717. Susceptibility to Cryptococcosis in Bruton's Tyrosine Kinase Knockout Model
Authors: Messina Julia A , Messina Julia A , Giamberardino Charles D , Tenor Jennifer , Toffaletti Dena , Schell Wiley , Lionakis Michail , Perfect John R 
Published: December 2021

Abstract

Background
Patients receiving the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib have an increased likelihood of systemic and central nervous system (CNS) fungal infections. Previous work has shown that BTK knockout (KO) mice have more severe Aspergillus infection compared to wild-type (WT) mice. We sought to determine: 1) if blocking BTK impacted Cryptococcus infection; 2) if the effect was strain-dependent; and 3) if the blood-brain barrier was impaired in BTK KO mice.

Methods
BTK KO C57 breeding pairs were obtained from Dr. Lionakis (NIH) and expanded in the Duke Breeding Core. We collected 4 clinical C. neoformans isolates from patients who developed cryptococcosis on ibrutinib and used virulent and avirulent control strains (H99 and A1-35-8, respectively). The following doses were used for infection: 1) 5x104 yeast for intranasal (IN); 2) 5x104 for oropharyngeal aspiration (OPA); and 3) 0.1 mL of 5x105 CFU/mL for tail vein injection. Mice were sacrificed on day 7 (IN infection, 6 infection strains; N=35 WT, 80 KO) and day 14 post-infection (OPA infection H99 only; N=15 WT, 20 KO). Lung and brain tissues were collected for yeast census. For tail vein injection, mice were sacrificed 48 hours post-infection (H99 only; N=10 WT, 8 KO). Yeast census was measured by colony forming units (CFUs) per gram of tissue weight. Survival experiments through day 28 were performed for OPA infection with H99 (N=12 WT, 17 KO) and analyzed by Kaplan Meier Curve.

Results
We observed no difference in infection severity as measured by lung and brain yeast census at days 7 and 14 post-infection or difference in survival between BTK KO and WT mice (Figure 1). We also did not observe a distinct pattern based on Cryptococcus strain to suggest that infection severity was strain-dependent (Figure 1A-B). For tail vein infection, there was no difference in brain yeast census at 48 hours post-infection (Figure 1F).

Figure 1. Yeast Census and Survival. A) Lung yeast census day 7 post-intranasal infection with 6 clinical strains and 2 control strains; B) Brain yeast census day 7 post-intranasal infection with 6 clinical strains and 2 control strains; C) Lung yeast census day 14 post infection by oropharyngeal aspiration with H99; D) Brain yeast census day 14 post infection by oropharyngeal aspiration with H99; E) 28 day survival post-infection by oropharyngeal aspiration with H99; F) Brain yeast census 48 hours post infection by tail vein injection.

Conclusion
Our results in mice suggest that Ibrutinib target BTK is not a major contributing factor for controlling Cryptococcus, and that human susceptibility to cryptococcosis and CNS infection may be due to an off-target effect of ibrutinib. Future work will focus on pharmacologic inhibition of BTK with ibrutinib to determine if the off-target effects of the drug increase risk for cryptococcosis.

Disclosures
Julia A. Messina, MD, MHS, MS, Uptodate (Other Financial or Material Support) Julia A. Messina, MD, MHS, MS, Uptodate (Individual(s) Involved: Self): Author, Other Financial or Material Support John R. Perfect, MD, Astellas Pharma, Inc. (Consultant, Grant/Research Support, Other Financial or Material Support, Honorarium)Basilea (Consultant, Grant/Research Support)Enzon (Consultant, Grant/Research Support)F2G (Consultant, Grant/Research Support)Merck (Consultant, Research Grant or Support)MethylGene (Consultant, Grant/Research Support)Pfizer, Inc. (Consultant, Grant/Research Support)Schering-Plough Corp. (Consultant, Grant/Research Support)