P-1865. Immunotherapy with Nebulized Toll-Like Receptor Agonists Restores Severe Immune Paralysis and Improves Outcomes in Mice with Influenza-Associated Pulmonary Aspergillosis
Authors: Wurster Sebastian , García Jezreel Pantaleón , Albert Nathaniel D , Bhoda Keerthi , Wang Yongxing , Kontoyiannis Dimitrios P , Evans Scott 
Published: January 2025

Abstract

Background
Given the poor outcomes of influenza-associated pulmonary aspergillosis (IAPA), there is a major unmet need for innovative therapeutic strategies. Impairment of pathogen recognition receptor (PRR) signaling is a known pathomechanism and prognostic factor in IAPA patients. Therefore, we performed immunotherapy with nebulized PRR agonists in a murine IAPA model and studied its impact on infection outcomes and pulmonary immunopathology.

Timeline of experimental interventions. Abbreviations: AF = Aspergillus fumigatus, CA = cortisone acetate (10 mg intraperitoneally), IAV = nebulization with influenza A virus (7.5% of the 90% lethal dose), PBS = phosphate-buffered saline (mock therapy), P/O = Pam2/ODN, VPS = viral pneumonia score (0 = healthy – 12 = moribund).

Methods
8-week-old BALB/c mice were infected with influenza A virus (IAV, H3N2), immunosuppressed with cortisone acetate (CA) on days (d) 5 and 8 after IAV infection, and infected intranasally with 50,000 Aspergillus fumigatus (AF) conidia on d9 (Fig. 1). Mice received nebulized Pam2+ODN (P/O, 4 µM/1 µM) or phosphate-buffered saline (mock therapy) before and after AF super-infection (d8/d12). Therapeutic outcomes were assessed using a combined morbidity (viral pneumonia score ≥ 7/12) and mortality endpoint. On d14, nCounter-based transcriptomics with Ingenuity pathway enrichment analysis was performed on lung tissue, along with histopathology and flow cytometry.

Event-free survival (A; Mantel-Cox log-rank test) and median viral pneumonia scores of survivors (B) depending on the treatment arm.

Results
All mock-treated mice with IAPA reached the morbidity/mortality endpoint by d13 (Fig. 2). Compared to CA-immunosuppressed mice with IAV infection only (100% survival), nCounter analysis revealed minimal incremental inflammatory response and suppression of several PRR pathways, interferon signaling, and neutrophil activation in the IAPA group. P/O therapy led to 80% event-free survival until d21 (p &lt; 0.001 vs. mock therapy) and full recovery of all survivors (Fig. 2). Histopathology revealed significantly reduced fungal burden and hemorrhagic lesions in P/O-treated animals. nCounter analysis showed induction of PRR signaling, several key effector cytokine pathways, and epithelial signaling after P/O therapy. Moreover, nCounter and flow cytometry revealed enhanced recruitment of macrophages, natural killer cells, and T cells in P/O-treated mice (Fig. 3).

Summary of nCounter and flow cytometry results.

Conclusion
Consistent with previous studies in human IAPA patients, our data revealed severe immune paralysis in mice with IAPA. Immunotherapy with nebulized P/O strongly improved infection outcomes, enhanced mobilization of key immune effector cells, and re-invigorated PRR and cytokine signaling.

Disclosures
Sebastian Wurster, MD, MSc, Astellas Pharma: Grant/Research Support|Gilead Sciences: Grant/Research Support Dimitrios P. Kontoyiannis, MD, AbbVie: Advisor/Consultant|Astellas Pharma: Advisor/Consultant|Astellas Pharma: Grant/Research Support|Astellas Pharma: Honoraria|Cidara Therapeutics: Advisor/Consultant|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Gilead Sciences: Honoraria|Knight: Advisor/Consultant|Merck: Advisor/Consultant|Scynexis: Advisor/Consultant