Liberia, to training of key personnel, to accelerated research efforts on diagnostic, vaccine and treatment approaches, the U.S. Army Medical Research Institute of Infectious Diseases is playing a significant role in assisting the Ebola Virus Disease outbreak response in West Africa.

Ebola virus causes a severe, often fatal hemorrhagic disease in humans and non-human primates. Currently there are no licensed vaccines or drugs to fight the disease, and case fatality rates as high as 90 percent have been reported in past outbreaks. As of Oct. 15, the World Health Organization reported at least 8,997 cases and 4,493 deaths in seven affected countries. These include Guinea, Liberia, Nigeria, Senegal, Sierra Leone and Spain, as well as the first-ever case of Ebola diagnosed in the U.S.

That patient, a man who had recently traveled from Liberia to the U.S., died Oct. 8.

The U.S. Department of Defense is supporting the U.S. Agency for International Development as part of a U.S. whole of government response effort to the Ebola virus outbreak, as announced by President Barack Obama on Sept. 16. U.S. military personnel are deploying to West Africa in support of the effort, called Operation United Assistance. In addition to setting up a regional staging base to facilitate transportation of equipment, supplies and personnel, the U.S. military is establishing additional treatment centers in Liberia and providing medical personnel to train health-care workers in the region.

At the U.S. Army Medical Research Institute of Infectious Diseases, known as USAMRIID, the response effort spans the institute's research and support divisions and there is no sign of the operational tempo slowing any time soon, according to Col. Erin P. Edgar, commander of the institute.

"This is definitely not business as usual," he said.

Late September, USAMRIID was asked to provide training to deploying U.S. forces, according to Lt. Col. Neal E. Woollen, who directs the institute's biosecurity program. Several personnel have volunteered to serve on mobile training teams that travel to deploying units to train and certify troops who will be working in Ebola-affected areas of West Africa. Training is focused on proper wearing of protective equipment, as well as decontamination procedures.

ON-SITE LABORATORY SUPPORT

Since April 2014, USAMRIID and the National Institute of Allergy and Infectious Diseases-Integrated Research Facility have provided personnel, training and diagnostic laboratory support to the Liberian Institute for Biomedical Research on a continuous rotational basis, according to Randal J. Schoepp, Ph.D., chief of USAMRIID's Applied Diagnostics branch. He and several others helped to set up an Ebola virus testing laboratory in Liberia and trained local personnel to run diagnostic tests on suspected Ebola hemorrhagic fever clinical samples.

Schoepp said USAMRIID has been working on a collaborative project in West Africa since 2006 (see sidebar article below). Because the team was working on disease identification and diagnostics in the region, he added, "We had people on hand who were already evaluating samples and volunteered to start testing right away when the current Ebola outbreak started."

In addition to providing laboratory testing and training support for the current outbreak, USAMRIID has provided more than 10,000 Ebola laboratory tests, referred to in the medical community as assays, to support laboratory capabilities in Liberia and Sierra Leone. The institute also supplied personal protective equipment to Metabiota Inc., a non-government organization involved in the testing.

Edgar called the project "a great example of medical diplomacy at work."

"This collaboration allows USAMRIID to bring our expertise to bear in responding to an international health crisis," he said. "In addition, it enables us to test the medical diagnostics that we develop in a real-world setting where these diseases naturally occur."

DIAGNOSTIC TOOLS

USAMRIID research led to the only assay currently authorized to diagnose Ebola in U.S. citizens, according to David A. Norwood, Ph.D., chief of USAMRIID's Diagnostic Systems Division. The assay, which detects the Zaire strain of Ebola virus in patient samples, is called the Ebola Zaire Real-Time PCR Assay Test Kit. It was developed, manufactured and tested with help from the U.S. Army Medical Materiel Development Activity.

While the test has not been approved by the U.S. Food and Drug Administration, the FDA has authorized its use under an Emergency Use Authorization, granted in August 2014. According to Norwood, the EUA provides a legal basis for the use of unapproved medical products, including diagnostics, in a declared emergency when there are no alternatives. The test is available at authorized DOD laboratories in the U.S. and overseas, as well as select CDC Laboratory Response Network state public health labs throughout the country for testing U.S citizens.

"This assay is also being used in West Africa for rapid diagnosis of host nation patients," said Norwood. "So there is no disparity between the diagnostic capabilities that are being used in-country and those that are available for testing U.S. citizens. While the labeling and execution is somewhat different for regulatory purposes for testing U.S. citizens, the same capability is available for diagnostic testing for everyone."

Issuance of the EUA was a collaborative effort among several agencies: Medical Countermeasure Systems, U.S. Army Medical Command; Health Affairs, Readiness Division, Health Care Operations Directorate; Joint Program Executive Office Critical Reagents Program; the DOD Clinical Laboratory Improvement Program Office; and the recipient laboratories, including five DOD labs and 15 CDC-LRN state public health laboratories.

DRUG AND VACCINE RESEARCH

USAMRIID is leading the evaluation of several promising Ebola medical countermeasure candidates, including therapeutics and vaccines, according to scientific director [Sina A Bavari (born 1959)], Ph.D.

Bavari, an expert at building public-private partnerships, says the current outbreak offers researchers an opportunity to accelerate the development of medical products to prevent and treat the disease through collaboration with pharmaceutical companies and other government agencies.

Among the products being evaluated by USAMRIID are four potential therapies, including synthetically made, small-molecule drugs that have shown efficacy against a broad range of viral diseases, according to Bavari. One of these drugs, known as BCX4430, has been tested in animal models at USAMRIID; its parent company is in the process of filing an Investigational New Drug application with the FDA to begin Phase I clinical trials in humans.

Two other compounds of interest are oral favipiravir, dubbed T-705, which is already in Phase III clinical trials as a potential influenza treatment, and AL-8176, currently is in Phase II clinical trials for Respiratory Syncytial Virus.

"If we can evaluate a drug that's already in development for another use, and show that it has potential against Ebola virus, that saves us years of research and development," [Sina A Bavari (born 1959)] explained.

The fourth therapeutic candidate being studied at USAMRIID is Z-Mapp, a "cocktail" of three antibodies, one of which was developed by USAMRIID. This drug made headlines when it was used to treat a handful of people infected during the current outbreak, including two American aid workers who contracted Ebola in Liberia and recovered at Emory University Hospital in Atlanta, Georgia.

Previous studies at USAMRIID with an earlier version of Z-Mapp showed that it could protect monkeys from Ebola even when administered five days after infection, according to John M. Dye, Ph.D., branch chief for viral immunology. He said additional studies of Z-Mapp in nonhuman primates will begin at USAMRIID later this month. Those efforts will help to determine dosing -- the optimal amounts of antibody that can be safely administered and still provide protection.

In addition, there are a number of Ebola virus vaccine platforms in various stages of development, Dye said. Two that have been studied extensively at USAMRIID are the VLP (virus-like particle) and the VRP (virus replicon particle) vaccine approaches. Other vaccine approaches include those based on adenovirus (currently in Phase I clinical trials) as well as the rVSV (recombinant vesicular stomatitis virus) platform.

USAMRIID's Division of Medicine is providing medical monitor support to the Phase I clinical trial of the rVSV vaccine, scheduled to begin this month at the Walter Reed Army Institute of Research.

According to [Sina A Bavari (born 1959)], USAMRIID is continuing to investigate potential treatments and vaccine candidates for Ebola, with several laboratory and nonhuman primate studies scheduled for the near future. The success of these research efforts will depend, in part, on future funding levels.

REWARDING EXPERIENCE

It's not often that USAMRIID scientists get to take their expertise out of the laboratory and into a field setting. For Schoepp, the experience has been "rewarding," though he says he'll be ready to stay home for a while after completing his fourth trip to West Africa in just six months.

"What makes me really proud is that the laboratory staff we trained [in West Africa] jumped right into the fray, and thanks to the training we provided, they didn't even blink," said Schoepp. "They started testing right away; they knew what to do."

While the scientists at the Liberian Institute for Biomedical Research put in long, hot hours wearing protective gear in the laboratory, their work environment is far from the only challenge they face, according to Schoepp. Diagnostics personnel are under a great deal of pressure to run the tests accurately, because the results they provide to the health care team literally can mean the difference between life and death for a patient.

"It's critical to diagnose Ebola-infected individuals, of course, but it's also important to tell people they're not infected," he said. "Being able to give them an answer -- so they can go home and not worry -- that's pretty satisfying."

[...]

SIDEBAR NEWS ARTICLE:

Sierra Leone Samples: Evidence of Ebola in West Africa in 2006

A study published in July 2014, in the journal, Emerging Infectious Diseases, showed that Ebola virus has been circulating in the region since at least 2006 -- well before the current outbreak.

According to first author Randal J. Schoepp, Ph.D., of USAMRIID, between 500 and 700 samples are submitted each year to the Kenema Government Hospital Lassa Diagnostic Laboratory in Sierra Leone. Generally, only 30 to 40 percent of the samples test positive for Lassa fever, so the aim of this study was to determine which other viruses had been causing serious illnesses in the region.

Using assays developed at USAMRIID that detect the presence of IgM, an early protein produced by the body to ward off infection, the research team found evidence of dengue fever, West Nile, yellow fever, Rift Valley fever, chikungunya, Ebola and Marburg viruses in the samples collected between 2006 and 2008. About two-thirds of the patients had been exposed to these diseases, and nearly 9 percent tested positive for Ebola virus.

In addition, of the samples that tested positive for Ebola, the vast majority reacted to the Zaire strain, which was unexpected, according to the authors.

"Prior to the current outbreak, only one case of Ebola had ever been officially reported in this region, and it was from the Ivory Coast strain," said Schoepp. "We were surprised to see that Zaire -- or a variant of Zaire -- was causing infection in West Africa several years ago."

The laboratory testing site in Kenema has been supported by the Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance and Response System. In collaboration with the host country, the site enables collection of samples that can be used in research toward new medical countermeasures, and allows USAMRIID to evaluate the performance of previously developed laboratory tests using samples collected on site. USAMRIID hopes to eventually obtain viral isolates for medical countermeasure development and receive data on the performance of the diagnostic assays.

Other contributors to the work include the Department of Defense Joint Program Executive Office-Critical Reagents Program, the Defense Threat Reduction Agency Cooperative Biological Engagement Program and the DTRA Joint Science and Technology Office.

[...]

When the Pan American Health Organization put out an alert last May about the first confirmed cases of Zika virus infection in Brazil, the news barely registered. After all, compared with other mosquito-borne viruses, such as potentially life-threatening dengue and yellow fever, Zika seemed pretty harmless. Only 20% of people infected with Zika even become ill, and their symptoms tend to be mild—fever, rash, joint pain, and conjunctivitis.

But in January, nine months after the organization raised the alarm, doctors in Brazil reported a disturbing trend that coincided with Zika’s spread across the country. Since October 2015, more than 4,000 babies in Brazil had been born with abnormally small heads and brains—a rare condition known as microcephaly. Although further analysis lowered that figure by 462 cases, the sharp rise nonetheless has experts worried that Zika could be to blame. For comparison, Brazil reported just 147 cases of microcephaly in 2014.

Zika is also being blamed for an uptick in cases of Guillain-Barré syndrome, a potentially life-threatening disorder in which the body’s immune system attacks the central nervous system and causes paralysis. As with microcephaly, the evidence connecting Zika and Guillain-Barré is still circumstantial. Nevertheless, the link is strong enough for the World Health Organization to declare the Zika outbreak a public health emergency of international concern.

Margaret Chan, WHO’s director-general, said earlier this month that the virus is “spreading explosively” through the Americas, with cases of active virus transmission in at least 26 countries and territories in the Americas. Panic over the virus has prompted health officials in some countries to take the drastic measure of advising women to delay pregnancy for months or longer. In El Salvador, Deputy Health Minister Eduardo Espinoza asked women to avoid becoming pregnant until 2018.

With Zika making headlines for the past month, scientists have been scrambling to get a handle on the virus. Industry, government, and academic scientists have all announced efforts to develop and test treatments and vaccines. But the path ahead for these researchers is long and full of pitfalls. Even though Zika has been around for almost 70 years, surprisingly little is known about the virus and its basic biology. A PubMed search for “Zika virus” turns up mostly case studies.

What we do know is that Zika is a flavivirus, a member of the same family as dengue, yellow fever, and West Nile virus. Zika is primarily transmitted via bites from infected mosquitoes, but in recent weeks doctors have reported that the virus can be sexually transmitted as well.

It was first identified in a monkey in Uganda’s Zika forest in 1947, but only a handful of human Zika cases were reported until a 2007 outbreak in Micronesia’s Yap Island. An outbreak in French Polynesia followed six years later. Last November officials in that country reexamined the cases of microcephaly that followed the outbreak. Before the outbreak, about one case of microcephaly was reported each year. In 2014–15, officials found 17 cases of fetuses and infants with “central nervous system malformations,” which includes microcephaly.

As the case connecting Zika to serious health effects builds, the world would love a vaccine or treatment for the virus. But because so few have studied Zika, drug developers currently have few tools to work with. For example, there’s no commercially available, U.S. Food & Drug Administration-approved test to screen for Zika virus.

Tracking Zika in people is hard because it’s difficult to determine that they’re infected with Zika and not a related flavivirus or that they’re not infected with more than one virus, says Priscilla L. Yang, a flavivirus expert at Harvard Medical School. Simultaneous infection with Zika and another virus could cause health effects that haven’t been seen before.

Scientists can use polymerase-chain-reaction-based methods to distinguish Zika from other flaviviruses. But those tests are accurate only during the short window patients still have the virus in their system—about seven days after infection. By the time a patient has symptoms that warrant a visit to the doctor, the virus is no longer circulating in their bloodstream, Yang notes.

Another option is to look for antibodies against the virus. But Zika and dengue are closely enough related that antibodies to Zika also recognize dengue and vice versa. Making a definitive diagnosis based on antibodies is possible but becomes time-consuming and laborious, Yang says.

For scientists who have compounds that might be effective against Zika, actually testing them has been tough. “We have small molecules that seem to be broadly acting against dengue and West Nile virus,” Yang says. “We want to test them, but getting access to the live virus has been hard.” She’s heard that certain labs known to have the Zika virus have been bombarded with hundreds of requests from researchers.

Even if someone manages to access the live virus and can find a compound that kills it in cells, the researcher will hit another roadblock: To date, no one has published practical animal models of Zika virus to screen potential therapies against. Yang points to a paper from the 1970s in which scientists did an intracranial injection of Zika virus in newborn mice, but she notes that is a poor model because many small molecules can’t slip past the blood-brain barrier.

“We’re basically starting from scratch on this one, unfortunately,” says [Sina A Bavari (born 1959)], chief scientific officer (CSO) at the U.S. Army Medical Research Institute of Infectious Diseases. Bavari and colleagues are currently working with pharma companies to see if they have any compounds that inhibit Zika replication in cells.

They’re primarily interested in compounds that have passed the hurdles of Phase I or Phase II clinical trials but are sitting idle for business reasons. That’s because it can take upward of a year and a half just to get a new compound ready for Phase I. “My worry is that by the time we get something out the door, this outbreak will have already burned out,” Bavari says.

Scientists are also grappling with this question: If only 80% of people infected with Zika have symptoms, who would get the treatment? The most vulnerable patients are pregnant women, but Bavari points out, there’s a high bar when it comes to approving a medication that can be given to them. “They don’t even want to drink caffeine,” he says.

Other scientists are working to develop a vaccine against the Zika virus. Earlier this month President Barack Obama said he would ask Congress for $1.8 billion to combat Zika at home and abroad. Of those funds, $200 million would be used for vaccine development. The U.S. National Institute of Allergy & Infectious Diseases, Sanofi Pasteur, and [NewLink Genetics Corporation] are among the heavy hitters in the vaccine field who’ve said they’ll step up to the plate.

Even so, it could take three to five years before a vaccine is ready, experts say. [Dr. Thomas Patrick Monath (born 1940)], CSO of [NewLink Genetics Corporation]’s infectious disease division, led that firm’s efforts to develop an Ebola vaccine and was CSO at Acambis, where he worked on vaccines for dengue and yellow fever. Monath tells C&EN he thinks a large field trial of 10,000 to 20,000 people across multiple sites will be necessary to determine efficacy once a Zika vaccine is developed. “Only after those trials would you contemplate doing studies in pregnant women,” he says.

Monath also says because so many people who are infected with Zika never show any symptoms, it is more difficult to determine whether the vaccine has actually prevented infections. Still, he thinks a large enough trial should be conclusive.

But some scientists say the emphasis on vaccines is misplaced. “We just don’t know enough about Zika virus right now to run around and vaccinate people,” Bavari says. “Understanding the immunopathology and immunology behind it would be really prudent before starting a full vaccination program.”

Harvard’s Yang says developing a vaccine for every emerging virus is impractical. “Vaccines are, for the most part, specific. You have one virus, and you have one vaccine for it,” she explains. “I don’t think we’ll ever have the luxury of enough resources to get a vaccine against every single possible emerging virus or enough time to do it in a reactive way.”

One area that’s not getting as much attention, she says, is development of broadly acting antivirals that could keep a virus in check while the immune system fights it off. Classical antivirals go after a single viral enzyme, but viruses are quick to develop resistance to them. “If people could identify targets that have the potential to be effective against multiple viral pathogens, it could be game-changing,” Yang says.

[Dr. Hugh Alexander "H. Alex" Brown Jr. (born 1960)], a Vanderbilt University professor who works on antivirals, agrees. “There are so many viruses out there. We need to be working on a much more broad-spectrum approach to infectious disease,” he says. “If we can develop more tools to combat broad categories of viruses, I think we would be much better off than we are today.”  [ Note - [Dr. Hugh Alexander "H. Alex" Brown Jr. (born 1960) passed July 25, 2017 ]

In the meantime, scientists agree that the research community needs to be more organized if it’s going to have a real shot at combating Zika. Yang thinks the first steps should be figuring out how to get the necessary reagents to the labs that need them and agreeing on standards so they can compare results and learn from each other’s work. “If you actually want to have some sort of impact, we all need to work together,” she says. In an encouraging sign, earlier this month, major scientific institutions and top research journals agreed to share data relevant to Zika virus.

Bavari agrees scientists need to be better at organizing their efforts, but he has doubts about the direction the community is taking. “The outbreak is moving so quickly that I am worried people will jump and we won’t do the correct research,” he says. 

Recalcitrant Mosquito Blamed For Zika’s Spread

With a treatment or vaccine for Zika potentially years away, countries are relying on mosquito control to curb the virus’s spread. Aedes aegypti mosquitoes, which inhabit tropical and subtropical regions, have been named as the culprit in transmitting the virus.

But getting rid of Aedes aegypti is extremely difficult because the mosquitoes don’t seem to be affected by most spraying regimens, says Joseph M. Conlon, an entomologist and technical adviser to the American Mosquito Control Association. According to Conlon, Aedes aegypti feed during the day, but pesticides must be sprayed at dawn or dusk. Also, mosquitoes like to come indoors to feed. So, unless pesticides are sprayed inside homes, chances are good they’re not getting to the insects.

These mosquitoes are very small, and you can’t feel the bites. “Oftentimes you don’t even know you’ve been bitten,” Conlon says.

To get rid of the biting bugs, it’s critical to eliminate any standing water. “I’ve seen Aedes aegypti breeding in discarded soda bottle caps,” Conlon says. “They’re survivors.”

Despite Aedes aegypti’s survival skills, the mosquitoes actually have a fairly limited flight range of about 150 meters. That has made some scientists suspect that because Zika has spread so quickly, the more common Culex mosquito may be transmitting the virus as well. The theory is currently being investigated.

“If that is true, that brings this to a whole different level,” Conlon says. Culex mosquitoes have a much larger range, he notes, but they can usually be controlled through common mosquito abatement programs. ◾

• Purpose of the study: Antiviral agent development.  Clinical care for Zika virus infection is supportive, and there are no prophylactic or therapeutic drugs, vaccines, or other biologicals licensed for use to prevent or treat Zika virus infection and disease. A Zika virus threat assessment and evaluation of medical countermeasure development options has been completed; re-purposing existing licensed drugs was identified as the most efficient strategy for rapid development of licensed medical countermeasures suitable for prevention, treatment, or containment of the pathogen (1,2).

• Methods/summarized description of the project: Hypothesis-driven high throughput re-purposed drug screening.  An iterative multi-step drug selection and screening algorithm was established; 1) drug targets involving inhibition of virus-host cell interactions were identified, 2) compounds with significant clinical pharmacokinetic and safety data (preferably including in pregnancy) which inhibit the pathways were selected, 3) selected pharmaceuticals were tested for inhibition of Zika virus infection and replication using multiple cell types and Zika viral isolates, 4) pharmaceuticals with single-digit micromolar to nanomolar IC50 and CC50/IC50 ratios consistent with anti-viral specificity were selected for subsequent development as prophylactic and therapeutic anti-Zika drug candidates. 

• Results:  Re-purposed licensed drugs with anti-Zika activity which are safe for use in pregnancy. Three general mechanisms of action (including autophagy inhibition) have been identified and corresponding compounds have been screened.  Multiple re-purposed drugs have been identified which meet selection criteria for subsequent development. 

• Conclusion: Hypothesis-driven high throughput re-purposed drug selection can expedite identification of emerging infectious disease medical countermeasure candidates.  A summary of the pathways targeted, drugs identified and viral inhibition results obtained will be presented. 

• Discussion: Zika infection of the recipient host requires viral envelope protein binding and particle uptake into susceptible cells, is mediated by specific receptors which include DC-SIGN, AXL, Tyro3, and TIM-1, and triggers transcriptional activation of Toll-like receptor 3 (TLR3), RIG-I, MDA5, interferon stimulated genes including OAS2, ISG15, and MX1, and beta interferon (4). Primarily infected cells include skin fibroblasts, epidermal keratinocytes, and skin dendritic cells. Zika virus subsequently exploits autophagy to facilitate uptake and replication8, and pharmacologic manipulation of Zika infected cells with 3-Methyladenine (3-MA), an inhibitor of autophagosome formation, strongly reduces viral copy numbers in infected fibroblasts (3). Based on prior murine studies involving Zika virus inoculation in mouse brain (5), autophagy of Zika virus has been postulated as playing a key role in the pathogenesis of Zika-associated primary microcephaly (6).  Pharmacological mechanisms of currently licensed 4-Aminoquinoline anti-malarial drugs include inhibition of autophagy and broad-spectrum cathepsin B-mediated inhibition of viruses which require endosomal acidification (7).

References:

•  1. Malone RW, Homan J, Callahan MV, Glasspool-Malone J, Damodaran L, Schneider Ade B, Zimler R, Talton J, Cobb RR, Ruzic I, Smith-Gagen J, Janies D, Wilson J; Zika Response Working Group.  Zika Virus: Medical Countermeasure Development Challenges.  PLoS Negl Trop Dis. 2016 Mar 2;10(3):e0004530. doi: 10.1371/journal.pntd.0004530. PMID: 26934531

• 2. Longini IM Jr, Nizam A, Xu S, Ungchusak K, Hanshaoworakul W, Cummings DA, Halloran ME.  Containing pandemic influenza at the source.  Science. 2005 Aug 12;309(5737):1083-7. PMID: 16079251

• 3. Hamel R, Dejarnac O, Wichit S, Ekchariyawat P, Neyret A, Luplertlop N, et al. Biology of Zika Virus Infection in Human Skin Cells. J Virol. 2015; 89(17):8880–96. doi: 10.1128/JVI.00354-15 PMID: 26085147.

• 4. Carneiro LA, Travassos LH. Autophagy and viral diseases transmitted by Aedes aegypti and Aedesalbopictus. Microbes Infect. 2016. doi: 10.1016/j.micinf.2015.12.006 PMID: 26774331.

• 5. Bell TM, Field EJ, Narang HK. Zika virus infection of the central nervous system of mice. Arch Gesamte Virusforsch. 1971; 35(2):183–93. PMID: 5002906.

• 6. Tetro JA. Zika and microcephaly: Causation, correlation, or coincidence? Microbes Infect. 2016. doi: 10.1016/j.micinf.2015.12.010 PMID: 26774330.

• 7. Zilbermintz L, Leonardi W, Jeong SY, Sjodt M, McComb R, Ho CL, Retterer C, Gharaibeh D, Zamani R, Soloveva V, Bavari S, Levitin A, West J, Bradley KA, Clubb R3, Cohen SN, Gupta V, Martchenko M. Identification of agents effective against multiple toxins and viruses by host-oriented cell targeting.  Sci Rep. 2015 Aug 27;5:13476. doi: 10.1038/srep13476. PMID: 26310922

A controversial leader is creating a culture of fear and is stifling cutting-edge research at the U.S. Army Medical Research Institute of Infectious Diseases, according to a newly released Army report.

The Army’s investigating officer urged that the leader, science director Sina Bavari, be removed from USAMRIID and re-assigned to a job without supervisory duties.

Bavari continues in the position, however, and there is no evidence the institute has acted on these recommendations.

At USAMRIID, Bavari oversees the majority of the Fort Detrick institute’s research departments, including staff members who work in immunology, genomics, bacteriology and virology.

The Army found in a 2015 investigation that since Bavari took that position in 2014, he has created an “environment of fear” at the prestigious lab.

“Workers are genuinely afraid for their jobs and scientific careers,” the investigating officer stated in the report. In June, The Frederick News-Post obtained a copy of the Army report through a Freedom of Information Act request.

The News-Post spoke to multiple sources, including current and former institute researchers, about the Army report. Those who declined to be named feared Bavari would fire them, damage their reputations as researchers, or both.

The Army’s investigation of Bavari did not find any illegal activity.

Bavari declined an interview with The News-Post, but issued a statement through a USAMRIID spokeswoman.

In the statement, he said he is honored to serve as science director and looks forward to “enabling USAMRIID’s expanded capabilities and increased collaborations.”

‘Hot potato’

Statements from multiple current and former USAMRIID employees in the Army report showed that Bavari “fostered a negative or toxic leadership climate,” the investigating officer stated.

“There is a duality in Dr. Bavari’s tactics,” a former USAMRIID employee wrote in an email to The News-Post. “Those who are loyal and follow him unquestioningly are richly rewarded, while anyone who questions him or whom he otherwise perceives as a threat does not have a long future at USAMRIID, and will usually be made to suffer in the time that they are still there.”

The science director tarnished reputations, falsely accused employees of scientific misconduct and threatened them with termination, according to sworn statements in the Army investigation.

Many researchers at the institute work closely with toxins and biological agents such as anthrax and the Ebola virus in maximum-containment laboratories where protective, full-body suits are mandatory.

Henry Heine, who worked at USAMRIID from 1998 until 2010, said in an interview that Bavari was already working at the institute when Heine started there.

According to Caree Vander Linden, a spokeswoman for the institute, Bavari started at USAMRIID as a post-doctoral fellow in 1991.

Heine described Bavari as a “hot potato” who caused problems and was passed from one department to another before he became science director.

In a sworn statement provided for the Army investigation, a colonel said Bavari preferred to hire contractors, so he could terminate them for any reason.

Heine said Bavari took advantage of his ability to terminate contractors.

“When somebody would get into something that crossed him, or crossed something he was trying to do, they would disappear,” Heine said.

Heine is currently program director at the University of Florida’s Institute for Therapeutic Innovation. He did not work directly with Bavari while at USAMRIID, but felt familiar with the director’s modus operandi.

He said Bavari used other employees to further his own goals.

“He’s a consummate user,” Heine said.

Transformational leader

Current and former USAMRIID employees have described Bavari as condescending, manipulative and disrespectful.

Some also described him as an ambitious, resourceful leader who has attracted diligent, loyal workers to his research team.

Travis Warren, a principal investigator at USAMRIID, noted that the Army investigator did not ask some of Bavari’s closest colleagues to provide statements for the Army investigation.

Warren said he has been one of Bavari’s right-hand workers since 2007.

“I regard him as the single most influential mentor in my career development,” he wrote in an email to The News-Post.

Bavari has high expectations and little tolerance for maintaining the status quo, Warren wrote.

“While he often voices strong opinions, I have never felt these viewpoints were presented in an antagonistic or authoritarian manner and have found him to be highly receptive and encouraging of countering viewpoints,” he wrote in an email.

Gustavo Palacios, director of the institute’s Center for Genome Sciences, also works closely with the science director.

The center houses cutting-edge equipment for genome sequencing, which help researchers gain insight into the genes of bacteria, viruses and potential biowarfare agents. According to Warren, Bavari led efforts to get the Center for Genome Sciences off the ground.

Bavari has a sense of purpose that is “inspirational,” Palacios said, and he challenges his staff to think outside the box.

Bavari said in a statement to the investigator that taking care of employees “is of paramount importance to me and is at the centerpiece of my management style.”

Palacios described Bavari as a “transformational” leader whose goal is to ensure that USAMRIID is constantly growing and operating as a first-class lab.

“That’s a very tall task, given the restrictions in funding and the continuous appearances of new natural challenges like Ebola and Zika, or the potential risks associated with bioterrorism acts,” Palacios wrote in an email to The News-Post.

Bavari received an honorary doctorate degree from the University of Nebraska this year for leading the institute’s efforts to fight the Ebola outbreak.

“Honorary degrees are awarded by the University of Nebraska to recognize those who have attained achievements of extraordinary and lasting distinction,” according to a press release from the school.

Palacios said institutions such as the World Health Organization and the Centers for Disease Control and Prevention also praised USAMRIID for its efforts during the Ebola outbreak.

“It means that the institute is moving in the right direction,” he wrote in an email to The News-Post. “Obviously that is not the sole result of Dr. Bavari’s work, but he has taken a leadership role at a crucial time.”

A culture without checks

The Army started an investigation into Bavari last year, after a group of anonymous USAMRIID scientists submitted a letter to Army officials who oversee the institute.

Bavari, “while directing his own highly productive research program, is a detriment to the current and future state of medical research at USAMRIID and should be removed from this position,” the letter stated. “He is a person lacking in honesty, integrity and character, who in his short time as director has created a climate of distrust, intimidation, and fear of retribution.”

The letter writers called Bavari a “catastrophic threat” to the future of USAMRIID.

Some USAMRIID researchers commented to The News-Post that the frequent changes in command, in addition to the fact that their recently assigned commanders aren’t familiar with infectious disease research, have created a culture without checks or balances.

Fort Detrick installation commander Maj. Gen. Brian Lein and Army Surgeon General Lt. Gen. Patricia Horoho, who received the letter from the anonymous USAMRIID scientists, have since left their positions.

Army spokespeople who took media inquiries for Lein were unable to reach him for comment the week before he ended his two-year assignment at Fort Detrick. Lein’s last day as commander of the installation was July 28.

The Department of the Army generally assigns the U.S. Army Medical Research and Materiel Command, or USAMRMC, and Fort Detrick a new commander every two or three years.

Fort Detrick’s installation commander traditionally has a dual role as commander of USAMRMC, which manages USAMRIID.

USAMRIID has its own commander, Col. Thomas Bundt, who is also serving a two- to three-year assignment.

Bundt’s spokespeople did not respond to a request for comment about the Army investigation.

Conflict of interest

In the investigation, current and former USAMRIID researchers told the Army’s investigating officer that Bavari maintains a large research program that seems to be funded “at the expense of other projects,” according to the report.

Warren said Bavari has had a positive effect on USAMRIID’s research portfolio.

“I have personally observed multiple research programs move from an early discovery phase program to advanced development status due in large part to Dr. Bavari’s dedication to USAMRIID’s mission,” Warren wrote in an email.

But the fact that Bavari has a research program at all creates a conflict of interest, the anonymous USAMRIID researchers wrote in their letter.

“It is impossible for the Science Director to have [his or her] own research program and maintain independent judgment in an ethically responsible way,” the researchers wrote.

Hood College biology professor Ann Boyd teaches ethics and sits on the Institutional Review Board for the USAMRMC. The board reviews study proposals from USAMRIID and USAMRMC researchers to ensure that human subjects are treated ethically in experiments.

Directors who conduct research are not necessarily wrong to do so, Boyd said, but those who act in their own self-interest may display a lack of leadership.

“The problem with [a conflict of interest] in research is that it erodes trust,” she said.

Connie Dudley, director of graduate programs in science at Mount St. Mary’s University, said Bavari’s position as director and researcher leaves an “open door” for conflicts.

“It would certainly be a reasonable conclusion that a conflict of interest exists, if indeed this person had the ability to make programmatic changes that would benefit them,” she said.

Bavari told the investigating officer that he does not believe having his own research program creates a conflict of interest.

“In order to be an effective Science Director and to have the respect of other scientists, I feel the need to maintain a good scientific reputation in the field,” he said.

As a researcher, he said he can better relate to the challenges his staff faces.

Heine said that as a director, it can be difficult to be engaged with scientists if you don’t have some of your own research to refer to, but that is a difficult line to walk.

“In an environment where you have a very limited and restricted amount of funding, the temptation is to take care of yourself first, and that’s exactly what I think happened,” he said.

But, Warren said Bavari has intentionally reduced his role in programs to allow colleagues to step up.

“He intentionally empowers individuals like myself to assume greater responsibilities, while remaining available to advise or assist with challenges that may arise,” he wrote in an email.

Questions of support

The Army investigator determined that Bavari uses his power and authority to limit researchers from submitting grant proposals and getting funding from the [Defense Threat Reduction Agency], a Department of Defense agency that is a major USAMRIID funder.

The agency declined to comment.

“It would be inappropriate for the [Defense Threat Reduction Agency] to comment on an USAMRIID matter or employee,” agency spokesman Ron Lovas wrote in an email.

Bavari opted to submit a limited number of proposals to the agency, which were subject to his approval, instead of allowing researchers to submit their own proposals.

That may have kept researchers from securing funding to support their own staff and goals, according to the Army report.

In Bavari’s response to the Army investigation, he argued that his approach eliminated a process that created internal competition and replaced it with team collaboration.

“To ensure that the collective wisdom of USAMRIID’s outstanding scientists are fully applied to solutions for the Warfighter, I am slowly changing the way in which we work with [Defense Threat Reduction Agency],” he said.

Some USAMRIID employees who provided sworn statements for the Army investigation also were concerned about Bavari’s relationship with the nonprofit Geneva Foundation.

The foundation supports innovative medical research and works with military researchers as they create proposals for federal or other funding.

A dozen principal investigators at USAMRIID are supported by the Geneva Foundation. The foundation also employs 30 research team members at the institute.

The Army investigator asserted that the science director used his relationship with the Geneva Foundation to “bank” funding from pharmaceutical companies to achieve his own research goals without letting USAMRIID know about the funds.

That’s not how the Geneva Foundation works, President Elise Huszar said.

The foundation does not award funding; it works with researchers during the proposal stage and after funding has been awarded from another source, such as the federal government.

“We’re not a grant-making body; we don’t have a cache of money that we make discriminately to different people,” Huszar said.threat

Huszar said the foundation typically works closely with USAMRIID’s business office to make sure financial awards are reported according to Geneva’s requirements.

“There shouldn’t be any awards that USAMRIID doesn’t know about,” she said.

Huszar found multiple problems with the Army’s report.

“Geneva’s experience with Dr. Bavari did not equate with the report that was provided,” she said.

The Army’s findings will not affect Bavari’s relationship with the Geneva Foundation, Huszar said, unless USAMRIID decides to restrict his proposals.

Recommendations

The Army investigator recommended removing Bavari from USAMRIID and re-assigning him to a job without supervisory duties.

The investigator also recommended the following:

• Changing the science director position to minimize the possibility of a conflict of interest between that person and the institute’s research

• Auditing funding from nongovernmental sources

• Requiring detailed reporting of that funding

• Extending commanders’ assignments from two to three years to reduce the impact of turnover

• Requiring commanders to have subject matter expertise in the research their organizations conduct.

Vander Linden confirmed that Bavari continues to hold the position of science director at the institute, but could not comment whether the institute had acted on the investigator’s recommendations.

Current employees who spoke to The News-Post said they have not seen any indications that the institute has done so.

Officially, the institute itself was unable to comment on the report.

“Laws such as the Privacy Act severely restrict what can be discussed related to these matters,” Vander Linden wrote in an email. “Therefore, we are unable to comment further.”

Broad-spectrum coronavirus antiviral drug discovery

Allison L. Totura ORCID Icon & Sina Bavari

Pages 397-412 | Received 16 Aug 2018, Accepted 07 Feb 2019, Published online: 08 Mar 2019

1. Introduction

• 1.1. Coronaviridae phylogeny and emergence
  • • Highly pathogenic coronaviruses SARS-CoV and MERS-CoV recently emerged into human populations, but other human coronaviruses (HCoVs) including HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1 are estimated to have circulated in human populations for hundreds of years, causing mild respiratory illness to which approximately 5–30% of ‘common colds’ are attributed [7,8]. Within the Coronaviridae family (order Nidovirales) four genera are recognized: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. The six HCoVs (Table 1) currently identified belong to the genera alphacoronavirus (HCoV-229E and HCoV-NL63) and betacoronavirus (SARS-CoV, MERS-CoV, HCoV-OC43, and HCoV-HKU1). Gammacoronaviruses and deltacoronaviruses have no known viruses that infect humans, but contain important agricultural pathogens of livestock. Epizootic coronaviruses in animals cause a wide range of disease signs resulting from respiratory, enteric, and neurological tissue tropism. Although HCoVs cause primarily respiratory symptoms, the potential for a wide range of severe disease symptoms in humans caused by infection by future emergent coronaviruses cannot be excluded. Despite the severity and diversity of coronavirus disease signs and symptoms affecting a large number of important livestock species as well as humans, there are no proven therapies that specifically target CoVs.
    • In addition to CoVs known to cause disease in humans and livestock, a large number of highly diverse coronaviruses have been identified based on sequences collected from sampling bat species. Bat coronavirus (BatCoV) sequences recovered from sampling sites on different continents (Asia, Europe, Africa, North America) over the last decade contain putative BatCoVs from diverse branches of the betacoronavirus and alphacoronavirus phylogenetic tree [9–12]. Importantly, the two coronaviruses that cause the most severe disease in humans, SARS-CoV and MERS-CoV, emerged from BatCoVs that were not previously recognized to infect humans or animals other than bats [12–14]. Recent studies suggest that BatCoV-SHC014 and BatCoV-WIV1 are genetically similar to SARS-CoV and enter cells using human receptors [10,15,16]. Similarly, BatCoV-HKU4 and BatCoV-HKU5 are MERS-like BatCoVs that may also be circulating in bat populations, and some MERS-like BatCoVs may also be able to recognize human host cell receptors [17–19]. Such BatCoVs are now called ‘pre-emergent’, because they may have the potential to emerge into human populations. Importantly, therapeutics that rely on host memory responses to target CoV infection are often not effective against pre-emergent BatCoVs that differ antigenically from known HCoVs, highlighting the need for pan-coronavirus therapeutics that target conserved mechanisms utilized by HCoVs and BatCoVs [15].
    • SARS-CoV and MERS-CoV likely evolved from BatCoVs that infected other intermediate host animals in closer proximity to humans, resulting in SARS and MERS outbreaks (Figure 1) [20,21]. SARS-CoV was detected in small animals like civets and raccoon dogs that were present in live-animal markets [20]. Evolution of SARS-CoV evidenced by genomic sequence differences between zoonotic SARS-CoV strains infecting civets and epidemic SARS-CoV isolates likely resulted from viral adaptation, which is thought to be required for emergent CoVs to become transmissible from person-to-person [22,23]. MERS-CoV has been identified in dromedary camels, and is now known to be endemic in camel populations in the Middle East and Sub-Saharan Africa.
    • Emergence of coronaviruses into human populations, including highly pathogenic viruses like SARS-CoV and MERS-CoV, has occurred by spillover from bat reservoir hosts into intermediate hosts. The intermediate hosts during the 2003 SARS-CoV epidemic included civets and other small carnivore species located in wet animal markets. MERS-CoV has been identified in dromedary camels, and is particularly associated with active infection of juvenile camels. Novel emerging CoVs may occur in the future via infection from bat populations into other intermediate animal hosts. Additional evidence from BatCoVs indicates that pre-emergent CoVs with the ability to directly infect human cells may be poised for emergence into human populations. Based on prior research from SARS and MERS outbreaks, animal workers that have contact with intermediate animal host species and health-care workers that are exposed to nosocomial CoV infections are at increased risk of highly pathogenic coronavirus transmission. More severe disease in SARS and MERS cases resulted in patients that were over the age of 65 or had comorbidities such as obesity, heart disease, diabetes, renal disease, or hypertension.
• 1.2. Epidemiological features of CoV outbreaks
  • • Research on coronavirus-specific antiviral drugs has focused primarily on highly pathogenic coronaviruses SARS-CoV and MERS-CoV due to the major potential consequences of pandemics resulting from these pathogens. SARS-CoV and MERS-CoV did not transmit as efficiently from person-to-person compared to other respiratory pathogens like seasonal influenza, but mortality in patients of SARS (approximately 10%) and MERS (approximately 35%) greatly exceeded typical seasonal influenza case-fatality rates (2.4 deaths per 100,000 cases) [24]. Air travel facilitated these CoVs in seeding outbreaks in regions distant from initial localized viral spread: SARS-CoV emerged in the Guangdong province of southeastern China in late 2002, and then spread rapidly to other parts of the world, with outbreaks in major cities including Beijing, Hong Kong, Singapore, and Toronto, resulting in one of the first pandemics of the twenty-first century [5]. Since MERS-CoV emerged in 2012, MERS cases have been exported from the Middle East to Europe, North America, Africa, and South East Asia, including a major outbreak of 186 people in the Republic of Korea in 2015 [25]. Superspreaders (individuals that transmit SARS-CoV or MERS-CoV to a large number of people) played an important role in initiating and perpetuating CoV outbreaks: the 2015 MERS-CoV outbreak in the Republic of Korea started from a single traveler case, and just five cases were responsible for more than 80% of the transmission events [25]. SARS-CoV and MERS-CoV were transmitted by close contact, with known outbreaks occurring in hotels, apartment buildings, and hospitals or health-care centers. Health-care workers, in particular, were at risk for infection by SARS-CoV and MERS-CoV at high rates [5,26]. In addition, animal workers were more likely to come into contact with CoV infected animals, and a large percentage of MERS patients had contact with intermediate host camels [26,27]. Analysis of severe SARS or MERS disease identified disproportionately high case-fatality rates in elderly patients (age > 65 years) and patients with pre-existing comorbidities including diabetes, heart disease, hypertension, and renal disease [4,26,28]. Based on these epidemiological considerations, pan-coronavirus therapeutics are needed to i) protect populations with occupational risk for transmission of CoVs, ii) protect populations with susceptibility to severe disease from CoVs, iii) work in concert with public health measures like quarantine and contact tracing, and iv) be rapidly deployable to geographically distant regions from local HCoV epidemics.

2. In vitro systems for pan-coronavirus drug discovery

• 2.1. Reverse genetics systems
  • • Advances in the study of highly pathogenic coronaviruses and potential pan-coronavirus drug candidates partially depend on the technology to genetically manipulate CoVs to probe mechanisms of viral pathogenesis and antiviral drug activity. Reverse genetics systems synthetically generate viruses from known viral sequences [29]. In situations where clinical isolates of infectious material are unavailable due to restriction for collecting patient samples, shipping infectious materials, or availability of containment laboratories, reverse genetics systems provide essential research materials for studies on viral pathogenesis and model development. Prior to the SARS pandemic, robust reverse genetics systems to manipulate the genomes of CoVs had already been developed by systematic assembly of cDNA cassettes into full-length infectious clones, allowing precise and targeted genetic manipulation of viral genes [30,31]. Infectious clones allow the creation of near-homogenous viral stocks, whereas traditional viral stocks are prepared by amplification of infectious material in cell culture over many passages. Strategies to build reverse genetics systems were rapidly applied to both SARS-CoV and MERS-CoV within the first year of identification of these viruses [32,33].
    • In addition to reconstructing epidemic strains of CoVs, reverse genetic systems allow targeting of mutations to specific viral genes and assembly of viruses when infectious material is not available. As an example, the ability to isolate mutations in particular genes was applied to studies of the spike (S) glycoprotein of SARS-CoV, while maintaining the isogenic background of the viral replicase and other structural proteins. Mutations from zoonotic, early, middle, and late epidemic strains of the SARS-CoV outbreak were inserted into the S glycoprotein of the epidemic strain of SARS-CoV (Urbani) to determine the effect of evolution on viral entry into human cells as well as viral pathogenesis in rodent and primate models of disease [34–36]. By targeting mutations to a specific viral gene, reverse genetics systems allow researchers to probe cause-and-effect relationships of host pathogenic responses to viral genetic changes. In addition, reverse genetics techniques were utilized to study pre-emergent BatCoV strains: recombinant versions of BatCoV-HKU3, BatCoV-WIV1, and BatCoV-SHC014 (SARS-CoV-like), as well as BatCoV-HKU5 (MERS-CoV-like) viruses, were generated and used for in vitro and in vivo models of emerging coronavirus disease [15,16,37,38]. Panels of zoonotic, epidemic, and pre-emergent viruses synthesized by reverse genetics techniques encompass a diverse array for use in high-throughput platforms for the discovery of countermeasures that are effective against the broadest range of CoVs without being reliant on procuring clinical isolates.
• 2.2. Cell-based systems
  • • Like all other viruses, coronaviruses require host cell machinery to replicate their genomes, produce progeny virions, and cause disease. Cell lines require expression of the host cell receptor as well as expression of necessary proteases to facilitate viral entry, although additional host factors may also be important for infection. The S glycoprotein of coronaviruses, the main determinant of host cell attachment and viral entry, is not well conserved between HCoVs. Most human coronaviruses use different host cell receptors for viral entry, and may also require different host cell proteases that allow fusion of viral and cellular membranes (Table 1) [39]. Although all known HCoVs have viral tropism targeted at the human respiratory tract, lung cell lines infected by a broad range of HCoVs have not been defined. A key feature of SARS-CoV and MERS-CoV is that highly pathogenic coronaviruses grow to higher viral titer on a wider range of cell lines than the other mildly pathogenic coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 [40–44]. High throughput approaches to screen compound libraries for targeted activity against coronaviruses have been underdeveloped and limited in the number of viral strains used [45–52]. Infection of panels of cell lines from various animal species with HCoVs and BatCoVs informs on the potential host range of the pathogen, and may help to identify susceptible mammalian host involved in viral spread. However, productive infection of cell lines does not always translate to recapitulation of pathogenesis in the same animal model that the cell lines are derived from, which may be due to receptor availability in live animals or other biological and immunological factors during infection.
    • Infection of pseudostratified airway epithelium cultures from primary cells of the lung provides a cell culture model that simulates infection of cells in a more complex environment more closely resembling the human respiratory tract. Known as Human Airway Epithelia (HAE) or Normal Human Bronchial Epithelia (NHBE) cells, these cultures can be infected with all of the HCoVs identified thus far, including SARS-CoV and MERS-CoV, providing a potential platform to screen novel CoVs for emergence into human populations [42,53–56]. However, several limitations are associated with HAEs including difficulty in collection due to the scarcity of donors and difficulty in maintenance because of limited capacity for cell divisions. HAEs may be sourced from donors with a preexisting disease state, which could influence viral pathogenesis. In addition, because of the genetic variability of donors, HAEs cultures often differ in expression levels of genes crucial to infectivity, including the various host receptors for HCoVs, which leads to high variability in the infectivity of these cultures. Importantly, these in vitro methods fail to capture more complex viral interactions that occur with an intact immune system including infiltration of proinflammatory cells that may promote and contribute to ARDS in the most severe forms of SARS and MERS. Organ-on-a-chip models in development may provide the next generation of in vitro models that could capture these critical interactions between respiratory cells and immune cells, but infection of these novel culture systems has not been reported with coronaviruses [57].

3. In vivo systems for pan-coronavirus drug discovery

• 3.1. Small animal models for pan-coronavirus drug discovery
  • • Following the emergence of SARS-CoV in 2003, small animal model development was initiated by inoculating animals with human epidemic isolates of SARS-CoV that replicated in mice, hamsters, guinea pigs, and ferrets, but only ferrets exhibited disease signs resulting from infection (Table 2) [60–63]. SARS-CoV replicated in laboratory strains of mice, but did not cause disease signs, and virus was rapidly cleared from the lung in these models [60]. Serial passage of SARS-CoV in the lungs of mice by multiple research groups resulted in mouse-adapted SARS-CoV strains that caused lethal lung disease in wild-type mouse intranasal (IN) infection models [64,65]. Mouse-adapted SARS-CoV MA15 is the best characterized small animal model of CoV infection, and has been used to test several pan-coronavirus drug candidates [66,67]. The benefit of mouse-adapted models of SARS-CoV in wild-type inbred mouse strains includes reproducible susceptibility to disease assayed by survival, weight loss, and whole body plethysmography of individual mice as well as quantification of infiltrating cells, viral titers, histopathology, and transcriptomics and proteomics changes in target organs. To evaluate pathogenesis of emergent viruses in vivo, zoonotic SARS-CoV and pre-emergent BatCoV mutations have been incorporated into the SARS-CoV MA15 backbone, providing novel animal models for viruses that have the potential to emerge into humans [15,35]. Additional valuable avenues of research on variables known to impact severe CoV disease in the MA15 models of SARS-CoV include age, dose, and host genetic contributions to disease phenotypes [68,69]. The greatest limitations of SARS-CoV mouse-adapted models for drug discovery are the incorporation of mutations in the SARS-CoV genome (particularly for testing antiviral drugs that target viral genes with mutations) and acknowledged differences between mouse and human immune responses.
    • Unlike SARS-CoV, human clinical strains of MERS-CoV (Table 3) did not replicate in mice, hamsters, or ferrets, and further studies of the host receptor identified critical amino acid residue differences between the MERS-CoV receptor, DPP4, in laboratory animal model species that prevented entry into cells compared to human DPP4 [70–72]. MERS-CoV infection of rabbits resulted in viral replication in the upper respiratory tract, but no clinical disease signs that reflect more severe MERS-CoV disease symptoms were reported, although the model has been used for limited testing of MERS-CoV antiviral therapeutics [73,74]. However, due to the utility of the mouse-adapted SARS-CoV model, a mouse model continued to be pursued, and adenovirus-vectored transient expression of the human DPP4 receptor in mice and subsequent replication of MERS-CoV in these mice determined that MERS-CoV replication was dependent on human DPP4 expression in rodents [75]. Transgenic expression of human DPP4 in mice allowed MERS-CoV replication in mice, but resulted in lethal brain disease not representative of MERS-CoV infection in humans [76,77]. Replacing mouse DPP4 with the expression of human DPP4 in mice resulted in a humanized DPP4 mouse model that allows MERS-CoV replication within the lung and some MERS-associated lung pathology, but no lethal disease [78]. Similarly, knock-in expression of the human DPP4 exons 10–12 in mice allowed viral replication but no overt MERS disease signs [79]. Serial passage of MERS-CoV in the lungs of these mice resulted in a MERS-MA virus that caused lethal disease in mice, including weight loss and severe lung pathology [79]. Identification of amino acids in mouse DPP4 that prevent entry of MERS-CoV into mouse cells led to the rational design of the mouse DPP4 gene edited by CRISPR/Cas9 to express two human DPP4 mutations (288/330 DPP4) [80]. 288/330 DPP4 mice supported viral replication without severe disease or lethality, but serial passage of MERS-CoV (generating a mouse-adapted virus called MERS-15) produced lethal disease in the 288/330 DPP4 mice [80]. Although many of the same metrics of disease to MA15-SARS-CoV are available in the MERS-15 or MERS-MA models for drug discovery of coronavirus antivirals, an additional drawback is the requirement for both mouse-adapted virus and a modified rodent host. Despite these limitations, mouse models of adapted SARS-CoV and MERS-CoV are currently the best-developed models of highly pathogenic coronavirus infection available for pan-coronavirus drug discovery.
• 3.2. Primate models for pan-coronavirus discovery
  • • Small animal models have been more thoroughly developed as models of SARS-CoV and MERS-CoV infection, due to ease of manipulation with rodents and increased costs and ethical concerns associated with nonhuman primates (NHPs). However, NHP model development of highly pathogenic coronavirus infections is pivotal in the evaluation of pan-coronavirus therapeutics, because host immune responses from NHPs share greater homology with humans compared to rodents, and may more accurately indicate immunological biomarkers of severe disease needed to evaluate pan-coronavirus therapeutics. Disease signs are observed in NHP models of infection without adaptation of CoVs required in rodent models of SARS-CoV and MERS-CoV. Both SARS-CoV and MERS-CoV isolates from humans replicate in NHPs, indicating conservation of important aspects to coronavirus-induced diseases including respiratory tract biology, receptor homology, and pattern of expression of host receptor and proteases.
    • SARS-CoV infection of common laboratory primate species by the IT route including African green monkeys, rhesus macaques, cynomolgus macaques, and common marmosets, resulted in disease signs with differing degrees of severity, but none were reflective of the lethal SARS disease seen in humans (Table 2) [81–85]. Commonly reported disease signs included lethargy and increased respiratory rates following SARS-CoV infection in multiple NHP models of infection, but other acute signs of illness including fever or dyspnea were infrequently reported. The most severe disease phenotypes were observed in the histopathology of the lungs at acute times post-infection (3–6 days) with typical findings of pulmonary lesions and pneumonitis and occasional observations of diffuse alveolar damage [86]. Although none of the NHP species that were infected with SARS-CoV developed lethal respiratory disease reflective of SARS patients, NHP models did recapitulate enhanced disease in aged NHPs, including aberrant innate immune signaling programs [87]. However, lack of emulation of human SARS disease was never resolved in an NHP model that could be used for consistent evaluation of therapeutic candidates against SARS-CoV.
    • MERS-CoV infection of nonhuman primate models was reported, with the best characterized NHP models of MERS-CoV infection in rhesus macaques and common marmosets (Table 3) [88–91]. Administering MERS-CoV via the IT route to either rhesus macaques or common marmosets resulted in mild disease with very few observable disease phenotypes [89,91]. However, infecting rhesus macaques or common marmosets by multiple concurrent routes (IN, IT, oral, and ocular) resulted in moderate disease in rhesus macaques, but more severe disease in marmosets [88,90]. Infecting NHPs by multiple routes likely caused a systemic infection potentially not representative of human MERS-CoV infection. Although the marmoset is currently the best developed NHP model of MERS-CoV disease, discrepancies in disease severity of marmosets infected by multiple routes and marmosets infected by the IT route illuminate potential difficulties with using this model for drug development studies. Small size and fragility of marmosets precluded serial blood draws on multiple days following infection, and may confound experimental outcomes resulting from MERS-CoV infection or treatment [92]. Absence of reproducible clinical disease signs like fever, respiratory distress, or lethality that recapitulated human symptoms of SARS or MERS indicates that currently developed models present significant challenges to testing of pan-coronavirus antivirals in NHP models of infection.

4. Pan-coronavirus antivirals

• 4.1. Targeting CoV nonstructural proteins
  • • Coronavirus nsps are highly conserved components of the coronavirus lifecycle that mediate viral replication including 3C-like protease (3CLpro), papain-like Protease (PLpro), and RNA-dependent RNA polymerase (RdRp). The CoV RdRp replicates the viral RNA genome and generates viral RNA transcripts, essential functions that cannot be performed by cellular polymerases. Another essential element of the CoV lifecycle is proteolytic processing of viral polyproteins into functional nsps by two viral proteases, the 3CLpro and PLpro. In addition to polymerase and protease functions, other essential functions performed by the nsps of CoVs include immune antagonism, double membrane vesicle organization, scaffolding for replication complex formation, nucleic acid binding, helicase activity, and viral RNA proofreading which may be future targets of coronavirus specific antiviral drug discovery [93].
    • 4.1.1. GS-5734
      • • GS-5734 is a small molecule nucleoside analog that has demonstrated antiviral activity in vitro against several viral families of emerging infectious diseases including Filoviridae, Pneumoviridae, Paramyxoviridae, and Coronaviridae [45,66,94]. Efficacy of GS-5734 in post-exposure treatment of Ebola virus-infected nonhuman primates led to GS-5734 inclusion in an experimental therapy for an infant survivor of Ebola virus disease [45,95]. These encouraging results demonstrated that GS-5734 may be an acceptable therapeutic intervention to lethal viral disease, even days after viral exposure, and tolerated by patients with viral diseases that were previously treated primarily with supportive care. Based on activity against MERS-CoV within a larger panel targeting lethal viruses from multiple viral families, additional studies demonstrated that GS-5734 decreased viral titers and viral RNA in in vitro models of both SARS-CoV and MERS-CoV infection of HAEs [66]. Additionally, GS-5734 had similar effects against other diverse CoVs including HCoV-NL63 and Mouse Hepatitis Virus (MHV, betacoronavirus group 2a) [66,96]. Importantly, GS-5734 inhibited replication of pre-emergent BatCoVs including BatCoV-HKU5, BatCoV-HKU3, BatCoV-SHC014, and BatCoV-WIV1 [66]. Activity in vivo against CoVs was supported by ameliorated disease signs (weight loss, lung viral titers) in MA15 SARS-CoV infected mice treated prophylactically or therapeutically with GS-5734 [66]. Although viral resistance to GS-5734 was shown experimentally in vitro, mutations to conserved motifs in SARS-CoV and MHV resulted in decreased viral fitness in vitro and in vivo [96]. Altogether, in vitro and in vivo data support GS-5734 development as a potential pan-coronavirus antiviral based on results against several CoVs, including highly pathogenic CoVs and potentially emergent BatCoVs.
    • 4.1.2. Lopinavir–Ritonavir
      • • Lopinavir–ritonavir was initially developed as an HIV-1 protease inhibitor but in vitro activity also targeted SARS-CoV nonstructural protein 3CLpro [97]. During the SARS-CoV epidemic, lopinavir–ritonavir combination therapy with ribavirin in SARS patients was associated with decreased viral load and decreased adverse clinical outcomes of death or ARDS when compared with historical control cases [98]. Shortly after the emergence of MERS, high throughput screening approaches of known antiviral compounds identified lopinavir activity against MERS-CoV in vitro [51]. Oral treatment with lopinavir-ritonavir in the marmoset model of MERS-CoV infection resulted in modest improvements in MERS disease signs, including decreased pulmonary infiltrates identified by chest x-ray, decreased interstitial pneumonia, and decreased weight loss [92]. MERS patient case reports of treatment regimens including lopinavir-ritonavir were associated with positive disease outcomes including defervescence, viral clearance from serum and sputum, and