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 Aug. 25 and Sept. 1, investigators from the tested nearly 3,000 Jefferson County residents for the to detect the presence of the virus in participants’ nasal swabs by the polymerase chain reaction (PCR) method and for the presence of antibodies against the virus in their blood.

The results showed that approximately 1.1% of all the participants tested positive for active coronavirus infections. Among vaccinated participants only 0.7% had an active infection, while nearly 5% of unvaccinated participants were actively infected. This number would roughly correspond to 7,260 active infections in the county, a nearly tenfold increase in infection rates over the rates measured in April, despite a sharp increase in vaccinated residents, as shown in Figure 1.

As in previous testing rounds, the team also tested for antibodies in participants. They found that independent of their vaccination status, in both the sampled and volunteer groups, nearly 16% of the participants had natural infection antibodies against the virus suggesting that they must have been infected by the virus in the recent past. These data indicate that in the last few months, at least 100,000 adults in Jefferson County have had COVID-19.

“These results highlight the steep rise in coronavirus infections in our community and provide a startling snapshot of the current state of the pandemic,” said Aruni Bhatnagar, director of the Envirome Institute. “Our estimates suggest that the number of infected individuals may be twice as high as that indicated in public records.

“Our work shows the vaccine is working as intended. Our population was almost 90% vaccinated, much higher than the 64% of fully vaccinated county residents. In the entire cohort, vaccinated people were over 12 times less likely to be infected compared with unvaccinated people. Though in our volunteer group, 65% of the active infections were in fully vaccinated individuals who had received the vaccine earlier this year. Most reported no or mild allergy-like symptoms and did not recognize that it may be a COVID infection thus did not think they needed to get tested.”

The study also provided estimates of where in Jefferson County the infections are most prevalent. To identify infection rates in different areas, the researchers classified the participants into neighborhood zones, as shown in Figure 2.

The highest rate of active infection was found in Zone 3A, or far southwestern Jefferson County. The highest rate of those recently having had an infection was found in Zone 3B, central southern Jefferson County, as shown in Figure 3.

Participants from Zone 3B also reported lower rates of vaccination, although vaccination rates were lowest in Zone 1B in western Jefferson County, as shown in Figure 4.

“Even though nearly 90% of the participants in the entire study population were vaccinated, we had areas that reported as low as 60% vaccination, and the persistence of infection in some geographical areas seems to be related to lower rates of reported vaccination,” said Rachel Keith, associate professor of environmental medicine at UofL who conducted the study. “Our results show that much work remains to be done and that knowing that rates of infection are high in their community may be an added incentive for some individuals to get vaccinated.

“Additionally, knowing that fully vaccinated individuals may still get an active infection allows those individuals to take additional precautions such as masking or testing which helps keep the community safe, including any young or immunocompromised friends and families who may need extra protection.”

“The vaccine is very effective. Nearly 96% of vaccinated individuals had detectable levels of antibodies against the virus in their blood. However, in a small number of people (less than 0.6%) the levels of antibodies were undetectable in our assay, even though these individuals were fully vaccinated,” Keith said. “The lack of a measurable response in some individuals even after vaccination may be due to their health and immune status. We are analyzing our results to find out more about why some rare individuals do not develop high antibody levels in response to vaccination.”

Using the data from more than 7,000 individuals tested over the past year, the team is trying to identify personal and environmental characteristics that increase the risk for coronavirus infection and how vaccination reduces this risk.

For this round of testing, the team collected samples at 13 locations across Jefferson County. Active coronavirus infections were analyzed by Bluewater Labs and antibodies against the virus were assayed at at the .

To randomly sample people from all parts of the city and to include proportional number of individuals of different age and race/ethnicity, researchers at UofL partnered with Westat to mail approximately 30,000 letters asking people to participate in the study. Nearly 1,000 people who responded to this invitation were tested and an additional 1,886 booked their own appointments after hearing about the study in the news or on social media.

This study was supported in part by a contract with the Centers for Disease Control and Prevention.

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.

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“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.

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.”