METHODS AND COMPOSITIONS FOR PREVENTING OR TREATING ACUTE EXACERBATIONS WITH POLYCLONAL IMMUNOGLOBULIN

Abstract

This invention is in the field of preventing or treating acute exacerbations in chronic lung diseases, such as chronic obstructive pulmonary disease and non-cystic fibrosis bronchiectasis, by administration of polyclonal immunoglobulin to the respiratory tract, in particular by direct application of an aerosolized composition comprising polyclonal immunoglobulin.

Claims

1-25. (canceled)

26. A method for treating or preventing an acute exacerbation in a human subject with a chronic lung disease, comprising administering to the human subject a composition comprising a polyclonal immunoglobulin, wherein the composition is administered to the respiratory tract of the human subject.

27. The method of claim 26, wherein the chronic lung disease is chronic obstructive pulmonary disease (COPD).

28. The method of claim 27, wherein the COPD is moderate to severe COPD.

29. The method of claim 28, wherein the composition is administered for the prevention of an acute exacerbation of COPD.

30. The method of claim 26, wherein the chronic lung disease is non-cystic fibrosis bronchiectasis (NCFB).

31. The method of claim 30, wherein the composition is administered for the prevention of an acute exacerbation of NCFB.

32. The method of claim 26, wherein the human subject has a lower level of immunoglobulin G (IgG) than the normal range for a healthy adult.

33. The method of claim 32, wherein the human subject has a plasma IgG level less than 700 mg/dL.

34. The method of claim 32, wherein the human subject has a lower level of IgG in sputum than the normal range for a healthy adult.

35. The method of claim 26, wherein the human subject has experienced one or more acute exacerbations in the 12 months prior to prevention or treatment starting.

36. The method of claim 26, wherein the human subject has one or more detectable pro-inflammatory cytokines in his or her sputum.

37. The method of claim 36, wherein the one or more detectable pro-inflammatory cytokines are IL-1β and/or IL-6 and/or IL-8.

38. The method of claim 26, wherein the human subject suffers from pneumonia.

39. The method of claim 26, wherein the human subject has a viral respiratory tract infection.

40. The method of claim 39, wherein the human subject has a rhinovirus infection.

41. The method of claim 26, wherein the human subject has a bacterial respiratory tract infection.

42. The method of claim 41, wherein the human subject has a Pseudomonas aeruginosa infection.

43. The method of claim 26, wherein the polyclonal immunoglobulin reduces inflammation in the respiratory tract of the human subject.

44. The method of claim 43, wherein the polyclonal immunoglobulin reduces the level of one or more pro-inflammatory cytokines in the respiratory tract of the human subject.

45. The method of claim 44, wherein the one or more pro-inflammatory cytokines are IL-1β and/or IL-6 and or IL-8.

46. The method of claim 26, wherein the polyclonal immunoglobulin causes immune exclusion of at least one potentially pathogenic microbe infecting the respiratory tract of the human subject.

47. The method of claim 46, wherein the polyclonal immunoglobulin reduces direct damage to epithelial tissue in the human subject caused by at least one pathogen.

48. The method of claim 47, wherein the polyclonal immunoglobulin reduces the activity of exoenzymes, reduces loss of epithelial barrier integrity, and/or reduces viral shedding.

49. The method of claim 26, wherein the composition comprises human plasma-derived IgG.

50. The method of claim 49, wherein the composition is at least 95% IgG.

51. The method of claim 50, wherein the composition is at least 98% IgG.

52. The method of claim 49, wherein the composition comprises proline.

53. The method of claim 52, wherein the composition comprises from 210 to 290 mmol/L of L-proline.

54. The method of claim 53, wherein the composition comprises 250 mmol/L of L-proline.

55. The method of claim 26, wherein the composition is administered as an aerosol.

56. The method of claim 26, wherein the composition is an aqueous solution having a polyclonal immunoglobulin concentration of 50 mg/mL to 150 mg/mL.

57. The method of claim 56, wherein the composition has a polyclonal immunoglobulin concentration of 100 mg/mL.

58. The method of claim 26, wherein the composition is administered in 2-10 mL.

59. The method of claim 58, wherein the composition is administered once every 48 hours, or once every 24 hours, or once every 12 hours during the treatment.

60. The method of claim 59, wherein the composition is administered during the fall and winter months.

61. The method of claim 26, wherein the composition is administered in a combination therapy with one or more of an antibiotic, a corticosteroid, a beta2-agonist, and an anticholinergic bronchodilator.

62. The method of claim 55, wherein the composition is a dry powder.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0170] The invention will now be illustrated in the following non-limiting examples, with reference to the following figures:

[0171] FIG. 1. Plasma-derived Ab formulations interact with Pseudomonas aeruginosa (PA). Binding of increasing concentrations of plasma-derived Abs or secretory IgA/M to coated PA as determined by ELISA.

[0172] FIG. 2. Association of plasma-derived IgG formulation with PA promotes agglutination. Laser scanning confocal microscopy images of immune complexes of PA associated with plasma-derived IgG. Bacteria were labelled with CFSE and IgG with Cy3 dye. Images are representative of one observed field obtained from 5-10 observations from two independent slides.

[0173] FIG. 3. Plasma-derived immunoglobulin formulation PA-induced LDH tissue release. Tissue damage was assessed by measuring LDH release in the basolateral medium of the MucilAir™ Non-infected transwells receiving only the vehicle or the plasma-derived immunoglobulin formulations served as controls. PA-infected transwells served as positive controls. Data are representative of 3 independent experiments.

[0174] FIG. 4. Plasma-derived IgG formulation prevents loss of trans-epithelial electrical resistance in a dose-dependent manner. Tissue integrity was assessed by measuring trans-epithelial electrical resistance. Non-infected and proline treated transwells served as negative controls and PA-infected transwells as positive controls of tissue damage. Data are representative of 3 independent experiments.

[0175] FIG. 5. Plasma-derived IgG formulation prevent PA-induced tissue damages in a dose depend manner. Laser scanning confocal microscopy images of paraffin-fixed MucilAir™ sections were acquired and analyzed for the expression of cytokeratine and beta-tubulin. Non-infected transwells receiving only the vehicle or IgG formulations served as controls. PA-infected transwells treated with vehicle served as positive controls. Data are representative of 3 independent experiments.

[0176] FIG. 6. Plasma-derived immunoglobulin formulation reduces PA-induced IL-8 release by epithelial cells. IL-8 was measured in the basolateral medium of the MucilAir™. Non-infected transwells receiving only the vehicle or immunoglobulin formulations served as controls. PA-infected transwells treated with proline served as positive controls. Data are representative of 3 independent experiments.

[0177] FIG. 7. Plasma-derived IgG formulation reduces PA-induced IL-8 release by epithelial cells in a dose-dependent manner. Relative concentrations of IL-8 secretion were calculated in regards to IL-8 secreted by MucilAir™ when exposed for 24 h with 10 CFU of PA. Non-infected transwells receiving only the vehicle or immunoglobulin formulations served as controls. PA-infected transwells treated with proline served as positive controls. Data are representative of one experiment using MucilAir™ from 3 different donors per condition.

[0178] FIG. 8. Plasma-derived Ab formulation reduces PA-induced IL-6 release by epithelial cells. IL-6 was measured in the basolateral medium of the MucilAir™. Non-infected transwells receiving only the vehicle or immunoglobulin formulations served as controls. PA-infected transwells treated with vehicle served as positive controls. Data are representative of 3 independent experiments.

[0179] FIG. 9. Plasma-derived Ab formulations interact with human rhinovirus C15. Binding of increasing concentrations of plasma-derived Abs or secretory IgAM to coated HRV C15 as determined by ELISA.

[0180] FIG. 10. Plasma-derived Abs reduce HRV shedding. Copy number of HRV-C15 genome was measured in apical washes using q-PCR. HRV-infected transwells treated with proline served as positive controls of infection. For efficacy measurements, Rupintrivir treated transwells served as positive control.

[0181] FIG. 11. Plasma-derived Abs reduce HRV-induced tissue damage. Tissue integrity was assessed by measuring trans-epithelial electrical resistance. Non-infected transwells treated with proline served as negative controls. HRV-infected transwells treated with proline served as positive controls of infection. For efficacy measurements, Rupintrivir treated transwells served as positive control.

[0182] FIG. 12. Plasma-derived Abs prevent HRV-induced mucociliary clearance reduction. Mucociliary clearance was assessed by measuring the speed of polystyrene microbeads of 30 μm diameter added on the apical surface of MucilAir™. Non-infected transwells treated with proline served as negative controls. HRV-infected transwells treated with proline served as positive controls of infection. For efficacy measurements, Rupintrivir treated transwells served as positive control.

[0183] FIG. 13. Plasma-derived immunoglobulin formulations inhibit HRV proliferation in a dose-dependent manner. Copy number of HRV-C15 genome was measured in apical washes using q-PCR after treatment with 4 μg/well, 20 μg/well, 100 μg/well and 500 μg/well of the different immunoglobulin formulations. HRV-infected transwells treated with proline served as positive controls of infection. For efficacy measurements, Rupintrivir treated transwells served as positive control.

[0184] FIG. 14. Plasma-derived immunoglobulin formulations inhibit Influenza virus proliferation in a dose-dependent manner. Copy number of Influenza virus genome was measured in apical washes using q-PCR after treatment with 4 μg/well, 20 μg/well, 100 μg/well and 500 μg/well of the different immunoglobulin formulations. Influenza virus-infected transwells treated with proline served as positive controls of infection. For efficacy measurements, Oseltamivir-treated transwells served as positive control.

MODES FOR CARRYING OUT THE INVENTION

[0185] The following non-limiting examples serve to illustrate the invention.

[0186] The studies encompassed in the examples below show that immunoglobulin delivered onto an airway tissue can have a combined antimicrobial (immune exclusion) and anti-inflammatory effect, and is therefore an attractive option for an effective treatment or prevention of an exacerbation, in particular an infection-related exacerbation, in subjects suffering from chronic lung diseases, such as COPD and NCFB. In particular, it is suitable for maintenance therapy in subjects with these diseases to prevent chronic infections and acute exacerbations.

[0187] Protection of mucosal surfaces against colonization and possible entry and invasion by microbes is provided by a combination of constitutive, non-specific substances (mucus, lysozyme and defensins), and also by specific immune mechanisms including secretory Igs (Slgs) at the humoral level [20;21]. In vivo, experimental and clinical resistance to infection can be correlated with specific secretory IgA (SIgA) antibodies (Abs) serving as an immunological barrier at mucosal surfaces [22;23]. It is thought that aggregation, immobilization and neutralization of pathogens at mucosal surfaces is facilitated by the multivalency of SIgA [24;25]. SIgM serving as a surrogate of SIgA in IgA-deficient individuals appears to act via a similar protective mechanism [26].

[0188] For a few pathogens such as Poliovirus, Salmonella, or influenza, protection against mucosal infection can be induced by active mucosal immunization with licensed vaccines. However, for the majority of mucosal pathogens no active mucosal vaccines are available. Alternatively, protective levels of Abs might directly be delivered to mucosal surfaces by passive immunization. In nature this occurs physiologically in many mammalian species by transfer of maternal antibodies to their offspring via milk [27]. Human and animal studies using passive mucosal immunization have demonstrated that pIgA and SIgA antibody molecules administered by oral, intranasal, intrauterine or lung instillation can prevent, diminish, or cure bacterial and viral infections [28]. However, the secretory form of IgA naturally found at mucosal surfaces was rarely used, and large scale production of SIgA is not possible to date. Construction of SIgA with biotechnological methods is challenging but such molecules could have important clinical applications [29]. The same also applies to secretory component-containing IgM.

[0189] Plasma-derived immunoglobulins have been used for many decades to protect patients with immunodeficiencies from potentially lethal infections [30]. Plasma-derived immunoglobulins are generally highly pure for IgG. However, few IgG products exist with enriched IgM in their formulations (e.g. Pentaglobin™). Delivery of plasma-derived immunoglobulins is intravenous or sub-cutaneous, ensuring systemic distribution of the immunoglobulins through the body. While Ig replacement therapy has been shown to lower pneumonia incidences in patients with immunodeficiency, it seems that they have a limited impact on upper airway infections as well as bronchial infections. Topical application of plasma-derived immunoglobulins could support a higher Ig content at the mucosal surface without having to increase systemic Ig delivery.

[0190] HRV and PA were chosen to test efficacy of plasma-derived immunoglobulins to prevent epithelial tissue infection, because of their main roles in COPD and NCFB exacerbations. To mimic better the situation in human, human primary cell-based airway model, MucilAir™ (Epithelix Sarl, Geneva) was used. MucilAir™ is a cell model of the human airway epithelium reconstituted in vitro. MucilAir™-Pool is made of a mixture of nasal or bronchial cells isolated from 14 different- or a unique donor(s) respectively. Cultured at the air-liquid interface, the model displays high trans-epithelial electrical resistance, cilia beating as well as mucus production, demonstrating the full functionality of the epithelial tissue as it would exist in vivo. Cytokine release (e.g. IL-8 and IL-6) as well as Lactate Dehydrogenase (LDH) release can be detected during infection, reflecting how infection is associated with inflammation and tissue damage in this model.

[0191] Material and Methods

[0192] Infections of the airways starts with the deposition of pathogenic bacteria and viruses on the apical side of the airway epithelium. To reach out to the tissue, viruses will infect epithelial cells while bacteria tend to damage the cells through the secretion of exotoxins. A model of Pseudomonas aeruginosa infection was used to test the efficacy of plasma-derived immunoglobulins to prevent tissue damage.

[0193] Bacterial Strain

[0194] Pseudomonas aeruginosa (PA) used for this model is a clinical isolate obtained from the Institute of Infectious Disease (University of Bern, Switzerland)). PA is a pathogenic organism that causes disease in human and is responsible for pulmonary infections. PA were cultured on a blood agar petri dish. A colony was selected and cultured in Brain Heart Infusion (BHI) medium for 24 h at 37° C. and 400 revolutions per minute (RPM). On the following day, culture was diluted 1:10 with fresh BHI medium and placed for an additional hour at 37° C. and 400 rpm. OD was then measured and number of bacteria was estimated from an OD/bacterial load curve, which was generated with multiple cultures prior experiment. An aliquot was collected for further dilution before and dosing, and a second aliquot was collected for further plating on blood agar plates to verify bacterial load accurately.

[0195] Viral Strains

[0196] Rhinovirus C15 is a clinical isolate (name S07-09-08-U) obtained from the Hospital of Geneva. Virus stocks were produced in MucilAir™ cultures and diluted in culture medium, they were not purified nor concentrated.

[0197] For the dose-response studies, rhinovirus C15 (2009) and influenza A/Switzerland/7717739/2013 (H1N1) were isolated directly on MucilAir™ from clinical specimen as described in [31]. Viral stocks for the experiments were produced on MucilAir™ collecting apical washes with culture medium. Production of several days were pooled and quantified by qPCR, aliquoted and stored at −80° C.

[0198] Tissue

[0199] MucilAir™ (Epithelix Sarl, Geneva) was used to mimic human bronchial tissues. For each study group, 3 MucilAir™ transwells were used, with each transwell originating from either one distinct donor or a mix of 14 donors used in the dose-response studies. Culture of MucilAir™ was performed at air-liquid interface. Medium used at the basolateral side was MucilAir™ culture medium (Epithelix Sarl, Geneva), which contains growth factors and phenol red. It does not contain serum.

[0200] Pseudomonas aeruginosa Infection Model and Treatment

[0201] Infection model using PA is based on the deposition of as low as 10 Colony Forming Unit (CFU) of PA on the apical side of one MucilAir™ transwell under a volume of 10 μL. Over 24 h, PA will grow to reach >10.sup.9 CFU/transwell. Infection leads to the release of lactate dehydrogenase (LDH)(relating to tissue damage) and pro-inflammatory molecules such as IL-8 and IL-6. Damage of the tissue is also demonstrated by the appearance of holes in the tissue and the loss of trans-epithelial electrical resistance.

[0202] In some experiments, immunoglobulins were deposited 10 minutes prior to the bacteria or simultaneously. Immunoglobulins were applied in a 10 μL final volume. The effects of immunoglobulins were compared to the vehicle solution (25 mM Proline).

[0203] Human Rhinovirus C15 and Influenza H1N1 Infection Model and Treatment

[0204] At t=0, 15 μL of 3.8×10.sup.7 genome copies/mL HRV C15 (clinical strain: S07-09-08-U) stock solution in proof-of-concept experiment (FIG. 10) and 10 μL of 1.0×10.sup.8 genome copies/mL for both HRV and influenza H1N1 in the dose-response studies (FIGS. 13 & 14) was applied on the apical side of MucilAir™ for 3 h at 34° C. and 5% CO2. Immunoglobulins were applied at the same time as viruses in 5 μL on the apical surface of MucilAir™ and renewed at 3.5 and 24 hours. The effects of immunoglobulins were compared to the vehicle solution (25 mM Proline). Three hours after inoculation, epithelia were washed thrice with PBS (with Ca2+/Mg2+) in order to clean the inoculum.

[0205] Cell free, apical washes (20 minutes) with 200 μL MucilAir™ culture media were collected at 3.5 hours post-inoculation and then 24, 48 hours and stocked at −80° C.

[0206] Immunoglobulins

[0207] Human plasma-derived IgG preparations (IgPro10, Privigen) were prepared as reported [32]. Preparations containing IgA and IgM were obtained from an ion-exchange chromatographic side fraction used in the large-scale manufacture of IgG from human plasma. The elution fraction containing IgA and IgM was concentrated and re-buffered to 50 g/I protein in PBS by tangential-flow filtration (TFF; Pellicon XL Biomax 30, Merck Millipore). The resulting IgA/M solution, which contained IgA and IgM in a 2:1 mass ratio, was further processed to SCIgA/M, by combining in vitro IgA/M with recombinant human SC [33].

[0208] ELISA

[0209] Pro-inflammatory cytokines release by human bronchial tissue upon infection was measured in an aliquot of the basolateral medium collected 24 h post-infection. In particular, IL-8 (RnD Systems; DY208) and IL-6 (RnD Systems; DY206) were evaluated. Measurements were performed according to the user manual.

[0210] For PA ELISA, PA was cultured overnight at 37° C. in BBL Todd Hewitt Broth Medium. PA were pelleted by centrifugation (3220 g) for 10 minutes. Supernatant was removed and the pellet was washed twice with 0.1 M carbonate buffer (pH 9.6). Pellet was resuspended in carbonate buffer and 50 μl/well (4×10.sup.6 bacteria) were added onto a polysorbate plate. Coating was performed overnight at 2-8° C. The following day, wells were washed 3 times with PBS/Tween (0.05%) and blocked with PBS/FCS (2.5%) for 1.5 h at room temperature. Wells were then washed 3 times with PBS/Tween (0.05%). Ig formulations (0.7 μg/ml-500 μg/ml) were added for 2 h at room temperature to the wells. After washing twice with PBS/Tween (0.05%), a secondary antibody, Goat anti Human IgG/A/M-HRP (1 mg/ml, 1:2′000 in blocking buffer), was incubated for 2 h at room temperature on the samples. Final washings with PBS/Tween (0.05%) were done 3 times before the TMB substrate of peroxidase was used. Blue precipitate formation is linearly proportional to the amount of enzyme in each well. Enzymatic reaction was stopped with 50 μl/well HCl 1M. Absorbance was read at 450 nm (620 nm reference wavelength). Mean blank absorbance for each triplicates was subtracted from the bacteria coated absorbance.

[0211] For the rhinovirus ELISA, a Maxisorp plate (Nunc) was coated overnight with purified rhinovirus C stock (3×10.sup.6/ml; clinical name: S07-09-09-U) (2-4° C.) in 0.1M Carbonate buffer. A second Maxisorp plate was coated with 5% BSA in 0.1M Carbonate buffer, and served as “blank” plate. The following day, wells were washed 3 times with PBS/Tween (0.05%) and blocked with PBS/FCS (2.5%) for 1.5 h at room temperature. Wells were then washed 3 times with PBS/Tween (0.05%). Ig formulations (0.7 μg/ml-500 μg/ml) were added for 2 h at room temperature to the wells. After washing twice with PBS/Tween (0.05%), a secondary antibody, Goat anti Human IgG/A/M-HRP (1 mg/ml, 1:2′000 in blocking buffer), was incubated for 2 h at room temperature on the samples. Final washings with PBS/Tween (0.05%) were done 3 times before the TMB substrate of peroxidase was used. Blue precipitate formation is linearly proportional to the amount of enzyme in each well. Enzymatic reaction was stopped with 50 μl/well HCl 1M. Absorbance was read at 450 nm (620 nm reference wavelength). Mean blank absorbance for each triplicates was subtracted from the virus coated absorbance.

[0212] Immunohistology

[0213] Tissue damage was assessed using laser scanning confocal microscopy. Tissue were prepared as follow. MucilAir™ transwells were washed once in PBS and fixed overnight at 4° C. in 4% paraformaldehyde. The next day, transwells were washed 3 times with PBS and tissues were permeabilized with ice cold methanol for 30 minutes at −20° C. Tissues were then washed 3 times with PBS and a blocking step was conducted overnight at 4° C. using 3% Goat Serum in PBS. After another step of washes with PBS (3 times), staining was performed on the tissue for 48 h-72 h at 4° C. with an anti-cytokeratin antibody (Abcam; ab192643)(1/200), anti-beta tubulin antibody (Abcam; ab11309)(1/200) and DAPI (Sigma D9542)(1/2000), all diluted in PBS. Tissues were then washed 3 times in PBS and transwells were separated from the tissues. Tissues were then mount onto slides, covered with mounting medium and a cover slip. The slides were kept 24 h at room temperature to allow them to dry before being imaged on a Zeiss LSM800 confocal microscope.

[0214] Transepithelial Electrical Resistance (TEER)

[0215] TEER is a dynamic parameter, which reflects the state of epithelia. However, it can be affected by several factors. For example, if holes were present or if tight cellular junction are lost, TEER values will reach values below 100 Ω.Math.cm.sup.2. In contrast, when epithelia is healthy, TEER value is typically above 200 Ω.Math.cm.sup.2.

[0216] Non-treated samples as well as samples treated with vehicle without virus or bacteria served as negative controls while 10% Triton X-100 was used as positive control.

[0217] To measure TEER value, 200 μL of MucilAir™ medium was added to the apical compartment of the MucilAir™ cultures, and resistance was measured with an EVOMX volt-ohm-meter (World Precision Instruments UK, Stevenage) for each condition. Resistance values (0) were converted to TEER (Ω.Math.cm.sup.2) by using the following formula:

TEER (Ω.Math.cm.sup.2)=(resistance value (Ω)−100(Ω))×0.33 (cm.sup.2)

where 100Ω is the resistance of the membrane and 0.33 cm.sup.2 is the total surface of the epithelium.

[0218] Lactate Dehydrogenase (LDH) Assay

[0219] Lactate dehydrogenase is a stable cytoplasmic enzyme that is rapidly released into the culture medium upon rupture of the plasma membrane. 100 μL basolateral medium was collected at each time-point and incubated with the reaction mixture of the Cytotoxicity Detection KitPLUS, following manufacturer's instructions (Sigma, Roche, 11644793001). The amount of the released LDH was then quantified by measuring the absorbance of each sample at 490 nm with a microplate reader. Non-treated and vehicle (without virus or bacteria) served as negative control and correspond to the physiological release of LDH 5%). 10% Triton X-100 was used as a negative control and corresponds to a massive LDH release, (equal 100% cytotoxicity) To determine the percentage of cytotoxicity, the following equation was used (A=absorbance values):

Cytotoxicity (%)=(A (exp value)−A (low control)/A (high control)−A (low control))*100

[0220] Viral Shedding

[0221] At each time point of the study, apical washes were conducted with 200 μL MucilAir™ culture medium. 20 μL was further used to viral RNA extraction (QIAamp® Viral RNA kit (Qiagen)), resulting in 60 μL of RNA elution volume. Viral RNA was quantified by quantitative RT-PCR (QuantiTect Probe RT-PCR, Qiagen) using 5 μL of viral RNA. Two Picornaviridae family specific, a Pan-Picornaviridae primers and Picornaviridae as well as Influenza-A specific primers and probes with FAM-TAMRA reporter-quencher dyes were also used.

[0222] Four dilutions of known concentration of HRV-A16 or H3N2 RNAs as well as control for RT-PCR were included and the plates were run on either a TaqMan ABI 7000 from Applied Biosystems or a Chromo4 PCR Detection System from Bio-Rad. Ct data were reported to the standard curve, corrected with the dilution factor and presented as genome copy number per ml on the graphs.

[0223] Mucociliary Clearance

[0224] The mucociliary clearance was monitored using a Sony XCD-U100CR camera connected to an Olympus BX51 microscope with a 5× objective. Polystyrene microbeads of 30 μm diameter (Sigma, 84135) were added on the apical surface of MucilAir™. Microbead movements were video tracked at 2 frames per second for 30 images at room temperature. Three movies were taken per insert. Average beads movement velocity (μm/sec) was calculated with the ImageProPlus 6.0 software. Data are presented as mean+SEM (n=3 inserts).

Example 1: Plasma-Derived Immunoglobulins Interact with Pseudomonas aeruginosa

[0225] PA has been associated with many infections of the respiratory tract such as in subjects with cystic fibrosis or with severe COPD. Many different strains exist. A clinical isolate was used for its relevance to the clinical set-up. Commercially available plasma-derived immunoglobulins are mainly consisting of highly purified IgG, obtained from the fractionation of plasma pools collected from thousands of healthy adult donors. Due to its multi-donor origin, isolated immunoglobulins offer not only polyvalence and polyclonality, but also higher titers against certain pathogens as a result from vaccination. Monomeric IgA and a mix of pentameric IgM and monomeric/dimeric IgA can be isolated from the waste fraction. The inventors have previously established that polyreactive, serum-derived polymeric IgA, IgM and a mixture of the two isotypes (IgA/M) can be assembled into secretory Abs upon combination with recombinant secretory component (SC) [34]. In support of their use for local passive immunization, the molecules display high in vitro stability upon exposure to intestinal washes rich in proteases [35].

[0226] FIG. 1 presents the binding of plasma-derived immunoglobulins to PA in an ELISA assay. Importantly, all plasma-derived immunoglobulins are able to bind PA clinical isolate in this assay (see material and methods section). Binding to PA was dose-dependent, with immunoglobulin amounts varying from 0.7 μg/mL to 500 μg/mL. Comparison between the immunoglobulin formulations showed differences in binding capacity of PA. For instance, mixes of IgA and IgM with or without association to SC showed the highest affinity to PA, followed by IgG and IgA.

Example 2: Plasma-Derived Immunoglobulin IgG Form Large Aggregates with PA

[0227] Immunoglobulins may account for several roles at the mucosal surfaces. They may serve as opsonins, leading to enhanced phagocytic recognition or promoting the deposition of complement and subsequent lysis. They can bind and therefore tag infected cells for destruction through a mechanism called antibody-dependent cell-mediated cytotoxicity (ADCC). Immunoglobulins can neutralize a pathogen by binding to its surface antigens and inhibiting its growth. It can also coat a pathogen and prevent its adherence to the mucosal epithelia, a mechanism called immune exclusion. At last, immunoglobulins, because of their di- or multivalent binding properties, may agglutinate microbes into larger clusters allowing for more effective recognition by the immune system and mechanical clearance by the host [36]. Secretory IgA and IgM present at the mucosal sites present 4 valences and 10 to 12 valences respectively. In contrast, IgG display only 2 valences. IgA and IgM are more prone to lead to microbe agglutination than IgG [37;38].

[0228] FIG. 2 shows the analysis of immune complexes formed between IgG and PA by confocal microscopy. Using CFSE-labelled PA and Cy3-labelled plasma-derived IgG, it was surprising to detect large immune complexes of IgG-PA. Antigen binding by immunoglobulins is largely dependent on their antigen-binding (Fab) fragment. As IgG is only divalent, it is not expected to see such aggregates. This result may point out that IgG may additionally bind PA outside of the Fab region, potentially through its sugars. IgG may therefore be more potent at signaling PA to the immune system as expected.

Example 3: Plasma-Derived Immunoglobulins Prevent Tissue Damage Induced by PA

[0229] PA is a pathogenic organism known for its involvement in biofilm formation as well as for its resistance to many antibiotics [39]. PA presents with many virulence factors. Some of those are exoenzymes, such as elastase A and B, Protease IV, exotoxin A, exoenzyme S or hemolysin. Exoenzymes serve at defending PA against components of the immune system as well as at participating into its toxicity and associated tissue damage.

[0230] To assess how much PA was inducing tissue damage in our infection model, we measured the release of lactate dehydrogenase (LDH), which is associated to the rupture of the plasma membrane. Experiment was run in our primary 3D cell culture system and LDH was measured in samples collected at 24 h post-infection. All the immunoglobulin formulations (e.g., IgG, IgA, IgAM and sIgAM) proline (vehicle) were tested. FIG. 3 demonstrates that PA infection is inducing the release of LDH at a level above normal LDH level found in the medium at steady state. Importantly, when immunoglobulins were given with PA, all immunoglobulin formulations were shown to be able to prevent the release of LDH and therefore prevent tissue damage.

[0231] Another way to evaluate tissue damage is to measure trans-epithelial electrical resistance (TEER) of the tissue in vitro. Indeed, this parameter reflects the integrity of tight junction dynamics in cell culture models of an epithelial mono or multi-layer [40]. As a consequence, when tissue integrity is affected, TEER is decreased. To understand how PA infection is affecting the barrier which represents a primary epithelial tissue, TEER pre- and 24 h post-infection was measured. FIG. 4 shows how tissue integrity is affected by the infection and how IgG play a role in preventing it. Maximal dose of IgG and proline did not affect TEER when no bacteria is present on the apical side of the transwell. Upon PA infection, not only LDH is released as seen in FIG. 3, but TEER is also decreased (proline sample). This result points out the loss of tissue integrity of the MucilAir™ when PA is added. To evaluate the activity of IgG in this context, increasing doses of IgG in combination with PA (dose ranging from 5 to 500 μg) were used. While the lowest IgG dose didn't show a good protection against tissue damage, increasing doses (50 to 500 μg) showed a good protection of the tissue with the best effect for the 2 highest doses.

[0232] In addition to LDH release and TEER measurements, the MucilAir™ tissue was viewed using microscopy to evaluate the damage occurring during PA infection. As MucilAir™ is a multi-layer epithelial tissue, confocal microscopy was used. The same set-up as described for FIG. 4 was used. 24 h post-infection, tissues were fixed and cut onto slides for staining and analysis. FIG. 5 demonstrates IgG efficacy at preventing PA-induced tissue damage. Healthy tissues (controls) are represented by the sections appearing on the lower row, on the far right. Upon PA infections, large holes are appearing in the tissue (transwell treated with proline; upper row, left). Increasing doses of IgG were applied with PA (dose ranging from 5 to 500 μg). A dose-dependent effect of IgG in preventing tissue damage was observed. The lowest IgG dose did not prevent tissue damage but seemed to have an effect as holes present with a smaller surface. Increasing IgG doses were associated with no holes. However, for doses ranging from 50 to 250 μg, tissue injuries were still observable. 500 μg IgG gave the best result with a tissue looking as good as in control wells.

[0233] Altogether, all plasma-derived immunoglobulin formulations are able to prevent LDH release. Detailing the mechanism of action behind this result using IgG, it has been shown that immunoglobulin can prevent loss of tissue integrity as well as tissue damage.

Example 4: Plasma-Derived Immunoglobulins Prevent Pseudomonas aeruginosa-Induced Tissue Release of Pro-Inflammatory Cytokines

[0234] Epithelial tissues in the mucosal environment function as barriers to the external world to physically prevent microbes to enter the tissues. However, once damaged, microbes can freely enter. Therefore, to signal when there is potential infection and damage of these barriers, epithelial tissues interact with the immune system through the secretion of “danger” signals or cytokines to alert cellular components of the immune system to migrate to the tissue and offer a second layer of defense.

[0235] IL-6 and IL-8 are pro-inflammatory cytokines, which can be secreted by epithelial tissues when these tissues are insulted. In a next set of experiments, plasma-derived immunoglobulins were evaluated in the prevention of pro-inflammatory cytokines release upon PA infection. FIG. 6 shows IL-8 release by MucilAir™ 24 h post-PA infection. All the immunoglobulin formulations (e.g., IgG, IgA, IgAM and sIgAM) were tested, along with proline (vehicle). FIG. 6 demonstrates that IL-8 secretion is highly increased during PA infection, reaching almost a 3-fold increase. None of the immunoglobulin formulations had a significant effect on IL-8 release by the tissue at steady state. However, when applied with PA, all immunoglobulin formulations could prevent PA-induced secretion of IL-8.

[0236] To depict the effect of IgG in preventing PA-induced IL-8 release, increasing doses of IgG (dose ranging from 5 to 500 μg) were tested in combination to PA. FIG. 7 detailed the dose-response of IL-8 secretion to IgG. Experiment was conducted on MucilAir™ generated from 3 different donors. To account for the donor-to-donor variability, IL-8 secretion post-infection was set in combination with proline as the 100% release condition. Additional conditions were calculated in relation to the 100% release. As shown in FIG. 7, maximal dose of IgG and proline did not affect IL-8 release. Interestingly, IgG decreased substantially PA-induced IL-8 secretion in a dose dependent manner with the best effect obtained for the maximal dose (500 μg).

[0237] In the same way, IL-6 secretion post-PA infection was studied. FIG. 8 shows IL-6 release by MucilAir™ 24 h post-PA infection. All the immunoglobulin formulations (e.g., IgG, IgA, IgAM and sIgAM) were tested, along with proline (vehicle). FIG. 8 demonstrates that IL-6 secretion is highly increased by PA, reaching almost a 6-fold increase. None of the immunoglobulin formulations had a significant effect on IL-6 release by the tissue at steady state. However, when applied with PA, all immunoglobulin formulations could prevent PA-induced secretion of IL-6.

[0238] Altogether, this data set demonstrates that all immunoglobulin formulations prevent the release of pro-inflammatory cytokines such as IL-6 and IL-8 and would potentially reduce local inflammation in PA-infected subjects who received topically applied immunoglobulins as prophylaxis. Prevention of IL-8 and IL-6 secretion upon PA infection may actually translate the prevention of tissue damage by topically applied immunoglobulins against PA. Plasma-derived immunoglobulins may act via immune exclusion against PA as well as by inhibiting exoenzyme activities.

Example 5: Plasma-Derived Immunoglobulins Interact with Human Rhinoviruses

[0239] HRV are mainly known to be responsible for more than half of cold-like illness [41]. However, there are also involved in the exacerbations of chronic obstructive pulmonary disease (COPD) as well as of asthma. More than 100 serotypes exist. To assess if nebulized plasma-derived immunoglobulins could protect individuals from HRV infections, binding from a clinical isolate of HRV by different plasma-derived immunoglobulins was tested. FIG. 9 shows that all immunoglobulins formulations were able to bind HRV in an ELISA assay (see material and methods section) in a dose dependent manner, with dose ranging from 0.7 μg/mL to 500 μg/mL. Binding was however different between each immunoglobulin formulation. IgG was the less potent binder while IgAM and IgA show good binding. Addition of SC to IgAM seems to decrease the potency of IgAM to bind HRV. It may point out that some of the binding is not Fab dependent.

Example 6: Plasma-Derived Immunoglobulins Prevent Shedding and Tissue Damage Induced by Human Rhinoviruses

[0240] Like other viruses, HRV are infecting cells to be able to replicate. In a following step, virions are then assembled and packaged prior to cellular export/shedding via cell lysis. FIG. 10 demonstrates the effect of plasma-derived immunoglobulins to prevent HRV shedding following infection of MucilAir™. When vehicle control (proline) was used, a high shedding (˜10.sup.9 HRV C15 genome copy number/mL) of PA was detected on the apical side of the MucilAir™. As a positive control, Rupintrivir, a rhinovirus 3C protease inhibitor against human rhinovirus, was used. As observed, application of Rupintrivir reduced effectively HRV shedding by 3 logs. Surprisingly, applying plasma-derived immunoglobulins at the time of infection completely reduced HRV shedding to a level which could not be detected with our assay. All immunoglobulin formulations but IgAM could show such a reduction. However, and importantly, IgAM could still decrease HRV shedding by at least 4 logs.

[0241] Plasma-derived immunoglobulins were therefore able to prevent the entrance of HRV in the epithelium and thus its subsequent replication and spreading.

[0242] While replicating, HRV can lead to cell lysis. In the context of an epithelium, we assessed if plasma-derived immunoglobulins would be able to protect epithelial cells against HRV-induced tissue damage. To evaluate this, the TEER parameter was used as a mean to assess tissue integrity post-HRV infection (FIG. 11). At steady state (no infection), TEER measurement was of around 260 Ohm.Math.cm.sup.2 after treatment with vehicle (negative control). Upon HRV infection and application of the vehicle on the tissues (positive control), TEER decreased by almost 5-fold, pointing out the loss of tissue integrity following infection. As showed in FIG. 11, Rupintrivir had a positive effect by preventing HRV-induced tissue damage. All plasma-derived formulations were able to prevent loss of tissue integrity when given with HRV. Immune exclusion of HRV by plasma-derived immunoglobulins proves to be sufficient to protect pulmonary tissues against PA invasion as well as PA-induced cellular damage.

Example 7: Plasma-Derived Immunoglobulins Reduce Human Rhinoviruses-Induced Mucociliary Clearance Decline

[0243] Mucociliary clearance is a key function of the bronchial tissue. Pathogens trapped into the mucus are exported out of the lungs and expectorated to prevent pathogens to stagnate onto the lungs tissues and replicate. Mucociliary clearance can be affected by different mechanisms, one of them being tissue damage.

[0244] As HRV infection is associated with tissue damage, the effect of plasma-derived immunoglobulins on mucociliary clearance 48 h after HRV infection was assessed (FIG. 12). At steady state (no infection), mucociliary clearance was of 40 mm/s after treatment with vehicle (negative control). Upon HRV infection and application of the vehicle on the tissues (positive control), mucociliary clearance decreased by 2-fold. As showed in FIG. 12, Rupintrivir had a positive effect by preventing HRV-induced reduction of mucociliary clearance. All plasma-derived formulations were able to prevent the decrease of mucociliary clearance with the best effect obtain for IgA and IgM, for which no loss of mucociliary clearance was observed.

Example 8: Dose-Dependency of Effects of Human Plasma-Derived Immunoglobulin Formulations on Human Rhinovirus Infection

[0245] Next it was investigated whether the observed effects were dose-dependent. Immunoglobulin formulations were added at 4, 20, 100 and 500 μg/well, and their effect on HRV expansion was assessed by measuring HRV genome copy number. FIG. 13 demonstrates that plasma-derived human immunoglobulin formulations were able to inhibit HRV expansion in a dose-dependent manner.

[0246] Effect on TEER was also investigated, as described above. The immunoglobulin formulations delayed HRV-induced TEER decrease at 4 μg/well and 20 μg/well, at 100 μg/well. At 500 μg/well, the decrease was completely prevented.

[0247] Furthermore, the effect of the different doses of the immunoglobulin formulations on cilia beating frequency and on mucociliary clearance were assessed, using the same doses as specified above. Again, a dose-dependent effect was observed on cilia beating frequency and on mucociliary clearance with all immunoglobulin formulations tested.

[0248] HRV-induced IL-8 secretion on day 2 post infection was also inhibited by the plasma-derived immunoglobulin formulations; even at 4 μg/well IgAM and SIgAM achieved complete inhibition; IgG and IgA achieved a very significant reduction at 4 μg/well, and all immunoglobulin formulations achieved complete inhibition at the higher doses. HRV-induced production of RANTES was also significantly inhibited by the lowest dose of immunoglobulins used (4 μg/ml), and completely inhibited by the higher doses of all immunoglobulin formulations at day 2.

[0249] Altogether, plasma-derived immunoglobulins were able to protect in vitro pulmonary tissues against HRV infection and its associated tissue damage. Prophylactic application of plasma-derived immunoglobulins topically into the lungs of subjects at risk of pulmonary infections will give them a protection against microbes of viral or bacterial origins.

Example 9: Effect of Human Plasma-Derived Immunoglobulin Formulations on Influenza Virus Infection

[0250] The experiments were set up using the same protocol as for rhinovirus infection of MucilAir™ cultures, using Influenza strain H1N1. Oseltamivir was used as positive control at 10 μg/well. It was shown that the immunoglobulin formulations all reduced Influenza expansion in a dose-dependent manner, as shown in FIG. 14.

[0251] TEER disruption by Influenza virus was also reduced by 4 μg/well and 20 μg/well of each of the immunoglobulin formulations, and completed prevented by 100 μg/ml and 500 μg/well.

[0252] An Influenza virus-induced reduction in cilia beating frequency was observed at day 4 post infection. IgG showed the best rescue effect on cilia beating frequency, showing complete restoration already with 4 μg/well. The IgA, IgAM and sIgAM formulations were also able to rescue the cilia beating frequency, albeit only at 20 μg/well and higher concentrations. The Ig formulations were also able to restore mucociliary clearance, reduce Influenza-induced IL-8 secretion and Influenza-induced RANTES secretion.

Example 10: Prevention of Respiratory Tract Infection-Driven Exacerbations in Subjects with Chronic Obstructive Pulmonary Disease (COPD) and/or with Non-Cystic Fibrosis

[0253] Bronchiectasis (NCFB) with nebulized plasma-derived immunoglobulins Subjects with COPD and/or NCFB are subject to chronic respiratory tract infections which can participate into the exacerbation of their disease. Chronicity of these infections is driving the remodeling of their tissues, increasing the severity of disease.

[0254] As shown in the examples above, topically applied plasma-derived immunoglobulins are preventing adhesion and invasion of bacteria and viruses in primary human respiratory tract tissues in vitro. Immune exclusion of these microbes prevented tissue damage and indirectly, the release of pro-inflammatory cytokines as well as the loss of mucociliary clearance.

[0255] To break the chronicity of these infections, subjects with NCFB or mild to severe COPD, potentially in association with NCFB, are treated once or twice daily with nebulized plasma-derived immunoglobulins. Plasma-derived immunoglobulins, formulated in solution at 50 mg/mL up to 150 mg/mL, are nebulized using an active vibrating mesh nebulizer. 2-10 mL of plasma-derived immunoglobulin formulation is applied in the morning and/or in the evening on a daily basis.

[0256] Reduction of infection-driven exacerbations will reduce local inflammation in COPD and NCFB subjects and will delay the progression of the disease.

[0257] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention

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