Thomas Sécher

Elsa Bodier-Montagutelli

Christelle Parent

Laura Bouvart

Mélanie Cortes

Marion Ferreira

Ronan Macloughlin

Guy Ilango

Otmar Schmid

Renaud Respaud

Thomas Secher

Nathalie Heuzé-Vourc'h
email: nathalie.vourch@med.univ-tours.fr

Aggregates Associated with Instability of Antibodies during Aerosolization Induce Adverse Immunological Effects

Keywords: therapeutic antibody, aerosol, aggregates, immunogenicity 40 41

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Introduction

Therapeutic antibodies (Ab), which mainly consist of monoclonal IgG, represent the fastest growing class of protein therapeutics, accounting for 90% of proteins on the market [START_REF] Anselmo | Non-invasive delivery strategies for biologics[END_REF]. As recently highlighted during the COVID-19 pandemic, Ab have a tremendous potential to provide a rapid neutralizing response to emerging viral respiratory pathogens, augmenting vaccines for control of respiratory infections. Interestingly, both pulmonary and nasal delivery of therapeutic proteins are receiving increasing interest, as the airways are a relevant non-invasive entry portal to the respiratory tract for local-acting protein therapeutics [START_REF] Dall'acqua | Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn)[END_REF][START_REF] Hart | Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys[END_REF][START_REF] Koleba | Pharmacokinetics of intravenous immunoglobulin: a systematic review[END_REF]. For the treatment of respiratory diseases, several preclinical studies demonstrated that inhaled protein therapeutics, including Ab, are efficacious and display a better pharmacokinetic profile, as compared to other routes [START_REF] Bodier-Montagutelli | Designing inhaled protein therapeutics for topical lung delivery: what are the next steps?[END_REF][START_REF] Secher | In a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration[END_REF][START_REF] Secher | Inhalation of Immuno-Therapeutics/-Prophylactics to Fight Respiratory Tract Infections: An Appropriate Drug at the Right Place![END_REF]. Despite recent clinical development of oral anti-infective Ab, in the context of the global response to SARS-CoV2 pandemic, the inhalation route remains unexploited for Ab. One of the unknowns is the biological consequence of Ab instability during aerosolization. Inhalation requires transformation of a (bulk) protein formulation in an aerosol, i.e., dispersion of a solution/suspension or a dry-powder dispersed into micron-sized particles suspended in a gaseous medium. As with 75% of the inhaled protein therapeutics in clinical development, nebulization of liquid formulations is often the primary technique used for inhalation of proteins [START_REF] Bodier-Montagutelli | Designing inhaled protein therapeutics for topical lung delivery: what are the next steps?[END_REF]. Nebulization generates a huge air-liquid interface which, combined with the potential for nebulization-induced temperature increase and/or shear forces, can be deleterious for proteins. In response to such stresses, proteins are prone to unfolding, aggregating and, in some cases, being partly inactivated [START_REF] Depreter | Inhaled proteins: challenges and perspectives[END_REF][START_REF] Hertel | Protein stability in pulmonary drug delivery via nebulization[END_REF]. Aggregation is a key marker of instability of full-length Ab during nebulization [START_REF] Respaud | Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF][START_REF] Bodier-Montagutelli | Protein stability during nebulization: mind the collection step![END_REF] and its extent mainly depends on the type of Ab, the aerosol generator type and the formulation characteristics.

Aggregates are associated with Ab-related adverse immunogenicity [START_REF] Braun | Protein aggregates seem to play a key role among the parameters influencing the antigenicity of interferon alpha (IFN-alpha) in normal and transgenic mice[END_REF][START_REF] Joubert | Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses[END_REF][START_REF] Uchino | Immunogenicity of protein aggregates of a monoclonal antibody generated by forced shaking stress with siliconized and nonsiliconized syringes in BALB/c mice[END_REF]. Immunogenicity refers to the inherent properties of a molecule to stimulate an immune response. Adverse immunogenicity is due to uncontrolled and protracted immune responses and has major consequences on product safety and pharmacology [START_REF] Mahlangu | Changes in the amino acid sequence of the recombinant human factor VIIa analog, vatreptacog alfa, are associated with clinical immunogenicity[END_REF][START_REF] Holland | Autoantibodies to variable heavy (VH) chain Ig sequences in humans impact the safety and clinical pharmacology of a VH domain antibody antagonist of TNF-alpha receptor 1[END_REF][START_REF] Ponce | Immunogenicity of biologically-derived therapeutics: assessment and interpretation of nonclinical safety studies[END_REF]. For instance, adverse immunogenicity is associated with patient's immunization and production of anti-drug antibodies (ADAs) and affects protein therapeutics pharmacokinetics (PK), pharmacodynamics, efficacy, and safety, sometimes resulting in extremely harmful side effects [START_REF] Schellekens | Bioequivalence and the immunogenicity of biopharmaceuticals[END_REF][START_REF] Li | Thrombocytopenia caused by the development of antibodies to thrombopoietin[END_REF]. To date, many factors have been implicated in adverse immunogenicity [START_REF] Demeule | Where disease pathogenesis meets protein formulation: renal deposition of immunoglobulin aggregates[END_REF][START_REF] Kiese | not stirred: mechanical stress testing of an IgG1 antibody[END_REF][START_REF] Fathallah | Immunogenicity of subcutaneously administered therapeutic proteins--a mechanistic perspective[END_REF][START_REF] Mahler | Protein aggregation: pathways, induction factors and analysis[END_REF] and they include the content of aggregates and the route of administration, which are important parameters of Ab inhalation.

After inhalation, aggregates from aerosolized Ab will encounter the airways immune system, which has evolved over time to recognize, and prevent foreign particles, including aggregates, from penetrating the body. The airway mucosa is sentinelled by a high density of antigen-presenting cells (APC) that quickly and efficiently orchestrate local immune responses against inhaled antigens [START_REF] Lamichhane | The mucosal immune system for vaccine development[END_REF]. In the present study, we analyzed, both in vitro and in vivo, the immunological consequences of Ab aggregates generated during aerosolization and delivered through the airways. First, we screened the potency of IgG aggregates generated by nebulization to activate APC in vitro, using a standard human monocyte-derived dendritic cell (MoDC) assay. To take into consideration the complexity of the immune system and the lung mucosal environment, we evaluated the impact of nebulization-mediated IgG aggregates on immune cells in vivo after pulmonary administration. Our findings show that aggregation attributable to Ab nebulization induced immune cell activation, in a dose-dependent manner and delivering high-level of aggregates through the pulmonary route had a dramatic effect on immune cell homeostasis but also that this was avoidable through appropriate formulation approaches.

Materials and Methods

Mice

Adult male C57BL/6jrj (B6) mice (5 to 7 weeks old) were obtained from Janvier (France). All mice were housed under specific-pathogen-free conditions at the PST Animaleries animal facility (France) and had access to food and water ad libitum. All animal experiments complied with the current European legislative, regulatory and ethical requirements and were approved by the local animal care and use committee (reference: APAFIS#10200-2017061311352787).

Antibodies

Abs 1, 2 and 3 are full-length IgG1 (named hIgG1-1, hIgG1-2, hIgG1-3) used in the clinics, supplied in their commercial formulation with endotoxin levels meeting acceptance thresholds (according to their certificate of analysis). To avoid any interference on the aggregation propensity of each Ab, excipients were removed by hydroxyapatite chromatography and subsequent dialysis against phosphate-buffered saline (PBS). Protein concentration was then evaluated for each Ab: hIgG1-1 [1.7mg/mL], hIgG1-2 [1.9mg/mL], hIgG1-3 [2.38mg/mL] mAb166 (mIgG2b-1) is a murine monoclonal IgG2b,κ Ab against pcrV, a component of a type three secretion system (T3SS) of P. aeruginosa [START_REF] Frank | Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV[END_REF]. MPC11 (mIgG2b-2) is the control isotype of mAb166. Both were supplied as sterile, pyrogen-free solution in phosphate-buffered saline (PBS), in accordance with good manufacturing practice, by BioXcell (USA) with endotoxin level < 1 EU/ml (according to their certificate of analysis).

They were supplied as follows: mIgG2b-1 [2.5mg/mL], mIgG2b-2 [8.5mg/mL]. In some experiments, Ab formulations were supplemented with Polysorbate 80 (Sigma-Aldrich, France) at 0.01 or 0.05% (final volume) prior to nebulization.

Antibody nebulization

For sterility purposes, this procedure was performed under a cell-culture hood. All antibodies, in suspension were filtered on a 0.22µm syringe filter (Millipore, France). For in vitro assay, all antibodies were diluted in PBS1X to a final concentration of ~1.7 mg/mL before nebulization. For each Ab, 1 mL were nebulized using a clinically used Aeroneb Pro TM vibrating-mesh nebulizer (Aerogen, Ireland), connected with a 13 mL polypropylene tube (Dutscher, France) or the VITROCELL Cloud 12 system (VITROCELL Systems, Germany), which is the commercial version of the Air-Liquid Interface Cell Exposure-Cloud (ALICE-cloud) system, described by Lenz and colleagues [START_REF] Lenz | Efficient bioactive delivery of aerosolized drugs to human pulmonary epithelial cells cultured in air-liquid interface conditions[END_REF]. Nebulization duration was measured and lasted ~5min for each sample (i.e., 0.4 ml/min liquid output rate). For in vitro aerosol-cell exposures, using the VITROCELL Cloud 12 system, the aerosol cloud was allowed to settle for 20min before removing cells. When necessary, nebulized Ab solutions were filtered on a 0.45µm syringe filter (Millipore, France) prior to further analysis. Nebulizers were washed extensively between each nebulization session using 0.22µm autoclaved water and PBS. Subsequently, nebulized PBS was analyzed by flow cell microscopy and the nebulizer was considered cleaned and operating for Ab nebulization when the nebulized PBS samples contained less than 300 particles/mL. To rule out that cell activation may be attributable to contamination released during nebulization, the activation potency of nebulized PBS (vehicle solution) was also investigated in vitro. Our analysis revealed that nebulized PBS had no impact on MoDC activation (data not shown).

Dynamic Light Scattering (DLS) analysis of antibodies

Native and nebulized Ab were analyzed by dynamic light scattering with a Dynapro Nanostar® (Wyatt Technology, USA) after appropriate dilution to 100 μg/ml in PBS1X1X in UVette (Eppendorf, France). For each sample, the acquisition was performed 10 times for 7 seconds each, at a temperature of 25°C with a detection angle of 90°. Particle diameter -(nm) and polydispersion index (pdi) were analyzed using DynamicsTM software (Wyatt Technology, USA). Samples with less than 70% of successful analyses were con-sidered multimodal and non-analyzable as recommended by the manufacturer (depicted as n/a).

Flow cell microscopy (FCM) of antibodies

Native and nebulized Ab were analyzed by flow cell microscopy with an Occhio® FC200S+ (Occhio, Belgium) after appropriate dilution to 100 μg/ml in medium in the flowcell. Briefly, 250 µL of each antibody solution passed continuously in the flow cell, where particles were automatically detected, sized and counted by a camera. Particle counting, size assessment and distribution were analyzed using CallistoTM software (Occhio, France).

Monocyte-derived dendritic cells (MoDC) preparation

Human peripheral mononuclear cells (PBMC) were purified from cytapheresis, obtained from naïve donors (Etablissement Français du Sang, Hôpital Bretonneau, France) by centrifugation on a Ficoll density gradient (Eurobio, France). MoDC were prepared from PMBC as previously described [START_REF] Spadaro | Generation and Maturation of Human Monocyte-derived DCs Bio-protocol[END_REF]. Briefly, CD14+ monocytes were isolated using magnetic cell sorting (Miltenyi Biotec, Germany) and cultured during 6 days, at 1x106cells/mL in RPMI1640-GlutamaxTM (Gibco, France) supplemented with 10% of FCS (Dutscher, France), 1X Penicillin/Streptomycin (Gibco, France) in the presence of 25ng/mL of IL-4 (Miltenyi Biotec, Germany) and 100 ng/mL of GM-CSF. On day 6, homogeneity and viability of MoDC population was checked by flow cytometry based on their physical characteristics (forward scatter (FSC) and side scatter (SSC)) and incorporation of vital dye (LiveDeadTM, Invitrogen, France). The immature phenotype of MoDC was checked based on their DC-SIGN+, CD80low, CD83low and HLA-DR low phenotype.

MoDC stimulation with antibody preparation

On day 6, immature MoDC were harvested and washed in complete medium (RPMI1640-Glutamax™ supplemented with 10% of FCS (Dutscher, France) and 1X Penicillin/Streptomycin). For experiments using Ab aerosols collected in a 13 mL polypropylene tube, 1x10 5 cells/well were plated in 75µL in a U-bottom 96-well plate (Falcon, BectonDickinson, France) for 4 hours. 75µL of nebulized or native Ab were then added to a final concentration of 1, 10, 100 and 200 µg/mL depending on the experiments. For experiments using the VITROCELL Cloud 12 system, 1x10 5 cells/well were plated in 150µL on a 24-well plate permeable insert (Corning, France) for 18 hours. 200µL of complete was added in the basolateral compartment to prevent cell drying. The insert was then placed in the VITROCELL Cloud 12 system with 200µL in the basolateral compartment and cells were exposed to Ab aerosol as describe above. Inserts were put back on a companion 24-well plate.

Cells were incubated for 18 hours at 37°C with 5% CO2. Lipopolysaccharide (LPS, from Escherichia coli O111:B5, Sigma-Aldrich, France) was used at 1µg/mL as a positive control. Untreated and nebulized PBS1X (Gibco, France) were used as vehicle controls.

Each condition was tested in 3-9 replicates.

Analysis of MoDC activation by flow cytometry

After 18 hours of stimulation, MoDC were harvested and saturated in PBS1X supplemented with 2% of FCS, 2 mM EDTA and 1X of human Fc-Block (Becton Dickinson, France) for 15 min at 4°C. Cells were washed and stained in FACS buffer (PBS1X supplemented with 2% FCS and 2 mM EDTA) with antibodies described in Supplementary table 2, for 20min at 4°C in the dark. Negative FMO controls were generated for each parameter analyzed. All antibodies were from Biolegend (UK). Data acquisition was made on the 8-colors MACSQuant flow cytometer (Miltenyi Biotec, Germany) on a minimum of 10,000 living cells. MoDC analysis was performed using VenturiOne software (Applied Cytometry, USA). Analysis was made on singlet (FSC-A / FSC-H gating) and CD45+ live cells (negative for LiveDead staining). Costimulatory protein expression was expressed as a ratio of the median fluorescence intensity (MFI) of cells treated with nebulized Ab / MFI of cells treated with native Ab.

Cytokine, chemokine and protein assays

All assays were perfomed on Immulon TM 96-well plates (ThermoFischer, France).

Concentrations of CXCL8 (IL-8), human IL-6 in cell-free MoDC supernatants and TNF, IL6, IL1b and CXCL1 (KC) were measured using specific ELISAs (Biolegend, UK; limit of detection: at 15.6 pg/mL) according to the manufacturer's instructions and were normalized based on the mean concentration of total protein. Total protein concentration was determined using a BCA assay (ThermoFischer Scientific, France), according to the manufacturer's instructions (limit of detection: 15 μg/mL).

Animal experiments

Animal experiments have been performed 2-3 times. For each experiments, 5 mice per groups were used. Nebulized or native mIgG2b-1 and mIgG2b-2 (100 µg/animal, in 40µL) were administered orotracheally or through intravenous injection using a restraining tube at days 0, 7, 14, 21 and 28. For orotracheal administration, mice were anesthetized with isofluorane 4% and an operating otoscope fit with intubation specula was introduced to both to maintain tongue retraction and to visualize the glottis. A fiber optic wire threaded through a 20G catheter and connected to torch stylet (Harvard Apparatus, France) was inserted into the mouse trachea. Correct intubation was confirmed using lung inflation bulb test and 40µL of the bacterial solution was applied using an ultrafine pipette tip. For the acute treatment assessment, animals were euthanized using a lethal dose of ketamine/xylazine at 4 hours, 1 day and 14 days after a single administration. In the chronic treatment groups, animals were euthanized at day 29.

At the time of necropsy, blood was recovered by intracardiac puncture.

Bronchoalveolar lavage fluid (BALF) was then collected by cannulating the trachea and washing the lung twice with 1.2 mL of PBS1X at room temperature. The lavage fluid was centrifuged at 400 g for 10 min at 4°C, and the supernatant was stored at -20°C until analysis. The cell pellet was resuspended in FACS buffer and counted in a hemocytometer chamber. Peripheral blood was washed out by intracardiac perfusion with 10 mL of PBS1X. Lung and spleen homogenates were then prepared in 2mL of RPMI1640 containing 125 µg/mL of Liberase (Sigma-Aldrich, France) and 100 µg/mL of DnaseI (Sigma-Aldrich, France) using a GentleMACS tissue homogenizer (Miltenyi Biotec, Germany). Cells were isolated through a 100µm cell strainer and purified with a 20% Percoll (GE Healthcare, France) density gradient. Cell preparation was centrifuged at 400 g for 10 min at 4°C, and the pellet was resuspended in FACS buffer, and counted in a hemocytometer chamber.

Immune cell phenotyping by flow cytometry BALF, lung and spleen cells isolated as described above were saturated in PBS1X supplemented with 2% of FCS, 2 mM EDTA and 1X of human Fc-Block (BectonDickinson, France) for 15 min at 4°C. Cells were washed and stained with antibodies described in Supplementary table 2 for 20min at 4°C in the dark. Data acquisition was made on the 8-colors MACSQuant flow cytometer (Miltenyi Biotec, Germany) on a minimum of 100,000 living cells. Analysis was performed using VenturiOne software (Applied Cytometry, USA). Analysis was made on singlet (FSC-A / FSC-H gating) and CD45+ live cells (negative for LiveDead staining). Immune cell phenotypes were determined as described in the Supplementary table 2. Data were normalized based on the mean concentration of respective native Ab conditions or expressed as a number of cell calculated as follow: % of the total CD45+ immune cell population x total number of cell.

For Annexin-V/PI staining, data were normalized as follows: % of the positive population for nebulized Ab / % of the positive population for native Ab.

Histology

Lungs were fixed in 10% buffered formalin (Shandon), dehydrated in ethanol and embedded in paraffin. Serial sections (3 mm) were stained with haematoxylin and eosin (HE) or Congo Red. Ten HE sections per mouse were randomly evaluated to count cell nucleus using a machine-learning pixel classification plugin for Image J (https://imagej.net/plugins/tws/), as described previously [START_REF] Arganda-Carreras | Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification[END_REF].

Statistical analysis

Differences between experimental groups were determined using Kruskal-Wallis or t-test comparing two groups, using one or two-way analysis of variance (ANOVA) followed by Newman-Keuls or Bonferroni post-test (for comparison between more than two groups), after confirmation that the data were normally distributed. All statistical tests were performed with GraphPad Prism software (version 4.03 for Windows, GraphPad Software Inc., USA). All data are presented as mean ± standard error of the mean (SEM). The threshold for statistical significance was set to p<0.05.

Results

Aggregation of antibodies is heterogeneous during nebulization

Nebulization promotes Ab aggregation [START_REF] Respaud | Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF][START_REF] Bodier-Montagutelli | Protein stability during nebulization: mind the collection step![END_REF]. In this study, mesh-nebulization was used since it is less deleterious on Ab and often considered for aerosolization of proteins [START_REF] Bodier-Montagutelli | Designing inhaled protein therapeutics for topical lung delivery: what are the next steps?[END_REF]. Full-length Ab commercially available or under preclinical development were reformulated in PBS and nebulized with the Aeroneb Pro™ vibrating-mesh nebulizer.

Ab aggregation results in a broad range of particles, from dimers (several nanometers) to micron-sized, and even visible particles in some cases [START_REF] Zolls | Particles in therapeutic protein formulations, Part 1: overview of analytical methods[END_REF], requiring the combination of different complementary analytical techniques. The aggregation profiles of Ab were assessed using DLS and FCM to report particles at the submicrometric and micrometric scales, respectively (Figure 1). Nebulization led to an increase in the concentration of particles > 2 µm for hIgG1-1, mIgG2b-1 and mIgG2b-2, as compared to their native counterparts. Conversely, no significant increase in particles was observed for hIgG1-2, hIgG1-3 (Figure 1). Furthermore, comparing nebulized Ab, we observed that hIgG1-1 and mIgG2b-1 had significantly more particles than the other human and murine Ab, respectively. In addition to differences in total particle concentration, there were also differences in particle size distribu-tion. Nebulized hIgG1-1 and -2 had more than 78% of their particles smaller than 5 µm, while hIgG1-3, mIgG2b-1 and mIgG2b-2 comprised more than 33% of their particles above 5 µm (Table 1). For murine mIgG2b-1 and mIgG2b-2, aggregation was also observed at the submicron scale, most notably for nebulized mIgG2b-1, which could not even be analyzed by DLS due to the large heterogeneity of the particle size distribution (most likely all-sized Ab aggregates), which cannot be clearly structured in defined size modes -a prerequisite for DLS measurements. Nebulized mIgG2b-2 exhibited a significant reduction (7%) of monomeric Ab amount (Supplementary Table 1). Overall, our results highlight the huge heterogeneity of Ab aggregation during mesh nebulization, most likely depending on Ab sequence/structure. Table 1. Particle size distribution (%) of nebulized antibodies.

Ab aggregates, produced during mesh-nebulization, activate antigen-presenting cells

The dramatic consequences of adverse immunogenicity have prompted regulatory authorities to establish a guidance to test the immunogenicity of therapeutic protein products and industry to propose screening approaches [START_REF] Ema | Guideline on Immunogenicity assessment of therapeutic proteins[END_REF][START_REF] Fda | Immunogenicity Assessment for Therapeutic Protein Products -Guidance for Industry[END_REF]. They include investigating the ability of protein aggregates to activate immune cells, in vitro. Here, we incubated human monocyte-derived dendritic cells (MoDC) overnight with native or nebulized Abs (hIgG1-1 to -3) and analyzed both the release of pro-inflammatory cytokines and expression of co-stimulatory proteins involved in the DC-T synapse (CD25, CD83, CD86 and CD80). Neither the native Ab, nor the nebulized buffer without Ab (data not shown) promoted IL-6 or IL-8 production by MoDC (Figures 2A and2B) and modulated cell markers as compared to untreated MoDC (Figures 2E-H).

Nebulized and aggregated hIgG1-1 induced a significant and dose-dependent increase of cytokine production (Figures 2A-D) whereas the other nebulized Ab solutions had only a minor and inconsistent impact on cytokine level. The nebulized and aggregated hIgG1-1 induced a slight but significant increase in all cell markers, whereas the effect of other nebulized Ab was limited (Figures 2E-H). Interestingly, the filtration of Ab solutions after nebulization, removing micrometric particles (data not shown), resulted in the abrogation of both cytokine release and expression of co-stimulatory markers by MoDC (Figures 2A-H, gray bars). In addition, when comparing nebulized antibodies, our analysis revealed that activation potency of hIgG1-1 was higher than for hIgG1-2 and hIgG1-3. Altogether, our results suggest that activation of APC was attributable to the presence of aggregates and that the extent of this activation was correlated to the number of particles (Figure 1). Since aerosol collection system may modulate the amount and the size distribution of Ab aggregates after nebulization [START_REF] Bodier-Montagutelli | Protein stability during nebulization: mind the collection step![END_REF], we determined whether collection might induce a bias in APC activation. We exposed directly MoDC to hIgG1-1 aerosols using the VITROCELL Cloud 12 system [START_REF] Lenz | Efficient bioactive delivery of aerosolized drugs to human pulmonary epithelial cells cultured in air-liquid interface conditions[END_REF]. As observed in Supplementary Figure 1, we obtained similar results on MoDC activation independent of whether the cells were directly exposed to hIgG1-1 aerosols (through use of the VITROCELL Cloud 12 system) or exposed to them after collection as a bulk solution supporting our decision of adopting the latter approach. Taken together, our results showed that nebulized Ab induced MoDC maturation and activation as compared to native Ab, and confirmed the involvement of Ab aggregates in this response.

High-level of nebulization-mediated antibody aggregates impair lung cell homeostasis after lung delivery

Historically, in vitro assays have been widely used to describe the potential immunogenicity of biotherapeutic aggregates [START_REF] Joubert | Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses[END_REF]. However, they display several limitations: (i) the amount of aggregates inducing a response in vitro may not directly translate into in vivo response and (ii) they may not predict the impact of the pulmonary delivery route.

To gain insight into the broad effects of Ab aggregates produced by aerosolization within the lung compartment, we selected two murine IgG2b -mIgG2b-1 and mIgG2b-2-producing different amounts of aggregates during mesh-nebulization (Figure 1) which differentially activate MoDC (Supplementary figures 2). We administered them through the airways in naïve mice. Remarkably, the nebulized and aggregated mIgG2b-1 resulted in a dramatic reduction in the total cell number in the airway compartment of mice (BAL), as compared to native antibodies administered by the same route (Figure 3A). Moreover, nebulized mIgG2b-2, producing 10-fold less aggregates after mesh-nebulization than mIgG2b-1 (Figure 1), showed no statistically significant reduction in BAL cell number (Figures 3G). Of note, the animals that received the native Ab through the airways had similar cell count as sham animals (data not shown), which implies that the oro-tracheal application itself did not obfuscate our results. Analysis of the cellular phenotype of the BAL cells revealed an analogous result for immune (CD45+ leukocytes) cells (Figures 3B and3H). Further analysis did not reveal any impact on specific lineage as almost all immune cell lineages were affected after the administration of aggregated Ab (Supplementary Figures 3A-E). In the lung tissue, nebulized mIgG2b-1 caused a 2-fold reduction in total cell and leukocyte counts relative to controls (Figures 3C-D), while nebulization of mIgG2b-2 did not modify leukocyte counts (Figures 3I andJ). This decrease affected both myeloid (neutrophil, monocyte, dendritic cell) (Supplementary Figures 3F-I) and lymphoid lineages (B, T CD4+/CD8+ cells) (Supplementary Figures 3J-L). The modification of cell homeostasis in the airway compartment was not associated with an alteration of the lung epithelial barrier (Supplementary figure 4), but was mostly attributable to micron-sized particles as 0.45µm-filtration of nebulized mIgG2b-1 prevented these adverse effects (Figures 3A-F, gray bars). The filtration step did not significantly modify Ab particle size distribution (Supplementary Tables 4 and5) or concentration (Supplementary Table 6), as compared to native Ab. Interestingly, we observed protein aggregates (appearing as apple-green birefringence structures under polarized light) on Congo-red stained lung sections from animals treated with nebulized mIgG2b-1 (Figure 3L). Unsupervised machine learning, which was used to quantify cell nucleus on HE-stained lung section, showed that local administration of nebulized mIgG2b-1 was associated with a significant reduction of lung cells after 18 hours (Figures 3K and3M). Unexpectedly, the number of total and immune cells was also diminished, even though to a lesser extent, in the spleen (Figures 3E-F), indicating that the impairment of cellular homeostasis reached the systemic compartment.

Contraction of cell number 18h after a single airway administration of nebulized Ab primarily occurred in the airways, and then probably extended through the lung tissue and systemically as it was restricted to the airway compartment after 4 hours (Figures 4A-4F). Moreover, this effect was sustained for at least up to 14 days after Ab administration (Figures 4G and4H), or after repeated administrations (Figures 4I-4N). For either single or repeated administrations of nebulized mIg2b-1, we did not observe any sign of general toxicity, including body-weight loss (data not shown). Overall, our results suggest that airway administration of aggregated IgG (>0.45 µm) profoundly affected cellular homeostasis, in a time-dependent manner, both locally and systemically.

Nebulized aggregated antibody induced immunologically silent cell death after lung administration

Next, we investigated the mechanisms accounting for host cell contraction and hypothesized that it was associated with cell death. Cell death occurs in multiple forms and can be divided in accidental cell death (ACD; necrosis) or regulated cell death (RCD; apoptosis) [START_REF] Galluzzi | Regulated cell death and adaptive stress responses[END_REF]. ACD is characterized by a dramatic and instantaneous collapse of cells and can be triggered in response to different stresses, including chemical, physical or mechanical insults, whereas RCD relies on committed molecular machinery [START_REF] Galluzzi | Essential versus accessory aspects of cell death: recommendations of the NCCD 2015[END_REF]. Using Annexin-V/propidium iodide (PI) staining, which allows the discrimination of early apoptotic cells (Annexin-V+/PI-), late apoptotic cells (Annexin-V+/PI+) and necrotic cells (Annexin-V-/PI+) [START_REF] Vermes | Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V[END_REF], we quantified the proportion of each cell death phenotypes in both total cells and CD45+ leukocytes population, 18 hours after a single administration of mIgG2b-1. We observed that administration of nebulized mIgG2b-1 provoked a significant increase of both late apoptotic and necrotic spleen cells while lung and airway cells were suffering from necrosis.as compared to mice treated with native Ab (Figures 5A-5F) or with nebulized mIgG2b-2 (Supplementary Figure 5). The type of cell death could also be determined by the analysis of mediators released in the environment. When comparing BALs from animals treated with native and nebulized mIgG2b-1, we did not observe any significant difference in the production of TNF, IL-6, IL-1b or CXCL1 (KC) (Figures 5G-J). These data suggested that the cell contraction occurring after single or multiple airway administration of nebulized and aggregated IgG was associated with an inflammatory silent cell death process.

The effect of nebulization-mediated antibody aggregates on immune cell homeostasis is specific of the pulmonary route

Immunogenicity of Ab is also dependent of their route of administration [START_REF] Demeule | Where disease pathogenesis meets protein formulation: renal deposition of immunoglobulin aggregates[END_REF][START_REF] Kiese | not stirred: mechanical stress testing of an IgG1 antibody[END_REF][START_REF] Fathallah | Immunogenicity of subcutaneously administered therapeutic proteins--a mechanistic perspective[END_REF]. Thus, we investigated the effect of native or nebulized-mIgG2b-1 after intravenous injection. In contrast to what was observed after airway administration, there were no significant differences in the BAL or lung cell counts in the animals who received native or nebulized mIgG2b-1 intravenously (Figures 6A-6D). Interestingly, these results were substantiated when analyzing cell number after repeated administration of nebulized mIgG2b-1 (Supplementary Figure 6), where no differences were noticed in the airways or lungs of animals treated by repeated intravenous injections as compared to animals, which received the IgG in the lungs. Our data suggested that the route of administration played an important role on the adverse effect of IgG aggregates produced during nebulization on cell homeostasis.

Reducing aggregation limits pulmonary cytotoxicity associated to lung administration of nebulized Ab

Pharmaceutical development aims to design a high-quality product ensuring an efficacious and safe treatment along the life of the product. Hence, formulation and protein engineering are often adapted to limit Ab aggregation, especially considering chronic-based therapies. Different parameters, including addition of surfactant, have a protective effect, limiting Ab aggregation during nebulization [START_REF] Respaud | Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF]. Here, we added polysorbate80 (PS80) in mIgG2b-1 formulation to significantly reduce its aggregation during nebulization (Supplementary Figure 7). The addition of surfactant did not significantly modify Ab particle size distribution (Supplementary Tables 4 and5) or concentration (Supplementary Table 6) as compared to unformulated Ab. Single administration of nebulized mIgG2b-1 supplemented with 0,05% of PS80 abrogated the reduction of lung cell number (Figures 7C andD) and to a lesser extent of airway cell (Figures 7A andB) as compared to non-formulated mIgG2b-1. This was likely attributable with a reduction of cell death in the same compartment (Supplementary Figure 8). These results suggest that optimizing IgG formulation improved its molecular stability and might limit adverse effects on cell homeostasis.

Discussion

Ab are highly sensitive to stresses encountered during their product development, manufacturing, storage or clinical use, and they often require high therapeutic doses, necessitating high concentration drug products with a higher risk of aggregation.

Tracking aggregates prior and after Ab bioprocessing is essential to avoid risks for patients, treatment failure and ultimately termination of drug development/commercialization.

Protein aggregation has been widely demonstrated as an influential factor in the induction of adverse immunogenicity of biotherapeutics [START_REF] Moussa | Immunogenicity of Therapeutic Protein Aggregates[END_REF][START_REF] Ratanji | Immunogenicity of therapeutic proteins: influence of aggregation[END_REF]. However, some of the stress conditions used in the literature are not representative of those experienced during product development, manufacturing, storage or clinical use, making the aggregates generated far different from those found in marketed products. This may hamper conclusions about the exact potency of protein aggregation in the occurrence of drug immunogenicity. Several intrinsic or extrinsic attributes of aggregates might work synergistically to induce immunogenicity. Among them, parameters related to the delivery route can affect the immune response associated with the administration of drugs. For example, subcutaneous delivery has been associated with higher immunogenicity than the intravenous route, for several biotherapeutics [START_REF] Ross | Immunogenicity of interferon-beta in multiple sclerosis patients: influence of preparation, dosage, dose frequency, and route of administration. Danish Multiple Sclerosis Study Group[END_REF][START_REF] Hermeling | Micelle-associated protein in epoetin formulations: aA risk factor for immunogenicity?[END_REF]. To our knowledge, there is not any study conducted so far to assess the immunogenicity associated with lung administration of an aerosolized and aggregated Ab. We set out a dual experimental approach to investigate the quality and the extent of the immune responses induced by a nebulized and aggregated Ab using both in vitro and in vivo models. Here, we showed for the first time that the aggregates resulting from IgG nebulization induced immune cell over-activation and that their delivery to the lung markedly and durably impaired cell homeostasis.

Protein aggregation is a process of non-specific association of monomers through multiple physical and chemical pathways which are well documented [START_REF] Cromwell | Protein aggregation and bioprocessing[END_REF]. The characteristics of protein aggregates are variable in terms of particle size, number, morphology, chemical modifications, reversibility, conformation or hydrophobicity [START_REF] Joubert | Classification and characterization of therapeutic antibody aggregates[END_REF]. This phenotypic heterogeneity results from the various stresses applied to proteins and require specific analytical methods [START_REF] Wang | Protein aggregation and its inhibition in biopharmaceutics[END_REF]. Aerosol generation involves the dispersion of liquid droplets into a gas. This process may be associated with physical stresses including temperature variations and the generation of a massive air-liquid interface, which ultimately induce changes in protein conformation and lead to its aggregation [START_REF] Respaud | Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF]. We measured the aggregation of several human IgG submitted to aerosolization using a vibrating-mesh nebulizer, which is expected to be less deleterious than other nebulizers [START_REF] Lightwood | The discovery, engineering and characterisation of a highly potent anti-human IL-13 fab fragment designed for administration by inhalation[END_REF][START_REF] Maillet | immunological and pharmacological properties of the anticancer antibody cetuximab following nebulization[END_REF]. Flow cell microscopy and dynamic light scattering revealed that the number and size of aggregates were Ab dependent, confirming the results in the literature on the necessity of a case-by-case approach. Besides, drastic differences were observed between commercial Abs (hIgG1-1 to 3) and murine Abs (mIgG2b-1 and -2), where the latter displayed higher aggregation upon nebulization. This may be explained by the fact that commercial Abs underwent advanced development programs and were thus selected for their limited aggregation potency. We next determined whether the aggregates found in the therapeutic product after nebulization may induce immunogenicity using a MoDC-based assay, which has been widely used to describe the potential immunogenicity of biotherapeutics or unwanted products [START_REF] Kraus | Test models for the evaluation of immunogenicity of protein aggregates[END_REF]. Our analysis revealed that nebulized and aggregated hIgG1 -in particular hIgG1-1, were able to induce MoDC activation and maturation, as evidenced by the enhanced secretion of cytokines and increased expression of co-stimulatory proteins on MoDC. This response correlated with the fraction of micron-sized aggregates in the Ab aerosol. There are still discrepancies regarding the size and type of aggregates involved in the generation of immunogenic responses [START_REF] Carpenter | Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality[END_REF]. This depends on the type and strength of the stress applied and the multiple experimental protocols which have been described [START_REF] Kiese | not stirred: mechanical stress testing of an IgG1 antibody[END_REF][START_REF] Telikepalli | Characterization of the physical stability of a lyophilized IgG1 mAb after accelerated shipping-like stress[END_REF]. Here, hIgG1-1 aerosol is mainly composed of small-sized particles (2-10µm), which have been shown to enhance the immune response and be the most immunogenic [START_REF] Carpenter | Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality[END_REF][START_REF] Xiang | Pathogen recognition and development of particulate vaccines: does size matter?[END_REF].

One potential bias regarding Ab aggregation could be attributed to the aerosol collection step, which uses a polypropylene tube to re-pool aerosol droplets into a bulk liquid. Indeed, it has been evidenced that the aerosol collection device could influence protein stability, generating different aggregation profiles [START_REF] Bodier-Montagutelli | Protein stability during nebulization: mind the collection step![END_REF][START_REF] Hertel | Comparison of Aerosol Collection Methods for Liquid Protein Formulations[END_REF]. In this context, we used the VITROCELL technology, which was developed to improve reliability of toxicological studies of aerosolized compounds on air-liquid interface cell cultures [START_REF] Brandenberger | Quantitative evaluation of cellular uptake and trafficking of plain and polyethylene glycol-coated gold nanoparticles[END_REF][START_REF] Brandenberger | Effects and uptake of gold nanoparticles deposited at the air-liquid interface of a human epithelial airway model[END_REF]. This system, which allows direct deposition of aerosols on cells, avoids the collection step [START_REF] Rohm | A comprehensive screening platform for aerosolizable protein formulations for intranasal and pulmonary drug delivery[END_REF].

Our results showed that direct hIgG1-1 deposition on MoDCs resulted in cell activation, as evidenced by the similar increase in both cytokine production and expression of costimulatory proteins than those obtained with the nebulized-collected Ab. Thus, the collection system used here was considered relevant for these assays and was kept for the in vivo experiments.

To take into account the complexity of the lung mucosal-associated immune system, it was necessary to use an animal model to predict the immunogenicity of nebulized Ab in vivo [START_REF] Shomali | Antibody responses in mice to particles formed from adsorption of a murine monoclonal antibody onto glass microparticles[END_REF][START_REF] Freitag | Investigation of the immunogenicity of different types of aggregates of a murine monoclonal antibody in mice[END_REF]. We chose murine antibodies to avoid a high immunogenic background response due to the non-specific activation of the mouse immune system by foreign proteins. In our study, we observed that single or repeated administration of a nebulized antibody in the lungs induced a significant contraction of the total and immune cell number in the airways starting quickly after the administration as compared to native Ab or saline controls. This was dependent on the number of aggregates as low aggregated Ab, filtered or PS80 preparation of nebulized Ab did not have any impact on cell number.

Interestingly, at later time-points this contraction reached the lung parenchyma and the spleen, reducing significantly the number of both myeloid and lymphoid cells. We wondered whether this cell number reduction induced by the administration of nebulized Ab was associated with cell death. We observed that apoptosis were significantly increased in lung total cells and in leukocytes after airway administration of nebulized Ab. Interestingly, these cellular injuries were dependent on the presence of aggregates, as Ab formulated with surfactant, known to limit aggregation [START_REF] Respaud | Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF], and even filtered nebulized Ab preparation did not promote cell death. These adverse effects were also dependent on the route of administration as nebulized Ab administered through the intravenous route was not associated with the same cell number reduction. The complete understanding of the molecular and cellular mechanism associated with the massive cytotoxicity of nebulized and aggregated antibodies requires further investigations.

Protein aggregation underlies many chronic diseases where aggregates are thought to elicit injury including cell apoptosis [START_REF] Lim | Neuronal aggregates: formation, clearance, and spreading[END_REF][START_REF] Bucciantini | Prefibrillar amyloid protein aggregates share common features of cytotoxicity[END_REF]. To the best of our knowledge, this is the first study reporting that extracellular therapeutic protein aggregates may sensitize the host to cytotoxicity. This cellular injury occurred in the absence of a pro-inflammatory response which is contradictory with the current paradigm regarding the induction of innate immune responses by protein aggregates [START_REF] Joubert | Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses[END_REF]. This discrepancy may come from the attributes of Ab aggregates associated with nebulization, as compared to the ones associated with other aggregation stresses, including the formation of neoepitopes, the immunomodulatory properties of the aggregates, the exposure of post-translational modifications or the generation of danger signals [START_REF] Dingman | Immunogenicity of Protein Pharmaceuticals[END_REF]. A complete understanding of the physical mechanisms accounting for the immunogenic properties of nebulized Ab aggregates is beyond the scope of this study. Immunogenicity may also be associated with the breakdown of self-tolerance rather than an active immune response [START_REF] Dingman | Immunogenicity of Protein Pharmaceuticals[END_REF]. It is particularly concordant with repeated exposure which may occur during dosing regimen for chronic disease [START_REF] Ratanji | Immunogenicity of therapeutic proteins: influence of aggregation[END_REF].

In conclusion, we demonstrated that aerosolization using a clinically relevant nebulizer induced Ab aggregation and resulted in immune cell activation and immunocytotoxicity in vivo. Although there are still many questions to address to understand better the relationship between Ab aggregates and immunogenicity, our findings point a significant role of the route of administration in the immunogenic/biological response associated to Ab aggregates. Further investigations will be required to determine the types and the amount of aggregates and the role of the Fc domain in the immunocytotoxic response of Ab aggregates produced during nebulization. Our findings also highlight the importance to explore further the different methods (protein engineering, aerosolization process, formulations) to stabilize further Ab during aerosolization to minimize risks for the patients.

Figure 1 .

 1 Figure 1. Antibodies were either nebulized using an Aeroneb Pro TM vibrating-mesh nebulizer and collected (Nebulization +) or left untreated (Nebulization -). The total number of particles (with diameter >2µm) was quantified using a flow microscope. The data are quoted as the mean ± SEM. *, ** : p<0.05, and p<0.01 respectively, in a one-way ANOVA with Newman-Keuls's correction for multiple comparisons. The results represent three to eight independent nebulizations.

Figure 2 .

 2 Figure 2. MoDC were stimulated using equal final concentration of Ab at 100 µg/mL either native (white bars), nebulized (black bars) or nebulized and 0.45µm-filtered (gray bars) for 18h. IL6 (A) and IL8 (B) were quantified in cell-free supernatant. MoDC were stimulated with 1, 10, 100 or 200µg/mL (gray to black bars) of nebulized hIgG1 or 100µg/mL of nebulized and 0.45µm-filtered hIgG1 (last bar) or left untreated (white bars) for 18h. IL6 (C) and IL8 (D) were quantified in cell-free supernatant. MoDC were stimulated using equal final concentration of Ab at 100 µg/mL either native (white bars), nebulized (black bars) or nebulized and 0.45µm-filtered (gray bars) for 18h.CD25 (E), CD83 (F), CD86 (G), CD80 (H) expression were measured using flow cytometry. The data are quoted as the mean ± SEM. *, **, *** : p<0.05, p<0.01 and p<0.001 respectively, in a one-way ANOVA with Newman-Keuls's correction for multiple comparisons. The results are representative of six independent experiments (n=6-9 technical replicates/experiment).

Figure 3 .

 3 Figure 3. B6 mice received a 40 µL orotracheal instillation of mIgG2b-1 at 100 µg/mL either native (white bars), nebulized (black bars) or nebulized and 0.45µm-filtered (gray bars). The total number of cells (A, C and E) and CD45+ cells (B, D and F) were quantified in BAL (A and B), in the lungs (C and D) and the spleen (E and F) using flow cytometry, 18h after the administration. B6 mice received a 40 µL orotracheal instillation of mIgG2b-2 at 100 µg/mL either native (white bars) or nebulized (black bars). The total number of cells (G and I) and CD45+ cells (H and J) were quantified in BAL (G and H), in the lungs (I and J) using flow cytometry, 18h after the administration. Lung tissues of mice treated with either native (white bars) or nebulized (black bars) mIgG2b-1 were histologically examined 18h after the administration. (K) Hematoxylin-eosin sections were used to

Figure 4 .

 4 Figure 4. B6 mice received a 40 µL orotracheal instillation of mIg2b-1 at 100 µg/mL either native (white bars) or nebulized (black bars) through the airways at D0, or D+0, D+7, D+14, D+21 and D+28. The total number of cells (A , C, E, G, I, K and M) and CD45+ cells (B, D, F, H, J, L and N) were quantified in BAL (A, B, I and J), in the lungs (C, D, G, H, K and L) and the spleen (E, F, M and N) using flow cytometry, 4h, 14 days or 29 days after the first administration. The data are quoted as the mean ± SEM. *, ** : p<0.05 and p<0.01 respectively, in a t-test. The results are representative of two independent experiments (n=5 mice /experiment).

Figure 5 .

 5 Figure 5. B6 mice received a single 40 µL orotracheal instillation of mIg2b-1 at 100 µg/mL either native (white bars) or nebulized (black bars). The proportion of early apoptotic cells (Annexin-V+/PI-), late apoptotic cells (Annexin-V+/PI+) and necrotic cells (Annexin-V-/PI+) were quantified in total cell (A, C and E) or CD45+ cell (B, D and F), 18h after the administration in BAL (A and B), lungs (C and D) and spleen (E and F) relative to mice treated with native Ab using flow cytometry. The concentrations of TNF (G), IL6 (H), IL1b (I) and KC (J) in BALF were determined 18h after the administration. The data are quoted as the mean ± SEM. *, **: p<0.05 and p<0.01 respectively, in a t-test. The results are representative of three independent experiments (n=5 mice/experiment).

Figure 6 .

 6 Figure 6. B6 mice received a 40 µL orotracheal instillation or 100µL intravenous injection of mIgG2b-1 at 100 µg/mL either native (white bars) or nebulized (black bars). The total number of cells (A and C) and CD45+ cells (B and D) were quantified in BAL (A and B) and in the lungs (C and D) using flow cytometry, 18h after the administration. The data are quoted as the mean ± SEM. *, **, ***: p<0.05, p<0.01 and p<0.001 respectively, in a t-test. The results are representative of two independent experiments (n=5 mice/experiment).

Figure 7 .

 7 Figure 7. B6 mice received a 40 µL orotracheal instillation of mIgG2b-1 at 100 µg/mL either native (white bars), nebulized (black bars) or nebulized + Polysorbate80 0.05% (PS80 0.05%, gray bars). The total number of cells (A and C) and CD45+ cells (B and D) were quantified in BAL (A and B) and in the lungs (C and D) using flow cytometry, 18h after the administration. The data are quoted as the mean ± SEM. *, **, ***: p<0.05, p<0.01 and p<0.001 respectively, in a t-test. The results are representative of two independent experiments (n=5 mice/experiment).

Acknowledgments:

We sincerely thank David Fraser PhD (Biotech Communication SARL, France) for copy-editing services and Dr. Roxane Lemoine (Université of Tours, EA4245) for providing MoDC.

Funding: This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (LabEx MAbImprove, ANR-10-LABX-53-01). TS is funded by a fellowship from ANR-10-LABX-53-01.

Data Availability Statement:

The data supporting this article will be shared on reasonable request to the corresponding author.

Supplementary Materials:

The following supporting information can be downloaded at: Lung cell count after administration of nebulized mIgG2b-1 supplemented with PS80-0.05%; Table S1: Dynamic light scattering analysis of nebulized antibodies; Table S2: List of antibodies used for flow cytometry; Table S3: Phenotype of analyzed cells; Table S4: Particle size distribution (%) of nebulized antibodies; Table S5: Dynamic light scattering analysis of nebulized antibodies; Table S6: Nanodrop analysis of antibody concentration.

Author Contributions: TS and NHV conceived the study. All authors substantially contributed to the acquisition, analysis or interpretation of data. GI performed machine-learning analysis of histological sections. OS loaned the VITROCELL Cloud 12 system and provided expertise regarding the ALICE-cloud system. TS and NHV contributed to manuscript drafting, revising and critically reviewing. All authors approved the final version of this manuscript to be published.

Conflicts of Interest:

NHV is co-founder and scientific expert for Cynbiose Respiratory. In the past two years, she received consultancy fees from Eli Lilly, Argenx, Novartis and research support from Sanofi and Aerogen Ltd. RML is Head of R&D, Science and Emerging Technologies in Aerogen. All other authors have no competing interests to declare.


Guillaume Desoubeaux

Carolina Le-Bert

Vanessa Fravel

Tonya Clauss

Alexa Delaune

Jeny Soto

Eric Jensen

Jennifer Flower

Randall Wells

Gregory Bossart

Evaluation of a genus-specific ELISA and a commercial Aspergillus Western blot IgG® immunoblot kit for the diagnosis of aspergillosis in common bottlenose dolphins (Tursiops truncatus)

Keywords: marine mammals, blood test, cetaceans, immunoblot, serology, galactomannan

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

1

Evaluation of a genus-specific ELISA and a commercial Aspergillus Western blot IgG ® immunoblot kit for the diagnosis of aspergillosis in common bottlenose dolphins (Tursiops

truncatus)

Guillaume DESOUBEAUX [START_REF] Sherif | Pulmonary aspergillosis: clinical presentation, diagnostic tests, management and complications[END_REF][START_REF] Lortholary | Epidemiological trends in invasive aspergillosis in France: the SAIF network (2005-2007)[END_REF][START_REF] Bitar | Population-based analysis of invasive fungal infections, France, 2001-2010[END_REF] , Carolina LE-BERT [START_REF] Desoubeaux | Diagnosis of invasive pulmonary aspergillosis: updates and recommendations[END_REF] , Vanessa FRAVEL [START_REF] Tell | Aspergillosis in mammals and birds: impact on veterinary medicine[END_REF] , Tonya CLAUSS 6 , Alexa J. DELAUNE [START_REF] Cray | Serosurvey and diagnostic application of antibody titers to Aspergillus in avian species[END_REF] , Jeny SOTO 1 , Eric D. JENSEN [START_REF] França | Serologic testing for aspergillosis in commercial broiler chickens and turkeys[END_REF] , Jennifer E. FLOWER [START_REF] Fischer | Comparison of diagnostic tools for the detection of aspergillosis in blood samples of experimentally infected falcons[END_REF] , Randall WELLS [START_REF] Abdo | Disseminated mycosis in a killer whale (Orcinus orca)[END_REF] , Gregory D.

BOSSART [START_REF] Sherif | Pulmonary aspergillosis: clinical presentation, diagnostic tests, management and complications[END_REF][START_REF] Cray | Serosurvey and diagnostic application of antibody titers to Aspergillus in avian species[END_REF] , and Carolyn CRAY 1,*

Introduction

Aspergillosis is a fungal airborne infection caused by ubiquitous molds belonging to the Aspergillus genus. In humans, aspergillosis is responsible for a wide array of distinct clinical conditions categorized as invasive form, chronic disease e.g. aspergilloma, and/or allergic affection e.g. allergic bronchopulmonary aspergillosis. [START_REF] Sherif | Pulmonary aspergillosis: clinical presentation, diagnostic tests, management and complications[END_REF][START_REF] Lortholary | Epidemiological trends in invasive aspergillosis in France: the SAIF network (2005-2007)[END_REF] Mortality rates of invasive aspergillosis in humans are estimated at 30-100% and depend on both the underlying medical condition and the delay in achieving a definitive diagnosis. [START_REF] Lortholary | Epidemiological trends in invasive aspergillosis in France: the SAIF network (2005-2007)[END_REF][START_REF] Bitar | Population-based analysis of invasive fungal infections, France, 2001-2010[END_REF] In spite of recent development of new laboratory tools like quantitative polymerase chain reaction (qPCR) or measurement of galactomannan (GM) antigen, the routine diagnosis of invasive aspergillosis remains difficult: [START_REF] Desoubeaux | Diagnosis of invasive pulmonary aspergillosis: updates and recommendations[END_REF] it is indubitably achieved when suggestive histopathological findings are reported from biopsy samples. Such procedures, however, are invasive and often not readily achievable. [START_REF] Desoubeaux | Diagnosis of invasive pulmonary aspergillosis: updates and recommendations[END_REF] Evaluating new noninvasive methodologies are thus essential in aiding the diagnosis of aspergillosis.

Definitive diagnosis of aspergillosis in animals is also challenging, [START_REF] Tell | Aspergillosis in mammals and birds: impact on veterinary medicine[END_REF] especially in avian species. 6- 8 In marine mammals, Aspergillus-related disease is assumed to be rare, but has been reported with increasing frequency: 66.7% of the 18 reported cases have been published after the year 2000. [START_REF] Abdo | Disseminated mycosis in a killer whale (Orcinus orca)[END_REF][START_REF] Barley | Encephalitis in a northern bottlenose whale[END_REF][START_REF] Dagleish | Fatal mycotic encephalitis caused by Aspergillus fumigatus in a northern bottlenose whale (Hyperoodon ampullatus)[END_REF][START_REF] Lamoth | Aspergillus fumigatus-Related Species in Clinical Practice[END_REF] The major species responsible for infection in cetaceans belong to the Fumigati section, [START_REF] Lamoth | Aspergillus fumigatus-Related Species in Clinical Practice[END_REF] Nigri section and Terrei section in which A. fumigatus stricto sensu (ss), A. niger ss and A. terreus ss are the most frequently isolated, [START_REF] Balajee | Aspergillus terreus complex[END_REF] respectively. In marine mammals, development of aspergillosis is thought to be based on a chronic invasive process which is generally indicative of another disease and/or (sub-)acute physiologic stress, [START_REF] Bunskoek | Successful treatment of azole-resistant invasive aspergillosis in a bottlenose dolphin with high-dose posaconazole[END_REF] but it is rarely associated with severe immunosuppression and profound neutropenia like in humans. [START_REF] Seyedmousavi | Aspergillus and aspergilloses in wild and domestic animals: a global health concern with parallels to human disease[END_REF] Underlying pulmonary disease may affect host defense mechanisms, leading to colonization and potential invasion of bronchial tissue by Aspergillus spp. [START_REF] Reidarson | Bronchoscopic and serologic diagnosis of Aspergillus fumigatus pulmonary infection in a bottlenose dolphin (Tursiops truncatus)[END_REF] However, other organs, including the brain, may also be infected following systemic dissemination. [START_REF] Abdo | Disseminated mycosis in a killer whale (Orcinus orca)[END_REF][START_REF] Dagleish | Fatal mycotic encephalitis caused by Aspergillus fumigatus in a northern bottlenose whale (Hyperoodon ampullatus)[END_REF][START_REF] Dagleish | Intracranial granuloma caused by asporogenic Aspergillus fumigatus in a harbour porpoise (Phocoena phocoena)[END_REF] In marine mammals, and especially in dolphins, laboratory tools are largely less developed than for humans. [START_REF] Dagleish | Fatal mycotic encephalitis caused by Aspergillus fumigatus in a northern bottlenose whale (Hyperoodon ampullatus)[END_REF][START_REF] Cassle | Coinfection by Cetacean morbillivirus and Aspergillus fumigatus in a juvenile bottlenose dolphin (Tursiops truncatus) in the Gulf of Mexico[END_REF][START_REF] Delaney | Occlusive fungal tracheitis in 4 captive bottlenose dolphins (Tursiops truncatus)[END_REF] For instance, qPCR has not be implemented in veterinary laboratories, and advanced medical imaging is not as easily accessible. Also, as reported in humans, sensitivity of blood cultures in bottles placed in automate is very low for Aspergillus spp. [START_REF] Desoubeaux | Diagnosis of invasive pulmonary aspergillosis: updates and recommendations[END_REF] Positive culture from respiratory specimens does not definitively diagnose a true infection, but may reflect a simple colonization of the upper airways or represent an environmental contaminant. [START_REF] Desoubeaux | Diagnosis of invasive pulmonary aspergillosis: updates and recommendations[END_REF] Furthermore, although it was not specifically reported in cetaceans, GM detection was demonstrated to be not totally reliable in some other animal species. [START_REF] Cray | Galactomannan assay and plasma protein electrophoresis findings in psittacine birds with aspergillosis[END_REF] Therefore, alternative non-invasive methods derived from human medicine need to be developed in order to increase the accuracy of the diagnosis in animals, and to make it readily available at lower costs. In such a context of chronic aspergillosis with no associated immunocompromised status, serological assays to detect antibodies directed against Aspergillus would be relatively easy and relevant to perform in animals, like dolphins. [START_REF] Persat | Aspergillus serology, from yesterday to today for tomorrow[END_REF][START_REF] Kurup | Aspergillus antigens: which are important?[END_REF] In this study, we assessed the diagnostic performance of a new commercial immunoblot assay for detection of anti-Aspergillus antibodies, the Aspergillus Western blot IgG ® kit (LDBio Diagnostics, Lyon, France), in comparison with a novel genus-specific enzyme-linked immunosorbent assay (ELISA).

Material and methods

Study samples

Case definitions. Classification of fungal disease in dolphins was derived from the definition utilized in human medicine, [START_REF] Sherif | Pulmonary aspergillosis: clinical presentation, diagnostic tests, management and complications[END_REF][START_REF] Seyedmousavi | Aspergillus and aspergilloses in wild and domestic animals: a global health concern with parallels to human disease[END_REF][START_REF] Marchetti | ECIL recommendations for the use of biological markers for the diagnosis of invasive fungal diseases in leukemic patients and hematopoietic SCT recipients[END_REF][START_REF] De Pauw | Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group[END_REF][START_REF] Arendrup | ECIL-3 classical diagnostic procedures for the diagnosis of invasive fungal diseases in patients with leukaemia[END_REF] which included cases subdivided into "proven" and "probable" infections. The definitive diagnosis of "proven" aspergillosis was based upon histopathological evidence of fungal disease, using Periodic Acid-Schiff or Gomori-Grocott's Methenamine silver staining (i.e. for Aspergillus-diseased cases: presence of invasive, 45°branched, septate filaments within tissues). [START_REF] Desoubeaux | Diagnosis of invasive pulmonary aspergillosis: updates and recommendations[END_REF] The diagnosis of "probable" fungal infection was based upon positive mycological culture on selective fungal media (Sabouraud dextrose agar) at 30 °C and/or 37 °C for seven days, in conjunction with examination findings, clinical signs (e.g. wheezing, changes in vocalizations, weight loss, elevated respiration pace, anorexia, …), imaging investigations (granuloma on celioscopy/endoscopy, infiltrates and peri-bronchial lesions on computed tomography and X-ray radiography), response to antifungal treatment, and any medical records that corroborated the diagnosis. GM antigen detection was performed according to the manufacturer's recommendations (Bio-Rad Clinical Diagnostics, Hercules, CA, U.S.A.), [START_REF] Cray | Serosurvey and diagnostic application of antibody titers to Aspergillus in avian species[END_REF][START_REF] Fischer | Comparison of diagnostic tools for the detection of aspergillosis in blood samples of experimentally infected falcons[END_REF][START_REF] Cray | Galactomannan assay and plasma protein electrophoresis findings in psittacine birds with aspergillosis[END_REF][START_REF] Cray | Application of galactomannan analysis and protein electrophoresis in the diagnosis of aspergillosis in avian species[END_REF] but it was not considered to be a pivotal criterion for the diagnosis of "probable" aspergillosis in dolphins in this study, since it has not been validated so far in such a context. [START_REF] Desoubeaux | Diagnosis of invasive pulmonary aspergillosis: updates and recommendations[END_REF] Dolphins that fell into the category of "possible" aspergillosis (i.e. dolphins with positive mycological cultures or clinical signs of invasive fungal infection, but not both simultaneously [START_REF] Sherif | Pulmonary aspergillosis: clinical presentation, diagnostic tests, management and complications[END_REF][START_REF] Marchetti | ECIL recommendations for the use of biological markers for the diagnosis of invasive fungal diseases in leukemic patients and hematopoietic SCT recipients[END_REF][START_REF] De Pauw | Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group[END_REF] ) were excluded. Clinically-normal dolphins served as the healthy controls in this study.

Observation cohort. Samples from this group were retrospectively assessed to set the diagnostic parameters/cutoffs of the serological assays. They had been opportunistically collected during routine or clinical health assessments from common bottlenose dolphins (Tursiops truncatus) under human care in the United States of America (U.S.A.). Tubes had been totally fulfilled with blood drawn from the tail fluke veins (with no usage of alcohol for preliminary cutaneous disinfection) through an 18-20 gauge, 1-1.5 inch needle (or butterfly set). Samples were thereafter archived in the Department of Pathology & Laboratory Medicine, Division of Comparative Pathology (University of Miami -Miller School of Medicine, Miami FL, U.S.A.) and in the facility of the National Marine Mammal Foundation (San Diego CA, U.S.A.) until testing. There were no non-inclusion criteria based on sex/age or underlying disease.

Overall, 32 samples had been obtained from Aspergillus-diseased cases with "proven" or "probable" diagnosis. Most were attributable to species of the Fumigati section, except eight characterized by species of the Flavi, Nigri and Terrei sections. Nine samples were drawn from dolphins affected by other invasive fungal infections, sometimes concurrent in certain individuals, including candidiasis (n=6), cryptococcosis (n=2), histoplasmosis (n=2), Coccidioides immitis infection (n=2), mucormycosis (n=1, in association with "proven" aspergillosis), and served as non-Aspergillus controls (except for the dolphin with mixed infection which was included in the case group). Forty-seven samples were obtained from clinically-normal dolphins, with no biological evidence of invasive fungal infection.

Validation cohort. Ninety-nine distinct serum samples were subsequently acquired from 21 animals (17.8 years ± 9.8; 30.6% males) with suspected aspergillosis or undergoing postdiagnostic follow-up. They were sent to the investigator center (Division of Comparative Pathology, University of Miami -Miller School of Medicine, Miami FL, U.S.A.) and prospectively tested for serological methods. Concomitantly, these dolphins underwent other routine and specialized diagnostic procedures to establish a definitive diagnosis during their clinical care. For example, all were sampled for mycological cultures.

Another subset of serum samples was obtained from 32 free-ranging dolphins (estimated mean age = 14.0 years ± 3.0; 36.4% males) living in Sarasota Bay (FL, U.S.A.), and archived in the collection of the National Marine Fisheries Service Scientific Research, Mote Marine Laboratory before diagnostic testing. [START_REF] Wells | Bottlenose dolphins as marine ecosystem sentinels: developing a health monitoring system[END_REF]

Serological tests

Pre-processing step. Upon arrival to the investigator center (Division of Comparative Pathology, University of Miami -Miller School of Medicine, Miami FL, U.S.A.), all samples were stored at -80C with no intervention until they were thawed, and centrifuged for 10 minutes (min) at 3000 g just before being utilized for testing.

ELISA. Briefly, each serum sample was systematically tested in eight serial dilutions performed in phosphate buffer saline (PBS) buffer, ranging from 1: 64 to 1: 8,192 dilutions. Fifty microliters of each dilution was placed into one well of a 96 well microtiter plate that had been manually coated with bulk Aspergillus ID antigens made of pooled mycelial-phase culture filtrates of A. fumigatus, A. flavus, A. niger, and A. terreus (IMMY, Norman OK, U.S.A.).

Samples were incubated at 37 C for 30 min. After a washing step with Tween-saline solution, 50 L of horseradish peroxidase-(HRP-) conjugated rabbit anti-bottlenose dolphin IgG h+l (Bethyl, Montgomery TX, U.S.A.), diluted 1: 10,000 in PBS, was added to each well, and incubated at 37 C for 30 min. Finally, 50 L of chromogen ABTS ® solution (KPL, Gaithersburg MA, U.S.A.) was added to each well after an additional washing step. The optical density (OD), related to reaction intensity after 0, 5, 10 and 15 min, was read at 405 nm, in a SPECTRAmax ® microplate spectrophotometer (Molecular Devices, Downingtown, PA, U.S.A.), following a five second-long auto-shaking sequence. An OD of 0.2 or higher was considered positive. For each run, a highly positive serum, tested previously with positive titer over 1: 1,024, was used as a control.

Immunoblot testing. Each serum sample was tested using the Aspergillus Western Blot IgG ® kit (LDBio Diagnostics, Lyon, France) according to the manufacturer's recommendations. For immunodetection, 1.2 mL of alkaline phosphatase-(AP-) conjugated rabbit anti-bottlenose dolphin IgG h+l (diluted 1: 1,000, Bethyl, Montgomery TX, U.S.A.) was distributed into each channel. The immunoblot patterns were compared to a positive control supplied by the manufacturer that was revealed by anti-IgG conjugate (LDBio Diagnostics, Lyon, France). Each sample was examined in triplicate and independently read by two experts. According to previously published works, [START_REF] Oliva | Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis[END_REF] a specific focus was made on four immunoblot bands, located at 30, 22, 18-20, and 16 kDa, that have been shown to be specific for Aspergillus sensitization in humans. By adding the intensity results of each abovementioned band (which was individually scored from 0 to 4, according to comparison with the score of the positive control), a global Western blot (WB) score was ranked on 16 points for each sample.

Data analysis

Statistical analyses were performed using XLStat Premium v.18.07 ® software (Addinsoft, Paris, France). Methods validation was achieved according to prior recommendations. [START_REF] Jensen | Method comparison in the clinical laboratory[END_REF] Receiver operating characteristic ROC curves and areas under the curve AUC were generated in order to establish positive cutoff with the WB score of the commercial kit. Lower and upper cutoff values were calculated using Youden's J statistic [START_REF] Li | Weighted Youden index and its two-independentsample comparison based on weighted sensitivity and specificity[END_REF] and ROC curves. [START_REF] Jensen | Method comparison in the clinical laboratory[END_REF] A true positive was defined as a positive result that was in agreement with the definitive diagnosis of "proven" or "probable" aspergillosis in the observation cohort. Diagnostic measures were then calculated to assess performance: sensitivity, specificity, positive and negative predictive values, and likelihood ratios. The comparison of diagnostic performance was made using the Wilcoxon signed-rank test. The Pearson's rank correlation coefficient was used to assess the correlation between results of the different serology assays. Statistical association was tested by regression models, expressed in odds ratio (OR) with 95% confidence interval ( 95% CI). Missing data, e.g. when the total volume of sample was insufficient to complete all the analyses, were managed by the method of mean imputation. The -risk was adjusted at 0.05.

Ethics

No animal was anesthetized, euthanized, or sacrificed for the purposes of this study. The serum samples utilized were prospectively banked during veterinary assessments in normal and clinical dolphins.

This study was conducted using archived samples collected from Six Flags Aquarium, Georgia

Results

Assessment of the serological assays within the observation cohort

Results are displayed in Table 1. Notably, no differences were observed between the Aspergillusdiseased cases and the controls (index 0.2 ± 0.1 versus 0.3 ± 0.1, P > 0.05).

ELISA. The ROC curve showed AUC at 0.844, sensitivity and specificity at 96.9%, [82.6 -100.0%] and 36.4%, [24.9 -49.6%] by the lower cutoff value set at 1: 256 titer, and at 90.6%, [74.8 -97.4%] and 69.1%, [55.9 -79.7%] by the upper cutoff established at 1: 512 titer (Figure 1A, upper left panel). The upper positive threshold allowed correct separation of Aspergillusdiseased dolphins and controls (Figure 1A, upper middle panel), but cross-reaction was noticed to be frequent, especially for cetaceans infected with C. immitis and Cryptococcus sp. (Table 2).

In these dolphins, the respective titers ranged from 1: 1,024 to 1: 8,192. For this reason, the positive predictive value was found moderate, at 63.0%. There was no significant impact of the Aspergillus species responsible for the infection on ELISA results (P = 0.8).

Immunoblot testing. Overall, immunoblot analysis confirmed that antibodies against

Aspergillus proteins of 30, 22, 18-20, and 16 kDa were consistently observed in dolphins with "proven" or "probable" aspergillosis (Figure 2). Presence of the 22 kDa band showed the highest sensitivity, but the lowest specificity (100.0% and 10.3%, respectively). The band at 16 kDa was rarely present during "proven" or "probable" aspergillosis, and had sensitivity estimated at 53.10%, but was the most specific (100.0%). Sensitivity was correct for band at 30 kDa (94.4%), and specificity was high for the band at 18-20 kDa (97.1%). When evaluating the four abovementioned immunoblot bands together by adding their respective intensities, the lower positive cutoff values for the WB score was determined to be 5 out of a total of 16, while the upper value was set at 6 out of 16, respectively (Figure 1B, lower left and middle panels). ROC curve showed AUC at 0.855, sensitivity and specificity at 78.1%, [60.9 -89.1%] and 78.2%, [65.4 -87.1%] by the lower cutoff value, and at 65.6%, [48.2 -79.6%] and 92.7%, [82.1 -97.5%] by the upper threshold, respectively (Table 2). Overall, positive results were strongly associated with diagnosis of aspergillosis (OR=21.3, 95% CI [6.1-73.7], P<0.0001). There was no significant influence upon the WB score from the Aspergillus species which was found to be involved in the infectious process (P = 1.0). The invasive fungal infections due to non-Aspergillus genera, e.g. Cryptococcus, Candida, Mucor, Rhizopus…, provided WB scores below the positive cutoff. Notably, the dolphin with aspergillosis secondary to mucormycosis, had a WB score value just below the upper positive threshold (5/ 16).

Diagnostic performance of the immunoblot testing was compared with that of the ELISA in Table 2. The two methods were statistically correlated (Pearson's coefficient R 2 = 0.577, P < 0.0001). Of all the dolphins with "proven" or "probable" diagnosis of aspergillosis, none were positive in immunoblot testing, while concurrently negative with the ELISA method.

Assessment of the serological assays within the validation cohort

Dolphins under human care. The ELISA was sporadically falsely-positive for three of them in which the diagnosis of aspergillosis was not concomitantly/subsequently confirmed. In contrast in six cases, ELISA was positive earlier than mycological cultures (in average 88 ± 55 days earlier). Besides, it resulted in the diagnosis in another case, while concomitant cultures constantly remained sterile. The antifungal treatment had a significant impact upon decreasing the ELISA titers from 1: 1,024 to 1: 512 (P = 0.039) (Figure 1A, upper right panel).

The immunoblot testing confirmed the suspected diagnosis of aspergillosis in 16 out of the 21 bottlenose dolphins. Importantly, it provided a positive diagnosis three cases in which the mycological culture remained sterile throughout the study period. In addition, the immunoblot test indicated diagnosis prior to the Aspergillus culture in six dolphins, in average 103 ± 66 days earlier. It also reflected positive response to antifungal treatment (P = 0.002), with a decrease in the mean WB score from 8.9 to 6.7/ 16 (Figure 1B, lower right panel). Conversely, one animal showed no response on immunoblot testing to antifungal therapy over a 3.5 year-period following diagnosis, i.e. WB scores were stable at 13-14/ 16, whereas ELISA titers were fluctuating from 1: 512 to 1: 16,384. While this animal had substantial clinical improvement on antifungal therapy, necropsy revealed rare fungal hyphae characterized by regular septa, parallel walls, and acute angle branching present only within devitalized airway cartilage. The animal died of causes unrelated to pulmonary aspergillosis. An example of serological follow up of another animal is shown in Figure 3.

Free-ranging dolphins. All but six were negative with ELISA method: one was positive at a 1: 2,048 titer, and showed multiple papilloma lesions on the rectal mucosa, the vulva, and the urinary papilla. This dolphin also had multiple pinpoint depressed dark brown skin lesions on the right side and mild peritoneal effusion in the left abdomen. Another dolphin, exhibiting a 1:

1,024 titer in ELISA, had skin lesions of unknown cause above the right pectoral flipper.

Due to low volume samples, only 13 free-ranging dolphins were tested using the immunoblot.

All but one were negative. For this one positive animal, the WB score was estimated at 7/ 16, and ELISA results were below 1: 64 titer, while no clinical signs were reported.

Discussion

Although ELISA has been widely used to detect anti-Aspergillus antibodies in serum, [START_REF] Persat | Société Française de Mycologie Médicale -SFMM Study group. Aspergillus antibody detection: diagnostic strategy and technical considerations from the Société Française de Mycologie Médicale (French Society for Medical Mycology) expert committee[END_REF] precipitin detection by immunodiffusion remains the reference standard for serological diagnosis of chronic or subacute aspergillosis in immunocompetent or slightly-immunocompromised human patients. [START_REF] Kauffmann | Antibodies against Aspergillus fumigatus II. Identification and quantification by means of crossed immunoelectrophoresis[END_REF][START_REF] Baxter | Performance of two Aspergillus IgG EIA assays compared with the precipitin test in chronic and allergic aspergillosis[END_REF][START_REF] Ohba | Clinical characteristics and prognosis of chronic pulmonary aspergillosis[END_REF] However, as they are less labor-intensive and allow detailed study of the immune response, immunoelectrophoresis and immunoblotting techniques have been progressively developed, often replacing immunodiffusion for diagnostic confirmation testing. [START_REF] Oliva | Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis[END_REF][START_REF] Persat | Société Française de Mycologie Médicale -SFMM Study group. Aspergillus antibody detection: diagnostic strategy and technical considerations from the Société Française de Mycologie Médicale (French Society for Medical Mycology) expert committee[END_REF][START_REF] Ohba | Clinical characteristics and prognosis of chronic pulmonary aspergillosis[END_REF] Increasing availability of commercial standardized kits approved for diagnostic purposes has reduced the usual limitations that were noticed with in-house immunoelectrophoresis and immunoblotting, such as poor intra-and inter-laboratory reproducibility and extended turnaround times. [START_REF] Persat | Aspergillus serology, from yesterday to today for tomorrow[END_REF][START_REF] Kauffmann | Antibodies against Aspergillus fumigatus II. Identification and quantification by means of crossed immunoelectrophoresis[END_REF] For example, ready-to-use immunoblot kits have already been utilized in human medicine for the diagnosis of HIV infection, [START_REF] Zöller | Validity of western immunoblot band patterns in the serodiagnosis of Lyme borreliosis[END_REF] Lyme borreliosis, [START_REF] Burke | Variations in Western blot banding patterns of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus[END_REF] and toxoplasmosis. [START_REF] Franck | LDBio-Toxo II immunoglobulin G Western blot confirmatory test for anti-toxoplasma antibody detection[END_REF] Nonetheless, such assays have been rarely applied in veterinary medicine, and not evaluated so far, [START_REF] Desoubeaux | Application of Western blot analysis for the diagnosis of Encephalitozoon cuniculi infection in rabbits: example of a quantitative approach[END_REF] with the exception of a few in-lab techniques like that for the diagnosis of Encephalitozoon cuniculi infection in rabbits. [START_REF] Desoubeaux | Application of Western blot analysis for the diagnosis of Encephalitozoon cuniculi infection in rabbits: example of a quantitative approach[END_REF] As aspergillosis can be a serious medical condition in dolphins, and its diagnosis can also be challenging, it seems vital to evaluate novel diagnostic tools, such as commercial immunoblot kit.

From this study, the Aspergillus Western blot IgG ® kit (LDBio Diagnostics, Lyon, France), as well as the genus-specific ELISA method with which it was compared, appeared convenient to process, and easy to interpret, for the diagnosis of aspergillosis in common bottlenose dolphins.

It is noteworthy that the multi-well plate reading for the ELISA testing is objective with spectrophotometer devices, but results of the immunoblot testing were more detailed for studying immunoreactivity. Addressing the lower molecular weight bands at 30, 22, 18-20, and 16 kDa through a quantitative approach, [START_REF] Oliva | Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis[END_REF] we found similar patterns as those reported for human samples, e.g. the 16 kDa band was often very slightly visible and the least sensitive of all, but very specific. Likewise, there were no obvious alternate patternsin either banding or intensity of bandsin the dolphin samples in comparison with the human ones. Altogether, our findings highlighted a high specificity of the immunoblotting, i.e. 92.7% vs. 94.0% in previous study carried out in humans, [START_REF] Oliva | Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis[END_REF] especially because the positive cutoff was herein voluntarily set at a high value. Furthermore in this reagent kit, the manufacturer prioritized obtaining standardized purified antigens, although the nature of these antigens has not been disclosed thus far.

Importantly, the results from the immunoblot testing showed no cross-reaction in blood samples from dolphins infected with fungi belonging to non-Aspergillus genera like Coccidioides sp. In contrast, and as previously reported, [START_REF] Powers-Fletcher | Nonculture Diagnostics in Fungal Disease[END_REF] several cross-reactions to Coccidioides and Cryptococcus spp. occurred when using the bulk antigens in the ELISA. Conversely to the purified proteins in the immunoblot kit, these bulk antigens are compound of mixture of unpurified proteins and other moieties like carbohydrates that are common among many fungal species. One should also note that both the ELISA and the immunoblot testing were reliable for the diagnosis of fungal infection due to most of the Aspergillus species, including those that do not belong to the Fumigati section, like A. niger and A. flavus. In this study, although the respective sensitivities were lower than those observed in some groups of human patients (100.0% and 41.7% in cystic fibrosis and non-cystic fibrosis patients for the immunoblot testing), [START_REF] Oliva | Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis[END_REF] the two tests appeared to rival mycological cultures, at least when considering serology as a preliminary screening step among the panel of tools dedicated to diagnosis. The current study showed a high positive predictive value of the immunoblot testing for detecting Aspergillus infection in a screening approach. It also demonstrated the diagnostic benefits of repeated blood measures over the course of disease.

Results of this work need to be carefully interpreted, because animals are more likely to be colonized and/or chronically infected than humans, with variable clinical symptoms depending on the species. In animal-based studies involving natural infection, there may be a poor reliability due to the lack of consensus regarding the disease classification. It is particularly hard to classify correctly aspergillosis in cetaceans, because the disease features are somewhat different from invasive aspergillosis in neutropenic humans. In dolphins, aspergillosis is only moderately invasive, but its evolution is more chronic than in immunocompromised patients. In light of these considerations, pathology and immune response of aspergillosis in cetaceans should be rather compared to those of subacute / chronic human aspergillosis, like the form observed in patients with morbidity factors, e.g. tuberculosis, sarcoidosis, autoimmune diseases, chronic obstructive pulmonary disease… [START_REF] Bunskoek | Successful treatment of azole-resistant invasive aspergillosis in a bottlenose dolphin with high-dose posaconazole[END_REF] Nonetheless, the concept based on categorization of fungal infection used for humans (who represent a single mammal species) should not be correctly applicable for all the other animals. However, to reduce misclassification of our study animals, we were forced to use a composite criterion based on clinical data and histopathology / mycological culture, as it is consensually approved for use in human infections. [START_REF] Desoubeaux | Diagnosis of invasive pulmonary aspergillosis: updates and recommendations[END_REF] Notably, unlike in clinical trials recently reported in human patients with aspergillosis, [START_REF] Bodey | Fungal infections in cancer patients: an international autopsy survey[END_REF][START_REF] Dimopoulos | Disseminated aspergillosis in intensive care unit patients: an autopsy study[END_REF] there were more "proven" cases than "probable" ones in our observation cohort. One possible explanation relies on post-mortem evaluation which allows reliable diagnosis of "proven" aspergillosis through gross and histopathology observations. [START_REF] Desoubeaux | Diagnosis of invasive pulmonary aspergillosis: updates and recommendations[END_REF][START_REF] Mason | Performing the field necropsy examination[END_REF] Conversely, the negative controls in the observation cohort could appear as a possible limitation due to underestimation: this group of dolphins was considered apparently healthy based on normal bloodwork and the lack of clinical signs consistent with aspergillosis, but only a few had been tested via mycological cultures before their inclusion. For these reasons, subclinical fungal infection or colonization cannot be definitely ruled out in these animals. In addition, it has been shown in humans that persistent colonization in clinically-asymptomatic humans is a pathway to infection, and can induce sustainable antibody response. [START_REF] Persat | Aspergillus serology, from yesterday to today for tomorrow[END_REF][START_REF] Oliva | Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis[END_REF] Since Aspergillus spp. is ubiquitous in the terrestrial and aquatic environment, exposure may be common in diverse populations of cetaceans in the wild and under human care. [START_REF] Waltzek | Marine mammal zoonoses: a review of disease manifestations[END_REF] It is however debatable if seroconversion is a sufficient argument to start antifungal therapy or if instead, this should reinforce the need for more frequent monitoring or additional diagnostics. [START_REF] Tashiro | Diagnostic significance of Aspergillus species isolated from respiratory samples in an adult pneumology ward[END_REF] In this study, antibodies appeared sustainable over time, including after long antifungal treatment, although their rates statistically decreased. Additional studies are now required with more cases that will undergo repeated measures after treatment to better assess antibody longevity.

In conclusion, studies focusing on specific diagnostic approaches, like this one, help to improve basic knowledge about fungal disease in wildlife and to overcome the lack of standardization, together with the inability to invest in expensive diagnostic tools. Herein, the novel ELISA and the immunoblot testing appeared valuable to detect anti-Aspergillus antibodies in dolphins. To our knowledge, this is the first time that a commercial immunoblot test initially intended for humans has been successfully used in animals. The uniformity of the reagents, as well as the availability of free software allowing scanning and easy reading of the blot patterns like ImageJ ® software (https://imagej.nih.gov/ij/) or Image Studio Lite ® software (https://www.licor.com/), [START_REF] Domínguez | IJ-OpenCV: Combining ImageJ and OpenCV for processing images in biomedicine[END_REF] would allow reproducibility, specificity to fungal genus, and reliable inter-laboratory results. ELISA and immunoblot testing (Aspergillus Western blot IgG ® kit,LDBio Diagnostics, Lyon, France) were both positive twelve weeks before the mycological culture (D-83). Worsening in clinical condition, especially appearance of abnormal vocalizations, marked the start for antifungal therapy (D166). Serological status returned negative two months after withdrawing posaconazole (D309), although non-specific increase of antibody rates was transiently noticed with ELISA testing (D248). Notably, as shown in the right column, measurements of GM antigen were never consistent with the actual medical condition at time of sampling. D, Day (D0 being the first day when mycological culture became positive); ELISA, Enzymelinked immunosorbent assay; GM, Galactomannan antigen; kDa, Kilo Dalton; NA, not available.

  Aquarium, and U.S. Navy marine mammals as part of their routine care. The U.S. Navy Marine Mammal Program (MMP) houses and cares for a population of dolphins in San Diego Bay (CA, U.S.A.). The MMP is Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited and adheres to the national standards of the United States Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the Animal Welfare Act. Samples obtained from free ranging dolphins in Sarasota Bay (FL, U.S.A.) were collected under National Marine Fisheries Service Scientific Research Permit No. 15543 and Mote Marine Laboratory IACUC approval, with support from Dolphin Quest, Inc., the Chicago Zoological Society, and the Office of Naval Research. Georgia Aquarium is a USDA/APHIS approved and inspected facility, and a member of the Association of Zoos and Aquariums and the Alliance of Marine Mammal Parks and Aquariums. Georgia Aquarium has been awarded the Humane Conservation certification by the American Humane Association. Biologic samples for this study were taken as part of a preventive medicine program or for medical monitoring purposes.

Figure 1 :

 1 Figure 1: Diagnostic performance of the novel Aspergillus genus-specific ELISA (A) and the immunoblot testing (Aspergillus Western blot IgG ® kit, LDBio Diagnostics, Lyon,

Figure 2 :

 2 Figure 2: Examples of anti-Aspergillus immunoblot testing (Aspergillus Western blot IgG ®

Figure 3 :

 3 Figure 3: Example of anti-Aspergillus serological follow up in a 15 year-old-male common

Acknowledgement

This publication constitutes Sea Research Foundation #279.

The authors are grateful to LDBio Diagnostics for the valuable discussions about the analytical material, and to the multi-institutional animal care and biological staff dedicated to the care and monitoring of the bottlenose dolphins in this study.

Disclosure of transparency

No conflict of interest.