Chung, associate professor in the UofL Department of Microbiology and Immunology and the , and colleagues at the University of Wisconsin–Madison’s School of Pharmacy and the University of Tennessee Health Sciences Center (UTHSC) described their work with BDGR-49 in a in Science Translational Medicine.

ChungÌęled chemical virology studies, Jennifer E. Golden, associate professor in the UW–Madison School of Pharmacy and synthetic medicinal chemist, led the discovery and optimization effort and Colleen Jonsson, a professor at UTHSC, performed animal efficacy studies.

The team found that BDGR-49 potently inhibited EEEV and VEEV and was well tolerated. The compound provided significant protection to EEEV-infected animals. Meanwhile, it not only fully protected VEEV-infected animals, but could also be used as a therapeutic treatment days after infection.

An important feature of this antiviral compound is its ability to access the brain where these viruses cause damage, while other critical attributes include its improved stability, potency and efficacy compared to earlier prototypes. Based on resistance studies, BDGR-49 efficiently prevents these viruses from copying themselves, meaning it operates by disrupting the viral machinery needed for replication.Ìę

Classified as New World alphaviruses, equine encephalitis viruses are transmitted by the bite of a mosquito and can infect the brain, causing neurological effects, serious illness and death in humans as well as horses. There currently are no FDA-approved vaccines or treatments available specifically for preventing or treating alphavirus infection in humans.

Symptoms of EEEV infection include fever, headache, chills and vomiting. Severe infection can result in seizure, coma and death. About one-third of individuals who develop encephalitis (brain inflammation) from EEEV infection die, and many of those who do recover suffer permanent neurological effects.

Although outbreaks of eastern equine encephalitis (EEE) are rare, with an average of 11 cases per year in the United States, a 2019 outbreak of EEE across nine states resulted in 38 confirmed cases, 19 deaths and neurological effects in survivors.

VEE has a much lower mortality rate of 1%, but outbreaks can affect thousands of people, most often occurring in Central and South America. While insect bites are the typical cause of these infections, there is also concern the viruses could be leveraged as bioweapons.

“What we are trying to do is to develop a drug that can be used to treat infected people or as a prophylactic in case of bioterrorism,” Chung said. “Now we are seeing that it protects from lethal infection. This is a big milestone in terms of the therapeutic development.”

Chung, Golden and Jonsson have been developing chemical structures against VEEV and EEEV for more than a decade. They are co-investigators in the Center of Excellence for Encephalitic Alphavirus Therapeutics, based at UTHSC. The center was created to refine the properties and activity of early-stage small molecule compounds discovered in the Golden lab and to develop them into clinical candidates for VEEV and EEEV that could be studied in humans. This work received a five-year, $21-million grant from the National Institutes of Health in 2019.

The team now is evaluating BDGR-49 in advanced preclinical studies and expanding the understanding of its antiviral properties. As RNA viruses such as EEEV and VEEV are prone to develop mutations, they can potentially evolve into more lethal or transmissible versions without warning, resulting in widespread infections.

“It is essential that we develop these countermeasures for viruses of pandemic potential so we don’t find ourselves unprepared to respond to an outbreak,” Golden says. “We can do better, and we intend to leverage this discovery as broadly as possible with respect to VEEV, EEEV and other viruses of concern.”

This research was supported by the National Institute of Allergy and Infectious Diseases (U19AI142762 and R01AI118814) and by a grant (S10OD016226) from the Office of the Director of the NIH.

Between Nov. 9 and 16, researchers at the tested samples from 2,800 individuals representing all parts of Jefferson County for both active infection and antibodies, indicating previous infection. From those test results, project researchers estimate that during these dates, 1 in 50 Louisville residents were infected and that the rates of infection were nearly five times higher than the publicly reported number of cases, estimated at 0.4% of the population.

“At this rate, as many as 13,000 Louisville residents likely are infected today, many of them asymptomatic and who unwittingly may be spreading the virus,” said Aruni Bhatnagar, director of the institute. “These rates are startling and should make every person living in Louisville re-evaluate their personal precautions to avoid coronavirus, especially as we approach the holidays.”

Other key findings from the project’s latest round of testing:

• Antibody testing indicates a 150% increase in antibody presence compared to documented cases.
• Nearly 13,000 Louisville residents likely were infected between September and November.
• About 45,000 people in Louisville likely have had a coronavirus infection at some point since the beginning of the pandemic based on antibody testing.
• Shively and Northeastern Jefferson County currently show the highest rates of infection in the city.

Benefits of representative sampling

The is a series of studies to estimate the true prevalence of SARS-CoV-2, the virus causing COVID-19, in Jefferson County. This phase of the project involves testing a representative sample of individuals from different areas in the city in proportion to the age and race of the population of the area. Researchers say this approach provides a more reliable estimate of the breadth and spread of coronavirus infection in different parts of the city than testing only those who have reason to believe they may have the virus. The team tested its first community sample in June, a second in September and the most recent in November.

In addition to the 2% infection rate among randomized participants, individuals who participated without an invitation showed a 3.3% rate of infection. This is higher than the random sampling because individuals self-selecting for testing are more likely to have been exposed to the virus.

“Most of the individuals we identified as having coronavirus infection did not have overt symptoms, which indicates that a large number of cases are likely to remain undetected,” said Rachel Keith, assistant professor of environmental medicine at UofL, who conducted the study. “We do not know for sure, but it seems likely that the recent increase in infections may be in part due to asymptomatic individuals.”

The project also tested for antibodies against the virus and found a one-and-a-half-fold increase in the number of individuals who previously had been exposed to the virus. Study researchers estimate that by Nov. 20, more than 45,000 individuals had been infected by the virus, rather than the 20,500 known cases documented so far. These data also suggest that approximately 15,000 individuals became infected between September and November.

Ìę

“One reason for the recent increase in coronavirus infection may be the recent drop in temperature,” Bhatnagar said. “ shows that low temperatures promote the spread of the virus. Hence, we were expecting the rates of infection to rise in winter, but this increase is much more than we thought.

“Unfortunately, things are likely to get much worse in the coming months as temperatures dip even further. Therefore, we urgently need collective action, maybe just for a few months more. An effective vaccine is on the horizon so it seems that there is clear hope ahead that might hearten us to make the necessary sacrifices for a little longer.”

In an effort to obtain a uniform sample of city residents, investigators at the institute mailed 30,000 letters to households across Louisville for the November round of testing. The invitations were sent to individuals selected using addresses derived fromÌęU.S. Census Bureau tract boundaries in proportion to the total population in each geographic area.

In addition, any adult resident of Jefferson County was invited to participate through news and social media messages.

A total of 2,800 individuals were tested, 1,091 in response to the invitations and an additional 1,709 who booked their own appointments. The testing took place at 10 community drive-up or walk-up locations. Participants were tested both for the presence of the virus in participants’ nasal swabs and for antibodies against the virus in their blood, indicating a previous infection. Samples were analyzed at UofL’s Regional Biocontainment Laboratory (RBL) by assistant professor Krystal Hamorsky, and Amanda Lasnik, at the Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases.

The random sampling of different neighborhoods also allowed the team to identify areas with higher prevalence of infection. Although infections were spread throughout the county, the highest rates were in the Shively area as well as northeastern Jefferson County.

The researchers are planning to conduct a fourth round of randomized coronavirus testing in Jefferson County Dec. 9-14.

This study was supported in part by the City of Louisville, the James Graham Brown Foundation, the Owsley Brown Family Foundation, Foundation for a Healthy Kentucky and others.

The technology is based on a piece of synthetic DNA – an “aptamer” – Ìęwhich targets and binds with a human protein called nucleolin. Early tests show that this aptamer may stop viruses, including novel coronavirus, from “hijacking” nucleolin to replicate inside the body.ÌęÌę

UofL is seeking to fast-track development, including application to the Food and Drug Administration for approval to start treating patients seriously affected with COVID-19.ÌęÌę

The aptamer was discovered by UofL’s Paula Bates, John Trent and Don Miller, who have applied it in a variety of ways, most notably as a potential therapeutic drug against multiple types of cancer.ÌęWith the current global pandemic of coronavirus and the COVID-19 disease it causes, Bates partnered with fellow researcher Kenneth Palmer to apply the technology once again.

“Like many scientists, as soon as I heard about the new coronavirus, I wanted to help and started to think about how my area of research might intersect with coronavirus research efforts,” said Bates, a professor of medicine. “I am fortunate to be at UofL, which is one of the few places in the country where we have the facilities to do experiments using the SARS-CoV-2 virus.”

Palmer, director of UofL’s Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases (CPM), conducted proof-of-concept experiments showing the aptamer was effective against the virus at doses previous research has shown to be safe in patients. Palmer also is working on another potential COVID-19 treatment, Q-Griffithsin, developed at UofL in partnership with the National Cancer Institute and the University of Pittsburgh.Ìę

The CPM houses UofL’s Regional Biocontainment Laboratory, one of only 12 regional and two national biocontainment labs in the United States and the only one in Kentucky. Established with support from the NIH to conduct research with infectious agents, the lab includes Biosafety Level 3 facilities built to the most exacting federal safety and security standards. The stringently secure facilities protect researchers and the public from exposure to the pathogens being investigated.Ìę

UofL is providing financial support for COVID-19 research, but additional funds are needed to continue the work over time. Donations specifically for the research .

The center, headquartered at the University of Tennessee Health Science Center in Memphis, will develop therapeutic drugs to treat three mosquito-borne alphaviruses that cause serious illness in humans and horses: Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV). Although a vaccine is available for horses, there are no FDA-approved treatments or preventive vaccines for these viruses in humans.

Chung, an associate professor in the and Department of Microbiology & Immunology at UofL, said the center’s goal is to refine potent small molecule compounds the researchers previously identified as promising as antiviral drugs for treating the encephalitis viruses, and enable those compounds to move to the next step of research, clinical trials in humans.

“In my previous research I found that these compounds inhibit the viral replication cycle. However, we want to further understand which target molecules are interacting with the compound,” Chung said. He described the mechanism of the compounds the group is investigating as similar to the way the drug nevirapine, a non-nucleoside reverse transcriptase inhibitor, works in treating human immunodeficiency virus (HIV).

“This project is to develop a new antiviral compound to treat these diseases in humans,” Chung said. “However, we do not limit our applications of this drug to only humans, as it may be possible to adapt it for treating horses as well.”

These equine encephalitis viruses infect humans and horses through the bite of an infected mosquito. According to the Centers for Disease Control and Prevention, symptoms of infection include fever, chills, headache and vomiting. Outbreaks of the Western and Venezuelan viruses are uncommon, and an average of only seven human cases of have been reported annually in the United States throughout the past ten years. However, the disease may leave people with permanent neurological symptoms, and approximately 30% of people who contract the Eastern equine encephalitis virus will die from the disease.

According to Chung, the potential for the viruses to be used in bioterrorism is perhaps even more worrisome. The CDC recognizes viral encephalitis as a Category B human biothreat, making the development of a treatment important.

Chung’s co-principal investigators in the center are Colleen Jonsson, PhD, of the University of Tennessee Health Science Center who directs the center, and Jennifer E. Golden, PhD, of the University of Wisconsin-Madison School of Pharmacy.

“The goal of our new Center of Excellence is to further develop novel therapeutic molecules discovered by our team that are highly potent across all three viruses, moving the optimal ones forward into pre-clinical development,” said Jonsson, formerly director of the UofL Center for Predictive Medicine.

Chung will test the compounds for toxicity against human cells in vitro, deciphering their molecular mechanisms and determining their resistance threshold utilizing the facilities and staff at the at UofL. He also will test the compounds’ resistance threshold that inevitably develops over time, along with Juw Won Park, PhD, assistant professor of computer engineering in the at UofL.

Once their work is completed, the researchers expect the new drugs would be ready to begin clinical trial testing in humans.

Researchers at the University of Louisville Ìęare working at the forefront in combating these pathogens. The CPM has been testing the effectiveness of new drugs against P. aeruginosa under a contract with the National Institutes of Health since 2013, and a new contract from the U.S. Food and Drug Administration will expand the center’s work in testing new drugs against this pathogen. Under the new two-year, $933,606 contract, CPM will develop a validated model for screening antimicrobial drugs against P. aeruginosa.

“This model likely will play an important role in drug development pipelines leading to identification of new antimicrobial drugs,” said Matthew Lawrenz, PhD, associate professor of microbiology and immunology who is leading the research. “Researchers at UofL and from around the world will use the model to screen new antimicrobials against multi-drug resistant bacteria prior to clinical trials.”

Forest Arnold, DO, hospital epidemiologist for UofL Hospital and associate professor in the Division of Infectious Diseases in the UofL School of Medicine, said multi-drug resistant bacteria and XDR bacteria, those with resistance to all existing antibiotics, are evolving faster than the drugs to kill them.

“The germs get smarter as we make new drugs. If we are going to stay on top of them, we need new antibiotics, especially new classes of antibiotics — those with a new mechanism of action that the germ hasn’t seen before,” Arnold said.

Infections with MDR bacteria are particularly threatening for patients with weakened immune systems, those who have had multiple rounds of treatment with antibiotics, and in patients using devices such as ventilators and blood catheters. Since these bacteria are now resistant to many of the antibiotic drugs used to treat them, they can lead to severe infections and death.

“If you have an infection with a bacterium we don’t have an antibiotic to treat, it could kill you,” Arnold said.

P. aeruginosa is common in the environment and in otherwise healthy people, it may cause relatively minor of the ear, skin or eye. However, in people with weakened immune systems or in hospital settings, P. aeruginosa can cause serious, life-threatening infections of the blood, lungs, digestive tract or tissue. Infected wounds will have a green pus or discharge and a fruity smell.

The validated animal model, to be developed by UofL researchers with collaborators at the University of Kentucky and the University of Wisconsin, will be used to test new compounds developed by drug companies and research labs around the world against P. aeruginosa. This model will allow testing against multiple strains of pseudomonas and will give more detailed information about the effectiveness of the drugs being tested.

“The previous methods we used for testing the drugs provided basic information about a compound’s effectiveness. This new model will allow us to test anything from older classes of antibiotics to brand new classes and will provide information on dosing and scheduling. In addition, we will be able to test different strategies, such as immunomodulation – targeting the host to better respond to the infection as opposed to directly killing the bacteria,” Lawrenz said.

The CPM’s new contract with the FDA will take advantage of the sophisticated resources at the , located on the UofL ShelbyHurst Campus, which provide the environment necessary for this work.

“This new contract from the Food and Drug Administration supports the development of a model for understanding how bacteria build resistance to current commercially available antibiotics, which in turn, will lead to the discovery of new drugs or methods to combat a variety of infectious diseases,” said Robert Keynton, PhD, interim executive vice president for research and innovation at UofL. “The UofL Center for Predictive Medicine and the Regional Biocontainment Laboratory represents a significant investment in infrastructure, faculty and staff by the universityÌęin the field of emerging infectious diseases, whichÌęis one of our research and training strategic priorities.”