Preclinical Evaluation of Recombinant Microbial Glycoside Hydrolases in the Prevention of Experimental Invasive Aspergillosis

ABSTRACT Aspergillus fumigatus is a ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses. Aspergillus biofilm formation requires the production of a cationic matrix exopolysaccharide, galactosaminogalactan (GAG). In this study, recombinant glycoside hydrolases (GH)s that degrade GAG were evaluated as antifungal agents in a mouse model of invasive aspergillosis. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that although GHs have short half-lives, GH prophylaxis resulted in reduced fungal burden in leukopenic mice and improved survival in neutropenic mice, possibly through augmenting pulmonary neutrophil recruitment. Combining GH prophylaxis with posaconazole treatment resulted in a greater reduction in fungal burden than either agent alone. This study lays the foundation for further exploration of GH therapy in invasive fungal infections.

Here, the tolerability and anti-Aspergillus activity of recombinant GH therapy with Sph3 h , PelA h , and Ega3 h was evaluated in vivo using immunocompromised mouse models of invasive aspergillosis (6,18). Single-dose intratracheal Sph3 h , PelA h , and Ega3 h administration was well tolerated by uninfected mice. Prophylaxis with a single dose of GH at the time of infection attenuated A. fumigatus virulence in two immunocompromised mouse models of invasive pulmonary aspergillosis. In addition, prophylaxis with Sph3 h in combination with posaconazole treatment enhanced the antifungal activity of posaconazole against A. fumigatus in a neutropenic mouse model of invasive pulmonary aspergillosis. These results suggest that GH therapy is a promising approach for the prevention of invasive aspergillosis.

RESULTS
Intratracheal GH treatment is well tolerated by mice. Previously, structure-function studies of Sph3 h and PelA h produced in Escherichia coli and Ega3 h produced in Pichia pastoris (Ega3 h -Pp) demonstrated that these soluble recombinant GH domains can disrupt A. fumigatus biofilms by degrading GAG (11)(12)(13). In addition, we found that intratracheal administration of a single dose of up to 500 mg Sph3 h was generally well tolerated by mice (14). To extend these findings, the tolerability of intratracheal Sph3 h , PelA h , and Ega3 h -Pp was examined in greater detail. Immunocompetent BALB/c mice were administered up to 500 mg of intratracheal Sph3 h , PelA h , or Ega3 h -Pp and monitored for changes in weight and temperature and then euthanized 7 days later for measures of pulmonary injury and inflammation. Treatment with a single dose of up to 500 mg of Sph3 h , PelA h , or Ega3 h -Pp was well tolerated by mice, without signs of respiratory distress or mortality. GH-treated mice exhibited no difference in body weight and temperature compared with mice treated with buffer alone (see Fig. S1 and S2 in the supplemental material).
To test if intratracheal GH therapy induced pulmonary injury, pulmonary damage was assessed by measuring lactate dehydrogenase activity in bronchoalveolar lavage fluid from mouse lungs. No significant increase in lactate dehydrogenase activity was detected in the bronchoalveolar lavage fluid from mice treated with 500 mg Sph3 h , PelA h , or Ega3 h -Pp compared with mice treated with buffer alone (Fig. 1) (14), suggesting that single-dose GH treatment does not induce pulmonary injury in mice. Consistent with these findings, histological examination of pulmonary sections did not reveal any differences between GH-treated and buffer-treated mice (Fig. S3).
To further probe the host response to intratracheal GH treatment, pulmonary leukocytes from GH-treated mice were quantified by flow cytometry (Fig. 2). There was no significant difference in pulmonary lymphocyte numbers between mice treated with FIG 1 Intratracheal GH therapy does not induce pulmonary damage. Lactate dehydrogenase activity was quantified in bronchoalveolar lavage (BAL) fluid from mouse lungs of immunocompetent BALB/c mice 7 days after intratracheal administration of a single dose of 100 or 500 mg of Sph3 h , PelA h , or Ega3 h -Pp. Bars represent the means 6 standard errors from at least 2 independent experiments with $9 mice per group. No significant differences were found between any test condition and the buffer-treated group (P . 0.05) as determined by two-way ANOVA with Dunnett's multiple-comparison test. either the 100-or 500-mg dose of Sph3 h and mice treated with buffer alone. Sph3 h treatment also had no effect on macrophage or eosinophil numbers at the 100-mg dose, while a small but significant increase in the number of these cells was detected in mice treated with 500 mg Sph3 h . Although a significant increase in pulmonary neutrophils was observed following treatment with 100 mg Sph3 h , this was not observed in mice treated with the higher 500-mg dose of Sph3 h . In the case of PelA h treatment, increases in pulmonary lymphocytes and macrophages were observed at the higher GH dose. In contrast to Sph3 h or PelA h , treatment with all doses of Ega3 h -Pp was associated with a significant increase in leukocyte populations, including lymphocytes, macrophages, eosinophils, and neutrophils. Taken together, these data suggest that while 500 mg of Sph3 h or PelA h is near the maximal tolerated intratracheal dose, Ega3 h -Pp is significantly more inflammatory and may be less tolerated by mice.
Ega3 h -Pp was produced in yeast, unlike Sph3 h and PelA h , which were produced in bacteria. As eukaryotic proteins differ from bacterial proteins in their glycosylation patterns (19) and contaminating fungal b-glucan could be copurified with Ega3 h -Pp (20), we hypothesized that the inflammatory response to Ega3 h -Pp treatment is a consequence of fungal glycosylation patterns or trace amounts of b-glucan. We therefore turned to the production of the soluble domain of Ega3 h in the human embryonic cell line HEK293-S (Ega3 h -HEK). In contrast to treatment with yeast-derived Ega3 h -Pp, no significant increase in pulmonary lymphocyte and eosinophil numbers was observed in mice treated with either 100 or 500 mg Ega3 h -HEK. Although a significant increase in pulmonary neutrophil numbers was detected following treatment with 100 mg Ega3 h -HEK, this was not seen in mice treated with 500 mg Ega3 h -HEK. As with Ega3 h -Pp, a significant increase in pulmonary macrophage numbers was detected at both 100 and 500 mg Ega3 h -HEK. These data suggest that production of Ega3 h in a mammalian cell line induced a lower inflammatory response than production in yeast.

FIG 2
Pulmonary leukocyte numbers following intratracheal GH therapy. Immunocompetent BALB/c mice were treated intratracheally with a single dose of 100 or 500 mg of Sph3 h , PelA h , Ega3 h -Pp, or Ega3 h -HEK. Pulmonary leukocyte populations, including lymphocytes, macrophages, eosinophils, and neutrophils, were quantified by flow cytometry 7 days after GH treatment. Bars represent the means 6 standard errors from 2 independent experiments with $9 mice per group. A significant difference is indicated (*, P , 0.05) relative to the buffer-treated group as determined by two-way ANOVA with Dunnett's multiple-comparison test.
GHs exhibited short pulmonary half-lives. To inform the design of further efficacy studies, the pharmacokinetics of the GHs were determined in leukopenic mice. Cyclophosphamide-and cortisone acetate-treated mice were given a single intratracheal dose of 500 mg Sph3 h , PelA h , Ega3 h -Pp, or Ega3 h -HEK. At select time points the mice were euthanized and their lungs were harvested and homogenized in a cocktail of protease inhibitors to prevent degradation of GHs. Lung homogenates were assessed by Western blotting using rabbit anti-GH antibodies, and the half-lives of the GHs in the lungs were determined by densitometry. Ega3 h -Pp and Ega3 h -HEK displayed longer half-lives of approximately 9 h compared to those of PelA h or Sph3 h , with halflives of approximately 5 and 3 h, respectively (Fig. 3). These data suggest that Ega3 h -Pp and Ega3 h -HEK are more stable in the lungs than either PelA h or Sph3 h . Given that Ega3 h -HEK had a similar half-life but induced a lower inflammatory response than Ega3 h -Pp, Ega3 h -HEK was used in all subsequent in vivo experiments.
Pulmonary GH prophylaxis attenuates fungal virulence in an immunocompromised mouse model of invasive pulmonary aspergillosis. To test the antifungal activity of GH prophylaxis in vivo, the effects of a single GH dose on survival of A. fumigatus-infected mice were assessed in a neutropenic model of invasive aspergillosis. Mice were rendered neutropenic with anti-Ly6G antibody treatment and then infected with A. fumigatus with or without the coadministration of 500 mg of Sph3 h , PelA h , or Ega3 h -HEK (Fig. S4A). GH prophylaxis was well tolerated in infected mice, and there was no clinical evidence of central nervous system dissemination (head tilt or leg drag) in GH-or buffer-treated mice. GH prophylaxis with a single dose of Sph3 h , PelA h , or Ega3 h -HEK resulted in a significant increase in survival of infected mice compared to untreated infected mice (Fig. 4).
To confirm and extend these findings, the effects of GH prophylaxis on A. fumigatus virulence were assessed in a second mouse model of invasive pulmonary aspergillosis. Mice were rendered leukopenic with cyclophosphamide and cortisone acetate treatment and then intratracheally infected with A. fumigatus conidia and coadministered a single dose of 500 mg of Sph3 h, PelA h , or Ega3 h -HEK (Fig. S4B). Four days following infection, pulmonary galactomannan content was determined as a measure of fungal burden (5,14,21). A. fumigatus-infected mice treated with Sph3 h or Ega3 h -HEK were found to have a significantly lower fungal burden than buffer-treated, infected mice (Fig. 5). Indeed, the pulmonary fungal burden of Sph3 h -or Ega3 h -HEK-treated mice was not statistically different from that observed in mice infected with the GAG-deficient Duge3 A. fumigatus strain (Fig. 5) (14). A similar trend in the reduction of pulmonary fungal burden was seen in mice receiving PelA h , although this difference failed to reach statistical significance (Fig. 5). Collectively the results of our studies in these two models suggest that GH administration can protect against invasive aspergillosis in mice.
GH prophylaxis enhances the susceptibility of A. fumigatus to the antifungal posaconazole in vitro (14). To determine if GH prophylaxis can enhance the antifungal activity of posaconazole in vivo, the combination of Sph3 h with the antifungal posaconazole was compared to monotherapy with either agent alone in the neutropenic model of invasive aspergillosis (Fig. S4C). Neutropenic mice were intratracheally infected with A. fumigatus conidia and administered a single dose of 500 mg Sph3 h , a submaximal dose of posaconazole (2.5 mg/kg every 12 h), or a combination of the two. Two days following infection, pulmonary galactomannan content was determined as a measure of  fungal burden (5,14,21). At this time point, prophylaxis with Sph3 h or treatment with posaconazole alone resulted in a trend toward reduced fungal burden compared with buffer-treated mice. In contrast, a significant reduction in fungal burden was observed in infected mice receiving Sph3 h -posaconazole in combination (Fig. 6). These findings suggest that, as was seen in vitro, Sph3 h prophylaxis enhances the antifungal activity of posaconazole and that GH-antifungal therapy is more effective than either therapy alone.
The catalytic activity of Sph3 h prophylaxis is dispensable for antifungal activity. GAG is absent from resting and swollen spores of A. fumigatus and is only produced following germination and during hyphal growth. Given the short pulmonary half-lives of GHs, it is possible that these enzymes augment host resistance through activating host inflammatory responses rather than via GAG degradation. To investigate this hypothesis, the early immune response to GH therapy was probed by pulmonary leukocyte quantification in Sph3 h -treated mice during early A. fumigatus infection (Fig. 7). Leukopenic mice were intratracheally infected with A. fumigatus conidia coadministered with a single dose of 500 mg Sph3 h (Fig. S4B), and the lungs were harvested after 24 and 48 h for quantification of pulmonary leukocyte populations. A significant increase in neutrophil numbers was detected in both uninfected and A. fumigatusinfected Sph3 h -treated mice at both 1 and 2 days after Sph3 h administration. A trend toward increased eosinophil populations was observed in uninfected Sph3 h -treated mice on day one; however, this failed to reach significance. No significant increase in pulmonary lymphocyte or macrophage numbers was observed. Together, these observations suggest that Sph3 h -driven recruitment of pulmonary neutrophils contributes to the activity of this agent in the prevention of invasive aspergillosis.
To confirm that the degradation of GAG was not required for the antifungal effects of GH prophylaxis, the antifungal activity of a catalytically inactive Sph3 h variant, D166A AC , was evaluated in A. fumigatus-infected neutropenic mice (Fig. 4A). Similar levels of protection were observed in A. fumigatus-infected mice receiving prophylaxis with wild-type Sph3 h and variant D166A AC (Fig. 8). Collectively, these data suggest that Sph3 h -antifungal activity is not mediated through enzymatic degradation of GAG.

DISCUSSION
In this study, pulmonary administration of the GHs Sph3 h , PelA h , or Ega3 h -HEK was demonstrated to be well tolerated and induced minimal immune response in uninfected mice. Although Sph3 h , PelA h , and Ega3 h -HEK exhibited relatively short half-lives in vivo, intratracheal Sph3 h or Ega3 h -HEK limited A. fumigatus growth in two in vivo Single-dose GH therapy was well tolerated and resulted in minimal changes in the pulmonary inflammatory response in the absence of infection. However, the GHs exhibited short half-lives, and the effects of repeated GH administration remain to be evaluated. Repeated GH dosing has been previously reported with a recombinant form of another P. aeruginosa GH with biofilm-disrupting activity, PslG h , and was found to be well tolerated in a chronic P. aeruginosa infection wound model (22). While these FIG 7 Single-dose intratracheal Sph3 h prophylaxis is associated with early pulmonary neutrophil recruitment. Leukopenic mice were intratracheally infected with 5 Â 10 3 wild-type (WT) A. fumigatus conidia coadministered with or without 500 mg Sph3 h . Pulmonary leukocyte populations were quantified by flow cytometry at 1 day and 2 days after Sph3 h prophylaxis. Bars represent the means 6 standard errors from 3 independent experiments for uninfected buffer-treated (UI 1 buffer), wild-type-infected buffer-treated (WT 1 buffer), and wild-type-infected Sph3 h -treated (WT 1 Sph3 h ), and 2 independent experiments for uninfected Sph3 h -treated (UI 1 Sph3 h ) group, with $6 mice per group. A significant increase in leukocyte populations is indicated (*, P , 0.0001) relative to the uninfected buffer-treated (UI 1 buffer) group at 1 day and 2 days as determined by two-way ANOVA with Tukey's multiple-comparison test.

FIG 8
Catalytic activity of Sph3 h is dispensable for antifungal activity in a neutropenic mouse model of pulmonary invasive aspergillosis. Shown is survival of neutropenic mice that were intratracheally infected with 5 Â 10 6 wild-type (WT) A. fumigatus conidia coadministered with a single dose of 500 mg of Sph3 h or catalytically inactive Sph3 h variant (D166A Ac ). Kaplan-Meier curves represent 3 independent experiments with $4 mice per group. A significant difference in survival between both Sph3 h -and D166A Ac -treated groups compared with the uninfected (UI) group is indicated (*, P , 0.0065). No significant difference was found between the wild-type-infected Sph3 h -treated (WT 1 Sph3 h ) and the wild-type-infected D166A Ac -treated (WT 1 D166A Ac ) groups as determined by Wilcoxon rank test (P = 0.6975).

Ostapska et al.
® results are promising, more detailed immunotoxicity studies of multiple GH dosing and anti-GH antibody response are required to advance these agents toward use in clinical trials.
Individual GH enzymes exhibited differences in efficacy and half-life. Although intratracheal Sph3 h , Ega3 h -HEK, and PelA h all enhanced survival after fungal challenge, only prophylaxis with Sph3 h and Ega3 h -HEK limited A. fumigatus growth in vivo, while a trend toward reduced fungal burden was observed with PelA h . This observation is unlikely to be driven by more rapid PelA h degradation, given that PelA h exhibited a slightly longer half-life than Sph3 h . Although this observation may simply reflect normal biological variability, it is possible that PelA h is less active than the other GH enzymes. Consistent with this hypothesis, previous studies of A. fumigatus biofilm disruption in vitro reported that Sph3 h and Ega3 h -Pp exhibited lower half-maximal effective concentrations than PelA h (0.45 nM and 0.85 nM versus 2.80 nM, respectively) (12,14).
The mechanism whereby GH enzyme therapy mediates improved survival and augments antifungal activity in invasive aspergillosis remains to be elucidated. Pharmacokinetics revealed that GHs have short half-lives in vivo relative to the growth rate of A. fumigatus (23), suggesting that there is a limited window where GAG and active GH enzymes are both present. Indeed, the efficacy of prophylaxis with the catalytically inactive Sph3 h variant and the early recruitment of neutrophils after Sph3 h therapy suggest that augmentation of innate pulmonary inflammatory responses underlie the antifungal activity of these agents. However, an important caveat to these studies is the fact that catalytically inactive GH enzymes can function as lectins that retain their ability to bind to their cognate polymers and can interfere with polymer function in vitro (17). It therefore remains possible that GH enzymes do mediate some of their effects in vivo via lectin-like interactions with GAG. GAG has been reported to mediate a wide variety of immunosuppressive and other effects in vivo, including cloaking cell wall glycans from host pattern recognition receptors, augmenting antifungal resistance, resisting damage by neutrophil extracellular traps, mediating hyphal adhesion, inducing neutrophil apoptosis, and inducing immunosuppressive IL-1 receptor antagonist secretion (5,(8)(9)(10). The relative contributions to virulence of each of these GAG-related phenotypes has not been well defined. These effects may become more apparent in investigations evaluating the mechanisms of GH efficacy with A. fumigatus isolates of various levels of virulence (24,25) or with mouse strains with differences in innate or acquired immunity (26)(27)(28). BALB/c mice used in this study are intrinsically polarized toward a type two helper T-cell response following intravenous administration of A. fumigatus conidia (26). The use of additional strains of mice with differential helper T-cell protective responses against A. fumigatus could provide insight into the mechanism of action of the GHs. Further studies are required to probe the relative contribution of pulmonary inflammatory responses and other anti-GAG effects of GH enzymes on these mechanisms of fungal pathogenesis in vivo.
An important concern with biofilm-targeting therapies is the potential for dispersion of organisms following degradation of the extracellular matrix and a worsening of infection. This phenomenon has been reported in vivo with manipulation of P. aeruginosa biofilm regulatory pathways (29) as well as enzymatic treatment of P. aeruginosa biofilms with a-amylase and cellulase (30). In this study, GH therapy was associated with improved outcomes in A. fumigatus infection, and no clinical evidence of dissemination to the central nervous system was observed, although necropsy was not performed. These findings may reflect the early immune action of GH enzymes prior to the production of significant amounts of GAG but could also reflect fundamental differences in morphology and motility between molds and bacteria. In contrast to unicellular bacteria, molds grow as long multicellular filamentous hyphae that are intertwined and, thus, are less likely to be able to passively detach and disseminate (31). Further, unlike P. aeruginosa and many other bacteria, A. fumigatus hyphae are not motile and therefore are unable to actively disseminate from the site of infection (32)(33)(34)(35). Consistent with the latter hypothesis, a-amylaseand cellulase-mediated dispersal of biofilms formed by a nonmotile bacterial species, Staphylococcus aureus, did not result in systemic bacterial dissemination in a chronic wound model (30). Together, these data suggest that morphology and the capacity for motility are important determinants that may partly drive the outcomes of biofilm-directed therapies.
For these initial studies of GH efficacy in vivo, a model of invasive aspergillosis in which GAG-covered hyphae invade and form fungal biomass around and within the lung parenchyma was used (4). One important future direction will be to investigate GH efficacy against fungal biofilms in a chronic pulmonary aspergillosis model. Concerns remain, however, about the use of the agar bead A. fumigatus chronic airway infection model (36), as the presence of a foreign extracellular matrix may compromise GH activity and penetration of GHs to the hyphal surface.
The results of this study demonstrate that intratracheal administration of Sph3 h , PelA h , or Ega3 h -HEK can improve survival during experimental invasive aspergillosis. The results of these studies lay the foundation for future work to elucidate the mechanism by which the GHs limit fungal growth in vivo, to test the efficacy of GHs in established infection, perform detailed immunotoxicity studies, and extend studies into a chronic pulmonary aspergillosis model. Further, nebulizable formulations for aerosolized GH deposition into the airways will need to be explored for eventual clinical use.
Recombinant GH expression and purification. His-tagged PelA h , Sph3 h , and catalytically inactive Sph3 h variant D166A AC were expressed in Clearcoli cells grown in Terrific broth (Bioshop) with 50 mg/ml kanamycin (Biobasic) as previously described (11,17,37). Bacterial cultures were induced with 0.5 mM isopropyl-b-D-thiogalactopyranoside (IPTG) (Biobasic) when the cells reached an optical density at 600 nm (OD 600 ) of 1.2 to 1.4. The cells were incubated postinduction overnight at 18°C with shaking at 200 rpm before being harvested by centrifugation at 5,000 Â g for 30 min at 4°C. Both proteins were purified using Ni-nitrilotriacetic acid columns (GE Healthcare) followed by buffer exchange as previously described (37). The protein yield using this method was an average of approximately 80 to 100 mg/liter of culture.
Expression of Ega3 h -Pp in the PichiaPink system was optimized as previously described (12). To generate a glycosylated Ega3 h that would mimic mammalian-like glycosylation patterns (38), Ega3 h -HEK was expressed in a cell line of human embryonic kidney cells (HEK293). The region of the ega3 68-318 gene was cloned using primers ega3-fwd (59-GGGACCGGTGGTAATTATACCACCGCAAAATGG) and ega3-rev (59-GGGGGTACCGCAATATTCCACCCA) from a pET28a vector (12) into a pHLsec vector under the control of a mammalian promoter. The plasmids were then transiently transfected into Freestyle 293 S (HEK293-S) cell lines for expression trials using FectoPro transfection reagent (VWR). The culture supernatants containing the secreted proteins were harvested at 3 and 6 days to measure protein yield. Six days was established as the essential incubation time for maximum protein expression. The cells were spun down, and the secreted His-tagged protein was purified from the supernatant by affinity chromatography followed by gel filtration using a HiLoad 16/600 Superdex 200 prep-grade column (GE Healthcare).
Mice. Six-to 8-week-old BALB/c female mice (Charles River Laboratories Inc., Senneville, QC, Canada, and Kingston, NY) were used for animal studies. Mice were anaesthetized with 4% isoflurane prior to intratracheal infection or prophylaxis/treatment with GHs/drug. Mice were monitored daily for signs of stress (ruffled fur, inactivity, and hunched posture), and body weights and temperatures were taken. Body weight was measured using a top-loading balance, and surface body temperature was taken on the abdomen using a digital infrared thermometer. Moribund animals were euthanized by isoflurane and CO 2 overdose.
Tolerability studies. Immunocompetent mice were treated intratracheally with a single dose of Sph3 h , PelA h , or Ega3 h -Pp at 1, 5, 10, 100, or 500 mg in 50 ml PBS or PBS alone. Mice were monitored daily for 7 days for signs of illness, and body weights and temperatures were recorded. For histopathology studies, lungs from immunocompetent mice were inflated with 10% buffered formalin (Fisher Scientific) and fixed in formalin as previously described (14). Lungs were then embedded in paraffin, and 4-mmthick sections were stained with hematoxylin and eosin. Scanned sections (Leica, Aperio) were analyzed with QuPath 0.1.2 software (39).
Densitometry and antibody production. SDS-PAGE and Western blotting techniques were used to assess pulmonary GH pharmacokinetics. Rabbit polyclonal antibodies specific to each of the GHs were produced by Cedarlane (Burlington, Canada) as previously described (37). Mice were treated intratracheally with a single dose of 500 mg of each GH and then euthanized, and their lungs were harvested at the indicated time points. Lungs were homogenized in a cocktail of protease inhibitors (Roche), and pulmonary GH concentrations were quantified by Western blotting with rabbit anti-GH antibodies. Goat-anti-rabbit Ostapska et al. ® horseradish peroxidase-conjugated secondary antibody (Bio-Rad) was detected with a chemiluminescent substrate (Thermo-Fisher). The half-life of each GH was determined by densitometric analysis using ImageJ software. Band intensity at each time point was normalized to the intensity at the zero-hour time point. Halflife was determined as 50% of the relative intensity of the bands compared to the zero-hour time point.
Pulmonary damage. Mice were treated intratracheally with a single dose of Sph3 h , PelA h , or Ega3 h -Pp at 100 or 500 mg in 50 ml PBS or PBS alone. Seven days after treatment, mice were euthanized and their lungs lavaged twice with 1 ml PBS as previously described (14). Lactate dehydrogenase activity was measured in the pooled bronchoalveolar lavage fluid with a commercial assay (CytoTox 96 nonradioactive cytotoxicity assay; Promega) per the manufacturer's instructions.
Pulmonary leukocyte quantification. Immunocompetent mice were treated intratracheally with a single dose of Sph3 h , PelA h , Ega3 h -Pp, or Ega3 h -HEK at 100 or 500 mg in 50 ml PBS or PBS alone. Seven days after treatment, mice were euthanized and their lungs were washed in PBS, minced in RPMI medium 1640 (Wisent) containing 5% (vol/vol) fetal bovine serum (FBS; Wisent), and then digested with 150 U/ml collagenase (Sigma) (14,36). The resulting suspension was passed through a 70-mm cell strainer and treated with ACK buffer (Gibco). Approximately 1 Â 10 6 leukocytes were resuspended in a fixable viability dye (eBioscience) and washed, and their Fc receptors were blocked by unlabeled anti-CD16/32 antibodies (FcBlock; BD Pharmingen) as previously described (14). Cell surface components were then stained with fluorescently labeled antibodies (BD Biosciences) as previously described (14). Leukocytes were washed, fixed with paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), and then resuspended in PBS as previously described (14). Data were acquired on an LSR Fortessa flow cytometer with FACSDiva software (BD Biosciences) and analyzed with FlowJo software version 10 (FlowJo, LLC). Immune cell subsets were defined as previously described (14). Total cell populations were calculated by using the CountBright absolute counting beads (Invitrogen).
(ii) Neutropenic mouse model. Mice were rendered neutropenic by intraperitoneal injection with anti-Ly6G antibody (clone 1A8; Bio X Cell) at 200 mg, starting 1 day prior to infection, every 48 h as previously described (18). Neutrophil depletion was confirmed by blood smear and differential staining. Mice were intratracheally infected with a 50-ml suspension of 5 Â 10 6 A. fumigatus conidia in PBS-T.
Effects of GH prophylaxis in leukopenic and neutropenic mouse models of invasive pulmonary aspergillosis. Leukopenic mice were intratracheally infected with a 50-ml suspension of 5 Â 10 3 A. fumigatus conidia in PBS-T and concomitantly treated with either a single dose of Sph3 h , PelA h , or Ega3 h -HEK at 500 mg in 50 ml of the corresponding buffer or buffer alone. At 1 and 2 or 4 days after prophylaxis, mice were euthanized and their lungs harvested for pulmonary leukocyte quantification as described above or pulmonary fungal burden determination as described below. Neutropenic mice were intratracheally infected and concomitantly treated with Sph3 h , PelA h , Ega3 h -HEK, or catalytically inactive Sph3 h variant D166 AC as described above and then monitored daily and euthanized upon reaching clinical endpoints.
Sph3 h -posaconazole combination prophylaxis in a neutropenic mouse model of invasive pulmonary aspergillosis. Neutropenic mice were intratracheally infected with a suspension of 5 Â 10 3 A. fumigatus conidia and concomitantly treated with either a single dose of Sph3 h at 500 mg or buffer alone as described above, and beginning 12 h after infection they were treated by oral gavage with 2.5 mg/kg of body weight posaconazole or buffer alone every 12 h. Two days after the initiation of prophylaxis, mice were euthanized and their lungs were harvested for fungal burden determination as described below.
Pulmonary fungal burden. Lungs were harvested and homogenized in 5 ml PBS with a Polytron tissue homogenizer, and homogenates were stored at 280°C, modified from what was previously done (5). Pulmonary galactomannan content was determined by using the Platelia Aspergillus immunoassay kit (Bio-Rad) according to the manufacturer's instructions as previously described (5,14). The galactomannan values were then normalized to a highly infected lung homogenate standard.
Statistical analysis. Data are presented and statistical significance calculated as indicated. All graphs were generated and statistical analyses were performed in GraphPad Prism version 9.0.0 software. Significant differences between values were compared by two-way analysis of variance (ANOVA) with Dunnett's multiple-comparison test, two-way ANOVA with Tukey's multiple-comparison test, Kruskal-Wallis test with Dunn's multiple-comparison test, or Wilcoxon rank test. Data availability. We declare that the data supporting the findings of this study are available within the paper and its supplemental material.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.