124. Establishment of a Post-Influenza Aspergillosis Model in Corticosteroid-Immunosuppressed Mice

Abstract Background Post-influenza aspergillosis (PIA) is a feared complication in patients with severe influenza, especially those receiving corticosteroids. However, validated murine models of PIA in a background of corticosteroid immunosuppression are lacking, compounding efforts to better characterize the immunopathology and treatment of this emerging entity. Methods 8-week-old female BALB/c mice were infected with ~5% of the lethal dose of a mouse-adapted influenza A/Hong Kong/1968 (H3N2) strain (flu), delivered by aerosolization, versus control (aerosolized saline). Mice then received two intraperitoneal injections of 10 mg cortisone acetate (CA) or mock injections on days 5 and 8 after flu infection. On day 9, mice were intranasally challenged with 50,000 A. fumigatus AF-293 conidia or mock-infected with saline. Survival was monitored until day 16 and infection severity was scored using the modified murine sepsis score (MSS, 0 = healthy to 3 = moribund). Pulmonary fungal burden was determined by an 18S quantitative PCR assay on day 16 or upon death. 15-16 mice per group were assessed across 3 independent experiments. Results Flu infection alone caused modest early morbidity, followed by full recovery of the mice until day 16. Treatment with CA after flu infection led to 12% mortality and increased morbidity that persisted until day 16 (median MSS = 0.8). Similarly, mice infected with AF after CA treatment had 12% mortality and a median MSS of 0.7. Combination of all 3 challenges (flu, CA, and AF) led to 40% mortality and severe morbidity in surviving mice (median MSS = 2.7). Likewise, prior flu infection of CA-treated, AF-infected mice increased the pulmonary fungal burden from 27k to 80k median conidial equivalents. In contrast, mice not receiving CA treatment showed consistently low morbidity (median day-16 MSS = 0.5) and minimal fungal burden after AF challenge, regardless of prior flu infection. Conclusion We have established a model of PIA in CA-immunosuppressed mice that underscores the detrimental effect of corticosteroid therapy on the outcomes of PIA. In the future, we will employ this model to study the impact of various pharmacological interventions on the natural history of PIA in the background of corticosteroid immunosuppression. Disclosures Dimitrios P. Kontoyiannis, MD, Astellas (Consultant)Cidara Therapeutics (Advisor or Review Panel member)Gilead Sciences (Consultant, Grant/Research Support, Other Financial or Material Support, Honoraria)


Support)Melinta
Background. Post-influenza aspergillosis (PIA) is a feared complication in patients with severe influenza, especially those receiving corticosteroids. However, validated murine models of PIA in a background of corticosteroid immunosuppression are lacking, compounding efforts to better characterize the immunopathology and treatment of this emerging entity.
Methods. 8-week-old female BALB/c mice were infected with ~5% of the lethal dose of a mouse-adapted influenza A/Hong Kong/1968 (H3N2) strain (flu), delivered by aerosolization, versus control (aerosolized saline). Mice then received two intraperitoneal injections of 10 mg cortisone acetate (CA) or mock injections on days 5 and 8 after flu infection. On day 9, mice were intranasally challenged with 50,000 A. fumigatus AF-293 conidia or mock-infected with saline. Survival was monitored until day 16 and infection severity was scored using the modified murine sepsis score (MSS, 0 = healthy to 3 = moribund). Pulmonary fungal burden was determined by an 18S quantitative PCR assay on day 16 or upon death. 15-16 mice per group were assessed across 3 independent experiments.
Results. Flu infection alone caused modest early morbidity, followed by full recovery of the mice until day 16. Treatment with CA after flu infection led to 12% mortality and increased morbidity that persisted until day 16 (median MSS = 0.8). Similarly, mice infected with AF after CA treatment had 12% mortality and a median MSS of 0.7. Combination of all 3 challenges (flu, CA, and AF) led to 40% mortality and severe morbidity in surviving mice (median MSS = 2.7). Likewise, prior flu infection of CA-treated, AF-infected mice increased the pulmonary fungal burden from 27k to 80k median conidial equivalents. In contrast, mice not receiving CA treatment showed consistently low morbidity (median day-16 MSS = 0.5) and minimal fungal burden after AF challenge, regardless of prior flu infection.

Conclusion.
We have established a model of PIA in CA-immunosuppressed mice that underscores the detrimental effect of corticosteroid therapy on the outcomes of PIA. In the future, we will employ this model to study the impact of various pharmacological interventions on the natural history of PIA in the background of corticosteroid immunosuppression.

Session: O-26. New Insights into Microbial Pathogenesis
Background. Rifampin, has potent, dose-dependent sterilizing activity against Gram-positive bacteria with the area under the concentration-time curve (AUC) being the most predictive of bactericidal activity. Combination therapy with rifampin (10-15 mg/kg/day) is recommended to treat S. aureus orthopedic implant associated infections. Recently, high-dose rifampin (35 mg/kg/day) has been shown to be safe in humans and is being evaluated to shorten tuberculosis treatments.
Methods. Dynamic 11 C-rifampin (chemically identical to rifampin) positron emission tomography (PET) was used to determine rifampin bone exposures (AUC) in mice and patients with S. aureus bone infection (Figure 1). PET data facilitated a pharmacokinetic model to predict rifampin concentration-time profiles in bone tissues, which were used to design studies in a mouse model of S. aureus orthopedic implant infection utilizing advanced imaging. (A) A validated mouse model of S. aureus orthopedic implant associated infection was used to determine bone concentrations following administration of escalating rifampin oral dosing (human equipotent dosing is indicated). m/z, mass/charge ratio. (B) Human patients and mice with S. aureus orthopedic infection were imaged using 11C-rifampin PET / CT. PET signal was quantified in infected and uninfected bone to generate time-activity curves (TACs) used to calculate area under the concentration time curve (AUC) over 45-90 minutes. These data were used to develop a pharmacokinetic model to predict rifampin exposures in bone. (C) Efficacy studies in mice compared vancomycin alone to vancomycin with either standard-dose or high-dose rifampin. Readouts included weekly in vivo bioluminescence, bacterial load and broth cultures at completion of follow up and CT to evaluate adverse bone remodeling. Bacterial isolates were evaluated for phenotypic resistance as well as subjected to whole genome sequencing. Human studies were approved by the Johns Hopkins University Institutional Review Board Committee. Patients were prospectively recruited at the Johns Hopkins Hospital and 11C-rifampin PET was performed under the U.S. FDA Radioactive Drug Research Committee program guidelines for investigational drugs. Animal studies were approved by the Johns Hopkins Animal Care and Use Committee. Approvals from the Johns Hopkins Biosafety, and Radiation Safety were also obtained for all studies.