Antibody-guided in vivo imaging of Aspergillus fumigatus lung infections during antifungal azole treatment

Abstract

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of immunocompromised humans, caused by the opportunistic fungal pathogen Aspergillus fumigatus. Inadequacies in current diagnostic procedures mean that early diagnosis of the disease, critical to patient survival, remains a major clinical challenge, and is leading to the empiric use of antifungal drugs and emergence of azole resistance. A non-invasive procedure that allows both unambiguous detection of IPA and its response to azole treatment is therefore needed. Here, we show that a humanised Aspergillus-specific monoclonal antibody, dual labelled with a radionuclide and fluorophore, can be used in immunoPET/MRI in vivo in a neutropenic mouse model and 3D light sheet fluorescence microscopy ex vivo in the infected mouse lungs to quantify early A. fumigatus lung infections and to monitor the efficacy of azole therapy. Our antibody-guided approach reveals that early drug intervention is critical to prevent complete invasion of the lungs by the fungus, and demonstrates the power of molecular imaging as a non-invasive procedure for tracking IPA in vivo.

Conflict of interest statement

C.R.T. is director of ISCA Diagnostics Ltd. The remaining authors declare no competing interests.

Figures

Fig. 1. Development of invasive pulmonary aspergillosis under healthy and immunosuppressed conditions.

a Schematic experimental workflow. Neutropenic groups were depleted with Gr-1 antibody 18 h prior to intratracheal (i.t.) inoculation with Aspergillus fumigatustdTomato conidia. Lungs were subsequently removed at different time postinfection (p.i.), cleared and imaged using light sheet fluorescence microscopy (LSFM). b Lung tissue (gray) and fungal biomass (red) is depicted for time points 0, 0.5, 6, 24, and 48 h during disease progression. Images were constructed using surface tool from Imaris. Scale bar = 1 mm. c Magnified two-photon microscopy images of the same lungs as in b, with lung tissue (gray), and fungal biomass (red). Scale bar = 50 µm. d Plot of fungal load per whole lung lobe for the different treatments over time. Sham-depleted, Aspergillus fumigatus (A.f.) infected group (PBS-A.f., gray circle, n = 2 per time point), Gr-1 neutrophil-depleted and infected group (Gr1-A.f., red triangles, n = 3 per time point), isotype-depleted and infected group (Iso-A.f., orange squares, n = 1 per time point). Results are plotted as individual values with average and standard deviation where applicable.

Fig. 2. ImmuoPET/MR imaging of invasive pulmonary aspergillosis in response to voriconazole (VCZ) treatment.

a Experimental workflow depicting neutrophil depletion, Aspergillus fumigatus (A.f.) infection, tracer injection, voriconazole administration (VCZ) and in vivo imaging. b ImmunoPET/MR images of PBS-deposited animals (Control, left column, n = 4), A. fumigatus-infected animals without VCZ treatment (A. fumigatus, middle column, n = 4), and with late-stage VCZ treatment (A.f. VCZ (24 h), right column, n = 3). Images acquired 24 h after inoculation (p.i.) are presented in the top row, the bottom row are images acquired 48 h p.i.. MIP is a Maximum Intensity Projection of the PET image; MRI is a T2-weighted MRI single slice MRI image; Fusion is a single slice PET image overlaid on the corresponding MRI slice. Magnification of the fusion image in the lung area is displayed below. c Quantification of 64Cu-hJF5 accumulation in the lung tissue at 24 h (top) and 48 h (bottom) after radiotracer injection in control (Ctrl), infected (A. fumigatus) and voriconazole treated animals (A.f., VCZ (24 h)). 48 h p.i.: Ctrl vs. A. fumigatus, p = 0.0152; Ctrl vs. A.f., VCZ (24 h), p = 0.0033. d Ex vivo Biodistribution data of 64Cu-hJF5 in major organs 48 h after radiotracer injection obtained after perfusion of the animal by 0.4% PFA. Blood: Ctrl vs. A. fumigatus, p = 0.0032; Ctrl vs. A.f., VCZ (24 h), p = 0.0002. Lungs: Ctrl vs. A. fumigatus, p < 0.0001; Ctrl vs. A.f., VCZ (24 h), p < 0.0001. Spleen: Ctrl vs. A. fumigatus, p = 0.0005; Ctrl vs. A.f., VCZ (24 h), p = 0.0004. Results are plotted as individual values with average and standard deviation and are expressed as percent injected dose per cubic centimeter (%ID/cc) for PET and injected dose per gram (ID/g) for Biodistribution. p values were generated using one way ANOVA (c) or two way ANOVA (d) with Tukey’s multiple comparison test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 3. Impact of antifungal treatment measured with PET/MRI and LSFM using dual-labeled 64Cu-hJF5-DyLight650.

a Experimental workflow depicting Aspergillus fumigatus (A.f.) infection, tracer injection, voriconazole administration (VCZ) and imaging using immunoPET/MR and LSFM. b ImmunoPET/MR images of PBS-deposited animals (Control, left, n = 2), A. fumigatus-infected animals without treatment (A. fumigatus, middle, n = 2) and animals with voriconazole treatment 24 h after inoculation (A.f. VCZ (24 h), right, n = 3). Images were acquired 48 h postinoculation (p.i.). MIP is a maximum intensity projection of the PET image; MRI is a T2-weighted MRI single slice MRI image; Fusion is a single slice PET image overlaid on the corresponding MRI slice. A close-up of the fusion image in the lung area is displayed below. PET data are presented as percent injected dose per cubic centimeter (%ID/cc). c Detection of 64Cu-hJF5-DyLight650 with LSFM. Surface view of lungs from same animals as in b imaged with LSFM, and with magnified detail shown below. Lung tissue (gray), A. fumigatus (red) and 64Cu-hJF5-DyLight650 signal (green). Scale bar first row = 1.5 mm, and second row = 300 µm. d High-resolution confocal images of same lungs as in b. Scale bar = 50 µm.

Fig. 4. Quantification and volumetric analysis of immunoPET and LSFM.

a Ratio of infected lung volume as measured by immunoPET to total lung volume measured via MRI as a function of 64Cu-hJF5-DyLight650 accumulation threshold in percent injected dose per volume (%ID/cc) at 48 h postinoculation in PBS-deposited animals (Control, left, n = 2), A. fumigatus-infected animals without treatment (A. fumigatus, middle, n = 2) and animals with voriconazole treatment 24 h after inoculation (A.f. VCZ (24 h), right, n = 3). 5%: Ctrl vs. A.f., VCZ (24 h), p = 0.0062. 10%: Ctrl vs. A.f., VCZ (24 h), p = 0.0013. b Volumetric ratio of A. fumigatus or 64Cu-hJF5-DyLight650 in the lung as measured by LSFM for each group. c Quantification of the co-localization of 64Cu-hJF5-DyLight650 and A. fumigatustdTomato measured by LSFM in untreated (A. fumigatus, red) and treated (A.f., VCZ (24 h)). d Correlation of A. fumigatus invasion and 64Cu-hJF5-DyLight650 distribution in the lungs of A. fumigatus-infected animals untreated (A. fumigatus, red) and VCZ treated 24 h after infection (A.f., VCZ (24 h) as measured by LSFM. e Correlation of 64Cu-hJF5-DyLight650 volumetric distribution in the lungs of A. fumigatus-infected animals untreated (A. fumigatus, red) and VCZ treated 24 h after infection (A.f., VCZ (24 h) as measured in LSFM and PET using the 15%ID/cc threshold. r2 represents Pearson’s correlation coefficient. Results are plotted as individual values with average and standard deviation. p values were generated using two way ANOVA with Tukey’s multiple comparison test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 5. ImmunoPET/MR evaluation of early- and late-stage voriconazole treatment on A. fumigatus lung infection.

a Experimental workflow depicting Aspergillus fumigatus (A.f.) infection, tracer injection, voriconazole administration (VCZ), and in vivo imaging using immunoPET/MR. b ImmunoPET/MR images of A. fumigatus-infected animals receiving voriconazole (VCZ) treatment initiated 24 h post inoculation (p.i.) (A.f. VCZ (24 h), left, n = 6), or when initiated at 3 h postinoculation and at 24 h p.i. (A.f. VCZ (3 + 24 h), right, n = 4). Images acquired 24 h p.i. are shown in the top row, while images in the bottom row show images acquired 48 h p.i.. MIP is a maximum intensity projection of the PET image; MRI is a T2-weighted MRI single slice MRI image; Fusion is a single slice PET image overlaid on the corresponding MRI slice. Magnifications of the fusion images of the lung are displayed below each image. c Quantification of 64Cu-hJF5 accumulation in the lung tissue at 24 h (top) and 48 h (bottom) p.i. of A. fumigatus in animals left untreated (A. fumigatus, red), receiving voriconazole treatment initiated 24 h after infection (A.f. VCZ (24 h), dark blue), or when initiated at 3 h postinfection and at 24 h postinfection (A.f. VCZ (3 + 24 h), light blue). 48 h p.i.: A. fumigatus vs. A.f., VCZ (3 h + 24 h), p = 0.0046; A.f., VCZ (24 h) vs. A.f., VCZ (3 h + 24 h), p = 0.0025. d Percentage of lung volume occupied by thresholded PET signal at 24 h (left) and 48 h (right) postinfection in the same animals. At 24 h, 10%: A. fumigatus vs. A.f., VCZ (3 h + 24 h), p = 0.0023; A.f., VCZ (24 h) vs. A.f., VCZ (3 h + 24 h), p = 0.0063. At 48 h, 10%: A. fumigatus vs. A.f., VCZ (3 h + 24 h), p < 0.0001; A.f., VCZ (24 h) vs. A.f., VCZ (3 h + 24 h), p < 0.0001 and 15%: A. fumigatus vs. A.f., VCZ (3 h + 24 h), p = 0.0321; A.f., VCZ (24 h) vs. A.f., VCZ (3 h + 24 h), p = 0.0009. Results are plotted as individual values with average and standard deviation and are expressed as percent injected dose per cubic centimeter (%ID/cc) for PET. p values were generated using one way ANOVA (c) or two way ANOVA (d) with Tukey’s multiple comparison test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).