Pharmaceutical compositions and methods for treating viral acute lung injury using lactoferrin

Abstract

This invention relates to the therapeutic use of lactoferrin (LTF), a multifunctional glycoprotein enriched in mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs), for the treatment of acute lung injury (ALI) caused by respiratory viral infection. LTF restores alveolar fluid clearance, reduces alveolar protein permeability, and downregulates pro-inflammatory cytokines in H5N1-infected human alveolar epithelial cells. In vivo administration of LTF significantly improved the survival rate of infected mice, suppressed lung inflammation, and reduced viral load. The therapeutic efficacy of LTF is comparable to that of MSC-EVs. This invention provides a novel, cell-free, and scalable strategy that bypasses the limitations of stem cell-based therapies while retaining potent antiviral and immunomodulatory effects. As an FDA-approved supplement with established safety, LTF can be rapidly repurposed for clinical use in respiratory diseases involving epithelial barrier dysfunction, inflammation, and viral infections, including influenza-induced ALI.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 730,422, filed December 10, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention utilizes FDA-approved supplement lactoferrin to treat respiratory diseases, the potential therapeutic effects of which have been found due to its enrichment in mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs). Background Technology

[0003] Acute lung injury (ALI) is a life-threatening condition characterized by severe lung inflammation, disruption of the alveolar-capillary barrier, and impaired alveolar fluid clearance, often caused by respiratory pathogens such as highly pathogenic influenza viruses. Current treatment strategies for ALI are largely supportive, including mechanical ventilation and fluid management, with limited pharmacological interventions targeting the underlying mechanisms of inflammation and epithelial damage.

[0004] In recent years, cell-based therapies using mesenchymal stromal cells (MSCs) have attracted attention due to their immunomodulatory and regenerative properties. However, the clinical application of MSCs faces several challenges, including the need for in vitro expansion, potential loss of cell function, and safety concerns such as genetic instability or immune responses. As an alternative, MSC-EVs carrying functional proteins and RNA that mediate paracrine effects have been explored to overcome these limitations.

[0005] Among the protein cargoes of MSC-EVs, lactoferrin (LTF) is a multifunctional glycoprotein known for its anti-inflammatory, antibacterial, and antioxidant activities in various disease models, including SARS-CoV-2 and chronic obstructive pulmonary disease (COPD). However, given the limited number of available preliminary studies and the lack of research exploring the role of LTF in influenza infection, its therapeutic potential in highly pathogenic influenza virus infections has been demonstrated through more in-depth preclinical studies before potential evaluation in subsequent clinical trials.

[0006] Therefore, there is a need in the field for a safe, scalable, and cell-free therapeutic strategy to alleviate acute lung injury caused by respiratory viral infections. This strategy should ideally combine anti-inflammatory and antiviral properties, preserve the integrity of the alveolar epithelial barrier, and be easily and rapidly translatable into clinical practice without the risks and limitations associated with cell-based therapies such as mesenchymal stromal cell transplantation. Summary of the Invention

[0007] Given the limitations associated with existing cell-based therapies for ALI, including immunogenicity risks, manufacturing complexity, and regulatory hurdles, this invention aims to develop a safe, cell-free, and easily translatable therapeutic strategy. A key objective of this invention is to identify and validate functional bioactive components derived from MSC-EVs that can exert equivalent therapeutic effects without requiring intact cells.

[0008] This invention provides a method for treating acute lung injury caused by respiratory viral infection, the method comprising administering an effective amount of LTF, an LTF being a multifunctional glycoprotein abundant in MSC-EV protein cargo. The method may further include evaluating the therapeutic efficacy of LTF in restoring alveolar fluid clearance, reducing epithelial protein leakage, downregulating inflammatory cytokines, and inhibiting viral replication.

[0009] According to one aspect of the invention, a method for treating acute lung injury in a subject infected with a respiratory virus is provided, the method comprising administering to the subject a composition comprising lactoferrin, said lactoferrin being a functional protein enriched in extracellular vesicles (UC-MSC-EV) derived from umbilical cord-derived mesenchymal stromal cells. Compared to an untreated control, the administration resulted in an increase of at least 50% in alveolar fluid clearance and a decrease of at least 50% in alveolar epithelial protein permeability.

[0010] According to one aspect of the invention, the respiratory virus includes a highly pathogenic avian influenza virus.

[0011] According to one aspect of the invention, the highly pathogenic avian influenza virus includes A / HK / 483 / 97 or A / HK / 486 / 1997H5N1 virus.

[0012] According to one aspect of the invention, the composition comprises lactoferrin, wherein the dose of lactoferrin is from about 100 mg / kg to 1000 mg / kg per day based on body weight.

[0013] According to one aspect of the invention, the lactoferrin is selected from recombinant human lactoferrin or purified bovine lactoferrin.

[0014] According to one aspect of the invention, the composition is administered intravenously, intranasally, or orally, or by inhalation.

[0015] According to one aspect of the invention, the administration reduces the expression of one or more cytokines selected from the group consisting of: MCP-1, IL-6, IL-8, RANTES, TNF-α, and MIP-1α.

[0016] According to one aspect of the invention, the composition inhibits the attachment or internalization of viruses into alveolar epithelial cells.

[0017] According to one aspect of the invention, the composition inhibits the expression of the influenza virus matrix (M) gene.

[0018] According to one aspect of the invention, the lactoferrin is formulated with one or more excipients selected from: stabilizers, surfactants, or buffers suitable for parenteral or pulmonary delivery.

[0019] According to one aspect of the invention, the composition further comprises extracellular vesicles derived from mesenchymal stromal cells.

[0020] According to one aspect of the invention, the composition is administered in combination with an antiviral compound selected from oseltamivir, zanamivir, or baloxavir.

[0021] According to one aspect of the invention, a pharmaceutical composition is provided comprising lactoferrin present at a concentration of about 0.01% to 10% (w / v); and a pharmaceutically acceptable carrier suitable for intravenous, intranasal, oral, or inhalation delivery. Proteomic analysis has identified the lactoferrin as a functional protein enriched in extracellular vesicles of umbilical cord-derived mesenchymal stromal cells (UC-MSCs). The composition is formulated for intravenous, intranasal, or inhalation administration in the treatment of acute lung injury caused by respiratory viral infection.

[0022] According to one aspect of the invention, the lactoferrin is selected from recombinant human lactoferrin or purified bovine lactoferrin.

[0023] According to one aspect of the invention, the pharmaceutical composition further comprises one or more excipients selected from the group consisting of surfactants, isotonic agents, buffers, or viscosity modifiers.

[0024] According to one aspect of the invention, the pharmaceutical composition is packaged in a unit-dose vial, nebulizer cartridge, or nasal spray bottle.

[0025] According to one aspect of the invention, the lactoferrin is dissolved in phosphate-buffered saline or citrate buffer.

[0026] According to one aspect of the invention, the pharmaceutical composition is lyophilized and reconstituted before administration.

[0027] According to one aspect of the invention, the pharmaceutical composition further comprises an antiviral drug selected from zanamivir, oseltamivir phosphate, or baloxavirmarboxil.

[0028] According to one aspect of the invention, the respiratory virus includes a highly pathogenic avian influenza virus.

[0029] To demonstrate therapeutic efficacy, this invention utilizes physiologically relevant in vitro human lung injury models and a murine influenza A (H5N1) virus infection model. In both models, LTF alone showed restoration of alveolar epithelial barrier function and reduction of pro-inflammatory cytokines, performing comparable to or better than its performance in MSC-EV. This highlights the feasibility of using LTF as a standalone agent.

[0030] By isolating specific protein components from the MSC-EV secretome, this invention circumvents the challenges associated with the expansion, storage, and administration of live stromal cells. The cell-free approach eliminates variability and enhances scalability while maintaining the desired anti-inflammatory and antiviral effects. Compared to current therapies and MSC-based products, this invention provides a novel, pharmaceutically acceptable, and FDA-approved supplement for respiratory disease intervention. It represents a significant advancement over the prior art by providing a clinically available, well-tolerated, and mechanistically supported strategy for treating viral-induced ALI. Attached Figure Description

[0031] Embodiments of the invention are described in more detail below with reference to the accompanying drawings, in which:

[0032] Figure 1A An experimental setup is shown in which human alveolar epithelial cells (AECs) cultured in transwell grooves are infected with influenza A (H5N1) virus to monitor changes in alveolar fluid clearance (AFC) and alveolar protein permeability (APP). Figure 1B This indicates that MSC-derived extracellular vesicle (MSC-EV) treatment of H5N1-infected AECs restored damaged AFCs compared to untreated infected cells. Figure 1C This indicates that MSC-EV treatment significantly reduced APP caused by H5N1 infection. Figure 1D This demonstrates the classification of cellular components associated with proteins identified in MSC-EVs through proteomics analysis. Figure 1E This illustrates the biological processes enriched in proteins carried by MSC-EVs, as revealed by proteomics profiling analysis.

[0033] Figure 2AA schematic representation of an in vitro lung injury model used to evaluate the therapeutic effects of MSC-EV and LTF on AEC in people infected with H5N1 virus is shown. Figure 2B This indicates that both MSC-EV treatment and LTF treatment attenuate H5N1-induced AFC damage in infected AECs. Figure 2C This indicates that both MSC-EV treatment and LTF treatment reduced the increased APP levels caused by H5N1 infection. Figure 2D This indicates that both MSC-EV treatment and LTF treatment exert anti-inflammatory effects on H5N1-infected AECs, as indicated by the reduced levels of pro-inflammatory cytokines. Figure 2E This indicates that MSC-EV treatment and LTF treatment inhibit the attachment and internalization of H5N1 virus particles into the AEC. Figure 2F This indicates that both treatments upregulated the expression of antiviral genes in infected AECs. Figure 2G This indicates that MSC-EV treatment and LTF treatment significantly inhibited H5N1 virus replication, as demonstrated by the reduced expression of the influenza virus M gene;

[0034] Figure 3A A schematic diagram of the in vivo experimental protocol is shown, in which mice are infected with H5N1 influenza virus intranasally, followed by intravenous administration of MSC-EV or LTF as treatment. Figure 3B This indicates that there was no significant difference in weight loss between the treated and untreated groups in H5N1-infected mice. Figure 3C This indicates that treatment with MSC-EV or LTF improves the survival rate of H5N1-infected mice. Figure 3D This indicates that the levels of pro-inflammatory cytokines in lung homogenates were significantly reduced in mice treated with MSC-EV or LTF. Figure 3E This indicates that the levels of pro-inflammatory cytokines in bronchoalveolar lavage (BAL) fluid were significantly suppressed by treatment with MSC-EV or LTF. Figure 3F The results showed that the expression level of the influenza virus M gene in the lungs of treated mice was reduced, indicating a decrease in viral load. Figure 3G This indicates that the tissue culture infectious dose (TCID) assay confirms that MSC-EV treatment and LTF treatment have comparable antiviral activity in vivo; and

[0035] Figure 4A This indicates that there was no significant difference in weight loss between the treated and untreated groups in H5N1-infected mice. Figure 4B The results showed that treatment with LTF or zanamivir improved the survival rate of H5N1-infected mice, but not when combined. Figure 4C This indicates that the levels of pro-inflammatory cytokines in lung homogenates were significantly reduced in mice treated with LTF, zanamivir, or a combination thereof. Figure 4D The results indicate that the tissue culture infection dose (TCID) assay confirms the potent antiviral activity of LTF, zanamivir, and the combination in vivo. Detailed Implementation

[0036] definition

[0037] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations such as “comprises” or “comprising” should be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes. It should also be noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” and “comprising” may have the meaning attributed to them under U.S. patent law, for example, allowing for elements not expressly listed but excluding elements present in the prior art or affecting the essential or novel characteristics of the invention.

[0038] Furthermore, throughout this specification and claims, unless the context otherwise requires, the word "include" or variations thereof, such as "includes" or "including," shall be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes.

[0039] As used herein and unless otherwise defined, the terms “substantially,” “basically,” “approximately,” and “about” are used to describe and explain small variations. When used in conjunction with an event or situation, the terms may cover instances where the event or situation occurs precisely or instances where the event or situation is close to occurring. For example, when used in conjunction with a numerical value, the terms may cover a range of variation less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%.

[0040] References to "an embodiment," "an example embodiment," "exemplary embodiment," etc., in this specification indicate that the described embodiment may include a specific feature, structure, or characteristic, but not every embodiment necessarily includes that specific feature, structure, or characteristic. Furthermore, such phrases do not necessarily refer to the same embodiment. Moreover, when a specific feature, structure, or characteristic is described in connection with an embodiment, it is assumed that the effect of such feature, structure, or characteristic in conjunction with other embodiments is within the knowledge of those skilled in the art, whether explicitly described or not.

[0041] As used in this article, the term "acute lung injury" (ALI) refers to a clinical syndrome characterized by diffuse alveolar damage, disruption of the alveolar-capillary barrier, pulmonary edema, impaired gas exchange, and an inflammatory response in the lungs. ALI can be caused by infectious agents such as respiratory viruses (including highly pathogenic avian influenza viruses) and is considered a precursor to acute respiratory distress syndrome (ARDS).

[0042] As used herein, the term "alveolar fluid clearance" (AFC) refers to the net transport of fluid from the alveolar cavity to the interstitium and blood, primarily mediated by active ion transport across the alveolar epithelium. An increase in AFC indicates improved epithelial function and reduction of pulmonary edema. In vitro, AFC can be measured based on the dilution of FITC-glucan over time in a transwell culture system.

[0043] As used herein, the term "alveolar epithelial protein permeability" (APP) refers to the extent to which macromolecules (such as proteins) can cross the alveolar epithelial barrier. Increased APP indicates disruption of tight junction integrity and barrier dysfunction. In vitro, APP is quantified by the translocation of fluorescently labeled dextran across the monolayer of alveolar epithelial cells from the apical compartment to the basolateral compartment.

[0044] The lactoferrin used in this invention is a naturally occurring iron-binding glycoprotein with a molecular weight of approximately 80 kDa and containing approximately 700 amino acid residues. Lactoferrin can be derived from human or bovine sources and can be produced through a recombinant expression system. Commercially available formulations (e.g., recombinant human lactoferrin from rice or yeast expression systems) can be used.

[0045] As used herein, the term "mesenchymal stromal cell-derived extracellular vesicle" (MSC-EV) refers to nanoscale membrane-bound vesicles secreted by mesenchymal stromal cells. MSC-EVs contain a variety of bioactive molecules, including proteins, lipids, and RNA, and mediate intercellular communication. In the context of this invention, MSC-EVs can be derived from umbilical cord-derived MSCs and can be isolated using ultracentrifugation or equivalent techniques.

[0046] As used herein, the term "highly pathogenic avian influenza virus" refers to a class of influenza A virus that causes severe illness and high mortality in birds and humans. Examples include the H5N1 and H7N9 subtypes. In this invention, representative viruses include the H5N1 strains A / Hong Kong / 483 / 97 and A / Hong Kong / 486 / 1997.

[0047] Other definitions of the selected terms used herein can be found in the specific description of the invention and apply throughout. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] Highly pathogenic avian influenza viruses, such as the H5N1 subtype, are known to cause severe lower respiratory tract infections and are considered a major threat to global public health. Conventional antiviral treatments, such as oseltamivir, have shown limited efficacy in reducing mortality associated with H5N1 infection. Severe respiratory viral infections often progress to acute lung injury (ALI), or its more advanced form, acute respiratory distress syndrome (ARDS), characterized by lung inflammation, alveolar-capillary barrier disruption, and impaired fluid homeostasis in the lungs. Clinical and preclinical studies have shown that H5N1 virus infection may directly impair AFC and increase APP, both key pathogenic features of ALI. Recovery of AFC and stabilization of barrier function are considered essential for slowing disease progression. Although various pharmacological interventions for ARDS have been explored in clinical trials, no effective drug-based therapies have been approved to date.

[0049] Cell-based therapies have recently gained attention as promising alternatives for treating ALI and other inflammatory lung diseases. Specifically, mesenchymal stromal cells (MSCs) and their secreted extracellular vesicles (EVs) have shown therapeutic potential due to their immunomodulatory, anti-inflammatory, and regenerative properties. EVs are nanoscale vesicles released from living cells that mediate intercellular communication through the delivery of bioactive proteins, RNA, and other molecular cargoes. Among these EVs, MSC-EVs have been reported to suppress inflammation, promote tissue repair, and modulate immune responses. Despite the increasing research on MSC-EVs, studies on their efficacy in highly pathogenic respiratory viral infections remain limited. Furthermore, the complexity of MSC-EV production and the heterogeneity of vesicle contents present logistical and regulatory challenges for clinical translation.

[0050] LTF is a naturally occurring iron-binding glycoprotein that has been extensively studied for its antibacterial, antiviral, and immunomodulatory functions. Initially discovered in cow's milk and later identified in human secretions, LTF is synthesized by exocrine glands and plays a crucial role in innate immune defense. It works by isolating iron, thereby limiting microbial growth, and by modulating immune responses at mucosal surfaces. Notably, human and bovine LTF share high sequence homology (approximately 69%) and exhibit comparable biological activity, making bovine variants suitable for a wide range of preclinical applications. LTF has been granted "generally recognized as safe" (GRAS) status by the US Food and Drug Administration and is approved for use as a dietary supplement in the US and Europe. Despite its well-established safety profile and multifunctional biological activity, the role of LTF in combating H5N1-induced lung injury remains unexplored.

[0051] The pathophysiology of H5N1 infection includes the activation of a pro-inflammatory cytokine cascade that disrupts epithelial integrity and impairs ion transporter function, leading to fluid accumulation in the alveolar spaces.

[0052] To investigate this mechanism, this invention employs a physiologically relevant in vitro lung injury model consisting of primary human aerosolized cells (AECs) cultured on transwell membranes and infected with the H5N1 virus. This model enables quantitative assessment of virus-induced changes in AFC, APP, and inflammatory responses.

[0053] This invention found that treatment with MSC-EV or recombinant human LTF restored damaged AFC in the AEC of H5N1-infected patients and reduced epithelial permeability. Both treatments also downregulated the expression of key pro-inflammatory cytokines and significantly reduced viral replication. Proteomic profiling of MSC-EV revealed that LTF was one of the most abundant protein carriers, suggesting it may be an important contributor to the biological activity of MSC-EV. In vivo studies using a mouse model further confirmed the therapeutic potential of LTF, which exhibited anti-inflammatory and antiviral effects comparable to those of MSC-EV, as well as improved survival outcomes. Caution should be exercised when administering LTF in combination with existing antiviral agents (e.g., zanamivir), as LTF has shown superiority over zanamivir in improving survival and suppressing dysregulated pro-inflammatory cytokines in H5N1; however, unexpected effects may occur when administered in combination. These findings support the application of LTF as a standalone, cell-free therapeutic agent for the treatment of influenza-induced acute lung injury.

[0054] In one embodiment, the present invention provides a method for treating viral-induced acute lung injury, the method comprising administering a therapeutically effective amount of lactoferrin to a subject in need, wherein the lactoferrin restores alveolar fluid clearance and reduces alveolar protein leakage.

[0055] In one embodiment, lactoferrin is derived from bovine or human sources and may be in the form of a recombinant protein or a purified natural product.

[0056] In one embodiment, lactoferrin is administered at a dose of about 100 mg / kg to about 1000 mg / kg per day by weight, preferably at a dose of about 100 mg / kg to about 500 mg / kg, depending on the subject’s condition, route of administration and severity of disease.

[0057] In one embodiment, lactoferrin is formulated as a pharmaceutical composition selected from the group consisting of: a sterile injectable solution, an inhalable powder, a nasal spray, an atomized suspension, or an oral capsule or tablet.

[0058] In one embodiment, the method further comprises reducing the expression of pro-inflammatory cytokines in infected alveolar epithelial cells, the pro-inflammatory cytokines being selected from the group consisting of MCP-1, IL-6, IL-8, RANTES, and TNF-α.

[0059] In one embodiment, the method includes inhibiting the attachment and internalization of respiratory viruses into alveolar epithelial cells by administering lactoferrin.

[0060] In one embodiment, the therapeutic agent is formulated for systemic administration, such as intravenous injection, or for local delivery by inhalation or intranasal instillation.

[0061] In one embodiment, the method is used in combination with existing antiviral agents or supportive care, thereby enhancing the overall treatment outcomes for patients with ALI or ARDS.

[0062] In one embodiment, the pharmaceutical composition comprises lactoferrin in combination with one or more pharmaceutically acceptable excipients, stabilizers, or delivery agents suitable for parenteral, intranasal, or pulmonary administration.

[0063] In one embodiment, lactoferrin is administered in combination with one or more therapeutic agents selected from the following: antiviral agents, anti-inflammatory agents, corticosteroids, cytokine inhibitors, or MSC-EV.

[0064] In another aspect, the present invention provides a combination therapy comprising lactoferrin and an antiviral agent, wherein co-administration results in enhanced inhibition of viral replication and improved survival outcomes.

[0065] In summary, this invention provides a method for treating acute lung injury caused by respiratory viral infections, including but not limited to highly pathogenic H5N1, using lactoferrin as a therapeutic agent. By directly targeting viral attachment, epithelial barrier dysfunction, and cytokine-mediated inflammation, LTF provides a novel therapeutic mechanism distinct from conventional antiviral agents or immunosuppressants. This invention further demonstrates that specific bioactive proteins derived from MSC-EVs, such as LTF, can be isolated and formulated for clinical use, thereby circumventing the complexities and risks associated with cell-based therapies.

[0066] Example

[0067] Example 1 - Materials and Methods

[0068] Virus

[0069] H5N1 was used for in vitro infection. All influenza viruses were passaged in Madin-Darby Canine Kidney (MDCK) cells. The median tissue culture infection dose (TCID) was used. 50 Virus titer was determined. All experiments were conducted in a biosafety level 3 facility.

[0070] Cultivating UC-MSCs

[0071] Umbilical cord MSCs were isolated by HealthBaby Biotech (Hong Kong, China) Company Ltd. and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) supplemented with 10% HyClone fetal bovine serum (FBS) (SV30160.03, Thermo Fisher Scientific) at low glucose levels (1.0 g / L), using 1% P / S for cell culture. UC-MSCs were cultured in a humid environment (37°C, 5% CO2), with the growth medium changed every 48–72 hours. Cells were trypsinized and seeded at 70% confluence for experimental use.

[0072] EV separation from UC-MSC

[0073] At 70% confluence, UC-MSCs were washed three times with HBSS to remove HyClone FBS, and then replenished with 10% EV-depleted FBS-low glucose DMEM. The supernatant was collected at 72 hours and stored at -20°C until EV separation. EV separation was achieved using differential centrifugation via the following steps: 1) centrifugation at 3,000 rpm for 15 minutes at 4°C; 2) collection of the supernatant and centrifugation again at 20,000 g for 30 minutes at 4°C; 3) collection of the supernatant, passing it through a 0.22 μM filter and centrifugation at 100,000 g for 2 hours at 4°C; 4) discarding the supernatant, washing the EV particles with PBS, and centrifuging the EV suspension at 100,000 g for an additional 2 hours at 4°C; 5) removing the supernatant, resuspending the EV particles in PBS, and storing at -20°C until use.

[0074] Isolation and culture of primary human AEC

[0075] AECs were isolated from resected non-malignant lung tissue obtained from the Department of the Cardiothoracic Surgery, Queen Mary Hospital, Hong Kong, China. First, visible bronchi were removed, and the lung tissue was minced using scissors into pieces >0.5 mm thick. The minced lung pieces were washed with Hank's balanced salt solution (Invitrogen, USA) at pH 7.4 to partially remove macrophages and blood cells. The washed lung pieces were then digested in a shaking water bath at 37°C for 40 minutes using 0.5% trypsin (GibcoBRL, USA) and 4 U / ml elastase (Worthington Biochemical, USA). Digestion was terminated using 40% FBSDMEM / F12 medium and DNase I (350 units / ml) (Sigma, USA). Incompletely digested lung cells were separated using a 50 μM pore size cell sieve (BD Bioscience, USA). Cell clumps were dispersed by repeated pipetting for 10 minutes. After downward spin, cells were incubated with a 1:1 mixture of DMEM / F12 medium and small airway growth medium (SAGM) containing 5% FBS and 350 units / ml DNase I (Lonza, USA). Cells were seeded at 37°C in new tissue culture flasks (Corning, USA) for adhesion. Unadhered cells were collected for centrifugation and resuspended in new tissue culture flasks with SAGM. The medium was changed daily for the first 4 days of plating. AECs were trypsinized for inoculation at 75% confluence.

[0076] In vitro acute lung injury model

[0077] To investigate the effects of influenza virus on alveolar fluid clearance and protein permeability in human alveolar epithelial cells, a physiologically relevant 24-well Transwell in vitro acute lung injury model was used. Alveolar epithelial cells were cultured at 1 x 10⁶ cells per well. 5Cells were plated at a density of 1,000 cells per cell in the top side chamber of a 24-well Costa Transwell membrane (Corning) with a pore size of 0.4 μm. The microporous transwell membrane established a liquid-liquid interface similar to that of human lung epithelium and kept the plated cells in a humid environment (5% CO2, 37°C). Transepithelial electrical resistance remained ≥800 Ω / cm. 2 This indicates that there is good tight junction integrity between cells.

[0078] Measurement of alveolar fluid clearance and alveolar protein permeability

[0079] At 24 hours post-infection, net alveolar fluid transport was measured from the apical lateral chamber of the transwell culture groove (containing a monolayer of influenza virus-infected alveolar epithelial cells) to the basal lateral chamber of the transwell. Alveolar epithelial cells were seeded with the corresponding influenza A virus at a multiplicity of infection (MOI) of 0.1 for 1 hour. Then, 200 μl of FITC-labeled 70 kDa dextran (Sigma-Aldrich) was added to the cells (final concentration 500 ng / μl), along with 1 × 10⁻⁶ saturated ... 8 One MSC-EV or 100 μg / ml of recombinant human LTF was added to the top-side medium for 24-hour treatment. After 5 minutes, 100 μl of sample was collected from the top-side chamber for initial FITC measurement and then transferred back to the top-side chamber for overnight incubation. After 24 hours of dextran incubation, 100 μl was collected from both the top-side and basal-side chambers for final FITC measurement. Fluorescence of each sample was measured using a modulus fluorometer (FLUOstar OPTIMA, BMG Labtech) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. A standard curve with known FITC concentrations was constructed to calculate the presence of FITC-glucan in the transwell chamber at different time points. Net alveolar fluid transport across the epithelial monolayer was measured as [1 – (FITC concentration in the initial top-side sample / FITC concentration in the final top-side sample)] / 200 μl / 0.33 cm⁻¹ 2 / 24 hours. After 24 hours of incubation with dextran, the final basal-side readings were collected to measure protein permeability based on the unidirectional flux of fluorescently labeled dextran from the top side chamber to the basal-side chamber of the transwell culture well.

[0080] Virus infection mice

[0081] Use 10 6 TCID log 50The A / Hong Kong / 486 / 1997 (H5N1) influenza virus was administered intranasally to 6-8 week old female BALB / c mice at a volume of 25 μl. On days 3, 5, and 7 post-infection, 1×10⁻⁶ cytidine diphosphate iodide (CDI) was injected intravenously into the mice at a volume of 100 μl. 8 MSC-EVs, 100 μg / ml recombinant human LTF, 10 mg / kg zanamivir, or PBS were administered. Survival and body weight were monitored for 10 days. On day 10 post-infection, viral titers were determined in the lungs of three mice from each group. BAL fluid was collected for mouse Luminex assays according to the manufacturer's instructions (R&D Systems, USA). Additionally, the lungs of three mice from each group were fixed for histopathological analysis.

[0082] Quantitative RT-PCR was used to detect influenza matrix genes, inflammatory cytokines, antiviral genes, and ion transporters. Quantification of mRNA

[0083] Total RNA and cellular proteins were extracted from infected AECs 24 hours post-infection. RNA extraction was performed, and RNA was reverse transcribed into complementary DNA using the PrimeScript RT kit (TaKaRa, Dalian), both according to the manufacturer's instructions. AECs infected with influenza virus at MOI 2 were lysed using 350 μl of buffer RLT (Qiagen, Germany) and β-mercaptoethanol (Sigma-Aldrich, USA). RNA extraction was performed using the MiniBEST Universal RNA Extraction Kit (TaKaRa, Dalian) with DNase treatment according to the manufacturer's instructions. Reverse transcription was performed using the PrimeScript RT kit (TaKaRa, Dalian). ViiA was used... TM 7. Real-time PCR was performed using an Applied Biosystems (USA) system. Gene expression profiles were normalized using the housekeeping gene β-actin mRNA. Standard plasmids with known copy numbers were bound to the study gene and run to generate a standard curve to determine the absolute copy number. Amplification was performed using an ABI 7500 PCR system (Applied Biosystems) via real-time PCR, consisting of a Power SYBR Green PCR master mix (Applied Biosystems) and a master mix of cDNA reverse transcribed from 500 ng of total RNA, for 40 cycles. Gene expression was calculated and statistical analysis was performed according to the manufacturer's instructions.

[0084] Statistical analysis

[0085] All in vitro experiments were performed in triplicate independently. Groups were compared using an unpaired two-tailed t-test. A difference was considered significant if p ≤ 0.05.

[0086] Example 2 - Evaluation of MSC-EV-mediated protection against H5N1-induced epithelial barrier dysfunction

[0087] In this case, a physiologically relevant in vitro lung injury model was used to evaluate the therapeutic efficacy of MSC-EV in alleviating H5N1-induced alveolar epithelial barrier dysfunction.

[0088] Primary human AECs were inoculated into transwell grooves to establish a tight epithelial monolayer under air-liquid interface conditions. After fusion was achieved, the parietal lateral chambers were inoculated with influenza A / HK / 483 / 97 (H5N1) virus at a fold increase in infection (MOI) of 0.1. Figure 1A As shown, the virus and MSC-EV were administered via the top-side compartment. The control group was treated with a mixture of cytokines (Cytomix: IL-1β, TNF-α, and IFN-γ) to induce barrier damage.

[0089] Twenty-four hours after H5N1 infection, a significant decrease in AFC and a significant increase in APP were observed, confirming the disruption of epithelial barrier function. Figure 1B As shown in the figure, compared to the control group infected with the simulant (approximately 1.5 μL / cm²), 2 / hour), H5N1-infected AEC showed a significant decrease in AFC (approximately 0.5 μL / cm). 2 / hour), while MSC-EV treatment significantly restored AFC to near baseline levels (P<0.0001).

[0090] Similarly, FITC-dextran leakage assays showed that MSC-EVs reduced the increased protein permeability induced by H5N1. Figure 1C The study described that the percentage increase in FITC-glucan transport across the epithelial layer was significantly elevated in H5N1-infected cells (approximately 1.0 arbitrary unit), but was significantly suppressed after MSC-EV treatment (approximately 0.2 unit), indicating that tight junction integrity was preserved.

[0091] To identify the functional protein components responsible for therapeutic effects, proteomic analysis was performed on isolated MSC-EVs. Subcellular classification revealed that most EV cargo proteins were located in exosomes, lysosomes, and extracellular compartments. Figure 1D This is consistent with its secretory and signal transduction functions. Functional enrichment analysis further indicates that EV proteins are mainly involved in protein metabolism, signal transduction, and cell communication pathways. Figure 1E All of these are related to tissue repair and antiviral responses.

[0092] Table 1 shows the top five most abundantly expressed proteins identified in MSC-EVs, with “LTF” identified as a key component contributing to the observed therapeutic effects. Among the top five proteins in MSC-EVs, “lactoferrin” is of particular interest due to its reported antiviral and anti-inflammatory properties in other diseases.

[0093] Table 1 protein Function lactoferrin Iron binding, antiviral, anti-inflammatory Function, cytoplasm 2 Cell migration, cytoskeleton maintenance albumin Protein transport Decorin (core proteoglycan) Connective tissue components, matrix assembly α-2-macroglobulin Broad-spectrum protease inhibitors

[0094] Overall, these findings suggest that MSC-EVs play a protective barrier and immunomodulatory role in H5N1-infected alveolar epithelial cells. In particular, the identification of lactoferrin as the major active cargo supports the feasibility of using selected MSC-EV-derived proteins instead of whole EV formulations to treat virus-induced acute lung injury.

[0095] Example 3 - The therapeutic effect of lactoferrin in H5N1-infected alveolar epithelium in vitro

[0096] This case study used an in vitro lung injury model to investigate the therapeutic efficacy of recombinant human LTF, a major functional protein present in MSC-EV, in attenuating H5N1-induced lung epithelial injury.

[0097] like Figure 2A As shown, primary human AECs were cultured in transwell wells and infected with influenza A / HK / 483 / 97 (H5N1) virus (MOI = 0.1). After infection, the cells were loaded with MSC-EVs (1 × 10⁶ cells per well). 8 Treat with 100 μg / mL of human lactoferrin or 100 μg / mL for 24 hours.

[0098] like Figure 2B As shown, H5N1 infection leads to a significant decrease in AFC, while treatment with MSC-EV or LTF partially restores the damaged AFC. Similarly, Figure 2C The results showed that both treatments significantly reduced the increase in APP caused by viral infection, indicating that the integrity of epithelial tight junctions was protected.

[0099] The anti-inflammatory effects of MSC-EV and LTF were evaluated by quantifying cytokine secretion in the culture supernatant. Figure 2D The study described that LTF treatment significantly suppressed the secretion of RANTES, MCP-1, IL-6, and IL-8 in infected AECs, but the extent of suppression was generally less than that observed with MSC-EV treatment. These findings confirm the immunomodulatory role of LTF.

[0100] To evaluate antiviral activity, the ability of LTF to interfere with viral entry was assessed. For example... Figure 2EAs shown, both MSC-EV and LTF significantly reduced the attachment and internalization of H5N1 virus particles to AECs. Furthermore, treatment with LTF significantly upregulated the expression of endogenous antiviral genes (e.g., IFITM3, MX1). Figure 2F As shown, this indicates the activation of the host's antiviral defense pathways.

[0101] Consistent with these findings, viral replication was significantly suppressed by LTF treatment, as measured by influenza M gene expression levels. Figure 2G The reduction in viral RNA levels was comparable to that observed with MSC-EV treatment, suggesting that LTF alone has antiviral efficacy in the infected lung epithelial model.

[0102] In summary, these results indicate that lactoferrin exerts multiple protective effects, including restoring alveolar fluid clearance, maintaining epithelial barrier function, inhibiting inflammatory cytokines, and suppressing viral replication in human alveolar epithelial cells infected with H5N1. The therapeutic efficacy observed in lactoferrin, comparable to that of MSC-derived extracellular vesicles, supports its application as a standalone cell-free therapy candidate for the treatment of virus-induced acute lung injury.

[0103] Example 4 - In vivo evaluation of the therapeutic activity of MSC-EV and lactoferrin against H5N1-induced lung injury.

[0104] This example uses a mouse model of acute lung injury induced by H5N1 virus to evaluate the in vivo therapeutic potential of MSC-EV and recombinant human LTF.

[0105] Infect female BALB / c mice (6-8 weeks old) with 10 nasal tract infections 6 TCID 50 The H5N1 (A / HK / 486 / 1997) virus was detected. Mice were intravenously administered MSC-EV (1×10⁻⁶ in 100 μL PBS) on days 3, 5, and 7 post-infection, following the onset of clinical symptoms. 8 (each particle) or LTF (100 μg in 100 μL PBS), such as Figure 3A As shown in the figure. Mice that received only PBS served as the untreated control group.

[0106] like Figure 3B As shown, no statistically significant difference in weight loss was observed between the treated and untreated groups during the 10-day monitoring period, indicating that treatment did not alter the overall weight dynamics under infection conditions. However, Figure 3C Survival analysis showed that mice treated with MSC-EVs had a significantly increased survival rate compared to untreated controls, while mice treated with LTFs also showed a moderate increase in survival rate.

[0107] Pro-inflammatory cytokines in lung homogenates and BAL solution were measured using multiplex Luminex assays. Figure 3D and Figure 3E As shown, both MSC-EV treatment and LTF treatment significantly reduced inflammatory mediators. Specifically, MSC-EV inhibited a broad spectrum of cytokines, including IL-6, MCP-1, and TNF-α, while LTF showed a more significant effect in reducing MCP-1, IL-6, and MIP-1α levels, indicating its potent anti-inflammatory activity.

[0108] Viral replication was assessed by quantifying the expression of the influenza M gene in lung tissue samples. Figure 3F As described, both MSC-EV and LTF significantly reduced viral gene expression levels compared to untreated mice. Additionally, Figure 3G The TCID assay shown in the figure confirms that both treatment groups significantly suppressed viral titers, indicating that their antiviral efficacy was comparable.

[0109] Example 5 - Combination therapy of LTF and zanamivir in H5N1-infected mice

[0110] To examine whether the combination therapy of LTF with existing antiviral agents could further enhance the treatment potential, a mouse model of acute lung injury induced by H5N1 virus, identical to the model described in Example 4, was used.

[0111] Infect female BALB / c mice (6-8 weeks old) with 10 nasal tract infections 6 TCID 50 The H5N1 (A / HK / 486 / 1997) virus was detected. Mice were administered LTF (100 μg in 100 μL PBS), zanamivir (10 mg / kg body weight), or a combination thereof intravenously on days 3, 5, and 7 post-infection, following the onset of clinical symptoms. Mice receiving only PBS served as an untreated control group.

[0112] like Figure 4A As shown, no statistically significant difference in weight loss was observed between the treated and untreated groups during the 10-day monitoring period, indicating that treatment did not alter the overall weight dynamics under infection conditions. However, Figure 4B Survival analysis showed that mice treated with LTF had a significantly increased survival rate compared to untreated controls, while mice treated with zanamivir also showed a moderate increase in survival rate.

[0113] Pro-inflammatory cytokines in BAL solution were measured using a multiplex Luminex assay. Figure 4CAs shown, all three treatments resulted in a significant reduction in inflammatory mediators. Specifically, LTF inhibited a broad spectrum of cytokines, including RANTES, TNF, IFN-γ, MCP-1, IL-6, and MIP-1α, while the other two treatments exhibited lower pro-inflammatory activity.

[0114] The viral replication in lung tissue samples was assessed using TCID assays. Figure 4D As described in the study, all treatment groups significantly suppressed viral titers, indicating comparable antiviral efficacy.

[0115] These in vivo results collectively support the therapeutic benefits of both MSC-EV and LTF in alleviating H5N1-induced lung pathology. Treatment reduced viral load and inflammatory cytokine levels, leading to improved survival outcomes. Notably, LTF exhibited an anti-inflammatory spectrum that differed from and was superior to that of MSC-EV in several parameters, further supporting its feasibility as a standalone cell-free therapeutic candidate for respiratory viral infections.

[0116] The foregoing description of the present invention has been provided for purposes of illustration and description. The foregoing description is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations will be apparent to those skilled in the art.

[0117] Examples have been selected and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention with respect to the various embodiments and with various modifications suitable for the particular intended use.

Claims

1. A method for treating acute lung injury in a subject infected with a respiratory virus, characterized in that, The method comprises administering to the subject a composition containing lactoferrin, which is a functional protein enriched in extracellular vesicles (UC-MSC-EV) derived from umbilical cord-derived mesenchymal stromal cells, and wherein the administration increases alveolar fluid clearance by at least 50% and reduces alveolar epithelial protein permeability by at least 50% relative to an untreated control.

2. The method of claim 1, wherein the respiratory virus comprises a highly pathogenic avian influenza virus.

3. The method according to claim 2, wherein the highly pathogenic avian influenza virus comprises A / HK / 483 / 97 or A / HK / 486 / 1997H5N1 virus.

4. The method of claim 1, wherein the composition comprises lactoferrin, and the dose of lactoferrin is from 100 mg / kg to 1000 mg / kg per day based on body weight.

5. The method according to claim 1, wherein the lactoferrin is selected from recombinant human lactoferrin or purified bovine lactoferrin.

6. The method of claim 1, wherein the composition is administered intravenously, intranasally, or orally, or by inhalation.

7. The method of claim 1, wherein the administration reduces the expression of one or more cytokines, the one or more cytokines being selected from the group consisting of: MCP-1, IL-6, IL-8, RANTES, TNF-α, and MIP-1α.

8. The method according to claim 1, wherein the composition inhibits the attachment or internalization of the virus into alveolar epithelial cells.

9. The method of claim 1, wherein the composition inhibits the expression of the influenza virus matrix (M) gene.

10. The method of claim 1, wherein the lactoferrin is formulated with one or more excipients selected from: stabilizers, surfactants, or buffers suitable for parenteral or pulmonary delivery.

11. The method of claim 10, wherein the composition further comprises extracellular vesicles derived from mesenchymal stromal cells.

12. The method of claim 1, wherein the composition is administered in combination with an antiviral compound selected from oseltamivir, zanamivir, or baloxavir.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: Lactoferrin, present at a concentration of 0.01% to 10% (w / v), wherein, by proteomics analysis, the lactoferrin is a functional protein enriched in extracellular vesicles derived from umbilical cord-derived mesenchymal stromal cells (UC-MSCs); and Pharmaceutically acceptable carriers, wherein the pharmaceutically acceptable carriers are suitable for intravenous, intranasal, oral, or inhalation delivery, The composition is formulated for intravenous, intranasal, oral, or inhalation administration in the treatment of acute lung injury caused by respiratory viral infection.

14. The pharmaceutical composition according to claim 13, wherein the lactoferrin is selected from recombinant human lactoferrin or purified bovine lactoferrin.

15. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition further comprises one or more excipients selected from surfactants, isotonic agents, buffer solutions, or viscosity modifiers.

16. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is packaged in a unit dose vial, nebulizer cartridge, or nasal spray bottle.

17. The pharmaceutical composition of claim 13, wherein the lactoferrin is dissolved in phosphate-buffered saline or citrate buffer.

18. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is lyophilized and reconstituted prior to administration.

19. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition further comprises an antiviral drug selected from zanamivir, oseltamivir phosphate, or baloxavir marboxil.

20. The pharmaceutical composition of claim 13, wherein the respiratory virus comprises a highly pathogenic avian influenza virus.

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