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WO2022272132A2 - Compositions et procédés pour le traitement d'une infection virale - Google Patents

Compositions et procédés pour le traitement d'une infection virale Download PDF

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Publication number
WO2022272132A2
WO2022272132A2 PCT/US2022/034997 US2022034997W WO2022272132A2 WO 2022272132 A2 WO2022272132 A2 WO 2022272132A2 US 2022034997 W US2022034997 W US 2022034997W WO 2022272132 A2 WO2022272132 A2 WO 2022272132A2
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Prior art keywords
lekti
subject
viral
seq
protein
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PCT/US2022/034997
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WO2022272132A3 (fr
Inventor
Trudy H. Grossman
Mark N. Sampson
Roger Leger
Dorival MARTINS, Jr.
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Azitra Inc
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Publication of WO2022272132A2 publication Critical patent/WO2022272132A2/fr
Publication of WO2022272132A3 publication Critical patent/WO2022272132A3/fr

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    • 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
    • A61K38/55—Protease inhibitors
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8135—Kazal type inhibitors, e.g. pancreatic secretory inhibitor, ovomucoid

Definitions

  • the present disclosure relates to methods, kits, and compositions for preventing or treating viral infections in a subject using one or more therapeutic LEKTI domains.
  • Proteases or proteolytic enzymes are essential in organisms, from bacteria and viruses to mammals. Proteases digest and degrade proteins by hydrolyzing peptide bonds. Serine proteases (EC. 3.4.21) have common features in the active site, primarily an active serine residue. There are two main types of serine proteases; the chymotrypsin/trypsin/elastase-like and subtilisin-like, which have an identical spatial arrangement of catalytic His, Asp, and Ser but in quite different protein scaffolds. Over twenty families (S1-S27) of serine proteases have been identified that are grouped into 6 clans on the basis of structural similarity and other functional evidence, SA, SB, SC, SE, SF & SG.
  • the family of chymotryp sin/trypsin/ elastase-like serine proteases have been subdivided into two classes.
  • the "large” class (ca 230 residues) includes mostly mammalian enzymes such as trypsin, chymotrypsin, elastase, kallikrein, and thrombin.
  • the "small” class (ca 190 residues) includes the bacterial enzymes.
  • serine proteases examples include trypsin, tryptase, chymotrypsin, elastase, thrombin, plasmin, kallikrein, Complement Cl, acrosomal protease, lysosomal protease, cocoonase, a- lytic protease, protease A, protease B, serine carboxypeptidase t, subtilisin, urokinase (uPA), Factor Vila, Factor IXa, and Factor Xa.
  • the serine proteases have been investigated extensively for many years and are a major focus of research as a drug target due to their role in regulating a wide variety of physiological processes.
  • SPINK serine protease inhibitors
  • the lymphoepithelial kazal-type inhibitor (LEKTI) is encoded by SPINK5 (Serine Proteinase Inhibitor Kazal type 5) (Magert et al. (1999) J Biol. Chem. 274; 21499 -21502).
  • the SPINK5 gene is located on chromosome 5q32 among a cluster of other SPINK genes (e.g., SPINK1, SPINK6, SPINK7, SPINK9 and SPINK13), and comprises 33 exons encoding 15 inhibitory domains separated by linker regions.
  • SPINK5 has shown to be expressed in the skin, oral mucosa, tonsils, parathyroid gland, thymus, and lung (Magert et ah, Int J Biochem Cell Biol. 2002;34(6):573-6; Magert et ah, Eur J Med Res. 2002;7(2):49-56).
  • SPINK5 stands out among the other SPINK genes for the large number of inhibitory domains it encodes.
  • the SPINK5 gene is transcribed into three different transcripts, resulting in three different LEKTI proteins that differ in the C-terminal region; i.e., a 145 kDa full length protein having inhibitory domains D1-D15, a 125 kDa (short) protein having inhibitory domains D1-D12, and a 148 kDa (long) protein having an extended linker region 13.
  • LEKTI is expressed as high molecular mass precursors, which are rapidly processed into several proteolytic fragments secreted in the intercellular space (Bitoun et al. (2003) Hum. Mol. Genet.
  • the Kazal motif of LEKTI is defined by the presence of six cysteine residues positioned at specific distances to allow formation of three disulfide bonds in a 1-5, 2-4, and 3-6 pattern. Two of the domains of LEKTI (D2 and D5) form this six cysteine motif, while other domains share four cysteine residues, which produce a rigid inhibitory loop believed to mimic the substrate of target proteases and inactivate the target protease catalytic site.
  • the LEKTI protein requires proteolytic cleavage for activation of its inhibitory function against many proteases. The full length protein is cleaved into domains D1-D5 and D6-D15.
  • the D6-D15 domains are then further cleaved in multiple steps into D6-D9 and D10-D15, -> D6 and D7-D9 -> D7 and D8-D9 -> D8. This process results in LEKTI proteins comprising between one and six inhibitory domains, with each protein having different protease targets.
  • KLKs Human kallikrein-related peptidases
  • chymo (chymo)-trypsin-like serine proteases that are expressed in a variety of tissues such as prostate, ovary, breast, testis, brain, and skin.
  • Most KLKs are produced by the epithelia of the upper and lower respiratory tracts (nose, paranasal sinuses, larynx, trachea, and bronchial tree) and by the submucosal glands of the main airways (Shaw& Diamandis, Clin Chem. 2007 Aug; 53(8):1423-32; Petraki et al., Biol. Chem. 2006 Jun; 387(6):653-63).
  • KLKs belong to a subgroup of the chymotrypsin-like serine protease family S1A of clan PA(S).
  • the 15 human KLK genes are located on chromosome 19ql3.4 and constitute the largest contiguous serine protease cluster in the human genome. These genes, generally composed of five coding exons and in some cases one or two 5’ non- coding exons, encode the kallikrein-related peptidases KLK1 to KLK15. All KLK genes encode single-chain pre-pro- proteins containing a chymotrypsin- or trypsin- like catalytic domain of 224-237 residues with an amino acid sequence identity of approximately 40% among KLK4 to KLK15.
  • KLK1 and its close homologs KLK2 and KLK3 form a clade of their own, KLK4, 5, and 7 belong to another subgroup, whereas KLK6 shares more similarity with KLK 13 and KLK14. See Debela et al. (2008) Biol Chem 389, 623-632.
  • LEKTI recombinant protein has been shown to inhibit trypsin, subtilisin A, plasmin, cathepsin G, and neutrophil elastase, but not chymotrypsin (Mitsudo et al. (2003) Biochemistry 42, 3874 -3881).
  • a partial recombinant form of LEKTI containing domains 6 -9 (rLEKTI6 -9) has been shown to inhibit trypsin, subtilisin A, chymotrypsin, kallikrein 5 (KLK5), and kallikrein 7 (KLK7), but not plasmin, cathepsin G, or elastase (Jayakumar et al. (2004) Protein Expr. Purif. 35, 93-101; Schechter et al. (2005) Biol.
  • aprotinin is a protein with unfavorable pharmacokinetics whereas camostat is a covalent binder; both are non-specific protease inhibitors with potential side-effects.
  • Coronavimses cause respiratory and enteric diseases in humans and animals. Seven coronaviruses infect humans, four of which (human coronavirus [HCoV]-229E, HCoV- NL63, HCoV-OC43, and HCoV-HKUl) cause relatively mild upper and lower respiratory tract disease and two (SARS-CoV and MERS-CoV) are associated with severe, life- threatening respiratory infections and multiorgan failure (Fields virology. (Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia, ed.
  • a coronavirus contains four structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins.
  • S protein plays the most important roles in viral attachment, fusion and entry.
  • Coronaviral infection is initiated by interaction between the trimeric spike (S) protein and its receptor, which is expressed on the surface of the susceptible cell (Du et al, Expert Opin. Ther. Targets 21, 131-143 (2017).; Lu et al, Nat. Commun. 5, 3067 (2014)).
  • Coronaviral S proteins are class I fusion proteins comprising a large N-terminal ectodomain, a hydrophobic trans-membrane region, and a small C-terminal endodomain.
  • the ectodomain is highly glycosylated and is composed of S 1 and S2 domains.
  • the globular SI domain is highly variable and carries the receptor-binding site, whereas the more conserved rod-like S2 domain undergoes structural rearrangement during entry, which brings the cellular and viral membranes into close proximity.
  • Such a structural switch may be triggered by different stimuli, including receptor binding, proteolytic cleavage of the S protein, and/or a reduction in pH.
  • Host proteases prime coronaviral S proteins. For example, trypsin-mediated cleavage in the small intestine is required for entry of porcine epidemic diarrhea virus (Wicht et al, J Virol.
  • cathepsin L processes the S proteins of SARS-CoV, MERS-CoV, HCoV-229E, and MHV-2 (Shirato et al., Journal of Virology Nov 2013, 87 (23) 12552-12561).
  • recent reports show that, in vivo, respiratory coronaviruses may be activated by the TMPRSS2 protease, which enables endocytosis-independent internalization, thereby re shaping the entry process (45, 47-50).
  • KLK13 is required for the infection of the human respiratory epithelium, and is sufficient to mediate the entry of HCoV-HKUl to non-permissive RD cells.
  • Viral proteases play critical roles in the life cycle of many different vims families, including proteases encoded by the herpesvirus, retrovims, hepatitis C vims (HCV), and human rhinovims (HRV) families (Patick and Potts, Clin Microbiol Rev. 1998 Oct; 11(4): 614-627). Anti-viral activities of certain serine proteinase inhibitors, such as alphal- proteinase inhibitor, have been previously described. Congote LF (Vims Res. 2007 May;
  • serpin Al alphal-antitrypsin, alpha 1 -proteinase inhibitor
  • Palesch et al (Med J Indones Vol. 22, No. 3, August 2013) have shown that LEKTI domain 15 is a potent inhibitor of HIV infection.
  • KLKs are well-reported to be inhibited by proteins of the lympho-epithelial Kazal-type-related inhibitor (LEKTI) family (Magert el ah, 2002; Borgono l ah, 2007; Fischer el ah, 2014), which are encoded by SPINK genes.
  • LEKTI lympho-epithelial Kazal-type-related inhibitor
  • Such inhibitors would have therapeutic potential as protease inhibitors, particularly as serine protease inhibitors, and more particularly as KLK serine protease inhibitors. Specifically, such compounds may be useful as antiviral agents.
  • the present disclosure is based, in part, on treating viral infection, by inhibiting pathways activated by protease targets of LEKTI.
  • the disclosure provides a method of treating or preventing a viral infection in a subject, comprising administering one or more LEKTI protein domains to the subject to provide a therapeutic effect.
  • the disclosure provides a method of treating or preventing a viral infection in a subject, comprising delivering a microbe genetically modified to express one or more protein domains encoded by one or more SPINK genes to the subject in need thereof.
  • the one or more SPINK genes are selected from the group consisting of SPINK1, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINK13, and SPINK14.
  • the one or more SPINK genes encodes a LEKTI protein, and protein domains thereof, selected from LEKTI, LEKTI-2 and LEKTI-3.
  • the disclosure provides a method of treating or preventing a viral infection in a subject, comprising administering to the subject one or more microbes comprising one or more LEKTI protein domains to provide a therapeutic effect.
  • the viral infection is a respiratory infection.
  • the viral infection is a dermal infection.
  • the viral infection causes cancer in the subject.
  • the therapeutic effect is a decrease in viral load in the subject.
  • the disclosure provides a method of treating or preventing a viral infection in a subject, comprising administering an effective amount of a viral fusion inhibitor; and/or an effective amount of a viral replication inhibitor, wherein the viral fusion inhibitor and/or the viral replication inhibitor is selected from one or more LEKTI protein domains.
  • both the viral fusion inhibitor and the viral replication inhibitor are administered to the subject.
  • the viral fusion inhibitor and the viral replication inhibitor are not the same.
  • the viral infection is a respiratory infection.
  • the viral infection is a dermal infection.
  • the viral infection causes cancer in the subject.
  • the disclosure provides a method of treating or preventing a cancer caused by a virus in a subject, comprising administering one or more LEKTI protein domains to the subject to provide a therapeutic effect.
  • the disclosure provides a method of inhibiting viral fusion in a subject, comprising administering one or more LEKTI protein domains to the subject.
  • the disclosure provides a method of inhibiting viral replication in a subject, comprising administering one or more LEKTI protein domains to the subject.
  • the disclosure provides a method of decreasing viral load in a subject, comprising administering one or more LEKTI protein domains to the subject.
  • the subject has been diagnosed with a viral infection.
  • the viral infection is a respiratory infection.
  • the viral infection is a dermal infection.
  • the subject has been diagnosed with cancer.
  • the one or more LEKTI protein domains are administered as part of a microbial composition.
  • the microbial composition is adapted for topical or mucosal administration. According to some embodiments, the microbial composition is administered as a nasal spray.
  • the method is part of a therapeutic regimen combining one or more additional treatment modalities.
  • the microbe is genetically modified by transfection/transformation with a recombinant DNA plasmid encoding the LEKTI protein domains.
  • the LEKTI domains are operably linked to one or more recombinant protein domains that are effective to enhance secretion from the microbe.
  • at least one LEKTI domain is operably linked to a Sec A domain.
  • At least one LEKTI domain is operably linked to an RMR domain.
  • at least one LEKTI domain comprises an amino acid sequence according at least one of SEQ ID NOs 103-118.
  • the disclosure provides a recombinant microorganism capable of secreting a polypeptide, wherein the recombinant microorganism comprises an expression vector comprising a first coding sequence comprising a gene capable of expressing the polypeptide and a second coding sequence comprising a gene capable of expressing a cell penetrating peptide.
  • the disclosure provides a pharmaceutical composition comprising the recombinant microorganism of any of the aspects and embodiments herein.
  • FIG. 1 shows the pUBTR119 vector.
  • FIG. 2 shows the chromosomal hLEKTI-d6 expression cassette.
  • FIG. 3 is a schematic showing the domains of the full length LEKTI polypeptide.
  • the LEKTI protein-producing bacteria are able to treat or prevent viral infection, by expressing and, optionally, secreting a therapeutic protein.
  • the LEKTI protein is a purified and isolated LEKTI protein.
  • the therapeutic protein comprises one or more LEKTI domains that are effective to inhibit serine proteases.
  • the protein being cleaved can directly affect viral replication (i.e., the viral spike protein, involved in cellular uptake).
  • the protein being cleaved can indirectly affect replication (i.e., affecting a host response that interferes with viral pathology.
  • the bacteria are able to self-replicate while retaining the ability to produce the recombinant protein, thereby providing a continuous supply of therapeutic agent.
  • administration is meant to refer to contact of a pharmaceutical composition, therapeutic composition, diagnostic agent or composition to a recipient, preferably a human.
  • the terms “disease” or “disorder” are meant to refer to an impairment of health or a condition of abnormal functioning. According to some embodiments, the disease or disorder is a viral infection.
  • an “effective amount” of an agent e.g., a pharmaceutical formulation, is meant to refer to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • “effective amount” refers to an amount of a compound as described herein that may be therapeutically effective to prevent, inhibit, reduce the severity of or treat the symptoms of one or more forms of viral infection.
  • the term “gene” as used herein refers to a region of DNA that controls a discrete hereditary characteristic, usually corresponding to a single protein or RNA. This definition includes the entire functional unit, encompassing coding DNA sequences, noncoding regulatory DNA sequences and introns.
  • the term “genetically modified” and grammatical variations thereof as used herein are meant to describe a microbial organism (e.g . bacteria) that has been genetically modified or engineered by the introduction of DNA prepared outside the microbe.
  • a microbial organism e.g . bacteria
  • the introduction of plasmid DNA containing new genes into bacteria will allow the bacteria to express those genes.
  • the DNA containing new genes can be introduced to the bacteria and then integrated into the bacteria's genome, where the bacteria will express those genes.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • inhibitor is meant to refer to the amount of a pharmaceutical composition as described herein that is sufficient to cause, for example, a decrease in KLK production or activity, protease production or activity, or a reduction in symptoms associated with viral infection as compared to a control subject or sample.
  • pharmaceutical formulation as used herein is meant to refer to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” as used herein is meant to refer to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • progression refers to the course of a disease, such as viral infection, as it becomes worse or spreads in the body.
  • protease and “proteinase” as used herein are interchangeable, with both terms referring to an enzyme that performs proteolysis.
  • quality of life refers to the overall enjoyment of life, including aspects of an individual's sense of well-being and ability to carry out various activities.
  • recombinant and grammatical variations thereof are meant to relate to or denote an organism, protein, or genetic material formed by or using recombined DNA comprising DNA pieces from different sources or from different parts of the same source.
  • recombinant DNA means a DNA molecule formed through recombination methods to splice fragments of DNA from a different source or from different parts of the same source.
  • two or more different sources of DNA are cleaved using restriction enzymes and joined together using ligases.
  • the term “recombinant protein” or “recombinant domains” and grammatical variations thereof means a protein molecule formed through recombination methods originating from spliced fragments of DNA from a different source or from different parts of the same source.
  • the term “recombinant microbe” or “recombinant bacteria” and grammatical variations thereof mean a microbe/bacteria that comprises one or more recombinant DNA/protein molecules.
  • risk factor refers to anything that raises the chances of a person contracting and/or developing a viral infection.
  • secretory peptides or “secretory sequences” or “secretion tags” or “signal peptides” or “export signals” and grammatical variations thereof means any peptide sequence that is capable of targeting the synthesized protein to the secretory pathway of a cell.
  • the term “subject” as used herein is meant to refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g ., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). According to some embodiments, the subject is a human.
  • the term “subject” is used interchangeably with the term “patient” herein.
  • treat generally refer to palliative (e.g., therapeutic), preventative (e.g., prophylactic), inhibitory, and/or curative treatment.
  • the terms “treat,” “treatment,” and/or “treating” refer to palliative, inhibitory, and/or curative treatment, with palliative and inhibitory treatment being more preferred.
  • Desirable effects of treatment include one or more of preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, stabilized (i.e., not worsening) state of disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, prolonging survival as compared to expected survival if not receiving treatment and improved prognosis.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • viral fusion refers to the binding of the virus to specific molecules on the surface of a host cell.
  • An inhibitor of viral fusion refers to a molecule or compound that is capable of adversely affecting, interfering with or otherwise inhibiting, at least in part, at least one aspect of viral fusion to a host cell.
  • viral replication refers to the formation of biological viruses during the infection process in the target host cells.
  • the viral replication cycle begins with the infection of a host cell and ends with the release of mature progeny virus particles.
  • inhibition of viral replication may involve inhibition of cleavage of viral proteins (e.g ., viral spike protein).
  • inhibition of viral replication may involve inhibition of cleavage of host factors required for viral replication.
  • KLKs may be involved in the inhibition of viral protein cleavage and/or inhibition of cleavage of host cell factors required for viral replication.
  • the present disclosure relates to treating or preventing a viral infection in a subject, comprising administering one or more LEKTI protein domains to the subject to provide a therapeutic effect.
  • the methods of the present disclosure can be used to treat those viruses whose replication requires a protease cleavage that may be inhibited by LEKTI.
  • Coronaviruses the largest RNA viruses identified so far, belonging to the Coronaviridae family, are divided into 4 genera, a-, b-, d- and g-coronaviruses, while the b- coronaviruses are further divided into A, B, C, and D lineages (Woo et ah, J Virol. 2012 Apr; 86(7):3995-4008). CoVs can co-infect humans and other vertebrate animals.
  • HCoVs seven CoVs were known to infect humans (HCoVs), including HCoV-229E and HCoV-NL63 in the a-coronaviruses, HCoV-OC43 and HCoV-HKUl in the b-coronaviruses lineage A, SARS-CoV and SARS-CoV-2 in the b-coronaviruses lineage B (b-B coronaviruses), and MERS-CoV in the b-coronaviruses lineage C.
  • SARS-CoV-2 shares a highly similar gene sequence and behavior pattern with SARS-CoV (Chan et ah, Emerg Microbes Infect. 2020; 9(l):221-236).
  • SARS-CoV-2 and SARS-CoV are in the coronavirus family, b- coronavirus genera (Chan et ah, Id.).
  • the genome of SARS-CoV-2 is more than 85% similar to the genome of the SARS-like virus ZC45 (bat-SL-CoVZC45, MG772933.1), and together these types of viruses form a unique Orthocoronavirinae subfamily with another SARS-like virus ZXC21 in the sarbecovirus subgenus (Zhu et ah, N Engl J Med. 2020 Feb 20; 382(8):727-733). All the three viruses show typical b-coronavirus gene structure.
  • SARS-CoV-2 coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO). According to the data released by the National Health Commission of the People’s Republic of China, SARS-CoV-2 was most likely transmitted from wild bats to humans.
  • C capsid
  • E envelope
  • M membrane
  • NS5 non-stmctural
  • NS1 non-stmctural proteins
  • the E protein a major virion surface protein, is involved in receptor binding and membrane fusion, and induces neutralizing antibodies in the infected hosts. Id.
  • Flaviviruses include Zika virus (ZIKV), dengue virus (DENV), yellow fever vims (YFV), West Nile vims (WNV), Japanese encephalitis vims (JEV), hepatitis C vims (HCV) and tick-borne encephalitis vims (TBEV).
  • ZIKV Zika virus
  • DEV dengue virus
  • YFV yellow fever vims
  • WNV West Nile vims
  • JEV Japanese encephalitis vims
  • HCV hepatitis C vims
  • TBEV tick-borne encephalitis vims
  • Other related viruses include, without limitation, the classical swine fever vims in the Pestivirus genus, and the viruses in the Pegivirus, genus.
  • the Paramyxoviridae are enveloped, non- segmented, negative- strand RNA viruses that include major human pathogens belonging to two subfamilies.
  • the Pneumonvirinae subfamily includes respiratory syncytial vims (RSV) and the metapneumovimses, while the Paramyxovirinae subfamily includes, amongst others, measles vims (MeV), Morbillivims genus, mumps vims (MuV) of the Rubulavirus genus, human parainfluenza viruses (hPIVl- 4), and the recently emerged, highly pathogenic henipavimses Hendra (HeV) and Nipah (NiV).
  • MeV in particular, remains a major cause of childhood mortality worldwide despite the availability of a live-attenuated vaccine.
  • Filoviridae Viruses [0063] The family of Filoviridae is a family of enveloped, single-stranded RNA(-) vimses comprising three different genera: monospecific Cuevavirus, Ebolavirus (EBOV), and monospecific Marburgvirus (MARV). Filoviruses get their name from their filamentous or thread-like virions which exhibit extreme pleomorphism: virions may appear U-shaped, 6- shaped, or circular. Their lengths may vary but they generally have a uniform diameter of
  • the Orthomyxoviridae family is comprised of segmented single- stranded RNA viruses with five genera: Influenzavirus A, Influenzavirus B, Influenzavirus C, Isavirus, and Thogotovirus.
  • the family is named as such because it is characterized by the ability of the viruses to attach to mucous proteins on cell surfaces (“myxo” is Greek for mucous) but more “orthodox” in comparison to the Paramyxoviridae — another group of RNA viruses, also characterized by the ability to attach to mucus-producing cells.
  • the influenza A, B, and C viruses, representing three of the five genera of the family Orthomyxoviridae are characterized by segmented, negative-strand RNA genomes.
  • Poxviruses comprise a family of genetically related, large, enveloped, DNA viruses that replicate exclusively within the cytoplasm of vertebrate or invertebrate cells.
  • the most intensively studied poxviruses belong to the Orthopoxvirus genus, including variola vims (causative agent of smallpox, eradicated from nature), vaccinia vims (VACV; the modern smallpox vaccine, now endemic in Brazil), cowpox vims (the original smallpox vaccine, indigenous in Europe, occasionally infects humans) and monkeypox vims (indigenous in Africa, causes a smallpox-like disease of humans). (Moss, Bernard. “Poxvirus cell entry: how many proteins does it take?” Vimses vol. 4,5 (2012): 688-707).
  • Rhabdoviridae Viruses includes the Vesiculovirus, Ephemerovirus and Lyssavirus genera which infect a wide range of mammals, including man; transmission is commonly vector- borne, typically hematophagous insects or animals (meaning those that feed on blood). There are seven recognized genotypes or species of the Lyssavirus genus, the most important of which to humans is the rabies vims (RABV).
  • RABV rabies vims
  • the Herpesviridae family is a significant viral family comprising major pathogens of a wide range of hosts. Known as a DNA vims, this family includes at least eight species of vimses known to infect humans. The family further includes a number of species that infect other mammals important to economies worldwide, including the livestock industry and the competition industry, potentially causing severe economic losses. (Sharma, V., el ah, Comparative Genomics of Herpesviridae Family to Look for Potential Signatures of Human Infecting Strains. Int. J Genomics. 2016; 2016: 9543274).
  • the Herpesviridae family includes vimses from the Alphaherpesvirinae subfamily, such as Varicella-zoster vims (VZV), which causes diseases known colloquially as chicken pox and shingles, Human herpesvirus-1 (HHV-1), Herpes simplex virus-1 (HSV-1), Human herpesvirus-2 (HHV-2), Herpes simplex virus-2 (HSV-2), Human herpesvirus-3 (HHV-3), Herpes simplex virus-3 (HSV-3), Bovine herpesvirus- 1 (BHV-1), Bovine herpesvirus-5 (BHV-5), Equid herpes vims 1 (EHV-1), Equid herpes vims 3 (EHV-3), Equid herpes vims 4(EHV-4), Equid herpes vims 8 (EHV-8), and Equid herpes vims 9 (EHV-9).
  • VZV Varicella-zoster vims
  • the Herpesviridae family also includes vimses from the Gammaherpesvirinae subfamily, such as Human herpesvirus-4 (HHV-4) otherwise known as Epstein-Barr vims, Equid herpes vims 2 (EHV-2), Equid herpes vims 5 (EHV-5), and Equid herpes vims 7(EHV-7).
  • HHV-4 Human herpesvirus-4
  • EHV-2 Equid herpes vims 2
  • EHV-5 Equid herpes vims 5
  • Equid herpes vims 7(EHV-7) Equid herpes vims 7
  • Picornaviridae Viruses [0070] The Picomavirus family consists of a large number of small RNA viruses, many of which are significant pathogens of humans and livestock. Picornaviridae, one of the largest viral families, is composed of 14 genera, six of which include human pathogens. The best known picornavimses are enteroviruses (including polio, PV, and rhinoviruses), foot-and- mouth disease vims (FMDV), and hepatitis A virus (HAV). Although infections often are mild, certain strains may cause pandemic outbreaks accompanied with meningitis and/or paralysis. (Norder, H.
  • the family encompasses a variety of human and animal diseases, such as poliomyelitisk, the common cold, hepatitis A, foot and mouth disease and the like.
  • Hepadnaviridae family whose member genuses include Orthohepadnavirus and Avihepadnavirus, are spherical, occasionally pleomorphic, 42-50 nm in diameter, with no evident surface projections after negative staining. Hepadnavirus infection induces overproduction of surface proteins that are secreted into the blood as pleomorphic lipoprotein particles together with vims. Hepatitis B vims (HBV) is part of the Orthohepadnavirus genus.
  • Retroviridae is a large diverse group of enveloped RNA viruses which include the following genera: Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, and Spumavirus.
  • Retroviridae viruses include members of the Lentivims genus, which are complex retroviruses that include human pathogens such as Human Immunodeficiency Vims (HIV) and Equine Infectious Anemia (EIAV), and feline immunodeficiency vims.
  • Other Retrovirdae viruses include Human T- Lymphotrophic vimses (HTLV), and Hepadnaviridae which encompasses Hepatitis B (HVB).
  • Retroviridae are characterized by the ability to transcribe their RNA genome into linear double- stranded DNA during their replication cycle with a reverse transcriptase enzyme.
  • viral dsDNA is usually integrated into the host genome as a DNA provims which can remain silent (i.e., latent) or become transcriptionally active to produce virions (Fermin, Gustavo, and Paula Tennant. Vimses: Molecular Biology, Host Interactions and Applications to Biotechnology, edited by Jerome E. Foster, Elsevier Science & Technology (2016)).
  • All replication-competent retroviruses contain the following three genes: gag (group antigen, encoding the core and matrix proteins, p24 and pl7), pol (polymerase, encoding the enzymatic proteins, reverse transcriptase, RNAase, protease and integrase), and env (encoding the envelope and transmembrane glycoproteins, gp 120 and gp 41) (Welles, L. and Yarchoan, R., In Antimicrobial Therapy and Vaccines. Yu, VL, Merigan, Jr, TC and Barriere, SL Eds, Williams & Wilkins, Baltimore, (2005), pgs. 1264-1287). They share the presence of antigens, the ability to replicate, and a viral envelope.
  • the cancer is a cancer associated with an oncogenic virus, for example Epstein Barr virus (EBV), hepatitis B and C (HBV and HCV), human papilloma virus (HPV), Human Hepesvirus 8/ Kaposi sarcoma virus (KSV), Human T-cell Lymphotropic Virus 1 (HTLV) and polyoma viruses.
  • EBV Epstein Barr virus
  • HBV and HCV hepatitis B and C
  • HPV human papilloma virus
  • HPV Human Hepesvirus 8/ Kaposi sarcoma virus
  • HTLV Human T-cell Lymphotropic Virus 1
  • polyoma viruses for example Epstein Barr virus (EBV), hepatitis B and C (HBV and HCV), human papilloma virus (HPV), Human Hepesvirus 8/ Kaposi sarcoma virus (KSV), Human T-cell Lymphotropic Virus 1 (HTLV) and polyoma viruses.
  • compositions comprising a microbe genetically modified to express one or more protein domains encoded by one or more SPINK genes.
  • the one or more SPINK genes are selected from the group consisting of SPINK1, SPINK2, SPINK4, SPINK5, SPINK6,
  • the SPINK gene is SPINK5.
  • the SPINK gene can be obtained from any mammal, such as mouse, rat, rabbit, goat, sheep, horse, cow, dog, primate, or human gene sequences. According to some embodiments, the SPINK gene sequence is a human gene sequence.
  • the present disclosure also provides recombinant vectors containing a nucleotide sequence encoding one or more SPINK genes or portions thereof.
  • Recombinant vectors include but are not limited to vectors useful for the expression of the open reading frames (ORFs) in E. coli, other bacteria, yeast, viral, baculovims, plants or plant cells, as well as mammalian cells.
  • ORFs open reading frames
  • the disclosure provides a microbe that is genetically modified to express one or more protein domains encoded by one or more SPINK genes to the subject in need thereof.
  • the recombinant microbe is engineered to comprise a SPINK gene, or a fragment of the SPINK gene.
  • the viral vector is derived from a virus that is an oncolytic vims.
  • Oncolytic viruses OVs
  • OVs can replicate in cancer cells but not in normal cells, leading to lysis of the tumor mass. Beside this primary effect, OVs can also stimulate the immune system.
  • Suitable expression vectors for expression in a suitable host are known to one skilled in the art and appropriate expression vectors can be obtained from commercial sources or from the American Type Culture Collection (ATCC).
  • Useful embodiments include, for example, promoter sequences operably linked to the ORF, regulatory sequences, and transcription termination signals.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other or is not hindered by the other.
  • a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.
  • two proteins can be operably linked, such that the function of either protein is not compromised.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • the nucleic acids have been appropriately modified, for example, by site directed mutagenesis, to remove sequences responsible for N- glycosylation not needed for biological activity.
  • N-glycosylation sites in eukaryotic peptides are characterized by the amino acid sequence Asn-X-Ser/Thr where X is any amino acid except Pro. Modification of glycosylation sites can improve expression in for example bacterial, yeast or mammalian cell cultures.
  • the nucleic acids have been modified to improve the production and solubility of recombinant protein in a suitable host which includes, but is not limited to removing cysteine residues unnecessary for intramolecular disulfide bond formation. Cysteine residues may be changed by mutagenesis to another amino acid, for example serine, or removed from the sequence without affecting the biological activity or tertiary structure of the recombinant polypeptide.
  • nucleic acids may be necessary to improve the stability and accumulation of the recombinant production of protein include but are not limited to mutations altering protease cleavage sites recognized by a suitable expression host. Such modifications can be made that will not adversely affect the biological activity or tertiary structure of the recombinant protein.
  • nucleic acids that result in alterations in enzyme activity, substrate specificity, and/or biological activity. Such modifications may be preconceived based on specific knowledge relating to the protein or may be introduced by a random mutagenesis approach, for example error prone polymerase chain reaction (PCR). Additionally, it is also envisioned that one skilled in the art could generate chimeric nucleotide sequence comprising specific domains that can functionally replace stretches of nucleotide sequences that may add new function or improve the specificity or activity of the produced recombinant protein. According to some embodiments, modification resulting in changed biological activity of LEKTI may be necessary to improve the therapeutic effectiveness of the protein or to minimize potential side effects. Modification of the nucleic acid sequences can also be made that alter potential immunogenic sites that may result in allergic reactions to patients' administered with recombinant LEKTI protein.
  • Silent modifications can be made to the nucleic acids that do not alter, substitute or delete the respective amino acid in the recombinant protein. Such modification may be necessary to optimize, for example, the codon usage for a specific recombinant host.
  • the nucleotide sequence of SPINK genes or portions thereof can be modified to replace codons that are considered rare or have a low frequency of appropriate t-RNA molecules to a more suitable codon appropriate for the expression host. Such codon tables are known to exist and are readily available to one skilled in the art.
  • silent modification can be made to the nucleic acid that minimizes secondary structure loops at the level of mRNA that may be deleterious to recombinant protein expression.
  • the one or more SPINK genes encodes a LEKTI protein, and protein domains thereof, selected from LEKTI, LEKTI-2 and LEKTI-3.
  • the present disclosure provides compositions comprising a therapeutically effective amount of a LEKTI polypeptide or a portion thereof.
  • a portion of a LEKTI polypeptide comprises one or more LEKTI protein domains.
  • the present disclosure provides compositions comprising one or more LEKTI protein domains.
  • Some non-limiting examples include one or more of domains Dl, D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14, and D15, and combinations thereof.
  • the LEKTI protein requires proteolytic cleavage for activation of its inhibitory function against many proteases.
  • the full length protein is cleaved into domains D1-D5 and D6-D15.
  • the D6-D15 domains are then further cleaved in multiple steps into D6-D9 and D10-D15, D8.
  • a schematic of the full-length LEKTI polypeptides, the domains and the naturally cleaved products is shown in FIG. 3 of International Patent Application No. PCT/US2018/037850.
  • International Patent Application No. PCT/US2018/037850 incorporated by reference in its entirety herein, discloses various LEKTI recombinant proteins expressed by an engineered microbe.
  • the amino acid sequence of full length LEKTI protein is set forth as SEQ ID NO: 103, as well as each of the 15 individual domains are shown below:
  • LEKTI Domains (and residues corresponding to the numbering of SEQ ID NO: 103) are set forth below:
  • LEKTI Domain 1 (residues 23-77; SEQ ID NO: 104)
  • LEKTI Domain 2 (residues 91-153; SEQ ID NO: 105)
  • LEKTI Domain 3 (residues 155-216; SEQ ID NO: 106)
  • LEKTI Domain 4 (residues 219-285; SEQ ID NO: 107)
  • LEKTI Domain 5 (residues 291-352; SEQ ID NO: 108)
  • LEKTI Domain 6 (residues 356-423; SEQ ID NO: 109)
  • LEKTI Domain 7 (residues_431-489; SEQ ID NO: 110) AS FEELC S E YRKS RKN GRLFCTRENDPIQGPDGKMHGNTC S MCE AFF QQEERARAK AKR
  • LEKTI Domain 8 (residues 490-550; SEQ ID NO: 111)
  • LEKTI Domain 10 (residues 626-688; SEQ ID NO: 113)
  • LEKTI Domain 11 (residues 701-757; SEQ ID NO: 114)
  • LEKTI Domain 12 (residues 768-830; SEQ ID NO: 115)
  • LEKTI Domain 13 (residues 843-905; SEQ ID NO: 116)
  • LEKTI Domain 14 (residues 910-970; SEQ ID NO: 117)
  • LEKTI Domain 15 (residues 987-1048; SEQ ID NO: 118) SLDSEMCKDYRVLPRIGYLCPKDLKPVCGDDGQTYNNPCMLCHENLIRQTNTHIRST
  • LEKTI nucleic acid sequence is set forth below as SEQ ID NO: 119.
  • the present disclosure relates the full length LEKTI molecule, one or more of domains Dl, D2, D3, D4, D5, D6, D7, D8, D9, DIO, Dll, D12, D13, D14, and D15, as well as isolated fragments, oligonucleotides, and truncations maintaining biological activity, for example N-terminal deletions, C-terminal deletions, or deletions at both N and C-termini derived from SEQ ID NO: 119 and deduced amino acid sequence SEQ ID NO: 103.
  • the present disclosure also relates to allelic variants of LEKTI, or portions thereof (one or more of domains Dl, D2, D3, D4, D5, D6, D7, D8, D9, DIO, Dll, D12, D13, D14, and D15), as well as synthetic or mutated genes of SPINK (e.g ., SPINK5 ) that have been modified to change, for example, the expression or activity of the recombinant protein.
  • SPINK5 synthetic or mutated genes of SPINK
  • degeneracy of the nucleic acid code can be considered variations in the nucleotide sequences that encode the same amino acid residues. Accordingly, the disclosure includes nucleic acid residues that are able to hybridize under moderately stringent conditions.
  • SPINK e.g., SPINK5
  • SPINK5 SPINK5 nucleic acids that encode polypeptides having at least about 70% to 80% identity, 80% to 85%, preferably 90% to 95% identity (90%, 91%, 92%, 93%, 94%, 95%), more preferably 98% to 99% identity to LEKTI set forth in SEQ ID NO: 103 or portions thereof (one or more of domains Dl, D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14, and D15).
  • the present disclosure also provides for recombinant cloning and expression vectors useful for the production of biologically active LEKTI.
  • Such expression plasmids may be used to prepare recombinant LEKTI polypeptides or portions thereof (one or more of domains Dl, D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14, and D15) encoded by the nucleic acids in a suitable host organism.
  • Suitable host organisms for the production of LEKTI or portions thereof include, but are not limited to bacteria, yeast, insect cells, mammalian cells, plants and plant cells.
  • cell free systems may also be employed for the production of recombinant proteins.
  • One skilled in the art can readily prepare plasmids suitable for the expression of recombinant LEKTI in the suitable host organism. Appropriate cloning and expression vectors are readily available to one skilled in the art and can be obtained from commercial sources or from the ATCC.
  • the recombinant protein can be produced in the within the host cell or secreted into the culture medium depending on the nature of the vector system used for the production of the recombinant protein.
  • plasmids useful for the expression of the recombinant LEKTI or portions thereof comprise necessary operable linked regulatory elements such as a promoter sequence (including operators, enhancers, silencers, ribosomal binding sites), transcriptional enhancing sequences, translational fusions to signal peptides (native or heterologous) or peptide sequences useful for the purification of recombinant protein (for example His Tag, FLAG® (a convenient binding moiety), MBP, GST), transcription termination signals and polyadenylation signals (if necessary).
  • the recombinant plasmid may also be necessary for the recombinant plasmid to replicate in the host cell. This requires the use of an origin of replication suitable for the host organism.
  • the recombinant expression plasmid may be stably integrated into the host's chromosome. This may require homologous recombination or random integration into the host chromosomes. Both instances require the use of an appropriate selection mechanism to distinguish transformed host cells from non-transformed host cells.
  • Useful selection schemes include the use of, for example, antibiotics (for example, G418, Zeocin® (a glycopeptide antibiotic of the bleomycin family), kanamycin, tetracycline, gentamicin, spectinomycin, ampicillin), complementation of an auxotroph (for example Trp-, DHFR-), and scorable markers (for example b-glucoronidase, b-galactosidase, GFP).
  • antibiotics for example, G418, Zeocin® (a glycopeptide antibiotic of the bleomycin family), kanamycin, tetracycline, gentamicin, spectinomycin, ampicillin
  • an auxotroph for example Trp-, DHFR-
  • scorable markers for example b-glucoronidase, b-galactosidase, GFP.
  • Expression systems useful in the present invention include yeast systems. Plasmid vectors particularly useful for the transformation and expression of protein in recombinant K. lactis have been descried (Chen, X-J., Gene (1996) 172:131-136). Other yeast expression systems based on Saccharomyces cerevisiae or Pichia pastoris or Pichia methanolica may also be useful for the recombinant production of LEKTI or portions thereof (one or more of domains Dl, D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14, and D15).
  • Expression plasmid suitable for the expression of LEKTI or portions thereof (one or more of domains Dl, D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14, and D15) in S. cerevisiae , P. pastoris, or P. methanolica may be obtained from a commercial source or ATCC. Plasmids described above may also be modified by one skilled in the art to optimize, for example, promoter sequences and or secretion signals optimal for the host organism and recombinant production of LEKTI. Established methods are also available to one skilled in the art for introducing recombinant plasmid into the yeast strains.
  • the cells may be transfected for transient expression or stable expression of the protein of interest.
  • Other factors for the production of foreign protein in mammalian cells including regulatory considerations have been reviewed (Bendig, M., Genetic Engineering (1988) 7:91-127).
  • One useful mammalian expression system is based on the EF-la promoter (Mizushima, S and Nagata Nucleic Acids Res (1990) 18:5322) and Human embryonic kidney (EK) 293T cell line (Chen, P., el al., Protein Expression and Purification (2002) 24:481-488).
  • Variants of the commercially available CHO and 293T cells lines and their suitable growth and expression media may be used to further improve protein production yields.
  • Variants of commercially available expression vectors including different promoters, secretion signals, transcription enhancers, etc., may also be used to improve protein production yields.
  • Another useful expression system includes expression in E. coli.
  • E. coli There are several expression systems known to one skilled in the art for production of recombinant proteins in E. coli. Expression of mammalian protein in E. coli has not been particularly useful due to the fact that many mammalian proteins are post translationahy modified by glycosylation or may contain intra or inter di-sulfide molecular bonds.
  • Particular E. coli expression plasmid useful in the present invention may include, for example, fusions with signal peptides to target the protein to the periplasmic space. Additionally, E.
  • coli host strains that contain mutations in both the thioredoxin reductase ( trxB ) and glutathione reductase (gor) genes greatly enhance disulfide bond formation in the cytoplasm (Prinz, W. A., et al, J. Biol. Chem. (1997) 272:15661-15667).
  • the addition of thioredoxin fused to the N-terminus or C-terminus of LEKTI may also aid in the production of soluble protein in E. coli cells. (LaVahie, E. R., et al, Bio/Technology (1993) 11:187-193).
  • LEKTI or portions thereof may be purified from the recombinant expression system using techniques known to one normally skilled in the art.
  • Expression of the LEKTI protein or portions thereof can either be intracellular or secreted in the media fraction. Secretion of LEKTI into the media simplifies protein purification.
  • Expression of intracellular LEKTI or portions thereof (one or more of domains Dl, D2, D3, D4, D5, D6,
  • D7, D8, D9, DIO, Dll, D12, D13, D14, and D15 requires disruption of the cell pellets by any convenient method including freeze-thaw, mechanical disruption, sonication, or use of detergents or cell lysing enzymes or agents. Following disruption or concentration of secreted protein, purification can be accomplished by a number of methods know to one skilled in the art.
  • affinity chromatography may be used to purify recombinant LEKTI or portions thereof (one or more of domains Dl, D2, D3, D4, D5, D6, D7, D8, D9, DIO, Dll, D12, D13, D14, and D15) fused with affinity tags such as: 6xHIS, FLAG® (a convenient binding moiety), GST, or MBP.
  • affinity tags such as: 6xHIS, FLAG® (a convenient binding moiety), GST, or MBP.
  • antibodies specific to LEKTI or portions thereof one or more of domains Dl, D2, D3, D4, D5, D6, D7, D8, D9, DIO, Dll, D12, D13, D14, and D15
  • affinity purification may be used for affinity purification.
  • matrices chemically modified with a ligand having strong affinity to LEKTI or portions thereof may also be used for affinity purification.
  • LEKTI may also be purified with the use of an affinity tag or antibodies following conventional protein purification methods know to one skilled in the art.
  • LEKTI may be purified using high performance liquid chromatography (HPLC), for example, reverse phase HPLC or normal phase HPLC.
  • LEKTI may be purified using capture chromatography (e.g., ion exchange) purification.
  • non-viral gene delivery can also be used. Examples include diffusion of DNA in the absence of any carriers or stabilizers (“naked DNA”), DNA in the presence of pharmacologic stabilizers or carriers (“formulated DNA”), DNA complexed to proteins that facilitate entry into the cell (“Molecular conjugates”), or DNA complexed to lipids.
  • naked DNA DNA in the absence of any carriers or stabilizers
  • formulated DNA DNA in the presence of pharmacologic stabilizers or carriers
  • Molecular conjugates DNA complexed to proteins that facilitate entry into the cell
  • DNA complexed to lipids lipids.
  • the disclosure provides microbial compositions comprising one or more of a wide range of bacteria suitable for administration to a subject.
  • the microbial compositions are administered by inhalation.
  • the microbial compositions are administered by nasal application, including nasopharyngeal administration.
  • the nasal application includes application to the back of the throat.
  • the microbial compositions are administered by a parenteral route of administration.
  • the microbial compositions are administered by a parenteral route of administration. Examples include, but are not limited to, non-pathogenic and commensal bacteria.
  • Bacteria suitable for use in the present invention include, but are not limited to, Bifidobacterium spp., Brevibacterium spp., Propionibacterium spp., Lactococcus spp., Streptococcus spp., Salmonella spp., Clostridium spp.,
  • Staphylococcus spp. e.g., S. epidermidis and/or S. hominis
  • Lactobacillus spp. e.g., L. acidophilus
  • Pediococcus spp. e.g., Leuconostoc spp.
  • microbial compositions comprise one or more of Staphylococcus warneri, Streptococcus pyogenes, Streptococcus mitis, Propionibacterium acnes,
  • Corynebacterium spp. Acinetobacter johnsonii, Moraxella spp Alloiococcus spp., Haemophilus spp., and Pseudomonas aeruginosa and mixtures thereof. According to some embodiments, other related or similar species found on the skin are used.
  • Certain embodiments involve the use of bacterium Staphylococcus epidermidis.
  • the strain of S. epidermidis to be used is incapable of producing biofilms.
  • An example of this is S. epidermidis strain ATCC 12228 or ARS Culture Collection (NRRL) B-4268.
  • the microbe is engineered to express a mammalian gene encoding LEKTI protein.
  • the recombinant microbe is adapted to live indefinitely or for a controlled duration on an epithelial or mucosal surface of a mammal (e.g., nasal cavity) to provide a continuous supply of LEKTI protein domains.
  • the continuous supply of LEKTI protein domain is provided by constitutively expressed LEKTI.
  • the continuous supply of LEKTI protein domain is provided by LEKTI that is inducibly expressed.
  • the recombinant microbe lives alongside commensal microorganisms naturally occurring on the epithelial or mucosal surface.
  • the recombinant microbe lives to the exclusion of commensal microorganisms that naturally occur on the epithelial or mucosal surface. According to some embodiments, the recombinant microbe is adapted to multiply on the epithelial or mucosal surface of the mammal. According to some embodiments, the recombinant microbe is no longer alive, but contains effective amounts of a therapeutic polypeptide, e.g. LEKTI or therapeutically effective domain(s) thereof. Such cells may be intact or not depending upon the particulars of delivering the therapeutic peptide (or domain(s) thereof) to the target site.
  • a therapeutic polypeptide e.g. LEKTI or therapeutically effective domain(s) thereof.
  • the microbe is selected from the group consisting of Bifidobacterium spp., Brevibacterium spp., Propionibacterium spp., Lactococcus spp., Streptococcus spp., Salmonella spp., Clostridium spp., Staphylococcus spp. (e.g., S. epidermidis and/or S. hominis), Lactobacillus spp. (e.g., L. acidophilus), Pediococcus spp., Leuconostoc spp., or Oenococcus spp..
  • microbial compositions comprise one or more of Staphylococcus warneri, Streptococcus pyogenes, Streptococcus mitis, Propionibacterium acnes, Corynebacterium spp., Acinetobacter johnsonii, Moraxella spp ., Alloiococcus spp., Haemophilus spp., and Pseudomonas aeruginosa and mixtures thereof.
  • the LEKTI protein (or domains thereof) is recombinantly produced and administered.
  • the LEKTI protein (or domains thereof) is administered in a composition not including a microbe.
  • the recombinant protein expressed by the engineered microbe comprises one or more protease inhibitory domains of the LEKTI protein.
  • Some non-limiting examples include one or more of domains Dl, D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14, and D15.
  • the recombinant protein expressed by the engineered microbe comprises a peptide sequence selected from any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO:
  • the recombinant protein expressed by the engineered microbe comprises a peptide sequence that is at least 85% identical to any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118.
  • the recombinant protein expressed by the engineered microbe comprises a peptide sequence that is at least 90% identical to any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118.
  • the recombinant protein expressed by the engineered microbe comprises a peptide sequence that is at least 95% identical to any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO:
  • SEQ ID NO: 107 SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:
  • the recombinant protein expressed by the engineered microbe comprises a peptide sequence that is at least 96% identical to any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118.
  • the recombinant protein expressed by the engineered microbe comprises a peptide sequence that is at least 97% identical to any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118.
  • the recombinant protein expressed by the engineered microbe comprises a peptide sequence that is at least 98% identical to any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118.
  • the recombinant protein expressed by the engineered microbe comprises a peptide sequence that is at least 99% identical to any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118.
  • the recombinant protein expressed by the engineered microbe consists of any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118.
  • the recombinant microbe comprises a sequence as disclosed herein that has at least about 75% identity, or 80% identity, or 85% identity, or 90% identity, or 95% identity to any one or more of the SEQ ID NOS listed herein.
  • identity and grammatical versions thereof means the extent to which two nucleotide or amino acid sequences have the same residues at the same positions in an alignment. Percent (%) identity is calculated by multiplying the number of matches in a sequence alignment by 100 and dividing by the length of the aligned region, including internal gaps.
  • the therapeutic LEKTI domain is operably linked to one or more secretion signals or export signals that tag the protein for transport through the secretory pathway.
  • the secretory peptide may be positioned on the N-terminal end of a recombinant protein, and may co-translationally or post-translationally target the tagged protein for secretion.
  • at least one LEKTI domain is operably linked to a SecA domain (SEQ ID NO: 3). Any secretion signal that facilitates exit of the LEKTI protein out of the bacterial cell may be used as a secretion peptide.
  • secretion peptides signals are set forth in Table 1, below:
  • the therapeutic LEKTI domain is operably linked to one or more signal sequences derived from endogenous proteins of Staphylococcus epidermidis.
  • signal sequences derived from endogenous proteins of Staphylococcus epidermidis are set forth in Table 2 below:
  • the therapeutic LEKTI domain is operably linked to one or more secretion signal sequences derived from endogenous proteins of other bacteria.
  • secretion signal sequences derived from endogenous proteins of other bacteria are known in the art.
  • one or more cell penetrating peptides are used to mediate delivery of therapeutic proteins in vivo without using cell surface receptors and without causing significant membrane damage.
  • the recombinant LEKTI domain is operably linked to a cell penetration peptide sequence that enhances the ability of the LEKTI domain to pass through a cell membrane.
  • the term “enhance” as used to describe the cell penetration peptide/LEKTI means that the cell penetration sequence improves the passage of recombinant LEKTI domain through a cell membrane relative to a recombinant LEKTI domain lacking the cell penetration sequence.
  • one or more cell penetrating peptides are operably linked to therapeutic proteins to facilitate entry into skin cells (e.g . keratinocytes). Non limiting examples are set forth in Table 3, below:
  • cell penetrating peptides comprise periodic amino acid sequences.
  • periodic cell penetrating sequences include: Polyarginines, R x n (wherein 4 ⁇ n ⁇ 17); Polylysines, K x n (wherein 4 ⁇ n ⁇ 17); arginine repeats interspaced with 6-aminocaprotic acid residues (RAca), wherein there are 2 to 6 arginine repeats; arginine repeats interspaced with 4-aminobutyric acid (RAbu), wherein there are 2 to 6 arginine repeats; arginine repeats interspaced with methionine, wherein there are 2 to 6 arginine repeats; arginine repeats interspaced with threonine, wherein there are 2 to 6 arginine repeats; arginine repeats interspaced with serine, wherein there are 2 to 6 arginine repeats; and arginine repeats interspaced with alanine, where
  • the LEKTI domain is operably linked to an RMR domain (SEQ ID NO: 4).
  • expression of the LEKTI domain is controlled by an operon and the amount of LEKTI provided to the mammal (e.g., to an epithelial surface) is proportional to the availability of an extrinsic factor.
  • the recombinant LEKTI gene may be under the control of a xylose inducible promoter (e.g.
  • xylose repressor xylR
  • xylose operator xylO
  • xylose isomerase gene xylA
  • CRE cis-acting catabolite-responsive element
  • the amount of recombinant LEKTI protein is controlled by the amount of exogenous xylose available to the recombinant microbe.
  • the expression of the LEKTI domain is controlled by a promoter that is constitutively active.
  • the expression of the LEKTI domain is controlled by a Cm R promoter according to SEQ ID NO: 8.
  • the microbe is genetically modified by transfection/transformation with a recombinant DNA plasmid encoding one or more of the LEKTI protein domains and one or more antibiotic resistance genes.
  • a recombinant DNA plasmid encoding one or more of the LEKTI protein domains and one or more antibiotic resistance genes.
  • some embodiments of the recombinant DNA plasmid comprise a kanamycin resistance gene and/or a trimethoprim resistance gene; e.g. dfrA (SEQ ID NO: 5).
  • treatment of the skin of the mammal with an antibiotic may be used to bias the population of commensal microbes toward a larger proportion of LEKTI producing microbes.
  • recombinant DNA plasmid include, without limitation, a replication protein gene, such as a member of the Rep superfamily of replication proteins.
  • a replication protein gene such as a member of the Rep superfamily of replication proteins.
  • the recombinant DNA plasmid comprises a rep gene (SEQ ID NO: 6).
  • the recombinant DNA plasmid comprises one or more sequences of the pUBTR vector.
  • the recombinant LEKTI is operably linked to an inducible promoter, ribosome binding site, export signal, and/or cell penetrating peptide in the pUBTR vector.
  • the recombinant LEKTI is operably linked to an inducible promoter, ribosome binding site, export signal, and/or cell penetrating peptide in the pUBTR vector.
  • the pUBTR vector is pUBTRl 19 as shown in FIG. 1.
  • the recombinant DNA plasmid comprises one or more sequences of the pJB38 vector.
  • the recombinant DNA plasmid comprises the pKK30-LEKTI-complete sequence according to SEQ ID NO: 7.
  • the present disclosure provides a composition for the treatment of a viral infection comprising a microbe comprising the pKK30-LEKTI-complete plasmid construct.
  • the present disclosure provides a composition for the prevention of a viral infection comprising a microbe comprising the pKK30-LEKTI-complete plasmid construct.
  • the microbe is selected from the group consisting of Bifidobacterium spp., Brevibacterium spp., Propionibacterium spp., Lactococcus spp., Streptococcus spp., Salmonella spp., Clostridium spp., Staphylococcus spp. (e.g., S. epidermidis and/or S. hominis), Lactobacillus spp. (e.g., L. acidophilus ), Pediococcus spp., Leuconostoc spp., or Oenococcus spp..
  • microbial compositions comprise one or more of Staphylococcus warneri, Streptococcus pyogenes, Streptococcus mitis, Propionibacterium acnes, Corynebacterium spp. , Acinetobacter johnsonii, Moraxella spp Alloiococcus spp., Haemophilus spp., and Pseudomonas aeruginosa and mixtures thereof.
  • the amount or durations of availability of therapeutic LEKTI protein is controlled by the stability of the vector harboring the LEKTI in a microbe.
  • the persistence of a recombinant vector may be controlled by one or more elements of a plasmid including those that provide host-beneficial genes, plasmid stability mechanisms, and plasmid co-adaptation.
  • some plasmid may provide for stable replication, active partitioning mechanisms, and mechanisms that insure reliable inheritance of plasmids to daughter cells over generations. (See, e.g., J.C. Baxter, B.E. Funnell, Plasmid partition mechanisms, Microbiol.
  • the present invention includes the use of all conventional selection and stability methods known to a person of skill in the art.
  • aspects of the present disclosure include one or more of the LEKTI protein domains described herein, in combination with a pharmaceutically acceptable carrier.
  • the compounds are preferably combined with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice as described, for example, in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980), the disclosures of which are hereby incorporated herein by reference, in their entirety.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • Pharmaceutical compositions as described herein may be administered to a mammalian host in a variety of forms adapted to the chosen route of administration, e.g., orally or parenterally.
  • Parenteral administration includes administration by the following routes: intravenous; intramuscular; subcutaneous; intraocular; intrasynovial; transepithelial including transdermal, ophthalmic, sublingual and buccal; topically, including ophthalmic, dermal, ocular, and rectal; and nasal inhalation via insufflations and aerosols, including nasopharyngeal and throat installation
  • the compounds may be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents.
  • they may be administered as the sole active agents in a pharmaceutical composition, or they can be used in combination with other therapeutically active ingredients.
  • Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum mono stearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the active compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the compositions are formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • compositions comprising a microbe comprising one or more LEKTI protein domains to for use according to the present invention can comprise any pharmaceutically effective amount of the recombinant bacteria to produce a therapeutically effective amount of the desired polypeptide or therapeutically effective domain(s) thereof, for example, at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about.
  • the composition for use according to the present invention can comprise, for example, at least about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 5%, about 0.1 % to about 30%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1 % to about 10%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 1% to about 5%, or more by weight of recombinant bacteria.
  • the composition is a topical formulation.
  • the topical formulation can be in any form suitable for application to the body surface, such as a cream, lotion, sprays, solution, gel, ointment, paste, plaster, paint, bioadhesive, suspensions, emulsions, or the like, and/or can be prepared so as to contain liposomes, micelles, and/or microspheres, polymeric and solid lipid nanoparticles and microparticles.
  • a formulation can be used in combination with an occlusive overlayer so that moisture evaporating from the body surface is maintained within the formulation upon application to the body surface and thereafter.
  • the formulation can include a living cell culture composition and can comprise at least one engineered bacterial strain that produces a therapeutically effective recombinant polypeptide or therapeutically effective domain(s) thereof.
  • Topical formulations include those in which any other active ingredient(s) is (are) dissolved or dispersed in a dermatological vehicle known in the art (e.g . aqueous or nonaqueous gels, ointments, water-in-oil or oil-in-water emulsions).
  • a dermatological vehicle known in the art
  • Constituents of such vehicles can comprise water, aqueous buffer solutions, non-aqueous solvents (such as ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycofurol or glycerol), oils (e.g.
  • the dermatological vehicle employed can contain one or more components (for example, when the formulation is an aqueous gel, components in addition to water) selected from the following list: a solubilizing agent or solvent (e.g. a b-cyclodextrin, such as bydroxypropyl b- cyclodextrin, or an alcohol or polyol such as ethanol, propylene glycol or glycerol); a thickening agent (e.g.
  • a solubilizing agent or solvent e.g. a b-cyclodextrin, such as bydroxypropyl b- cyclodextrin, or an alcohol or polyol such as ethanol, propylene glycol or glycerol
  • a thickening agent e.g.
  • hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose or carbomer e.g. a gelling agent (e.g. a polyoxyethylene- polyoxypropylene copolymer); a preservative (e.g. benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorbutol, a benzoate, potassium sorbate or EDTA or salt thereof); and pH buffering agent(s) (such as a mixture of dihydrogen phosphate and hydrogen phosphate salts, or a mixture of citric acid and a hydrogen phosphate salt), a plant based or bacterial extract containing hydrating or protein component (oligosaccharides, oatmeal or other common ingredients).
  • a gelling agent e.g. a polyoxyethylene- polyoxypropylene copolymer
  • a preservative e.g. benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorbutol, a benzoate, potassium
  • a pharmaceutically acceptable carrier can also be incorporated in the compositions of the present invention and can be any carrier conventionally used in the art. Examples thereof include water, lower alcohols, higher alcohols, polyhydric alcohols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water- based mixtures and emulsion-based mixtures of such carriers.
  • pharmaceutically acceptable or “pharmaceutically acceptable carrier” is used herein to refer to a compound or composition that can be incorporated into a pharmaceutical formulation without causing undesirable biological effects or unwanted, interaction with other components of the formulation
  • Carriers or “vehicles” as used herein refer to carrier materials suitable for incorporation in a topically applied composition. Carriers and vehicles useful herein include any such materials known in the art, which are non-toxic and do not interact with other components of the formulation in which it is contained in a deleterious manner.
  • aqueous refers to a formulation that contains water or that becomes water-containing following application to the skin or mucosal tissue.
  • the pharmaceutical compositions described herein are administered in the form of a mucosal spray, such as a nasal spray.
  • a mucosal spray such as a nasal spray.
  • the pharmaceutical compositions described herein are co-administered as a mucosal spray, in combination with an oral form of a pharmaceutical composition of the present invention for the prevention, inhibition, reduction of the severity of, and/or treatment of a viral infection.
  • the nasal spray further comprises a beta-glucan, an immune system modulator compound that may prime the innate immune system to protect the body.
  • compositions of the invention comprise one or more active ingredients, e.g. therapeutic agents, in admixture with one or more pharmaceutically- acceptable diluents or carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.).
  • diluents or carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and poly
  • Each pharmaceutically acceptable diluent or carrier used in a pharmaceutical composition of the invention must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • Diluents or carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
  • compositions of the present invention suitable for parenteral administrations may comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.
  • suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.
  • Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants
  • compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
  • the present disclosure provides methods of preventing, inhibiting or treating a viral infection in a subject.
  • Various viruses causing viral infection have been described herein.
  • the disclosure provides a method of treating a viral infection in a subject, comprising administering one or more LEKTI protein domains to the subject to provide a therapeutic effect.
  • the disclosure provides a method of treating or preventing a viral infection in a subject, comprising administering one or more LEKTI protein domains to the subject to provide a therapeutic effect.
  • the disclosure provides a method of treating a viral infection in a subject, comprising administering to the subject one or more microbes comprising one or more LEKTI protein domains to provide a therapeutic effect.
  • the viral infection is a respiratory infection.
  • the viral infection is a dermal viral infection.
  • the subject is infected with a virus that causes cancer.
  • the disclosure provides a method of preventing a viral infection in a subject, comprising administering to the subject one or more microbes comprising one or more LEKTI protein domains to provide a therapeutic effect.
  • the viral infection is a respiratory infection.
  • the viral infection is a dermal viral infection.
  • the subject is infected with a virus that causes cancer.
  • the subject has not been exposed to the virus. According to some embodiments, the subject has not been infected with the virus.
  • the methods of treating or preventing a viral infection described herein result in a decrease in viral load in the subject.
  • the viral load is in the lung.
  • the methods of treating or preventing a viral infection described herein result in a decrease in the severity and/or duration of symptoms of the viral infection. According to some embodiments, the methods of treating or preventing a viral infection described herein mitigate the spread of the viral infection.
  • the methods of treating or preventing a viral infection described herein result in an increase in the induction of an immune response.
  • the methods of treating or preventing a viral infection described herein work in synergy with the natural anti-viral response. That is, administering to the subject one or more microbes comprising one or more LEKTI protein domains when combined with the natural anti- viral response provides a synergistic effect on the immune response (see, e.g., Chen el al. Scientific Reports volume 6: 27870 (2016); Kim et al, Microbiome 7, 80 (2019)).
  • the immune response is an innate immune response.
  • the innate arm of the immune system is a nonspecific fast response to pathogens that is predominantly responsible for an initial inflammatory response via a number of soluble factors, including the complement system and the chemokine/cytokine system; and a number of specialized cell types, including mast cells, macrophages, dendritic cells (DCs), and natural killer cells (NKs).
  • Type I interferons IFNs
  • administering one or more LEKTI protein domains to the subject results in an increase in type I interferon.
  • the disclosure provides a method of treating or preventing a viral infection in a subject, comprising administering an effective amount of a viral fusion inhibitor; and/or an effective amount of a viral replication inhibitor, wherein the viral fusion inhibitor and/or the viral replication inhibitor is selected from one or more LEKTI protein domains.
  • both the viral fusion inhibitor and the viral replication inhibitor are administered to the subject.
  • the viral fusion inhibitor and the viral replication inhibitor are not the same.
  • the viral infection is a respiratory infection.
  • the viral infection is a dermal viral infection.
  • the subject is infected with a virus that causes cancer.
  • the disclosure provides a method of inhibiting viral fusion in a subject, comprising administering one or more LEKTI protein domains to the subject.
  • the disclosure provides a method of inhibiting viral replication in a subject, comprising administering one or more LEKTI protein domains to the subject.
  • the disclosure provides a method of decreasing viral load in a subject, comprising administering one or more LEKTI protein domains to the subject.
  • the subject has a viral infection.
  • the viral infection is a respiratory infection.
  • the subject shows a decrease in viral load after administering the one or more LEKTI protein domains.
  • the subject shows a decrease in the severity and/or duration of symptoms of the viral infection compared to a subject not administered the one or more LEKTI protein domains.
  • decrease in viral load can be determined using, e.g., Reverse transcription polymerase chain reaction (RT-PCR) or quantitative polymerase chain reaction (qPCR) methodology.
  • RT-PCR Reverse transcription polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • “decrease” as it refers to viral load of a subject means at least about 1- fold (for example 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000-fold or more) less than the viral load of a control subject. “Decrease” as it refers to the viral load of a subject also means at least about 5% less than (for example 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%) the viral load of a control subject.
  • an immune response elicited after administering the one or more LEKTI protein domains, either alone or as part of a microbial composition may be measured by measuring anyone of viral load, T-cell proliferation, T-cell survival and cytokine secretion by T-cells.
  • the method for preventing, inhibiting or treating a viral infection comprises multiple treatments (e.g ., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 20, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more treatments).
  • the appropriate number of treatments, and the duration of each treatment can be determined by a health care provider based on, for example, the severity of the disorder, and the overall health of the subject.
  • the present disclosure provides a kit for the preventing, inhibiting or treating a viral infection in a mammal in need thereof comprising: (1) a composition comprising a microbe that is genetically modified to express one or more LEKTI protein domains; and (2) instructions for use.
  • the present disclosure provides a kit for the preventing, inhibiting or treating a viral infection in a mammal in need thereof comprising: (1) a composition comprising one or more recombinant LEKTI protein domains; and (2) instructions for use.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the kits will generally be packaged to include at least one vial, test tube, flask, bottle, syringe or other container means, into which the described reagents may be placed, and preferably, suitably aliquoted. Where additional components are provided, the kit will also generally contain a second, third or other additional container into which such component may be placed.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • a sterile access port for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.
  • the original pUBTRl 19 plasmid has a kanamycin-resistance gene and triple promoter expression cassette including: 1) hpall native promotor essential for plasmid replication, 2) phosphate-starvation inducible yxiE promoter, and 3) constitutive sarA promoter with the open reading frame (ORF) of SsaAl secretion signal fused to the expressed protein downstream.
  • FIG. 1 is a schematic of the pUBTR119 plasmid.
  • transformed cells were fractionated and analyzed via SDS-PAGE electrophoresis and western blotting.
  • Bacterial cells expressing recombinant FEKTI and bacterial control cells were pelleted and lysed with CelFytic B Cell Fysis Reagent (Sigma- Aldrich, St. Fouis, MO). The supernatant from the induced sample was collected and concentrated. Samples were resuspended in a reduced sample buffer and then electrophoresed on a 4-15% Tris-acrylimide gel with Tris-HCF running buffer. Following electrophoresis, the gel was transferred to a PVDF membrane, and sequentially probed with a primary goat monoclonal antibody against FEKTI domains of interest, e.g., D6 or a His tag.
  • a primary goat monoclonal antibody against FEKTI domains of interest e.g., D6 or a His tag.
  • a horseradish peroxidase-conjugated donkey anti-goat antibody (sc-2020) was then probed and the secondary antibodies detected through autoradiography (Syngene GeneGnome Bio Imaging System) using enhanced chemiluminescence substrate (SuperSignal West Pico, Thermo Scientific).
  • the protein sequence was verified using a combined HPFC enrichment consisting of isolation of active fractions and a proteomic analysis of the digested media.
  • the proteomics methodology is based on known methods (Universal sample preparation method for proteome analysis, Wisniewski et al., Nature Methods, 6, 359-362, 2009). Briefly, the active HPLC fractions were prepared for bottom-up peptides sequencing by reduction and alkylation. The FASP (filter-aided sample preparation)! strategy was applied to clean-up and digest the active fractions. The high concentration of Urea and co -fractionated small molecular weight species were reduced using 3 kDa MWCO filters in spin format.
  • Reconstituted peptide digests were injected onto a Thermo Q-exactive HF Biopharma instrument using an ultra-high pressure nano-LC for high sensitivity peptide detection and sequencing.
  • a Top 12 method was used to detect and sequence peptide ions in data dependent acquisition mode. Once acquired, the RAW files were searched using PEAKS software using the LEKTI sequence containing the signal-, linker-, and propeptides, as well as the secreted form of the protein. The peptide sequences were transferred to Scaffold software.
  • a dose of S. epidermidis containing recombinant LEKTI can be added to a pharmaceutically acceptable carrier.
  • the foregoing composition is useful for treating or preventing viral infection in a subject in need thereof.
  • the composition can be administered at least once per day, up to for example about 3 to 4 times per day, or as needed or prescribed. According to some embodiments, only a single application is required to achieve a therapeutic effect. According to some embodiments, more than one application is required to achieve a therapeutic effect.
  • the composition can be used for as long as needed to ensure treatment of the condition or to continue to prevent the condition.
  • the duration of treatment can vary from about 1 day up to about 10 to 14 days or longer. In certain instances, long term or chronic treatment can be administered.
  • the protease inhibition activity of recombinant LEKTI is tested for differences achieved when operably linked to various secretion peptides and cell penetration peptides. According to some embodiments, specific combinations of secretion peptides and cell penetration peptides may have unpredictable effects on the protease inhibition function of the LEKTI domains, and therefore may be determined empirically.
  • one or more LEKTI domains described herein are cloned into an insect expression vector for large scale production of purified recombinant protein and assessed for inhibitory activity on one or more proteases (e.g. plasmin, cathepsin G, elastase, and trypsin).
  • proteases e.g. plasmin, cathepsin G, elastase, and trypsin.
  • the following reagents may be obtained commercially as indicated: Fall Army worm cell line Spodoptera frugiperda (Sf9), low-melting point agarose, cellFECTIN, pFASTBACl, pCRITTOPO, Escherichia colicompetent DH10BAC, cabbage looper egg cell line Trichoplusia ni 5B1-4 (High Five), and ultimate serum-free insect medium from Invitrogen (Carlsbad, CA); restriction endonucleases from New England Biolabs (Beverly, MA); TALON Superflow from Clontech Laboratory (Palo Alto, CA); Insect-XPRESS medium and fetal bovine serum from Bio Whittaker (Walkers ville, MD); YM10 Centriplus from Millipore Corp. (Bedford, MA); precast SDS-PAGE gels, protein assay kit, SEC-250 size column, and prestained markers from Bio-Rad (Hercules, CA); BSA from Kabi
  • 6xHis tagged LEKTI domains (e.g. SEQ ID NO: 1) operably linked to various permutations of secretion peptides and cell penetration peptides may be cloned into the pFASTBACl vector according to the manufacturers’ instructions. Recombinant LEKTI composite viruses are then generated as previously described by Gao, M. et ah, (1996) J.
  • the recombinant LEKTI protein may be produced on a large scale by infecting spinner cultures of Sf9 cells (1.6 billion cells) in 10% serum containing Insect- XPRESS medium at a multiplicity of infection of 8 plaque forming units (PFU). Three days after infection, the cell pellet may be harvested and the recombinant LEKTI selectively purified from the cell lysate using a Co 2+ -charged Sepharose affinity column (TALON) followed by SEC-250 size column chromatography, as previously described in Jayakumar, A. el ah, (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 8695-8699. Fractions containing homogeneous LEKTI may be pooled and concentrated by ultrafiltration. Protein may be quantified using the Bio-Rad Protein Assay Kit II.
  • the following enzymes, chromogenic substrates, and reagents may be obtained commercially as indicated: human plasmin, human cathepsin L, human cathepsin S, human trypsin, human cathepsin G, human chymotrypsin, and human neutrophil elastase (HNE) from Athens Research & Technology, Inc.
  • human plasmin human cathepsin L
  • human cathepsin S human trypsin
  • human cathepsin G human chymotrypsin
  • HNE human neutrophil elastase
  • subtilisin A from Calbiochem- Novabiochem (San Diego, CA); papain from Roche Molecular Biochemicals (Indianapolis, IN); furin from New England BioLabs; succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Succ- AAPF-pNA), succinyl- Ala-Ala- V al-pNA (Succ-AAVpNA), andD-Val-Leu-Lys-pNA (VLK- pNA) from Sigma Chemical Co. (St.
  • H-Glu-Gly-Arg-pNA EGRpNA
  • benzyloxycarbonyl-Phe-Arg-pNA Z-FR-pNA
  • methoxy-Succ-Arg-Pro-Tyr-pNA MeO-Succ-RPY-pNA
  • PBS reaction buffer 137 mM NaCl, 27 mM KC1, and 10 mM phosphate buffer (pH 7.4) may be used with trypsin, plasmin, cathepsin G, HNE, and chymotrypsin.
  • Cathepsin reaction buffer (0.1% CHAPS, 50 mM sodium acetate (pH 5.5), 1 mM EDTA) may be used with cathepsins K, L, and S and papain.
  • a unique reaction buffer may be used with subtilisin A (PBS and 0.1% Tween 20).
  • Proteinase inhibitory activity may be detected by the ability of recombinant LEKTI to block the cleavage of small, chromogenic peptide substrates as determined by a spectroscopy technique described previously in Schick, C. et ah, (1998) Biochemistry 37, 5258-5266, which is incorporated herein by reference in its entirety.
  • Inhibition of proteinase may be assessed after preincubating the enzyme with recombinant LEKTI for 2 min at 25 °C in 100 uL of assay buffer. This mixture may be added to 890 or 880 uL of assay buffer in a 1 mL quartz cuvette.
  • the proteinase activity may be initiated by adding 10-20 uL of the appropriate pNA substrate.
  • the rate changes (AA405/min) of inhibited and control reactions may be determined from velocity plots.
  • fluorescence based assays are commercially available that may be used to determine proteinase activity by the ability of recombinant LEKTI to block the cleavage of florescent peptide substrates.
  • different combinations of secretory tag and cell penetration tag may cause differing LEKTI protease activity on each of the tested proteases (e.g . trypsin, plasmin, cathepsin G, HNE, subtilisin A, and chymo trypsin). Furthermore, discrete combinations of secretory tag and cell penetration tag may cause differing LEKTI protease activity among individual proteases.
  • various combinations of secretory tag and cell penetration tag may affect the ability of the recombinant LEKTI protein to penetrate into a tissue or pass through cellular or tissue membrane or barrier or a cell membrane to a greater or lesser degree.
  • the various recombinant LEKTI products may be tested in cell culture to assess the effect of the various combinations of secretory tag and cell penetration tag.
  • adherent fibroblastic HS-68, NIH-3T3, 293, Jurkat T, or Cos-7 cell lines may be cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% (vol/vol) 200 mM glutamine, 1% (vol/vol) antibiotics (streptomycin, 10,000 pg/ml; penicillin, 10,000 IU/ml), and 10% (wt/vol) FBS, at 37°C in a humidified atmosphere containing 5% C02.
  • DMEM Dulbecco’s modified Eagle’s medium
  • purified recombinant LEKTI product (as obtained above) may be loaded in DMEM or PBS (500 pi of DMEM containing 0.25 pg of protein) and incubated for 30 min at 37°C. Cells grown to 75% confluency are then overlaid with these recombinant LEKTI protein media. After 30 min incubation at 37°C, 1 ml of fresh DMEM supplemented with 10% FBS is added to the cells, without removing the overlay of recombinant LEKTI protein, and cells are returned to the incubator for another 30 min. Cells are then extensively washed with PBS and examined for recombinant LEKTI protein.
  • Cells could be observed by immunofluorescence by first fixing with 2% formalin (Sigma), permeabilizing, then incubating with primary anti-6xHis tag antibody and secondary antibody according to the manufacturers’ instruction.
  • cells lysates could be obtained and the presence of His tagged recombinant LEKTI observed via Western blot, as described above.
  • certain combinations of secretory protein and penetrating peptide have differing effects on the ability of the recombinant LEKTI protein’s ability to pass through the cell membrane.
  • Candidate LEKTI therapies may be assayed in vitro for inhibition of purified target serine proteases involved in viral replication. Quantification of enzyme substrates/reaction products may be done by fluorescence, luminescence, colorimetry, or by analytical methodology, all of which are known to one of skill in the art.
  • Antiviral activity of candidate therapies may be demonstrated in vitro by quantifying inhibition of viral replication in laboratory tissue culture cell lines such as Vero or MDCK cells.
  • Specific antiviral readouts may include cytopathic effect (host cell viability), cell fusion (counting syncytia), plaque formation (counting plaques), and viral genome replication (as measured by qPCR).
  • candidate therapies protein or bacteria
  • animal models of respiratory viral replication which may include, but not limited to, mouse, hamster, ferret and non-human primate.
  • KLK13 has recently been shown to be required for the infection of the human respiratory epithelium and is sufficient to mediate the entry of Coronavirus, in particular HCoV-HKUl, to non-permissive RD cells (Milewska el ah, biorxiv.org/content/

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Abstract

La présente invention concerne, entre autres, des microbes génétiquement modifiés exprimant des domaines LEKTI recombinants qui sont efficaces pour traiter ou prévenir une infection virale. Dans certains modes de réalisation, l'invention concerne des compositions, des procédés et des kits comprenant des microbes exprimant le domaine LEKTI.
PCT/US2022/034997 2021-06-24 2022-06-24 Compositions et procédés pour le traitement d'une infection virale WO2022272132A2 (fr)

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