Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/395—Alveolar surfactant peptides; Pulmonary surfactant peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses

Definitions

• Some embodiments of the methods and compositions provided herein relate to the use of surfactant protein D (SP-D) to treat or ameliorate a viral infection in a subject.
• the viral infection comprises a coronavirus, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• Some embodiments include the use of certain formulations comprising a recombinant human SP-D (rhSP-D).
• SARS-CoV-2 belongs to a family of coronaviruses which also includes severe acute respiratory syndrome coronavirus (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
• COVID-19 was first identified in December 2019 in Wuhan, the capital of China’s Hubei province, and has since spread globally, resulting in a coronavirus pandemic.
• Common symptoms include fever, cough, and shortness of breath.
• Other symptoms may include fatigue, muscle pain, diarrhea, sore throat, loss of smell, and abdominal pain. While the majority of patients result m mild symptoms, some cases progress to viral pneumonia and multi-organ failure.
• Patients are managed with supportive care, which may include fluid therapy, oxygen support, and supporting other affected vital organs. There is a need for treatments for COVID-19 and related viral disorders.
• Some embodiments of the methods and compositions include a method of treating or ameliorating a viral infection in a subject, comprising: administering an effective amount of a recombinant human surfactant protein D (rhSP-D) or active fragment thereof to the subject.
• rhSP-D recombinant human surfactant protein D
• the viral infection comprises a respiratory tract infection.
• the viral infection comprises a coronavirus.
• 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- related coronavirus (MERS-CoV), HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKUl .
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• SARS-CoV-1 severe acute respiratory syndrome coronavirus
• MERS-CoV Middle East respiratory syndrome- related coronavirus
• HCoV-229E HCoV-NL63
• HCoV-OC43 Middle East respiratory syndrome- related coronavirus
• HCoV-HKUl Middle East respiratory syndrome- related coronavirus
• the viral infection comprises SARS-CoV-2.
• the SARS-CoV-2 comprises an SI protein variant.
• the SI protein variant comprises a mutation selected from N501Y, D614G, HV69-70del, K417N, and E484K.
• the SI protein lacks a mutation selected from K4G7N, and E484K.
• the administration comprises administering a pharmaceutical composition comprising the rhSP-D or active fragment thereof.
• the pharmaceutical composition comprises a buffer, a sugar, and a calcium salt.
• the buffer is selected from the group consisting of acetate, citrate, glutamate, histidine, succinate, and phosphate. In some embodiments, the buffer is histidine. [0013] In some embodiments, the concentration of the histidine is from about 1 mM to about 10 mM.
• the sugar is selected from the group consisting of sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabmose, xylose, ribose, rhamnose, trehalose, sorbose, melezitose, raffinose, thioglucose, thiomaimose, thiofructose, octa-Q-acetyl-thiotrehalose, thiosucrose, and tluomaltose.
• the sugar is lactose.
• the concentration of the lactose is from 200 mM to 300 mM. In some embodiments, the concentration of the lactose is about 265 mM.
• the calcium salt is selected from the group consisting calcium chloride, calcium bromide, calcium acetate, calcium sulfate, and calcium citrate. In some embodiments, the calcium salt is calcium chloride.
• the concentration of the calcium chloride is from about 1 niMto about 10 mM. In some embodiments, the concentration of the calcium chloride is about 5 mM
• the pharmaceutical composition has a pH from about 5.0 to about 7.0. In some embodiments, the pharmaceutical composition has a pH about 6.0
• the concentration of the rhSP-D is from about 0.1 mg/ml to about 10 mg/ml.
• the pharmaceutical composition comprises a population of rhSP-D polypeptides having oligomeric forms, wherein greater than 30% of the oligomeric forms comprise dodecamers of rh8P-D. In some embodiments, greater than 35% of the oligomeric forms comprise dodecamers of rhSP-D. In some embodiments, greater than 40% of the oligomeric forms comprise dodecamers of the rbSP-D.
• the pharmaceutical composition comprises a bulking agent.
• the bulking agent is selected from the group consisting of mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, arabitol, glycine, alanine, threonine, valine, and phenylalanine.
• the pharmaceutical composition lacks a chelating agent in some embodiments, the chelating agent is selected from EDTA and EGTA.
• the rhSP-D comprises an amino acid sequence having at least 95% identity to the amnio acid sequence of SEQ ID NO:02.
• the subject is mammalian. In some embodiments, the subject is human.
• Some embodiments of the methods and compositions include a pharmaceutical composition for use in treating or ameliorating a viral infection in a subject, wherein the pharmaceutical composition comprises a recombinant human surfactant protein D (rhSP-D) or active fragment thereof.
• rhSP-D recombinant human surfactant protein D
• the viral infection comprises a respiratory tract infection.
• the viral infection comprises a coronavirus.
• 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- related coronavirus (MERS-CoV), HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKUl .
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• SARS-CoV-1 severe acute respiratory syndrome coronavirus
• MERS-CoV Middle East respiratory syndrome- related coronavirus
• HCoV-229E HCoV-NL63
• HCoV-OC43 Middle East respiratory syndrome- related coronavirus
• HCoV-HKUl Middle East respiratory syndrome- related coronavirus
• the viral infection comprises SARS-CoV-2.
• FIG 1 A depicts a schematic overview of an FITS A assay to detect binding between immobilized SP-D and an SI subunit of a spike protein of SARS-CoV-2 (S 1 -protein).
• FIG. IB depicts a line graph of absorbance with increasing concentration of SI -protein in an assay for binding between immobilized SP-D and the SI -protein with a first sample of immobilized SP-D in the presence of calcium, of EDTA, or of maltose, in which plates were coated using 5 gg/mL SP-D.
• FIG. 1C depicts a line graph of absorbance with increasing concentration of SI -protein in an assay for binding between immobilized SP-D and the SI -protein with a second sample of immobilized SP-D in the presence of calcium, of EDTA, or of maltose, in which plates were coated using 5 iig/mL SP-D.
• FIG. ID depicts a line graph of absorbance with increasing concentration of SI -protein in an assay for binding between immobilized SP-D and the SI -protein with a first sample of immobilized SP-D in the presence of calcium, of EDTA, or of maltose, in which plates were coated using 2 pg/mL SP-D.
• FIG. IE depicts a line graph of absorbance with increasing concentration of SI -protein in an assay for binding between immobilized SP-D and the SI -protein with a second sample of immobilized SP-D in the presence of calcium, of EDTA, or of maltose, in which plates were coated using 2 gg/mL SP-D.
• FIG. 2 A depicts a schematic overview' of an ELISA assay to detect binding between SP-D and immobilized SI -protein.
• FIG. 2B depicts a graph of absorbance with increasing concentration of SP- D in an assay for binding between SP-D and immobilized SI -protein with a first sample of immobilized SP-D in the presence of calcium, or of EDTA.
• FIG. 2C depicts a graph of absorbance with increasing concentration of SP- D in an assay for binding between SP-D and immobilized S I -protein with a second sample of immobilized SP-D m the presence of calcium, or of EDTA.
• FIG. 3 is a graph of SP-D concentration in bronchoalveolar lavage fluid obtained from COVTD-I9 patients, and also in control subjects previously reported in literature. Error bars represent 1 5 times the interquartile rate (Q I to Q3).
• FIG. 4A is a graph of absorbance units for various concentrations of rhSP- D in an ELIS A to measure rhSP-D binding to immobilized S I -protein of SARS-CoV-2 (Wuhan variant).
• FIG. 4B is a graph of absorbance units for various concentrations of 81- protein (Wuhan variant) in an ELISA to measure SARS-CoV-2 SI -protein binding to immobilized rh8P-D.
• FIG. 4C is a graph of absorbance units for various concentrations of rhSP- D in an ELISA to measure rhSP-D binding to immobilized SI -protein variants of SARS-CoV- 2 (Wuhan variant; U.K. variant, and South Africa variant).
• FIG. 4D is a graph of absorbance units for various concentrations of rliSP- D in an ELISA to measure rhSP-D binding to an immobilized SI -protein variant of 8ARS- CoV-2 containing a single mutation (N501 Y).
• FIG. 4E is a graph of absorbance units for various concentrations of rhSP- D in an ELISA to measure rhSP-D binding to an immobilized SI -protein variant of SARS- CoV-2 containing a single mutation (D614G).
• FIG. 5 A depicts a scheme for a bridge assay between SI -protein and maltose-coated beads via rhSP-D in winch rhSP-D is pre-mixed with SI -protein before addition of maltose-coated beads.
• FIG. 5B depicts a scheme for a bridge assay between S 1 -protein and maltose beads via rhSP-D in which rhSP-D is pre-incubated with maltose-coated before addition of Sl- protein.
• FIG. 5 € depicts a SDS-PAGE gel for the scheme shown in FIG. 5A in which the gel was developed by silver-staining to detect SI -protein (migrates as 100-140 kDa) and rhSP-D (43 kDa).
• FIG. 5D depicts a SDS-PAGE gel for the scheme shown in FIG. 5B in which the gel was developed by silver-staining to detect SI -protein (migrates as 100-140 kDa) and rhSP-D (43 kDa).
• FIG. 6.4 depicts a line graph for the results of an ELISA to determine binding of ACE2 to immobilized SI -protein in the presence of various concentrations of rhSP- D.
• FIG. 6B depicts a bar chart for the results of an ELISA to determine binding of ACE-2 to immobilized SI -protein in the presence of various concentrations of rhSP-D.
• FIG. 6C depicts a line graph for the results of an ELISA to determine binding of SI -protein to immobilized rhSP-D in the presence of various concentrations of ACE2.
• FIG. 6D depicts a bar chart for the results of an ELISA to determine binding of SI -protein to immobilized rhSP-D in the presence of various concentrations of ACE2.
• FIG. 7 depicts a graph of CCID50 (50% ceil culture infectious dose) of SARS-CoV-2 at various concentrations of rhSP-D. Individual data points represent the average of three replicates. DETAILED DESCRIPTION
• Some embodiments of the methods and compositions provided herein relate to the use of surfactant protein D (SP-D) to treat or ameliorate a viral infection m a subject.
• the viral infection comprises a coronavirus, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• Some embodiments include the use of certain formulations comprising a recombinant human SP-D (rhSP-D).
• SP-D plays a role m innate defense against some viruses, such as influenza A virus (IAV) in the lungs (Hartshorn K.L. et al. (1994) J. Clin. Invest. 94:311-319 which is incorporated herein by reference in its entirety) ⁇ Multivalent lectin-mediated interactions of SP-D with lAVs result in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phagocytic cells (VanEijk, M. et al, (2019) Front Immunol. 10:2476 which is incorporated herein by reference in its entirety).
• viruses such as influenza A virus (IAV) in the lungs (Hartshorn K.L. et al. (1994) J. Clin. Invest. 94:311-319 which is incorporated herein by reference in its entirety) ⁇ Multivalent lectin-mediated interactions of SP-D with lAVs result in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phago
• SP-D binds to viral hemagglutinin (HA) and in particular, mannosylated glycans on the HA in a calcium dependent manner (Hsieh I.N. et al (2016) Front Immunol. 9:1368 which is incorporated herein by reference in its entirety').
• HA hemagglutinin
• 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 holds the RNA genome, and the S, E, and M proteins together create the viral envelope.
• the spike glycoprotein (S-protein) is responsible for allowing the virus to attach to and fuse with the membrane of a host cell. Coronavirus entry' into host cells is mediated by the S-protein that forms homotrimers protruding from the viral surface (Walls A.C. et al. (2020) Cell 181 :281-292 which is incorporated herein by reference in its entirety).
• S-protein includes two functional subunits responsible for binding to the host cell receptor (SI subunit) and fusion of the viral and cellular membranes (82 subunit). For many coronaviruses, S-protein is cleaved at the boundary between the SI and 82 subunits, which remain non-covalently bound in the prefusion conformation.
• the distal SI subunit comprises the receptor-binding domain(s) and contributes to stabilization of the prefusion state of the membrane-anchored S2 subunit that contains the fusion machinery.
• the SI subunit of the S-protein comprises a receptor binding domain that interacts with the human angiotensin-converting-enzyme-2 (ACE2) receptor in type II pneumocytes.
• ACE2 human angiotensin-converting-enzyme-2
• Viral recognition of the S-protein by the ACE2 receptor leads to the internalization of the vims by the host cells, resulting in viral replication.
• New copies of SARS- CoV-2 are externalized to infect more cells, increasing the viral load in lungs, exacerbating the pro-inflammator response, and extending the cellular and epithelial lung damage.
• pathologic events in the lungs trigger the clinical symptoms of COVID-19: fever, cough, shortness of breath, fatigue and dyspnea m mild to moderate manifestations.
• variants enclose different mutations but, the three of them share two common mutations in the SI -protein: N501Y and D614G (Liu, Y , et a! (2021) ‘The N501Y spike substitution enhances SARS-CoV-2 transmission’ bioRxiv; and Rees-Spear, C , et ai, (2021) Cell Rep 34: 108890). More examples of variants are disclosed in Fi!ipe Pereira (2021) Biochem Biophys Res Commun. 550: 8-14 which is incorporated by reference in its entrirety.
• Pulmonary' surfactant contains four different surfactant proteins. Two hydrophobic proteins, surfactant protein B and surfactant protein C, are involved in the reduction of surface tension at the air-water interface; while two hydrophilic proteins, surfactant protein A and SP-D, are members of the collectin family and are involved in the modulation of the host immune response and in surfactant pool recycling.
• SP-D is a C-type (Ca 2+ - dependent) lectin that includes four domains: a cysteine- linked N -terminal region required for the formation of intermolecular disulfide bonds; a triple-helical collagen region; an a-helical-coiled-coil trimerizing neck peptide; and a C-terminal calcium-dependent carbohydrate-recognition domain (CRD) (Crouch E. et a!. (1994) J Biol Chem 269:17311-9). Monomers form trimers through folding of the collagenous region into triple helices and the assembly of a coiled-coil bundle of a-helices in the neck region.
• CRD calcium-dependent carbohydrate-recognition domain
• the SP-D trimer has a total molecular weight of 129 kDa which includes three identical 43-kDa polypeptide chains.
• SP-D trimers can form higher order oligomerization states which vary by size and conformation. Higher order 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. etal. (2006) J Biol Chem 281:18008-14).
• compositions of SP-D should have an appropriate oligomerization state for optimal activity including binding to carbohydrate ligands on the surface of pathogens, and achieving potent bacterial and viral agglutination effects (White M, etal., J Immunol (2008) 181:7936-43).
• An appropriate oligomerization state also has a role in optimal receptor recognition and receptor-mediated signal transduction for modulation of the host immune response (Yamoze M et a!., J Biol Chem (2008) 283:35878- 35888) as well as for maintenance of surfactant homeostasis (Zhang L et al, J Biol Chem (2001) 276:19214-19219).
• SP-D binds to glycosylated ligands on pathogens such as IPS m bacteria, hemagglutinin (HA) in influenza virus, and F-protein in respiratory syncytial virus. Binding triggers opsonization, aggregation, and direct killing of microbes, which facilitates their clearance from the lungs by phagocytic cells such as macrophages. SP-D dodecamers and higher order oligomers have shown an increased activity and potency in this anti-microbial function.
• pathogens such as IPS m bacteria, hemagglutinin (HA) in influenza virus, and F-protein in respiratory syncytial virus. Binding triggers opsonization, aggregation, and direct killing of microbes, which facilitates their clearance from the lungs by phagocytic cells such as macrophages.
• SP-D dodecamers and higher order oligomers have shown an increased activity and potency in this anti-microbial function.
• SP-D has also shown an anti-inflammatory effect in animal models of bacterial and viral respiratory infections as well as in lung injury induced by mechanical ventilation; in both cases, SP-D has decreased the levels of pro- inflammatory cytokines (e.g. IL-6), the neutrophilic response and NETosis, and lung tissue damage.
• Animal models have consistently demonstrated an association between higher levels of pulmonary SP-D and improved outcomes following viral, bacterial, or mechanical lung injury.
• human studies have demonstrated lower mortality rates in ARDS patients with high levels of pulmonary SP-D.
• Full length recombinant hSP-D has been successfully produced in mammalian cells, showing comparable structure and activity to human native SP D. Therefore, rhSP-D could be a novel class of antiviral therapeutic for COVlD-l 9.
• Pathogen recognition and binding to glycosylated determinants is the first step and hallmark action of SP-D to opsonize infectious agents (e.g. viruses and bacteria) and facilitate their fast clearance by phagocytic cells in the lungs, as it has been shown in in vivo animal models of SP-D reduction or exogenous SP-D supplementation (Wright JR. (2005) Nat Rev Immunol 2005; 5: 58-68; and Kmgma PS, et a! (2006) Curr Opm Pharmacol 6:277-283; LeVine AM, et al.
• infectious agents e.g. viruses and bacteria
• SP-D has shown calcium- dependent binding to the S-protem of the previous SARS-CoV strain and high glycosylation of the current SARS-CoV-2 S-protein has been confirmed and mapped suggesting 8ARS- CoV-2 S-protein may be a target of SP-D.
• rhSP-D binds to the antigen of the current SARS-CoV-2 (FIG. 4A, FIG. 4B) via a process that mimics opsonization and the critical first step of clearance of SARS-CoV-2 by SP-D in vivo.
• rhSP-D could increase viral clearance and reduce viral load in COVID-19 patients.
• binding affinity' of SP-D for the spike protein of the original variant from Wuhan was very similar to the variant emerged in U.K. (B.1.1.7.) which has widespread worldwide quickly.
• binding affinity to the S-protein from the South African variant (B.1.351) w3 ⁇ 4s significantly decreased.
• the N501 Y spike mutation enhances virus transmission.
• SP-D had decreased binding affinity' to the spike protein with the N501 Y spike mutation
• binding of pathogens by rhSP-D leads to their aggregation, forming clusters where multiple viral molecules that are removed at once by phagocytic cells, thus making viral clearance more effective.
• the critical first step of aggregation is driven by the ability of SP D (hexarners, dodecamers or higher order multi mers to bind more than one virus and form a protein bridge linking multiple pathogens.
• SP-D was able to form protein bridges between S-proteins (FIG. 5 A, FIG. 5B, FIG 5C, FIG. 5D, FIG 5E).
• Studies disclosed herein demonstrated a first step of viral aggregation (i.e. binding) and the subsequent formation of the rhSP-D protein bridge.
• rhSP-D inhibited SARS-CoV-2 life cycle by inhibiting virus replication in cells with an EC3 ⁇ 4>o of 3.7 gg/niL (FIG. 7).
• a first mechanism for rhSP-D inhibition of virus replication may include a steric blockage on the interaction between the receptor binding domain within S-protem and ACE2 by the rhSP-D bound to the gly cosy lated S-protem, which could restrict the accessibility of key domains in the presence of the bound SP-D molecule.
• this effect was not evident when experiments were performed with isolated SI -protein, ACE2 and rhSP-D (FIG. 6A, FIG.
• a second mechanism for rhSP-D inhibition of virus replication may include, potential aggregation of SARS-CoV-2 induced by rhSP-D by reducing the number of viral molecules available to interact with the host cell.
• the first and second mechanisms may not be mutually exclusive, and may be cooperative with one another.
• Some embodiments of the methods and compositions provided herein include aspects disclosed in U.S. Pat No. 10975389, U.S. Pat No. 10752914, U.S. Pat No. 9492503, U.S. Pat. No. 6838428, U.S 2021/0010988, and WO 2019/191247, which are each incorporated herein by reference in its entirety.
• compositions and methods provided herein include methods of treating or ameliorating a viral infection in a subject.
• the viral infection comprises a respiratory viral infection.
• symptoms of a viral infection are prevented, relieved and/or ameliorated.
• symptoms of a viral infection include fever, cough, and shortness of breath. More symptoms include tiredness, aches, runny nose, sore throat, headache, diarrhea, vomiting, and a loss of smell or taste in some embodiments, a therapeutically effecti v e amount of a pharmaceutical composition and/of SP-D is sufficient to prevent, relieve and/or ameliorate symptoms of a viral infection.
• the viral infection comprises a corona virus.
• coronavirus examples include severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory sy ndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome-related coronavirus (MERS-CoV), HCoV-229E, HCoV- NL63, HCoV-OC43, and HCoV-HKUl.
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• SARS-CoV-1 severe acute respiratory sy ndrome coronavirus
• MERS-CoV Middle East respiratory syndrome-related coronavirus
• HCoV-229E HCoV- NL63
• HCoV-OC43 Middle East respiratory syndrome-related coronavirus
• HKUl Middle East respiratory syndrome-related coronavirus
• Some embodiments include a method of treating or ameliorating a viral infection in a subject, comprising administering an effective amount of a recombinant human surfactant protein D frhSP-D) or active fragment thereof to the subject.
• the viral infection comprises a respiratory tract infection.
• the viral infection comprises a coronavirus.
• 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-related coronavirus (MERS-CoV).
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• SARS-CoV-1 severe acute respiratory syndrome coronavirus
• MERS-CoV Middle East respiratory syndrome-related coronavirus
• the viral infection comprises SARS-CoV-2.
• the SARS-CoV-2 comprises a wildtype SI protein. In some embodiments, the SARS-CoV-2 comprises a SI protein of a Wuhan wildtype or variant; a II K. variant; or a South Africa variant. In some embodiments, the SARS-CoV-2 comprises an SI protein variant. In some embodiments, the SI protein variant comprises a mutation selected from N501Y, D614G, HV69-70del, K417N, and E484K In some embodiments, the SI protein lacks a mutation selected from K417N, and E484K
• the administration comprising administering a pharmaceutical composition comprising the recombinant human surfactant protein D (rhSP- D) or active fragment thereof.
• the pharmaceutical composition comprises a buffer, a sugar, and a calcium salt.
• the buffer is selected from the group consisting of acetate, citrate, glutamate, histidine, succinate, and phosphate.
• the buffer is histidine.
• the concentration of the histidine is from about 1 mM to about 10 mM.
• the sugar is selected from the group consisting of sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabinose, xylose, ribose, rhamnose, trehalose, sorbose, melezitose, raffmose, thioglucose, thiomannose, thiofructose, octa-O-acetyl-thiotrehalose, thiosucrose, and thiomaltose.
• the sugar is lactose in some embodiments, the concentration of the lactose is from 200 niM to 300 mM. In some embodiments, the concentration of the lactose is about 265 mM.
• the calcium salt is selected from the group consisting 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 chloride is from about 1 mM to about 10 mM. In some embodiments, the concentration of the calcium chloride is about 5 mM.
• the pharmaceutical composition has a pH from about
• the pharmaceutical composition has a pH about 6.0.
• the concentration of the rhSP-D is from about 0.1 mg/ml to about 10 mg/ml.
• the pharmaceutical composition comprises a population of rhSP-D polypeptides having oligomeric forms, wherein greater than 30% of the oligomeric forms comprise dodecamers of rhSP-D. In some embodiments, greater than 35% of the oligomeric forms comprise dodecamers of rhSP-D. In some embodiments, greater than 40% of the oligomeric forms comprise dodecamers of the rhSP ⁇ D
• the pharmaceutical composition comprises a bulking agent.
• the bulking agent is selected from the group consisting of mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, arabitol, glycine, alanine, threonine, valine, and phenylalanine.
• the pharmaceutical composition lacks a chelating agent.
• the chelating agent is selected from EDTA and EGTA.
• the rhSP-D comprises an ammo acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:Q2.
• the subject is mammalian. In some embodiments, the subject is human.
• compositions and methods provided herein include pharmaceutical compositions of recombinant human surfactant protein D (rhSP-D) or an active fragment thereof
• rhSP-D or an active fragment thereof has activity in a bacterial aggregation assay, or in a TLR4 inhibition assay.
• the pharmaceutical composition can be an aqueous solution, a suspension, or a solid form.
• the pharmaceutical composition of rhSP-D or an active fragment thereof is suitable for lyophilization to a solid form.
• a solid form such as a lyophile or powder
• a pharmaceutical composition comprising the aqueous solution or suspension of rhSP-D or an active fragment thereof is suitable for administration to a lung.
• Certain activities of rhSP-D, or a fragment thereof can be readily determined using bacterial aggregation assays, Toll-like receptor 4 (TLR4) inhibition assays, and/or an asymmetric flow field-flow fractionation with multi-angle laser light scattering (AF4-MALLS) analysis.
• the activity of rhSP-D, or an active fragment thereof can include a biological activity, such as activity measured in a bacterial aggregation assays, or a TLR4 inhibition assay.
• the activity of rhSP-D, or an active fragment thereof can include the activity of a population of the rhSP-D, or active fragments thereof, to form certain oligomeric forms of the rhSP-D and/or to form a certain distribution of oligomeric forms of the rhSP-D.
• Example methods to identify the distribution of oligomeric forms of rhSP-D in a sample are provided in WO 2019/191254 wirich is incorporated herein by reference in its entirety.
• the pharmaceutical composition can include a buffer.
• buffers include acetate, citrate, glutamate, histidine, succinate, and phosphate.
• the buffer is histidine.
• the concentration of the buffer, such as histidine is 0.1 mM, 1 mM, 2 mM, 3 niM, 4mM, 5 mM, 6 rnM, 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 rnM, or a concentration in a range between any two of the foregoing concentrations in some embodiments, the concentration of the buffer, such as histidine, is about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, 4 mM, about 5 mM, about 6 m
• the pharmaceutical composition can include a sugar.
• sugars include trehalose, sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabinose, xylose, nbose, rhamnose, trehalose, sorbose, melezitose, raffmose, thioglucose, thiomannose, thiofmctose, octa-O-acetyl-thiotrehalose, thiosucrose, and thiomaltose.
• the sugar is lactose.
• the concentration of the sugar is 0.1 niM, 1 liiM, 10 mM, 20 niM, 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 concentration in a range between any two of the foregoing concentrations.
• the concentration of the sugar 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 concentration in a range between any two of the foregoing concentrations.
• the pharmaceutical composition can include a calcium salt.
• calcium salts include calcium chloride, calcium bromide, calcium acetate, calcium sulfate, and calcium citrate.
• the calcium salt is calcium chloride.
• the concentration of the calcium salt, such as calcium chloride is 0.1 mM, 1 M, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 M, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 M, 100 mM, or a concentration in a range between any two of the foregoing concentrations.
• the concentration of the calcium salt, such as 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 M, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 M, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or a concentration in a range between any two of the foregoing concentrations.
• the pharmaceutical composition can include an inorganic salt or organic salt.
• inorganic salts include sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium hydrogen carbonate.
• organic salts include sodium citrate, potassium citrate and sodium acetate.
• the inorganic salt is sodium chloride.
• the concentration of the inorganic salt or organic salt, such as sodium chloride is 0.1 niM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 nxM, 7 mM, 8 inM, 9 mM, 10 liiM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a concentration in a range between any two of the foregoing concentrations.
• the concentration of the inorganic salt or organic salt, such as 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 concentration in a range between any two of the foregoing concentrations.
• the pharmaceutical composition can lack an inorganic salt or organic salt, such as sodium chloride.
• the pharmaceutical composition can include a surface-active agent.
• surface-active agents include hexadecanol, tyloxapol, dipalmitoylphosphatidylcholine (DPPC), PG, palmitoyl-oleoyl phosphatidylglyceroi, palmitic acid, tripalmitin, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-21, po!ysorbate-40, po!ysorhate-60, polysorbate-65, polysorbate-81, and poiysorbate-85.
• DPPC dipalmitoylphosphatidylcholine
• PG palmitoyl-oleoyl phosphatidylglyceroi
• palmitic acid tripalmitin
• polysorbates such as polysorbate-20, polysorbate-80, polysorbate-21, po!ysorbate-40, po!ysorhate-60, polysorbate-65, polysorbate-81, and poi
• surface active agents include poloxamer such as po!oxamer 188, Triton such as Triton X-iOG, sodium dodecyl sulfate (8DS), sodium laurel sulfate, sodium octyl glycoside, lauryl-su!fobetame, myristyi-sulfohetame, Imoleyl-sulfobetame, stearyl-su!fobetame, lauryl- sareosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl- betaine, cetyl-betaine, lauroamidopropyl-betaine, cocamidopropyl-, linoleamidopropyl- betaine, myristamidopropyl-betaine, pal id
• the surface-acti e agent is tyloxapol.
• the concentration of the surface-active agent, such as tyloxapol is 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, (v/v) or a concentration in a range between any two of the foregoing concentrations.
• the concentration of the surface-active agent, such as tyloxapol is about 0.0001%, about 0.0005%, about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, (v/v) or a concentration in a range between any two of the foregoing concentrations.
• the pharmaceutical composition can lack a surface-active agent, such as tyloxapol.
• the pharmaceutical composition can 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 pH in a range between any two of the foregoing values.
• the pharmaceutical composition can 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 pH m a range between any two of the foregoing values.
• the concentration of protein, such as rhSP-D or an active fragment thereof, m 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 concentration in a range between any two of the foregoing concentrations.
• the concentration of protein, such as 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 concentration in a range between any two of the foregoing concentrations.
• the pharmaceutical composition can include a bulking agent.
• bulking agents include a sugar disclosed herein. More examples of bulking agents include mannitol, xylitoi, sorbitol, maltitol, iaetitol, glycerol, erythritoi, arabitol, glycerine, glycine, alanine, threonine, valine, and phenylalanine.
• the concentration of the bulking agent is 0.1 uiM, 1 mM, 2 mM, 3 rnM, 4 mM, 5 rnM, 6 mM, 7 rnM, 8 mM, 9 rnM, 10 mM, 20 rnM, 30 mM, 40 mM, 50 mM, 60 mM, 70 M, 80 mM, 90 mM, 100 mM, or a concentration in a range between any two of the foregoing concentrations.
• 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 concentration m a range between any two of the foregoing concentrations.
• the pharmaceutical composition can include a chelating agent in some embodiments, the pharmaceutical composition can lack a chelating agent.
• chelating agents include EOT A, and EGTA.
• the rhSP-D comprises a wild-type human SP-D polypeptide.
• the rhSP-D includes a polymorphism of the human SP-D polypeptide.
• Example SP-D polypeptide sequences are provided in TABLE 1.
• Polymorphisms in the human SP-D polypeptide 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) in which the positions relate to a position in a mature SP-D polypeptide, such as the example polypeptide of SEQ ID NO: 02.
• the rhSP-D comprises a certain residue at a polymorphic position in winch the residue selected from Metll/31, Thrl60/180, Ser 270/290, and Ala 286/306 in which residue positions relate to a position in the mature SP-D polypeptide, such as example SEQ ID NO:02, and a position in the SP-D polypeptide with its leader polypeptide, such as example SEQ ID NO:01.
• the rhSP-D comprises MetH/31.
• the rhSP-D comprises Metl 1/31, Thrl60/180, Ser 270/290, and Ala 286/306.
• the rhSP-D polypeptide has an identity with a polypeptide of SEQ ID NO:02 over the entire length of the polynucleotide of at least 80%, 90%, 95%, 99% and 100%, or any percentage in a range between any of the foregoing percentages.
• the rhSP-D is derived from a human myeloid leukemia cell line expressing the rhSP-D from an integrated transgene.
• Example expression vectors, rhSP-D polypeptides, cell-lines, and methods of purifying rhSP-D from such cells, are provided m U.S Patent Publications 2019/0071693 and U.S. 2019/0071694 each of which is expressly incorporated by reference herein in its entirety.
• a pharmaceutical composition such as a solution or suspension, comprising a population of rhSP-D polypeptides can have a certain distribution of oligomeric forms of the rhSP-D.
• a composition of rhSP-D can include different rhSP-D oligomeric forms including: trimers with a mass of about 130-150 kDa on SDS-PAGE which include 3 monomers and which together can have a rod-like appearance as visualized by atomic force microscopy (AFM); hexamers with a mass of about 250 kDa on SDS-PAGE which include 6 monomers; dodecamers with a predicted mass of about 520 kDa, as measured by AF4-MALLS and which include 12 monomers and can have an X-like appearance as visualized by AFM; larger heterogeneous oligomeric species which comprise multiples of more than four trimers and can have a star-like- or star- shaped appearance with a radius of about 70
• more than about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or a percentage within a range between any two of the foregoing percentages, of the oligomeric forms of rhSP-D can be a dodecameric oligomeric form of rhSP-D as measured as a relative peak area (RPA) in an AF4-MALLS analysis.
• RPA relative peak area
• more than about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or a percentage within a range between any two of the foregoing percentages, of the mass of the oligomeric forms, such as in a solution or suspension, of rhSP-D can be a dodecameric oligomeric form of rhSP-D.
• more than about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or a percentage within a range between any two of the foregoing percentages, of the number of molecules of the oligomeric forms, such as in a solution or suspension, of rhSP-D can be a dodecameric oligomeric form of rhSP-D.
• less than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 50%, or a percentage within a range between any two of the foregoing percentages, of the oligomeric forms of rhSP-D can be an aggregate oligomeric form of rhSP-D as measured as an RPA or an adjusted RPA m an AF4-MALLS analysis.
• less than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 50%, or a percentage within a range between any two of the foregoing percentages, of the mass of the oligomeric forms, such as in a solution or suspension, of rhSP-D can be an aggregate oligomeric form of rhSP-D.
• less than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 50%, or a percentage within a range between any two of the foregoing percentages, of the number of molecules of the oligomeric forms, such as m a solution or suspension, of rhSP-D can be an aggregate oligomeric form of rhSP-D.
• a pharmaceutical composition consists of, consists essentially of, or comprises 1 mg/ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, having a pH of 6.0.
• a pharmaceutical composition consists of consists essentially of or comprises 1 mg/ml rhSP-D, 5 mM histidine, 265 mM lactose, 1 mM calcium chloride, having a pH of 6.0.
• a pharmaceutical composition consists of, consists essentially of, or comprises 2 rng/'ml rhSP-D, 5 mM Histidine, 265 mM Lactose, 1 mM CaCb, pH 6.0. In some embodiments, a pharmaceutical composition consists of, consists essentially of, or comprises 2 mg/ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, having a pH of 6.0.
• a pharmaceutical composition consists of, consists essentially of, or comprises 4 mg/ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, having a pH of 6.0.
• the pharmaceutical compositions provided herein can include an admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like, and can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
• a suitable carrier diluent, or excipient
• auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
• such preparations can include complexing agents, metal ions, polymeric compounds such as polyacetic acid, polygiycoiie acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
• Suitable lipids for liposomal formulation include monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.
• Such additional components can influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus can be chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration, such as pulmonary' delivery, such as delivery to a lung, such as delivery' to a neonate lung.
• compositions are suitable for intratracheal, intrabronchial or bronchoalveolar administration to a lung.
• intratracheal, intrabronchial or bronchoalveolar administration can include spraying, lavage, inhalation, flushing or installation, using as fluid a physiologically acceptable composition in which the pharmaceutical composition has been dissolved.
• Methods of administration can include the use of continuous positive airway pressure (CPAP).
• Methods of administration can include direct intubation in some embodiments, pharmaceutical compositions provided herein can be delivered to the lungs while inhaling. Example forms that can be delivered include dry powders, and aerosols.
• a wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be employed, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
• These devices employ formulations suitable for the dispensing of a pharmaceutical composition.
• each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants, and/or earners useful in therapy. Kits
• kits can include a pharmaceutical composition provided herein. Some embodiments include a sterile container comprising a pharmaceutical composition provided herein. Some embodiments include a pharmaceutical composition provided herein in lyophilized form, and a sterile reconstituting solution. In some embodiments, a kit can include a device for administering a pharmaceutical composition provided herein, such as an inhaler, and a nebulizer.
• Example 1 In vitro binding of S- protein to immobilized rhSP-D
• FIG. 1 A depicts a schematic overview of the assay.
• Recombinant SI -protein was produced in HEK293 cells with a mouse Fc IgG tag on the C-terminal end (SinoBiologicals, #40591-V05H1).
• a first sample of rhSP-D was produced from human myeloid leukemia ceils, and a second sample of rhSP-D was obtained from CHO cells.
• the wells of microtiter plates were coated with 200 gL of a suspension of rhSP-D at 5 pg/mL or at 2 pg/mL in a carbonate-bicarbonate coating buffer (50 rnM NaHCOs-NaAlCb (pH 9.6)). The plate was incubated overnight at 4 °C.
• a second set of SI -protein samples was prepared where maltose was added to the SI -protein samples to obtain a final concentration of 200 niM maltose and it was incubated 10 minutes before being added to the plate wells.
• a third set of SI -protein samples was prepared with the same purpose, in this case, using 100 ml EDTA in the dilution buffer instead of 5 mM calcium to inhibit the calcium-dependent binding of rhSP-D. In all the cases, once added to the wells, the SI -protein was incubated for 1 hour at room temperature.
• HRP horseradish peroxidase
• FIG. IB and FIG. 1C summarize results for wells coated with solutions of rhSP-D at 5 pg/mL, for a first sample of SP-D and a second sample of SP-D, respectively.
• FIG ID and FIG. IE summarize results for wells coated with solutions of rhSP-D at 2 pg/mL, for a first sample of SP-D and a second sample of SP-D, respectively.
• the S I -protein bound to SP-D in the presence of calcium. The binding was inhibited by the presence of EDTA or maltose. Thus, the SI -protein bound to SP-D in a calcium dependent manner, and this binding was inhibited by a competitor, maltose.
• FIG. 2A depicts a schematic overview ' - of the assay.
• Recombinant Sl-protein w3 ⁇ 4s produced in HEK293 cells with a mouse Fe IgG tag on the C-terminai end (SinoBioiogicals, #40591-V05HT).
• a first sample of rhSP-D was produced from human myeloid leukemia cells; and a second sample of rhSP-D was obtained from CHO ceils.
• the wells of microtiter plates were coated with 200 mE of a suspension of SI -protein at 2,5 pg/rnL in a carbonate-bicarbonate coating buffer (50 mM NaHCG -NasCOs (pH 9.6)). The plate was incubated overnight at 4 °C.
• rhSP-D carbohydrate recognition domain of rhSP-D
• a second set of rhSP-D samples was prepared using 100 mM EDTA in the dilution buffer instead of 5 mM calcium to inhibit the calcium-dependent binding of rhSP-D.
• the rhSP-D was incubated for 1 hour at room temperature. After washing the plate, 50 m ⁇ , of rabbit anti-SP-D antibody (dilution 1:5000) were incorporated and incubated for 1 hour at room temperature.
• HRP horseradish peroxidase
• FIG. 2B and FIG. 2C summarize results for wells coated with SI -protein, for a first sample of SP-D and a second sample of SP-D, respectively.
• the S i -protein bound to SP-D in the presence of calcium.
• the binding was inhibited by the presence of EDTA.
• the SI -protein bound to SP-D in a calcium dependent manner.
• This example shows determination of SP-D levels in bronchoalveolar lavage of COVID- 19 patients.
• CV cardiovascular disease
• HAV human immunodeficiency virus
• Example 4 -Recombinant hSP-D binds to the S-protein of SARS-CoV-2
• Recombinant SARS-CoV-2 spike protein variants (Sl- 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), Aero Biosystems (#S1N-C52H3, #SlN-C52Hk, #SlNN-C52Hg), The NativeAntigen Company (#REC31806-100-HRP) and from Biomart Creative (#ACE2- 736H).
• a first ELISA assay was developed in winch microtiter plates were coated with a SI -spike-protein variant (0.4 pg in 200 pL/well). Washes and dilutions w r ere performed with 0.05% TBS-tween, 5 mM CaCh. Weils were blocked with 2% BSA and serially diluted rhSP-D (10 gg/mL to 9.8 ng/mL) was added to the wells.
• Bound rhSP-D was detected with a mouse anti-SP-D antibody (#2D12-A-88, Seven Hills Bioreagents), followed by an anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibody (#7076, Cell Signaling).
• HRP horseradish peroxidase
• the plates were developed w th TMB (#TMBS010001, Surmodics) for 10 minutes and the reaction was stopped with 2N H2SQ4. Plates -were read for absorption at 450 ran.
• 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 SI -protein.
• wells were coated with 1% BSA instead of SI -protein.
• a second ELISA assay was also developed in which the wells were coated with rhSP-D instead of SI -protein.
• Serially diluted SI -protein samples with a mouse Fc tag (10 pg/mL to 9.8 ng/mL) were added to the wells.
• Bound SI -protein was detected with the same anti-mouse IgG HRP-conjugated antibody.
• Analysis of the binding isotherms was performed with GraphPad Prism 8, considering total binding and one site to determine the apparent dissociation constant (kd) and the apparent maximum number of binding sites (Bmax).
• the apparent number of maximum binding sites was higher when rhSP-D was the ligand (Bmax :::: L35, FIG. 4A) compared to SI -protein (Bmax ::::: 0.81, FIG.
• Example 5 rhSP-D forms protein bridges with the S-protein of SARS-CoV-2
• rhSP-D (2 gg or 4 gg) and SI -protein (2 gg, Wuhan variant) were pre-mixed and incubated for 2 hours to favor binding and aggregation of SI -protein by rhSP-D. Then, the mix was added to the beads. After incubation at room temperature for 30 min, the beads were centrifuged and the supernatant (SI ) was saved. Then, the beads were washed and eluted as previously described, saving the eluted fraction (P) for analysis.
• SI supernatant
• rhSP-D (2 gg or 4 gg) was incubated at room temperature for 30 nun with maltose-coated agarose beads in 50 gL TBS (150 mM NaCl, 20 mM Tris (pH 7.4))- 10 ml CaCk buffer.
• TBS 150 mM NaCl, 20 mM Tris (pH 7.4)
• SI supernatant
• the supernatant (SI) with the excess unbound rhSP-D was separated by centrifugation and saved. The beads were washed with TBS-CaCb.
• the spike protein of SARS-CoV-2 interacts with ACE2 receptors in epithelial cells. Binding of ACE2 to SI -protein (Wuhan variant) in the presence of rhSP-D was examined. Plates were coated with purified SI -protein (Wuhan variant). RhSP-D (0.1 to 1 pg/mL) in TBS-Ca 5 niM or buffer (negative control) were added to the wells and incubated for 2 hours. Without washing, human ACE2 protein (0.186 to 1.5 pg/mL) was added to the wells at each of the rhSP-D concentrations, a control with TBS buffer instead of ACE2 w3 ⁇ 4s also included.
• FIG. 6A Binding of SI -protein to rhSP-D in the presence of ACE2 was examined. Plates were coated with rhSP-D (5 pL/mL, 200 pL/well). SI -protein HRP-tagged at different concentrations or buffer (negative control), were added to the wells and incubated for 2 hours. Without washing, human ACE2 protein His-tagged was added to the wells to reach 3, 0.375 or 0.045 pg/mL at each of the SI -protein concentrations. After incubation for 30 minutes, bound Sl-protein-HRP was detected directly with TMB and the reaction was stopped with 2N H2SO4 (FIG. 6C, FIG. D)
• Example 7 rhSP-D inhibits SARS-CoV-2 replication m host ceils
• Ceil toxicity of rhSP-D was evaluated m additional plate wells by using a neutral red dye that penetrated into living cells and allows quantification of viable ceils.
• the more intense the red color the larger the number of viable cells present m the wells.
• the dye content in each well was quantified using a spectrophotometer at 540 nm wavelength.
• rhSP-D inhibited viral replication in a dose-dependent manner with higher concentrations of rhSP-D leading to greater inhibition of viral replication, which was observed by measuring the virus titer in the cell supernatant at the different rhSP-D concentrations tested and reported as CCID50 (50% cell culture infectious dose) (FIG. 7).
• the concentration of rhSP-D necessary to inhibit viral replication by 90% (EC90) was 3.7 gg/rnL.
• rhSP- D did not show any cell toxicity even at the highest rhSP-D tested (100 pg/ nL) when compared to control (non-treated and non-infected) cells.
• Example 8 Treatment of a SARS-CoV-2 infection with rh8P-D
• a patient having a SARS-CoV-2 infection is administered a pharmaceutical solution comprising rhSP-D, 5 mM Histidine, 265 mM Lactose, and 5 niM CaCb.
• the patient has symptoms including fever, cough, shortness of breath, fatigue, muscle pain, diarrhea, sore throat, loss of smell, and abdominal pain.
• On administration of the pharmaceutical solution one or more symptoms of the SARS-CoV-2 infection in the patient are reduced.
• the term “composing” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

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