Effects of serine protease inhibitors of SERP-1 and SERP-1RCL-derived peptides on microbiota composition and uses thereof

Abstract

The present disclosure relates to the effects of serine protease inhibitors (SERP-1) and SERP-1 RCL-derived peptides on the microbiota composition and uses thereof. Specifically, disclosed herein are methods of preventing, reducing, or inhibiting a viral or immune disorder comprising administering to a subject at risk or having suffered from a viral or immune disorder an effective amount of a composition comprising a polypeptide derived from a reactive central ring of a serine protease inhibitor, or a biologically active variant thereof, wherein the subject is not administered an antibiotic for at least one day of administration of the composition. Compositions and kits for preventing, reducing or inhibiting viral or immune disorders are also disclosed.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 2020800173841, application date January 13, 2020, and invention name “Effects of Serpins: SERP-1 and SERP-1RCL-derived peptides on microbiota composition and their uses”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of an earlier filing date of U.S. Provisional Application No. 62 / 792,201, filed on January 14, 2019, the entire contents of which are incorporated herein by reference.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on January 12, 2020, is named 131849-252219_SL.txt, and is 6,265 bytes in size. Technical Field

[0006] The present disclosure relates to serine protease inhibitors and methods of use, and in particular, to Serp-1 compositions and derivatives thereof, including active fragments, such as Serp-1 RCL-derived peptide S-7, and methods of improving survival in vasculitis (e.g., lethal vasculitis caused by murine gammaherpesvirus 68 (MHV68)).

[0007] Thank you for the government's support

[0008] This invention was made with government support under Grants AI100987 and HL100202 from the National Institutes of Health. The government has certain rights in this invention. Background Art

[0009] Viral infections induce a potent immune response, an immunopathogenesis that can lead to severe complications with sepsis or capillary leak syndrome, excessive coagulation and bleeding, and very high mortality, with limited effective treatments and a real unmet clinical need. Sepsis is associated with the risk of disseminated intravascular coagulation (DIC) with thrombosis, hemorrhage, and shock. One class of viruses with known severe complications is the gammaherpesviruses (GHV). Murine gammaherpesvirus-68 (MHV-68) is a widely used, well-controlled laboratory model of GHV host-pathogen interactions that has genetic similarities to the human viruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV).

[0010] Inflammatory vasculitis syndromes (IVS) are a group of rare, heterogeneous, devastating inflammatory disorders of the body's widespread vasculature with increased morbidity, including sudden loss of vision, aneurysms, aortic arch syndrome, stroke, and associated increased mortality. The etiology of many systemic vasculitides is currently unknown, with proposed mechanisms ranging from induction by fungal spores to infection with herpes viruses (e.g., herpes zoster). IVS can be treated by intraperitoneal infection of interferon gamma receptor knockout (IFNγR - / - ) mice, inducing a lethal IVS large vessel arteritis model that closely resembles human giant cell arteritis, Kawasaki disease, and Takayasu's arteritis. Many infected mice exhibit extensive pulmonary hemorrhage and consolidation, resembling DIC in viral sepsis, with a very high concomitant mortality rate. In addition to an aggressive inflammatory cell invasion with hemorrhagic pulmonary consolidation, a significant number of mice also have marked colonic dilatation reminiscent of toxic megacolon. Herpesvirus infection has been associated with inflammatory bowel disease, and gastrointestinal involvement has been reported in systemic vasculitis. Summary of the invention

[0011] Disclosed herein are compositions, methods and kits for preventing, reducing or inhibiting viral or immune disorders.

[0012] In some embodiments, a method of preventing, reducing or inhibiting a viral or immune disorder comprises administering to a subject at risk for or already suffering from a viral or immune disorder an effective amount of a composition comprising a polypeptide derived from a reactive center loop of a serine protease inhibitor or a biologically active variant thereof, wherein the subject has not been administered an antibiotic within at least one day of administering the composition.

[0013] In some embodiments, the polypeptide has an amino acid sequence that has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:1.

[0014] In some embodiments, the polypeptide has an amino acid sequence that has at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:1.

[0015] In some embodiments, the polypeptide has an amino acid sequence as shown in GTTASSDTAITLIPR (SEQ ID No: 2).

[0016] In some embodiments, the composition further comprises a carrier, such as 0.9% saline, or citrate buffered saline (CBS), or phosphate buffered saline (PBS), or Ringer's lactate solution (RL), or Ringer's acetate solution (AR), or 5% or 10% glucose in saline (D5NS, D10NS), or 5% or 10% glucose in half-normal saline (D5HNS, D10HNS), or Hank's balanced salt solution (HBSS), or Earle's balanced salt solution (EBSS), or Gey's balanced salt solution (GBSS), or TRIS buffered saline (TBS), or HEPES buffered saline (HBS).

[0017] In some embodiments, administration comprises one or more of the following routes of administration: subcutaneous, intravenous, or intramuscular.

[0018] In some embodiments, the virus or immune disorder is gammaherpesvirus (GHV), Epstein-Barr virus (EBV), and / or Kaposi's sarcoma-associated herpesvirus (KSHV).

[0019] In some embodiments, the GHV is murine gammaherpesvirus-68 (MHV-68).

[0020] In some embodiments, the GHV is an acute or chronic GHV infection.

[0021] In some embodiments, the immune disorder is inflammatory vasculitis syndrome (IVS).

[0022] In some embodiments, the composition increases protective gut microbiota and / or decreases patho-exacerbative gut microbiota.

[0023] In some embodiments, the protective gut microbiota includes an ASV4 microbiota, and the pathology-exacerbating gut microbiota includes ASV1 and ASV123.

[0024] In some embodiments, a kit for preventing, reducing or inhibiting a viral or immune disorder in a subject at risk of or already suffering from a viral or immune disorder is disclosed, the kit comprising a composition comprising a polypeptide derived from a reactive center loop of a serine protease inhibitor or a biologically active variant thereof, and instructions for use, wherein the instructions indicate that the subject should not be administered antibiotics within at least one day of using the kit.

[0025] In some embodiments, a method of altering a gut microbiota is disclosed, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a polypeptide derived from a reactive center loop of a serine protease inhibitor or a biologically active variant thereof, wherein the subject has not been administered an antibiotic within at least one day of administering the composition, and the composition increases a protective gut microbiota and / or decreases a pathology-exacerbated gut microbiota.

[0026] In some embodiments, the protective gut microbiota includes an ASV4 microbiota, and the pathology-exacerbating gut microbiota includes ASV1 and ASV123.

[0027] The foregoing and other features of the present disclosure will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A The chemical structure of Serp-1 is provided, with the A-beta sheet and reactive center loop (RCL) highlighted. The RCL-derived peptide S-7 is also provided.

[0029] Figure 1B Figure 2 is a Kaplan-Meier curve depicting IFNγR in mice infected with MHV-68, with or without antibiotics, and treated with saline (left; N = 12 and 5), Serp-1 (middle; N = 5 and 5), or S-7 (right; N = 5 and 5). - / - Survival of mice.

[0030] Figure 2A-2E Antibiotic treatment was shown to accelerate MHV-68 lethality and alter the intestinal bacterial microbiota. Figure 2A An overview of the experimental design is provided. Mice were treated with a broad-spectrum antibiotic cocktail in drinking water for 10 days, placed in normal water for 1 day, infected with MHV-68, with or without treatment, and followed for survival. Figure 2B It was shown that the medicated water containing antibiotics completely eliminated the bacterial microbiota content during the 10-day pretreatment process, which was restored three days after the removal of antibiotics. Figure 2C Indicates the relative abundance (genus level) of the 16S microbiome, Figure 2D Shown is the bacterial alpha diversity (Shannon index) in saline-treated mice with and without antibiotics. Figure 2E is a heat map showing the abundance of ASVs in mice treated with saline in the presence or absence of antibiotic pretreatment.

[0031] Figure 3A-Figure 3F Analysis of the 16S microbiota. Figure 3A The relative abundance (genus level) of the 16S microbiota is shown. Figure 3BBacterial alpha diversity (Shannon index) is shown. Statistical significance was assessed by the Mann-Whitney test. Figure 3C A principal coordinate analysis (PCoA) plot of the weighted UniFrac distance is provided. Figure 3D A heat map is provided showing the abundance of ASVs in mice treated with saline, Serp-1, or S7 in the absence of antibiotics. The ASVs shown were identified by differential analysis relative to Serp-1 or S7 (top) or saline (bottom). Figure 3E Provided Figure 3D Heatmap of differentially expressed ASVs comparing Serp-1-treated mice (left) or S-7-treated mice (right) in the presence or absence of antibiotics. Figure 3F The relative abundance of ASV4 (identified as differentially associated with Serp-1 and S-7 treatment) and ASV1 and 123 (identified as differentially associated with saline) in the microbiota of individual mice is shown. The ASV taxonomic classification of the most closely related bacterial taxa is shown. Saline + Abx, N = 6; Saline no Abx, N = 6; Serp-1 + Abx, N = 6; Serp-1 no Abx, N = 5; S7 + Abx, N = 6; S7 no Abx, N = 5.

[0032] Figure 4A-4G Microbiota-dependent activation of Serp-1 and S-7 was associated with lung inflammation and increased occupancy of CD3+ and CD8+ cells. Figure 4A Representative H&E sections of lungs from mice followed for 3 days after MHV-68 infection and treatment with saline, Serp-1, or S-7 without antibiotic pretreatment (top row) or after antibiotic pretreatment (bottom row) are shown. Scale bar, 100 μm. Figure 4B Show in Figure 4A The alveolar wall thickness of the mouse lungs after 3 days of follow-up is shown. Figure 4C Shown are the alveolar cavity areas of mouse lungs. Figure 4D Representative CD3 IHC micrographs are shown for lungs of mice treated with saline, Serp-1, or S-7 without antibiotic pretreatment (top row) or after antibiotic pretreatment (bottom row) following MHV-68 infection and followed for 3 days. Scale bar, 20 μm. FIG. 4E to FIG. 4G CD3+ ( Figure 4E )、CD4+( Figure 4F ) and CD8+( Figure 4G ) cells. Two-way ANOVA with Fisher's LSD post hoc analysis was performed Figure 4B , Figure 4C and Figure 4E-Figure 4GStatistical analysis in. *p<0.05, **p<0.01, ***p<0.001; ns not significant. N=3 for all conditions.

[0033] Figure 5A-Figure 5B These results suggest that Serp-1 and S-7 reduce the presence of MHV-68 in an intestinal bacterial microbiota-dependent manner. Figure 5A Representative IHC images at 100× fields are shown, depicting MHV-68 antigen staining in the lungs of mice without antibiotic pretreatment (top row) or after antibiotic pretreatment (bottom row) followed for 3 days after MHV-68 infection and treatment with saline, Serp-1, or S-7. Figure 5B Quantification of the percentage of MHV-68 positively stained cells per 100x field from 3 fields per mouse is shown. N=3 for all conditions.

[0034] Figure 6 is a schematic diagram according to an embodiment disclosed herein. (Left) In serial lethal MHV-68 infection of IFNγR- / - mice, the gut microbiota and immune response interact to ultimately initiate inadequate cellular (e.g., T cell) responses to affected tissues (e.g., lungs), leading to severe disease and death. Antibiotics inhibit the immunostimulatory effects of the bacterial microbiota, further reducing the immune response and worsening the disease. (Right) In protection mediated by immunomodulators such as Serp-1 or S-7 treatment, the interactions that lead to adequate initiation of the immune response are enhanced, promoting increased infiltration of T cells into affected tissues, reducing disease pathology and leading to survival.

[0035] Fig. 7A The ASV readings for each sample are shown. One saline no abx sample was removed due to low readings (12,107 readings), as marked in grey. There were no statistically significant differences between the groups as assessed by the Mann-Whitney test.

[0036] Figure 7B Plots showing abundance (number of bacterial ASVs observed) (n=33).

[0037] Figure 7C Shown are the results of differential analysis by likelihood test, DeSeq2, and LefSe between saline null abx mice and Serp-1 null abx mice.

[0038] Fig.7D Shown are the results of differential analysis by likelihood test, DeSeq2, and LefSe between saline null abx mice and S7 null abx mice.

[0039] Fig. 7EShown are the results of differential analysis by likelihood test, DeSeq2 and LefSe between saline null abx mice and combined Serp1 / S7 null abx mice. Figure 7C-Figure 7E As shown, ASVs identified as significantly different in each analysis are indicated by "x", and ASVs without significant differences are indicated by "-".

[0040] Figure 8 is a bar graph showing that the inhibition of uPA activity by Serp-1 in an in vitro substrate conversion assay is not affected by the presence of a broad-spectrum antibiotic cocktail.

[0041] Fig. 9 is a bar graph indicating that antibiotics increase the viral load of MHV68 in the lung. DETAILED DESCRIPTION

[0042] In the following description, the technology disclosed herein is described in one or more exemplary embodiments with reference to the accompanying drawings, in which like numbers represent like or similar elements. Reference throughout the specification to "one embodiment," "an embodiment," or similar language means that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the technology disclosed herein. Therefore, the phrases "in one embodiment," "in an embodiment," and similar language appearing throughout the specification may, but do not necessarily, all refer to the same embodiment.

[0043] In one or more embodiments, the features, structures or characteristics of the technology disclosed herein described may be combined in any suitable manner. In the following description, many specific details are described to provide a thorough understanding of the embodiments of the technology disclosed herein. However, those skilled in the relevant art will recognize that the technology disclosed herein can be practiced without one or more specific details, or can be practiced using other methods, components, materials, etc. In other cases, well-known structures, materials or operations are not shown or described in detail to avoid confusing various aspects of the technology disclosed herein.

[0044] abbreviation:

[0045] DIC: Disseminated intravascular coagulation

[0046] GHV: Gammaherpesvirus

[0047] MHV-68: Murine gammaherpesvirus-68

[0048] MYXV: Myxoma virus

[0049] IVS: Inflammatory vasculitis syndrome

[0050] GCA: giant cell arteritis

[0051] TD: Takayasu disease

[0052] KD: Kawasaki disease

[0053] RCL: reactive center ring

[0054] IFNγ: interferon gamma

[0055] TNFα: tumor necrosis factor alpha

[0056] IFNγR: interferon gamma receptor

[0057] IP: intraperitoneal

[0058] The following explanations of terms and methods are provided to better describe the compounds, compositions and methods of the present invention, and to guide those of ordinary skill in the art to practice the present disclosure. It should also be understood that the terms used in the present disclosure are only used for the purpose of describing specific embodiments and examples, and are not intended to be limiting.

[0059] As used herein, the singular forms a, an and the are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0060] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as combinations not involved when interpreted in the alternative (or).

[0061] As used herein, "one or more" or at least one may mean one, two, three, four, five, six, seven, eight, nine, ten or more, up to any number.

[0062] As used herein, the term "comprising" means "including". Thus, "comprising A or B" means including A, B, or A and B. It should also be understood that all base sizes and all molecular weight or molecular mass values ​​for nucleic acids given are approximate and are provided for description. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood.

[0063] As used herein, "amplicon sequence variant" (ASV) is a term used to refer to individual DNA sequences that are recovered from high-throughput marker gene analysis after removing spurious sequences generated during PCR amplification and sequencing. Therefore, ASV is an inferred sequence of true biological origin. The term was introduced to distinguish between traditional methods, which depict operational taxonomic units (OTUs) generated by clustering sequences based on a shared similarity threshold, and newer alternative methods that resolve individual sequences without clustering. Because the ASV method is able to resolve sequences that differ by as little as one nucleotide and avoids similarity-based clustering, ASV is also called exact sequence variant (ESV) or zero radius OTU (zOTU).

[0064] As used herein, "clinical outcome" refers to the health status of a patient after treatment of a disease or disorder, or in the absence of treatment. Clinical outcomes include, but are not limited to: increased time to death, decreased time to death, increased chance of survival, increased risk of death, survival, disease-free survival, chronic disease, advanced or aggressive disease, disease recurrence, death, and good or poor response to treatment.

[0065] "Effective amount" or "therapeutically effective amount" refers to the amount of the compounds of the present invention or composition sufficient to produce the desired effect, which can be therapeutic and / or beneficial effects. The effective amount will vary with the age of the subject, the general condition, the severity of the disease being treated, the specific agent being administered, the duration of treatment, the nature of any simultaneous treatment, the pharmaceutically acceptable carrier used, etc., within the knowledge and expertise of those skilled in the art. When appropriate, the effective amount or therapeutically effective amount in any individual case can be determined by those of ordinary skill in the art with reference to relevant texts and documents. (For example, see Remington, The Science and Practice of Pharmacy (latest edition)). In general, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral preparations generally include injectable fluids, including pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc. as carriers. For solid compositions (for example, powders, pills, tablets or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0066] An "isolated" biological component (e.g., a nucleic acid molecule, protein, or cell) has been substantially separated or purified from other biological components in the cells of an organism, or from the organism itself in which the component naturally occurs, such as from other chromosomal and extrachromosomal DNA and RNA, proteins, and cells. Nucleic acid molecules and proteins that have been "isolated" include cytokine molecules (e.g., DNA or RNA) and proteins purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[0067] As used herein, the terms "subject" and "patient" are used interchangeably herein and refer to humans and non-human animals. The term "non-human animal" of the present disclosure includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, rodents (e.g., mice, rats, etc.), etc. Preferably, the subject is a human patient. In a specific embodiment, the subject of the present disclosure is a human subject. A "subject in need thereof" or "subject in need thereof" is a subject known to have or suspected of having a surface wound, such as a wound in the skin and surrounding tissue.

[0068] As used herein, the terms "treating" or "treatment" refer to any type of effect that imparts a modulatory effect, which can be, for example, a beneficial and / or therapeutic effect on a subject suffering from a condition, disorder, disease or disease, including, for example, improvement of the subject's condition (e.g., one or more symptoms), delay in progression of the condition, disorder or disease, delay in the onset of the disease, disorder or disease, and / or changes in clinical parameters of the condition, disorder, disease or disease, as is known in the art, etc.

[0069] "Peptide" refers to any compound consisting of amino acids or amino acid analogs chemically bound together. Peptides as used herein include oligomers of amino acids, amino acid analogs, or small and large peptides, including polypeptides, or "protein" refers to any amino acid chain, regardless of length or post-translational modification (such as glycosylation or phosphorylation). In one example, a peptide is two or more amino acids connected by peptide bonds. Typically, a peptide consists of less than fifty amino acids; for example, from about 7 to about 40 amino acids, from about 7 to about 30 amino acids, from about 7 to about 20 amino acids. In one example, a peptide consists of 4 amino acids and is called a tetrapeptide.

[0070] "Peptide" applies to amino acid polymers, both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers as well as those in which one or more amino acid residues is a non-natural amino acid, eg, an artificial chemical mimetic of a corresponding naturally occurring amino acid.

[0071] A "polypeptide" is a polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acid is an α-amino acid, either the L-optical isomer or the D-optical isomer can be used. As used herein, the term "polypeptide" or "protein" is intended to encompass any amino acid sequence, including modified sequences, such as glycoproteins. The term "polypeptide" is specifically intended to encompass naturally occurring proteins as well as recombinantly or synthetically produced proteins. The term "residue" or "amino acid residue" includes reference to an amino acid incorporated into a protein, polypeptide, or peptide. The term "soluble" refers to a form of the polypeptide that is not inserted into a cell membrane.

[0072] Conservative amino acid substitutions are those substitutions that, when made, interfere least with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and is not significantly altered by such substitutions. Examples of conservative substitutions are shown below.

[0073]

[0074]

[0075] Conservative substitutions generally preserve (a) the structure of the polypeptide backbone in the area of ​​the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0076] Substitutions that are generally expected to produce the greatest changes in protein properties are non-conservative, such as changes in (a) a hydrophilic residue, such as seryl or threonyl, is substituted for (or replaced by) a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline is substituted for (or replaced by) any other residue; (c) a residue having a positively charged side chain, such as lysyl, arginyl, or histidyl, is substituted for (or replaced by) a negatively charged residue, such as glutamyl or aspartyl; or (d) a residue having a bulky side chain, such as phenylalanine, is substituted for (or replaced by) a residue without a side chain, such as glycine.

[0077] Regarding the use of any plural and / or singular terms herein, those skilled in the art may translate the plural into the singular and / or the singular into the plural according to the context and / or application. For clarity, various singular / plural permutations and combinations may be explicitly described herein.

[0078] Unless otherwise indicated, technical terms are used conventionally. Definitions of commonly used terms in molecular biology can be found in: Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); and other similar references.

[0079] Suitable methods and materials for practicing or testing the present disclosure are described below. Such methods and materials are illustrative only and not restrictive. Although methods and materials similar or equivalent to those methods or materials described herein can be used to practice or test the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood. Other methods and materials similar or equivalent to those methods or materials described herein can be used. For example, conventional methods well known in the art to which the present disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and supplements in 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999. In addition, the materials, methods, and examples are illustrative only and not limiting.

[0080] Compositions and methods of use

[0081] Serine protease inhibitors, or serpins, are ubiquitous, complex, and highly active regulatory molecules that potently control a variety of coagulation, inflammatory, and neuroendocrine pathways. Serp-1 is a secreted myxoma virus-derived purified protein that binds to and inhibits urokinase- and tissue-type plasminogen activators (uPA and tPA, respectively), plasmin (piasmin), factor X (fXa), and thrombin (in the presence of defined concentrations of heparin).

[0082] As disclosed herein, the inventors have discovered that compositions comprising Serp-1 polypeptides, such as compositions comprising full-length Serp-1 polypeptides or biologically active fragments thereof, improve survival in vasculitis (e.g., MHV-68 lethal vasculitis). In particular, Serp-1 and Serp-1 RCL-derived peptide S-7 have been shown to improve survival in MHV-68 vasculitis. In addition, gut microbiota elimination accelerates the lethality of MHV-68 and eliminates the effectiveness of Serp-1 and S-7. In addition, the effect of microbiota elimination on early MHV-68-induced lung pathology is also demonstrated. The effect of MHV-68 and Serp-1 or S-7 on microbiota composition is also provided.

[0083] Disclosed herein are formulations comprising Serp-1 polypeptides and / or biologically active fragments and derivatives thereof (e.g., Serp-1 polypeptides and fragments, such as Serp-1 RCL-derived peptide S-7) that improve survival, reduce, decrease, prevent and / or inhibit one or more signs or symptoms associated with vasculitis in mammalian subjects to whom the Serp-1 polypeptides or fragments thereof are topically administered. In one embodiment, the Serp-1 polypeptide comprises amino acids available at GENBANK Accession No. NP_051722 having an amino acid sequence as shown below: MKYLVLVLCL TSCACRDIGL WTFRYVYNESDNVVFSPYGLTSALSVLRIA AGGNTKREID VPESVVEDSD AFLALRELFV DASVPLRPEF TAEFSSRFNT SVQRVTFNSENVKDVINSYV KDKTGGDVPR VLDASLDRDT KMLLLSSVRM KTSWRHVFDP SFTTDQPFYS GNVTYKVRMMNKIDTLKTET FTLRNVGYSV TELPYKRRQT AMLLVVPDDL GEIVRALDLS LVRFWIRNMR KDVCQVVMPKFSVESVLDLR DALQRLGVRD AFDPSRADFG QASPSNDLYV TKVLQTSKIE ADERGTTASSDTAITLIPRNALTAIVANKP FMFLIYHKPT TTVLFMGTIT (SEQ ID NO: 1). Serp-1 polypeptides include polypeptides having at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and biologically active fragments thereof. In an embodiment, the Serp-1 polypeptide comprises a Serp-1 reactive center loop derived peptide or a series of modified RCL peptides. In some embodiments, the Serp-1 reactive central loop derived peptide has amino acids that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identical to the amino acid sequence shown in GTTASSDTAITLIPR (SEQ ID NO: 2).

[0084] Disclosed isolated peptides include synthetic embodiments of the peptides described herein. In addition, analogs (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting from the disclosed peptide sequences) and variants (homologues) of these peptides can be used in the compositions and methods described herein. Each peptide disclosed herein consists of an amino acid sequence, which can be naturally occurring and other L- and / or D-amino acids.

[0085] Peptides can be modified by a variety of chemical techniques to produce derivatives that have substantially the same activity as the unmodified peptide and optionally have other desired properties. In another example, the carboxylic acid group of the protein, whether at the carboxyl terminus or the side chain, can be provided in the form of a pharmaceutically acceptable cationic salt, or esterified to form a C1-C16 ester, or converted to an amide of the formula NR1R2, wherein R1 and R2 are each independently H or a C1-C16 alkyl, or combined to form a heterocycle, such as a 5- or 6-membered ring. The amino group of the peptide, whether at the amino terminus or the side chain, can be in the form of a pharmaceutically acceptable acid addition salt, such as HCl, HBr, acetic acid, benzoic acid, toluenesulfonic acid, maleic acid, tartaric acid and other organic salts, or can be modified to a C1-C16 alkyl or dialkylamino group or further converted to an amide.

[0086] The hydroxyl groups of the peptide side chains can be converted into C1-C16 alkoxy groups or C1-C16 esters using recognized techniques. The phenyl and phenol rings of the peptide side chains can be substituted by one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or by C1-C16 alkyl groups, C1-C16 alkoxy groups, carboxylic acids and their esters or amides of these carboxylic acids. The methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylene groups. Thiols can be protected with any of many recognized protecting groups, such as acetamide groups. Those skilled in the art also know methods for introducing cyclic structures into peptides to select and provide conformational constraints on the structure to improve stability. Although the peptides disclosed herein can be linear or cyclic, cyclic peptides generally have advantages over linear peptides because their cyclic structures are more rigid, and therefore their biological activity can be higher than that of the corresponding linear peptides. Any method for cyclizing peptides can be applied to the serpin-derived peptides or fragments described herein.

[0087] As indicated, the length of the Serp-1 polypeptide can vary, and can be or include contiguous amino acid residues that naturally occur in Serp-1, or that differ to some extent from the naturally occurring Serp-1 sequence (but retain biological activity). When the fragment includes amino acid residues that do not naturally occur in Serp-1 at its N-terminus or C-terminus (or both), the length of the additional sequence can be about 200 amino acid residues, and these residues can be evenly or unevenly distributed between the N-terminus and the C-terminus. For example, both the N-terminus and the C-terminus can include about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues. Alternatively, one terminus may include about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 20 150, 160, 170, 180, 190, or 200 residues, and one terminus may not include any residues (e.g., it may end in an amino acid sequence identical to a naturally occurring Serp-1 sequence).

[0088] More specifically, the N- or C-terminus may include 1 to about 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100) positively charged amino acid residues (e.g., basic amino acid residues, such as arginine, histidine and / or lysine residues); 1 to about 100 negatively charged amino acid residues (e.g., acidic amino acid residues, such as aspartic acid or glutamic acid residues); 1 to about 100 glycine residues; 1 to about 100 hydrophobic amino acid residues (e.g., hydrophobic aliphatic residues, such as alanine, leucine, isoleucine or valine, or hydrophobic aromatic residues, such as phenylalanine, tryptophan or tyrosine); or 1 to about 100 (e.g., 1-4) cysteine ​​residues. When a biologically active variant of a Serp-1 fragment is used, the variant may be varied by substituting one or more amino acid residues in these groups. Variants may include conservative amino acid substitutions.

[0089] Embodiments of peptide mimetics and organomimetics are envisioned whereby the three-dimensional arrangement of the chemical components of such peptide mimics and organomimetics mimics the three-dimensional arrangement of the peptide backbone and component amino acid side chains, such that such peptide mimics and organomimetics of the peptide have measurable Serp-1 activity. For computer modeling applications, a pharmacophore is an idealized three-dimensional definition of the structural requirements for biological activity. Using current computer modeling software, peptide mimics and organomimetics can be designed to fit each pharmacophore.

[0090] In an embodiment, the Serp-1 polypeptide is included in a fusion protein. Thus, the fusion protein may include a Serp-1 polypeptide and a second heterologous moiety covalently linked to the Serp-1 polypeptide, such as a myc protein, an enzyme, or a carrier (e.g., a hepatitis carrier protein or bovine serum albumin). The second heterologous moiety may be covalently or non-covalently linked to the Serp-1 polypeptide. The Serp-1 polypeptide may be included in a fusion protein or may include a heterologous sequence.

[0091] Nucleic acids encoding one or more Serp-1 polypeptides are contemplated. These polynucleotides include DNA, cDNA and RNA sequences encoding the target peptides. Using the amino acid sequences and genetic codes provided herein, one skilled in the art can easily generate nucleic acid molecules encoding these peptides. In addition, one skilled in the art can easily construct multiple clones containing functionally equivalent nucleic acids, such as nucleic acids with different sequences but encoding the same peptide.

[0092] Nucleic acid sequences encoding one or more Serp-1 polypeptides can be prepared by any suitable method, including, for example, cloning of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using, for example, Needham-Van Devanter et al., Nucl. Acids Res. 12: 6159-6168, 1984; and the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides. This can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization by a DNA polymerase using the single strand as a template.

[0093] Exemplary nucleic acids comprising sequences encoding one or more Serp-1 polypeptides disclosed herein can be prepared by cloning techniques or chemical synthesis. Examples of suitable cloning and sequencing techniques and instructions sufficient to guide the skilled artisan through cloning can be found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provides useful information. Such manufacturers include SIGM Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), PharmaciaAmersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to skilled artisans.

[0094] Once nucleic acids encoding one or more Serp-1 polypeptides are isolated and cloned, the peptides can be expressed in recombinant engineered cells using appropriate expression vectors, such as in bacteria, plants, yeast, insects and mammalian cells, or in viral vectors for therapeutic methods, for example, in adeno-associated virus (AAV) vectors. One or more DNA sequences encoding one or more immunogenic peptides can be expressed in vitro by transferring the DNA into suitable host cells. The cells can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It should be understood that all progeny may be different from the parent cell because mutations may occur during the replication process. Methods for stable transfer, i.e., methods for the continued maintenance of exogenous DNA in a host, are known in the art. In one example, the vector is an AAV vector.

[0095] The polynucleotide sequence encoding one or more Serp-1 polypeptides can be operably linked to an expression control sequence (e.g., a promoter). The expression control sequence operably linked to the coding sequence is linked so that expression of the coding sequence is achieved under conditions compatible with the expression control sequence. The expression control sequence includes, but is not limited to, an appropriate promoter, an enhancer, a transcription terminator, a start codon (i.e., ATG) before a protein coding gene, a splicing signal for an intron, maintenance of the correct reading frame of the gene to allow correct translation of mRNA, and a stop codon.

[0096] The polynucleotide sequence encoding one or more Serp-1 polypeptides can be inserted into an expression vector, including but not limited to a plasmid, virus or other vector that can be manipulated to allow insertion or incorporation of the sequence and can be expressed in a prokaryotic or eukaryotic organism. Hosts can include microorganisms, yeast, insects and mammalian organisms. Methods for expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.

[0097] In one aspect, the compositions disclosed herein comprise a nucleic acid molecule encoding a serpin-derived peptide or fragment thereof disclosed herein in an expression construct or in a single or separate cassette.Disclosed herein is an expression construct capable of expressing a serpin-derived peptide or fragment thereof.

[0098] The disclosed expression cassette may include 5' and 3' regulatory sequences operably connected to the polynucleotide disclosed herein. "Operably connected" is intended to refer to the functional connection between two or more elements. For example, the operably connected between the polynucleotide disclosed herein and the regulatory sequence (e.g., promoter) is a functional connection that allows the expression of the polynucleotide disclosed herein. The operably connected elements can be continuous or non-continuous. When used to refer to the connection of two protein coding regions, operably connected refers to that the coding regions are in the same reading frame. The expression cassette may further include at least one additional polynucleotide to be co-transformed into an organism. Alternatively, one or more polypeptides can be expressed on one or more expression cassettes. An expression cassette may be provided having a plurality of restriction sites and / or recombination sites for inserting polynucleotides under the transcriptional regulation of the regulatory region.

[0099] Regulatory regions (i.e., promoters, transcriptional regulatory regions, and translation termination regions) and / or polynucleotides disclosed herein may be natural / similar to a host cell or to each other. Alternatively, the regulatory regions and / or polynucleotides used in the present invention may be heterologous to a host cell or to each other. As used herein, a "heterologous" sequence is a sequence derived from an alien species, or, if from the same species, it is a sequence substantially modified from its native form in composition and / or locus by intentional human intervention. For example, a promoter operably connected to a heterologous polynucleotide is from a species different from the species from which the polynucleotide is derived, or, if from the same / similar species, one or both are substantially modified compared to their original form and / or locus, or the promoter is not a natural promoter for a polynucleotide operably connected. As used herein, a chimeric gene comprises a coding sequence operably connected to a transcription initiation region, and the transcription initiation region is heterologous to the coding sequence.

[0100] In preparing the expression cassette, the various DNA fragments may be manipulated to provide the DNA sequence in the correct orientation and, where appropriate, the correct reading frame. To this end, aptamers or linkers may be used to connect the DNA fragments, or other manipulations may be included to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. In vitro mutagenesis, primer repair, restriction, annealing, re-substitutions such as conversions and transversions may be included for this purpose.

[0101] Many promoters can be used to practice the present invention. Promoters can be selected according to the desired results. The selection of promoters depends on several factors, including but not limited to efficacy, selectivity, inducibility, desired expression level and cell or tissue preference expression. Nucleic acids can be combined with constitutive, tissue preference, inducible or other promoters to express in host organisms. Those skilled in the art can appropriately select and position promoters and other regulator regions relative to the encoding sequence.

[0102] In addition to the Serp-1 polypeptide and / or nucleic acid encoding the Serp-1 polypeptide, the formulation may further include one or more carriers, excipients and additional active agents. The dosage and administration regimen for the method depends on the nature and condition, such as the nature and condition of vasculitis. The subject treated by the formulation can be a human or non-human mammal.

[0103] In embodiments, methods for preventing, treating, reducing and / or suppressing one or more disorders of a subject, such as a virus and / or inflammatory disorder, are provided, thereby improving the survival of the subject. In some embodiments, methods for treating, preventing, suppressing and / or alleviating herpes virus infection, such as a gamma herpes virus (GHV), including MHV-68, are provided. In some embodiments, methods for treating, preventing, suppressing and / or alleviating one or more signs and / or symptoms associated with inflammatory vasculitis syndromes are provided.

[0104] In embodiments, the method comprises administering to a subject an effective amount of a composition comprising one or more peptides disclosed herein. In some embodiments, the method comprises administering to a subject an effective amount of a composition comprising one or more Serp-1 and the Serp-1 RCL-derived peptide S-7. In some embodiments, methods of using the disclosed peptides to treat other disorders are provided, such as juvenile rheumatoid arthritis, polymyalgia rheumatica, reactive arthritis, relapsing polychondritis, polymyositis, undifferentiated connective tissue disease, dermatomyositis, dermatitis herpetiformis, celiac disease, inflammatory bowel disease, ulcerative colitis, Crohn's disease, uveitis, macular degeneration, dry eye, systemic lupus erythematosus with or without diffuse alveolar hemorrhage, pulmonary hypertension, sarcoidosis, glomerulonephritis, diabetes, transplant vascular disease, portal hypertension, Takayasu disease, giant cell arteritis, Kawasaki disease, polyarteritis nodosa, Behcet's disease, Raynaud's disease, Chagas disease, myocarditis, post-infarction disease, inflammatory cardiomyopathy and Buerger's disease (thromboangiitis obliterans). The disclosed peptides can be used alone or in combination therapy with other compositions or drugs for the treatment of the aforementioned disorders. Such combination therapies include, but are not limited to, simultaneous or sequential administration of drugs for preventing, treating, inhibiting and / or alleviating disorders such as GHV and / or inflammatory vasculitis syndrome. These additional therapies do not include antibiotics or other compositions that destroy microbiota associated with protecting intestinal bacterial microbiota (such as ASV4), or increase microbiota, or increase microbiota associated with aggravated phenotypes of pathology (such as ASV1 and ASV123).

[0105] In some embodiments, the methods comprise administering an effective amount of a composition having one or more disclosed peptides instead of an antibiotic to a subject having or at risk of having a herpesvirus infection (e.g., gammaherpesvirus (GHV), including MHV-68) or an inflammatory vasculitis syndrome.

[0106] In some embodiments, the method includes selecting a subject who has or is at risk of having a herpes virus infection (such as gamma herpes virus (GHV), including MHV-68), or is at risk of developing an inflammatory vasculitis syndrome or some autoimmune and autoinflammatory diseases. In some embodiments, selecting a subject who has or is at risk of having a herpes virus infection (e.g., gamma herpes virus (GHV), including MuHV4 / MHV-68, HHV4 / EBV, HHV8 / KSHV) or an inflammatory vasculitis syndrome includes characterizing the subject's intestinal microbiota, wherein the presence of ASV4 indicates that the subject is a candidate for treatment with one or more disclosed compositions.

[0107] The disclosed peptides can be administered by any means known to those skilled in the art, for example, it is expected that administration is by systemic, intramuscular, subcutaneous, intraperitoneal, intravenous, oral, nasal or anal administration. In one embodiment, administration is by subcutaneous or intramuscular injection. In order to extend the time when the peptide can be used to suppress or treat the disease, the peptide can be provided as an implant, an oily injection or as a particle system. The particle system can be microparticles, microcapsules, microspheres, nanocapsules or similar particles (Banga, "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995).

[0108] In some example embodiments, the peptides provided are combined with pharmaceutically acceptable carriers (e.g., phospholipids or other types of lipids) or carriers for administration to human or animal subjects. In some embodiments, more than one disclosed peptide may be combined to form a single formulation. The peptides may be conveniently provided in unit dosage form and prepared using conventional pharmaceutical techniques. Such techniques include the step of combining the active ingredient with a pharmaceutical carrier or excipient. Typically, the formulation is prepared by uniformly and closely combining the active ingredient with a liquid carrier. Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and / or solutes that make the preparation isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The preparation may be present in unit dose or multi-dose containers, such as sealed ampoules and vials, and may be stored under freeze-drying (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, e.g., water for injection, before use. Extemporaneous injection solutions and suspensions may be prepared by sterile powders, granules, and tablets commonly used by those of ordinary skill in the art.

[0109] In certain embodiments, unit dose formulations are those that contain a dose or unit of the administered ingredient, or an appropriate portion thereof. It should be understood that, in addition to the ingredients specifically mentioned above, the formulations encompassed herein may also include other agents commonly used by those of ordinary skill in the art, including, but not limited to, 0.9% saline, or citrate buffered saline (CBS), or phosphate buffered saline (PBS), or Ringer's lactate solution (RL), or Ringer's acetate solution (AR), or 5% or 10% glucose in saline (D5NS, D10NS), or 5% or 10% glucose in semi-normal saline (D5HNS, D10HNS), or Hank's balanced salt solution (HBSS), or Earle's balanced salt solution (EBSS), or Gey's balanced salt solution (GBSS), or TRIS buffered saline (TBS), or HEPES buffered saline (HBS).

[0110] The pharmaceutical compositions provided herein can be administered by different routes, such as systemic, oral (including buccal and sublingual), rectal, parenteral, aerosol, nasal, intramuscular, intraperitoneal, subcutaneous, intradermal and topical administration. They can be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles and liposomes.

[0111] In an embodiment, it can be administered by direct injection at the site (or pre-site) of the tissue to be treated. In another embodiment, the pharmaceutical composition is delivered in a vesicle, particularly a liposome (see, e.g., Langer, Science 249: 1527-1533, 1990; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, NY, pp. 353 365, 1989). In one embodiment, it is administered by IP injection over the course of several days. In another embodiment, the agent can be delivered by a viral vector (e.g., AAV or lentivirus).

[0112] In certain embodiments, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see, e.g., Langer Science 249: 1527-1533, 1990; Sefton Crit. Rev. Biomed. Eng. 14: 201-240, 1987; Buchwald et al., Surgery 88: 507-516, 1980; Saudek et al., N. Engl. J. Med. 321: 574-579, 1989). In another embodiment, polymeric materials can be used (see, e.g., Ranger et al., Macromol. Sci. Rev. Macromol. Chem. 23:61-64, 1983; Levy et al., Science 228:190-192, 1985; During et al., Ann. Neurol. 25:351-356, 1989; and Howard et al., J. Neurosurg. 71:105-112, 1989). Other controlled release systems, such as those discussed in the review by Langer (Science 249:1527-1533, 1990), can also be used.

[0113] The effective amount of the pharmaceutical composition depends on the nature of the disease or condition to be treated, and the stage of the disease or condition. The effective amount can be determined by standard clinical techniques. The exact dosage used in the preparation also depends on the route of administration and should be determined according to the judgment of the health care practitioner and the circumstances of each subject. An example of such a dosage range is a single or divided dose of 10 ng / kg to 0.001 to 200 mg / kg body weight. Another example of a dosage range is a single or divided dose of 1.0 μg to 100 mg / kg body weight.

[0114] In some examples, an effective amount of a disclosed pharmaceutical composition is an amount in which one or more signs or symptoms associated with a viral and / or inflammatory disorder are reduced or inhibited, e.g., at least 10% less active than in the absence of the composition, e.g., about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%. For example, an effective amount of a disclosed pharmaceutical composition is one in which one or more signs or symptoms associated with infection with a herpes virus, such as a gammaherpes virus (GHV), including MHV-68, are reduced or inhibited, e.g., at least 10% less active than in the absence of the composition, e.g., from about 15% to about 98%, from about 30% to about 95%, from about 40% to about 80%, from about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%. In one example, an effective amount of a disclosed pharmaceutical composition is an amount in which one or more signs or symptoms associated with an inflammatory vasculitis syndrome are reduced or inhibited, e.g., at least 10% less active than in the absence of the composition, e.g., about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0115] The specific dose level and frequency of administration for any particular subject may vary and will depend on a variety of factors, including the activity of the specific compound, the metabolic stability and duration of action of the compound, age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and the severity of the condition of the subject being treated.

[0116] The pharmaceutical composition of the present disclosure can be administered at approximately the same dose throughout the treatment period, in an increasing dosage regimen, or in a loading dose regimen (e.g., wherein the loading dose is about 2 to 5 times the maintenance dose). In some embodiments, during the course of treatment, the dosage is changed based on the severity of the condition, disease, or condition of the subject being treated, the apparent response to treatment, and / or other factors determined by a person of ordinary skill in the art. The volume of administration is changed according to the route of administration.

[0117] Compositions containing one or more disclosed conjugates may be administered single or multiple times using the dosage level and pattern selected by the treating physician. Typically, multiple doses are administered.

[0118] In a specific example, the composition is administered once a day by IV, IM, SC or IP, or continuously by an infusion pump. It is contemplated that the composition may be administered daily, every other day, twice a week, weekly or monthly. Treatment typically continues for at least one month, more commonly for two or three months, sometimes for six months or a year, and may even continue indefinitely, i.e., for a long period of time. Repeated courses of treatment are also possible.

[0119] In one embodiment, administration is by IP injection, for example, about 1 ng / kg to about 100 μ g / kg, including 100 ng / g dosage, for a course of treatment for several days, for example, for at least one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen weeks, sixteen weeks, seventeen weeks, eighteen weeks, nineteen weeks, twenty weeks or more, for example, for one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months. For example, intramuscular injection can be in the range of about 0.5 ml to about 3 ml. In some other examples, the method is by IV.

[0120] Reagent test kit

[0121] The kit provided by the present disclosure can be used for preventing, treating, reducing or suppressing one or more diseases in the subject, such as viruses and / or inflammatory diseases, so as to improve the survival of the subject. In some embodiments, disclosed herein is a kit for treating, preventing, suppressing and / or alleviating herpes virus infection, such as gamma herpes virus (GHV), including MHV-68. In some embodiments, a kit for treating, preventing, suppressing and / or alleviating one or more signs and / or symptoms associated with inflammatory vasculitis syndrome is provided. An exemplary kit includes at least one disclosed pharmaceutical composition. The disclosed kit may include illustrative materials, which disclose methods for using the composition in the kit. The illustrative materials may be written, electronic (such as computer floppy disks or optical disks) or visual (such as video files).

[0122] The following examples are provided to illustrate specific features of certain embodiments. However, the specific features described below should not be construed as limitations on the scope of the disclosure, but rather as examples of equivalents that would be recognized by those of ordinary skill in the art.

[0123] Example

[0124] Immunomodulatory proteins from myxoma virus (MYXV) offer a new class of anti-inflammatory therapeutics when delivered as recombinant proteins or by ectopic expression following adeno-associated viral (AAV) delivery. MYXV, the causative agent of lethal myxomatosis in European rabbits (Oryctolagus cuniculus), expresses a highly potent immune evasion protein that functions as a virulence factor in MYXV infection. MYXV is not a pathogen in mice or humans. When purified as isolated recombinant proteins, these immunomodulatory biologics can alter disease progression in a wide range of inflammatory diseases in preclinical animals and humans. MYXV-derived serine protease inhibitor (serpin) Serp-1 and the Serp-1 reactive center loop (RCL) peptide S-7 (G 305 TTASSDTAITLIPR 319 –SEQ ID NO: 2) significantly improved survival and reduced pulmonary and aortic inflammation and hemorrhagic lung consolidation after MHV-68 infection. Serp-1 targets the coagulation pathway, including thrombolysis and thrombosis, and has been shown to have efficient anti-inflammatory functions. - / - Antibiotic treatment of mice abolished the efficacy of S-7 treatment against lethal MHV-68 infection and resulted in a significant decrease in survival. These modified peptides are very important.

[0125] Therapeutic modified S-7 peptides (MPS7-8, GTTASSDTAITLEPR, (SEQ ID NO: 5) and MPS7-9, GTTASSDTAITDEPR (SEQ ID NO: 6)), designed based on the serpin crystal structure, maintained efficacy after MHV-68 infection in this model and improved survival. When compared to the native S-7 peptide (Mahon et al., Biochemistry, 57, 1096-1107 (2018)), the MPS7 peptide had the expected increased hydrogen bonding.

[0126] The pathophysiological role of the gut bacterial microbiota in gammaherpesvirus infection and immunomodulatory therapy has not been previously investigated. In this example, the role of the gut bacterial microbiota in MHV-68 infection and the effects of Serp-1 and Serp-1-derived S-7 peptides on the treatment of MHV-68 infection were systematically examined.

[0127] method

[0128] Animal.IFNγR - / - Mouse (B6.129S7-Ifngr 1tm1Agt / J) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Animals were housed under barrier conditions in the Animal Care Services Animal House at the University of Florida and maintained under specific pathogen-free conditions. Mice were weaned at week 3, maintained on a 12-hour light-dark cycle, and had free access to food, water, and standard rodent chow.

[0129] Antibiotic treatment. At 4 weeks of age, IFNγR - / - The mouse colony was transferred from the ABSL1 colony to a separate ABSL2 colony. Intestinal microbiota suppression was achieved by replacing standard drinking water with autoclaved reverse osmosis water (obtained from the same animal care facility) for 10 days containing an antibiotic cocktail (Table 1) consisting of streptomycin (2 g / L), gentamicin (0.5 g / L), bacitracin (1 g / L), and ciprofloxacin (0.125 g / L). One day before infection (24 h), the medicated water was replaced with standard animal care facility water, which was maintained for the remainder of the experimental period.

[0130] Table 1 - Composition of Antibiotic Mixture

[0131]

[0132] MHV-68 infection. On day 11 (after 10 days of antibiotics), 12.5 × 10 6 0.1mL DMEM of MHV-68 of PFU was used to infect mice (5 weeks old). The mice were returned to the colony and monitored for signs of pain during the experiment. Mice were followed up for 150 days to determine survival, or euthanized 3 days after infection, and organs were collected in formalin for histology or RNAlater (Thermo Scientific, USA) for microbiome analysis. Table 2 details the number of MHV-68-infected mice in this study.

[0133] Table 2 - MHV-68-infected mice (73 mice in total)

[0134]

[0135] Sample processing of intestinal bacterial microbiota. Intestinal samples stored in RNAlater were processed using a miniprep kit (Zymo Research) for total genomic DNA isolation. Isolated DNA was quantified using a NanoDrop 2000C (Thermo Scientific) and stored at −80°C. Samples were analyzed at the KED Genomics Core at Arizona State University for whole-sample 16S rRNA gene amplicon sequencing. The DNA was prepared according to the protocol of the Earth Microbiome Project (World Wide Web address - earthmicrobiome.org / emp-standard-protocols / 16s). DNA library preparation for MiSeq platform. 16S primer set 515f-806r 86 was used for 2 × 150 paired-end sequencing.

[0136] 16S rRNA gene amplicon sequencing and analysis. Illumina MiSeq sequencing reads (2×150 bp) of the 16S rRNA gene V4 region87 of 34 samples were analyzed using QIIME 2 (version 2017.12)88: saline + Abx (n=6), saline no Abx (n=6), Serp-1 + Abx (n=6), Serp-1 no Abx (n=5), S7 + Abx (n=6), S7 no Abx (n=5). Sequencing reads were processed using DADA2, and amplicon sequence variants (ASVs) at a 97% identity threshold were inferred using the Greengenes database (version 13.8)89. To address the depth of inter-sample variability, all samples were pruned to 55,000 reads per sample (10 iterations) ( Figure 7A-7B ). One sample (12,107 reads; saline no Abx group) was removed due to insufficient reads, and the remaining 33 samples were retained for further analysis. ASV abundance, alpha diversity (Shannon diversity index), and beta diversity (UniFrac distance) were calculated using QIIME2. Statistical analysis (read depth, ASV abundance, and Shannon index comparison) was performed using the Mann-Whitney test. PCoA was performed using weighted unifrac distance in QIIME 2. To identify discriminant ASVs associated with the corresponding treatments, LEfSe (linear discriminant analysis effect size), DESeq2 (version 1.20.0), and likelihood ratio tests ( Figure 7C-Figure 7E). Discriminant analyses were first performed comparing Serp-1(no Abx) vs saline(no Abx) mice, S-7(no Abx) vs saline(no Abx) mice, and finally comparing the combined group of Serpin-treated mice [Serp-1(no Abx) and S-7(no Abx)] vs saline(no Abx mice). Discriminant ASVs identified in these analyses were pooled and validated in heatmap and abundance analyses ( Figure 4D-4F The total 16S sequence data for these samples have been deposited in the NCBI Sequence Read Archive under BioProject accession number PRJNA517927.

[0137] Histopathology. Mice were euthanized by carbon dioxide asphyxiation and subsequent cervical dislocation at 3 days of follow-up after infection. Organs were collected and fixed in 10% neutral buffered formalin. Samples were dehydrated by graded alcohol, embedded in paraffin, sectioned into 4-6 μm strips, captured on charged glass and dried overnight at 37°C before processing for histopathology. Slides were rehydrated, stained with Gill's hematoxylin No. 3 and eosin Y (H&E) according to standard procedures, dehydrated and mounted in Cytoseal XYL (Thermo Fisher Scientific, USA).

[0138] Quantitative Morphology. H&E-stained aorta, lung, and colon sections were imaged using a 20× / 0.5NA objective on an Olympus BX51 microscope equipped with an Olympus DP74 camera operated by cellSens Dimensions v1.16 software. Objective-calibrated measurements of alveolar septum thickness and luminal area were collected using cellSens Dimensions. At least 50 measurements were collected per mouse and averaged, and at least three mice were examined per group. Images were processed using ImageJ / FIJI v1.52i92 for visualization in the figures.

[0139] Immunohistochemistry. FFPE blocks containing lung tissue were cut into 4-6 μm strips, captured on loaded glass and dried overnight at 37°C before processing. The slides were rehydrated and epitopes were restored by boiling in sodium citrate buffer. Endogenous peroxidase was quenched with 3% hydrogen peroxide, slides were blocked in TBS / 0.1% Tween20 containing 5% bovine serum albumin, and sections were detected with rabbit polyclonal antibodies against CD3 (Abcam ab5690; 1:200), CD4 (Abcam ab183685; 1:1000) or CD8 (Abcam ab209775; 1:2000). For MHV-68 detection, 1:500 rabbit antiserum or preimmune serum (as a control) was used for immunostaining (a gift from Dr. HW, Virgin III9). 1:500 goat anti-rabbit HRP was used for secondary staining (Jackson Immuno Research 111-035-144). Antigens were visualized with ImmPACT DAB (Vector Labs, USA) and mounted with Cytoseal XYL. Sections were imaged using a 40× / 0.75NA objective.

[0140] Viral load was determined by qPCR. Total DNA was isolated from each FFPE sample (4 × 5 μm thick sections were used for each sample) using the QIAamp DNA FFPE Tissue Kit according to the manufacturer's instructions. Samples were quantified using a DS-11 series spectrophotometer / fluorimeter (Denovix, USA). Quantitative PCR (qPCR) was performed to investigate relative viral load using the following reactions for each sample: 10 μL SsoAdvanced Universal SYBR Green Supermix (Bio-radLaboratories Inc., USA), 0.4 μL each of primers 65F (5'-GTCAGGGCCCAGTCCGTA-3'; SEQ ID NO: 3) and 65R (5'-TGGCCCCTCTACCTTCTGTTGA-3'; SEQ ID NO: 4), 200 ng DNA, and a total volume of 20 μL of water. Reactions were performed in triplicate, including controls and standard curves, using the following cycling conditions for a CFX96 (Bio-rad Laboratories, Inc., USA) instrument: 95°C for 20 sec, 40 cycles (95°C for 15 sec, 60°C for 20 sec), followed by melting curve analysis.

[0141] Statistical analysis. Survival and pathological statistics were analyzed using GraphPad Prism v8.0.1. Kaplan-Meier survival statistics were calculated using the Log-rank (Mantel-Cox) test. For visualization, curves for each comparison are presented. Lung pathological statistics were compared using two-way ANOVA with Fisher's LSD or Tukey post hoc analysis test. Bars are mean ± standard error. P-values ​​less than 0.05 were considered significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns not significant.

[0142] result

[0143] Serp-1 protein and S-7 peptide treatment improved the survival of IFNγR- / - mice after MHV-68 infection.

[0144] MHV-68 infection establishes latency and chronic infection in wild-type mice, causing only flu-like symptoms. In contrast, intraperitoneal infection of mice with underlying immunodeficiencies, such as IFNγR- / - mice, results in lethal large vessel vasculitis, colitis, and hemorrhagic pneumonia, leading to early death. Intranasal infection is an alternative model for MHV-68 that is not a model of severe vasculitic syndromes. This intraperitoneal infection vasculitis model has been used to study the pathogenesis of gammaherpesvirus host-pathogen interactions and the potential for anti-inflammatory treatments for related inflammatory vasculitic syndromes. 12.5×10 6 pfu were intraperitoneally infected in IFNγR- / - mice and followed for 150 days to determine survival. MHV-68 infection with control saline alone for 30 days was lethal with a median survival of 41 days. IP injection of Serp-1 (100 ng / g) or S-7 (100 ng / g) for 30 days improved the survival rate at 150 days to 60% (p=0.0022 and p=0.0218, respectively) ( Figure 1B ). Thus, Serp-1 and S-7 protect IFNγR- / - mice after lethal MHV-68 infection.

[0145] Depletion of intestinal bacteria accelerates MHV-68-induced disease.

[0146] It has been hypothesized that the intestinal bacterial microbiota may play a role in the host-pathogen interaction in MHV-68-induced disease through cross-kingdom interactions. A systematic analysis of the effect of microbiota depletion on MHV-68-induced disease progression was completed ( Figure 2AThe mice were maintained on a medicated drinking water containing a broad-spectrum cocktail of four antibiotics: bacitracin, gentamicin, streptomycin, and ciprofloxacin (Table 1) to suppress the intestinal bacterial flora. During this period, the density of intestinal bacteria (as measured by colony-forming units [CFU] per gram of fecal pellets) decreased significantly, confirming a reduction in bacterial load ( Figure 2B Ten days later, mice were allowed to resume normal drinking water for 24 hours, during which time antibiotic treatment maintained suppression of the microbiota ( Figure 2B ). Mice were subsequently infected with MHV-68 by daily IP injections, as well as control saline injections daily for 30 days, and survival was assessed until day 150.

[0147] Suppression of intestinal bacteria by antibiotic treatment significantly accelerated the progression of MHV-68 infection. Saline-treated mice showed a median survival of 41 days, while antibiotic-treated mice had a median survival of 19 days (p = 0.0411; Figure 1B ). DNA extracts from the large intestine of individual mice were subsequently subjected to 16S rRNA gene amplicon sequencing 3 days after MHV-68 infection to assess early changes in the gut microbiota. Compared with mice with a stable bacterial microbiota before infection, a pronounced dysbiosis was detected in mice pretreated with antibiotics before MHV-68 infection ( Figure 2C This dysbiosis was associated with a non-statistically significant trend toward increased ASV abundance and diversity ( Figure 2D ; p = 0.329). Subgroup analysis showed that candidate ASVs were significantly altered in MHV-68-infected mice, which was consistent with antibiotic treatment ( Figure 2E , upper panel; increased abundance after antibiotics) or without antibiotic treatment ( Figure 2E , bottom panel; decreased abundance after antibiotics). These results suggest that the intestinal bacterial microbiota is an important determinant of MHV-68-induced disease progression.

[0148] The therapeutic efficacy of Serp-1 and S-7 peptides depends on the intestinal bacterial microbiota.

[0149] The possibility of a dependence of the therapeutic efficacy of Serp-1 and S-7 on the intestinal bacterial microbiota in MHV-68-infected IFNγR- / - mice was investigated. Mice were treated with medicated drinking water (Table 1) for 10 days, placed in normal water for 24 h, and then infected with MHV-68 and treated with Serp-1 or S-7 by IP injection for 30 days. Mice were followed for survival or 3 days follow-up ( Figure 2A ).

[0150] Serp-1 and S-7 peptides improved survival, with 60% of mice surviving up to 150 days after MHV-68 infection. In contrast, antibiotic-treated mice had accelerated disease, with a median survival of 19 days (Serp-1; p = 0.0025; Figure 1B ) and 18 days (S-7; p = 0.0339; Figure 1B ). The efficacy of Serp-1 or S-7 after antibiotic treatment was not significantly different from saline treatment after antibiotics (Serp-1+Abx vs saline+Abx, p=0.2012; S-7+Abx vs saline+Abx, p=0.9164). Therefore, the efficacy of Serp-1 or S-7 treatment and protection against lethal MHV-68 infection depends on the intestinal bacterial microbiota. Even with antibiotic treatment, the modified S-78 peptide once again improved survival.

[0151] Microbiome analysis identified candidate taxa associated with Serp-1 and S-7 treatment.

[0152] Based on the findings that (1) antibiotic-treated mice had accelerated and exacerbated MHV-68 disease, shortened survival, and increased mortality, and (2) immune protection conferred by Serp-1 and S-7 was highly sensitive to antibiotic treatment, the intestinal bacterial microbiota of mice treated with Serp-1 or S-7 was analyzed. DNA extracts from the large intestine of individual mice were subjected to 16S rRNA gene amplicon sequencing ( Figure 3A In the absence of antibiotics, Serp-1 and S-7 treated mice had higher microbiota diversity (mean = 1.270, p = 0.0079; mean = 0.862, p = 0.1508) compared to saline treated mice (mean = 0.385) ( Figure 3B ). Principal coordinate analysis showed that although the intestinal bacterial microbiota of Serp-1 and S-7 treated mice was different from that of saline treated mice, there were differences within Serp-1 and S-7 treated mice ( Figure 3C , Figure 7B ). This finding is consistent with the phenotypic variability observed in vivo. Specifically, treatment with Serp-1 and S-7 conferred partial protection, with 60% survival.

[0153] It was hypothesized that responses to Serp-1 and S-7 treatment could be due to the presence of “protective” ASVs (differentially associated with Serp-1 and / or S-7 treatment) or the absence of “enhancing” ASVs (differentially associated with saline treatment). Therefore, to identify potential microbes responsible for microbiota-mediated protection by Serp-1 and S-7, the gut bacterial microbiota was analyzed to identify discriminant ASVs that differentially appeared in Serp-1 and / or S-7 treated mice compared with saline treated mice in the absence of antibiotics. Figure 7C-Figure 7E Of the 15 ASVs identified as differentially associated with Serp-1 / peptide treatment, ASV4 was consistently associated with both Serp-1 and S-7 treatment ( Figure 3D ). ASV4 was most closely related to sequences from Bacillus massiliogorillae. In contrast, ASV1 (most closely related to Frondihabitans peucedani) and ASV123 (most closely related to Enterococcus saigonensis) were identified from eight candidate ASVs as being associated with saline treatment when compared to Serp-1 or S-7 treatment. Based on the fact that Serp-1 and S-7 protected mice in an antibiotic-dependent manner, it was reasonable to infer that the relative abundance of the distinguishing ASVs would be altered in mice pretreated with antibiotics. As predicted, the relative abundance of Serp-1 or S-7-associated ASVs was reduced in antibiotic-pretreated mice, although the reduction was more modest in S-7 ( Figure 3E and Figure 3F , comparing "no Abx" to "+Abx"). These results suggest that crosstalk between bacterial microbiota associated with a protective phenotype (such as ASV4) and microbiota associated with an exacerbated phenotype (such as ASV1 and ASV123) can influence the outcome of immunomodulatory therapy. In conclusion, the response to immunomodulatory therapy in MHV-68-induced disease is associated with specific alterations in the intestinal bacterial microbiota.

[0154] The sequence of ASV is as follows:

[0155] ASV1

[0156] (k_bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Enterococcaceae; g_Enterococcus; s_Enterococcus saigonensis):

[0157]

[0158] ASV4

[0159] (k = bacteria; p = Firmicutes; c = Bacilli; o = Bacillales; f = Bacillaceae; g = Bacillus; s = Bacillus massiliogorillae):

[0160]

[0161] ASV123

[0162] (k_bacteria; p_Actinobacteria; c_Actinobacteria; o_Micrococcales; f_Microbacteriaceae; g_Frondihabitans; s_Frondihabitans peucedani):

[0163]

[0164] Antibiotic-dependent exacerbation of MHV-68-induced lung pathology early in the disease.

[0165] Persistent infection with MHV-68 results in severe hemorrhagic and consolidative lung pathology. Therefore, it was investigated whether the lung pathology induced by MHV-68 infection reflects the antibiotic-dependent deterioration observed in the survival analysis. Quantitative morphometric analysis of the lungs was performed at an early follow-up visit 3 days after infection. In the absence of antibiotics, considerable early lung consolidation and inflammation were observed in MHV-68 infected lung tissues in IFNγR- / - mice treated with control saline alone. Treatment with Serp-1 or S-7 peptides greatly reduced this severe lung pathology ( Figure 4A Early lung pathology was significantly worse after antibiotic treatment in all conditions (saline, Serp-1, or S-7), with the majority of animals in the saline group also showing hemorrhages in the affected areas.

[0166] Thickening of alveolar walls / septa and reduction in alveolar space area have been shown to be reliable indicators of lung inflammation in acute laboratory models. In the present study, quantitative lung morphometry revealed a significant reduction in alveolar wall thickness in MHV-68-infected mice treated with Serp-1 or S-7 following antibiotic treatment, compared with no significant reduction in saline-treated controls ( Figure 4BIn the absence of antibiotic pretreatment, alveolar space area was significantly increased in S-7 treated mice compared with saline treated controls (p = 0.0067), while there was a trend toward an increase in Serp-1 treated mice. Antibiotic treatment negated any level of protection from S-7 treatment ( Figure 4C ). At the early follow-up of 3 days, no significant changes in diagnostic pathology or inflammatory cell infiltration were noted in the intestine or aorta in histological analysis. These results suggest that the interaction between bacterial microbiota associated with a protective phenotype (such as ASV4) and microbiota associated with an exacerbated phenotype (such as ASV1 and ASV123) can affect the outcome of immunomodulatory therapy. In summary, the response to immunomodulatory therapy in MHV-68-induced disease is associated with specific changes in the intestinal bacterial microbiota. These results suggest that the immune protection against lung inflammation and early bleeding promoted by Serp-1 and S-7 treatment is microbiota-dependent.

[0167] Serp-1 and S-7 promote gut microbiota-dependent increase in CD3+ occupancy in the lung.

[0168] T cell responses play a crucial role in limiting active infection and have been reported as central mediators in the management of chronic MHV-68 infection. Therefore, recruitment of early acute phase CD3+ T cells to the lungs during MHV-68 infection was examined. Antibiotic treatment significantly reduced CD3+ cells in the lungs of saline-treated mice ( Figure 4E , p = 0.0155). In addition, S-7 increased significantly, Serp-1 tended to increase, and CD3+ cells ( Figure 4D , Figure 4E ), indicating that serpin-mediated protection against MHV-68-induced disease is closely associated with increased lung T cell activity. Further staining showed that Serp-1 and S-7 had no significant effect on lung CD4+ cells ( Figure 4F ), while CD8+ staining showed a significant increase with S-7 treatment (p=0.0312), and a strong increasing trend with Serp-1 treatment (p=0.1734) ( Figure 4G Notably, antibiotic treatment to deplete the gut microbiota resulted in loss of this observed CD3 infiltration-promoting effect as well as loss of the CD8 bias of Serp-1 and S-7 ( Figure 4E-Figure 4G Taken together, these results suggest that Serp-1- or S-7-enhanced protection against MHV-68-induced disease via CD3 recruitment and surveillance in the lung via CD8 tropism, through a microbiota-dependent mechanism.

[0169] Serp-1 and S-7 reduce lung MHV-68 levels in a gut bacterial microbiota-dependent manner.

[0170] Since T cells are involved in controlling MHV-68 infection, it was hypothesized that the promotion of CD3 T-cell infiltration would result in a reduction in viral levels in the lungs. Antibiotic treatment did not alter the amount of MHV-68 antigen staining in the lungs of mice treated with saline controls alone ( Figure 5A , Figure 5B However, Serp-1 and S-7 significantly reduced the level of MHV-68 antigen in the lungs of mice not treated with antibiotics ( Figure 5B ; Serp-1, p=0.0009; S-7, p=0.0003). These reductions in detectable MHV68 were partially reversed after antibiotic pretreatment in MHV-68-infected mice ( Figure 5A , Figure 5B and Figure 8 ). Thus, Serp-1 and S-7 initiate the reduction of MHV-68 levels in the lung in a gut bacterial microbiota-dependent manner.

[0171] discuss

[0172] Compelling evidence has established an important role for the gut microbiota in immune responses and in the pathogenesis of a variety of diseases, including those with predominantly extraintestinal involvement, such as respiratory, liver, kidney, neurologic, autoimmune, rheumatoid, and cardiovascular conditions. Several groups have demonstrated that targeted manipulation of the gut microbiota to correct dysbiosis, such as with fecal transplants, may be helpful in treating certain diseases. For example, a small randomized clinical trial (NCT02636647) found that fecal microbiota transplantation from a single, rationally selected donor enriched in Lachnospiraceae and Ruminococcaceae (taxa identified as eliminated in patients with hepatic encephalopathy) improved outcomes in patients with treated recurrent hepatic encephalopathy. A second trial (NTR1776) found that the transfer of fecal microbiota from lean donors to obese donors partially reversed symptoms of the metabolic syndrome, including improvements in insulin resistance. In addition to correcting dysbiosis, recent evidence supports the hypothesis that microbiota composition also plays an important role in determining the efficacy of certain treatments. Low gut microbiota levels of Akkermansia muciniphila were recently found to result in nonresponse in patients receiving PD-1 blockade immunotherapy for epithelial tumors, reinforcing the importance of the microbiota in treatment efficacy, as highlighted by an earlier key study identifying a positive association between Bacteroides fragilis and efficacy of anti-CTLA4 therapy for melanoma. Alternatively, certain treatments may inadvertently alter the gut microbiota and predispose it to increased pathogen infection. For example, it was recently demonstrated that proton pump inhibitors significantly reduced microbial diversity, altering more than 20% of bacterial taxa, leading to an increase in enterococci, streptococci, staphylococci, and potentially pathogenic Escherichia coli species. Therefore, it is of great interest to understand how the gut microbiota influences new treatments, and how it can be influenced.

[0173] Cross-kingdom interactions may also play a key role in determining disease pathogenesis in mammalian hosts. For viruses that require intestinal bacteria for replication, antibiotics may reduce viral loads. Many studies have demonstrated such virus-bacteria interactions in laboratory systems, such as murine and human norovirus infections. However, for other viruses, loss of the bacterial microbiota may lead to worse clinical disease. A recent study reported that oral antibiotic (ampicillin) treatment exacerbated disease severity in flavivirus infections by modulating flavivirus-specific CD8 responses, suggesting an interaction between the microbiota, the immune system, and viruses. These different observations, which largely depend on (a) the virus being studied, (b) the host system, and (c) the antibiotic regimen, highlight why the virome remains a poorly understood and therefore underestimated component of health and disease.

[0174] The researchers found that suppressing the gut microbiota prior to MHV-68 infection accelerated disease, with increased lethality and early death occurring at about twice the rate of untreated mice ( Figure 2C This acceleration of MHV-68 disease was accompanied by significant dysbiosis following antibiotics, with a panel of candidate ASVs with increased or decreased relative abundance associated with worse disease outcomes.

[0175] Furthermore, our studies demonstrate that the protective immune responses induced by Serp-1 or S-7 require bacterial-microbiota interactions. Depletion of the microbiota completely abolished the therapeutic efficacy and improved survival ( Figure 1B ). Further exploration revealed that Serp-1 and S-7 promoted protection against lung inflammation and consolidation, which was associated with increased CD3+ cell invasion (with a CD8+ bias) and reduced MHV-68 staining in the lungs ( Figure 4A-4G and Figure 5A , Figure 5B This finding suggests that T cell surveillance (affected by CD8+ cells) is important for limiting acute GHV infection, and that the loss of efficacy of Serp-1-based treatments is not expected to be due to a direct interaction between antibiotics and immunomodulatory treatments for a number of reasons: (1) the antibiotics used in this study have relatively short half-lives (1–7 h), and previous studies have reported negligible concentrations 24 h after administration—a timeframe incorporated into the design of this study; (2) the microbial burden was observed to recover rapidly within three days ( Figure 2B); (3) the antibiotics in the current mixture are not known to suppress immune responses on their own and have been observed to enhance T cell activity in some reports; (4) the loss of activity is not expected to be due to a direct interaction with the antibiotics, since in vitro inhibition of uPA activity by Serp-1 is not affected by the presence of the same antibiotic mixture ( Fig. 9 ). A recent report by Yang et al. suggests that in the short term, antibiotics can induce metabolite changes by altering respiratory activity, which have a systemic inhibitory effect on immune function in the peritoneal space (Yang, JH et al., Cell Host Microbe 22, 1-9 (2017)). However, Yang et al. used Escherichia coli infection, and the antibiotic ciprofloxacin was given within 4 hours after infection and maintained during the infection. Here, a gammaherpesvirus infection with longer pathogenic kinetics was used, and the antibiotics were removed 24 hours before infection, with no trace of ciprofloxacin detected, and the inventors are not aware of any other reports indicating that other antibiotics in the currently used mixture have immunosuppressive effects. In summary, these data provide the first evidence that microbiota interactions are essential for protective immune responses, which are induced here by Serp-1 and S-7 treatment. We found that Serp-1, acting through the urokinase-type plasminogen activator receptor (uPAR) in this system, is involved in immune cell infiltration (e.g., regulating the levels of binding partners) that is essential for the function of Serp-1 and S-7 in MHV-68 infection.

[0176] The inventors believe that host innate immunity requires cross-kingdom interactions with the intestinal bacterial microbiota to generate a protective response against lethal gammaherpesvirus disease ( Figure 6 ). When MHV-68 infection leads to viral lung inflammation and sepsis, as induced in the MHV-68 infection model, the host innate immune response mediated by gut bacterial microbiota interactions leads to the recruitment of immune cells (e.g. T cells) to the lungs. However, antibiotic elimination of the gut bacterial microbiota alters the pathological outcome by preventing an effective immune response, leading to an acceleration of the MHV-68 inflammatory pathogenesis. Immunomodulatory treatments (e.g. Serp-1 and S-7) can induce stronger innate immune responses (e.g. T cell recruitment) through (other) bacterial microbiota interactions. Therefore, the outcome of GHV infection depends on the mutual influence of host innate immunity and microbiota interactions.

[0177] Interestingly, the intestinal microbiota of all Serp-1 and S-7 treated mice did not cluster clearly apart from saline treated mice. Thus, the dynamics of selected increased or decreased microbiota ASVs were associated with exacerbated pathology or protective immunomodulation. Specific ASVs were found to be increased in response to Serp-1 / peptide treatment (e.g. ASV4) or decreased compared to saline treatment (e.g. ASV1 and ASV123). Overall, these findings suggest that the response to immunomodulation in MHV-68 disease involves a combination of interactions with both the protective microbiota and the pathology exacerbating microbiota.

[0178] In this example, we demonstrate that the pathogenicity of MHV-68 infection is substantially accelerated by the use of antibiotics to suppress the intestinal bacterial microbiota, and that immunomodulatory serpins (Serp-1) and serpin peptides (S-7) are effective treatments. MHV-68 is a model virus used to study GHV pathogenesis and severe viral sepsis, so this finding has important implications for the pathogenesis and treatment of other GHVs (such as KSHV and EBV), which have been clinically recognized to have adverse reactions to antibiotics. We further report here the regulation of the composition of the intestinal microbiota by immunomodulatory proteins and peptides. In summary, (1) changes in the microbiota have a significant impact on therapeutic immunomodulatory proteins and peptides, and (2) antibiotic treatment increases the severity of GHV infection.

[0179] Although the present disclosure has been described with emphasis on specific embodiments, it will be apparent to those of ordinary skill in the art that variations of the specific embodiments may be used, and it is intended that the present disclosure may be practiced in a manner other than that specifically described herein. Features, characteristics, compounds, or embodiments described in conjunction with a particular aspect, embodiment, or example of the present invention should be understood to apply to any other aspect, embodiment, or example of the present invention. Therefore, the present disclosure includes all modifications contained within the spirit and scope of the present disclosure as defined by the following claims. Therefore, we claim all that falls within the scope and spirit of these claims as our invention.

Claims

1. Use of a composition for the preparation of a medicament, the composition comprising a polypeptide derived from the reactive center loop of a serine protease inhibitor or a biologically active variant thereof, the medicament being used to prevent, reduce or inhibit a viral or immune disorder in a subject at risk of or already suffering from a viral or immune disorder, wherein the subject's intestinal microbiota comprises bacteria expressing 16S rRNA comprising the sequence of amplicon sequence variant 4 (ASV4), and the subject has not been administered antibiotics within at least one day of administering the composition.

2. The use according to claim 1, wherein the polypeptide has an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

1.

3. The use according to claim 1, wherein the polypeptide has an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

1. The use according to claim 1, wherein the polypeptide has an amino acid sequence as shown in GTTASSDTAITLIPR (SEQ ID No: 2).

5. The use according to any one of claims 1 to 4, wherein the composition further comprises a carrier.

6. The use according to claim 5, wherein the carrier is 0.9% saline, or citrate buffered saline (CBS), or phosphate buffered saline (PBS), or Ringer's lactate solution (RL), or Ringer's acetate solution (AR), or 5% or 10% glucose in saline (D5NS, D10NS), or 5% or 10% glucose in semi-normal saline (D5HNS, D10HNS), or Hank's balanced salt solution (HBSS), or Earle's balanced salt solution (EBSS), or Gey's balanced salt solution (GBSS), or TRIS buffered saline (TBS), or HEPES buffered saline (HBS).

7. The use according to any one of claims 1 to 4, wherein the composition is formulated for subcutaneous, intravenous or intramuscular administration.

8. The method of claim 1, wherein the virus or immune disorder is gammaherpesvirus (GHV), Epstein-Barr virus (EBV) and / or Kaposi's sarcoma-associated herpesvirus (KSHV).

9. The method according to claim 8, wherein the GHV is murine gammaherpesvirus-68 (MHV-68).

10. The use according to claim 8, wherein the GHV is an acute or chronic GHV infection.

11. The use according to any one of claims 1 to 4, wherein the immune disorder is inflammatory vasculitis syndrome (IVS).

12. A kit for preventing, reducing or inhibiting a viral or immune disorder in a subject at risk of or already suffering from a viral or immune disorder, the kit comprising a composition and instructions for use, the composition comprising a polypeptide derived from the reactive center loop of a serine protease inhibitor or a biologically active variant thereof, wherein the instructions indicate that the subject must not be administered antibiotics within at least one day of using the kit, and the subject's intestinal microbiota has been characterized as comprising bacteria expressing 16S rRNA comprising the sequence of amplicon sequence variant 4 (ASV4).

13. The kit of claim 12, wherein the polypeptide has an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

1.

14. The kit of claim 12, wherein the polypeptide has an amino acid sequence that has at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

1. The kit according to claim 12 , wherein the polypeptide has an amino acid sequence as shown in GTTASSDTAITLIPR (SEQ ID No: 2).

16. The kit according to any one of claims 12 to 15, wherein the composition further comprises a carrier.

17. The kit according to claim 16, wherein the carrier is 0.9% saline, or citrate buffered saline (CBS), or phosphate buffered saline (PBS), or Ringer's lactate solution (RL), or Ringer's acetate solution (AR), or 5% or 10% glucose in saline (D5NS, D10NS), or 5% or 10% glucose in half-normal saline (D5HNS, D10HNS), or Hank's balanced salt solution (HBSS), or Earle's balanced salt solution (EBSS), or Gey's balanced salt solution (GBSS), or TRIS buffered saline (TBS), or HEPES buffered saline (HBS).

18. Use of a composition for the preparation of a medicament, the composition comprising a polypeptide derived from a reactive center loop of a serine protease inhibitor or a biologically active variant thereof, the medicament being used to alter the intestinal microbiota in a subject, wherein the subject has not been administered antibiotics within at least one day of administering the composition, and the composition increases the amplicon sequence variant 4 (ASV4) microbiota in the subject's intestinal microbiota and / or decreases the amplicon sequence variant 1 (ASV1) and amplicon sequence variant 123 (ASV123) microbiota in the subject's intestinal microbiota.

19. The use according to claim 18, wherein the polypeptide has an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

1.

20. The use according to claim 18, wherein the polypeptide has an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

1.

21. The use according to claim 18, wherein the polypeptide has an amino acid sequence as shown in GTTASSDTAITLIPR (SEQ ID No: 2).

22. The use according to any one of claims 18 to 21, wherein the subject is a subject at risk of or already suffering from a viral or immune disorder.

23. The use according to claim 22, wherein the virus or immune disorder is gammaherpesvirus (GHV), Epstein-Barr virus (EBV) and / or Kaposi's sarcoma-associated herpesvirus (KSHV).

24. The use according to claim 23, wherein the GHV is murine gammaherpesvirus-68 (MHV-68).

25. The use according to claim 23, wherein the GHV is an acute or chronic GHV infection.

26. The use according to claim 22, wherein the immune disorder is inflammatory vasculitis syndrome (IVS).

Images