Use of pulmonary surfactant protein D for the treatment of viral infections

Abstract

Some embodiments of the methods and compositions provided herein relate to the use of pulmonary surfactant protein D (SP-D) for the treatment or amelioration of a viral infection in a subject. In some embodiments, the viral infection comprises a coronavirus, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Some embodiments involve the use of certain formulations comprising recombinant human SP-D (rhSP-D).

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 072,354, filed August 31, 2020, and entitled "USE OF SURFACTANT PROTEIN D TO TREAT VIRAL INFECTIONS," and U.S. Provisional Application No. 63 / 013,726, filed April 22, 2020, and entitled "USE OF SURFACTANT PROTEIN D TO TREAT VIRAL INFECTIONS," each of which is incorporated by reference in its entirety.

[0002] Sequence Listing Reference This application has been submitted with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled AIRWY017SEQLIST, created on April 14, 2021, and is approximately 6 kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] Some embodiments of the methods and compositions provided herein relate to the use of pulmonary surfactant protein D (SP-D) for the treatment or amelioration of a viral infection in a subject. In some embodiments, the viral infection comprises a coronavirus, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Some embodiments involve the use of certain formulations comprising recombinant human SP-D (rhSP-D). [Background technology]

[0004] A novel human disease, coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged. SARS-CoV-2 belongs to the Coronaviridae family, which also includes severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), which cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), respectively. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 10,975,389 [Patent Document 2] U.S. Patent No. 1,0752,914 [Patent Document 3] U.S. Patent No. 9,492,503 [Patent Document 4] National Patent No. 6838428 [Patent Document 5] U.S. Patent Application Publication No. 2021 / 0010988 [Patent Document 6] WO2019 / 191247 [Patent Document 7] WO2019 / 191254 [Patent Document 8] U.S. Patent Application Publication No. 2019 / 0071693 [Patent Document 9] U.S. Patent Application Publication No. 2019 / 0071694 [Non-patent literature]

[0006] [Non-Patent Document 1] Hartshorn KL et al. (1994) J. Clin. Invest. 94:311-319 [Non-patent document 2] VanEijk, M.ら, (2019) Front Immunol. 10: 2476 pages [Non-licensed Document 3] Hsieh INら(2018) Front Immunol. 9:1368 pages [Non-licensed Document 4] Walls ACら(2020) Cell 181:281~292 pages [Non-licensed Document 5] Tegally, H.ら(2021) Nature 592:438~443 pages [Non-licensed Document 6] Voloch, CMら(2021) J Virol., doi: 10.1128 / jvi.00119-21 [Non-licensed Document 7] Liu, Y., (2021) "The N501Y spike substitution enhances SARS-CoV-2 transmission" bioRxiv [Non-licensed Document 8] Rees-Spear, C.ら, (2021) Cell Rep 34: 108890 [Non-licensed Document 9] Filipe Pereira (2021) Biochem Biophys Res Commun、550: pages 8~14 [Non-licensed Document 10] Crouch E.ら(1994) J Biol Chem 269:17311~page 9 [Non-licensed Document 11] Hakansson Kら, Protein Sci (2000) 9:1607~17 [Non-licensed Document 12] Crouch E. Respir Res (2000) 1: 93-108 [Non-licensed Document 13] Crouch E.ら(2006) J Biol Chem 281:18008~14 pages [Non-licensed Document 14] White Mら, J Immunol (2008) 181:7936~43 pages [Non-licensed Document 15] Yamoze Mら, J Biol Chem (2008) 283:35878~35888 pages [Non-licensed Document 16] Zhang Lら, J Biol Chem (2001) 276:19214~19219 pages [Non-licensed Document 17] White M.ら、(2008) J. Immunol 181:7937~7942 pages [Non-licensed Document 18] Greene KEら(1999). Am J Respir Crit Care Med 160:1843~1850 pages [Non-licensed Document 19] Wright JR. (2005) Nat Rev Immunol 2005; 5: 58-68 [Non-licensed Document 20] Kingma PSら(2006) Curr Opin Pharmacol 6:277~283 pages [Non-licensed Document 21] LeVine AMら(2004) Am J Respir Cell Mol Biol 31:193~199 pages [Non-licensed Document 22] Ikegami Mら(2006) Am J Respir Crit Care Med 173: 1342~1347 pages [Non-licensed Document 23] Hartshorn KL (1998). Am J Physiol 274: pp. 958-969 [Non-licensed Document 24] LeVine AM (2001) J Immunol 167:5868~5873 [Non-licensed Document 25] "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins; 20th Edition (June 1, 2003) [Non-Patent Document 26] "Remington's Pharmaceutical Sciences," Mack Pub. Co.; 18th and 19th editions (December 1985 and June 1990, respectively) [Non-Patent Document 27] Pandolfi, L. et al. (2020) BMC Pulm Med 20: 301 [Non-patent document 28] Sorensen, GL et al. (2007) Immunobiology 212:381-416 [Non-Patent Document 29] Hermans C et al. (1999). Am J Respir Crit Care Med 159:646-678 [Non-Patent Document 30] Honda Y et al. (1995) Am J Respir Crit Care Med 152:1860~1866 [Non-Patent Document 31] Arroyo, R. et al. (2018) J Mol Biol 430:1495-1509 [Non-Patent Document 32] Reed LJ et al. (1938) American Journal of Epidemiology 27:493-497 Summary of the Invention [Problem to be solved by the invention]

[0007] COVID-19 was first identified in Wuhan, the capital of Hubei Province, China, in December 2019. It has since spread worldwide, causing a coronavirus pandemic. Common symptoms include fever, cough, and shortness of breath. Other symptoms include fatigue, muscle pain, diarrhea, sore throat, loss of smell, and abdominal pain. While the majority of patients experience mild illness, some cases progress to viral pneumonia and multiple organ failure. Patients are managed with supportive care, which can include fluid therapy, oxygen support, and support for other affected vital organs. Treatment for COVID-19 and related viral disorders is needed. [Means for solving the problem]

[0008] Some embodiments of the methods and compositions include a method of treating or ameliorating a viral infection in a subject, comprising administering to the subject an effective amount of recombinant human pulmonary surfactant protein D (rhSP-D) or an active fragment thereof.

[0009] In some embodiments, the viral infection comprises a respiratory infection.

[0010] In some embodiments, the viral infection comprises a coronavirus. In some embodiments, the viral infection comprises a virus selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV-1), and Middle East respiratory syndrome-associated coronavirus (MERS-CoV), HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1. In some embodiments, the viral infection comprises SARS-CoV-2.

[0011] In some embodiments, the SARS-CoV-2 comprises an S1 protein variant. In some embodiments, the S1 protein variant comprises a mutation selected from N501Y, D614G, HV69-70del, K417N, and E484K. In some embodiments, the S1 protein lacks a mutation selected from K417N and E484K.

[0012] In some embodiments, administering comprises administering a pharmaceutical composition comprising rhSP-D or an active fragment thereof.

[0013] In some embodiments, the pharmaceutical composition comprises a buffer, a sugar, and a calcium salt.

[0014] In some embodiments, the buffering agent is selected from the group consisting of acetate, citrate, glutamate, histidine, succinate, and phosphate, hi some embodiments, the buffering agent is histidine.

[0015] In some embodiments, the concentration of histidine is from about 1 mM to about 10 mM.

[0016] In some embodiments, the sugar is selected from the group consisting of sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabinose, xylose, ribose, rhamnose, trehalose, sorbose, melezitose, raffinose, thioglucose, thiomannose, thiofructose, octa-O-acetyl-thiotrehalose, thiosucrose, and thiomaltose, hi some embodiments, the sugar is lactose.

[0017] In some embodiments, the lactose concentration is between 200 mM and 300 mM, hi some embodiments, the lactose concentration is about 265 mM.

[0018] In some embodiments, the calcium salt is selected from the group consisting of calcium chloride, calcium bromide, calcium acetate, calcium sulfate, and calcium citrate, hi some embodiments, the calcium salt is calcium chloride.

[0019] In some embodiments, the concentration of calcium chloride is about 1 mM to about 10 mM, hi some embodiments, the concentration of calcium chloride is about 5 mM.

[0020] In some embodiments, the pharmaceutical composition has a pH of about 5.0 to about 7.0, hi some embodiments, the pharmaceutical composition has a pH of about 6.0.

[0021] In some embodiments, the concentration of rhSP-D is from about 0.1 mg / ml to about 10 mg / ml.

[0022] In some embodiments, the pharmaceutical composition comprises a population of rhSP-D polypeptides having oligomeric forms, wherein more than 30% of the oligomeric forms comprise a rhSP-D dodecamer, in some embodiments, more than 35% of the oligomeric forms comprise a rhSP-D dodecamer, in some embodiments, more than 40% of the oligomeric forms comprise a rhSP-D dodecamer.

[0023] In some embodiments, the pharmaceutical composition comprises a bulking agent, hi some embodiments, the bulking agent is selected from the group consisting of mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, arabitol, glycine, alanine, threonine, valine, and phenylalanine.

[0024] In some embodiments, the pharmaceutical composition lacks a chelating agent, hi some embodiments, the chelating agent is selected from EDTA and EGTA.

[0025] In some embodiments, rhSP-D comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:02.

[0026] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0027] Some embodiments of the methods and compositions include a pharmaceutical composition for use in treating or ameliorating a viral infection in a subject, the pharmaceutical composition comprising recombinant human pulmonary surfactant protein D (rhSP-D) or an active fragment thereof.

[0028] In some embodiments, the viral infection comprises a respiratory infection.

[0029] In some embodiments, the viral infection comprises a coronavirus. In some embodiments, the viral infection comprises a virus selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV-1), and Middle East respiratory syndrome-associated coronavirus (MERS-CoV), HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1. In some embodiments, the viral infection comprises SARS-CoV-2. [Brief explanation of the drawings]

[0030] [Figure 1A] FIG. 1 is a schematic diagram showing an ELISA assay for detecting binding of immobilized SP-D to the S1 subunit of the spike protein of SARS-CoV-2 (S1 protein). [Figure 1B] 1 is a line graph of absorbance with increasing S-protein concentration in an assay of binding between immobilized SP-D and S-protein for a first sample of immobilized SP-D in the presence of calcium, EDTA, or maltose. Plates were coated using 5 μg / mL SP-D. [Figure 1C]1 is a line graph of absorbance with increasing concentrations of S1 protein in an assay of binding between immobilized SP-D and S1 protein for a second sample of immobilized SP-D in the presence of calcium, EDTA, or maltose. Plates were coated using 5 μg / mL SP-D. [Figure 1D] 1 is a line graph of absorbance with increasing concentrations of S1 protein in an assay of binding between immobilized SP-D and S1 protein for a first sample of immobilized SP-D in the presence of calcium, EDTA, or maltose. Plates were coated using 2 μg / mL SP-D. [Figure 1E] 1 is a line graph of absorbance with increasing concentrations of S1 protein in an assay of binding between immobilized SP-D and S1 protein for a second sample of immobilized SP-D in the presence of calcium, EDTA, or maltose. Plates were coated using 2 μg / mL SP-D. [Figure 2A] FIG. 1 is a schematic diagram showing an ELISA assay for detecting binding of SP-D to immobilized S1 protein. [Figure 2B] 1 is a graph of absorbance with increasing concentrations of SP-D in an assay of binding of SP-D to immobilized S1 protein for a first sample of immobilized SP-D in the presence of calcium or EDTA. [Figure 2C] 10 is a graph of absorbance with increasing concentrations of SP-D in an assay of binding of SP-D to immobilized S1 protein for a second sample of immobilized SP-D in the presence of calcium or EDTA. [Figure 3] Graph showing SP-D concentrations in bronchoalveolar lavage fluid obtained from COVID-19 patients and control subjects previously reported in the literature. Error bars represent 1.5 times the interquartile ratio (Q3 vs. Q1). [Figure 4A] Graph showing absorbance units of various concentrations of rhSP-D in an ELISA to measure binding of rhSP-D to immobilized S1 protein of SARS-CoV-2 (Wuhan variant). [Figure 4B]Graph showing absorbance units of various concentrations of SARS-CoV-2 S1 protein (Wuhan variant) in an ELISA to measure binding of S1 protein to immobilized rhSP-D. [Figure 4C] Graph showing absorbance units of various concentrations of rhSP-D in an ELISA to measure binding of rhSP-D to immobilized S1 protein variants of SARS-CoV-2 (Wuhan variant, UK variant, and South Africa variant). [Figure 4D] Graph showing absorbance units of various concentrations of rhSP-D in an ELISA to measure binding of rhSP-D to immobilized S1 protein variants of SARS-CoV-2 containing a single mutation (N501Y). [Figure 4E] Graph showing absorbance units of various concentrations of rhSP-D in an ELISA to measure binding of rhSP-D to immobilized S1 protein variants of SARS-CoV-2 containing a single mutation (D614G). [Figure 5A] FIG. 1 shows a scheme of a cross-linking assay between S1 protein and maltose-coated beads via rhSP-D, in which rhSP-D is premixed with S1 protein before the addition of maltose-coated beads. [Figure 5B] FIG. 1 shows a scheme of a cross-linking assay between S1 protein and maltose beads via rhSP-D, in which rhSP-D is pre-incubated with maltose-coated beads before the addition of S1 protein. [Figure 5C] Figure 5B shows an SDS-PAGE gel for the scheme shown in Figure 5A. The gel was developed by silver staining to detect S1 protein (migrating as 100-140 kDa) and rhSP-D (43 kDa). [Figure 5D] Figure 5B shows an SDS-PAGE gel for the scheme shown in Figure 5B. The gel was developed by silver staining to detect S1 protein (migrating as 100-140 kDa) and rhSP-D (43 kDa). [Figure 5E]1 is a bar graph showing the relative densitometry of the eluted (P) bands from the premixing approach and the first rhSP-D at 4 μg of rhSP-D in the presence of S1 protein or buffer. Error bars represent the standard deviation of the densitometry (n=2). [Figure 6A] 1 is a line graph showing the results of an ELISA to determine the binding of ACE2 to immobilized S1 protein in the presence of various concentrations of rhSP-D. [Figure 6B] 1 is a bar graph showing the results of an ELISA to determine the binding of ACE2 to immobilized S1 protein in the presence of various concentrations of rhSP-D. [Figure 6C] 1 is a line graph showing the results of an ELISA to determine binding of S1 protein to immobilized rhSP-D in the presence of various concentrations of ACE2. [Figure 6D] 1 is a bar graph showing the results of an ELISA to determine binding of S1 protein to immobilized rhSP-D in the presence of various concentrations of ACE2. [Figure 7] 1 is a graph showing the CCID50 (50% cell culture infectious dose) of SARS-CoV-2 at various concentrations of rhSP-D. Each data point represents the average of three replicates. DETAILED DESCRIPTION OF THE INVENTION

[0031] Some embodiments of the methods and compositions provided herein relate to the use of pulmonary surfactant protein D (SP-D) for the treatment or amelioration of a viral infection in a subject. In some embodiments, the viral infection comprises a coronavirus, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Some embodiments involve the use of certain formulations comprising recombinant human SP-D (rhSP-D).

[0032] SP-D is involved in innate defense against some viruses in the lungs, such as influenza A virus (IAV) (Hartshorn KL et al. (1994) J. Clin. Invest. 94:311-319, which is incorporated herein by reference in its entirety). Multivalent lectin-mediated interaction of SP-D with IAV results in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phagocytes (VanEijk, M. et al. (2019) Front Immunol. 10:2476, which is incorporated herein by reference in its entirety). SP-D binds to viral hemagglutinin (HA), particularly mannosylated glycans on HA, in a calcium-dependent manner (Hsieh IN et al. (2018) Front Immunol. 9:1368, which is incorporated herein by reference in its entirety).

[0033] Coronaviruses, including SARS-CoV-2, have four structural proteins known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins. The N protein carries the RNA genome, while the S, E, and M proteins together form the viral envelope. The spike glycoprotein (S protein) is responsible for attaching the virus to and fusing with the host cell membrane. Coronavirus entry into host cells is mediated by the S protein, which forms a homotrimer that protrudes from the viral surface (Walls AC et al. (2020) Cell 181:281-292, incorporated herein by reference in its entirety). The S protein contains two functional subunits: the S1 subunit, which is responsible for binding to host cell receptors, and the S2 subunit, which is responsible for fusing viral and cellular membranes. In many coronaviruses, the S protein is cleaved at the interface between the S1 and S2 subunits, which remain noncovalently associated in the prefusion conformation. The distal S1 subunit contains the receptor-binding domain and contributes to stabilizing the prefusion state of the membrane-anchored S2 subunit, which contains the fusion machinery.

[0034] The S1 subunit of the S protein contains a receptor-binding domain that interacts with the human angiotensin-converting enzyme 2 (ACE2) receptor on type II pneumocytes. Viral recognition of the S protein by the ACE2 receptor leads to internalization of the virus by host cells, resulting in viral replication. New copies of SARS-CoV-2 are externalized and infect more cells, increasing the viral load in the lungs, exacerbating the proinflammatory response, and amplifying cellular and epithelial lung injury. These pathological events in the lungs trigger the clinical symptoms of COVID-19: fever, cough, shortness of breath, fatigue, and mild to moderate respiratory distress. In severe cases, pneumonia progresses to combined ALI / ARDS, respiratory failure, septic shock, and even death. To date, a vaccine for this disease is still in clinical trials. Remdesivir has shown efficacy by shortening patient recovery time by 4 days, and dexamethasone has reduced mortality in critically ill patients by 33%. However, there remains a need for more effective treatments that specifically target the virus and the exacerbated inflammatory response.

[0035] New variants of SARS-CoV-2 have emerged due to mutations in certain amino acids in the viral sequence, some of which are located in the spike protein. B.1.1.7 (the so-called UK variant), B.1.351 (the South African variant), and P.1 (Brazil) are some of the most concerning due to their global spread and / or the severity of the resulting clinical disease (Tegally, H. et al. (2021) Nature 592:438-443; and Voloch, C.M. et al. (2021) J Virol., doi: 10.1128 / jvi.00119-21). These variants harbor different mutations, but three of them share two common mutations in the S1 protein: N501Y and D614G (Liu, Y. et al., (2021) "The N501Y spike substitution enhances SARS-CoV-2 transmission," bioRxiv; and Rees-Spear, C. et al., (2021) Cell Rep 34: 108890). Further examples of variants are disclosed in Filipe Pereira (2021) Biochem Biophys Res Commun, 550: 8-14, which is incorporated by reference in its entirety.

[0036] Pulmonary surfactant comprises four distinct pulmonary surfactant proteins. Two hydrophobic proteins, pulmonary surfactant protein B and pulmonary surfactant protein C, are involved in reducing surface tension at the air-water interface; two hydrophilic proteins, pulmonary surfactant protein A and SP-D, are members of the collectin family and are involved in regulating the host immune response and recycling the surfactant pool. SP-D is a C-type (Ca 2+SP-D is a calcium-dependent lectin containing four domains: a cysteine-bonded N-terminal region required for intermolecular disulfide bond formation; a triple-helical collagen region; an α-helical coiled-coil trimerization neck peptide; and a C-terminal calcium-dependent carbohydrate recognition domain (CRD) (Crouch E. et al. (1994) J Biol Chem 269:17311-9). Monomers form trimers by folding the collagen region into a triple helix and assembling a coiled-coil bundle of α-helices in the neck region. These trimers are stabilized by two disulfide bonds in the cysteine-rich N-terminal domain. SP-D trimers have a total molecular weight of 129 kDa and contain three identical 43 kDa polypeptide chains. SP-D trimers can form higher-order oligomerization states that differ in size and conformation. Higher oligomerization states may be important for SP-D function (Hakansson K et al., Protein Sci (2000) 9:1607-17; Crouch E. Respir Res (2000) 1:93-108; Crouch E. et al. (2006) J Biol Chem 281:18008-14). Therefore, pharmaceutical compositions of SP-D should have an oligomerization state appropriate for optimal activity, including binding to carbohydrate ligands on the surface of pathogens (White M et al., J Immunol (2008) 181:7936-43). The proper oligomerization state is also involved in optimal receptor recognition and receptor-mediated signaling for regulating host immune responses (Yamoze M et al., J Biol Chem (2008) 283:35878-35888) and maintaining surfactant homeostasis (Zhang L et al., J Biol Chem (2001) 276:19214-19219). Deletion studies with the rat SP-D protein demonstrated that the rat cysteine-linked N-terminal region is responsible for efficient virus neutralization and opsonization. See White M. et al., (2008) J. Immunol 181:7937-7942, which is incorporated herein by reference in its entirety.

[0037] SP-D binds to glycosylated ligands on pathogens, such as LPS on bacteria, hemagglutinin (HA) on influenza viruses, and F protein on respiratory syncytial viruses. Binding triggers opsonization, aggregation, and direct killing of the microorganisms, promoting their clearance from the lungs by phagocytes, such as macrophages. SP-D dodecamers and higher oligomers have shown increased activity and efficacy in this antibacterial function. In addition to its role in pathogen clearance, SP-D has also demonstrated anti-inflammatory effects in animal models of bacterial and viral respiratory infections and in mechanical ventilation-induced lung injury; in both cases, SP-D reduced levels of inflammatory cytokines (e.g., IL-6), neutrophil responses and nephrosis, and lung tissue damage. Animal models have consistently demonstrated an association between higher levels of pulmonary SP-D and improved outcomes after viral, bacterial, or mechanical lung injury. Similarly, human studies have demonstrated lower mortality in ARDS patients with high levels of pulmonary SP-D. Full-length recombinant hSP-D has been successfully produced in mammalian cells and displays structure and activity comparable to human native SP-D. Therefore, rhSP-D may represent a novel class of antiviral therapeutic agents for COVID-19.

[0038] Disclosed herein are studies that demonstrate the importance of SP-D in COVID-19 and the potential of antiviral molecules, such as rhSP-D, as antiviral therapeutics for COVID-19. As discussed in more detail below, SP-D levels have been found to be substantially reduced in COVID-19 patients. Administration of rhSP-D may compensate for the reduced pulmonary SP-D levels observed in the lungs of COVID-19 patients. Additionally, binding of rhSP-D to the SARS-CoV-2 spike protein has been found to inhibit viral replication in host cells. Such binding may also lead to viral aggregation, resulting in more effective clearance of the virus by phagocytes.

[0039] Consistent with the clinical significance of SP-D activity, a positive correlation has been shown between survival from ARDS and higher levels of lung SP-D early in this syndrome (Greene KE et al. (1999). Am J Respir Crit Care Med 160:1843-1850). Herein, we show that COVID-19 patients have a 3- to 4-fold decrease in lung SP-D concentrations compared with non-COVID-19 controls (Figure 3). It was unclear whether low lung SP-D levels in COVID-19 patients are a result of severe SARS-CoV-2 infection or whether SP-D levels increase the risk of developing severe COVID-19. A previous study in patients at risk for developing ARDS found that lower SP-D concentrations in BALF before the onset of ARDS were associated with worse outcomes. This suggests that the latter explanation is more likely and that low lung SP-D levels lead to more severe disease. It is also unclear whether other comorbidities affect lung SP-D levels in COVID-19 patients. Therefore, supplementing COVID-19 patients with exogenous SP-D to reestablish normal and functional levels of SP-D in the lungs may improve outcomes.

[0040] Pathogen recognition and binding to glycosylated determinants is the first step and hallmark action of SP-D, which opsonizes infectious agents (e.g., viruses and bacteria) and promotes their rapid clearance by phagocytes in the lung, as shown in in vivo animal models of SP-D depletion or exogenous SP-D supplementation (Wright JR. (2005) Nat Rev Immunol 2005; 5: 58-68; and Kingma PS et al. (2006) Curr Opin Pharmacol 6: 277-283; LeVine AM et al. (2004) Am J Respir Cell Mol Biol 31: 193-199; Ikegami M et al. (2006) Am J Respir Crit Care Med 173: 1342-1347; Hartshorn KL et al. (1998). Am J Physiol 274:L958-969; and LeVine AM et al. (2001) J Immunol 167:5868-5873). SP-D has shown calcium-dependent binding to the S protein of earlier SARS-CoV strains, and the hyperglycosylation of the current SARS-CoV-2 S protein has been confirmed and mapped. This suggests that the SARS-CoV-2 S protein may be a target of SP-D. Herein, we demonstrate that rhSP-D binds to the current SARS-CoV-2 antigen through a process resembling opsonization and a critical initial step in the clearance of SARS-CoV-2 by SP-D in vivo (Figures 4A and 4B). Thus, rhSP-D may increase viral clearance and reduce viral load in COVID-19 patients.

[0041] The binding affinity of SP-D to the spike protein of the initial variant from Wuhan was very similar to that of the variant that emerged in the UK (B.1.1.7), which spread rapidly and widely around the world. However, its binding affinity to the S protein from the South African variant (B.1.351) was significantly reduced. Many factors determine the infectivity and severity of disease caused by a virus. While recognizing its limitations, it is tempting to speculate that the reduced binding affinity of SP-D to the spike protein may be one of the factors affecting the higher pathogenicity observed in this new South African variant, which may more easily bypass innate immune defenses and translate into viral transmission. Consistent with this, the N501Y spike mutation enhances viral transmission. As disclosed herein, SP-D exhibits reduced binding affinity to spike proteins carrying the N501Y spike mutation.

[0042] Binding of pathogens by rhSP-D causes them to aggregate to form clusters, and multiple viral molecules in the clusters can be removed by phagocytes at once, resulting in more effective viral clearance. The critical initial step of aggregation is driven by the ability of SP-D (hexamers, dodecamers, or higher multimers) to bind more than one virus and form protein bridges linking multiple pathogens. As disclosed herein, SP-D was able to form protein bridges between S proteins (Figures 5A, 5B, 5C, 5D, and 5E). The studies disclosed herein demonstrate the initial step of viral aggregation (i.e., binding) and the subsequent formation of rhSP-D protein bridges. Furthermore, the presence of multiple spike proteins on the surface of intact viruses may further promote viral aggregation and clearance.

[0043] As disclosed herein, rhSP-D has an EC 90The SARS-CoV-2 life cycle was inhibited by blocking intracellular viral replication (Figure 7). Without wishing to be bound by any one theory, a first mechanism for rhSP-D inhibition of viral replication may involve steric blocking of the interaction between the receptor-binding domain within the S protein and ACE2 by rhSP-D bound to the glycosylated S protein, which may limit the accessibility of critical domains in the presence of bound SP-D molecules. However, this effect was not evident when experiments were performed with isolated S1 protein, ACE2, and rhSP-D (Figures 6A, 6B, 6C, and 6D). It is possible that steric blocking may be observed even when the conformation and location of the S protein and ACE2 receptor are restricted on the viral envelope or cellular membrane, respectively. A second mechanism for rhSP-D inhibition of viral replication may involve potential aggregation of SARS-CoV-2 induced by rhSP-D by reducing the number of viral molecules available to interact with host cells. The first and second mechanisms are not mutually exclusive and may be cooperative with each other.

[0044] As disclosed herein, lung levels of SP-D were reduced in patients with COVID-19. Recombinant hSP-D inhibited the viral life cycle by binding SARS-CoV-2 S proteins from various viral variants and inhibiting viral replication. SP-D formed protein cross-links with the S proteins, which may correspond to a step in viral aggregation that enhances viral clearance from the lungs by phagocytes. In addition, SP-D has previously demonstrated anti-inflammatory and lung-protective roles in several viral and bacterial infections. SP-D may represent a novel class of antiviral therapeutics targeting multiple stages of SARS-CoV-2 infection.

[0045] Some embodiments of the methods and compositions provided herein include aspects disclosed in U.S. Pat. No. 10,975,389, U.S. Pat. No. 10,752,914, U.S. Pat. No. 9,492,503, U.S. Pat. No. 6,838,428, U.S. Patent Application Publication No. 2021 / 0010988, and WO2019 / 191247, each of which is incorporated by reference in its entirety.

[0046] Certain treatment methods Some embodiments of the compositions and methods provided herein include methods of treating or ameliorating a viral infection in a subject. In some embodiments, the viral infection includes a respiratory viral infection. In some embodiments, symptoms of the viral infection are prevented, alleviated, and / or ameliorated. In some embodiments, symptoms of the viral infection include fever, cough, and shortness of breath. Other symptoms include fatigue, aches, runny nose, sore throat, headache, diarrhea, vomiting, and loss of smell or taste. In some embodiments, the therapeutically effective amount of the pharmaceutical composition and / or SP-D is sufficient to prevent, alleviate, and / or ameliorate symptoms of the viral infection. In some embodiments, the viral infection includes a coronavirus. Examples of coronaviruses include severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1.

[0047] Some embodiments include methods of treating or ameliorating a viral infection in a subject, comprising administering to the subject an effective amount of recombinant human pulmonary surfactant protein D (rhSP-D) or an active fragment thereof. In some embodiments, the viral infection comprises a respiratory infection. In some embodiments, the viral infection comprises a coronavirus. In some embodiments, the viral infection comprises a virus selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV-1), and Middle East respiratory syndrome-associated coronavirus (MERS-CoV). In some embodiments, the viral infection comprises SARS-CoV-2. In some embodiments, SARS-CoV-2 comprises a wild-type S1 protein. In some embodiments, SARS-CoV-2 comprises an S1 protein of the Wuhan wild-type or variant; the UK variant; or the South African variant. In some embodiments, SARS-CoV-2 comprises an S1 protein variant. In some embodiments, the S1 protein variant comprises a mutation selected from N501Y, D614G, HV69-70del, K417N, and E484K. In some embodiments, the S1 protein lacks a mutation selected from K417N and E484K.

[0048] In some embodiments, the administering step comprises administering a pharmaceutical composition comprising recombinant human pulmonary surfactant protein D (rhSP-D) or an active fragment thereof, hi some embodiments, the pharmaceutical composition comprises a buffer, a sugar, and a calcium salt.

[0049] In some embodiments, the buffering agent is selected from the group consisting of acetate, citrate, glutamate, histidine, succinate, and phosphate. In some embodiments, the buffering agent is histidine. In some embodiments, the concentration of histidine is about 1 mM to about 10 mM.

[0050] In some embodiments, the sugar is selected from the group consisting of sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabinose, xylose, ribose, rhamnose, trehalose, sorbose, melezitose, raffinose, thioglucose, thiomannose, thiofructose, octa-O-acetyl-thiotrehalose, thiosucrose, and thiomaltose. In some embodiments, the sugar is lactose. In some embodiments, the concentration of lactose is 200 mM to 300 mM. In some embodiments, the concentration of lactose is about 265 mM.

[0051] In some embodiments, the calcium salt is selected from the group consisting of calcium chloride, calcium bromide, calcium acetate, calcium sulfate, and calcium citrate. In some embodiments, the calcium salt is calcium chloride. In some embodiments, the concentration of calcium chloride is about 1 mM to about 10 mM. In some embodiments, the concentration of calcium chloride is about 5 mM.

[0052] In some embodiments, the pharmaceutical composition has a pH of about 5.0 to about 7.0, hi some embodiments, the pharmaceutical composition has a pH of about 6.0.

[0053] In some embodiments, the concentration of rhSP-D is from about 0.1 mg / ml to about 10 mg / ml.

[0054] In some embodiments, the pharmaceutical composition comprises a population of rhSP-D polypeptides having oligomeric forms, wherein more than 30% of the oligomeric forms comprise a rhSP-D dodecamer, in some embodiments, more than 35% of the oligomeric forms comprise a rhSP-D dodecamer, in some embodiments, more than 40% of the oligomeric forms comprise a rhSP-D dodecamer.

[0055] In some embodiments, the pharmaceutical composition comprises a bulking agent, hi some embodiments, the bulking agent is selected from the group consisting of mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, arabitol, glycine, alanine, threonine, valine, and phenylalanine.

[0056] In some embodiments, the pharmaceutical composition lacks a chelating agent, hi some embodiments, the chelating agent is selected from EDTA and EGTA.

[0057] In some embodiments, rhSP-D comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:02.

[0058] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0059] Pharmaceutical Composition Some embodiments of the compositions and methods provided herein include pharmaceutical compositions of recombinant human pulmonary surfactant protein D (rhSP-D) or an active fragment thereof. In some embodiments, the rhSP-D or an active fragment thereof has activity in a bacterial agglutination assay or a TLR4 inhibition assay. In some embodiments, the pharmaceutical composition can be in the form of an aqueous solution, a suspension, or a solid. In some embodiments, the pharmaceutical composition of rhSP-D or an active fragment thereof is suitable for lyophilization into a solid form. In some embodiments, the solid form, e.g., the lyophilized material or powder, can be administered to the lungs and / or reconstituted to form certain solutions suitable for pulmonary administration. In some embodiments, a pharmaceutical composition comprising an aqueous solution or suspension of rhSP-D or an active fragment thereof is suitable for pulmonary administration.

[0060] A specific activity of rhSP-D or a fragment thereof can be readily determined using a bacterial agglutination assay, a Toll-like receptor 4 (TLR4) inhibition assay, and / or asymmetric flow field-flow fractionation-multi-angle laser light scattering (AF4-MALLS) analysis. In some embodiments, the activity of rhSP-D or an active fragment thereof can include biological activity, e.g., activity measured in a bacterial agglutination assay or a TLR4 inhibition assay. In some embodiments, the activity of rhSP-D or an active fragment thereof can include the activity of a population of rhSP-D or an active fragment thereof that forms a specific oligomeric form of rhSP-D and / or forms a specific distribution of oligomeric forms of rhSP-D. An exemplary method for determining the distribution of oligomeric forms of rhSP-D in a sample is provided in WO2019 / 191254, which is incorporated herein by reference in its entirety.

[0061] In some embodiments, the pharmaceutical composition may contain a buffer. Examples of buffers include acetate, citrate, glutamate, histidine, succinate, and phosphate. In some embodiments, the buffer is histidine. In some embodiments, the concentration of the buffer, e.g., histidine, is 0.1 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a range between any two of the above concentrations. In some embodiments, the concentration of the buffering agent, e.g., histidine, is about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or a range between any two of the foregoing concentrations.

[0062] In some embodiments, the pharmaceutical composition may contain a sugar. Examples of sugars include trehalose, sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabinose, xylose, ribose, rhamnose, trehalose, sorbose, melezitose, raffinose, thioglucose, thiomannose, thiofructose, octa-O-acetyl-thiotrehalose, thiosucrose, and thiomaltose. In some embodiments, the sugar is lactose. In some embodiments, the concentration of the sugar, e.g., lactose, is 0.1 mM, 1 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 265 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or a range between any two of the foregoing concentrations. In some embodiments, the concentration of the sugar, e.g., lactose, is about 0.1 mM, about 1 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 265 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, or a range between any two of the foregoing concentrations.

[0063] In some embodiments, the pharmaceutical composition may contain a calcium salt. Examples of calcium salts include calcium chloride, calcium bromide, calcium acetate, calcium sulfate, and calcium citrate. In some embodiments, the calcium salt is calcium chloride. In some embodiments, the concentration of the calcium salt, e.g., calcium chloride, is 0.1 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a range between any two of the foregoing concentrations. In some embodiments, the concentration of the calcium salt, e.g., calcium chloride, is about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or a range between any two of the foregoing concentrations.

[0064] In some embodiments, the pharmaceutical composition may contain an inorganic salt or an organic salt. Examples of inorganic salts include sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate. Examples of organic salts include sodium citrate, potassium citrate, and sodium acetate. In some embodiments, the inorganic salt is sodium chloride. In some embodiments, the concentration of the inorganic salt or organic salt, for example, sodium chloride, is 0.1 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a range between any two of the foregoing concentrations. In some embodiments, the concentration of an inorganic or organic salt, e.g., sodium chloride, is about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or a range between any two of the foregoing concentrations. In some embodiments, the pharmaceutical composition may lack an inorganic or organic salt, e.g., sodium chloride.

[0065] In some embodiments, the pharmaceutical composition may contain a surfactant. Examples of surfactants include hexadecanol, tyloxapol, dipalmitoylphosphatidylcholine (DPPC), PG, palmitoyl-oleoylphosphatidylglycerol, palmitic acid, tripalmitin, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-81, and polysorbate-85. Other examples of surfactants include poloxamers such as poloxamer 188, Triton, and the like. X-100, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glycoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium cocoyl methyl taurate, disodium oleoyl methyl taurate, polyethylene glycol, polypropylene glycol glycol), and copolymers of ethylene and propylene glycol. In some embodiments, the surfactant is tyloxapol. In some embodiments, the concentration of the surfactant, e.g., tyloxapol, is 0.0001% (v / v), 0.0005% (v / v), 0.001% (v / v), 0.005% (v / v), 0.01% (v / v), 0.05% (v / v), 0.1% (v / v), 0.5% (v / v), 1% (v / v), or a range between any two of the foregoing concentrations.In some embodiments, the concentration of the surfactant, e.g., tyloxapol, is about 0.0001% (v / v), about 0.0005% (v / v), about 0.001% (v / v), about 0.005% (v / v), about 0.01% (v / v), about 0.05% (v / v), about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), or a range between any two of the foregoing concentrations. In some embodiments, the pharmaceutical composition may lack a surfactant, e.g., tyloxapol.

[0066] In some embodiments, the pharmaceutical composition may have a pH of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, or a range between any two of the foregoing values. In some embodiments, the pharmaceutical composition may have a pH of about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or a range between any two of the foregoing values.

[0067] In some embodiments, the concentration of the protein, e.g., rhSP-D or an active fragment thereof, in the pharmaceutical composition can be 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, or a range between any two of the foregoing concentrations. In some embodiments, the concentration of the protein, e.g., rhSP-D or an active fragment thereof, in the pharmaceutical composition can be about 0.01 mg / ml, about 0.05 mg / ml, about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, or a range between any two of the foregoing concentrations.

[0068] In some embodiments, the pharmaceutical composition may contain a bulking agent. Examples of bulking agents include the sugars disclosed herein. Other examples of bulking agents include mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, arabitol, glycerin, glycine, alanine, threonine, valine, and phenylalanine. In some embodiments, the bulking agent is present at a concentration of 0.1 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a range between any two of the foregoing concentrations. In some embodiments, the concentration of the bulking agent is about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or a range between any two of the foregoing concentrations.

[0069] In some embodiments, the pharmaceutical composition may include a chelating agent. In some embodiments, the pharmaceutical composition may lack a chelating agent. Examples of chelating agents include EDTA and EGTA.

[0070] In some embodiments, rhSP-D comprises a wild-type human SP-D polypeptide. In some embodiments, rhSP-D comprises a genetic polymorphism of a human SP-D polypeptide. Exemplary SP-D polypeptide sequences are shown in TABLE 1. Genetic polymorphisms in human SP-D polypeptides can include residue 11, ATG (Met) → ACG (Thr); residue 25, AGT (Ser) → AGC (Ser); residue 160, ACA (Thr) → GCA (Ala); residue 270, TCT (Ser) → ACT (Thr); and residue 286, GCT (Ala) → GCC (Ala), where the positions are relative to the positions in a mature SP-D polypeptide, e.g., the exemplary polypeptide of SEQ ID NO: 02. In some embodiments, rhSP-D comprises specific residues at genetic polymorphic positions, the residues being selected from Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306, with respect to the positions in a mature SP-D polypeptide, e.g., exemplary SEQ ID NO: 02, and the SP-D polypeptide with its leader polypeptide, e.g., exemplary SEQ ID NO: 01. In some embodiments, rhSP-D comprises Met11 / 31. In some embodiments, rhSP-D comprises Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306. In some embodiments, the rhSP-D polypeptide has at least 80%, 90%, 95%, 99%, and 100% identity, or any percentage range between any of the foregoing percentages, over the entire length of the polynucleotide, to the polypeptide of SEQ ID NO: 02.

[0071] [Table 1]

[0072] In some embodiments, the rhSP-D is derived from a human myeloid leukemia cell line that expresses rhSP-D from an integrated transgene. Exemplary expression vectors, rhSP-D polypeptides, cell lines, and methods for purifying rhSP-D from such cells are set forth in U.S. Patent Application Publication Nos. 2019 / 0071693 and 2019 / 0071694, each of which is expressly incorporated by reference in its entirety.

[0073] In some embodiments, a pharmaceutical composition, eg, a solution or suspension, comprising a population of rhSP-D polypeptides may have a particular distribution of oligomeric forms of rhSP-D. Compositions of rhSP-D may contain a variety of rhSP-D oligomeric forms, including: trimers, which contain three monomers and have a mass of approximately 130-150 kDa on SDS-PAGE, and which may have a rod-like appearance when visualized by atomic force microscopy (AFM); hexamers, which contain six monomers and have a mass of approximately 250 kDa on SDS-PAGE; dodecamers, which have a predicted mass of approximately 520 kDa as measured by AF4-MALLS and may have an X-like appearance when visualized by AFM; larger heterogeneous oligomeric species, which contain multiples of more than four trimers and may have a star-like or star-shaped appearance with a radius of approximately 70 nm when visualized and identified by AFM (such oligomers are known as star-shaped oligomers); and even larger oligomeric species, known as aggregates, which have a radius of greater than 70 nm as visualized by AFM and measured by AF4-MALLS.

[0074] In some embodiments, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or a range between any two of the foregoing percentages, of the oligomeric form of rhSP-D may be the dodecamer oligomeric form of rhSP-D, as measured as relative peak area (RPA) in an AF4-MALLS analysis. In some embodiments, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or a range between any two of the foregoing percentages, of the oligomeric form of rhSP-D, e.g., of the mass of the oligomeric form in a solution or suspension, may be the dodecamer oligomeric form of rhSP-D. In some embodiments, the percentage of the oligomeric form of rhSP-D, e.g., by number of molecules in the solution or suspension, may be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or a range between any two of the foregoing percentages, may be the dodecamer oligomeric form of rhSP-D.

[0075] In some embodiments, less than about 0.5%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 10%, less than about 20%, less than about 30%, less than about 50%, or a range between any two of the above percentages, of the oligomeric form of rhSP-D may be aggregated oligomeric form of rhSP-D, as measured as RPA or adjusted RPA in an AF4-MALLS analysis. In some embodiments, less than about 0.5%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 10%, less than about 20%, less than about 30%, less than about 50%, or a range between any two of the above percentages, of the mass of the oligomeric form of rhSP-D, e.g., the mass of the oligomeric form in a solution or suspension, may be aggregated oligomeric form of rhSP-D. In some embodiments, the number of molecules in oligomeric form of rhSP-D, e.g., less than about 0.5%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 10%, less than about 20%, less than about 30%, less than about 50%, or a range between any two of the foregoing percentages, of the number of molecules in oligomeric form in the solution or suspension may be aggregated oligomeric form of rhSP-D.

[0076] In some embodiments, the pharmaceutical composition consists of, consists essentially of, or comprises 1 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, and 5 mM calcium chloride, and has a pH of 6.0. In some embodiments, the pharmaceutical composition consists of, consists essentially of, or comprises 1 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, and 1 mM calcium chloride, and has a pH of 6.0. In some embodiments, the pharmaceutical composition consists of, consists essentially of, or comprises 2 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, and 1 mM CaCl2, and has a pH of 6.0. In some embodiments, the pharmaceutical composition consists of, consists essentially of, or comprises 2 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, and 5 mM calcium chloride, and has a pH of 6.0. In some embodiments, the pharmaceutical composition consists of, consists essentially of, or comprises 4 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, and has a pH of 6.0.

[0077] In some embodiments, the pharmaceutical compositions provided herein may include a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, etc., and may contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc., depending on the desired route of administration and formulation. See, e.g., "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins; 20th Edition (June 1, 2003); and "Remington's Pharmaceutical Sciences," Mack Pub. Co.; 18th and 19th Editions (December 1985 and June 1990, respectively). In some embodiments, such formulations may include complexing agents, metal ions, polymeric compounds, such as polylactic acid, polyglycolic acid, hydrogels, dextran, etc., liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Suitable lipids for liposomal formulation include monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, etc. The presence of such additional components can affect the physical state, solubility, stability, rate of in vivo release and rate of in vivo clearance, and therefore, depending on the intended use, the properties of the carrier can be selected to be tailored for a selected route of administration, e.g., pulmonary delivery, e.g., delivery to the lungs, e.g., neonatal lungs.

[0078] In some embodiments, the pharmaceutical composition is suitable for endotracheal, endobronchial, or bronchoalveolar administration to the lungs. In some embodiments, endotracheal, endobronchial, or bronchoalveolar administration can include nebulization, irrigation, inhalation, flushing, or installation using a physiologically acceptable composition in which the pharmaceutical composition is dissolved as a fluid. The administration method can include the use of continuous positive airway pressure (CPAP). The administration method can include direct intubation. In some embodiments, the pharmaceutical compositions provided herein can be delivered to the lungs via inhalation. Exemplary delivery forms include dry powder and aerosol formulations. A wide range of medical devices designed for pulmonary delivery of therapeutic products can be used. These medical devices include, but are not limited to, nebulizers, metered-dose inhalers, and dry powder inhalers, all of which are well known to those skilled in the art. These devices use formulations suitable for dispensing pharmaceutical compositions. Typically, each formulation is specific to the type of device used and can include the use of an appropriate propellant material in addition to diluents, adjuvants, and / or carriers useful in therapeutic agents.

[0079] kit Some embodiments provided herein include kits. In some embodiments, the kits can include a pharmaceutical composition provided herein. Some embodiments include a sterile container containing a pharmaceutical composition provided herein. Some embodiments include a pharmaceutical composition provided herein in lyophilized form and in a sterile reconstitution solution. In some embodiments, the kits can include a device for administering a pharmaceutical composition provided herein, such as an inhaler or nebulizer. [Example]

[0080] Example 1 In vitro binding of S protein to immobilized rhSP-D An ELISA-based binding assay was developed to determine the binding of immobilized recombinant human SP-D (rhSP-D) to the S1 subunit (S1 protein) of the spike protein of SARS-CoV-2. SP-D binding activity is enhanced by the presence of calcium, and SP-D binds maltose. Assays were performed in the presence of calcium, a calcium chelator, EDTA, or maltose. Figure 1A shows a schematic diagram of the assay.

[0081] Recombinant S1 protein was produced in HEK293 cells and contained a mouse Fc IgG tag at the C-terminus (Sino Biologicals, #40591-V05H1). The first sample of rhSP-D was produced from human myeloid leukemia cells, and the second sample of rhSP-D was obtained from CHO cells. Microtiter plate wells were coated with 200 μL of a 5 μg / mL or 2 μg / mL suspension of rhSP-D in carbonate-bicarbonate coating buffer (50 mM NaHCO3-Na2CO3, pH 9.6). The plates were incubated overnight at 4°C. The plates were washed five times between incubations; all washes and dilutions from this point on were performed with dilution buffer: 0.05% TBS-tween, 5 mM CaCl2 (TBS is 50 mM Tris, pH 7.4, 150 mM NaCl). Washing was performed by adding 200 μL / well of wash buffer, followed by aspirating the wells; this process was repeated five times. After washing the plate, the wells were blocked with 2% bovine serum albumin (BSA) in dilution buffer (200 μL / well) for 1 hour at room temperature to prevent nonspecific binding of rhSP-D to uncoated areas of the wells. The plate was washed, and serially diluted (1:2) samples of SARS-CoV-2 S protein (10 μg / mL to 9.8 ng / mL) were added to the wells to generate a standard curve.

[0082] To determine whether binding was mediated by the carbohydrate-recognition domain of rhSP-D, a second set of S1 protein samples was prepared in which maltose was added to the S1 protein samples to obtain a final concentration of 200 mM maltose, which was then incubated for 10 minutes before being added to the plate wells. For the same purpose, a third set of S1 protein samples was prepared, this time using 100 mM EDTA instead of 5 mM calcium in the dilution buffer to inhibit calcium-dependent binding. In both cases, once added to the wells, the S1 protein was incubated at room temperature for 1 hour.

[0083] After washing the plate, 100 μL of anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibody (diluted 1:5000) (#7076, Cell Signaling; Danvers, MA, USA) was added and incubated at room temperature for 1 hour. The plate was washed, and 100 μL of TMB / E (3,3',5,5'-tetramethylbenzidine) (#TMBS010001, Surmodics) was added and incubated at room temperature for 10 minutes. The reaction was stopped with 100 μL of 2N H2SO4. The plate was read for absorbance at 450 nm.

[0084] Figures 1B and 1C summarize the results for wells coated with a 5 μg / mL solution of rhSP-D for the first and second samples of SP-D, respectively. Figures 1D and 1E summarize the results for wells coated with a 2 μg / mL solution of rhSP-D for the first and second samples of SP-D, respectively. S1 protein bound to SP-D in the presence of calcium. Binding was inhibited by the presence of EDTA or maltose. Thus, S1 protein bound to SP-D in a calcium-dependent manner, and this binding was inhibited by the competitor maltose.

[0085] Example 2 In vitro binding of rhSP-D to immobilized S protein An ELISA-based binding assay was developed to determine the binding of rhSP-D to immobilized S1 subunit of the SARS-CoV-2 spike protein (S1 protein). The assay was performed in the presence of calcium or the calcium chelator, EDTA. Figure 2A shows a schematic diagram of the assay.

[0086] Recombinant S1 protein was produced in HEK293 cells and contained a mouse Fc IgG tag at the C-terminus (SinoBiologicals, #40591-V05H1). The first sample of rhSP-D was produced from human myeloid leukemia cells, and the second sample of rhSP-D was obtained from CHO cells. Microtiter plate wells were coated with 200 μL of a 2.5 μg / mL suspension of S1 protein in carbonate-bicarbonate coating buffer (50 mM NaHCO3-Na2CO3, pH 9.6). The plate was incubated overnight at 4°C. The plate was washed five times between incubations; all washes and dilutions from this point onward were performed with dilution buffer: 0.05% TBS-tween, 5 mM CaCl2. Washing was performed by adding 200 μL / well of wash buffer, followed by aspirating the wells; this process was repeated five times. After washing the plates, the wells were blocked with 2% BSA in dilution buffer (200 μL / well) for 1 h at room temperature to prevent nonspecific binding of rhSP-D to uncoated areas of the wells. The plates were washed as described, and serially diluted (1:2) rhSP-D (5 μg / mL to 4.9 ng / mL) samples were added to the wells to generate a standard curve.

[0087] To determine whether binding was mediated by the carbohydrate recognition domain of rhSP-D, a second set of rhSP-D samples was prepared using 100 mM EDTA instead of 5 mM calcium in the dilution buffer to inhibit calcium-dependent binding of rhSP-D. In both cases, once added to the wells, rhSP-D was incubated at room temperature for 1 hour. After washing the plate, 50 μL of rabbit anti-SP-D antibody (diluted 1:5000) was added and incubated at room temperature for 1 hour.

[0088] The plate was washed, and 100 μL of anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody (diluted 1:7500) (#7074, Cell Signaling; Danvers, MA, USA) was added and incubated at room temperature for 1 hour. The plate was washed, and 100 μL of TMB / E (3,3',5,5'-tetramethylbenzidine) (#TMBS010001, Surmodics) was added and incubated at room temperature for 5 minutes. The reaction was stopped with 100 μL of 2N H2SO4. The plate was read for absorbance at 450 nm.

[0089] Figures 2B and 2C summarize the results for the S1 protein-coated wells of the first and second samples of SP-D, respectively. S1 protein bound to SP-D in the presence of calcium. Binding was inhibited by the presence of EDTA. Thus, S1 protein bound to SP-D in a calcium-dependent manner.

[0090] Example 3 Lung SP-D concentrations in COVID-19 patients This example demonstrates the determination of SP-D levels in bronchoalveolar lavage fluid (BALF) from patients with COVID-19. Bronchoscopy and bronchoalveolar lavage fluid (BALF) were obtained as described in Pandolfi, L. et al. (2020) BMC Pulm Med 20: 301. Briefly, bronchoscopy was performed on sedated, paralyzed, and mechanically ventilated patients (n = 12) with PCR-confirmed COVID-19. BALF aliquots were collected after 5–6 bolus infusions of 20 mL of sterile saline. The first 20 mL was discarded. The suspension was centrifuged at 400 g for 10 min, and the supernatant was inactivated with 0.2% SDS and 0.1% Tween 20, followed by 15 min at 65°C. The resulting BALF was stored at -20°C until analysis. SP-D levels in BALF were quantified using a human anti-SP-D antibody (Biovendor) by an ELISA procedure. BALF samples were collected after approval by the Ethic Committee of Ospedale Luigi Sacco (experiment number 2020 / ST / 145). Bronchoalveolar lavage fluid samples were collected from COVID-19 patients with different ages, characteristics, and comorbidities, as shown in Table 2. A body mass index (BMI) of >30 was considered obese. Comorbidities were screened, including smoking, cardiovascular disease (CV), respiratory disease, immunosuppression, human immunodeficiency virus (HIV), type I and II diabetes, and cancer.

[0091] [Table 2]

[0092] Decreased SP-D levels have been observed in bronchoalveolar lavage fluids of several respiratory diseases indicative of acute lung injury (Sorensen, GL et al. (2007) Immunobiology 212:381-416). SP-D lung levels in COVID-19 patients were found to have a median concentration of 68.9 ng / mL (mean = 244.8 ng / mL, n = 12) (Figure 3). This compares with previously reported BALF SP-D levels of 900-1300 ng / mL in non-COVID-19 healthy control subjects, 940 ng / mL in surviving early ARDS patients, and 406 ng / mL in non-surviving early ARDS patients (Hermans C et al. (1999). Am J Respir Crit Care Med 159:646-678; and Honda Y et al. (1995) Am J Respir Crit Care Med 152:1860-1866). Thus, COVID-19 patients were found to have reduced lung SP-D levels when compared with levels reported in the literature for healthy subjects and ARDS patients.

[0093] Example 4 Recombinant hSP-D binds to the S protein of SARS-CoV-2 Binding experiments were carried out substantially similar to those described in Examples 1 and 2 above. Full-length recombinant human rhSP-D was produced in the human cell line GlycoExpress® (GEX), developed at Glycotope-GmbH. The rhSP-D variants were derived from Met 11 , Thr 160 , Ser 260The purification method for rhSP-D has been described elsewhere (Ikegami, M. et al. (2006) Am J Respir Crit Care Med 173:1342-1347; and Arroyo, R. et al. (2018) J Mol Biol 430:1495-1509). Recombinant SARS-CoV-2 spike protein variant (S1 subunit) and recombinant human ACE2 protein were expressed in HEK293 cells and purchased from SinoBiologicals (#40591-V08H, #40591-V05H1, #10108-H05H, #40591-V08H3, #40591-V08H10), Acro Biosystems (#S1N-C52H3, #S1N-C52Hk, #S1NN-C52Hg), The NativeAntigen Company (#REC31806-100-HRP), and Biomart Creative (#ACE2-736H).

[0094] Briefly, a first ELISA assay was developed in which microtiter plates were coated with S1 spike protein variants (0.4 μg in 200 μL / well). Washing and dilution were performed using 0.05% TBS-tween, 5 mM CaCl2. Wells were blocked with 2% BSA, and serially diluted rhSP-D (10 μg / mL to 9.8 ng / mL) was added to the wells. Bound rhSP-D was detected with a mouse anti-SP-D antibody (#2D12A-88, Seven Hills Bioreagents) followed by an anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibody (#7076, Cell Signaling). Plates were developed with TMB (#TMBS010001, Surmodics) for 10 minutes, and the reaction was stopped with 2N H2SO4. Plates were read for absorbance at 450 nm. Non-binding negative controls were included using 50 mM EDTA to prevent calcium-dependent binding or 200 mM maltose, also with 5 mM calcium, to create binding competition between maltose and S1 protein. To address non-specific binding to the plate, wells were coated with 1% BSA instead of S1 protein.

[0095] A second ELISA assay was also developed in which rhSP-D was coated onto the wells instead of S1 protein. Serially diluted S1 protein samples with a mouse Fc tag (10 μg / mL to 9.8 ng / mL) were added to the wells. Bound S1 protein was detected with the same anti-mouse IgG HRP-conjugated antibody. Analysis of the binding isotherms was performed using GraphPad Prism 8, taking into account total binding and one site, to determine the apparent dissociation constant (kd) and the apparent maximum number of binding sites (B). max ) was decided.

[0096] ELISA assays showed that rhSP-D recognized and bound to the S1 subunit of the spike protein from the first identified variant of SARS-CoV-2 (Wuhan variant) with similar apparent dissociation constants when rhSP-D was the ligand (Kd = 1.65) (Figure 4A) or when the S1 protein was the ligand (Kd = 2.02) (Figure 4B). The apparent maximum number of binding sites was 1.08 when rhSP-D was the ligand and 2.07 when the S1 protein was the ligand (B max = 0.81, Figure 4B) compared with max = 1.35, Figure 4A). This was expected because the higher oligomeric forms of rhSP-D (dodecameric and multimeric forms) contain several trimeric carbohydrate recognition domains (CRDs) that serve as binding sites for rhSP-D, whereas the S1 protein contains only one binding site for rhSP-D. Binding of rhSP-D to the S1 protein was inhibited by EDTA, confirming its calcium-dependent nature. Maltose also binds to the CRD of rhSP-D in a calcium-dependent manner, but binding competition by maltose abrogated rhSP-D binding to the S protein. Binding of rhSP-D to the S1 protein in the presence of calcium was significantly different from binding with EDTA or maltose (p < 0.0001). This strongly suggests that the CRD of rhSP-D mediates binding to the carbohydrates described for the S1 protein of SARS-CoV-2.

[0097] We tested the binding of rhSP-D to S1 proteins bearing mutations identified in the UK B.1.1.7. variant (HV69-70, N501Y, D614G) or the South African B.1.351 variant (K417N, E484K, N504Y, D614G). rhSP-D bound to all variants tested (Figure 4C). rhSP-D binding to the S1 protein from the UK variant was similar to that of the Wuhan variant, but binding was significantly reduced for the South African S1 protein variant. Specifically, binding to the South African variant was significantly reduced compared to the Wuhan variant (p=0.0002) and the UK variant (p=0.007), with no significant difference observed when comparing the Wuhan and UK variants (p>0.99) (Friedman test with Dunn's post hoc test).

[0098] The significance of two common mutations in the S1 protein found in new variants, N501Y and D614G, on rhSP-D binding was investigated separately. The mutation N501Y reduced rhSP-D binding compared to the original Wuhan variant (Figure 4D), while D614G had little effect on rhSP-D binding to the spike protein compared to the Wuhan variant (Figure 4E). rhSP-D binding to the Wuhan-derived S1 protein variant was compared with S1 proteins with the single mutation N501Y (p=0.04) or the single mutation D614G (D) (p=0.05) (t-test).

[0099] The following experiments were performed using the S1 protein from the Wuhan variant.

[0100] Example 5 rhSP-D forms a protein bridge with the S protein of SARS-CoV-2 To determine whether rhSP-D can aggregate SARS-CoV-2, we investigated its ability to link the S protein to a second molecule (maltose-coated beads). Protein cross-linking (aggregation) assays were performed, including a premixing approach (Figure 5A) and a first rhSP-D approach (Figure 5B).

[0101] In the premixing approach (Figure 5A), rhSP-D (2 μg or 4 μg) and S1 protein (2 μg; Wuhan variant) were premixed and incubated for 2 h to favor binding and aggregation of S1 protein by rhSP-D. The mixture was then added to the beads. After a 30-minute incubation at room temperature, the beads were centrifuged and the supernatant (S1) was saved. The beads were then washed and eluted as described above, and the eluted fraction (P) was saved for analysis.

[0102] In the first rhSP-D approach (Figure 5B), rhSP-D (2 μg or 4 μg) was incubated with maltose-coated agarose beads in 50 μL of TBS (150 mM NaCl, 20 mM Tris (pH 7.4))-10 mM CaCl2 buffer at room temperature for 30 minutes. The supernatant (S1), containing excess unbound rhSP-D, was separated by centrifugation and saved. The beads were washed with TBS-CaCl2. Then, 2 μg of S1 protein or buffer (negative control) was added to the beads, and the final volume was adjusted to 50 μL with TBS-CaCl2 or 20 mM TBS-EDTA for the unbound control. After 2 hours of incubation at room temperature, the beads were centrifuged, and the supernatant (S2) was saved. The beads (pellet) were washed with the appropriate buffer, and subsequently, bound rhSP-D was eluted with 20 mM TBS-EDTA. The eluted fraction from the pellet (P) was saved for analysis.

[0103] In both methods, the presence of rhSP-D and S1 proteins in fractions (S1, S2, and P) was determined by SDS-PAGE under reducing conditions and developed by silver staining. The intensity of the rhSP-D band from a sample containing 4 μg of rhSP-D was quantified in duplicate by densitometry using ImageJ software. The relative intensity of the rhSP-D band in the pellet fraction (P) was calculated by considering the intensity of the "S1" band in 5 mM calcium in the buffer control as 100%. Gel densitometry was performed twice.

[0104] The results demonstrated that rhSP-D formed protein cross-links with the Wuhan variant S1 protein (Figure 5C, "P": lanes 4 and 8; Figure 5D, "P": lane 9) and maltose-coated beads. The formation of protein cross-links by rhSP-D was inhibited in the presence of EDTA and was therefore calcium-dependent (Figure 5C, lane 10; Figure 5D, lane 12). The binding of S protein to rhSP-D was also confirmed in this second assay, as only fraction "S2" contained rhSP-D in the presence of S1 protein (Figure 5D, lane 2 vs. lane 8). Addition of S1 protein to rhSP-D pre-bound to maltose-coated beads shifted a portion of the rhSP-D, indicating that it preferentially bound to S protein (observed in the "S2" fraction). To determine whether rhSP-D could form aggregates of multiple S protein and rhSP-D molecules, we compared the premixing approach and the first rhSP-D approach. The premixing approach (Figure 5A) should allow the formation of higher-order aggregates of S protein and rhSP-D from multiple S protein and rhSP-D molecules. In contrast, first binding rhSP-D to maltose beads, followed by removal of unbound rhSP-D, and then binding to S protein should be limited to single units of rhSP-D bound to S protein and maltose (Figure 5B). The intensity of the rhSP-D band in the elution ("P") fraction in the premixing approach was stronger than its respective band in the first SP-D approach (Figure 5E), consistent with the formation of higher-order aggregates. Collectively, these data demonstrated the presence of rhSP-D-facilitated protein cross-linking and suggested rhSP-D-driven SARS-CoV-2 aggregation.

[0105] Example 6 Binding of S protein to rhSP-D in the presence of ACE2 receptor The spike protein of SARS-CoV-2 interacts with the ACE2 receptor on epithelial cells. Binding of ACE2 to the S1 protein (Wuhan variant) in the presence of rhSP-D was examined. Purified S1 protein (Wuhan variant) was coated onto plates. rhSP-D (0.1–1 μg / mL) or buffer (negative control) in 5 mM TBS-Ca was added to the wells and incubated for 2 hours. Without washing, human ACE2 protein (0.186–1.5 μg / mL) was added to each well at each rhSP-D concentration. A control containing TBS buffer instead of ACE2 was also included. After 30 minutes of incubation, bound ACE2-mFc was detected using an anti-mouse IgG HRP-conjugated antibody (Figure 6A and B). Binding of S1 protein to rhSP-D in the presence of ACE2 was examined. Plates were coated with rhSP-D (5 μL / mL, 200 μL / well). Various concentrations of HRP-tagged S1 protein or buffer (negative control) were added to the wells and incubated for 2 hours. Without washing, His-tagged human ACE2 protein was added to the wells to reach S1 protein concentrations of 3, 0.375, or 0.045 μg / mL, respectively. After 30 minutes of incubation, bound S1 protein-HRP was directly detected with TMB, and the reaction was stopped with 2N H2SO4 (Figures 6C and 6D).

[0106] A decrease in ACE2 binding to S1 protein was observed in the presence of 0.5 μg / mL rhSP-D compared to the control without rhSP-D (Figures 6A and 6B). The results also demonstrated that the addition of ACE2 did not inhibit rhSP-D binding to S1 protein, until a slight decrease in binding was observed at the highest concentration of ACE2 (3 μg / mL) (Figures 6C and 6D). Thus, rhSP-D and ACE2 bind to different regions of S1 protein, allowing simultaneous interaction of the three molecules.

[0107] Example 7 rhSP-D inhibits SARS-CoV-2 replication in host cells The effect of rhSP-D on SARS-CoV-2 replication in host cells was tested in vitro using a viral replication assay in human Caco-2 cells.

[0108] Twenty-four hours after preparation, human epithelial Caco-2 cell monolayers were infected with virus in 96-well microtiter plates at 37°C with 5% CO2. Growth medium was removed from the cells, and rhSP-D was applied and tested in triplicate at eight serial half-log10 dilutions starting at 100 μg / mL. 200 CCID50 (50% cell culture infectious dose) of SARS-CoV-2 (USA / WA1 / 2020 strain) was added to wells designated for virus infection at an MOI of 0.02. Controls were performed using infected, untreated (virus control) cells and untreated, uninfected (cell control) cells. Plates were incubated at 37°C for 72 hours. Supernatant samples were taken from each infected well for testing and virus titer determination (n = 3 replicates). Titration of previously collected virus samples was performed by end-point dilution as described by Reed LJ et al. (1938) American Journal of Epidemiology 27:493-497. Serial 10-fold dilutions of virus were made and plated into wells containing fresh monolayers of Vero 76 cells. Plates were incubated, and cells were scored for the presence or absence of virus after observing a clear cytopathic effect. CCID50 was calculated using the method of Reed et al. The 90% (1 log10) effective concentration (EC90) was calculated. The cytotoxicity of rhSP-D was assessed in additional plate wells by using neutral red dye, which penetrates viable cells and allows for quantification of viable cells. In the cytotoxicity assay, the more intense the red color, the greater the number of viable cells present in the well. The dye content in each well was quantified using a spectrophotometer at a wavelength of 540 nm.

[0109] rhSP-D inhibited viral replication in a dose-dependent manner, with higher concentrations of rhSP-D leading to greater inhibition of viral replication. This was observed by measuring the virus titer in the cell supernatant at various rhSP-D concentrations tested, reported as CCID50 (50% cell culture infectious dose) (Fig. 7). The concentration of rhSP-D required to inhibit viral replication by 90% (EC90) was 3.7 μg / mL. Furthermore, rhSP-D, even at the highest rhSP-D concentration tested (100 μg / mL), did not exhibit cytotoxicity compared to control (untreated, uninfected) cells.

[0110] Example 8 Treatment of SARS-CoV-2 infection with rhSP-D A patient with SARS-CoV-2 infection is administered a medicinal solution containing rhSP-D, 5 mM histidine, 265 mM lactose, and 5 mM CaCl. The patient has symptoms including fever, cough, shortness of breath, fatigue, muscle pain, diarrhea, sore throat, loss of smell, and abdominal pain. Administration of the medicinal solution reduces one or more symptoms of SARS-CoV-2 infection in the patient.

[0111] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0112] The above description discloses several methods and materials of the present invention. The invention is susceptible to modifications of the methods and materials, and to variations in the manufacturing methods and apparatus. Such modifications will become apparent to those skilled in the art from consideration of this disclosure or practice of the invention disclosed herein. Consequently, the invention is not intended to be limited to the specific embodiments disclosed herein, but rather is intended to cover all modifications and alternatives falling within the true scope and spirit of the invention.

[0113] All reference materials cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material.

Claims

1. 1. A pharmaceutical composition for use in treating or ameliorating a viral infection, including a coronavirus, in a subject, comprising: containing recombinant human pulmonary surfactant protein D (rhSP-D) or an active fragment thereof, viral infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); A pharmaceutical composition, wherein SARS-CoV-2 comprises the S1 protein of (1) the Wuhan wild type or (2) the Wuhan variant containing the N501Y or D614G mutation.

2. 2. The pharmaceutical composition of claim 1, wherein the S1 protein lacks a mutation selected from K417N and E484K.

3. 3. The pharmaceutical composition of claim 1, comprising a buffer, a sugar and a calcium salt.

4. 4. The pharmaceutical composition of claim 3, wherein the buffering agent is selected from the group consisting of acetate, citrate, glutamate, histidine, succinate, and phosphate.

5. 5. The pharmaceutical composition of claim 3, wherein the buffering agent is histidine.

6. 6. The pharmaceutical composition of claim 5, wherein the concentration of histidine is from about 1 mM to about 10 mM.

7. 7. The pharmaceutical composition of any one of claims 3 to 6, wherein the sugar is selected from the group consisting of sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabinose, xylose, ribose, rhamnose, trehalose, sorbose, melezitose, raffinose, thioglucose, thiomannose, thiofructose, octa-O-acetyl-thiotrehalose, thiosucrose and thiomaltose.

8. 8. The pharmaceutical composition of claim 7, wherein the sugar is lactose.

9. 9. The pharmaceutical composition of claim 8, wherein the concentration of lactose is 200 mM to 300 mM.

10. 10. The pharmaceutical composition of claim 9, wherein the concentration of lactose is about 265 mM.

11. 11. The pharmaceutical composition of claim 3, wherein the calcium salt is selected from the group consisting of calcium chloride, calcium bromide, calcium acetate, calcium sulfate and calcium citrate.

12. 12. The pharmaceutical composition of claim 11, wherein the calcium salt is calcium chloride.

13. 13. The pharmaceutical composition of claim 12, wherein the concentration of calcium chloride is from about 1 mM to about 10 mM.

14. 14. The pharmaceutical composition of claim 13, wherein the concentration of calcium chloride is about 5 mM.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, having a pH of about 5.0 to about 7.

0.

16. 16. The pharmaceutical composition of claim 15, having a pH of about 6.

0.

17. 17. The pharmaceutical composition of any one of claims 1 to 16, wherein the concentration of rhSP-D is from about 0.1 mg / ml to about 10 mg / ml.

18. 18. A pharmaceutical composition according to any one of claims 1 to 17, comprising a population of rhSP-D polypeptides having oligomeric forms, wherein more than 30% of the oligomeric forms comprise a dodecamer of rhSP-D.

19. 19. The pharmaceutical composition of claim 18, wherein more than 35% of the oligomeric forms comprise a dodecamer of rhSP-D.

20. 20. The pharmaceutical composition of claim 18 or 19, wherein more than 40% of the oligomeric forms comprise a dodecamer of rhSP-D.

21. 21. The pharmaceutical composition of any one of claims 1 to 20, comprising a filler.

22. 22. The pharmaceutical composition of claim 21, wherein the bulking agent is selected from the group consisting of mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, arabitol, glycine, alanine, threonine, valine, and phenylalanine.

23. 23. The pharmaceutical composition of any one of claims 1 to 22, which is devoid of a chelating agent.

24. 24. The pharmaceutical composition of claim 23, wherein the chelating agent is selected from EDTA and EGTA.

25. 25. The pharmaceutical composition of any one of claims 1 to 24, wherein rhSP-D comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

02.

26. 26. The pharmaceutical composition of any one of claims 1 to 25, wherein the subject is a mammal.

27. 27. The pharmaceutical composition of any one of claims 1 to 26, wherein the subject is a human.

28. A pharmaceutical composition described in any one of claims 1 to 27, wherein the S1 protein is Wuhan wild type.

29. A pharmaceutical composition described in any one of claims 1 to 27, wherein the S1 protein is the Wuhan variant containing the N501Y mutation.

30. A pharmaceutical composition described in any one of claims 1 to 27, wherein the S1 protein is the Wuhan variant containing the D614G mutation.

31. A pharmaceutical composition described in any one of claims 1 to 27, wherein the S1 protein is the Wuhan variant including the N501Y mutation, the D614G mutation, and the HV69-70del mutation.

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