Efficacy of a brain-penetrant antiviral in lethal Venezuelan and eastern equine encephalitis mouse models.

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× LD50 VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD50 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× LD50 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.

Cardiopulmonary Injury in the Syrian Hamster Model of COVID-19.

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.

Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infections During SARS-CoV-2.

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate infection with the SARS-CoV-2 USA-WA1/2020 strain increased the risk of pneumococcal (type 2 strain D39) coinfection in a time-dependent, but sex-independent, manner in the transgenic K18-hACE2 mouse model of COVID-19. Bacterial coinfection increased lethality when the bacteria was initiated at 5 or 7 d post-virus infection (pvi) but not at 3 d pvi. Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.

Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infection during SARS-CoV-2 Infection.

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate SARS-CoV-2 infection increased the risk of pneumococcal coinfection in a time-dependent, but sexindependent, manner in the transgenic K18-hACE mouse model of COVID-19. Bacterial coinfection was not established at 3 d post-virus, but increased lethality was observed when the bacteria was initiated at 5 or 7 d post-virus infection (pvi). Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.

Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes.

COVID-19 is characterized by excessive production of pro-inflammatory cytokines and acute lung damage associated with patient mortality. While multiple inflammatory cytokines are produced by innate immune cells during SARS-CoV-2 infection, we found that only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by inflammatory cell death, PANoptosis. Mechanistically, TNF-α and IFN-γ co-treatment activated the JAK/STAT1/IRF1 axis, inducing nitric oxide production and driving caspase-8/FADD-mediated PANoptosis. TNF-α and IFN-γ caused a lethal cytokine shock in mice that mirrors the tissue damage and inflammation of COVID-19, and inhibiting PANoptosis protected mice from this pathology and death. Furthermore, treating with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection, sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other infectious and autoinflammatory diseases by limiting tissue damage/inflammation.

Synergism of TNF-α and IFN-γ triggers inflammatory cell death, tissue damage, and mortality in SARS-CoV-2 infection and cytokine shock syndromes.

The COVID-19 pandemic has caused significant morbidity and mortality. Currently, there is a critical shortage of proven treatment options and an urgent need to understand the pathogenesis of multi-organ failure and lung damage. Cytokine storm is associated with severe inflammation and organ damage during COVID-19. However, a detailed molecular pathway defining this cytokine storm is lacking, and gaining mechanistic understanding of how SARS-CoV-2 elicits a hyperactive inflammatory response is critical to develop effective therapeutics. Of the multiple inflammatory cytokines produced by innate immune cells during SARS-CoV-2 infection, we found that the combined production of TNF-α and IFN-γ specifically induced inflammatory cell death, PANoptosis, characterized by gasdermin-mediated pyroptosis, caspase-8-mediated apoptosis, and MLKL-mediated necroptosis. Deletion of pyroptosis, apoptosis, or necroptosis mediators individually was not sufficient to protect against cell death. However, cells deficient in both RIPK3 and caspase-8 or RIPK3 and FADD were resistant to this cell death. Mechanistically, the JAK/STAT1/IRF1 axis activated by TNF-α and IFN-γ co-treatment induced iNOS for the production of nitric oxide. Pharmacological and genetic deletion of this pathway inhibited pyroptosis, apoptosis, and necroptosis in macrophages. Moreover, inhibition of PANoptosis protected mice from TNF-α and IFN-γ-induced lethal cytokine shock that mirrors the pathological symptoms of COVID-19. In vivo neutralization of both TNF-α and IFN-γ in multiple disease models associated with cytokine storm showed that this treatment provided substantial protection against not only SARS-CoV-2 infection, but also sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock models, demonstrating the broad physiological relevance of this mechanism. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other cytokine storm-driven syndromes by limiting inflammation and tissue damage. The findings also provide a molecular and mechanistic description for the term cytokine storm. Additionally, these results open new avenues for the treatment of other infectious and autoinflammatory diseases and cancers where TNF-α and IFN-γ synergism play key pathological roles.

Identification of Anti-tuberculosis Compounds From Aurone Analogs.

The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains has made tuberculosis (TB) control more difficult. Aurone derivatives have demonstrated promising anti-bacterial activities, but their effects against Mtb have not been thoroughly determined. In this study, we aimed to develop anti-TB compounds from aurone analogs. We used a fluorescent protein tdTomato labeled Mtb CDC1551 strain to screen 146 synthesized aurone derivatives for effective anti-TB compounds. The 9504, 9505, 9501, 9510, AA2A, and AA8 aurones inhibited the growth of Mtb with minimal inhibitory concentrations of 6.25, 12.5, 25, 25, 25, and 50 µM, respectively. We also examined cytotoxicities of the six leads against the human liver cell line HepG2, the primate kidney cell line Vero and human monocyte THP-1 derived macrophages. Three of the aurone leads (9504, 9501, and 9510) showed low cytotoxic effects on all three cell lines and high Mtb inhibitory efficacy (selectivity index > 10). Aurone 9504, 9501, AA2A, or AA8 significantly reduced the Mtb load in the lungs of infected mice after a 12-days treatment. We determined that the aurone leads inhibit Mtb chorismate synthase, an essential enzyme for aromatic acid synthesis. Our studies demonstrate the promise of synthetic aurones as novel anti-TB therapeutics.

Mycobacterium tuberculosis LipE Has a Lipase/Esterase Activity and Is Important for Intracellular Growth and In Vivo Infection.

Mycobacterium tuberculosis Rv3775 (LipE) was annotated as a putative lipase. However, its lipase activity has never been characterized, and its precise role in tuberculosis (TB) pathogenesis has not been thoroughly studied to date. We overexpressed and purified the recombinant LipE (rLipE) protein and demonstrated that LipE has a lipase/esterase activity. rLipE prefers medium-chain ester substrates, with the maximal activity on hexanoate. Its activity is the highest at 40°C and pH 9. We determined that rLipE hydrolyzes trioctanoate. Using site-directed mutagenesis, we confirmed that the predicted putative activity triad residues Ser97, Gly342, and His363 are essential for the lipase activity of rLipE. The expression of the lipE gene was induced under stressed conditions mimicking M. tuberculosis' intracellular niche. The gene-disrupting mutation of lipE led to significantly reduced bacterial growth inside THP-1 cells and human peripheral blood mononuclear cell-derived macrophages and attenuated M. tuberculosis infection in mice (with ∼8-fold bacterial load reduction in mouse lungs). Our data suggest that LipE functions as a lipase and is important for M. tuberculosis intracellular growth and in vivo infection.

Rv1075c of Mycobacterium tuberculosis is a GDSL-Like Esterase and Is Important for Intracellular Survival.

Mycobacterium tuberculosis lipid metabolism pathways facilitate access to carbon and energy sources during infection. M. tuberculosis gene Rv1075c was annotated as a conserved hypothetical protein. We identified that Rv1075c amino acid sequence shares similarities with other bacterial lipase/esterases and we demonstrated that it has esterase activity, with preference for short-chain fatty acids, particularly acetate, with highest activity at 45°C, pH 9. Site-direct mutagenesis revealed its activity triad as Ser80, Asp244, and His247. We further determined that rRv1075c hydrolyzed triacetin and tributyrin, and it was mainly distributed in cell wall and membrane. Its expression was induced at pH 4.5, mimicking the acidic phagosome of macrophages. Mutation of Rv1075c led to reduced bacterial growth in THP-1 cells and human peripheral blood mononuclear cell-derived macrophages, and attenuated M. tuberculosis infection in mice. Our data suggest that Rv1075c is involved in ester and fatty acid metabolism inside host cells.

Molecular basis for T cell response induced by altered peptide ligand of type II collagen.

Mounting evidence from animal models has demonstrated that alterations in peptide-MHC interactions with the T cell receptor (TCR) can lead to dramatically different T cell outcomes. We have developed an altered peptide ligand of type II collagen, referred to as A9, which differentially regulates TCR signaling in murine T cells leading to suppression of arthritis in the experimental model of collagen-induced arthritis. This study delineates the T cell signaling pathway used by T cells stimulated by the A9·I-A(q) complex. We have found that T cells activated by A9 bypass the requirement for Zap-70 and CD3-ζ and signal via FcRγ and Syk. Using collagen-specific T cell hybridomas engineered to overexpress either Syk, Zap-70, TCR-FcRγ, or CD3-ζ, we demonstrate that A9·I-A(q) preferentially activates FcRγ/Syk but not CD3-ζ/Zap-70. Moreover, a genetic absence of Syk or FcRγ significantly reduces the altered peptide ligand induction of the nuclear factor GATA3. By dissecting the molecular mechanism of A9-induced T cell signaling we have defined a new alternate pathway that is dependent upon FcRγ and Syk to secrete immunoregulatory cytokines. Given the interest in using Syk inhibitors to treat patients with rheumatoid arthritis, understanding this pathway may be critical for the proper application of this therapy.

Visualization of murine intranasal dosing efficiency using luminescent Francisella tularensis: effect of instillation volume and form of anesthesia.

Intranasal instillation is a widely used procedure for pneumonic delivery of drugs, vaccine candidates, or infectious agents into the respiratory tract of research mice. However, there is a paucity of published literature describing the efficiency of this delivery technique. In this report we have used the murine model of tularemia, with Francisella tularensis live vaccine strain (FTLVS) infection, to evaluate the efficiency of pneumonic delivery via intranasal dosing performed either with differing instillation volumes or different types of anesthesia. FTLVS was rendered luminescent via transformation with a reporter plasmid that constitutively expressed the Photorhabdus luminescens lux operon from a Francisella promoter. We then used an IVIS Spectrum whole animal imaging system to visualize FT dissemination at various time points following intranasal instillation. We found that instillation of FT in a dose volume of 10 µl routinely resulted in infection of the upper airways but failed to initiate infection of the pulmonary compartment. Efficient delivery of FT into the lungs via intranasal instillation required a dose volume of 50 µl or more. These studies also demonstrated that intranasal instillation was significantly more efficient for pneumonic delivery of FTLVS in mice that had been anesthetized with inhaled (isoflurane) vs. parenteral (ketamine/xylazine) anesthesia. The collective results underscore the need for researchers to consider both the dose volume and the anesthesia type when either performing pneumonic delivery via intranasal instillation, or when comparing studies that employed this technique.