COMPOSITIONS FOR PREVENTION AND TREATMENT OF RSV AND CORONAVIRUS INFECTION

Abstract

Compositions and methods are provided for the prevention or treatment of RSV infection and SARS-CoV2 virus related infections, such as COVID-19 (2019-nCoV), in a human. The methods include administering one or more doses of a composition comprising an encapsulated nano-metal oxide (NMO) or nano-metal oxide chelate (NMO-Ch). The dose can be formulated for topical or parenteral administration. Topical administration includes administration as a nasal spray, or by inhalation of respirable particles or droplets. Particles or droplets contain material systems that include composite particles having a core and one or more shells that enclose the core. In this case, the shell is a nonlamellar amorphous material, and the internal matrix core contains a metal oxide or metal oxide chelate.

Claims

1. A composition for inhibiting respiratory viruses including RSV and SARS-CoV2 related infections comprising; encapsulated metal oxides or chelated metal oxide molecules wherein said metal oxide(s) molecules are silver oxide molecules in solution and wherein said silver oxide(s) molecules are present in a concentration of between 5 and 250 parts per million in said solution.

2. The composition of claim 1, wherein a solvent provides a liquid solution of encapsulated metal oxides or chelated metal oxide molecules wherein said solvent is selected from one or more of a group consisting of water, alcohol, plant-derived glycerine, and aloe.

3. The composition of claim 1, wherein said silver oxide molecules are encapsulated with one or more biopolymers that have a core and one or more shells that enclose said core.

4. The composition of claim 2, wherein said shells are comprised of a nonlamellar amorphous material and wherein said core contains said silver oxide molecules.

5. The composition of claim 2, wherein said biopolymers are polyvinylpyrrolidone (PVP) as provided in Structure (1), or another water-soluble polymer of N-vinylpyrrolidone (NVP) or a derivative thereof as provided in structure (2). ##STR00003##

6. The composition of claim 4, wherein said silver oxide molecule encapsulated with said biopolymers are either silver oxide or chelated silver oxide nanoparticles with a particle size of between 1 nm and 20 nm.

7. The composition of claim 6, wherein said silver oxide nanoparticles exhibit a high degree of inhibition of said viruses specifically in a range of 8 nm to 11 nm.

8. The composition of claim 1, wherein said silver oxide nanoparticles reduce RSV replication and production of pro-inflammatory cytokines in epithelial cell lines and in mouse lung that is mediated by neutrophils.

9. A method of inhibiting respiratory viruses including RSV and SARS-CoV2 related infections comprising inhalation and/or intranasal administration of doses of encapsulated metal oxide or chelated metal oxide molecules wherein said metal oxides are silver oxides and are provided in a liquid solution wherein a solvent provides a solution of encapsulated metal oxides or chelated metal oxide molecules wherein said solvent is selected from one or more of a group consisting of water, alcohol, plant-derived glycerine, and aloe.

10. The method of claim 9, wherein said method includes parenteral administration of said molecules.

11. The method of claim 9, wherein said method is useful in reducing RSV mRNA levels, RSV protein levels and RSV viral titers in a subject, said subject including a mammal, and a human.

12. The method of claim 9, wherein administration of multiple doses of encapsulated silver oxide or chelated silver oxide molecules over a course of days provides a therapeutically effective amount of chelated silver oxide.

13. The method of claim 9, wherein a first of a plurality of doses is administered in a prophylactic manner before a subject is infected with RSV or a SARS-CoV2 virus related infection, such as COVID-19.

14. The method of claim 9, wherein an additional risk reduction benefit of an inhalation treatment of an encapsulated metal oxide or chelated metal oxide composition for ventilated patients includes suppression of biofilm formation inside an endotracheal or tracheostomy tube.

15. The method of claim 9, wherein said liquid solution is formulated to have an osmolality ranging from 200-400 mOsm/kg.

16. The method of claim 9, wherein said liquid solution is a buffered solution and wherein a pH of said liquid solution is between 5 and 8.

17. The method of claim 98, wherein a composition is administered as an aerosolized liquid such as a nasal spray.

18. The method of claim 9, wherein said aerosolized liquid is produced by a nebulizer.

19. The method of claim 9, wherein 0.1 ml to 0.6 ml of said aerosolized liquid comprising an encapsulated metal oxide or metal oxide chelate is administered to each nostril.

20. The method of claim 9, wherein a plurality of doses can be administered daily, wherein a plurality includes two, three, four, or five doses.

21. The method of claim 9, wherein administering of said plurality of doses reduces RSV protein, mRNA, or titer in a cell of a human or mammalian respiratory tract to at least a level of administering a single dose that equals a dose provided by said plurality of doses.

22. The method of claim 9 wherein administering of said plurality of doses by inhalation delivers a total dose of between 0.1 and 0.6 mg/kg of an encapsulated metal oxide or metal oxide chelate to a mammal and/or human.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0075] FIG. 1: In-vivo intranasal NAgC administration protected mice from H3N2 infection. (A) survival rate changes (%). (B) Changes in body weight (%). (Prior Art; Adapted from Xiang et al., “Inhibition of A/Human/Hubei/3/2005 (H3N2) influenza virus infection by silver nanoparticles in vitro and in vivo”. Int J Nanomedicine. 2013; 8(1):4103-4114, https://doi.org/10.2147/IJN.S53622).

[0076] FIG. 2A-2F: Graphical analysis of results showing silver nanoparticles (AgNPs) decreased RSV replication in epithelial lines.

[0077] FIG. 3A-3E: AgNPs decreased RSV replication in the lung tissue of experimentally infected mice.

[0078] FIG. 4A-4C: AgNPs decrease viral-induced cytokines, while increasing those associated with neutrophil recruitment and activation.

[0079] FIG. 5A-5F: AgNPs increase recruitment and activation of neutrophils to the lung, regardless of infection status.

[0080] FIG. 6A-6B: Neutrophil depletion results in a reversal of the antiviral effect noted in neutrophil immunocompetent AgNP-RSV mice.

DETAILED DESCRIPTION

[0081] The instant specification may refer to one or more of the following abbreviations whose meanings are defined in Table 1, below.

[0082] TABLE 1: List of Abbreviations

[0083] A Adenosine

[0084] AgNPs Silver Nanoparticles

[0085] BAL Bronchoalveolar Lavage

[0086] BALF Bronchoalveolar Lavage Fluid

[0087] C Cytidine

[0088] CXCL1 Chemokine (C-X-C motif) Ligand 1

[0089] CXCL8 Chemokine (C-X-C motif) Ligand 8

[0090] Da Daltons

[0091] ELISA Enzyme-Linked Immunosorbent Assay

[0092] F12K Nutrient Mixture Ham's F-12K (Kaighn's Modification)

[0093] g Gram

[0094] H1N1 Influenza A virus subtype H1N1; A/H1N1

[0095] H3N2 Influenza A virus subtype H3N2; A/H3N2

[0096] HCV Hepatitis C Virus

[0097] HIV Human Immunodeficiency Virus

[0098] IFN Interferon

[0099] IL Interleukin

[0100] kg Kilogram

[0101] LDH Lactate Dehydrogenase

[0102] LRTI Lower Respiratory Tract Illness

[0103] M Molar

[0104] MEM Minimum Essential Media

[0105] mg Milligram

[0106] mL Milliliter

[0107] mM Millimolar

[0108] MOI Multiplicity of Infection

[0109] mRNA Messenger Ribonucleic Acid

[0110] NAgC Nano-silver Colloids

[0111] ND Not Detected

[0112] nm Nanometer

[0113] NT Not Tested

[0114] OTC Over the counter

[0115] p.i. post-infection

[0116] PBS Phosphate-Buffered Saline

[0117] pH Potential of Hydrogen

[0118] ppm Parts Per Million

[0119] PCR Polymerase Chain Reaction

[0120] qRT-PCR Quantitative Reverse Transcription PCR

[0121] RSV Respiratory Syncytial Virus

[0122] VAP Ventilator Acquired Pneumonia

[0123] vol/vol Volume per Volume

[0124] w/v Weight by Volume

[0125] w/w Weight by Weight

[0126] In the anti-viral uses of the present disclosure, silencing of a target gene will result in a reduction in “viral titer” in the cell or in the subject. As used herein, “reduction in viral titer” refers to a decrease in the number of viable virus produced by a cell or found in an organism undergoing the silencing of a viral target gene. Reduction in the cellular amount of virus produced will preferably lead to a decrease in the amount of measurable virus produced in the tissues of a subject undergoing treatment and a reduction in the severity of the symptoms of the viral infection.

[0127] As used herein, a “subject” refers to a mammalian organism undergoing treatment for a disorder mediated by viral expression, such as RSV infection or undergoing treatment prophylactically to prevent viral infection. The subject can be any mammal, such as a primate, cow, horse, mouse, rat, dog, pig, goat. In the preferred embodiment, the subject is a human.

[0128] As used herein, treating viral infection refers to the amelioration of any biological or pathological endpoints that 1) is mediated in part by the presence of the virus in the subject and 2) whose outcome can be affected by reducing the level of viral gene products present.

Example

Materials and Methods

RSV Preparation

[0129] RSV Long strain was grown in HEp-2 cells and purified by centrifugation on discontinuous sucrose gradients. The titer of viral pools was determined by a methylcellulose plaque assay using HEp-2 cells, as described previously by Ueba (Ueba, O. Acta Med. Okayama 1978, 32, 265-272) and Kisch et al. (Proc. Soc. Exp. Biol. Med. 1963, 112, 583-589.). Virus pools were aliquoted, quick-frozen on dry ice-alcohol, and stored at −80 deg C. until needed.

PVP-Coated Silver Nanoparticles (AgNP)

[0130] 10 nm polyvinylpyrrolidone (PVP) coated BioPure™ silver nanospheres were purchased from NanoComposix Inc. (San Diego, Calif., USA). The PVP coating was chosen for its tight association with the silver particle, making the AgNP as stable as possible in a variety of different environments. Per the manufacturer, the AgNPs used in this study have a mass concentration of 1 mg/mL with a size distribution of 8-12 nm. The AgNPs exhibited an optimal density of 155 cm.sup.−1 and a peak wavelength of 390 nm. Endotoxin concentrations were less than 5 EU/mL, and silver purity was 99.99%. TEM images of the AgNPs provided by the manufacturer are available upon request. For in vitro analyses, AgNPs were diluted in F12K or MEM (110 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin) to a total volume of 1 mL. For in vivo analyses, AgNPs were diluted in sterile phosphate-buffered saline (PBS) prior to mice inoculation.

Studies In Vitro

[0131] A549 cells, a human alveolar type II-like epithelial cell line, and HEp-2 Cells (American Type Culture Collection, Manassas, Va., USA) were grown in F12K and MEM, respectively. Media contained 10% (vol/vol) FBS, 10 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Confluent monolayers were infected with RSV treated with varying doses of AgNPs (0, 10, 25 or 50 μg/mL). Samples were incubated with shaking for 1 h at room temperature prior to plating. A549 cells were infected at a multiplicity of infection (MOI) of 1 for 24 h. HEp-2 cells were infected at an MOI of 0.01 for 48 h. Supernatants were aliquoted and stored at −80 deg C. To evaluate viral titer, serial five-fold dilutions of infected supernatants were determined by plaque assay on HEp-2 cells under methylcellulose overlay. Plaques were visualized five days later, and viral titers were calculated as PFU/mL. Additionally, CXCL8 (IL-8) and CCL5 (RANTES) were also quantified by an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minn., USA).

[0132] The toxicity of AgNPs on epithelial cells was assessed in vitro with an A549 cell line, using lactate dehydrogenase (LDH) activity as an index of cellular damage. To measure LDH activity, A549 epithelial cells were exposed to varying doses of AgNPs (0, 10, and 50 μg/mL) for 24 h. LDH in the medium was measured by colorimetric assay using a commercially available kit (Cayman Chemical, Ann Arbor, Mich., USA) following the manufacturer's instructions. This assay measures cellular damage in response to chemical compounds or environmental factors using a coupled two-step reaction, as previously described.

[0133] A549 are adenocarcinomic human alveolar basal epithelial cells constituting a cell line that was first developed in 1972 by D. J. Giard, et al. through the removal and culturing of cancerous lung tissue in the explanted tumor of a 58-year-old Caucasian male. HEp-2 cells lines are from tumors which were produced in irradiated-cortisonised weanling rats after injection of epidermoid carcinoma tissue isolated from the larynx of a 56 year old male. STR (DNA)-profiling has revealed that the Hep-2 cell line is almost identical to the HeLa cell line.

Viral Infection of Balb/c Mice

[0134] Female, 10 to 12-week-old BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, Me., USA) and housed under pathogen-free conditions in the animal research facility of the University of Texas Medical Branch (UTMB), Galveston, Tex. BALB/c is an albino, laboratory-bred strain of the house mouse from which a number of common sub-strains are derived. Now over 200 generations from New York in 1920, BALB/c mice are distributed globally, and are among the most widely used inbred strains used in animal experimentation. All care and procedures involving mice in this study were in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and UTMB institutional guidelines for animal care. A mixture of Ketamine (90-150 mg/kg) and Xylazine (7.5-16 mg/kg) was administered by intraperitoneal (IP) injection for anesthesia and euthanasia. This protocol was approved by the Institutional Animal Care and Use Committee of UTMB (protocol number 90010021).

[0135] The dosage of AgNPs was calculated based on the weight of the animals. All inoculants were incubated with shaking for 1 h at room temperature prior to inoculation. Under light anesthesia, mice were intranasally inoculated with 100 μL of sterile PBS as a mock inoculation, AgNPs (2 mg/kg or 4 mg/kg) diluted in PBS (denoted as AgNP-PBS), RSV diluted in PBS at a dose of 5×10.sup.6 PFU (denoted as RSV), or RSV mixed with AgNPs (2 mg/kg or 4 mg/kg) diluted in PBS (denoted as AgNP-RSV). Animals from all groups were evaluated on a daily basis for weight loss, illness score, and presence of respiratory symptoms. The percentage of bodyweight change was plotted over time. Clinical illness scores were visually determined by two investigators using a standardized 0-5 grading system (0-no disease, 1-slightly ruffed fur, 2-full ruffed fur, 3-ruffed fur and hunched back, 4-ruffed fur, hunched back and inactive, and 5-death). These parameters have been shown to closely correlate with lung pathology in experimental infection of mice.

[0136] Determination of viral copy number and Mus musculus MX dynamin-like GTPase 1 (Mx1) in the mouse lung was done using quantitative real-time PCR (qRT-PCR). Total RNA was extracted using a ToTALLY RNA kit (catalog number AM1910; Ambion, Austin, Tex., USA). RNA samples were quantified by a NanoDrop spectrophotometer and quality was analyzed on an RNA Nano-drop by the Agilent 2100 bioanalyzer (Agilent Technologies). Synthesis of cDNA was performed with 1 μg of total RNA in a 20 μL reaction mixture by using TaqMan Reverse Transcription Reagents kit from ABI (catalog number N8080234; Applied Biosystems). Amplification was done using 1 μL of cDNA in a total volume of 25 μL using the Faststart Universal SYBR green Master Mix (Roche Applied Science #04913850001). The RSV N-specific reverse transcriptase (RT) primer contained a tag sequence from the bacterial chloramphenicol resistance (Cmr) gene to generate the cDNA, because of self-priming exhibited by RSV RNA. To detect the RSV genome (−) strand, RSV N RT primer 5′-CTGCGATGAGTGGCAGGCACTACAGTGTATTAGACTTRACAGCAGAAG-3′ was used. For PCR assays, we used RSV tag primer CTGCGATGAGTGGCAGGC and primer RSV P GCATCTTCTCCATGRAATTCAGG. To detect Mx1, the mRNA sequence reported under GenBank accession number NM_010846 was used to design amplification primers for qRT-PCR assays. 18S RNA was used as a housekeeping gene for normalization. PCR assays were run in the ABI Prism 7500 Sequence Detection System. Triplicate cycle threshold (C.sub.T) values were analyzed in Microsoft Excel by the comparative C.sub.T (ΔΔC.sub.T) method according to the manufacturer's instructions (Applied Biosystems). The amount of target (2.sup.−ΔΔCT) was obtained by normalization to the endogenous reference (18S) sample. RNA isolation, primer design, and qRT-PCR assays were performed at the Molecular Genomics Core, UTMB, Galveston.

Airway Obstruction

[0137] Airway obstruction was measured in unrestrained mice at days one and five post-infection (p.i.) using whole-body barometric plethysmography (Buxco Electronics, Troy, N.Y., USA) to record enhanced pause (Penh)., as previously described (Ivancuic, T., et al., Am J Respir Cell Mol Biol Vol 55, Iss 5, pp 684-696, November 2016). Penh is a dimensionless value that represents a function of the ratio of peak expiratory flow to peak inspiratory flow and a function of the timing of expiration. To establish baseline airway obstruction values, mice were acclimatized to the chambers for five minutes, and respiratory activity was recorded for five minutes. This protocol was designed by Buxco Electronics, and the laboratory staff was trained by the company on the use of this protocol.

Bronchoaveolar Lavage (Bal)

[0138] At days one or five p.i., mice were euthanized with an IP injection of ketamine and xylazine followed by exsanguination via the femoral vessels. An incision was made in the trachea, through which the lungs were flushed twice with 1 mL of cold sterile PBS to obtain BAL fluid (BALF). The chest cavity then was opened for lung collection. Total cell counts were determined by trypan blue staining, followed by counting of viable cells using a hemocytometer. Additionally, 100 μL of BALF was spun onto glass cytocentrifuge slides and stained with H&E (Hema 3 stain, Fisher Scientific) for differential cell counts. The remaining BALF was centrifuged and supernatants were collected, and stored at −80 deg C. until needed for further assays.

Measurement of Cytokines, Chemokines, Interferon, Total Protein and Elastase

[0139] Cytokines, chemokines, interferons, and elastase were all measured using BALF collected at day one p.i. Total proteins were measured using BALF collected at days one and five p.i. Levels of cytokines and chemokines in the BALF were determined with a Bio-Plex Pro Mouse Group I 23-plex panel (BioRad Laboratories, Hercules, Calif., USA). Interferon (IFN)-α and IFN-β were measured by ELISA, following the manufacturer's protocol (PBL Biomedical Laboratories, Piscataway, N.J., USA). Total protein concentrations were determined using the Bradford method (BioRad Laboratories, Hercules, Calif., USA). Neutrophil elastase was measured using a neutrophil elastase ELISA kit (R&D Systems, Minneapolis, Minn., USA). Absorbance for all microplate assays was measured on a SpectraMax 190 microplate reader (MDS Analytical Technologies, Sunnydale, Calif., USA).

Neutrophil Depletion in Balb/c Mice

[0140] In a separate set of experiments, 10 to 12-week-old female BALB/c mice were depleted of neutrophils with an IP injection of 200 μg anti-Ly6G (Clone 1A8; Bio X Cell, West Lebanon, N.H., USA) in a final volume of 100 μL, 12 h prior to infection. Anti-Ly6G was diluted in PBS immediately before being administered to mice. Control mice received the same volume of PBS via IP injection 12 h prior to infection. For infection, mice were intranasal inoculated with 100 μL RSV diluted in PBS at a dose of 5×10.sup.6 PFU or RSV treated with AgNPs (2 mg/kg or 4 mg/kg) diluted in PBS. Lungs were collected at day five p.i. for determination of viral titer CPE plaque assay using lung homogenate and by qRT-PCR.

Statistical Analysis

[0141] The data were analyzed by a one-way ANOVA followed by Tukey's post hoc test for samples with unequal variances (GraphPad Prism 7; GraphPad Software, Inc., San Diego, Calif., USA). Results are expressed as mean±SEM for each experimental group unless stated otherwise, and p≤0.05 value was selected to indicate significance.

Results

AgNPs Reduce RSV Replication in Epithelial Cell Lines

[0142] The effect of AgNPs on RSV infection was assessed in vitro by plaque assay in two epithelial cell lines, A549 (MOI: 1; FIG. 2A) and HEp-2 (MOI: 0.01; FIG. 2B). Following incubation of RSV with AgNPs (0, 10, 25, and 50m/mL), both cell lines demonstrated significant dose-dependent reductions in RSV replication. The dose of 50m/mL AgNP was the most effective in either cell type with a decrease of RSV replication by 79% in A549 cells and 78% in HEp-2 cells. This decrease was associated with a significant reduction in CXCL8 (FIG. 2C) and CCL5 (FIG. 2D) secretion by RSV-infected cells. Interestingly, the lowest dose of AgNP (10 μg/mL) induced a small increase in viral replication, coupled with increase secretion of CXCL8 and CCL5 (FIG. 2A-D). This represents a concentration as low as 10 ppm of the AgNPs.

[0143] In addition to viral replication, the potential toxicity of AgNPs on A549 cells was also assessed using LDH as an index of cellular damage. There were no notable cytotoxic effects following 24 h of exposure to either the lowest or highest dose of AgNPs (10 or 50 μg/mL) (FIG. 2E).

[0144] Additional toxicity testing of AgNPs on epithelial cells was assessed in vitro with a A549 cell line at varying doses of AgNPs (0.016, 0.05, 0.159, 0.501, 1.58, 5, 15.82, and 50m/mL), as shown in FIG. 2F. There were no notable cytotoxic effects following 24 h of exposure to either the lowest or highest dose of AgNPs (0.016 or 50m/mL).

AgNPs Reduces RSV Replication in the Lung Tissue of Experimentally Infected Mice

[0145] To understand the role of AgNPs in the context of RSV infection in vivo, BALB/c mice were intranasally inoculated with PBS, AgNP-PBS (2 mg/kg or 4 mg/kg), RSV (5×10.sup.6 PFU), or AgNP-RSV. Lung tissue was collected at day five p.i. to evaluate RSV copy number by qRT-PCR. Mice treated with AgNP-RSV had significant reductions in RSV copy number as compared to the RSV untreated mice (FIG. 3A). Of the two AgNP doses, 4 mg/kg AgNP was most significant, with a decrease of 55%. The dose of 2 mg/kg AgNP elicited a reduction of 45%.

[0146] Over the course of the disease, mice were monitored daily for changes in clinical parameters (i.e., bodyweight loss and illness score). Mice inoculated with AgNP-PBS did not display any signs of disease or weight loss over the five-day monitoring period, indicating that AgNPs per se do not lead to clinical illness in mice. Mice inoculated with either dose of AgNP-RSV did not differ in bodyweight loss as compared to RSV untreated mice (FIG. 3B). Mice inoculated with 4 mg/kg AgNP-RSV demonstrated a minor increase in illness score only at day five p.i. (FIG. 3C). No other group had any significant changes in illness score over the five-day period.

[0147] Next, to assess the effects of AgNPs on pulmonary function, airway obstruction was analyzed by whole-body plethysmography (Buxco Electronics, Inc., Sharon, Conn., USA) and expressed as enhanced pause (Penh). Mice inoculated with either dose of AgNP-PBS had no notable changes to baseline Penh at day one or day five p.i. (data not shown). Mice inoculated with AgNP-RSV showed an increasing trend in baseline Penh values, but were statistically similar to the RSV untreated mice at both time points (FIG. 3D). We also evaluated the concentration of total protein as a marker of epithelial damage and increased vascular permeability. At day 1 p.i., mice inoculated with 2 mg/kg AgNP-RSV demonstrated an average value of 0.89 mg/mL, which was a marginal increase compared to the RSV untreated group that demonstrated total protein levels of 0.66 mg/mL (FIG. 3E). Mice inoculated with 4 mg/kg AgNP-RSV had total protein levels comparable to the RSV untreated mice. At day 5 p.i., mice inoculated with 4 mg/kg AgNP-RSV demonstrated a significant increase in total protein concentration with a value of 2.53 mg/mL as compared to 1.11 mg/mL for RSV untreated mice. Mice inoculated with 2 mg/kg AgNP-RSV had, total protein levels comparable to the RSV untreated mice (FIG. 3E).

AgNPs Decrease Many RSV-Induced Cytokines and Chemokines, while Increasing Those Associated with Neutrophil Recruitment and Activation

[0148] To investigate the immunomodulatory effects of AgNPs during RSV infection, cytokine and chemokine concentrations of the BALF at day one p.i. were evaluated by a multiplex cytokine array. In mice inoculated with AgNPs, regardless of infection status, G-CSF and GM-CSF were significantly increased (FIG. 3A). Conversely, inflammatory and immunomodulatory cytokines, such as interleukin (IL)-1α, IL-6, IL-9, IL-10, IL-12p40, IL-12-p70, IL-13, and TNF-α were significantly decreased in mice inoculated with either dose of AgNP-RSV (FIG. 4A). In addition to G-CSF and GM-CSF, the chemokine CXCL1 (KC) was significantly increased in all mice inoculated with AgNPs, regardless of infection status. Chemokines associated with viral replication, such as CCL3 (MIP-1α) and CCL5 (RANTES), were significantly decreased (FIG. 4B).

[0149] To assess type-I IFN production, an ELISA (PBL Biomedical Laboratories, Piscataway, N.J., USA) was conducted at day one p.i. using the BALF of mice inoculated with AgNP-RSV and compared with RSV untreated mice. This was further supported by testing for the Mx1 gene within the lung tissue by qRT-PCR. IFN-α, IFN-β, and Mx1 were all significantly decreased following inoculation with either dose of AgNP-RSV as compared to the RSV untreated mice (FIG. 4C).

AgNPs Increase and Maintain Neutrophil Cell Counts in the BALF, Regardless of Infection Status

[0150] To determine whether AgNP treatment affected the cellular composition, BAL samples were collected from inoculated mice at days one and five p.i. for total and differential cell counts. The total cell count was significantly greater in the BALF of all AgNP inoculated mice at days one and five, regardless of infection status (FIG. 5A). Differential cell counts revealed this increase to be primarily due to a significant increase in the number of neutrophils at both time points (FIG. 5B,C). Macrophage cell counts were unaffected in either group at day one p.i. and had an increasing trend in cell count at day five p.i., regardless of infection status (FIG. 5D). Lymphocyte cell counts were comparable to the respective controls at both days one and five p.i. (FIG. 5E).

[0151] Next, we wanted to understand if the neutrophils present in the BALF of mice inoculated with either dose of AgNPs were activated using elastase as a marker of neutrophil activation. BALF collected at day one p.i. demonstrated significant increases in elastase in all AgNP inoculated mice, regardless of infection status. A dose-dependent increase in elastase production was observed in AgNP-RSV treated mice as compared to RSV untreated mice (FIG. 5F).

Neutrophils are a Primary Mechanism of the Antiviral Activity by AgNPs In RSV-Experimentally Infected Mice

[0152] Some studies have shown AgNPs to increase neutrophil cell counts within the BALF of treated mice, but few have investigated their activity following recruitment (Haberl, N, et al. Beilstein J. Nanotechnol. 2013, 4, 933-940; Luo, Y. et al. Bio Med Res. Int., 2015). Neutrophils have also been suggested to have antiviral capabilities during RSV infection, leading us to evaluate the function of neutrophils in AgNP-RSV treated mice. Mice were depleted of neutrophils with an injection of anti-Ly6G clone 1A8 12 h prior to inoculation with RSV (5×10.sup.6 PFU), or AgNP-RSV. In neutrophil-depleted mice inoculated with 2 mg/kg AgNP-RSV or 4 mg/kg AgNP-RSV, the viral copy number at day five p.i. was found to be similar to the viral copy number in neutrophil-depleted RSV untreated mice, indicating a reversal of the antiviral effect noted in immunocompetent mice (FIG. 6A). This was further supported by a viral plaque assay using the lung homogenate of infected mice (FIG. 6B). These results support neutrophils as a primary mechanism of the antiviral activity generated by AgNPs against experimental RSV infection in mice.

Discussion of Results

[0153] A significant dose-dependent reduction of RSV replication in both HEp-2 and A549 cell lines was reported, with the strongest antiviral effect elicited by a dose of 50 μg/mL AgNP. In addition, cells exposed to this dose of AgNPs were found to release levels of LDH similar to that of the control, demonstrating that AgNPs at the dose of 50 μg/mL are not toxic to epithelial cells. The effectiveness of 50 μg/mL AgNPs in epithelial cell lines is in agreement with work previously reported examining the effects of AgNPs on influenza strains H1N1 and H3N2. To confirm the findings in vivo, BALB/c mice were inoculated with AgNP-RSV, and lung tissue was collected at day five p.i. to evaluate viral titer. Inoculation with AgNP-RSV resulted in significant reductions in viral titer as compared to RSV untreated mice. This demonstrates, for the first time, the effectiveness of AgNPs against experimental RSV infection in mice.

[0154] The mechanism to the antiviral effect against RSV in epithelial cell lines is likely due to be the attachment of AgNPs to surface glycoproteins. By doing so, AgNPs would interfere with RSV's ability to initiate attachment with the proper receptors, preventing fusion of the virus to the host cell. This would leave RSV in the extracellular space where it is unable to propagate, resulting in the reduction of syncytia formation seen in the plaque assays. The basis of this mechanism has been investigated with other RNA viruses, such as human immunodeficiency virus type-1 (HIV-1). AgNPs were shown to directly associate with the surface glycoprotein gp120, preventing HIV-1 from interacting with host receptors. The gp120 glycoprotein has also been found to have some structural similarities with the RSV-F surface glycoprotein, supporting the hypothesis of a direct association of AgNPs with RSV. Interestingly, exposure of 10m/mL AgNP to RSV in A549 and HEp-2 cell lines resulted in a slight increase in RSV replication and secretion of CXCL8 and CCL5. Though it is not clear at the present time why a low dose of AgNPs would have such an impact on viral parameters, we believe that insufficient coating of the virus with AgNPs would allow the virus to continue infecting epithelial cells. This enhancement of replication was not appreciated with increasing doses of AgNPs, suggesting that RSV virions are more efficiently coated with higher doses of AgNPs (FIG. 1A-B).

[0155] Although a potential direct antiviral mechanism has been previously described, cell culture lacks the interaction of a complex immune system. Therefore, to elucidate potential antiviral mechanisms due to AgNPs in vivo, investigation of the cytokine, and cellular composition of the BALF collected from inoculated mice was undertaken. AgNP-RSV infected mice were found to have significant reductions in many pro-inflammatory and viral associated markers, such as IL-6, TNF-α, CCL5 (RANTES), and type I IFNs. Additionally, significant reductions were also noted with IL-1α, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, CCL2 (MCP-1), and CCL3 (MIP-1α). Previous data in the field of RSV has suggested that a downregulation in pro-inflammatory cytokines, such as TNF-α corresponds with significantly improved clinical disease (i.e., bodyweight loss, illness score, airway obstruction). Conversely, the study demonstrated no significant changes to clinical parameters in AgNP-RSV inoculated mice, despite significant decreases in an array of RSV-induced cytokines. Upregulations in cytokines and chemokines responsible for neutrophil recruitment and/or activation (i.e., elastase, CXCL1, G-CSF, and GM-CSF) have been linked to heightened airway inflammation and RSV bronchiolitis. As shown in FIG. 3, CXCL1, G-CSF, and GM-CSF were all found to be significantly increased following AgNP inoculation. Therefore, the beneficial effects typically associated with reductions in pro-inflammatory cytokines are likely counteracted by the strong presence of these neutrophil associated cytokines.

[0156] Consistent with increases in cytokines associated with neutrophil recruitment, the number of neutrophils in the BALF was also found to be significantly increased. These findings are in agreement with other reports demonstrating an upregulation of neutrophils in many rodent models following inoculation with AgNPs. The mechanism to this neutrophilia is believed to be an initial stimulation of macrophages to release CXCL1 (KC), but the role of neutrophils during AgNP exposure is still poorly understood. RSV is also known to induce neutrophilia during the early stages of the disease, but the activity of neutrophils during RSV infection has only recently begun to be properly elucidated, and reports remain contradictory. Therefore, to evaluate the role of neutrophils following AgNP-RSV exposure, we depleted mice of neutrophils using an injection of anti-Ly6G. AgNP-RSV treated mice that had been depleted of neutrophils were found to have viral titers similar to RSV untreated neutrophil-depleted mice, demonstrating a reversal of the antiviral effect observed in AgNP-RSV neutrophil immunocompetent mice. These findings suggest AgNPs modulate the function of neutrophils in RSV infection to enhance their antiviral activity.

[0157] Toxicity studies of AgNPs in the lung have been performed in a variety of rodent models and have shown overall that AgNPs induce minor airway mucosa thickening and cellular infiltration (primarily neutrophils), but generate no major alterations to lung function, even following 28 days of continuous exposure. This is consistent with our findings, which demonstrate a sizable neutrophil influx to the BAL fluid through day five in mice inoculated with PBS-AgNPs, but without evidence of clinical disease (i.e., bodyweight-loss, illness score, airway obstruction). Similarly, although both doses of AgNPs resulted in significantly increased neutrophil recruitment/activation in RSV-infected mice, clinical parameters were comparable to those observed in RSV-infected untreated mice at the peak of disease (day 2-4). At the time of recovery (day 5), we observed some delay in the regaining of bodyweight and clinical disease along with higher total protein content in the BAL only in the group of infected mice that had received the higher dose of AgNPs (4 mg/kg), a result of the higher number of activated neutrophils in the BALF (FIG. 2) as previously suggested. However, mice that received the 4 mg/kg AgNP dose also exhibited the largest reduction in RSV lung titers, as well as in all pro-inflammatory cytokines (i.e., TNF-α, IL-6), suggesting that AgNPs potentiate the neutrophil anti-RSV activity as their major antiviral function in the experimental mouse model.

[0158] In conclusion, the study demonstrates that AgNPs effectively reduce RSV replication and production of pro-inflammatory cytokines in epithelial cell lines and in mouse lung. In the mouse model, the antiviral activity appears to be mediated to a large extent by neutrophils, which are recruited in higher number to the airways and activated via a neutrophil-specific program of cytokines that include CXCL1, G-CSF, and GM-CSF. These findings contribute to the understanding of AgNP bioactivity in the lung while providing insights on the role that neutrophils play in the host response against infections caused by RSV.

[0159] This application incorporates all cited references, patents, and patent applications by references in their entirety for all purposes.