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Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...

University of Texas Houston

Efficient live-imaging methods are pivotal to understand fungal morphogenesis, especially as it relates to interactions with host immune cells and mechanisms of antifungal drugs. Due to the notable similarities in growth patterns of neuronal cells and mycelial networks, we sought to repurpose the NeuroTrack (NT) processing module of the IncuCyte time-lapse microscopy system as a tool to quantify mycelial growth and branching of pathogenic fungi. We showed the robustness of NT analysis to study  and five different molds and confirmed established characteristics of mycelial growth kinetics. We also documented high intra- and interassay reproducibility of the NT module for a spectrum of spore inocula and culture periods. Using GFP-expressing  and , the feasibility of fluorescence-based NT analysis was validated. In addition, we performed proof-of-concept experiments of NT analysis for several translational applications such as studying the morphogenesis of a filamentation-defective  mutant, the effects of different classes of antifungals (polyenes, azoles, and echinocandins), and coculture with host immune cells. High accuracy was found, even at high immune cell-to-fungus ratios or in the presence of fungal debris. For antifungal efficacy studies, addition of a cytotoxicity dye further refined IncuCyte-based analysis, facilitating real-time determination of fungistatic and fungicidal activity in a single assay. Complementing conventional MIC-based assays, NT analysis is an appealing method to study fungal morphogenesis and viability in the context of antifungal compound screening and evaluation of novel immune therapeutics. Pathogenic fungi remain a major cause of infectious complications in immunocompromised patients. Microscopic techniques are crucial for our understanding of fungal biology, host-pathogen interaction, and the pleiotropic effects of antifungal drugs on fungal cell growth and morphogenesis. Taking advantage of the morphological similarities of neuronal cell networks and mycelial growth patterns, we employed the IncuCyte time-lapse microscopy system and its NeuroTrack image analysis software package to study growth and branching of a variety of pathogenic yeasts and molds. Using optimized image processing definitions, we validated IncuCyte NeuroTrack analysis as a reliable and efficient tool for translational applications such as antifungal efficacy evaluation and coculture with host immune effector cells. Hence, the IncuCyte system and its NeuroTrack module provide an appealing platform for efficient  studies of antifungal compounds and immunotherapeutic strategies in medical mycology.

Live Monitoring and Analysis of Fungal Growth, Viability, and Mycelial Morphology Using the IncuCyte NeuroTrack Processing Module.

Flow cytometric quantification of CD154 mould specific T-cells in antigen-stimulated peripheral blood mononuclear cells (PBMCs) or whole blood has been described as a supportive biomarker to diagnose invasive mould infections and to monitor therapeutic outcomes. As patients at risk frequently receive immunosuppressive and antifungal medication, this study compared the matrix-dependent impact of representative drugs on CD154 T-cell detection rates. PBMCs and whole blood samples from healthy adults were pre-treated with therapeutic concentrations of liposomal amphotericin B, voriconazole, posaconazole, cyclosporine A (CsA) or prednisolone. Samples were then stimulated with an Aspergillus fumigatus lysate or a viral antigen cocktail (CPI) and assessed for CD154 T-helper cell frequencies. Specific T-cell detection rates and technical assay properties remained largely unaffected by exposure of both matrices to the studied antifungals. By contrast, CsA and prednisolone pre-treatment of isolated PBMCs and whole blood adversely impacted specific T-cell detection rates and caused elevated inter-replicate variation. Unexpectedly, the whole blood-based protocol that uses additional α-CD49d co-stimulation was less susceptible to CsA and prednisolone despite prolonged drug exposure in the test tube. Accordingly, addition of α-CD49d during PBMC stimulation partially attenuated the impact of immunosuppressive drugs on test performance. Translating these results into the clinical setting, false-negative results of CD154 antigen-specific T-cell quantification need to be considered in patients receiving T-cell-active immunosuppressive medication. Optimized co-stimulation regimes with α-CD49d could contribute to an improved feasibility of functional T-cell assays in immunocompromised patient populations.

Impact of immunosuppressive and antifungal drugs on PBMC- and whole blood-based flow cytometric CD154 Aspergillus fumigatus specific T-cell quantification.

Invasive pulmonary aspergillosis and mucormycosis are life-threatening complications in immunocompromised patients. A rapid diagnosis followed by early antifungal treatment is essential for patient survival. Given the limited spectrum of biomarkers for invasive mold infections, recent studies have proposed the use of radiolabeled siderophores or antibodies as molecular probes to increase the specificity of radiological findings by nuclear imaging modalities. While holding enormous diagnostic potential, most of the currently available molecular probes are tailored to the detection of  species, and their cost-intensive and sophisticated implementation restricts their accessibility at less specialized centers. In order to develop cost-efficient and broadly applicable tracers for pulmonary mold infections, this study established streamlined and high-yielding protocols to radiolabel amphotericin B (AMB) with the gamma emitter technetium-99m (Tc-AMB) and the positron emitter gallium-68 (Ga-AMB). The radiochemical purity of the resulting tracers consistently exceeded 99%, and both probes displayed excellent stability in human serum (>98% after 60 to 240 min at 37°C). The uptake kinetics by representative mold pathogens were assessed in an  Transwell assay using infected endothelial cell layers. Both tracers accumulated intensively and specifically in Transwell inserts infected with , , and other clinically relevant mold pathogens compared with their accumulation in uninfected inserts and inserts infected with bacterial controls. Inoculum-dependent enrichment was confirmed by gamma counting and autoradiographic imaging. Taken together, this pilot  study proposes Tc-AMB and Ga-AMB to be facile, stable, and specific probes, meriting further preclinical  evaluation of radiolabeled amphotericin B for molecular imaging in invasive mycoses.

 Evaluation of Radiolabeled Amphotericin B for Molecular Imaging of Mold Infections.

While mammalian models remain the gold standard to study invasive mycoses, mini-host invertebrate models have provided complementary platforms for explorative investigations of fungal pathogenesis, host-pathogen interplay, and antifungal therapy. Specifically, our group has established Toll-deficient Drosophila melanogaster flies as a facile and cost-effective model organism to study candidiasis, and we have recently expanded these studies to the emerging and frequently multidrug-resistant yeast pathogen Candida auris. Our proof-of-concept data suggest that fruit flies could hold a great promise for large-scale applications in antifungal drug discovery and the screening of C. auris (mutant) libraries with disparate pathogenic capacity. This chapter discusses the advantages and limitations of D. melanogaster to study C. auris candidiasis and provides a step-by-step guide for establishing and troubleshooting C. auris infection and antifungal treatment of Toll-deficient flies along with essential downstream readouts.

Sebastian Wurster

Department of Infectious Diseases,
Infection Control and Employee Health,
MD Anderson Cancer Center,
Department of Infectious Diseases, Infection Control and Employee Health, MD Anderson Cancer Center

University of Texas Houston

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Sebastian Wurster

Department of Infectious Diseases,Infection Control and Employee Health,MD Anderson Cancer Center,Department of Infectious Diseases, Infection Control and Employee Health, MD Anderson Cancer Center

University of Texas Houston

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Department of Infectious Diseases,
Infection Control and Employee Health,
MD Anderson Cancer Center,
Department of Infectious Diseases, Infection Control and Employee Health, MD Anderson Cancer Center