Jennifer Stabenow

Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV, respectively) are mosquito-borne, neuroinvasive human pathogens for which no FDA-approved therapeutic exists. Besides the biothreat posed by these viruses when aerosolized, arthropod transmission presents serious health risks to humans, as demonstrated by the 2019 outbreak of EEE disease in the United States that resulted in 38 confirmed cases, 19 deaths, and neurological effects in survivors. Here, we describe the discovery of a 2-pyrrolidinoquinazolinone scaffold, efficiently synthesized in two to five steps, whose structural optimization resulted in profound antiviral activity. The lead quinazolinone, BDGR-49, potently reduced cellular VEEV and EEEV titers by >7 log at 1 μM and exhibited suitable intravenous and oral pharmacokinetic profiles in BALB/c mice to achieve excellent brain exposure. Outstanding in vivo efficacy was observed in several lethal, subcutaneous infection mouse models using an 8-day dosing regimen. Prophylactically administered BDGR-49 at 25 mg kg ⁻¹ per day fully protected against a 10× LD ₅₀ VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD ₅₀ EEEV FL93-939–infected C57BL/6 mice were treated prophylactically with BDGR-49 at 50 mg kg ⁻¹ per day. Last, we observed 100% therapeutic efficacy when mice, challenged with 10× LD ₅₀ VEEV TrD, were dosed at 48 hours after infection with BDGR-49 at 25 mg kg ⁻¹ per day. Mouse brain viral titers at 96 hours after infection were reduced to values near the limit of detection. Collectively, these results underscore the substantial development potential of a well-tolerated, brain-penetrant lead compound that shows promise in preventing and treating encephalitic alphavirus disease.

The Syrian hamster has proved useful in the evaluation of therapeutics and vaccines for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). To advance the model for preclinical studies, we conducted serial sacrifice of lungs, large pulmonary vessels, and hearts from male and female Syrian hamsters for days 1–4, and 8 post-infection (dpi) following infection with a high dose of SARS-CoV-2. Evaluation of microscopic lung histopathology scores suggests 4 and 8 dpi as prime indicators in the evaluation of moderate pathology with bronchial hyperplasia, alveolar involvement and bronchiolization being key assessments of lung disease and recovery, respectively. In addition, neutrophil levels, red blood cell count and hematocrit showed significant increases during early infection. We present histological evidence of severe damage to the pulmonary vasculature with extensive leukocyte transmigration and the loss of endothelial cells and tunica media. Our evidence of endothelial and inflammatory cell death in the pulmonary vessels suggests endothelialitis secondary to SARS-CoV-2 epithelial cell infection as a possible determinant of the pathological findings along with the host inflammatory response. Lastly, pathological examination of the heart revealed evidence for intracardiac platelet/fibrin aggregates in male and female hamsters on 8 dpi, which might be indicative of a hypercoagulative state in these animals.

Working with infectious agents that require BSL 3 level containment agents offers many challenges for researchers. BSL 3 containment laboratories are usually not equipped with expensive specialty equipment that is needed for studies such as flow cytometric analysis, microscopy, and proteomic analyses. Therefore, for most researchers that are working with BSL 3 level infectious agents, removal of samples from BSL 3 laboratories for these types of studies is necessary, and methods for complete and dependable inactivation of the samples are required. In this report, we have carried out a thorough characterization of the effectiveness of paraformaldehyde fixation for inactivation of cell samples infected with the intracellular bacterial agents Burkholderia pseudomallei (Bp) and Francisella tularensis (Ft), both of which are Tier 1 select agent pathogens that require BSL 3 containment. We have demonstrated that cells infected with these pathogens are completely inactivated via 5 min treatment with 4% paraformaldehyde. Moreover, a 15 min treatment with 2% paraformaldehyde completely sterilized both Bp and Ft infected cells. These studies also revealed that Bp is significantly more sensitive to paraformaldehyde treatment than Ft. Our findings have clearly demonstrated that a 15 min treatment of Bp or Ft infected cells with 4% paraformaldehyde solution will allow for safe removal of the cell samples from BSL 3 laboratories for downstream studies.