WO2018085937A1 - Managing site specific ibd immunotherapies - Google Patents

Managing site specific ibd immunotherapies Download PDF

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Publication number
WO2018085937A1
WO2018085937A1 PCT/CA2017/051344 CA2017051344W WO2018085937A1 WO 2018085937 A1 WO2018085937 A1 WO 2018085937A1 CA 2017051344 W CA2017051344 W CA 2017051344W WO 2018085937 A1 WO2018085937 A1 WO 2018085937A1
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WIPO (PCT)
Prior art keywords
prr
treatment
snp
agonists
subject
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PCT/CA2017/051344
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French (fr)
Inventor
Harold David Gunn
David W. MULLINS
Shirin KALYAN
Momir BOSILJCIC
Monan Angela ZHANG
Mark BAZETT
Marcel THALEN
Dermot MCGOVERN
Boyko Traychev KABAKCHIEV
Ho Pan SHAM
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Qu Biologics Inc.
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Priority claimed from PCT/CA2017/050513 external-priority patent/WO2017185180A1/en
Application filed by Qu Biologics Inc. filed Critical Qu Biologics Inc.
Publication of WO2018085937A1 publication Critical patent/WO2018085937A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Methods are provided for predicting the degree to which a patient has an increased likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists.
  • Methods may include screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the subject has an increased likelihood of benefiting from said treatment if the diagnostic SNP comprises a selected response allele.
  • Methods are also provided for assessing the degree to which a patient is responding to a particular immunomodulatory treatment regimen.
  • Figure 1 is a series of graphs illustrating efficacy of SSI treatment in alternative model animals in the colitis model: a logarithmic Y axis scale illustrating relative levels of IFN-gamma (A) and IL-17A expression (B), and cumulative data for IL-17A expression (C), as well as site-specific evidence of QBECO efficacy in increasing I L-18 gene expression in colon tissue, compared to QBKPN (D).
  • A IFN-gamma
  • B IL-17A expression
  • C cumulative data for IL-17A expression
  • D site-specific evidence of QBECO efficacy in increasing I L-18 gene expression in colon tissue
  • Figure 2 is a series of graphs illustrating efficacy of SSI treatment in alternative model animals in the colitis model: microbiome (A and B) and histology (C).
  • Figure 3 includes three bar graphs illustrating the activation of pattern recognition receptors in HEK cells after QBECO or QBKPN stimulation, showing respectively: A) Toll-like receptor (TLR) activation as measured by ⁇ - ⁇ activation; B) NOD2 and C-type lectin receptors (CTL) as measured by ⁇ - ⁇ activation; and, C) RLR (Rig-1 -like receptors) as measured by IRF3 activation.
  • TLR Toll-like receptor
  • CTL C-type lectin receptors
  • Figure 4 is a PRR repertoire fingerprint bar graph, in which a PRR fingerprint was constructed for QBECO and QBKPN SSIs from the 1/10 dilution data, after subtracted the negative control data. Bars, in order, represent TLR2, 3, 4, 5, 7, 8, 9, NOD1 , NOD2, Dectin 1 a, Dectin 1 b and Mincle. RIG-1 and MDA5 are not shown. The positive control is specific for each PRR (i.e. LPS for TLR4).
  • Figure 5 is a PRR fingerprint radar graph, in which a PRR fingerprint was constructed for QBECO and QBKPN SSIs from the 1/10 dilution data, after subtracted the negative control data, and plotted on a radar graph.
  • Figure 6 is a collection of 3 bar graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure 7 is a collection of 4 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure 8 is a collection of 4 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure 9 is a column scatter graph illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure 10 is a collection of 5 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure 11 is a collection of 4 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure 12 is a collection of 3 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure 13 is a collection of 4 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure 14 is a collection of 4 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure 15 is a collection of 5 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure16 is a collection of 6 bar graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
  • Figure 17A is a violin plot representing the log distribution of risk scores, comparing last recorded response for all CD subjects using risk scores based on 1 12 IBD SNPs (P-value: 2.430E-05).
  • Figure 17B is a violin plot representing the log distribution of risk scores, comparing last recorded response for all CD subjects using risk scores based on 3 IBD SNPs (P-value: 1.385E-04).
  • Figure 18 is a violin plot representing the log distribution of risk scores, comparing last recorded response for all UC subjects using risk scores based on 84 IBD SNPs (P-value: 1.255E-02).
  • Figure 19 is a violin plot representing the log distribution of risk scores, comparing last recorded response for all CD and UC subjects using risk scores based on 1 12 IBD SNPs (P-value: 8.184E-07).
  • Figure 20 is a graph illustrating the change in serum IL-18 levels in patients treated with QBECO vs. Placebo.
  • Figure 21 is a set of 4 graphs illustrating serum immune cytokine changes with QBECO treatment that associated with clinical response.
  • Figure 22 is a set of 3 graphs illustrating baseline levels of Eotaxin-1 , IL- 10 and IL-12p40 by patient response to QBECO.
  • Figure 23 is a set of line graphs, illustrating changes in cytokine concentration over time in the sera of ulcerative colitis patients undergoing a QBECO SSI therapy. In each case, Responders are the top line.
  • Figure 24 is a graph illustrating the change in body weight over time in a murine DSS colitis model.
  • Figure 25 is a graph illustrating change in disease activity index over time in a murine DSS colitis model.
  • Figure 26 is a graph illustrating change in the FITC-dextran assay over time in a murine DSS colitis model.
  • an "immunogen” refers to a molecule, or a composition comprising the molecule, that is capable of eliciting an immune response by an organism's immune system.
  • An “antigen” refers to a molecule that is capable of binding to the product of an immune response.
  • Pathogenic agents are agents, such as microbes, such as bacteria or viruses, which are known to cause infection in a host in nature, and in this sense, "pathogenic” is used in the context of the present invention to mean “naturally pathogenic”. Although a wide variety of microbes may be capable of causing infection under artificial conditions, such as artificial inoculations of a microbe into a tissue, the range of microbes that naturally cause infection is necessarily limited, and well established by medical practice.
  • An "infection” is the state or condition in which the body or a part of it is invaded by a pathogenic agent (e.g., a microbe, such as a bacterium) which, under favorable conditions, multiplies and produces effects that are injurious (Taber's Cyclopedic Medical Dictionary, 14th Ed. , C.L. Thomas, Ed., F.A. Davis Company, PA, USA).
  • a pathogenic agent e.g., a microbe, such as a bacterium
  • An infection may not always be apparent clinically and may result in only localized cellular injury. Infections may remain subclinical, and temporary if the body's defensive mechanisms are effective. Infections may spread locally to become clinically apparent as an acute, a subacute, or a chronic clinical infection or disease state.
  • a local infection may also become systemic when the pathogenic agent gains access to the lymphatic or vascular. Infection is usually accompanied by inflammation, but inflammation may occur without infection.
  • Inflammation is the characteristic tissue reaction to injury (marked by swelling, redness, heat, and pain), and includes the successive changes that occur in living tissue when it is injured. Infection and inflammation are different conditions, although one may arise from the other (Taber's Cyclopedic Medical Dictionary, supra). Accordingly, inflammation may occur without infection and infection may occur without inflammation (although inflammation typically results from infection by pathogenic bacteria or viruses). Inflammation is characterized by the following symptoms: redness (rubor), heat (calor), swelling (tumour), pain (dolor). Localized visible inflammation on the skin may be apparent from a combination of these symptoms, particularly redness at a site of administration.
  • a subject is an animal, for e.g. , a vertebrate or a mammal. Accordingly, a subject may be a patient, e.g., a human, suffering from an immune dysregulation. A subject may also be an experimental animal, e.g., an animal model of an immune dysregulation. In some embodiments, the terms "subject” and “patient” may be used interchangeably, and may include a human, a non-human mammal, a non-human primate, a rat, mouse, or dog.
  • a healthy subject may be a human who is not suffering from a disease, such as immune dysfunction, or suspected of having the disease, or who is not suffering from a chronic disorder or condition.
  • a "healthy subject” may also be a subject who is not immunocompromised.
  • immunocompromised is meant any condition in which the immune system functions in an abnormal or incomplete manner. Immunocompromisation may be due to disease, certain medications, or conditions present at birth. Immunocompromised subjects may be found more frequently among infants, the elderly, and individuals undergoing extensive drug or radiation therapy.
  • sample from a subject may include any relevant biological material, including for example a cell, tissue or bodily fluid sample taken from a patient.
  • a sample may conveniently include samples of skin, cheek, blood, stool, hair or urine.
  • Sample nucleic acids for use in diagnostic and prognostic methods can for example be obtained from a selected cell type or tissue of a subject.
  • a subject's bodily fluid e.g. blood
  • nucleic acid tests can be performed on dry samples (e.g., hair or skin).
  • polymorphism refers to a location within a biological sequence, such as a genomic sequence, which varies within a population.
  • Polymorphisms are comprised of different "alleles".
  • the term "genotype” refers to the specific alleles in a genome, for example in a cell, tissue sample or an individual.
  • the location of a polymorphism may be identified by its position, for example within the genome or within a sequence such as a protein that is reflective of a genomic locus. This may for example be provided in the form of a
  • genotype is typically comprised of at least two alleles, which may be the same (homozygous) or different (heterozygous).
  • Individual polymorphisms are typically assigned unique identifiers in the art (such as "Reference SNP”, “refSNP” or “rs#”), for example in the Single Nucleotide Polymorphism Database (dbSNP) of Nucleotide Sequence Variation available on the NCBI website.
  • dbSNP Single Nucleotide Polymorphism Database
  • Characterization of polymorphisms, alleles or a genotype may be performed by any of very wide variety of methods. These methods may for example variously involve hybridization, labeling, cloning, sequencing and/or amplification of nucleic acids, such as genomic DNA, for example using PCR, LCR, xMAP, invader assays, mass spectrometry, pyrosequencing, selective oligonucleotide
  • probes includes naturally occurring or recombinant single- or double-stranded nucleic acids or chemically synthesized nucleic acids.
  • a probe can for example be a polynucleotide of a length suitable for selective hybridization to a nucleic acid containing a polymorphic region. Labeled probes also can be used in conjunction with amplification of a polymorphism.
  • DNA microarray technologies sometimes referred to as DNA chips or gene chips, may for example be used for genomic characterization, for example to characterize point mutations, single nucleotide polymorphisms (SNPs), and/or short tandem repeats (STRs).
  • SNPs single nucleotide polymorphisms
  • STRs short tandem repeats
  • the presence of the specific allele in DNA from a subject can in some case be characterized by restriction enzyme analysis.
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide
  • DNA/DNA, or RNA/DNA heteroduplexes using technique that may be described as "mismatch cleavage" assays. Alterations in electrophoretic mobility may be used to characterize allelic variants, for example to detect single strand conformation polymorphisms.
  • kits for example comprising at least one probe or primer nucleic acid, or one of more of the compositions described herein and instructions for use of the kit.
  • Kits can for example comprise at least one probe or primer which is capable of specifically hybridizing to a polymorphic region or adjacent to the polymorphic region, so that the oligonucleotides are "specific for" the polymorphic region.
  • Kits may also comprise at least one reagent necessary to perform a particular assay.
  • Kits can also include positive controls, negative controls, sequencing markers, or antibodies, for example for determining a subject's genotype or biological marker profile.
  • An "immune response” includes, but is not limited to, one or more of the following responses in a mammal: induction or activation of antibodies, neutrophils, monocytes, macrophages (including both M1 -like macrophages and M2-like macrophages as described herein), B cells, or T cells (including helper T cells, natural killer cells, cytotoxic T cells, gamma-delta ( ⁇ ) T cells), such as induction or activation by one or more immunogens in an immunogenic composition, following administration of the composition.
  • An immune response to a composition thus generally includes the development in the host animal of a cellular and/or antibody- mediated response to the composition.
  • the immune response is such that it will also result in slowing or stopping the progression of an immune dysregulation, or a disease characterized by immune dysregulation.
  • An immune response may accordingly include one or both of a cellular immune response and/or a humoral immune response, and may be an adaptive response or an innate immune response.
  • "Immune dysregulation" is an inappropriately regulated immune response, such as an inappropriately restrained or inappropriately robust immune response.
  • the immune dysregulation may for example be in the context of an autoimmune, inflammatory, or degenerative disease (such as Crohn's disease, inflammatory bowel disease or ulcerative colitis).
  • I BD Inflammatory bowel disease
  • I BD is a name frequently given to a group of inflammatory conditions of the colon and small intestine, generally characterized by similar symptoms of immune
  • IBD indeterminate etiology.
  • Major sub-types of IBD are recognized clinically as Crohn's disease and ulcerative colitis.
  • IBD may also include conditions recognized as any one of the following: collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome or indeterminate colitis. The difference between these conditions relate primarily to the location and nature of the inflammatory changes in the gastrointestinal tract (GIT).
  • GIT gastrointestinal tract
  • Crohn's disease for example, is generally recognized as potentially affecting any part of the gastrointestinal tract, from mouth to anus, with a majority of the cases marked by relapsing and remitting
  • Ulcerative colitis in contrast, is generally considered to be restricted to the colon and the rectum.
  • the various regions of the gastrointestinal tract in which these inflammatory conditions may exhibit symptoms include: the bowel or intestine, including: the small intestine (which has three parts: the duodenum, the jejunum , and the ileum); the large intestine (which has three parts: the cecum, the colon, which includes the ascending colon, transverse colon, descending colon and sigmoid flexure; and the rectum); and, the anus.
  • a "site specific immunotherapy” is an immunomodulatory treatment that is effective to therapeutically or prophylactically alter an aspect of the immune state, or immune system physiology, at an anatomical site or sites, such as an organ or tissue.
  • an SSI may be adapted to ameliorate an immune dysregulation, or to treat a condition characterized by an immune dysregulation.
  • PRR Liqands may for example be available commercially, for example in widely available preparations of attenuated or killed recombinant bacteria, which may for example be ligands for TLR2, TLR4 and TLR5.
  • compositions of pathogen-associated molecular patterns may include PAMPS that are recognized by PRRs, including: Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-l-like receptors (RLRs), C-type lectin receptors (CLRs) including Dectin-1 , cytosolic dsDNA sensors (CDSs) and NLRs involved in the formation of inflammasomes.
  • PRRs Toll-like receptors
  • NLRs NOD-like receptors
  • RLRs RIG-l-like receptors
  • CLRs C-type lectin receptors
  • Dectin-1 Dectin-1
  • CDSs cytosolic dsDNA sensors
  • NLRs cytosolic dsDNA sensors
  • Toll-like receptor 2 (TLR2) is involved in the recognition of a wide array of microbial molecules representing broad groups of species including Gram-positive and Gram-negative bacteria, as well as mycoplasma and yeast. TLR2 recognizes cell-wall components such as peptidoglycan, lipoteichoic acid and lipoprotein from Gram-positive bacteria, lipoarabinomannan from mycobacteria, and zymosan from the yeast cell wall. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA).
  • dsRNA double-stranded RNA
  • LPS Bacterial lipopolysaccharide
  • TLR4 Toll-like receptor 4
  • LBP LPS- binding protein
  • CD14 CD14
  • MD-2 myeloid differentiation protein 2
  • LPS generally consists of a polysaccharide region that is anchored in the outer bacterial membrane by a carbohydrate lipid moiety: lipid A, which is largely responsible for the immunostimulatory activity of LPS.
  • lipid A contains six fatty acyl groups, as for example may be found in pathogenic bacteria that are strains of Escherichia coli or Salmonella spp.
  • Toll-like receptor 5 (TLR5) recognizes flagellin from both Gram-positive and Gram-negative bacteria.
  • Toll-like receptor 7 (TLR7) and TLR8 recognize single stranded RNAs and small synthetic molecules such as imidazoquinolines and nucleoside analogs.
  • Tolllike receptor 9 (TLR9) recognizes specific unmethylated CpG motifs prevalent in microbial but not vertebrate genomic DNA.
  • NLRs are a family of at least 22 cytoplasmic innate immune sensors, including NOD1 (CARD4) and NOD2 (CARD15) which are intracellular pattern- recognition receptors involved in the recognition of peptidoglycan (PGN). These receptors detect specific motifs within PGN.
  • NOD1 senses the diaminopimelatic acid (DAP)-containing muropeptide (specifically d-Glu-meso-DAP dipeptide "iE- DAP” dipeptide) which is found primarily in PGN of Gram-negative bacteria, as well as certain Gram-positive bacteria.
  • NOD2 recognizes the muramyl dipeptide (MDP) structure found in almost all bacterial PGN.
  • DAP diaminopimelatic acid
  • MDP muramyl dipeptide
  • RIG-I-Like receptors particularly RIG-I and MDA-5, detect viral RNA species.
  • CLR ligands include Dectin-1 and Mincle (macrophage-inducible C-type lectin) agonists.
  • Dectin-1 is a specific receptor for ⁇ -glucans, which are glucose polymers found in the cell walls of fungi.
  • Mincle is a multi-tasking danger signal receptor that recognizes a wide variety of ligands such as damaged cells, fungal components, yeast components and components of mycobacteria.
  • Cytosolic DNA Sensors bind intracellular DNA from pathogens, and there are multiple CDSs which may display contextual preferences for the recognition of particular DNAs.
  • Cyclic dinucleotides and xanthenone derivatives, such as DMXAA, bind to and activate STING (STimulator of INterferon Genes).
  • the inflammasome is a multi-protein complex involved in the production of mature I L-1 ⁇ , specifically through cleavage of pro— I L-1 ⁇ and pro— I L-18 into active and secretable forms. Inflammasomes may be segregated into NLRP1 , NLRP3, NLRC4 and AI M2 subtypes, which are activated by a wide variety of microbial molecules, danger signals and crystalline substances.
  • NOD1 iE-DAP Acylated iE-DAP, D-gamma- Glu-mDAP, L-Ala-gamma-D-Glu- mDAP
  • NOD2 MDP (MurNAc-L-Ala-D-isoGIn,
  • muramyl dipeptide N-glycolylated muramyldipeptide, N-Acetyl-muramyl- L- Alanyl-D-Glutamin-n-butyl-ester, MurNAc-Ala-D-isoGln-Lys, N- Acetylmuramyl-L-Alanyl-D- Isoglutamine (L-D isoform), 6-0- stearoyl-N-Acetyl-muramyl-L-alanyl-D- isoglutamine, Pam2C-Aca-Benzyl- Murabutide,
  • PRR agonists derived from a selected microbial pathogen For example, peptidoglycan (PGN) may be obtained from a bacteria or bacterial strain that is pathogenic in a selected target tissue or organ, for use as a NOD1/NOD2 agonist.
  • PPN peptidoglycan
  • cell wall components may be obtained from a bacteria or bacterial strain that is pathogenic in a selected target tissue or organ, for use as a TLR2 agonist.
  • DNA including double stranded DNA, particularly repetitive double stranded DNA
  • a microbial pathogen such as a bacteria or bacterial strain that is pathogenic in a selected target tissue or organ, for use as a DAI, LRRFIP1 , RIG1 , TLR9, AIM2 or cytosolic DNA sensor (CDS) agonist.
  • Beta-glucan peptides may be obtained from fungi or yeast that are pathogenic in a selected target tissue or organ, for use as a Dectin-1 agonists.
  • Cyclic dinucleotides may be obtained from a microbial pathogen that is pathogenic in a selected target tissue or organ, for use as a STING agonist.
  • compositions that have a distinct PRR agonist signature, which connotes a repertoire of PRR agonists that are together collected in a therapeutic vehicle, so that the selected collection of PRR agonists is distinct.
  • a "therapeutic vehicle” in this context is a formulation that aggregates and retains the PRR agonists, for example in a pharmaceutically acceptable particle or vesicle, such as a recombinant microbe.
  • the PRR agonist signature may be different from a reference PRR agonist signature, for example different from the collection of PRR agonists that would be present on a microbe that is not pathogenic in the target tissue.
  • the PRR signature may also be distinct in the sense that it is different than a native PRR agonist signature of the microbial mammalian pathogen, for example altered by way of the recombinant expression of genes that alter what would otherwise be the wildtype PRR agonist signature of the pathogen.
  • the levels or kinds of PRR agonist may be directly measured, or may be measured for example by determining the activation or inhibition of a signaling pathway in a cell consequent to PRR agonist/receptor binding.
  • Immunomodulatory or immunogenic compositions are provided that constitute an artificial repertoire of mammalian pattern recognition receptor (PRR) agonists.
  • PRR pattern recognition receptor
  • the PRR agonist repertoire is selected so that it in effect recapitulates a distinct portion of a PRR agonist signature of a microbial pathogen, and more specifically a pathogen that is pathogenic in a selected target tissue.
  • the PRR agonist signature is distinct in the sense that it is different from PRR agonist signatures of microbes that are not pathogenic in the target tissue, and it is also distinct in the sense that it is different from the native PRR agonist signature of the wild-type pathogen.
  • This distinct artificial repertoire of PRR agonists may then be formulated so that the PRR agonists are presented together in a therapeutic vehicle, for example so that the PRR agonist repertoire may be presented in combination.
  • the therapeutic vehicle may for example be a recombinant microbe, a cellular fraction of a microbial cell, a microparticle or a liposome.
  • the composition may for example comprise microbial agonists for at least a minimum number of distinct mammalian PRRs, for example at least 5, as described in more detail herein.
  • the vehicle may then be delivered, for example systemically, so that the PRR agonist repertoire is presented to an innate immune cell resident in the target tissue in a host, such as a mammalian host.
  • the therapeutic vehicle may for example aggregate the artificial repertoire of PRR agonists, so that the proximity of the plurality of PRR agonists is maintained during systemic distribution in a host.
  • Compositions of this kind may be used to treat a wide variety of diseases characterized by immune dysregulation, including neoplastic diseases and autoimmune diseases.
  • compositions involve the use of an immunogenic composition in methods of treating an immune dysregulation in a target tissue in a mammalian host, wherein the composition comprises the foregoing artificial repertoire of mammalian PRR.
  • the artificial repertoire of PRR agonists may be formulated together in a therapeutic vehicle for combined presentation following administration to a mammalian host.
  • Composition may for example include components of the microbial mammalian pathogen that are agonists for a select number of distinct mammalian PRRs, as discussed in more detail below, for example at least 5.
  • Compositions may for example be adapted for use so as to modulate an innate immune response in the target tissue.
  • the therapeutic vehicle may for example include a recombinant microbe, a cellular fraction of the recombinant microbe, a cellular fraction of a microbial cell, a microparticle or a liposome, each comprising components of the microbial mammalian pathogen that provide the PRR agonists that together make up the artificial repertoire of PRR agonists.
  • a recombinant microbe may for example include a recombinant gene encoding a component of at least one of the PRR agonists.
  • the therapeutic vehicle may for example include a whole killed or attenuated cell of the recombinant microbe.
  • the cellular fraction of the microbial mammalian pathogen may be used, for example, a bacterial outer membrane fraction; a bacterial inner membrane fraction; a pellet from a gradient centrifugation of microbial cell components; or chromosomal DNA.
  • the therapeutic vehicle may for example be formulated for use for delivering the PRR agonists to the target tissue.
  • the therapeutic vehicle may for example include additional therapeutic moieties, such as one or more of: GMCSF, vitamin D, NOHA, alphl antitrypsin, glutathione, an isoprenoid, or a-galactosylceramide.
  • additional therapeutic moieties such as one or more of: GMCSF, vitamin D, NOHA, alphl antitrypsin, glutathione, an isoprenoid, or a-galactosylceramide.
  • the therapeutic vehicle may further include a heterologous PRR agonist, such as a PRR agonist that is not a component of the microbial mammalian pathogen.
  • the subject of treatment such as a mammalian host or human patient, may for example be suffering from a disease or condition characterized by the immune dysregulation in the target tissue, such as an inflammatory disorder.
  • the composition may be adapted for use in an amount effective to modulate a biomarker, for example one or more of PD1 , PDL1 , IP-10, MIG, RANTES, neutrophils, Ly6C monocytes, and NKG2D.
  • a biomarker for example one or more of PD1 , PDL1 , IP-10, MIG, RANTES, neutrophils, Ly6C monocytes, and NKG2D.
  • the composition may for example be adapted for use in an amount effective to down- regulate PD1 and/or PDL1 expression in cells present in the target tissue.
  • the composition may accordingly be adapted for use so as to modulate an adaptive immune response in the host, for example as a concomitant of modulating an innate immune response.
  • the therapeutic vehicle may be for administration at an administration site that is not the target tissue, and the site may for example be the skin, subcutaneous tissue, the respiratory tract. Administration may be enteric, or non-enteric.
  • the therapeutic vehicle may be formulated for systemic distribution of the PRR agonists following administration at a localized
  • the therapeutic vehicle may be administered in a plurality of doses over a dosage duration, and the dosage duration may for example be at least two weeks, or any of other wide range of dosage regimens disclosed herein or known in the art.
  • human patient treated in accordance with the invention may for example be immunosuppressed or immunocompromised, or may be geriatric or pediatric patients.
  • nucleic acid sequences of the invention may be recombinant sequences.
  • the term "recombinant” means that something has been recombined, so that when made in reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that are joined together or produced by means of molecular biological techniques.
  • Nucleic acid "constructs" are accordingly recombinant nucleic acids, which have been generally been made by aggregating interoperable component sequencers.
  • Recombinant when made in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques.
  • Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature.
  • nucleic acid construct as "recombinant” therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention (so that it is anthropogenic).
  • Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation.
  • Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species.
  • Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent
  • Recombinant constructs of the invention may include a variety of functional molecular or genomic components, as required for example to mediate gene expression or suppression in a transformed plant.
  • DNA regulatory sequences such as promoters, enhancers, polyadenylation signals, terminators, and protein degradation signals that regulate gene expression, as well as epigenetic regulatory signals for example involving methylation or acetylation of histones (e.g. histone methyltransferase or
  • promoter means a sequence sufficient to direct transcription of a gene when the promoter is operably linked to the gene.
  • the promoter is accordingly the portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription.
  • Promoter sequences are commonly, but not universally, located in the 5' non-coding regions of a gene.
  • a promoter and a gene are "operably linked” when such sequences are functionally connected so as to permit gene expression mediated by the promoter.
  • operably linked accordingly indicates that DNA segments are arranged so that they function in concert for their intended purposes, such as initiating transcription in the promoter to proceed through the coding segment of a gene to a terminator portion of the gene.
  • Gene expression may occur in some instances when appropriate molecules (such as transcriptional activator proteins) are bound to the promoter. Expression is the process of conversion of the information of a coding sequence of a gene into mRNA by transcription and subsequently into polypeptide (protein) by translation, as a result of which the protein is said to be expressed.
  • a gene or nucleic acid is "expressible” if it is capable of expression under appropriate conditions in a particular host cell.
  • an "isolated" nucleic acid or polynucleotide as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring).
  • An isolated nucleic acid or polypeptide may contain less than about 50%, less than about 75%, less than about 90%, less than about 99.9% or less than any integer value between 50 and 99.9% of the cellular or biological components with which it was originally associated.
  • a polynucleotide amplified using PCR so that it is sufficiently distinguishable (on a gel from example) from the rest of the cellular components is, for example, thereby "isolated".
  • the polynucleotides of the invention may be "substantially pure," i.e., having the high degree of isolation as achieved using a purification technique.
  • endogenous refers to a molecule such as a nucleic acid that is naturally found in and/or produced by a given organism or cell.
  • An "endogenous” molecule may also be referred to as a “native” molecule.
  • exogenous refers to a molecule, such as a nucleic acid, that is not normally or naturally found in and/or produced by a given organism or cell in nature.
  • heterologous refers to molecules or portions of molecules, such as DNA sequences, that are artificially introduced into a particular host cell, for example by transformation.
  • Heterologous DNA sequences may for example be introduced into a host cell by transformation.
  • Such heterologous molecules may include sequences derived from the host cell.
  • Heterologous DNA sequences may become integrated into the host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination events.
  • Various aspects of the present disclosure encompass nucleic acid or amino acid sequences that are homologous to other sequences.
  • nucleic acid sequence is "homologous" to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, sequence conservation or identity does not infer evolutionary relatedness). Nucleic acid sequences may also be homologous if they encode substantially identical amino acid sequences, even if the nucleic acid sequences are not themselves substantially identical, for example as a result of the degeneracy of the genetic code.
  • substantially identical is meant, in the alternative, a homology of greater than 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% up to 100% sequence identity.
  • Homology may refer to nucleic acid or amino acid sequences as the context dictates. In alternative embodiments, sequence identity may for example be at least 75%, at least 90% or at least 95%.
  • Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman (1981 ) Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al. (1990), J. Mol. Biol.
  • HSPs high scoring sequence pairs
  • T some positive-valued threshold score
  • Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1 , less than about 0.1 , less than about 0.01 , or less than about 0.001 .
  • an alternative indication that two amino acid sequences are substantially identical is that one peptide is specifically immunologically reactive with antibodies that are also specifically immunoreactive against the other peptide.
  • Antibodies are specifically immunoreactive to a peptide if the antibodies bind preferentially to the peptide and do not bind in a significant amount to other proteins present in the sample, so that the preferential binding of the antibody to the peptide is detectable in an immunoassay and distinguishable from non-specific binding to other peptides.
  • immunoassay formats such as solid-phase ELISA immunoassays for selecting monoclonal antibodies specifically immunoreactive with a protein (see Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York).
  • hybridize to each other under moderately stringent, or stringent, conditions Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHP0 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C, and washing in
  • hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHP0 , 7% SDS, 1 mM EDTA at 65° C , and washing in 0.1 xSSC/0.1 % SDS at 68° C. (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular
  • stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • the term "a polynucleotide that hybridizes under stringent (low, intermediate) conditions" is intended to encompass both single and double-stranded
  • washing in the specified solutions may be conducted for a range of times from several minutes to several days and those skilled in the art will readily select appropriate wash times to discriminate between different levels of homology in bound sequences.
  • substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
  • non-conserved amino acid substitutions refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution causes an appreciable loss or gain of function of the peptide, to obtain a polypeptide that is not biologically equivalent.
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar
  • hydrophilicity value e.g., within a value of plus or minus 2.0
  • hydrophilicity values are assigned to amino acid residues (as detailed in U.S. Pat. No. 4,554, 101 ): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn
  • Non-conserved amino acid substitutions may be made were the hydrophilicity value of the residues is significantly different, e.g. differing by more than 2.0.
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0).
  • each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: lie (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1 .9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1 .6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).
  • Non-conserved amino acid substitutions may be made were the hydropathic index of the residues is significantly different, e.g. differing by more than 2.0.
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, lie, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gin, Tyr.
  • Non-conserved amino acid substitutions may be made were the residues do not fall into the same class, for example substitution of a basic amino acid for a neutral or non-polar amino acid.
  • microorganisms such as bacteria, which exist in symbiotic or commensal relationships with the host animal.
  • bacteria many species of normally harmless bacteria are found in healthy animals, and are usually localized to the surface of specific organs and tissues. Often, these microbial communities aid in the normal functioning of the body, as members of what is termed the microbiome.
  • Microbes that are generally harmless, such as Escherichia coli, can cause infection in healthy subjects, with results ranging from mild infection to death.
  • Whether or not a microorganism is pathogenic depends on factors such as: the route of entry and access to specific host cells, tissues, or organs; the intrinsic virulence of the microorganism; the amount of the microorganism present at the site of potential infection; or the health of the host animal.
  • microorganisms that are normally harmless can become pathogenic given favorable conditions for infection, and even the most virulent microorganism generally requires specific circumstances to cause infection.
  • microbial species that are members of the normal flora can be pathogens when they move beyond their normal ecological role in the endogenous flora.
  • endogenous species can cause infection outside of their ecological niche in regions of anatomical proximity, for example by contiguous spread. When this occurs, these normally harmless endogenous bacteria are pathogenic.
  • Infections of the stomach are commonly caused by the following bacterial species: Streptococcus pyogenes or Helicobacter pylori; or viral pathogens:
  • cytomegalovirus herpes simplex, Epstein-Barr, rotaviruses, noroviruses, or adenoviruses.
  • Infections of the small bowel are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri; or viral pathogens: adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, or cytomegalovirus.
  • Infections of the colon/rectum are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis,
  • Bacteroides vulgatus Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri; or viral pathogens: adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, or cytomegalovirus.
  • Infections of the anus are commonly caused by the following bacterial species: Streptococcus pyogenes, Bacteroides spp., Fusobacterium spp., anaerobic streptococci, Clostridium spp., Escherichia coli, Enterobacter spp., Pseudomonas aeruginosa, or Treponema pallidum; or viral pathogens: herpes simplex.
  • Infections of the perineum are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterococcus spp., Bacteroides spp., Fusobacterium spp. , Clostridium spp., Pseudomonas aeruginosa, anaerobic streptococci, Clostridium spp., or Enterobacter spp.; or viral pathogens: herpes simplex.
  • Infections of the peritoneum are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Escherichia coli, Klebsiella spp. , Proteus spp. , enterococci, Bacteroides fragilis, Prevotella melaninogenica, Peptococcus spp., Peptostreptococcus spp. , Fusobacterium spp., or Clostridium spp.
  • Infections of the retroperitoneal area are commonly caused by the following bacterial species: Escherichia coli, or Staphylococcus aureus.
  • Bacterial species are classified operationally as collections of similar strains (which generally refers to groups of presumed common ancestry with identifiable physiological but usually not morphological distinctions, and which may be identified using serological techniques against bacterial surface antigens).
  • each bacterial species e.g., Streptococcus pneumoniae
  • Streptococcus pneumoniae has numerous strains (or serotypes), which may differ in their ability to cause infection or differ in their ability to cause infection in a particular organ/site.
  • serotypes e.g., a particular organ/site.
  • serotypes 1 , 3, 4, 7, 8, and 12 are most frequently responsible for pneumococcal disease in humans.
  • ETEC enterotoxigenic E. coli
  • EPEC enteropathogenic E. coli
  • EHEC enterohemorrhagic E coli
  • STEC Shiga toxin-producing E coli
  • EAEC enteroaggregative E. coli
  • EIEC enteroinvasive E coli
  • DAEC diffuse adhering E coli
  • ETEC ETEC
  • EPEC EHEC
  • STEC EAEC
  • EIEC EIEC
  • DAEC strains of E. coli may be chosen for a formulation to treat immune dysregulation in the colon.
  • compositions of the invention include immunogens of pathogenic microbial species (bacterial, viral or fungal) that are pathogenic in a specific tissue or organ, in which the immunogens are provided in the form of an artificial repertoire of mammalian PRR agonists that recapitulate a distinct portion of the PRR agonist signature of the microbial mammalian pathogen that is pathogenic in the target tissue.
  • the portion of the PRR agonist signature is distinct in the sense that it is both: different from a reference PRR agonist signature of a microbe that is not pathogenic in the target tissue; and, different than the native PRR agonist signature of the microbial mammalian pathogen.
  • This distinct artificial repertoire of mammalian PRR agonists are formulated together in a therapeutic vehicle for combined presentation to an innate immune cell resident in the target tissue in the mammalian host.
  • compositions of the invention may be provided alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to mammals, for example, humans (a "therapeutic vehicle”).
  • compounds for example, nucleic acid molecules, small molecules, peptides, or peptide analogues
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier can be suitable for any appropriate form of administration, including subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intramuscular, sublingual, inhalational, intratumoural or oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound (i.e., the specific bacteria, bacterial antigens, or compositions thereof of the invention), use thereof in the
  • compositions of the invention is contemplated.
  • Supplementary active compounds can also be incorporated into the compositions.
  • VLPs virus-like particles
  • membrane envelopes In general, VLPs lack genetic material. Production of VLPs may for example be by expression of viral proteins in mammalian, avian, insect, plant, yeast, or bacterial cells. Alternatively, fully synthetic VLPs may be produced.
  • NP/TLR ligand preparations that may be adapted for use to induce immune responses are ligands for TLR2
  • TLR9 Poly I: C
  • TLR4 (3- O-desacyl-4 0-monophosphoryl lipid A (MPL)
  • TLR7 (9-benzyl-8-hydroxyadenine
  • TLR7/8 resiquimod, R848)
  • TLR9 CpG DNA
  • Treatment with PRR ligands according to the invention may be combined with more traditional and existing therapies.
  • a therapy that stimulates the immune system, reduces inflammation or otherwise benefits the subject such as nutrients, vitamins and supplements.
  • a therapy that stimulates the immune system reduces inflammation or otherwise benefits the subject, such as nutrients, vitamins and supplements.
  • vitamin A, vitamin D, vitamin E, vitamin C, vitamin B complex, selenium, zinc, co-enzyme Q10, beta carotene, fish oil, curcumin, green tea, bromelain, resveratrol, ground flaxseed, garlic, lycopene, milk thistle, melatonin, other antioxidants, cimetidine, indomethacin, or COX-2 Inhibitors e.g., CelebrexTM [celecoxib] or VioxxTM [rofecoxib]
  • COX-2 Inhibitors e.g., CelebrexTM [celecoxib] or VioxxTM [rofecoxib]
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; for intranasal formulations, in the form of powders, nasal drops, or aerosols; and for sublingual formulations, in the form of drops, aerosols or tablets.
  • Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences” (20th edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, PA.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • the pathogenic bacterial species are administered to an individual in an amount effective to stop or slow progression of the disorder.
  • compositions or formulations may be packaged in a variety of ways depending upon the method used for administering the drug.
  • an article of manufacture or package may include a container having deposited therein the pharmaceutical formulation in an appropriate form.
  • Suitable containers may for example include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and vials.
  • the container may have a sterile access port, for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.
  • the package or container may also include a tamper-proof or multi-use mechanism adapted to control access to the contents of the package or the container, for example a multi dose vial adapter matched to a vial contained in the package.
  • the container or package may include a label, for example a label that describes the contents of the container, for example a drug label identifying the pharmaceutical composition therein and/or specifying modes or routes of administration.
  • the label may also include appropriate warnings, for example specifying storage conditions for the container or package, or setting out contraindications or adverse effects of a mode of treatment.
  • Articles of manufacture may accordingly take the form of a "kit" comprising pharmaceutical compositions or accessories adapted to facilitate use of pharmaceutical compositions.
  • Kits may include a label or package insert, where the term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. Kits may further include accessories associated with use of the pharmaceutical composition, including buffers, diluents, filters, needles, and syringes. Kits may also be adapted for the delivery of selected dosage forms of a pharmaceutical composition, for example including a number of unit dosages. Such kits can include a memory aid or mechanism, in the form of a physical or written indication of the intended timing of a treatment schedule in which the dosages are to be used.
  • a "companion diagnostic” may be associated with a pharmaceutical treatment or composition.
  • Companion diagnostics are assays that facilitate the associated treatment, by providing diagnostic or prognostic information, typically in the form of a diagnostic test to determine the applicability of a treatment to a specific patient.
  • Point-of-care companion diagnostics may for example involve providing diagnostic compositions and/or articles of manufacture in conjunction with providing a pharmaceutical formulation, for example as part of a kit.
  • companion diagnostics may be separately provided, as assays to monitor the therapy of subjects or to predict the therapeutic efficacy of an intended treatment.
  • a companion diagnostic may for example take the form of a medical device, such as an imaging tool, or a process carried out by such a device, for example for conducting assays in vitro, which provides information that is relevant for the safe and effective use of a corresponding drug or biological product.
  • Companion diagnostics may be used with therapies disclosed herein so as to provide diagnostic or prognostic information about therapeutic efficacy or evidence of undesirable side effects or risks.
  • the use of a companion diagnostic with a particular therapeutic may be stipulated in instructions, for example on the labeling of a diagnostic device and/or the labeling of the corresponding therapeutic product.
  • Types of companion diagnostic tests may for example include: screening and detection, in form of tests that screen for genetic patterns, such as genetic SSI response markers; prognosis and theranostics, such as assays for biochemical SSI response markers that help to predict the future course of a disease, or indicate a patient's response to a therapy; monitoring, for example to evaluate the
  • an "effective amount" of a composition according to the invention includes a therapeutically effective amount or a prophylactically effective amount.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction or elimination of the immune dysregulation.
  • a therapeutically effective amount of a composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • a therapeutically effective amount may also be one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as amelioration of immune dysregulation.
  • a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a
  • the timing and dose of treatments may be adjusted over time (e.g., timing may be daily, every other day, weekly, monthly) according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
  • the compositions may be administered every second day.
  • An initial dose of approximately 0.05 ml may be administered subcutaneously, followed by increases from 0.01 -0.02 ml every second day until an adequate skin reaction is achieved at the injection site (for example, a 1 inch to 2 inch diameter delayed reaction of visible redness at the injection site). Once this adequate immune reaction is achieved, this dosing is continued as a maintenance dose.
  • the maintenance dose may be adjusted from time to time to achieve the desired visible skin reaction (inflammation) at the injection site. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer.
  • Oral dosages may for example range from 4 times per day, daily or weekly. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer.
  • the invention may include compositions administered sublingually or by inhalation, or administered to one or more epithelial tissues (i.e., skin by intradermal or subcutaneous injection; lung epithelium by inhalation; gastrointestinal mucosa by oral ingestion; mouth mucosa by sublingual administration) simultaneously or sequentially. Accordingly, in some embodiments the compositions of the invention are administered so as to provoke an immune response in an epithelial tissue.
  • one or more epithelial routes of administration may be combined with one or more additional routes of administration, such as
  • intratumoural intramuscular or intravenous administration.
  • an immunogenically effective amount of a composition of the invention can be provided, alone or in combination with other compounds, for example with an immunological adjuvant.
  • the composition may for example include compounds linked with a carrier molecule, such as bovine serum albumin or keyhole limpet hemocyanin to enhance immunogenicity.
  • An immunogenic composition is a composition that includes materials that elicit a desired immune response.
  • An immunogenic composition may select, activate or expand, without limitation: memory B, T cells, neutrophils, monocytes or macrophages of the immune system.
  • An antigenic composition comprising killed recombinant bacteria for administration by injection may be made as follows.
  • the bacteria may be grown in suitable media, and washed with physiological salt solution.
  • the bacteria may then be centrifuged, resuspended in saline solution, and killed with heat.
  • the suspensions may be standardized by direct microscopic count, mixed in required amounts, and stored in appropriate containers, which may be tested for safety, shelf life, and sterility in an approved manner.
  • a killed bacterial vaccine suitable for administration to humans may include 0.4% phenol preservative and/or 0.9% sodium chloride.
  • the bacterial vaccine may also include trace amounts of brain heart infusion (beef), peptones, yeast extract, agar, sheep blood, dextrose, sodium phosphate and/or other media components.
  • medicaments may be administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least one week.
  • the medicament may be administered intradermally or subcutaneously.
  • the medicament may be administered in a dose so that each dose is effective to cause a visible localized inflammatory immune response at the administration site.
  • the medicament may be administered so that visible localized inflammation at the administration site occurs within 1 to 48 hours.
  • a visible localized inflammatory immune response may not always be present in all circumstances despite an immune response being initiated.
  • the mounting of an immune response can be monitored. For example, the profile (and relative change in characterization) of immune cells from a subject undergoing an immune reaction can be compared with those from a subject that is not undergoing an immune reaction.
  • a method of monitoring efficacy of a treatment regime in an individual being treated for an immune dysfunction in a specific organ or tissue involves measuring a characteristic of an immune response in a post-treatment immune sample obtained from the specific organ or tissue after the individual has been subject to the treatment regime for a period of time.
  • PRR agonists derived from bacteria that are members of the endogenous flora of a particular region of the GIT may be used to formulate immunogenic compositions of the invention.
  • the rows of Table 3 list a number of bacterial species, together with the biological regions in which each species may form a part of the endogenous flora. For example, Abiotrophia spp. are typically members of the endogenous flora of the mouth.
  • Table 3 Human Bacterial Normal Flora (Endogenous Bacterial Human Pathogens)
  • buccae Prevotella + + buccalis
  • Endogenous microbial flora such as bacteria
  • Endogenous microbial flora have access to tissues for pathogenesis either through contiguous spread or bacteremic spread. Under favorable conditions, endogenous organisms can become pathogenic and invade locally and spread by contiguous spread to adjacent tissues and organs.
  • Endogenous bacterial flora of the skin, mouth and colon are species that are understood to also be amenable to bacteremic spread. Bacteria that are members of a particular endogenous flora domain may therefore cause infection in tissues or organs to which these bacteria may spread. Accordingly, one aspect of the invention involves the use of PRR agonists derived from endogenous microbial pathogens to treat an immune dysregulation having symptoms localized to a region of the GIT in which the endogenous bacteria may spread to cause infection.
  • the columns of Table 2 list domains for endogenous flora.
  • the rows of Table 4 list regions of the GIT within which immune dysregulation may be symptomatic or etiologically located.
  • one aspect of the invention involves the use of PRR agonists derived from endogenous microbial pathogens to formulate immunogenic compositions for treating an immune dysregulation symptomatic or etiologically located in the region of the GIT to which the pathogen may spread to cause an infection.
  • an immune dysregulation symptomatic or etiologically located in the region of the GIT to which the pathogen may spread to cause an infection Accordingly, in alternative embodiments, an immune
  • dysregulation that is symptomatic in the region listed in the first column of Table 2 may be treated with immunogenic compositions comprising an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is a member of the endogenous flora of one or more of the endogenous flora domains listed in the first row of Table 2 and indicated with an X or a check mark in the appropriate row.
  • an immune dysregulation manifest in a particular region of the GIT set out in column 1 of Table 2 may be treated with antigenic compositions comprising an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is one of the
  • PRR agonists may be derived from
  • PRR agonists derived from the organisms listed in Table 5 may be used in an artificial repertoire of PRR agonists to treat an immune dysregulation that is symptomatic in the region of the GIT listed with the relevant organism in Table 5.
  • PRR agonists derived from both endogenous and exogenous microbial species may be used in combination.
  • Table 5 Exogenous Bacterial Human Pathogens, and their Sites of Infection in the GIT.
  • Vibrio fluvial is small bowel, colon
  • PRR agonists for use in the invention may be derived from viral pathogens.
  • Table 6 provides an exemplary list of viral pathogens together with the tissue and organ sites for which each viral species is reportedly a pathogen. Accordingly, one aspect of the invention involves utilizing immunogenic compositions of PRR agonists derived from the named viruses to treat an immune dysregulation that is symptomatic in the region of the GIT that is identified adjacent to the name of the virus in Table 6.
  • the pathogen from which PRR agonists are derived for use in immunogenic compositions of the invention may be one that is a common cause of acute infection in the region of the GIT in which the immune dysregulation to be treated is symptomatic.
  • Table 7 identifies bacterial and viral pathogens of this kind, together with the region of the GIT in which they commonly cause infection. Accordingly, in selected embodiments, an immune dysregulation that is symptomatic in a region of the GIT identified in the first column of Table 7 may be treated with an immunogenic composition that comprises an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of the PRR agonist signature of a pathogenic organism listed in the second column of Table 7
  • Table 7 Common causes of acute infection (bacteria and viruses) for selected regions of the GIT Oral Prevotella melaninogenicus, anaerobic streptococci,
  • Peptostreptococcus spp. Bacteroides spp., and other oral anaerobes
  • cytomegalovirus herpes simplex, Epstein-Barr, rotaviruses, noroviruses, adenoviruses
  • Bacteroides vulgatus Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri
  • adenoviruses adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, cytomegalovirus
  • Bacteroides vulgatus Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri
  • adenoviruses adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, cytomegalovirus
  • compositions of the invention may accordingly include PRR agonists of various protozoa, including for example: Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominus, Isospora belli, Sarcocystis species, Coccidian like bodies (Cyclospora species), Enterocytozoon bieneusi, Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Endolimax nana, lodamoeba butschlii, Dientameoba fragilis, Blastocystis hominus, Cyclospora cayetanensis, Microsporidia,
  • PRR agonists of various protozoa including for example: Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominus, Isospora belli, Sarcocystis species, Coccidian like bodies (Cyclospora species), Entero
  • compositions of the invention may include antigenic components of various helminths, including for example: Cestodes (tapeworms), Taenia saginata, Taenia solium, Diphyllobothrium species, Hymenolepis nana, Hymenolepis diminuta, Dipylidium caninum, Nematodes (round worms), Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus, Ancylostoma duodenale,
  • Schistosoma mansoni Schistosoma japonicum
  • Schistosoma mekongi Schistosoma mekongi
  • Schistosoma intercalatum Echinostoma species and Paragonimus species.
  • the invention may involve the treatment of an immune dysregulation with formulations of an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial pathogen that is an: Acidaminococcus fermentans; Acinetobacter spp. ; Actinobaculum spp.; Actinomyces spp. ;
  • Aeromonas spp. Anaerorhabdus furcosus; Anaerococcus hydrogenalis;
  • Anaerococcus lactolyticus Anaerococcus prevotii; Atopobium spp.; Bacillus spp.; Bacteroides caccae; Bacteroides distasonis; Bacteroides eggerthii; Bacteroides fragilis; Bacteroides merdae; Bacteroides ovatus; Bacteroides splanchnicus;
  • Bacteroides thetaiotaomicron Bacteroides vulgatus; Bifidobacterium adolescentis; Bifidobacterium bifidum; Bifidobacterium breve; Bifidobacterium catenulatum;
  • Campylobacter curvus Campylobacter gracilis; Campylobacter jejuni;
  • Campylobacter rectus Campylobacter showae; Capnocytophaga ochracea;
  • Cedecea spp Citrobacter freundii; Citrobacter koseri; Clostridium spp.;
  • Desulfomonas pigra Desulfomonas pigra; Dysgonomonas spp.; Eikenella corrodens; Enterobacter aerogenes; Enterobacter cloacae; Enterobacter gergoviae; Enterobacter sakazakii;
  • Enterobacter taylorae Enterococcus spp. ; Escherichia coli; Escherichia fergusonii; Escherichia hermannii; Escherichia vulneris; Eubacterium spp.; Finegoldia magnus;
  • Fusobacterium gonidiaformans Fusobacterium mortiferum; Fusobacterium naviforme; Fusobacterium necrophorum; Fusobacterium nucleatum; Fusobacterium russii; Fusobacterium varium; Gardnerella vaginalis; Gemella morbillorum;
  • Globicatella spp. Hafnia alvei; Helicobacter spp.; Klebsiella spp. ; Lactobacillus acidophilus; Lactobacillus fermentum; Lactobacillus reuteri; Lactobacillus salivarius;
  • Leclercia adecarboxylata Leminorella spp.; Megasphaera elsdenii; Mitsuokella multiacidus; Mobiluncus curisii; Mobiluncus mulieris; Moellerella wisconsensis;
  • Bacillus cereus Bacillus cereus; other Bacillus spp.; Borrelia recurrentis; Brucella spp.;
  • Campylobacter coli Campylobacter coli; Campylobacter fetus; Campylobacter jejuni; Campylobacter sputorum; Clostridium bifermentans; Clostridium botulinum; Clostridium difficile; Clostridium indolis; Clostridium mangenolii; Clostridium perfringens; Clostridium sordellii; Clostridium sporogenes; Clostridium subterminale; Edwarsiella tarda;
  • Francisella tularensis Listeria monocytogenes; Mycobacterium bovis; Mycobacterium tuberculosis; Pediococcus spp.; Plesiomonas shigelloides;
  • Rickettsia rickettsiae Salmonella spp.; Shigella boydii; Shigella dysenteriae;
  • zooepidemicus Tropheryma whipplei; Vibrio cholerae; Vibrio fluvialis; Vibrio furnissii; Vibrio hollisae; Vibrio parahaemolyticus; Yersinia enterocolitica; Yersinia pseudotuberculosis; Herpes Simplex virus (1 and 2); Cytomegalovirus; Adenovirus; Orthoreoviruses; Rotaviruses; Alphaviruses; Coronaviruses; Toroviruses; Human metapneumovirus; Vesicular stomatitis virus; Machupo virus; Junin virus;
  • Poliovirus Poliovirus; Coxsackieviruses; Echoviruses; Hepatitis A virus; Noroviruses and other Caliciviruses; Astroviruses; Picobirnaviruses; or Hepatitis E virus.
  • the invention may involve the treatment of an immune dysregulation with formulations of an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is a common small and larger bowel pathogens, for example: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri; adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, and cytomegalovirus.
  • an immune dysregulation with formulations of an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is a common
  • the invention involves diagnostic steps to assess a patient's previous exposure to an organism.
  • the diagnostic steps may include taking a medical history of exposure to selected pathogens, and/or evaluating a patient's immune response to a selected pathogen.
  • a serology test may be conducted to detect antibodies to selected pathogens in a patient's sera.
  • antigenic determinants of a selected pathogen may be chosen for use in an immunogenic composition on a selected patient based on a diagnostic indication that the patient has had one or more prior exposure(s) to the pathogen, for example by virtue of the presence of antibodies to antigenic determinants of that pathogen in the patient's sera.
  • the invention involves diagnostic steps to assess a patient's immunological response to treatment with a selected immunogenic composition.
  • the diagnostic steps may include evaluating a patient's immune response to the immunological determinants of that immunogenic composition, for example using a serological test to detect antibodies to those immunogenic determinants.
  • a treatment with a selected immunogenic composition may be continued if the evaluation indicates that there is an active immunological response to the immunogenic determinants of that composition, and the treatment may be discontinued, and an alternative treatment with a different immunogenic
  • composition may be initiated, if the evaluation indicates that there is not a sufficiently active immunological response to the immunogenic determinants of the immunogenic composition.
  • Patients may advantageously be screened for disorders of innate immunity, such as genetic disorders, for example by primary sequence analysis or by analysis of epigenetic changes.
  • disorders of innate immunity such as genetic disorders, for example by primary sequence analysis or by analysis of epigenetic changes.
  • a variety of genetic disorders have for example been identified that are associated with gene products involved in innate immunity (see Mogensen T., 2009, Clinical Microbiology Reviews, Vol. 22, No.2, p.240-273), such as TLR genes (TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9), signalling protein genes (MyD88, Mai, I RAKI , I RAK4, NEMO, ⁇ , I RF5), N LR genes (NOD2, NALP1 , NALP3) and others (CD14, U NC93B).
  • TLR genes TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9
  • signalling protein genes MyD88, Mai, I RAKI , I RAK4, NEMO, ⁇
  • Patients identified as having a Mendelian primary immunodeficiency associated with impaired TLR signaling or N F-KB activation may for example not benefit from some embodiments, or may require an approach adapted to their condition.
  • Patients having polymorphisms in genes encoding components of innate signalling pathways may also be identified prior to treatment with an SSI, for example having mutations in the gene encoding TIR-domain-containing adaptor-inducing beta interferon (TRIF).
  • TIR-domain-containing adaptor-inducing beta interferon TIR-domain-containing adaptor-inducing beta interferon
  • Gram negative bacterial formulation may advantageously comprise an LPS that is recognized by a TLR, such as TLR4, rather than a form of LPS that is not recognized by a TLR (Hornef, M. W. et al., 2002, Nat. Immunol. 3: 1033-1040).
  • bacterial formulations may advantageously include a class of flagellin that activates a TLR, such as TLR5, rather than one that does not (Andersen-Nissen, E. et al., 2005, Proc. Natl. Acad. Sci. USA 102:9247-9252).
  • peptidases that proteolytically degrade important components of the triggered innate response such as the amastigote-specific cysteine peptidases of Leishmania mexicana that proteolytically degrade ⁇ and NF- ⁇ (Cameron, P. et al. , 2004, J. Immunol. 173:3297-3304).
  • these undesireable components may be removed from a formulation by an appropriate step of manufacturing, for example to wash or fractionate a microbial preparation so as to remove a component.
  • Patients may be genotyped, for example by identifying polymorphisms in PRR genes (see WO 2009003905).
  • Genes associated with inflammation and immune related diseases and disorders may for example be the subject of screening, such as: AIDS (KIR3DL1 , NKAT3, NKB1 , AMB1 1 , KIR3DS1 ,
  • TNFRSF6, APT1 , FAS, CD95, ALPS1A Combined immunodeficiency, (I L2RG, SCI DX1 , SCI DX, I MD4); HIV-1 (CCL5, SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G, AICDA, AI D, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1 , IGM, FOXP3, IPEX, AND, XPID, PIDX, TNFRSF14B, TACI); Inflammation (I L-10, IL-1 (IL-1 a, I L-1 b), IL-13, IL- 17 (IL-17a (CTLA8), IL-17b, I L-17c, IL-17d,
  • TNFalpha, NOD2/CARD15 for IBD IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1 ); Severe combined immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1 C, ARTEMIS, SCI DA, RAG1 , RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, I L2RG,
  • SCIDs Severe combined immunodeficiencies
  • genes involved in selected signalling pathways may for example be screened, identifying for example patients that are more or less susceptible to an SSI treatment, such as: GM-CSF Signaling (LYN; ELK1 ; MAPK1 ; PTPN1 1 ; AKT2; PIK3CA; CAMK2A; STAT5B; PI K3CB; PIK3C3; GNB2L1 ; BCL2L1 ; MAPK3; ETS1 ; KRAS; RUNX1 ; PIM1 ; PIK3C2A; RAF1 ;
  • GM-CSF Signaling GM-CSF Signaling
  • patients may for example be genotyped for SNPs located in the non-coding regions of the genome that are linked to inflammatory disorders, such as SNP's identified through publicly available GWAS datasets, for example SNPs in genomic regions linked to sequences which serve a regulatory role in immune-function-related gene expression.
  • Standard reference works setting forth the general principles of recombinant DNA technology include Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 -3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. , 1989; Current Protocols in Molecular Biology, ed. Ausubel et al. , Greene Publishing and Wiley-lnterscience, New York, 1992 (with periodic updates) ("Ausubel et al. 1992”); the series Methods in Enzymology (Academic Press, Inc.); Innis et al. , PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990; PCR 2: A Practical Approach (M.J.
  • the invention excludes steps that involve medical or surgical treatment. Similarly, in some embodiments, the invention disclaims naturally occurring embodiments, so that aspects of the invention relate only to anthropogenic compositions. Further, in select aspects of the invention, previously known products, process of making products, or methods of using products are hereby disclaimed. [00127] General Codes and Abbreviations:
  • Example 1 Recombinant Microbes
  • TIR domain-containing proteins A family of virulence factors in Escherichia coli and Brucella melitensis, named TIR domain-containing proteins, impede TLR signalling through direct binding to MyD88, thus suppressing innate immunity and increasing bacterial virulence. Aspects of the invention accordingly provide recombinant bacteria that lack expression of TIR domain-containing proteins, or other virulence factors that interfere with an innate host immune response to the pathogen.
  • compositions may be prepared from recombinant E. coli strains specifically adapted from strains that are pathogenic within the peritoneum, or in intraperitoneal organs or in retroperitoneal organs.
  • Strains may totally lack resistance genes to the following classes of antibiotic: aminoglycoside, beta-lactam, fluoroquinolone, fosfomycin, fusidic acid, MLS - macrolide, lincosamide and streptogramin B, nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide, tetracycline, trimethoprim, and glycopeptide.
  • strains may or may not include one or more virulence factor genes, having for example at least 90%, 95%, 99% or 100% identity to selected database sequences (identified by accession number in the following tables). The strain may also lack stx holotoxin virulence factors.
  • the serotype of the E. coli strain may for example be 018ac:H7, for example representing the presence of H type serotype gene fliC (accession AF228492, and O type serotype genes wzx (accession GU299793), and wzy (accession GU299793).
  • Recombinant strains may include one or more plasmids, for example having 90%, 95%, 99% or 100% identity to plasmid IncFIB (accession AP001918) and/or plasmid lncFII(29) (accession CP003035), and/or plasmid ColRNAI
  • the recombinant E. coli may for example be, or be derived from an E. coli strain having at least 80%, 90% or 95% sequence identity to E. coli UT189 (see Chen et al., 2006, Proc Natl Acad Sci U S A 103:5977-82). Escherichia coli. (colon)
  • compositions may be prepared from recombinant E. coli strains specifically adapted from strains that are pathogenic in the colon or other regions of the Gl tract, for example for therapy of colon or Gl immune dysfunction, for example for treatment of IBD.
  • Strains may totally lack resistance genes to the following classes of antibiotic: aminoglycoside, beta-lactam, fluoroquinolone, fosfomycin, fusidic acid, MLS - macrolide, lincosamide and streptogramin B, nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide, tetracycline, trimethoprim, and glycopeptide.
  • antibiotics aminoglycoside, beta-lactam, fluoroquinolone, fosfomycin, fusidic acid, MLS - macrolide, lincosamide and streptogramin B, nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide, tetracycline, trimethoprim, and glycopeptide.
  • strains may have one or more resistance genes, such as the strB or strA aminoglycoside resistance genes (accession numbers M96392 or AF321551 ), and/or sul1 sulphonamide resistance gene (accession AY224185), and/or sul2 sulphonamide resistance gene (accession GQ421466), and/or dfrA5 trimethoprim resistance (accession X12868).
  • strains may or may not include one or more virulence factor genes, having for example at least 90%, 95%, 99% or 100% identity to selected database sequences (identified by accession number in the following tables). The strain may also have a gene that is at least 95% or 99% or 100% identical to the stx holotoxin virulence factor gene stxl (accession M19437).
  • the serotype of the E. coli strain may for example be 01 17: H7, for example representing the presence of H type serotype gene fliC (accession AF228492, and O type serotype genes wzx (accession EU694096).
  • Recombinant strains may include one or more plasmids, as set out in the following table.
  • the recombinant E. coli may for example be, or be derived from an E coli strain having at least 80%, 90% or 95% sequence identity to E coli SE15 or any 01 17:H7 serotype E. coli.
  • This Example illustrates embodiments in which an SSI is co- formulated with or co-administered with additional therapeutic components.
  • additional therapeutic components comprises molecules or compositions for activating or recruiting innate immune cells, and these include:
  • GMCSF for example in an amount that synergistically recruits and promotes the production of neutrophils and potentiates the SSI-induced innate immune response.
  • Vitamin D for inflammatory disease, such as I BD
  • Vitamin D for inflammatory disease, such as I BD
  • the vitamin D used in conjunction with SSIs may for example be one or more of vitamin D3, D 2 or calcitriol (1 ,25- dihydroxycholecalciferol).
  • vitamin D3 and/or D 2 may for example be given locally at a dosage that is effective to provide a locally effective amount of calcitriol at the site of SSI and vitamin D administration.
  • vitamin D precursors may be administered in an amount that is locally effective once it is converted into the calcitriol active form by local monocytes and/or macrophages (expressing CYP27B1 ) at the site of administration.
  • calcitriol may be administered in dose that is locally effective at the site of SSI administration, and this may for example be dose that is less than the dose required for other systemic effects.
  • An additional class of therapeutic components for co-formulation or co-administration comprise molecules or compositions that relieve
  • NOHA N(omega)- hydroxy-nor-L-arginine
  • an Arginase inhibitor - Arginase degrades arginine needed for immune activation.
  • NOHA may for example be used in an amount effective to relieve immune suppression by making available free arginine.
  • Alphal antitrypsin - for example in an amount effective to relieve immune suppression mediated by neutrophils secreting proteases.
  • An additional class of therapeutic components for co-formulation or co-administration comprise molecules or compositions that prevent oxidative damage and improve immune function under stress:
  • Glutathione and other antioxidants particularly for fibrotic diseases (such as I BD).
  • An additional class of therapeutic components for co-formulation or co-administration comprise co-stimulatory molecules for innate cytotoxic
  • lymphocytes
  • Phospho-antigens isoprenoid molecules, such as isopentenyl pyrophosphate
  • SSIs in co-formulation or co-administration with zoledronate increase markers of activation, for example CD25 and CD69, on human peripheral blood ⁇ 9 ⁇ / ⁇ 2 T cells.
  • Glycolipid molecules recognized by Type I NKT cells (such as synthetic a-galactosylceramide)
  • This Example illustrates results from a mouse spontaneous colitis model (Muc2 knockout "KO" mice) that mimics the underlying immune system defect and chronic bacterial infection associated with Crohn's disease and ulcerative colitis.
  • I BD patients typically display structural and/or functional defects in their normally protective colonic mucosal barriers.
  • the mucus barrier is largely dependent on the release of goblet cell-derived mucin (Muc2) which prevents microbes and luminal antigens from contacting the epithelial surface in the gastrointestinal tract.
  • Muc2 KO mice are healthy just after weaning (1 month old), as they age, they develop progressive diarrhea and sporadic rectal prolapse.
  • Muc2 KO mice Histological analysis of colonic tissue shows crypt hyperplasia, crypt abscesses, inflammatory cell infiltration and submucosal edema. Accordingly, the Muc2 KO mice have a defective gastrointestinal mucosal barrier and after time spontaneously develop colitis, resembling ulcerative colitis in humans. In this model, young (2 month old) Muc2 KO mice have less severe colitis, and older (3 month old) Muc2 KO mice have more severe colitis.
  • Results from this animal model illustrate that an E. coli SSI (QBECO) decreases pro-inflammatory markers in the colon (using qPCR gene expression data).
  • Figure 14D illustrates the site specific activity of QBECO in increasing IL-18 gene expression in the colon, compared to QBKPN.
  • the I FN-gamma expression data in particular illustrates how SSI efficacy can be affected by the stage of colitis (comparing expression data in old vs young mice).
  • IL17A data relating to a cytokine that is produced by activated T-cells (a marker of IBD inflammation), illustrates a significant decrease in this marker of inflammation after E. coli SSI treatment.
  • QBECO treatment substantially improved all components of the histopathology score, including infiltration, integrity, hyperplasia, and edema.
  • this Example illustrates that an SSI, such as QBECO, may be used to significantly decrease disease severity in IBD model, including so as to substantially dampen adaptive immune system over- response.
  • gastrointestinal microbiome Alterations in bacterial species in the intestinal microbiome can either be detrimental ('unhealthy' bacteria) or therapeutic ('healthy' bacteria) in IBD patients (and mouse models). Some bacteria promote a healthy immune environment and can improve symptoms (for example, Lactobacillus species), whereas others (for example, ⁇ -proteobacteria) can have detrimental effects in IBD.
  • Some bacteria promote a healthy immune environment and can improve symptoms (for example, Lactobacillus species), whereas others (for example, ⁇ -proteobacteria) can have detrimental effects in IBD.
  • QBECO SSI improved dysbiosis in the colon of Muc2 mice, increasing the relative proportion of Lactobacillus
  • aspects of the invention involve the use of an SSI , such as an E. coli derived SSI, for treating dysbiosis in IBD.
  • QBECO treatment significantly improved the overall histological score and reduced T cell infiltration in the colonic tissues. Furthermore, a reduction in pro-inflammatory mediators in the colon (IL-17A) and serum (KC) was observed. QBECO treatment did not impact regulatory T cell marker (FoxP3) and anti-inflammatory growth factor (TGF- ⁇ ) expressions in affected tissues. In addition, SSI treated mice demonstrated reduced levels of the antimicrobial lectins Reglll- ⁇ and Regll l- ⁇ . The changes in antimicrobial lectins brought on by QBECO allowed for a modulation of the gut microbiome causing a reduction in gamma- proteobacteria and a significant increase in lactobacilli.
  • This Example illustrates that efficacious treatment for Crohn's disease may be carried out over an extended period of periodic dosing of an SSI.
  • CDAI Crohn's Disease Activity Index
  • This example involved use of a whole killed E. coli SSI preparation, administered every second day by subcutaneous injection.
  • the dose was individualized to the patient by adjusting the dose so that each dose was effective to cause a visible localized inflammatory immune response at the administration site (a 1 inch to 2 inch diameter delayed reaction of visible redness at the injection site).
  • aspects of the invention involve use of an SSI over an extended duration period, with dosage intervals and dosage duration adapted to provide an increased therapeutic benefit over the entire dosage duration, such as a progressive reduction of CDAI score in Crohn's patients over a duration period of at last 16 weeks.
  • Example 5 Distinct SSIs Agonize Distinct PRRs
  • This Example illustrates that both QBECO and QBKPN SSIs activate multiple PRRs, and QBECO and QBKPN each activate different PRRs, with different PRR repertoire fingerprints being identified for each SSI.
  • This data in this Example was obtained from assays of QBKPN and QBECO PRR activation in cell lines that have a single PRR.
  • the cell lines used were HEK293 cells lines that express a single human Toll-Like Receptor (TLR2, 3, 4, 5, 7, 8 and 9), NOD-Like Receptor (NOD1 and NOD2), C-Type Lectin (Dectin 1 a, Dectin 1 b, and Mincle) or RIG-1 -like receptor (RIG-1 and MDA5).
  • TLR 2 and TLR4 Two TLRs were highly activated by both QBECO and QBKPN (TLR 2 and TLR4).
  • TLR5 One TLR was highly activated by just QBKPN (TLR5).
  • 1 PRR was moderately activated by bother QBECO and QBKPN (NOD2).
  • 4 were moderately activated by only QBECO (TLR3, TLR7, TLR8, TLR9) while 2 were moderately activated by only QBKPN (Dectin 1 a, Dectin 1 b).
  • NOD1 , Mincle, RIG-1 and MDA5 were not activated by either QBECO or QBKPN.
  • TLR2 and TLR4 are localized on the plasma membrane and primarily recognize lipoprotein and LPS respectively.
  • TLR5 is a plasma membrane receptor that responds to Flagellin.
  • TLR3 was only slightly activated by QBECO (and not by QBKPN).
  • TLR3 is primarily a dsRNA receptor for viral RNA.
  • TLR7 and 8 which are located in the endolysosome and also recognize RNA (bacterial and viral) were activated by only QBECO.
  • TLR9 which recognized CpG-DNA and is located in the endolysosome was also activated by only QBECO.
  • QBECO Nod-Like Receptors
  • NOD1 was not activated by either QBECO or QBKPN, but NOD2, which recognizes muramyl dipeptide (MDP) was activated by both QBECO and QBKPN.
  • MDP muramyl dipeptide
  • CLR C-type lectin receptors
  • This Example provides a genetic analysis of subjects with IBD undergoing treated with an SSI therapy, illustrating the use of genetic markers associated with IBD to identify patient populations amenable to SSI treatments.
  • this Example there were 48 subjects with IBD and approximately 2.4 million single nucleotide polymorphisms (SNPs) which were the subject of analyses following genotyping on the Infinium Omni2.5-8 bead chip. The end-points used for these genetic analyses were varied and encompassed both clinical response and also the use of object markers of disease activity. Following standard quality control measures (including call frequency, minor allele frequency, and Hardy-Weinberg equilibrium test) a total of 1 ,271 ,655 SNPs were available for analyses. 1 13 known IBD loci were represented on the chip and passed quality control. Of the study subjects there were 31 Crohn's disease (CD) and 12 ulcerative colitis (UC) cases included.
  • CD Crohn's disease
  • UC ulcerative colitis
  • HNF4A and GPR12 are also associated with drop in CRP in UC after 16 weeks of treatment.
  • an aspect of the present invention involves the provision of companion diagnostic genetic testing assays in association with an SSI therapy.
  • SNPs and genetic loci that may be used in such assays are set out below.
  • Table 12 SNP alleles associated (or negatively associated) with Response to SSI Therapy
  • CD - CRP rs2641348 2.20E-03 NOTCH2, ADAM30, REG4, NBPF7 Comparing
  • IBD associated SNPs are spatially associated with genes (Liu et al., Nature Genetics. 47.9 (Sept. 2015): p979), so that alternative markers, such as SNPs, associated with these genes may also serve as markers of SSI efficacy, as set out in Table 25:
  • IBD associated SNPs are spatially associated with genes (Jostins, et ai, Nature. 2012; 491 : 1 19-124), so that alternative markers, such as SNPs, associated with these genes may also serve as markers of SSI efficacy, as set out in Table 16:
  • IFNAR2 GART,(7)
  • IBD associated SNPs are spatially associated with genes (Jostins, et ai, Nature. 2012; 491 : 1 19-124), so that alternative markers, such as SNPs, associated with these genes may also serve as markers of SSI efficacy, as set out in Table 17:
  • HCP5 HCG26, MICB, MCCD1, DDX39B, SN0RD117, SNORD84, ATP6V1G2, NFKBIL1 rs9491697 rs2503322 RSP03, RNF146, ECHDC1
  • rs3764147 rs3764147 EN0X1, CCDC122, LACC1, LINC00284 rs16967103 rs 16967103 SPRED1, FAM98B, RASGRP1, C15orf53 rs2066847 ** rs5743289 ADCY7, BRD7, NKD1, SNX20, N0D2, CYLD rs2945412 rs2945412 WSB1, LOC440419, KSR1, LGALS9, N0S2 rs2024092 rs2024092 MED16, R3HDM4, KISS1R, ARID3A, WDR18,
  • GRIN3B C19orf6, CNN2, ABCA7, HMHA1, P0LR2E, GPX4, SBN02, STK11, C19orf26, ATP5D, MIDN, CIRBP-AS1, CIRBP, C19orf24, EFNA2, MUM1
  • FAM83E SPACA4, RPL18, SPHK2, DBP, CA11, SEC1, NTN5, FUT2, MAMSTR, RASIP1, IZUM01, FUT1, FGF21, BCAT2, HSD17B14, PLEKHA4, PPP1R15A, TULP2, NUCB1, DHDH rs2284553 rs2284553 C21orf54, IFNAR2, IL10RB, IFNAR1, IFNGR2,
  • r-squared a measure of the degree to which alternative genetic markers provide similar diagnostic or prognostic information.
  • the value of r 2 ranges between 0 and 1 (1 when two markers provide identical information, and 0 when they are in perfect equilibrium). Conventionally, markers with r 2 > 0.8 may be considered to be in high linkage disequilibrium, so that they may provide similar diagnostic or prognostic information.
  • an aspect of the assays described herein involves the use of makers that are in linkage disequilibrium with the markers identified above, having for example r 2 > 0.7, r 2 > 0.8, r 2 > 0.9 or r 2 > 0.95.
  • markers that provide related information may be characterized by physical proximity in the genome, for example being within 1 Mbp of each other, for example within 50Kb, 60Kb, 70Kb, 80Kb, 90Kb, 100Kb, 200Kb, 300Kb, 400Kb or 500Kb of each other.
  • a "genetic SSI response marker” means a genetic biomarker, the presence of which is correlated with the probability of response to a treatment with an SSI.
  • Exemplary genetic SSI response markers are disclosed in this Example, evidencing a correlation with response to an SSI in IBD patients.
  • Genetic SSI response markers may be detected by a wide range of genomic assays, and may also be detected by assays that interrogate the transcription or translation products of a genome, for example protein isoforms associated with a particular genomic allele.
  • biochemical SSI response markers are disclosed herein that provide a biochemical indication of response to an SSI therapy, these for example include temporal or special changes in cellular populations or in the abundance or concentration of biologically relevant molecules.
  • Biochemical and genetic SSI response markers may be used as diagnostic or prognostic indicators in the context of an SSI treatment, for example for IBD in general, or for specific forms of IBD such as Crohn's Disease and ulcerative colitis.
  • Exemplary genetic SSI response markers are set out in Table 18, as well as
  • rs9286879 A TNFSF18, TNFSF4, FASLG Crohn's rs7517810 G TNFSF18, TNFSF4, FASLG Crohn's rs17391694 G DNAJB4 (HSP-40 family member), GIPC2, Crohn's
  • Figure 6 includes bar graphs illustrating the frequency of selected SNP genotypes in responders and non-responders, for the SNPs rs9286879, rs7517810 and rs17391694, where "response" is indicative of the last recorded response for CD patients undergoing a QBECO SSI therapy.
  • Figure 7 shows similar data for 4 SNPs when response is measured by a drop in Crohn's Disease Activity Index (CDAI , Best et al. , 1976, Gastroenterology 70 (3): 439-444), after 8 weeks of QBECO SSI treatment, for the SNPs: rs1734907, rs7517810, rs9286879 and rs4836519.
  • CDAI Crohn's Disease Activity Index
  • Figure 8 shows similar data for 4 SNPs when response is measured by a drop in fecal calprotectin after 8 weeks of QBECO SSI treatment, for the SNPs: rs17391694, rs2413583, rs1 1209026 and rs2472649.
  • Figure 9 shows similar data for 1 SNPs when response is measured by a drop in CRP (serum levels of C-reactive protein) after 8 weeks of QBECO SSI treatment, for the SNP: rs2641348.
  • Figure 10 includes bar graphs illustrating the frequency of selected SNP genotypes in UC responders and non-responders, for the SNPs rs17085007, rs2024092, rs6017342, rs2188962 and rs941823, where "response" is indicative of a drop in Mayo score after 16 weeks of treatment for UC patients undergoing a QBECO SSI therapy.
  • Figure 11 shows similar data for 4 SNPs when response is measured in UC patients by a drop in CRP (serum levels of C-reactive protein) after 16 weeks of QBECO SSI treatment, for the SNPs: rs17085007, rs2024092, rs6017342 and rs17229285.
  • Figure 12 shows similar data for 3 SNPs when response is measured in UC patients by last recorded histological score (indicative of histological healing), for the SNPs: rs17085007, rs6017342, rs2024092.
  • Figure 13 shows similar data for 4 SNPs when response is measured in UC patients by a drop in histology score after 16 weeks of QBECO SSI treatment, for the SNPs: rs10065637, rs1 1739663, rs6017342 and rs2651244.
  • Figure 14 shows similar data for 4 SNPs when response is measured in UC patients by a drop in continuous score after 16 weeks of QBECO SSI treatment, for the SNPs: rs10065637, rs1 1739663, rs1 1083840 and rs2651244.
  • Figure 15 shows similar data for 5 SNPs when response is measured in UC patients by a drop in last recorded continuous score, for the SNPs: rs6017342, rs13277237, rs4802307, rs9847710 and rs2188962.
  • SNP rs17085007 is associated with the protein ubiquitin specific peptidase 12 (USP12) which is an important protein for macrophage mediated inflammatory response to bacterial endotoxin (LPS) and may mediate a key response to bacteria, and bacterial derived products (Nayak et al. , Biochem Biophys Res Commun 2017;483:69-74.).
  • SNP rs2188962 is located in the IBD genetic locus of susceptibility 5, which includes many important inflammatory genes for immune activation and modulation, including IL-13, II-4, I L- 5, and 11-13, among others
  • Figure 16 shows similar data for 6 SNPs when response is measured by the last recorded response for CD and UC patients undergoing a QBECO SSI therapy, for the SNPs: rs7517810, rs9286879, rs2413583, rs10758669,
  • some embodiments involve assays to distinguish homo- or
  • This Example provides an analysis of the PRR receptors that are the targets for QBECO SSIs.
  • Table 28 List of PRRs stimulated by QBECO SSI. Where a PRR is Optional", this indicates that some embodiments may be designed to include agonists for the specified PRR.
  • glycoproteins and/or
  • glycosphingolipids PMID 1580230
  • DC-SIGN CD- High-mannose-containing Optional 209 glycoproteins
  • DNGR1/CLEC9A Actin filaments (no microbial Optional ligands identified)
  • NLR Nod-Like Receptors
  • DAP diaminopimelatic acid
  • NLRC3 (NOD3) Cytosolic DNA, cyclic di-GMP, optionally
  • NLRC4 (NOD4) Flagellin components of the Optional type three secretion system
  • NALP1 -14 Pathway unclear (Anthrax and Optional muramyldipeptide for NALP1 )
  • RLR (Rig-1 Like Receptors)
  • RIG-1 dsRNA (viral), maybe bacterial Optional
  • Table 29 PRR agonists in fractionated QBECO SSIs, particularly in the DNA fractions Exemplified herein.

Abstract

Methods are provided for predicting the degree to which a patient has a likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists. Methods may include screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP. In some instances, the subject has an increased likelihood of benefiting from a treatment if the diagnostic SNP comprises a selected response allele.

Description

MANAGING SITE SPECIFIC IBD IMMUNOTHERAPIES
FIELD
[0001] Innovations are disclosed in the field of medical science, relating to managed therapeutic use of preparations that contain immunogens, such as microbial components.
BACKGROUND
[0002] There is growing recognition of the complexities of immunological dysregulation that are associated with a wide range of diseases, particularly autoimmune disease. This dysregulation is generally characterized by an imbalance between immune response and immune tolerance (see Immune Rebalancing, 1 st Edition: The Future of Immunosuppression, 2016, Boraschi and Penton-Rol Eds, Academic Press). In vertebrates, an important aspect of immunological regulation involves the concerted activity of the innate immune system and the adaptive immune system. This concerted activity involves metabolic, enzymatic and molecular genetic changes within immune cells, orchestrating an elaborate system of cellular, cytokine and chemokine
communication pathways mediating the coordinated activity of the disparate components of these complementary systems (see Iwasaki & Madzhitov, 2015, Nature Immunology 16:343-353; WO0209748; WO03051305; Turner et al. , 2014, BBA-Molecular Cell Research 1843: 1 1 2563-2582). A variety of therapies have been described that therapeutically modulate the immune system in an effort to restore a healthy equilibrium, including therapies that make use of tissue-targeted immunogenic anti-inflammatory composition (see WO/2012/012874).
SUMMARY
[0003] Methods are provided for predicting the degree to which a patient has an increased likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists. Methods may include screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the subject has an increased likelihood of benefiting from said treatment if the diagnostic SNP comprises a selected response allele. Methods are also provided for assessing the degree to which a patient is responding to a particular immunomodulatory treatment regimen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] Figure 1 is a series of graphs illustrating efficacy of SSI treatment in alternative model animals in the colitis model: a logarithmic Y axis scale illustrating relative levels of IFN-gamma (A) and IL-17A expression (B), and cumulative data for IL-17A expression (C), as well as site-specific evidence of QBECO efficacy in increasing I L-18 gene expression in colon tissue, compared to QBKPN (D).
[0005] Figure 2 is a series of graphs illustrating efficacy of SSI treatment in alternative model animals in the colitis model: microbiome (A and B) and histology (C).
[0006] Figure 3 includes three bar graphs illustrating the activation of pattern recognition receptors in HEK cells after QBECO or QBKPN stimulation, showing respectively: A) Toll-like receptor (TLR) activation as measured by ΝΚ-κΒ activation; B) NOD2 and C-type lectin receptors (CTL) as measured by ΝΚ-κΒ activation; and, C) RLR (Rig-1 -like receptors) as measured by IRF3 activation.
[0007] Figure 4 is a PRR repertoire fingerprint bar graph, in which a PRR fingerprint was constructed for QBECO and QBKPN SSIs from the 1/10 dilution data, after subtracted the negative control data. Bars, in order, represent TLR2, 3, 4, 5, 7, 8, 9, NOD1 , NOD2, Dectin 1 a, Dectin 1 b and Mincle. RIG-1 and MDA5 are not shown. The positive control is specific for each PRR (i.e. LPS for TLR4).
[0008] Figure 5 is a PRR fingerprint radar graph, in which a PRR fingerprint was constructed for QBECO and QBKPN SSIs from the 1/10 dilution data, after subtracted the negative control data, and plotted on a radar graph.
[0009] Figure 6 is a collection of 3 bar graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
[0010] Figure 7 is a collection of 4 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy. [0011] Figure 8 is a collection of 4 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
[0012] Figure 9 is a column scatter graph illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
[0013] Figure 10 is a collection of 5 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
[0014] Figure 11 is a collection of 4 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
[0015] Figure 12 is a collection of 3 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
[0016] Figure 13 is a collection of 4 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
[0017] Figure 14 is a collection of 4 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
[0018] Figure 15 is a collection of 5 column scatter graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
[0019] Figure16 is a collection of 6 bar graphs illustrating the frequency of a selected SNP genotype in responders and non-responders to an SSI therapy.
[0020] Figure 17A is a violin plot representing the log distribution of risk scores, comparing last recorded response for all CD subjects using risk scores based on 1 12 IBD SNPs (P-value: 2.430E-05).
[0021] Figure 17B is a violin plot representing the log distribution of risk scores, comparing last recorded response for all CD subjects using risk scores based on 3 IBD SNPs (P-value: 1.385E-04). [0022] Figure 18 is a violin plot representing the log distribution of risk scores, comparing last recorded response for all UC subjects using risk scores based on 84 IBD SNPs (P-value: 1.255E-02).
[0023] Figure 19 is a violin plot representing the log distribution of risk scores, comparing last recorded response for all CD and UC subjects using risk scores based on 1 12 IBD SNPs (P-value: 8.184E-07).
[0024] Figure 20 is a graph illustrating the change in serum IL-18 levels in patients treated with QBECO vs. Placebo.
[0025] Figure 21 is a set of 4 graphs illustrating serum immune cytokine changes with QBECO treatment that associated with clinical response.
[0026] Figure 22 is a set of 3 graphs illustrating baseline levels of Eotaxin-1 , IL- 10 and IL-12p40 by patient response to QBECO.
[0027] Figure 23 is a set of line graphs, illustrating changes in cytokine concentration over time in the sera of ulcerative colitis patients undergoing a QBECO SSI therapy. In each case, Responders are the top line.
[0028] Figure 24 is a graph illustrating the change in body weight over time in a murine DSS colitis model.
[0029] Figure 25 is a graph illustrating change in disease activity index over time in a murine DSS colitis model.
[0030] Figure 26 is a graph illustrating change in the FITC-dextran assay over time in a murine DSS colitis model.
[0031] Figure 27 is a graph illustrating blood neutrophil levels in disease free mice, over time, with or without an initial QBECO SSI treatment (mean +/- SEM, n = 10 mice per group).
[0032] Figure 28 is a collection of three graphs illustrating blood cytokine levels in disease free mice, over time, with or without an initial QBECO SSI treatment (mean +/- SEM, n = 10 mice per group).
DETAILED DESCRIPTION
[0033] In the following detailed description, various examples are set out of particular embodiments, together with experimental procedures that may be used to implement a wide variety of modifications and variations in the practice of the present invention. For clarity, a variety of technical terms are used herein in accordance with what is understood to be the commonly understood meaning, as reflected in definitions set out below. General Definitions
[0034] An "immunogen" refers to a molecule, or a composition comprising the molecule, that is capable of eliciting an immune response by an organism's immune system. An "antigen" refers to a molecule that is capable of binding to the product of an immune response.
[0035] "Pathogenic" agents are agents, such as microbes, such as bacteria or viruses, which are known to cause infection in a host in nature, and in this sense, "pathogenic" is used in the context of the present invention to mean "naturally pathogenic". Although a wide variety of microbes may be capable of causing infection under artificial conditions, such as artificial inoculations of a microbe into a tissue, the range of microbes that naturally cause infection is necessarily limited, and well established by medical practice.
[0036] An "infection" is the state or condition in which the body or a part of it is invaded by a pathogenic agent (e.g., a microbe, such as a bacterium) which, under favorable conditions, multiplies and produces effects that are injurious (Taber's Cyclopedic Medical Dictionary, 14th Ed. , C.L. Thomas, Ed., F.A. Davis Company, PA, USA). An infection may not always be apparent clinically and may result in only localized cellular injury. Infections may remain subclinical, and temporary if the body's defensive mechanisms are effective. Infections may spread locally to become clinically apparent as an acute, a subacute, or a chronic clinical infection or disease state. A local infection may also become systemic when the pathogenic agent gains access to the lymphatic or vascular. Infection is usually accompanied by inflammation, but inflammation may occur without infection.
[0037] "Inflammation" is the characteristic tissue reaction to injury (marked by swelling, redness, heat, and pain), and includes the successive changes that occur in living tissue when it is injured. Infection and inflammation are different conditions, although one may arise from the other (Taber's Cyclopedic Medical Dictionary, supra). Accordingly, inflammation may occur without infection and infection may occur without inflammation (although inflammation typically results from infection by pathogenic bacteria or viruses). Inflammation is characterized by the following symptoms: redness (rubor), heat (calor), swelling (tumour), pain (dolor). Localized visible inflammation on the skin may be apparent from a combination of these symptoms, particularly redness at a site of administration.
[0038] Various subjects may be treated or assayed or sampled in accordance with alternative aspects of the invention. As used herein, a "subject" is an animal, for e.g. , a vertebrate or a mammal. Accordingly, a subject may be a patient, e.g., a human, suffering from an immune dysregulation. A subject may also be an experimental animal, e.g., an animal model of an immune dysregulation. In some embodiments, the terms "subject" and "patient" may be used interchangeably, and may include a human, a non-human mammal, a non-human primate, a rat, mouse, or dog. A healthy subject may be a human who is not suffering from a disease, such as immune dysfunction, or suspected of having the disease, or who is not suffering from a chronic disorder or condition. A "healthy subject" may also be a subject who is not immunocompromised. By immunocompromised is meant any condition in which the immune system functions in an abnormal or incomplete manner. Immunocompromisation may be due to disease, certain medications, or conditions present at birth. Immunocompromised subjects may be found more frequently among infants, the elderly, and individuals undergoing extensive drug or radiation therapy.
[0039] A "sample" from a subject may include any relevant biological material, including for example a cell, tissue or bodily fluid sample taken from a patient. For example, a sample may conveniently include samples of skin, cheek, blood, stool, hair or urine. Sample nucleic acids for use in diagnostic and prognostic methods can for example be obtained from a selected cell type or tissue of a subject. For example, a subject's bodily fluid (e.g. blood) can be obtained by known techniques. Alternatively, nucleic acid tests can be performed on dry samples (e.g., hair or skin).
[0040] The term "polymorphism" refers to a location within a biological sequence, such as a genomic sequence, which varies within a population.
Polymorphisms are comprised of different "alleles". The term "genotype" refers to the specific alleles in a genome, for example in a cell, tissue sample or an individual. The location of a polymorphism may be identified by its position, for example within the genome or within a sequence such as a protein that is reflective of a genomic locus. This may for example be provided in the form of a
characterization of the different amino acids or bases that are found at a
designated location. For diploid genomes, the genotype is typically comprised of at least two alleles, which may be the same (homozygous) or different (heterozygous). Individual polymorphisms are typically assigned unique identifiers in the art (such as "Reference SNP", "refSNP" or "rs#"), for example in the Single Nucleotide Polymorphism Database (dbSNP) of Nucleotide Sequence Variation available on the NCBI website.
[0041] Characterization of polymorphisms, alleles or a genotype may be performed by any of very wide variety of methods. These methods may for example variously involve hybridization, labeling, cloning, sequencing and/or amplification of nucleic acids, such as genomic DNA, for example using PCR, LCR, xMAP, invader assays, mass spectrometry, pyrosequencing, selective oligonucleotide
hybridization, selective amplification, selective primer extension or probes. In this context, the term "probes" includes naturally occurring or recombinant single- or double-stranded nucleic acids or chemically synthesized nucleic acids. A probe can for example be a polynucleotide of a length suitable for selective hybridization to a nucleic acid containing a polymorphic region. Labeled probes also can be used in conjunction with amplification of a polymorphism. DNA microarray technologies, sometimes referred to as DNA chips or gene chips, may for example be used for genomic characterization, for example to characterize point mutations, single nucleotide polymorphisms (SNPs), and/or short tandem repeats (STRs). For example, several probes capable of hybridizing specifically to an allelic variant may be attached to a solid phase support by a variety of processes, including
lithography. Additional methods include laser capture microdissection (LCM), comparative genomic hybridization (CGH) and chromatin immunoprecipitation (ChiP). Allele specific hybridization may for example make use of probes
overlapping the polymorphic site and having about 5, or alternatively 10, or alternatively 20, or alternatively 25, or alternatively 30 nucleotides around the polymorphic region. Alternatively, the presence of the specific allele in DNA from a subject can in some case be characterized by restriction enzyme analysis.
Similarly, protection from cleavage agents (such as a nuclease, hydroxylamine or osmium tetroxide) can be used to detect mismatched bases in RNA/RNA
DNA/DNA, or RNA/DNA heteroduplexes, using technique that may be described as "mismatch cleavage" assays. Alterations in electrophoretic mobility may be used to characterize allelic variants, for example to detect single strand conformation polymorphisms.
[0042] Many of the methods described herein may be performed using kits, for example comprising at least one probe or primer nucleic acid, or one of more of the compositions described herein and instructions for use of the kit. Kits can for example comprise at least one probe or primer which is capable of specifically hybridizing to a polymorphic region or adjacent to the polymorphic region, so that the oligonucleotides are "specific for" the polymorphic region. Kits may also comprise at least one reagent necessary to perform a particular assay. Kits can also include positive controls, negative controls, sequencing markers, or antibodies, for example for determining a subject's genotype or biological marker profile.
[0043] An "immune response" includes, but is not limited to, one or more of the following responses in a mammal: induction or activation of antibodies, neutrophils, monocytes, macrophages (including both M1 -like macrophages and M2-like macrophages as described herein), B cells, or T cells (including helper T cells, natural killer cells, cytotoxic T cells, gamma-delta (γδ) T cells), such as induction or activation by one or more immunogens in an immunogenic composition, following administration of the composition. An immune response to a composition thus generally includes the development in the host animal of a cellular and/or antibody- mediated response to the composition. In some embodiments, the immune response is such that it will also result in slowing or stopping the progression of an immune dysregulation, or a disease characterized by immune dysregulation. An immune response may accordingly include one or both of a cellular immune response and/or a humoral immune response, and may be an adaptive response or an innate immune response. [0044] "Immune dysregulation" is an inappropriately regulated immune response, such as an inappropriately restrained or inappropriately robust immune response. The immune dysregulation may for example be in the context of an autoimmune, inflammatory, or degenerative disease (such as Crohn's disease, inflammatory bowel disease or ulcerative colitis). Inflammatory bowel disease (I BD) is a name frequently given to a group of inflammatory conditions of the colon and small intestine, generally characterized by similar symptoms of immune
dysregulation and indeterminate etiology. Major sub-types of IBD are recognized clinically as Crohn's disease and ulcerative colitis. In addition to Crohn's disease and ulcerative colitis, IBD may also include conditions recognized as any one of the following: collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome or indeterminate colitis. The difference between these conditions relate primarily to the location and nature of the inflammatory changes in the gastrointestinal tract (GIT). Crohn's disease, for example, is generally recognized as potentially affecting any part of the gastrointestinal tract, from mouth to anus, with a majority of the cases marked by relapsing and remitting
granulomatous inflammation of the alimentary tract in the terminal ileum and colon. Ulcerative colitis, in contrast, is generally considered to be restricted to the colon and the rectum. The various regions of the gastrointestinal tract in which these inflammatory conditions may exhibit symptoms include: the bowel or intestine, including: the small intestine (which has three parts: the duodenum, the jejunum , and the ileum); the large intestine (which has three parts: the cecum, the colon, which includes the ascending colon, transverse colon, descending colon and sigmoid flexure; and the rectum); and, the anus.
[0045] A "site specific immunotherapy" (SSI) is an immunomodulatory treatment that is effective to therapeutically or prophylactically alter an aspect of the immune state, or immune system physiology, at an anatomical site or sites, such as an organ or tissue. In some instances, for example, an SSI may be adapted to ameliorate an immune dysregulation, or to treat a condition characterized by an immune dysregulation.
PRR Liqands [0046] Aspects of the invention relate to the use of PRR ligands. PRR ligands may for example be available commercially, for example in widely available preparations of attenuated or killed recombinant bacteria, which may for example be ligands for TLR2, TLR4 and TLR5. Compositions of pathogen-associated molecular patterns (PAMPs) may include PAMPS that are recognized by PRRs, including: Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-l-like receptors (RLRs), C-type lectin receptors (CLRs) including Dectin-1 , cytosolic dsDNA sensors (CDSs) and NLRs involved in the formation of inflammasomes.
[0047] Toll-like receptor 2 (TLR2) is involved in the recognition of a wide array of microbial molecules representing broad groups of species including Gram-positive and Gram-negative bacteria, as well as mycoplasma and yeast. TLR2 recognizes cell-wall components such as peptidoglycan, lipoteichoic acid and lipoprotein from Gram-positive bacteria, lipoarabinomannan from mycobacteria, and zymosan from the yeast cell wall. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA). Bacterial lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) which interacts with at least three different extracellular proteins: LPS- binding protein (LBP), CD14 and, myeloid differentiation protein 2 (MD-2), to induce a signaling cascade leading to the activation of NF-κΒ and the production of proinflammatory cytokines. LPS generally consists of a polysaccharide region that is anchored in the outer bacterial membrane by a carbohydrate lipid moiety: lipid A, which is largely responsible for the immunostimulatory activity of LPS. Particularly active forms of lipid A contain six fatty acyl groups, as for example may be found in pathogenic bacteria that are strains of Escherichia coli or Salmonella spp. Toll-like receptor 5 (TLR5) recognizes flagellin from both Gram-positive and Gram-negative bacteria. Toll-like receptor 7 (TLR7) and TLR8 recognize single stranded RNAs and small synthetic molecules such as imidazoquinolines and nucleoside analogs. Tolllike receptor 9 (TLR9) recognizes specific unmethylated CpG motifs prevalent in microbial but not vertebrate genomic DNA.
[0048] NLRs are a family of at least 22 cytoplasmic innate immune sensors, including NOD1 (CARD4) and NOD2 (CARD15) which are intracellular pattern- recognition receptors involved in the recognition of peptidoglycan (PGN). These receptors detect specific motifs within PGN. NOD1 senses the diaminopimelatic acid (DAP)-containing muropeptide (specifically d-Glu-meso-DAP dipeptide "iE- DAP" dipeptide) which is found primarily in PGN of Gram-negative bacteria, as well as certain Gram-positive bacteria. NOD2 recognizes the muramyl dipeptide (MDP) structure found in almost all bacterial PGN.
[0049] The RIG-I-Like receptors (RLRs), particularly RIG-I and MDA-5, detect viral RNA species.
[0050] CLR ligands include Dectin-1 and Mincle (macrophage-inducible C-type lectin) agonists. Dectin-1 is a specific receptor for β-glucans, which are glucose polymers found in the cell walls of fungi. Mincle is a multi-tasking danger signal receptor that recognizes a wide variety of ligands such as damaged cells, fungal components, yeast components and components of mycobacteria.
[0051] Cytosolic DNA Sensors (CDS) bind intracellular DNA from pathogens, and there are multiple CDSs which may display contextual preferences for the recognition of particular DNAs.
[0052] Cyclic dinucleotides (CDNs) and xanthenone derivatives, such as DMXAA, bind to and activate STING (STimulator of INterferon Genes).
[0053] The inflammasome is a multi-protein complex involved in the production of mature I L-1 β, specifically through cleavage of pro— I L-1 β and pro— I L-18 into active and secretable forms. Inflammasomes may be segregated into NLRP1 , NLRP3, NLRC4 and AI M2 subtypes, which are activated by a wide variety of microbial molecules, danger signals and crystalline substances.
Table 1 : PRR Receptors and their Ligands
Figure imgf000013_0001
TLR4 Lipopolysacchande, Monophosphoryl
Lipid A
TLR5 Flagellin
TLR7/8 Single-stranded RNAs, Nucleoside analogs,
Imidazoquinolines/Thiazoquinolines
TLR9 unmethylated CpG DNA motifs
NOD1 iE-DAP, Acylated iE-DAP, D-gamma- Glu-mDAP, L-Ala-gamma-D-Glu- mDAP
NOD2 MDP (MurNAc-L-Ala-D-isoGIn,
muramyl dipeptide), N-glycolylated muramyldipeptide, N-Acetyl-muramyl- L- Alanyl-D-Glutamin-n-butyl-ester, MurNAc-Ala-D-isoGln-Lys, N- Acetylmuramyl-L-Alanyl-D- Isoglutamine (L-D isoform), 6-0- stearoyl-N-Acetyl-muramyl-L-alanyl-D- isoglutamine, Pam2C-Aca-Benzyl- Murabutide,
TLR2/NOD2 Pam2C-conjugated murabutide
NOD1/NOD2 PGN, Pam2C-conjugated murabutide
RIG1 /MDA5 5' triphosphate double stranded RNA
(18-20mer),
polyriboinosinic:polyribocytidylic acid
DAI, LRRFIP1 , AI M2, RIG1 dsDNA, poly(dA-dT poly(dT-dA)
Dectin-1 Beta-glucan peptide, fungal cell wall preparations
Mincle damaged microbial cells, fungus, yeast and mycobacteria, Trehalose- 6,6-dibehenate, trehalose-6,6- dimycolate
STING Cyclic dinucleotides (c-di-nucleotides), xanthenone derivatives, 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, 22'- cGAMP, c-di-AMP (cyclic di-adenylate monophosphate), c-di-GMP, c-di-I MP, c-di-UMP, c-di-AMP
Table 2: Cytosolic nucleic acid-sensing PRRs and their Ligands (Broz & Monack, 2013, Nature Reviews Immunology 13, 551-565).
Figure imgf000015_0001
NLRP3 ssRNA, dsRNA, bacterial mRNA and oxidized
mitochondrial DNA
AIM2 DNA
I FI 16 dsDNA
LRRFIP1 B-form DNA, Z-form DNA and dsRNA
DAI DNA
I FIT1 ,2,3 and 5 PPP-ssRNA
[0054] Aspects of the invention accordingly involve using PRR agonists derived from a selected microbial pathogen. For example, peptidoglycan (PGN) may be obtained from a bacteria or bacterial strain that is pathogenic in a selected target tissue or organ, for use as a NOD1/NOD2 agonist. Similarly, cell wall components may be obtained from a bacteria or bacterial strain that is pathogenic in a selected target tissue or organ, for use as a TLR2 agonist. Similarly, DNA, including double stranded DNA, particularly repetitive double stranded DNA, may be obtained from a microbial pathogen, such as a bacteria or bacterial strain that is pathogenic in a selected target tissue or organ, for use as a DAI, LRRFIP1 , RIG1 , TLR9, AIM2 or cytosolic DNA sensor (CDS) agonist. Beta-glucan peptides may be obtained from fungi or yeast that are pathogenic in a selected target tissue or organ, for use as a Dectin-1 agonists. Cyclic dinucleotides may be obtained from a microbial pathogen that is pathogenic in a selected target tissue or organ, for use as a STING agonist.
[0055] Aspects of the invention involve compositions that have a distinct PRR agonist signature, which connotes a repertoire of PRR agonists that are together collected in a therapeutic vehicle, so that the selected collection of PRR agonists is distinct. A "therapeutic vehicle" in this context is a formulation that aggregates and retains the PRR agonists, for example in a pharmaceutically acceptable particle or vesicle, such as a recombinant microbe. For example, the PRR agonist signature may be different from a reference PRR agonist signature, for example different from the collection of PRR agonists that would be present on a microbe that is not pathogenic in the target tissue. The PRR signature may also be distinct in the sense that it is different than a native PRR agonist signature of the microbial mammalian pathogen, for example altered by way of the recombinant expression of genes that alter what would otherwise be the wildtype PRR agonist signature of the pathogen. For purposes of determining the distinctiveness of a PRR agonist signature, the levels or kinds of PRR agonist may be directly measured, or may be measured for example by determining the activation or inhibition of a signaling pathway in a cell consequent to PRR agonist/receptor binding.
[0056] Immunomodulatory or immunogenic compositions are provided that constitute an artificial repertoire of mammalian pattern recognition receptor (PRR) agonists. The PRR agonist repertoire is selected so that it in effect recapitulates a distinct portion of a PRR agonist signature of a microbial pathogen, and more specifically a pathogen that is pathogenic in a selected target tissue. The PRR agonist signature is distinct in the sense that it is different from PRR agonist signatures of microbes that are not pathogenic in the target tissue, and it is also distinct in the sense that it is different from the native PRR agonist signature of the wild-type pathogen. This distinct artificial repertoire of PRR agonists may then be formulated so that the PRR agonists are presented together in a therapeutic vehicle, for example so that the PRR agonist repertoire may be presented in combination. The therapeutic vehicle may for example be a recombinant microbe, a cellular fraction of a microbial cell, a microparticle or a liposome. The composition may for example comprise microbial agonists for at least a minimum number of distinct mammalian PRRs, for example at least 5, as described in more detail herein. The vehicle may then be delivered, for example systemically, so that the PRR agonist repertoire is presented to an innate immune cell resident in the target tissue in a host, such as a mammalian host. The therapeutic vehicle may for example aggregate the artificial repertoire of PRR agonists, so that the proximity of the plurality of PRR agonists is maintained during systemic distribution in a host. Compositions of this kind may be used to treat a wide variety of diseases characterized by immune dysregulation, including neoplastic diseases and autoimmune diseases.
[0057] Aspects of the innovation involve the use of an immunogenic composition in methods of treating an immune dysregulation in a target tissue in a mammalian host, wherein the composition comprises the foregoing artificial repertoire of mammalian PRR. The artificial repertoire of PRR agonists may be formulated together in a therapeutic vehicle for combined presentation following administration to a mammalian host. Composition may for example include components of the microbial mammalian pathogen that are agonists for a select number of distinct mammalian PRRs, as discussed in more detail below, for example at least 5.
Compositions may for example be adapted for use so as to modulate an innate immune response in the target tissue. The therapeutic vehicle may for example include a recombinant microbe, a cellular fraction of the recombinant microbe, a cellular fraction of a microbial cell, a microparticle or a liposome, each comprising components of the microbial mammalian pathogen that provide the PRR agonists that together make up the artificial repertoire of PRR agonists. A recombinant microbe may for example include a recombinant gene encoding a component of at least one of the PRR agonists. In select aspects, the therapeutic vehicle may for example include a whole killed or attenuated cell of the recombinant microbe.
Alternatively, the cellular fraction of the microbial mammalian pathogen may be used, for example, a bacterial outer membrane fraction; a bacterial inner membrane fraction; a pellet from a gradient centrifugation of microbial cell components; or chromosomal DNA. The therapeutic vehicle may for example be formulated for use for delivering the PRR agonists to the target tissue.
[0058] The therapeutic vehicle may for example include additional therapeutic moieties, such as one or more of: GMCSF, vitamin D, NOHA, alphl antitrypsin, glutathione, an isoprenoid, or a-galactosylceramide. Alternatively, the therapeutic vehicle may further include a heterologous PRR agonist, such as a PRR agonist that is not a component of the microbial mammalian pathogen.
[0059] The subject of treatment, such as a mammalian host or human patient, may for example be suffering from a disease or condition characterized by the immune dysregulation in the target tissue, such as an inflammatory disorder.
[0060] The composition may be adapted for use in an amount effective to modulate a biomarker, for example one or more of PD1 , PDL1 , IP-10, MIG, RANTES, neutrophils, Ly6C monocytes, and NKG2D. In select embodiments, the composition may for example be adapted for use in an amount effective to down- regulate PD1 and/or PDL1 expression in cells present in the target tissue. The composition may accordingly be adapted for use so as to modulate an adaptive immune response in the host, for example as a concomitant of modulating an innate immune response.
[0061] In select embodiments, the therapeutic vehicle may be for administration at an administration site that is not the target tissue, and the site may for example be the skin, subcutaneous tissue, the respiratory tract. Administration may be enteric, or non-enteric. The therapeutic vehicle may be formulated for systemic distribution of the PRR agonists following administration at a localized
administration site. The therapeutic vehicle may be administered in a plurality of doses over a dosage duration, and the dosage duration may for example be at least two weeks, or any of other wide range of dosage regimens disclosed herein or known in the art.
[0062] In select embodiments, human patient treated in accordance with the invention may for example be immunosuppressed or immunocompromised, or may be geriatric or pediatric patients.
[0063] The therapeutic uses recited herein are reflected in corresponding methods of treatment, and vice versa.
Recombinant Embodiments
[0064] Various genes and nucleic acid sequences of the invention may be recombinant sequences. The term "recombinant" means that something has been recombined, so that when made in reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that are joined together or produced by means of molecular biological techniques. Nucleic acid "constructs" are accordingly recombinant nucleic acids, which have been generally been made by aggregating interoperable component sequencers. The term
"recombinant" when made in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques. The term "recombinant" when made in reference to the genetic composition or an organism or cell refers to new combinations of alleles that did not occur in the parental genomes. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Referring to a nucleic acid construct as "recombinant" therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention (so that it is anthropogenic). Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent
recombination and/or repair events.
[0065] Recombinant constructs of the invention may include a variety of functional molecular or genomic components, as required for example to mediate gene expression or suppression in a transformed plant. In this context, "DNA regulatory sequences," "control elements," and "regulatory elements," refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, and protein degradation signals that regulate gene expression, as well as epigenetic regulatory signals for example involving methylation or acetylation of histones (e.g. histone methyltransferase or
acetyltransferase), leading to conformational changes in the transcriptional landscape and gene expression differences. In the context of the present disclosure, "promoter" means a sequence sufficient to direct transcription of a gene when the promoter is operably linked to the gene. The promoter is accordingly the portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not universally, located in the 5' non-coding regions of a gene. A promoter and a gene are "operably linked" when such sequences are functionally connected so as to permit gene expression mediated by the promoter. The term "operably linked" accordingly indicates that DNA segments are arranged so that they function in concert for their intended purposes, such as initiating transcription in the promoter to proceed through the coding segment of a gene to a terminator portion of the gene. Gene expression may occur in some instances when appropriate molecules (such as transcriptional activator proteins) are bound to the promoter. Expression is the process of conversion of the information of a coding sequence of a gene into mRNA by transcription and subsequently into polypeptide (protein) by translation, as a result of which the protein is said to be expressed. As the term is used herein, a gene or nucleic acid is "expressible" if it is capable of expression under appropriate conditions in a particular host cell.
[0066] An "isolated" nucleic acid or polynucleotide as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring). An isolated nucleic acid or polypeptide may contain less than about 50%, less than about 75%, less than about 90%, less than about 99.9% or less than any integer value between 50 and 99.9% of the cellular or biological components with which it was originally associated. A polynucleotide amplified using PCR so that it is sufficiently distinguishable (on a gel from example) from the rest of the cellular components is, for example, thereby "isolated". The polynucleotides of the invention may be "substantially pure," i.e., having the high degree of isolation as achieved using a purification technique.
[0067] In the context of biological molecules "endogenous" refers to a molecule such as a nucleic acid that is naturally found in and/or produced by a given organism or cell. An "endogenous" molecule may also be referred to as a "native" molecule. Conversely, in the context of biological molecules "exogenous" refers to a molecule, such as a nucleic acid, that is not normally or naturally found in and/or produced by a given organism or cell in nature.
[0068] As used herein to describe nucleic acid or amino acid sequences, the term "heterologous" refers to molecules or portions of molecules, such as DNA sequences, that are artificially introduced into a particular host cell, for example by transformation. Heterologous DNA sequences may for example be introduced into a host cell by transformation. Such heterologous molecules may include sequences derived from the host cell. Heterologous DNA sequences may become integrated into the host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination events. [0069] Various aspects of the present disclosure encompass nucleic acid or amino acid sequences that are homologous to other sequences. As the term is used herein, an amino acid or nucleic acid sequence is "homologous" to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, sequence conservation or identity does not infer evolutionary relatedness). Nucleic acid sequences may also be homologous if they encode substantially identical amino acid sequences, even if the nucleic acid sequences are not themselves substantially identical, for example as a result of the degeneracy of the genetic code.
[0070] With reference to biological sequences "substantial homology" or
"substantial identity" is meant, in the alternative, a homology of greater than 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% up to 100% sequence identity. Homology may refer to nucleic acid or amino acid sequences as the context dictates. In alternative embodiments, sequence identity may for example be at least 75%, at least 90% or at least 95%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman (1981 ) Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al. (1990), J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (NCBI) at their Internet site. The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 1 1 , the BLOSUM62 scoring matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1 , less than about 0.1 , less than about 0.01 , or less than about 0.001 .
[0071] An alternative indication that two amino acid sequences are substantially identical is that one peptide is specifically immunologically reactive with antibodies that are also specifically immunoreactive against the other peptide. Antibodies are specifically immunoreactive to a peptide if the antibodies bind preferentially to the peptide and do not bind in a significant amount to other proteins present in the sample, so that the preferential binding of the antibody to the peptide is detectable in an immunoassay and distinguishable from non-specific binding to other peptides. Specific immunoreactivity of antibodies to peptides may be assessed using a variety of immunoassay formats, such as solid-phase ELISA immunoassays for selecting monoclonal antibodies specifically immunoreactive with a protein (see Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York).
[0072] An alternative indication that two nucleic acid sequences are substantially identical is that the two sequences hybridize to each other under moderately stringent, or stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C, and washing in
0.2xSSC/0.1 % SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1 , Green Publishing Associates, Inc. , and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHP0 , 7% SDS, 1 mM EDTA at 65° C , and washing in 0.1 xSSC/0.1 % SDS at 68° C. (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular
Biology— Hybridization with Nucleic Acid Probes, Part I, Chapter 2 Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, N.Y.). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. The term "a polynucleotide that hybridizes under stringent (low, intermediate) conditions" is intended to encompass both single and double-stranded
polynucleotides although only one strand will hybridize to the complementary strand of another polynucleotide. Washing in the specified solutions may be conducted for a range of times from several minutes to several days and those skilled in the art will readily select appropriate wash times to discriminate between different levels of homology in bound sequences.
[0073] It is well known in the art that some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. As used herein, the term "conserved amino acid substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without any appreciable loss or gain of function, to obtain a
biologically equivalent polypeptide. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing. Conversely, as used herein, the term "non-conserved amino acid substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution causes an appreciable loss or gain of function of the peptide, to obtain a polypeptide that is not biologically equivalent.
[0074] In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar
hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following hydrophilicity values are assigned to amino acid residues (as detailed in U.S. Pat. No. 4,554, 101 ): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn
(+0.2); Gin (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1 .0); Met (-1 .3); Val (-1 .5); Leu (-1 .8); lie (-1 .8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4). Non-conserved amino acid substitutions may be made were the hydrophilicity value of the residues is significantly different, e.g. differing by more than 2.0.
[0075] In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: lie (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1 .9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1 .6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5). Non-conserved amino acid substitutions may be made were the hydropathic index of the residues is significantly different, e.g. differing by more than 2.0.
[0076] In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, lie, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gin, Tyr. Non-conserved amino acid substitutions may be made were the residues do not fall into the same class, for example substitution of a basic amino acid for a neutral or non-polar amino acid. Microorganisms
[0077] Most animals are colonized to some degree by microorganisms, such as bacteria, which exist in symbiotic or commensal relationships with the host animal. Thus, many species of normally harmless bacteria are found in healthy animals, and are usually localized to the surface of specific organs and tissues. Often, these microbial communities aid in the normal functioning of the body, as members of what is termed the microbiome. Microbes that are generally harmless, such as Escherichia coli, can cause infection in healthy subjects, with results ranging from mild infection to death. Whether or not a microorganism is pathogenic (i.e., causes infection) depends on factors such as: the route of entry and access to specific host cells, tissues, or organs; the intrinsic virulence of the microorganism; the amount of the microorganism present at the site of potential infection; or the health of the host animal. Thus, microorganisms that are normally harmless can become pathogenic given favorable conditions for infection, and even the most virulent microorganism generally requires specific circumstances to cause infection. Accordingly, microbial species that are members of the normal flora can be pathogens when they move beyond their normal ecological role in the endogenous flora. For example, endogenous species can cause infection outside of their ecological niche in regions of anatomical proximity, for example by contiguous spread. When this occurs, these normally harmless endogenous bacteria are pathogenic.
[0078] Specific microbial species are known to cause infections in specific cells, tissues, or organs in otherwise healthy subjects. Examples of bacteria and viruses that commonly cause infections in specific organs and tissues of the body are listed below; and these examples are not limiting in the sense that a skilled person would be able to recognize and identify infectious or pathogenic bacteria that cause infections, or commonly cause infections, in various organs and tissues in otherwise healthy organisms (and recognize the relative frequency of infection with each bacterial species) based on the knowledge in the field as represented, for example, by the following publications: Manual of Clinical Microbiology 8th Edition, Patrick Murray, Ed., 2003, ASM Press American Society for Microbiology,
Washington DC, USA; Mandell, Douglas, and Bennett's Principles and Practice of
Infectious Diseases 5th Edition, G. L. Mandell, J.E. Bennett, R. Dolin, Eds., 2000, Churchill Livingstone, Philadelphia, PA, USA, all of which are incorporated by reference herein.
[0079] Infections of the stomach are commonly caused by the following bacterial species: Streptococcus pyogenes or Helicobacter pylori; or viral pathogens:
cytomegalovirus, herpes simplex, Epstein-Barr, rotaviruses, noroviruses, or adenoviruses.
[0080] Infections of the small bowel are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri; or viral pathogens: adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, or cytomegalovirus.
[0081] Infections of the colon/rectum are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis,
Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri; or viral pathogens: adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, or cytomegalovirus.
[0082] Infections of the anus are commonly caused by the following bacterial species: Streptococcus pyogenes, Bacteroides spp., Fusobacterium spp., anaerobic streptococci, Clostridium spp., Escherichia coli, Enterobacter spp., Pseudomonas aeruginosa, or Treponema pallidum; or viral pathogens: herpes simplex.
[0083] Infections of the perineum are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterococcus spp., Bacteroides spp., Fusobacterium spp. , Clostridium spp., Pseudomonas aeruginosa, anaerobic streptococci, Clostridium spp., or Enterobacter spp.; or viral pathogens: herpes simplex.
[0084] Infections of the peritoneum are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Escherichia coli, Klebsiella spp. , Proteus spp. , enterococci, Bacteroides fragilis, Prevotella melaninogenica, Peptococcus spp., Peptostreptococcus spp. , Fusobacterium spp., or Clostridium spp.
[0085] Infections of the retroperitoneal area are commonly caused by the following bacterial species: Escherichia coli, or Staphylococcus aureus.
[0086] Bacterial species are classified operationally as collections of similar strains (which generally refers to groups of presumed common ancestry with identifiable physiological but usually not morphological distinctions, and which may be identified using serological techniques against bacterial surface antigens).
Thus, each bacterial species (e.g., Streptococcus pneumoniae) has numerous strains (or serotypes), which may differ in their ability to cause infection or differ in their ability to cause infection in a particular organ/site. For example, although there are at least 90 serotypes of Streptococcus pneumoniae, serotypes 1 , 3, 4, 7, 8, and 12 are most frequently responsible for pneumococcal disease in humans.
[0087] Certain strains of Escherichia coli, referred to as extraintestinal pathogenic E. coli (ExPEC), are more likely to cause urinary tract infection or other extraintestinal infections such as neonatal meningitis, whereas other strains, including enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E coli (EHEC), Shiga toxin-producing E coli (STEC), enteroaggregative E. coli (EAEC), enteroinvasive E coli (EIEC) and diffuse adhering E coli (DAEC) are more likely to cause gastrointestinal infection/diarrhea. Even among the sub-category of ExPEC strains, specific virulence factors (e.g., production of type-1 fimbriae) enable certain strains to be more capable of causing infection of the bladder, while other virulence factors (e.g., production of P fimbriae) enable other strains to be more capable of causing infection in the kidneys. In accordance with the present invention, one or more of an ETEC, EPEC, EHEC, STEC, EAEC, EIEC or DAEC strains of E. coli (i.e., strains that cause colon infection), may be chosen for a formulation to treat immune dysregulation in the colon.
[0088] The compositions of the invention include immunogens of pathogenic microbial species (bacterial, viral or fungal) that are pathogenic in a specific tissue or organ, in which the immunogens are provided in the form of an artificial repertoire of mammalian PRR agonists that recapitulate a distinct portion of the PRR agonist signature of the microbial mammalian pathogen that is pathogenic in the target tissue. In select embodiments, the portion of the PRR agonist signature is distinct in the sense that it is both: different from a reference PRR agonist signature of a microbe that is not pathogenic in the target tissue; and, different than the native PRR agonist signature of the microbial mammalian pathogen. This distinct artificial repertoire of mammalian PRR agonists are formulated together in a therapeutic vehicle for combined presentation to an innate immune cell resident in the target tissue in the mammalian host. Formulations and Therapeutic Vehicles
[0089] Compositions of the invention may be provided alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to mammals, for example, humans (a "therapeutic vehicle"). As used herein
"pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable for any appropriate form of administration, including subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intramuscular, sublingual, inhalational, intratumoural or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound (i.e., the specific bacteria, bacterial antigens, or compositions thereof of the invention), use thereof in the
pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
[0090] Aspects of the invention involve the use of nanoparticle (NP)
formulations. For example, virus-like particles (VLPs) are in essence empty viral particles with an intact protein hull and, in some embodiments, membrane envelopes. In general, VLPs lack genetic material. Production of VLPs may for example be by expression of viral proteins in mammalian, avian, insect, plant, yeast, or bacterial cells. Alternatively, fully synthetic VLPs may be produced.
Alternative nanoparticle formulations emulsions, liposomes alginates, chitosan, and polylactide-coglycolide (PLGA) NPs. Examples of NP/TLR ligand preparations that may be adapted for use to induce immune responses are ligands for TLR2
(Pam(3)Cys), TLR9 (Poly I: C), TLR4 (3- O-desacyl-4 0-monophosphoryl lipid A (MPL)), TLR7 (9-benzyl-8-hydroxyadenine), TLR7/8 (resiquimod, R848), and TLR9 (CpG DNA).
[0091] In addition to selected co-formulations, a wide variety of adjuvants may be used to potentiate a desired immune response (see Levast et ai , 2014,
Vaccines, 2, 297-322).
[0092] Treatment with PRR ligands according to the invention may be combined with more traditional and existing therapies. For example, a therapy that stimulates the immune system, reduces inflammation or otherwise benefits the subject, such as nutrients, vitamins and supplements. For example, vitamin A, vitamin D, vitamin E, vitamin C, vitamin B complex, selenium, zinc, co-enzyme Q10, beta carotene, fish oil, curcumin, green tea, bromelain, resveratrol, ground flaxseed, garlic, lycopene, milk thistle, melatonin, other antioxidants, cimetidine, indomethacin, or COX-2 Inhibitors (e.g., Celebrex™ [celecoxib] or Vioxx™ [rofecoxib]) may be also be administered to the subject.
[0093] Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to subjects. Alternative routes of administration may be employed, for example, parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, inhalational, aerosol, topical, intratumoural, sublingual or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; for intranasal formulations, in the form of powders, nasal drops, or aerosols; and for sublingual formulations, in the form of drops, aerosols or tablets. [0094] Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences" (20th edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, PA. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. For therapeutic or prophylactic compositions, the pathogenic bacterial species are administered to an individual in an amount effective to stop or slow progression of the disorder.
[0095] Pharmaceutical compositions or formulations may be packaged in a variety of ways depending upon the method used for administering the drug. For example, an article of manufacture or package may include a container having deposited therein the pharmaceutical formulation in an appropriate form. Suitable containers may for example include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and vials. The container may have a sterile access port, for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The package or container may also include a tamper-proof or multi-use mechanism adapted to control access to the contents of the package or the container, for example a multi dose vial adapter matched to a vial contained in the package. The container or package may include a label, for example a label that describes the contents of the container, for example a drug label identifying the pharmaceutical composition therein and/or specifying modes or routes of administration. The label may also include appropriate warnings, for example specifying storage conditions for the container or package, or setting out contraindications or adverse effects of a mode of treatment. Articles of manufacture may accordingly take the form of a "kit" comprising pharmaceutical compositions or accessories adapted to facilitate use of pharmaceutical compositions. Kits may include a label or package insert, where the term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. Kits may further include accessories associated with use of the pharmaceutical composition, including buffers, diluents, filters, needles, and syringes. Kits may also be adapted for the delivery of selected dosage forms of a pharmaceutical composition, for example including a number of unit dosages. Such kits can include a memory aid or mechanism, in the form of a physical or written indication of the intended timing of a treatment schedule in which the dosages are to be used.
[0096] A "companion diagnostic" may be associated with a pharmaceutical treatment or composition. Companion diagnostics are assays that facilitate the associated treatment, by providing diagnostic or prognostic information, typically in the form of a diagnostic test to determine the applicability of a treatment to a specific patient. Point-of-care companion diagnostics may for example involve providing diagnostic compositions and/or articles of manufacture in conjunction with providing a pharmaceutical formulation, for example as part of a kit. Alternatively, companion diagnostics may be separately provided, as assays to monitor the therapy of subjects or to predict the therapeutic efficacy of an intended treatment. A companion diagnostic may for example take the form of a medical device, such as an imaging tool, or a process carried out by such a device, for example for conducting assays in vitro, which provides information that is relevant for the safe and effective use of a corresponding drug or biological product. Companion diagnostics may be used with therapies disclosed herein so as to provide diagnostic or prognostic information about therapeutic efficacy or evidence of undesirable side effects or risks. The use of a companion diagnostic with a particular therapeutic may be stipulated in instructions, for example on the labeling of a diagnostic device and/or the labeling of the corresponding therapeutic product.
Types of companion diagnostic tests may for example include: screening and detection, in form of tests that screen for genetic patterns, such as genetic SSI response markers; prognosis and theranostics, such as assays for biochemical SSI response markers that help to predict the future course of a disease, or indicate a patient's response to a therapy; monitoring, for example to evaluate the
effectiveness and appropriate dosing of a prescribed therapy; or, recurrence, involving tests that analyze the patient's risk for a recurrence of the disease.
[0097] An "effective amount" of a composition according to the invention includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction or elimination of the immune dysregulation. A therapeutically effective amount of a composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as amelioration of immune dysregulation.
Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a
therapeutically effective amount.
[0098] For any particular subject, the timing and dose of treatments may be adjusted over time (e.g., timing may be daily, every other day, weekly, monthly) according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. For example, in the context of subcutaneous or intradermal administration, the compositions may be administered every second day. An initial dose of approximately 0.05 ml may be administered subcutaneously, followed by increases from 0.01 -0.02 ml every second day until an adequate skin reaction is achieved at the injection site (for example, a 1 inch to 2 inch diameter delayed reaction of visible redness at the injection site). Once this adequate immune reaction is achieved, this dosing is continued as a maintenance dose. The maintenance dose may be adjusted from time to time to achieve the desired visible skin reaction (inflammation) at the injection site. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer.
[0099] Oral dosages may for example range from 4 times per day, daily or weekly. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer. In some embodiments, the invention may include compositions administered sublingually or by inhalation, or administered to one or more epithelial tissues (i.e., skin by intradermal or subcutaneous injection; lung epithelium by inhalation; gastrointestinal mucosa by oral ingestion; mouth mucosa by sublingual administration) simultaneously or sequentially. Accordingly, in some embodiments the compositions of the invention are administered so as to provoke an immune response in an epithelial tissue. In some embodiments, one or more epithelial routes of administration may be combined with one or more additional routes of administration, such as
intratumoural, intramuscular or intravenous administration.
[00100] In the case of immunogenic formulations, an immunogenically effective amount of a composition of the invention can be provided, alone or in combination with other compounds, for example with an immunological adjuvant. The composition may for example include compounds linked with a carrier molecule, such as bovine serum albumin or keyhole limpet hemocyanin to enhance immunogenicity. An immunogenic composition is a composition that includes materials that elicit a desired immune response. An immunogenic composition may select, activate or expand, without limitation: memory B, T cells, neutrophils, monocytes or macrophages of the immune system.
[00101 ] An antigenic composition comprising killed recombinant bacteria for administration by injection may be made as follows. The bacteria may be grown in suitable media, and washed with physiological salt solution. The bacteria may then be centrifuged, resuspended in saline solution, and killed with heat. The suspensions may be standardized by direct microscopic count, mixed in required amounts, and stored in appropriate containers, which may be tested for safety, shelf life, and sterility in an approved manner. In addition to the pathogenic bacterial species and/or antigens thereof, a killed bacterial vaccine suitable for administration to humans may include 0.4% phenol preservative and/or 0.9% sodium chloride. The bacterial vaccine may also include trace amounts of brain heart infusion (beef), peptones, yeast extract, agar, sheep blood, dextrose, sodium phosphate and/or other media components.
[00102] In select embodiments, medicaments may be administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least one week. Optionally, the medicament may be administered intradermally or subcutaneously. Optionally, the medicament may be administered in a dose so that each dose is effective to cause a visible localized inflammatory immune response at the administration site.
Optionally, the medicament may be administered so that visible localized inflammation at the administration site occurs within 1 to 48 hours. However, a visible localized inflammatory immune response may not always be present in all circumstances despite an immune response being initiated. There are other methods by which the mounting of an immune response can be monitored. For example, the profile (and relative change in characterization) of immune cells from a subject undergoing an immune reaction can be compared with those from a subject that is not undergoing an immune reaction.
[00103] In another aspect, a method of monitoring efficacy of a treatment regime in an individual being treated for an immune dysfunction in a specific organ or tissue is provided. The method involves measuring a characteristic of an immune response in a post-treatment immune sample obtained from the specific organ or tissue after the individual has been subject to the treatment regime for a period of time.
[00104] In some embodiments, PRR agonists derived from bacteria that are members of the endogenous flora of a particular region of the GIT may be used to formulate immunogenic compositions of the invention. The rows of Table 3 list a number of bacterial species, together with the biological regions in which each species may form a part of the endogenous flora. For example, Abiotrophia spp. are typically members of the endogenous flora of the mouth. Table 3: Human Bacterial Normal Flora (Endogenous Bacterial Human Pathogens)
Figure imgf000036_0001
Bacteroides + + distasonis
Bacteroides + + eggerthii
Bacteroides + + fragilis
Bacteroides + + merdae
Bacteroides + + ovatus
Bacteroides + + splanchnicus
Bacteroides + + thetaiotaomicron
Bacteroides + + vulgatus
Bifidobacterium + + + adolescentis
Bifidobacterium + + + bifidum
Bifidobacterium + + + breve
Bifidobacterium + + + catenulatum
Bifidobacterium + + + + dentium
Bifidobacterium + + + longum
Bilophila + + + + wadsworthia Burkholderia + + + cepacia
Butyrivibrio + + + fibrisolvens
Campylobacter + + + concisus
Campylobacter + + + curvus
Campylobacter + + + gracilis
Campylobacter + + + jejuni
Campylobacter + + + rectus
Campylobacter + + + + showae
Campylobacter +
sputorum
Capnocytophaga +
granulosum
Capnocytophaga +
gingivalis
Campylobacter +
haemolytica
Capnocytophaga + + + + ochracea
Capnocytophaga +
sputigena
Cardiobacterium +
hominis
Cedecea spp + Centipeda +
periodontii
Citrobacter + + + freundii
Citrobacter + + + koseri
Clostridium spp. + + +
Corynebacterium +
accolens
Corynebacterium +
afermentans
Desulfomonas + + + pigra
Dysgonomonas + + + spp.
Eikenella + + + + corrodens
Enterobacter + + + aerogenes
Enterobacter + + + cloacae
Enterobacter + + + gergoviae
Enterobacter + + + sakazakii
Enterobacter + + + taylorae
Enterococcus + + + spp.
Escherichia coli + + + Escherichia + + + fergusonii
Escherichia + + + hermannii
Escherichia + + + vulneris
Eubacterium + + + + spp.
Ewingella +
americana
Finegoldia + + + magnus
Fusobacterium +
alocis
Fusobacterium + + + gonidiaformans
Fusobacterium + + + mortiferum
Fusobacterium + + + navi forme
Fusobacterium + + + + necrophorum
Fusobacterium + + nucleatum
Fusobacterium +
sulci
Fusobacterium + + + russii
Fusobacterium + + + varium Gardnerella + + + vaginalis
Gemella +
haemolysans
Gemella + + + + morbillorum
Globicatella spp. + +
Granulicatella +
spp.
Haemophilus +
spp.
Hafnia alvei + + +
Helcococcus
kunzii
Helicobacter spp. + + +
Kingella spp. +
Klebsiella spp. + + + +
Lactobacillus + + + + + acidophilus
Lactobacillus +
breve
Lactobacillus +
casei
Lactobacillus + + + + + fermentum
Lactobacillus + + + + reuteri
Lactobacillus + + + + + salivarius
Leclercia + + + adecarboxylata Leminorella spp. + + +
Leptotrichia +
buccalis
Megasphaera + + + elsdenii
Micrococcus +
luteus
Micrococcus +
lylae
Micromonas +
micros
Mitsuokella + + + multiacidus
Mobiluncus + + + curisii
Mobiluncus + + + mulieris
Moellerella + + + wisconsensis
Moraxella +
catarrhalis
other Moraxella +
spp.
Morganella + + + morganii
Mycoplasma +
buccale
Mycoplasma +
fermentans
Mycoplasma +
hominis Mycoplasma +
lipophilum
Mycoplasma +
orale
Mycoplasma +
pneumoniae
Mycoplasma +
salivarium
Pantoea + + + agglomerans
Pasteurella +
mult odd a
Pediococcus + + spp.
Peptoniphilus + + + asaccharoiyticus
Peptostreptococc + + + + us anaerobus
Peptostreptococc + + + us productus
Porphyromonas + + + + asaccharolytica
Porphyromonas + +
catoniae
Porphyromonas + +
endodontalis
Porphyromonas + +
gingivalis
Prevotella + +
buccae Prevotella + + buccalis
Prevotella + + corporis
Prevotella + + dentalis
Prevotella + + denticola
Prevotella + + enoeca
Prevotella + + heparinolytica
Prevotella + + intermedia
Prevotella + + loescheii
Prevotella + + melaninogenica
Prevotella + + nigrescens
Prevotella oralis + +
Prevotella oris + +
Prevotella + + oulorum
Prevotella + + tannerae
Prevotella + + veroralis
Prevotella + + zoogieoformans Propionibacte u +
m propionicum
Proteus mirabilis + +
Proteus penneri + +
Proteus vulgaris + +
Providencia + + rettgeri
Providencia + + + stuartii
Pseudomonas + + + aeruginosa
Retortamonas + + + intestinalis
Rothia +
dentocariosa
Rothia +
mucilaginosa
Ruminococcus + + + productus
Selenomonas +
spp.
Serratia + + liquefaciens
Serratia + + marcescens
Serratia odorifera + +
Staphylococcus +
aureus
Staphylococcus +
epidermidis Streptococcus + + + agalactiae
Streptococcus + + + + anginosus
Streptococcus + + + bovis
Streptococcus + + + + constellatus
Streptococcus +
criceti
Streptococcus +
crista
Streptococcus +
equisimilis
Streptococcus +
gordonii
Streptococcus + + + intermedius
Streptococcus + +
mitis
Streptococcus +
mutans
Streptococcus +
oralis
Streptococcus +
parasanguis
Streptococcus + +
pyogenes
Streptococcus + +
salivarius Streptococcus + +
sanguis
Streptococcus +
sobrinus
Streptococcus +
vestibularis
Group C + G + +
Streptococci
Succinivibrio + + +
dextrinosolvens
Sutterella spp. + + +
Suttonella +
indologenes
Tissierella + + +
praeacuta
Treponema +
denticola
Treponema +
maltophilum
Treponema +
socranskii
Treponema +
vincentii
Ureaplasma +
urealyticum
Veillonella spp. + + + +
[00105] Endogenous microbial flora, such as bacteria, have access to tissues for pathogenesis either through contiguous spread or bacteremic spread. Under favorable conditions, endogenous organisms can become pathogenic and invade locally and spread by contiguous spread to adjacent tissues and organs.
Endogenous bacterial flora of the skin, mouth and colon are species that are understood to also be amenable to bacteremic spread. Bacteria that are members of a particular endogenous flora domain may therefore cause infection in tissues or organs to which these bacteria may spread. Accordingly, one aspect of the invention involves the use of PRR agonists derived from endogenous microbial pathogens to treat an immune dysregulation having symptoms localized to a region of the GIT in which the endogenous bacteria may spread to cause infection. The columns of Table 2 list domains for endogenous flora. The rows of Table 4 list regions of the GIT within which immune dysregulation may be symptomatic or etiologically located. Accordingly, one aspect of the invention involves the use of PRR agonists derived from endogenous microbial pathogens to formulate immunogenic compositions for treating an immune dysregulation symptomatic or etiologically located in the region of the GIT to which the pathogen may spread to cause an infection. Accordingly, in alternative embodiments, an immune
dysregulation that is symptomatic in the region listed in the first column of Table 2 may be treated with immunogenic compositions comprising an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is a member of the endogenous flora of one or more of the endogenous flora domains listed in the first row of Table 2 and indicated with an X or a check mark in the appropriate row.
Table 4: Tissue/Organ Pathogenicity of Endogenous Flora
Figure imgf000048_0001
Small bowel X X
Colon/
Rectum X
Anus X
[00106] In accordance with the combined information in Tables 1 and 2, an immune dysregulation manifest in a particular region of the GIT set out in column 1 of Table 2 may be treated with antigenic compositions comprising an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is one of the
corresponding bacterial species of Table 1 , so that the column headings in Table 2 are in effect replaced with the bacterial species of Table 1.
[00107] In some embodiments, PRR agonists may be derived from
exogenous bacterial pathogens. For example, PRR agonists derived from the organisms listed in Table 5 may be used in an artificial repertoire of PRR agonists to treat an immune dysregulation that is symptomatic in the region of the GIT listed with the relevant organism in Table 5. In some embodiments, PRR agonists derived from both endogenous and exogenous microbial species may be used in combination.
Table 5: Exogenous Bacterial Human Pathogens, and their Sites of Infection in the GIT.
Figure imgf000049_0001
Campylobacter small bowel, colon sputorum
Clostridium small bowel, colon, stomach bifermentans
Clostridium colon, small bowel botulinum
Clostridium difficile colon
Clostridium indolis small bowel, colon, stomach,
Clostridium small bowel, colon, stomach mangenolii
Clostridium small bowel, colon, stomach perfringens
Clostridium sordellii small bowel, colon, stomach
Clostridium small bowel, colon, stomach sporogenes
Clostridium small bowel, colon, stomach subterminale
Edwarsiella tarda small bowel, colon
Francisella small bowel
tularensis
Helicobacter pylori stomach
Leptospirosis spp. oral
Listeria small bowel, colon monocytogenes
Mycobacterium colon, small bowel bo vis
Mycobacterium small bowel, colon tuberculosis
Pediococcus spp. colon
Plesiomonas small bowel, colon shigelloides Rickettsia small bowel
rickettsiae
Salmonella spp. stomach, small bowel, colon
Shigella boydii colon
Shigella colon
dysenteriae
Shigella flexneri colon
Shigella sonnei colon
other Spirillum spp. colon
Streptococcus small bowel
zooepidemicus
Treponema oral, anus
pallidum
Tropheryma small bowel, colon
whipplei
Vibrio cholerae colon, small bowel
Vibrio fluvial is small bowel, colon
Vibrio furnissii small bowel, colon
Vibrio hollisae small bowel, colon
Vibrio colon, small bowel
parahaemolyticus
Yersinia small bowel, colon
enterocolitica
Yersinia small bowel, colon
pseudotuberculosis
[00108] In some embodiments, PRR agonists for use in the invention may be derived from viral pathogens. Table 6 provides an exemplary list of viral pathogens together with the tissue and organ sites for which each viral species is reportedly a pathogen. Accordingly, one aspect of the invention involves utilizing immunogenic compositions of PRR agonists derived from the named viruses to treat an immune dysregulation that is symptomatic in the region of the GIT that is identified adjacent to the name of the virus in Table 6.
Table 6: Viral Human Pathogens and Their Sites of Infection
Virus Region of the GIT
Herpes Simplex rectum, anus
virus (1 and 2)
Cytomegalovirus small bowel, colon/rectum
Epstein-Barr virus oral
Adenovirus oral, small bowel, colon
Human anus, oral
papillomavirus
Orthoreoviruses small bowel, colon, oral
Coltiviruses oral
Rotaviruses small bowel, colon
Alphaviruses small bowel, colon,
Coronaviruses oral, small bowel, colon
Toroviruses small bowel, colon
Parainfluenza oral
viruses
Respiratory syncytial oral
virus
Human oral, small bowel, colon
metapneumovirus
Vesicular stomatitis oral, small bowel, colon
virus
Rabies virus oral
Influenza virus oral
Hantaviruses oral Machupo virus small bowel, colon
Junin virus small bowel, colon
Poliovirus small bowel, colon
Coxsackie viruses small bowel, colon
Echovi ruses oral, small bowel, colon
Hepatitis A virus small bowel, colon
Rhinoviruses oral
Noroviruses and small bowel, colon
other Caliciviruses
Astrovi ruses small bowel, colon
Picobirnaviruses small bowel, colon
Hepatitis E virus small bowel, colon
[00109] In some embodiments, the pathogen from which PRR agonists are derived for use in immunogenic compositions of the invention may be one that is a common cause of acute infection in the region of the GIT in which the immune dysregulation to be treated is symptomatic. Table 7 identifies bacterial and viral pathogens of this kind, together with the region of the GIT in which they commonly cause infection. Accordingly, in selected embodiments, an immune dysregulation that is symptomatic in a region of the GIT identified in the first column of Table 7 may be treated with an immunogenic composition that comprises an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of the PRR agonist signature of a pathogenic organism listed in the second column of Table 7
Table 7: Common causes of acute infection (bacteria and viruses) for selected regions of the GIT
Figure imgf000053_0001
Oral Prevotella melaninogenicus, anaerobic streptococci,
viridans streptococci, Actinomyces spp.,
Peptostreptococcus spp., Bacteroides spp., and other oral anaerobes
herpes simplex, coxsackieviruses, Epstein-Barr
Stomach Streptococcus pyogenes, Helicobacter pylori
cytomegalovirus, herpes simplex, Epstein-Barr, rotaviruses, noroviruses, adenoviruses
Small bowel Escherichia coli, Clostridium difficile, Bacteroides fragilis,
Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri
adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, cytomegalovirus
Colon/Rectum Escherichia coli, Clostridium difficile, Bacteroides fragilis,
Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri
adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, cytomegalovirus
Anus Streptococcus pyogenes, Bacteroides spp., Fusobacterium spp. , anaerobic streptococci, Clostridium spp., E. coli, Enterobacter spp. , Pseudomonas aeruginosa, Treponema pallidum
herpes simplex
[00110] Humans are hosts to a wide range of gastrointestinal parasites, including various protozoa and helminths, which for purposes of the present invention constitute pathogens of the GIT (Schafer, T.W., Skopic, A. Parasites of the small intestine. Curr Gastroenterol Reports 2006;8:312-20; Jernigan, J., Guerrant, R.L., Pearson, R.D. Parasitic infections of the small intestine. Gut 1994;35:289-93; Sleisenger & Fordtran's Gastrointestinal and liver disease. 8th ed. 2006; Garcia, L.S. Diagnostic medical parasitology. 5th ed. 2007). Compositions of the invention may accordingly include PRR agonists of various protozoa, including for example: Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominus, Isospora belli, Sarcocystis species, Coccidian like bodies (Cyclospora species), Enterocytozoon bieneusi, Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Endolimax nana, lodamoeba butschlii, Dientameoba fragilis, Blastocystis hominus, Cyclospora cayetanensis, Microsporidia,
Trypanosoma cruzi, Chilomastix mesnili, Pentatrichomonas hominis, Balantidium coli. Similarly, compositions of the invention may include antigenic components of various helminths, including for example: Cestodes (tapeworms), Taenia saginata, Taenia solium, Diphyllobothrium species, Hymenolepis nana, Hymenolepis diminuta, Dipylidium caninum, Nematodes (round worms), Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus, Ancylostoma duodenale,
Ancylostoma caninum, Tichuris trichiura, Capillaria philippinensis, Trichostrongylus species, Trichinella species, Necator americanus, Anisakis and related species, Angiostrongylus costaricensis, Enterobius vermicularis, Trematodes (flukes), Fasciolopsis buski, Heterophyes speicies, Echinostoma species, Clonorchis sinensis, Opisthorchis species, Fasciola species, Metagonimus yokogawi,
Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi,
Schistosoma intercalatum, Echinostoma species and Paragonimus species.
[00111 ] In accordance with the foregoing, in various aspects, the invention may involve the treatment of an immune dysregulation with formulations of an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial pathogen that is an: Acidaminococcus fermentans; Acinetobacter spp. ; Actinobaculum spp.; Actinomyces spp. ;
Aeromonas spp.; Anaerorhabdus furcosus; Anaerococcus hydrogenalis;
Anaerococcus lactolyticus; Anaerococcus prevotii; Atopobium spp.; Bacillus spp.; Bacteroides caccae; Bacteroides distasonis; Bacteroides eggerthii; Bacteroides fragilis; Bacteroides merdae; Bacteroides ovatus; Bacteroides splanchnicus;
Bacteroides thetaiotaomicron; Bacteroides vulgatus; Bifidobacterium adolescentis; Bifidobacterium bifidum; Bifidobacterium breve; Bifidobacterium catenulatum;
Bifidobacterium dentium; Bifidobacterium longum; Bilophila wadsworthia;
Burkholderia cepacia; Butyrivibrio fibrisolvens; Campylobacter concisus;
Campylobacter curvus; Campylobacter gracilis; Campylobacter jejuni;
Campylobacter rectus; Campylobacter showae; Capnocytophaga ochracea;
Cedecea spp; Citrobacter freundii; Citrobacter koseri; Clostridium spp.;
Desulfomonas pigra; Dysgonomonas spp.; Eikenella corrodens; Enterobacter aerogenes; Enterobacter cloacae; Enterobacter gergoviae; Enterobacter sakazakii;
Enterobacter taylorae; Enterococcus spp. ; Escherichia coli; Escherichia fergusonii; Escherichia hermannii; Escherichia vulneris; Eubacterium spp.; Finegoldia magnus;
Fusobacterium gonidiaformans; Fusobacterium mortiferum; Fusobacterium naviforme; Fusobacterium necrophorum; Fusobacterium nucleatum; Fusobacterium russii; Fusobacterium varium; Gardnerella vaginalis; Gemella morbillorum;
Globicatella spp.; Hafnia alvei; Helicobacter spp.; Klebsiella spp. ; Lactobacillus acidophilus; Lactobacillus fermentum; Lactobacillus reuteri; Lactobacillus salivarius;
Leclercia adecarboxylata; Leminorella spp.; Megasphaera elsdenii; Mitsuokella multiacidus; Mobiluncus curisii; Mobiluncus mulieris; Moellerella wisconsensis;
Morganella morganii; Pantoea agglomerans; Pediococcus spp.; Peptoniphilus asaccharolyticus; Peptostreptococcus anaerobus; Peptostreptococcus productus; Porphyromonas asaccharolytica; Proteus mirabilis; Proteus penneri; Proteus vulgaris; Providencia rettgeri; Providencia stuartii; Pseudomonas aeruginosa;
Retortamonas intestinalis; Ruminococcus productus; Serratia liquefaciens; Serratia marcescens; Serratia odorifera; Streptococcus agalactiae; Streptococcus anginosus; Streptococcus bovis; Streptococcus constellatus; Streptococcus intermedius; Group C + G Streptococci; Succinivibrio dextrinosolvens; Sutterella spp.; Tissierella praeacuta; Veillonella spp.; Aerobacter spp. ; Bacillus anthracis;
Bacillus cereus; other Bacillus spp.; Borrelia recurrentis; Brucella spp.;
Campylobacter coli; Campylobacter fetus; Campylobacter jejuni; Campylobacter sputorum; Clostridium bifermentans; Clostridium botulinum; Clostridium difficile; Clostridium indolis; Clostridium mangenolii; Clostridium perfringens; Clostridium sordellii; Clostridium sporogenes; Clostridium subterminale; Edwarsiella tarda;
Francisella tularensis; Listeria monocytogenes; Mycobacterium bovis; Mycobacterium tuberculosis; Pediococcus spp.; Plesiomonas shigelloides;
Rickettsia rickettsiae; Salmonella spp.; Shigella boydii; Shigella dysenteriae;
Shigella flexneri; Shigella sonnei; other Spirillum spp.; Streptococcus
zooepidemicus; Tropheryma whipplei; Vibrio cholerae; Vibrio fluvialis; Vibrio furnissii; Vibrio hollisae; Vibrio parahaemolyticus; Yersinia enterocolitica; Yersinia pseudotuberculosis; Herpes Simplex virus (1 and 2); Cytomegalovirus; Adenovirus; Orthoreoviruses; Rotaviruses; Alphaviruses; Coronaviruses; Toroviruses; Human metapneumovirus; Vesicular stomatitis virus; Machupo virus; Junin virus;
Poliovirus; Coxsackieviruses; Echoviruses; Hepatitis A virus; Noroviruses and other Caliciviruses; Astroviruses; Picobirnaviruses; or Hepatitis E virus.
[00112] In alternative aspects, the invention may involve the treatment of an immune dysregulation with formulations of an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is a common small and larger bowel pathogens, for example: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri; adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, and cytomegalovirus.
[00113] In selected embodiments, the invention involves diagnostic steps to assess a patient's previous exposure to an organism. For example, the diagnostic steps may include taking a medical history of exposure to selected pathogens, and/or evaluating a patient's immune response to a selected pathogen. For example, a serology test may be conducted to detect antibodies to selected pathogens in a patient's sera. In connection with this aspect of the invention, antigenic determinants of a selected pathogen may be chosen for use in an immunogenic composition on a selected patient based on a diagnostic indication that the patient has had one or more prior exposure(s) to the pathogen, for example by virtue of the presence of antibodies to antigenic determinants of that pathogen in the patient's sera.
[00114] In further selected embodiments, the invention involves diagnostic steps to assess a patient's immunological response to treatment with a selected immunogenic composition. For example, the diagnostic steps may include evaluating a patient's immune response to the immunological determinants of that immunogenic composition, for example using a serological test to detect antibodies to those immunogenic determinants. In connection with this aspect of the invention, a treatment with a selected immunogenic composition may be continued if the evaluation indicates that there is an active immunological response to the immunogenic determinants of that composition, and the treatment may be discontinued, and an alternative treatment with a different immunogenic
composition may be initiated, if the evaluation indicates that there is not a sufficiently active immunological response to the immunogenic determinants of the immunogenic composition.
Screening
[00115] Patients may advantageously be screened for disorders of innate immunity, such as genetic disorders, for example by primary sequence analysis or by analysis of epigenetic changes. A variety of genetic disorders have for example been identified that are associated with gene products involved in innate immunity (see Mogensen T., 2009, Clinical Microbiology Reviews, Vol. 22, No.2, p.240-273), such as TLR genes (TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9), signalling protein genes (MyD88, Mai, I RAKI , I RAK4, NEMO, ΙκΒα, I RF5), N LR genes (NOD2, NALP1 , NALP3) and others (CD14, U NC93B). Patients identified as having a Mendelian primary immunodeficiency associated with impaired TLR signaling or N F-KB activation may for example not benefit from some embodiments, or may require an approach adapted to their condition. Patients having polymorphisms in genes encoding components of innate signalling pathways may also be identified prior to treatment with an SSI, for example having mutations in the gene encoding TIR-domain-containing adaptor-inducing beta interferon (TRIF).
[00116] There are a variety of microbial strategies for evading the innate immune system (Mogensen T., 2009, Clinical Microbiology Reviews, Vol. 22, No.2, p.240-273), and embodiments of the invention may accordingly be adapted to avoid the inhibitory effect of such strategies on the triggered innate response. Select embodiments provide recombinant microorganisms that lack virulence factors that impede TLR signalling, such as recombinant E. coli that lack TIR domain-containing proteins (Cirl, C. et al., 2008, Nat. Med. 14:399-406). Gram negative bacterial formulation may advantageously comprise an LPS that is recognized by a TLR, such as TLR4, rather than a form of LPS that is not recognized by a TLR (Hornef, M. W. et al., 2002, Nat. Immunol. 3: 1033-1040). Similarly, bacterial formulations may advantageously include a class of flagellin that activates a TLR, such as TLR5, rather than one that does not (Andersen-Nissen, E. et al., 2005, Proc. Natl. Acad. Sci. USA 102:9247-9252). In some embodiments, it may be advantageous to exclude peptidases that proteolytically degrade important components of the triggered innate response, such as the amastigote-specific cysteine peptidases of Leishmania mexicana that proteolytically degrade ΙκΒ and NF-κΒ (Cameron, P. et al. , 2004, J. Immunol. 173:3297-3304). In alternative embodiments, these undesireable components may be removed from a formulation by an appropriate step of manufacturing, for example to wash or fractionate a microbial preparation so as to remove a component.
[00117] Patients may be genotyped, for example by identifying polymorphisms in PRR genes (see WO 2009003905). Genes associated with inflammation and immune related diseases and disorders may for example be the subject of screening, such as: AIDS (KIR3DL1 , NKAT3, NKB1 , AMB1 1 , KIR3DS1 ,
IFNgamma, CXCL12, SDF1 ); Autoimmune lymphoproliferative syndrome
(TNFRSF6, APT1 , FAS, CD95, ALPS1A); Combined immunodeficiency, (I L2RG, SCI DX1 , SCI DX, I MD4); HIV-1 (CCL5, SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G, AICDA, AI D, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1 , IGM, FOXP3, IPEX, AND, XPID, PIDX, TNFRSF14B, TACI); Inflammation (I L-10, IL-1 (IL-1 a, I L-1 b), IL-13, IL- 17 (IL-17a (CTLA8), IL-17b, I L-17c, IL-17d, IL-17f), II-23, Cx3cr1 , ptpn22,
TNFalpha, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1 ); Severe combined immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1 C, ARTEMIS, SCI DA, RAG1 , RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, I L2RG,
SCI DX1 , SCI DX, I MD4). Alternatively, genes involved in selected signalling pathways may for example be screened, identifying for example patients that are more or less susceptible to an SSI treatment, such as: GM-CSF Signaling (LYN; ELK1 ; MAPK1 ; PTPN1 1 ; AKT2; PIK3CA; CAMK2A; STAT5B; PI K3CB; PIK3C3; GNB2L1 ; BCL2L1 ; MAPK3; ETS1 ; KRAS; RUNX1 ; PIM1 ; PIK3C2A; RAF1 ;
MAP2K2; AKT1 ; JAK2; PI K3R1 ; STAT3; MAP2K1 ; CCND1 ; AKT3; STAT1 ); IL-10 Signaling (TRAF6; CCR1 ; ELK1 ; IKBKB; SP1 ; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB; MAP3K7; JAK1 ; CHUK; STAT3; NFKB1 ; JUN; IL1 R1 ; IL6); Toll-like Receptor Signaling (IRAKI ; EIF2AK2; MYD88; TRAF6; PPARA; ELK1 ; I KBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; I KBKG; RELB; MAP3K7; CHUK; NFKB1 ; TLR2; JUN).
[00118] In addition, patients may for example be genotyped for SNPs located in the non-coding regions of the genome that are linked to inflammatory disorders, such as SNP's identified through publicly available GWAS datasets, for example SNPs in genomic regions linked to sequences which serve a regulatory role in immune-function-related gene expression.
Alternative embodiments
[00119] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way.
Numeric ranges are inclusive of the numbers defining the range, and inclusive of all numbers and fractions subsumed within the respective ranges. The word
"comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a
corresponding meaning. Terms such as "consisting essentially of" and "consists essentially of" allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to the present invention. Any priority document(s) and all publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. Nothing herein is intended as a promise of any specific utility for all embodiments.
[00120] The term "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-20% or less, preferably +/-10% or less, more preferably +1-5% or less, and still more preferably or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.
[00121 ] All references cited in the present specification are hereby
incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference, along with all documents cited in documents that are cited herein.
[00122] Standard reference works setting forth the general principles of recombinant DNA technology include Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 -3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. , 1989; Current Protocols in Molecular Biology, ed. Ausubel et al. , Greene Publishing and Wiley-lnterscience, New York, 1992 (with periodic updates) ("Ausubel et al. 1992"); the series Methods in Enzymology (Academic Press, Inc.); Innis et al. , PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990; PCR 2: A Practical Approach (M.J.
MacPherson, B.D. Hames and G.R. Taylor eds. (1995); Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual; and Animal Cell Culture (R.I. Freshney, ed. (1987). General principles of microbiology are set forth, for example, in Davis, B. D. et al., Microbiology, 3rd edition, Harper & Row, publishers, Philadelphia, Pa. (1980). [00123] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.
[00124] In this description of the invention, reference is made to the
accompanying drawings that form a part hereof, and in which are shown by way of illustration only of specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present invention. The description, therefore, is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims.
[00125] Preferred statements (features) and embodiments may be combined with any other features or embodiments unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features or statements indicated as being preferred or advantageous.
[00126] In some embodiments, the invention excludes steps that involve medical or surgical treatment. Similarly, in some embodiments, the invention disclaims naturally occurring embodiments, so that aspects of the invention relate only to anthropogenic compositions. Further, in select aspects of the invention, previously known products, process of making products, or methods of using products are hereby disclaimed. [00127] General Codes and Abbreviations:
SSI Site Specific Immunomodulator
OD Optical density
I P Intraperitoneal
SC Subcutaneous
SOP Standard operating protocol
RPM Revolutions per minute
EDTA Ethylenediaminetetraacetic acid
ANOVA Analysis of variance
SD Standard deviation
NA No value; not applicable; not present
EXAMPLES
Example 1 : Recombinant Microbes
[00128] A family of virulence factors in Escherichia coli and Brucella melitensis, named TIR domain-containing proteins, impede TLR signalling through direct binding to MyD88, thus suppressing innate immunity and increasing bacterial virulence. Aspects of the invention accordingly provide recombinant bacteria that lack expression of TIR domain-containing proteins, or other virulence factors that interfere with an innate host immune response to the pathogen.
Escherichia coli. (peritoneal, intraperitoneal or retroperitoneal)
[00129] In select embodiments, compositions may be prepared from recombinant E. coli strains specifically adapted from strains that are pathogenic within the peritoneum, or in intraperitoneal organs or in retroperitoneal organs. For example, strains of a sequence type having the following alleles, or homologous sequences being at least 99% identical thereto: adk-37, fumc-38, gyrb-19, icd-37, mdh-151 , pura-1 1 , reca-26 (sequence type 1231 ). Strains may totally lack resistance genes to the following classes of antibiotic: aminoglycoside, beta-lactam, fluoroquinolone, fosfomycin, fusidic acid, MLS - macrolide, lincosamide and streptogramin B, nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide, tetracycline, trimethoprim, and glycopeptide. Similarly, strains may or may not include one or more virulence factor genes, having for example at least 90%, 95%, 99% or 100% identity to selected database sequences (identified by accession number in the following tables). The strain may also lack stx holotoxin virulence factors.
Table 8: E. coli - Virulence factors
Figure imgf000064_0001
[00130] The serotype of the E. coli strain may for example be 018ac:H7, for example representing the presence of H type serotype gene fliC (accession AF228492, and O type serotype genes wzx (accession GU299793), and wzy (accession GU299793).
[00131 ] Recombinant strains may include one or more plasmids, for example having 90%, 95%, 99% or 100% identity to plasmid IncFIB (accession AP001918) and/or plasmid lncFII(29) (accession CP003035), and/or plasmid ColRNAI
(accession DQ298019) and/or plasmid Col156 (accession NC009781 ).
[00132] The recombinant E. coli may for example be, or be derived from an E. coli strain having at least 80%, 90% or 95% sequence identity to E. coli UT189 (see Chen et al., 2006, Proc Natl Acad Sci U S A 103:5977-82). Escherichia coli. (colon)
[00133] In select embodiments, compositions may be prepared from recombinant E. coli strains specifically adapted from strains that are pathogenic in the colon or other regions of the Gl tract, for example for therapy of colon or Gl immune dysfunction, for example for treatment of IBD. For example, strains of a sequence type having the following alleles, or homologous sequences being at least 99% identical thereto: adk-76, fumc-43, gyrb-9, icd-36, mdh-404, pura-14, reca-10 (sequence type ST-5292). Strains may totally lack resistance genes to the following classes of antibiotic: aminoglycoside, beta-lactam, fluoroquinolone, fosfomycin, fusidic acid, MLS - macrolide, lincosamide and streptogramin B, nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide, tetracycline, trimethoprim, and glycopeptide. Alternatively, strains may have one or more resistance genes, such as the strB or strA aminoglycoside resistance genes (accession numbers M96392 or AF321551 ), and/or sul1 sulphonamide resistance gene (accession AY224185), and/or sul2 sulphonamide resistance gene (accession GQ421466), and/or dfrA5 trimethoprim resistance (accession X12868). Similarly, strains may or may not include one or more virulence factor genes, having for example at least 90%, 95%, 99% or 100% identity to selected database sequences (identified by accession number in the following tables). The strain may also have a gene that is at least 95% or 99% or 100% identical to the stx holotoxin virulence factor gene stxl (accession M19437).
Table 9: E. coli - Virulence factors
Figure imgf000065_0001
Shiga toxin 1, sub unit B,
stxIB AM230663
variant a
Shiga toxin 1, sub unit A,
stxIA EF079675
variant a
astA EAST-1 heat-stable toxin AB042002
[00134] The serotype of the E. coli strain may for example be 01 17: H7, for example representing the presence of H type serotype gene fliC (accession AF228492, and O type serotype genes wzx (accession EU694096).
[00135] Recombinant strains may include one or more plasmids, as set out in the following table.
Table 10: E. coli plasmids
Figure imgf000066_0001
[00136] The recombinant E. coli (colon) may for example be, or be derived from an E coli strain having at least 80%, 90% or 95% sequence identity to E coli SE15 or any 01 17:H7 serotype E. coli.
Example 2: Co-Formulations and Co-Administration
[00137] This Example illustrates embodiments in which an SSI is co- formulated with or co-administered with additional therapeutic components. [00138] One class of additional therapeutic components comprises molecules or compositions for activating or recruiting innate immune cells, and these include:
GMCSF, for example in an amount that synergistically recruits and promotes the production of neutrophils and potentiates the SSI-induced innate immune response.
Vitamin D (for inflammatory disease, such as I BD), for example in an amount that is effective to differentiate and activate monocytes and play a role in regulating innate immune function. In alternative embodiments, the vitamin D used in conjunction with SSIs may for example be one or more of vitamin D3, D2 or calcitriol (1 ,25- dihydroxycholecalciferol). In some embodiments, vitamin D3 and/or D2 may for example be given locally at a dosage that is effective to provide a locally effective amount of calcitriol at the site of SSI and vitamin D administration. For example, vitamin D precursors (D3 and/or D2) may be administered in an amount that is locally effective once it is converted into the calcitriol active form by local monocytes and/or macrophages (expressing CYP27B1 ) at the site of administration. In alternative embodiments, calcitriol may be administered in dose that is locally effective at the site of SSI administration, and this may for example be dose that is less than the dose required for other systemic effects.
[00139] An additional class of therapeutic components for co-formulation or co-administration comprise molecules or compositions that relieve
immunosuppression:
NOHA (N(omega)- hydroxy-nor-L-arginine), an Arginase inhibitor - Arginase degrades arginine needed for immune activation. NOHA may for example be used in an amount effective to relieve immune suppression by making available free arginine. Alphal antitrypsin - for example in an amount effective to relieve immune suppression mediated by neutrophils secreting proteases.
[00140] An additional class of therapeutic components for co-formulation or co-administration comprise molecules or compositions that prevent oxidative damage and improve immune function under stress:
Glutathione and other antioxidants, particularly for fibrotic diseases (such as I BD).
[00141 ] An additional class of therapeutic components for co-formulation or co-administration comprise co-stimulatory molecules for innate cytotoxic
lymphocytes:
Phospho-antigens (isoprenoid molecules, such as isopentenyl pyrophosphate) - recognized by human peripheral blood Vv9V52 T cells. In exemplary embodiments, it has been found that SSIs in co-formulation or co-administration with zoledronate increase markers of activation, for example CD25 and CD69, on human peripheral blood νγ9\/δ2 T cells.
Glycolipid molecules recognized by Type I NKT cells (such as synthetic a-galactosylceramide)
Example 3: Colitis Animal Model, Anti-inflammatory Efficacy
[00142] This Example illustrates results from a mouse spontaneous colitis model (Muc2 knockout "KO" mice) that mimics the underlying immune system defect and chronic bacterial infection associated with Crohn's disease and ulcerative colitis. I BD patients typically display structural and/or functional defects in their normally protective colonic mucosal barriers. The mucus barrier is largely dependent on the release of goblet cell-derived mucin (Muc2) which prevents microbes and luminal antigens from contacting the epithelial surface in the gastrointestinal tract. Muc2 KO mice are healthy just after weaning (1 month old), as they age, they develop progressive diarrhea and sporadic rectal prolapse.
Histological analysis of colonic tissue shows crypt hyperplasia, crypt abscesses, inflammatory cell infiltration and submucosal edema. Accordingly, the Muc2 KO mice have a defective gastrointestinal mucosal barrier and after time spontaneously develop colitis, resembling ulcerative colitis in humans. In this model, young (2 month old) Muc2 KO mice have less severe colitis, and older (3 month old) Muc2 KO mice have more severe colitis.
[00143] Results from this animal model, as shown in Figures 14A, 14B and 14C illustrate that an E. coli SSI (QBECO) decreases pro-inflammatory markers in the colon (using qPCR gene expression data). Figure 14D illustrates the site specific activity of QBECO in increasing IL-18 gene expression in the colon, compared to QBKPN. The I FN-gamma expression data in particular illustrates how SSI efficacy can be affected by the stage of colitis (comparing expression data in old vs young mice). IL17A data, relating to a cytokine that is produced by activated T-cells (a marker of IBD inflammation), illustrates a significant decrease in this marker of inflammation after E. coli SSI treatment. Accordingly, QBECO treatment substantially improved all components of the histopathology score, including infiltration, integrity, hyperplasia, and edema. The infiltration of T lymphocytes in the colonic tissue, a hallmark of IBD in patients and mouse models, was markedly decreased with QBECO treatment. Accordingly, this Example illustrates that an SSI, such as QBECO, may be used to significantly decrease disease severity in IBD model, including so as to substantially dampen adaptive immune system over- response.
[00144] QBECO was also shown to have a positive impact on the
gastrointestinal microbiome. Alterations in bacterial species in the intestinal microbiome can either be detrimental ('unhealthy' bacteria) or therapeutic ('healthy' bacteria) in IBD patients (and mouse models). Some bacteria promote a healthy immune environment and can improve symptoms (for example, Lactobacillus species), whereas others (for example, γ-proteobacteria) can have detrimental effects in IBD. We analyzed the intestinal microbiome before and after QBECO SSI treatment. As illustrated in Figures 15A and 15B, QBECO SSI improved dysbiosis in the colon of Muc2 mice, increasing the relative proportion of Lactobacillus
(healthy bacterial species) and decreasing the relative proportion of gamma- proteobacteria (unhealthy bacterial species). As illustrated in Figure 15C, QBECO SSI also reduced all aspects of the histological inflammation/damage score
(infiltration, integrity, hyperplasia and edema) in the colon of MUC2 spontaneous colitis mice. These results illustrate that an SSI treatment using a formulation derived from a Gl pathogen, such as QBECO, has a therapeutic effect on the gastrointestinal microbiome. Accordingly, aspects of the invention involve the use of an SSI , such as an E. coli derived SSI, for treating dysbiosis in IBD.
[00145] To summarize, QBECO treatment significantly improved the overall histological score and reduced T cell infiltration in the colonic tissues. Furthermore, a reduction in pro-inflammatory mediators in the colon (IL-17A) and serum (KC) was observed. QBECO treatment did not impact regulatory T cell marker (FoxP3) and anti-inflammatory growth factor (TGF-β) expressions in affected tissues. In addition, SSI treated mice demonstrated reduced levels of the antimicrobial lectins Reglll-β and Regll l-γ. The changes in antimicrobial lectins brought on by QBECO allowed for a modulation of the gut microbiome causing a reduction in gamma- proteobacteria and a significant increase in lactobacilli.
Example 4: Durability of Treatment Response in IBD
[00146] This Example illustrates that efficacious treatment for Crohn's disease may be carried out over an extended period of periodic dosing of an SSI. In particular, in a phase 1 /2, randomized, placebo-controlled, double-blinded clinical trial involving adults with moderate to severe Crohn's disease, the Crohn's Disease Activity Index (CDAI , Best et al. , 1976, Gastroenterology 70 (3): 439-444) declined on average by significantly more on week 16 of treatment compared to week 8. More specifically, by week 8, the average reduction in CDAI score in SSI treated patients was approximately 80 points; by week 16, the average reduction in CDAI score was approx. 120 points. This illustrates continued clinical improvement through 16 weeks of SSI treatment.
[00147] This example involved use of a whole killed E. coli SSI preparation, administered every second day by subcutaneous injection. The dose was individualized to the patient by adjusting the dose so that each dose was effective to cause a visible localized inflammatory immune response at the administration site (a 1 inch to 2 inch diameter delayed reaction of visible redness at the injection site).
[00148] Accordingly, aspects of the invention involve use of an SSI over an extended duration period, with dosage intervals and dosage duration adapted to provide an increased therapeutic benefit over the entire dosage duration, such as a progressive reduction of CDAI score in Crohn's patients over a duration period of at last 16 weeks.
Example 5: Distinct SSIs Agonize Distinct PRRs
[00149] This Example illustrates that both QBECO and QBKPN SSIs activate multiple PRRs, and QBECO and QBKPN each activate different PRRs, with different PRR repertoire fingerprints being identified for each SSI.
[00150] This data in this Example was obtained from assays of QBKPN and QBECO PRR activation in cell lines that have a single PRR. The cell lines used were HEK293 cells lines that express a single human Toll-Like Receptor (TLR2, 3, 4, 5, 7, 8 and 9), NOD-Like Receptor (NOD1 and NOD2), C-Type Lectin (Dectin 1 a, Dectin 1 b, and Mincle) or RIG-1 -like receptor (RIG-1 and MDA5).
[00151 ] As illustrated in Figure 63, two TLRs were highly activated by both QBECO and QBKPN (TLR 2 and TLR4). One TLR was highly activated by just QBKPN (TLR5). 1 PRR was moderately activated by bother QBECO and QBKPN (NOD2). 4 were moderately activated by only QBECO (TLR3, TLR7, TLR8, TLR9) while 2 were moderately activated by only QBKPN (Dectin 1 a, Dectin 1 b). NOD1 , Mincle, RIG-1 and MDA5 were not activated by either QBECO or QBKPN.
[00152] TLR2 and TLR4 are localized on the plasma membrane and primarily recognize lipoprotein and LPS respectively. TLR5 is a plasma membrane receptor that responds to Flagellin. Of the RNA/DNA recognition TLR's, TLR3 was only slightly activated by QBECO (and not by QBKPN). TLR3 is primarily a dsRNA receptor for viral RNA. TLR7 and 8, which are located in the endolysosome and also recognize RNA (bacterial and viral) were activated by only QBECO. Finally,
TLR9 which recognized CpG-DNA and is located in the endolysosome was also activated by only QBECO. In this context, it is relevant that HEK cells are not known to highly uptake bacteria in endolysosomes. Therefore, the lack of QBKPN activation for TLR 7, 8 and 9 may be due to no interaction of the DNA/RNA with these receptors. Nod-Like Receptors (NLR) are cytoplasmic receptors. NOD1 was not activated by either QBECO or QBKPN, but NOD2, which recognizes muramyl dipeptide (MDP) was activated by both QBECO and QBKPN. The other
cytoplasmic receptors, RIG-1 and MDA5 which recognize short dsRNA and long dsRNA respectively, were not activated. The C-type lectin receptors (CLR) are located in the plasma membrane and primarily recognize carbohydrates. Mincle was not increased by either QBKPN or QBECO. Dectin 1 a and Dectin 1 b are primarily fungi receptors for beta-Glucans but can also see bacteria carbohydrates.
[00153] When graphed as either bar graphs (Figure 64) or radar graphs
(Figure 65), an overall PRR repertoire fingerprint appears. These results are all derived from the 1/10 dilution of the relevant SSI, with the negative control subtracted from the absorbance value.
Example 6: Genetic Markers for SSI Therapy Response
[00154] This Example provides a genetic analysis of subjects with IBD undergoing treated with an SSI therapy, illustrating the use of genetic markers associated with IBD to identify patient populations amenable to SSI treatments. In this Example there were 48 subjects with IBD and approximately 2.4 million single nucleotide polymorphisms (SNPs) which were the subject of analyses following genotyping on the Infinium Omni2.5-8 bead chip. The end-points used for these genetic analyses were varied and encompassed both clinical response and also the use of object markers of disease activity. Following standard quality control measures (including call frequency, minor allele frequency, and Hardy-Weinberg equilibrium test) a total of 1 ,271 ,655 SNPs were available for analyses. 1 13 known IBD loci were represented on the chip and passed quality control. Of the study subjects there were 31 Crohn's disease (CD) and 12 ulcerative colitis (UC) cases included.
[00155] A number of IBD-associated SNPs are associated with SSI treatment outcomes in IBD cases, using p = 0.05 as a nominal significance, for example: CD phenotype and IBD-associated SNPs
• Last recorded response in CD (response vs no response) - a SNP tagging the FASLG, TNFSF18 genes was the top association (p = 0.0033).
• The same FASLG, TNFSF18 locus was also associated with drop in CDAI in CD cases (p = 0.018).
• CD drop in calprotectin was associated with a number of SNPs tagging 4 loci:
o NEXN, FUBP1 ,DNAJB4,GIPC2, MGC27382;
o ATF4.TAB1 ;
o IL23R;
o IL8, CXCL1 , CXCL6, CXCL3, PF4, CXCL5, CXCL2 (all p < 0.05).
• 8 week drop in CRP (serum levels of C-reactive protein) was associated with a SNP tagging NOTCH2 (p = 0.002). UC phenotype and IBD-associated SNPs
• Mayo score drop at 16 weeks associated with SNPs tagging :
o HNF4A
o IRFI
o GPR12
o nd FOX01 (all p < 0.05)
• HNF4A, and GPR12 are also associated with drop in CRP in UC after 16 weeks of treatment.
IBD phenotype and IBD-associated SNPs
• Last response in all IBD cases combined showed associations with SNPs that tagged loci tagging FASLG, TNFSF18 (p = 0.02) and also JAK2 (p = 0.04).
[00156] An analyses of all the SNPs across the chip against the phenotypes listed above revealed a number of associations as summarized in the Table 22: Table 11 : Unbiased analyses of all SNPs across the genotyping platform
Figure imgf000074_0001
[00157] Using a cumulative gene-risk score (GRS; see Jostins et al., (2013) PLoS ONE 8(10): e76328) based on all known I BD associated SNPs, a highly significant association was identified with CD responders to SSI treatment having higher GRS developed from 1 12 I BD-associated SNPs (listed below) than non- responders (p = 2.43 x 10"5), as illustrated in Figure 71 A. Using just 3 of these SNPs, with raw p-values < 0.05 (rs9286879, rs7517810, rs17391694), also evidenced a significant association (P-value: 1 .385E-04) with CD responders, as illustrated in Figure 71 B. Similarly there was an association with higher GRSs observed in UC responders than non-responders, as illustrated in Figure 72 (p = 0.012), providing an independent verification of the CD phenotype and GRS finding summarized above. Given that the overwhelming majority of IBD-associated loci are shared between CD and UC combining these data as cumulative GRS in all IBD cases is valid. Despite the small number of cases there is a very significant association between GRS and response at last follow up, as illustrated in Figure 73 (p = 8.18 x 10-7).
[00158] The remarkable association of the cumulative GRS with last documented response in CD, UC, and combined I BD patient populations indicates that individuals with I BD that are genetically enriched for genetic markers associated with IBD are more likely to respond to SSI. Furthermore, since the majority of these genetic variants are associated with other immune-mediated diseases, this indicates that this approach may be extended to other patient cohorts beyond IBD when treated with an SSI. These findings indicate that it is possible to identify subjects, such as IBD subjects, more likely to respond to an SSI treatment.
Accordingly, an aspect of the present invention involves the provision of companion diagnostic genetic testing assays in association with an SSI therapy. SNPs and genetic loci that may be used in such assays are set out below.
• List of 243 IBD susceptibility SNPs: rs1748195, rs34856868, rs1 1583043, rs6025, rs10798069, rs7555082, rs1 1681525, rs4664304, rs31 16494, rs7556897, rs1 1 1781203, rs35320439, rs1 13010081 , rs616597, rs724016, rs2073505, rs4692386, rs6856616, rs2189234, rs395157, rs4703855, rs564349, rs7773324, rs13204048, rs7758080, rs1077773, rs2538470, rs17057051 , rs701 1507, rs3740415, rs7954567, rs653178, rs1 1064881 , rs9525625, rs3853824, rs17736589, rs9319943, rs7236492, rs727563, rs17391694, rs6679677, rs3897478, rs9286879, rs1728918, rs10865331 , rs6716753, rs12994997, rs6837335, rs13126505, rs10065637, rs7702331 , rs17695092, rs12663356, rs9264942, rs9491697, rs13204742, rs212388, rs10486483, rs864745, rs7015630, rs6651252, rs3764147, rs16967103, rs2066847, rs2945412, rs2024092, rs4802307, rs516246, rs2284553, rs10797432, rs6426833, rs2816958, rs1016883, rs17229285, rs9847710, rs3774959, rs1 1739663, rs254560, rs6927022, rs798502, rs4722672, rs4380874, rs4728142, rs483905, rs561722, rs28374715, rs1 1 150589, rs1728785, rs7210086, rs1 126510, rs6088765, rs6017342, rs12103, rs35675666, rs12568930, rs1 1209026, rs2651244, rs4845604, rs670523, rs4656958, rs1801274, rs2488389, rs755451 1 , rs3024505, rs6545800, rs925255, rs10495903, rs7608910, rs6740462, rs917997, rs21 1 1485, rs1517352, rs2382817, rs3749171 , rs4256159, rs3197999, rs2472649, rs7657746, rs2930047, rs1 1742570, rs1363907, rs4836519, rs2188962, rs686341 1 , rs1 1741861 , rs6871626, rs12654812, rs171 19, rs9358372, rs1847472, rs6568421 , rs3851228, rs6920220, rs12199775, rs1819333, rs1456896, rs9297145, rs1734907, rs38904, rs921720, rs1991866, rs10758669, rs4743820, rs4246905, rs10781499, rs12722515, rs1042058, rs1 1010067, rs2790216, rs10761659, rs2227564, rs1250546, rs6586030, rs791 1264, rs4409764, rs90761 1 , rs10896794, rs1 1230563, rs4246215, rs559928, rs2231884, rs2155219, rs6592362, rs630923, rs1 1612508, rs1 1564258, rs1 1 168249, rs7134599, rs17085007, rs941823, rs9557195, rs194749, rs4899554, rs8005161 , rs17293632, rs7495132, rs529866, rs7404095, rs26528, rs10521318, rs3091316, rs12946510, rs12942547, rs1292053, rs1893217, rs7240004, rs727088, rs1 1879191 , rs17694108, rs1 1672983, rs6142618, rs491 1259, rs1569723, rs913678, rs259964, rs6062504, rs2823286, rs2836878, rs7282490, rs2266959, rs2412970, rs2413583, rs2641348, rs7517810, rs1260326, rs7438704, rs10061469, rs2503322, rs5743289, rs6667605, rs1440088, rs3774937, rs477515, rs1 182188, rs17780256, rs1 1083840, rs3766606, rs13407913, rs6708413, rs2457996, rs10051722, rs4976646, rs7746082, rs3891 1 , rs13277237, rs2227551 , rs7097656, rs12778642, rs1 1229555, rs174537, rs568617, rs2226628, rs566416, rs1 1054935, rs3742130, rs1569328, rs2361755, rs3091315, rs1654644, rs4243971 , rs6087990, rs6074022, rs5763767.
Subset of 1 12 SNPs which together generated the GRS of Figure 71A:
rs10065637, rs1016883, rs1042058, rs10521318, rs10758669, rs1077773, rs10781499, rs10865331 , rs10896794, rs1 1054935, rs1 1083840, rs1 1 150589, rs1 1 168249, rs1 1209026, rs1 1583043, rs1 1672983, rs1 1739663, rs1 1742570, rs1 182188, rs1260326, rs12778642, rs13204048, rs13277237, rs1517352, rs1569723, rs1654644, rs17085007, rs171 19, rs17229285, rs1728918, rs1734907, rs17391694, rs1748195, rs17780256, rs1801274, rs1847472, rs1893217, rs194749, rs2024092, rs21 1 1485, rs212388, rs2155219, rs2188962, rs2189234, rs2227551 , rs2231884, rs2413583, rs2472649, rs2641348, rs2651244, rs26528, rs2816958, rs2823286, rs2836878, rs2930047, rs3024505, rs35320439, rs3742130, rs3764147, rs3766606, rs38904, rs395157, rs4243971 , rs4409764, rs4692386, rs4728142, rs477515, rs4802307, rs4836519, rs483905, rs4976646, rs516246, rs559928, rs564349, rs566416, rs568617, rs6017342, rs6088765, rs616597, rs6426833, rs6651252, rs6667605, rs6856616, rs686341 1 , rs6920220, rs7097656, rs7134599, rs7210086, rs7236492, rs7240004, rs724016, rs7282490, rs7495132, rs7517810, rs7702331 , rs7758080, rs864745, rs917997, rs921720, rs925255, rs9264942, rs9286879, rs9297145, rs9319943, rs941823, rs9491697, rs9847710, rs12199775, rs12654812, rs1292053, rs2227564, rs3197999, rs6074022.
[00159] The foregoing subset of 1 12 SNPs exhibited varying degrees of association with response to SSI therapy, as set out in Tables 12 and 13, which identifies the relevant allele for each SNP and the odds ratio reflecting the association of that allele with SSI response. In Table 12, odds ratios greater than 1 indicate that the designated allele is positively associated with response to SSI therapy, odds ratios less 1 indicate that alternative allele is positively associated with response to SSI therapy and the allele set out in the Table is negatively associated with response to SSI therapy. In Table 13, the odds ratios that are negative in Table 12 have been converted to positive odds ratios for the alternative allele, so that all odds ratios are greater than one and the Response Allele is the allele associated with response to SSI therapy.
Table 12: SNP alleles associated (or negatively associated) with Response to SSI Therapy
rsID Response Odds Ratio
Allele
rs7517810 G
19.83
rs17391694 G
12.00
rs2413583 G
5.67
rs13204048 G
3.84
rs1 1209026 G
3.64
rs1734907 G
3.56
rs212388 G
3.19
rs1 1739663 G
3.19
rs3742130 G
3.00
rs1 1672983 G
2.99
rs1801274 G
2.94
rs559928 G
2.68 rs1042058 G
2.68 rs9847710 G
2.66
TS4802307 C
2.49 rs4836519 G
2.37 rs194749 G 2.31 rs4243971 C
2.28 rs10781499 G
2.26 rs26528 G
1.99 rs864745 G
1.90 rs516246 G
1.87 rs2472649 G
1.87 rs12654812 G
1.85 rs3764147 G
1.85 rs2155219 C
1.79 rs12199775 G
1.69 rs4728142 G
1.63 rs1 182188 G
1.63 rs171 19 G
1.60 rs2189234 C
1.59 rs483905 G
1.58 rs925255 G
1.49 rs7702331 G
1.48 rs564349 G
1.44 rs35320439 G
1.43 rs10865331 G
1.37 rs7495132 G
1.33 rs1016883 G
1.33 rs1292053 G 1.29 rs1260326 G
1.27 rs724016 G
1.25 TS9264942 G
1.23 rs1 1742570 G
1.21 rs3024505 G
1.20 rs1 1083840 C
1.20 rs686341 1 T
1.18 rs1 1150589 G
1.16 rs2188962 G
1.15 rs38904 G
1.15 rs2231884 G
1.14 rs568617 G
1.14 rs566416 C 1.09 rs941823 G
1.09 rs2930047 G
1.08 rs1748195 G
1.06 rs2227564 G
1.05
TS9491697 G
1.00 rs7240004 G
0.98 rs3766606 C
0.96 rs2227551 C
0.95 rs1 1054935 G
0.95 rs7758080 G
0.95 rs477515 G
0.94 rs1847472 C
0.93 rs10896794 G
0.92 rs6426833 G
0.91
TS1893217 G
0.90 rs4409764 C
0.89 rs13277237 G
0.86 rs6017342 C
0.82 rs1517352 C
0.82 rs1 1583043 G
0.81 rs4692386 G
0.79 rs2823286 G
0.79 rs211 1485 G
0.77 rs395157 G
0.76 rs17780256 C
0.76 rs7210086 C
0.76 rs921720 G
0.75 rs616597 C
0.74 rs10521318 G
0.71 rs9319943 G
0.71 rs7282490 G
0.70 rs1569723 C
0.69 rs4976646 G
0.68 rs9297145 C
0.67 rs6074022 G
0.67 rs7097656 G
0.66 rs1077773 G
0.66 rs1 1168249 G
0.63 rs10758669 C
0.62 rs1728918 G
0.61
TS2651244 G
0.59 rs12778642 C
0.58 rs17229285 G
0.56 rs2836878 G
0.56 rs6667605 G
0.50 rs1654644 C
0.48 rs10065637 G
0.48 rs2641348 G
0.44 rs2816958 G
0.43 rs7134599 G
0.41
rs6651252 G
0.40
rs917997 G
0.38
rs6088765 C
0.38
rs2024092 G
0.38
rs3197999 G
0.34
rs7236492 G
0.32
rs17085007 G
0.24
rs6920220 G
0.21
rs9286879 G
0.05
Table 13: SNP alleles associated with Response to SSI Therapy
Figure imgf000081_0001
rs559928 G 2.68 rs1042058 G 2.68 rs2024092 A 2.67 rs6088765 T 2.66 rs9847710 G 2.66 rs917997 A 2.64 rs6651252 A 2.52 rs4802307 C 2.49 rs7134599 A 2.46 rs4836519 G 2.37 rs2816958 A 2.33 rs194749 G 2.31 rs2641348 A 2.29 rs4243971 C 2.28 rs10781499 G 2.26 rs10065637 A 2.10 rs1654644 T 2.06 rs26528 G 1.99 rs6667605 A 1.99 rs864745 G 1.90 rs516246 G 1.87 rs2472649 G 1.87 rs12654812 G 1.85 rs3764147 G 1.85 rs2836878 A 1.80 rs17229285 A 1.79 rs2155219 C 1.79 rs12778642 T 1.73 rs2651244 A 1.71 rs12199775 G 1.69 rs1728918 A 1.65 rs4728142 G 1.63 rs1 182188 G 1.63 rs10758669 T 1.60 rs171 19 G 1.60 rs2189234 C 1.59 rs483905 G 1.58 rs1 1168249 A 1.58 rs1077773 A 1.53 rs7097656 A 1.51 rs6074022 A 1.50 rs925255 G 1.49 rs9297145 T 1.48 rs7702331 G 1.48 rs4976646 A 1.46 rs1569723 T 1.45 rs564349 G 1.44 rs35320439 G 1.43 rs7282490 A 1.42 rs9319943 A 1.41 rs10521318 A 1.40 rs10865331 G 1.37 rs616597 T 1.35 rs7495132 G 1.33 rs1016883 G 1.33 rs921720 A 1.33 rs17780256 T 1.32 rs7210086 T 1.32 rs395157 A 1.32 rs211 1485 A 1.30 rs1292053 G 1.29 rs1260326 G 1.27 rs2823286 A 1.27 rs4692386 A 1.26 rs724016 G 1.25 rs1 1583043 A 1.23 rs9264942 G 1.23 rs1517352 T 1.21 rs6017342 T 1.21 rs1 1742570 G 1.21 rs3024505 G 1.20 rs1 1083840 C 1.20 rs686341 1 T 1.18 rs13277237 A 1.16 rs1 1150589 G 1.16 rs2188962 G 1.15 rs38904 G 1.15 rs2231884 G 1.14 rs568617 G 1.14 rs4409764 T 1.13 rs1893217 A 1.1 1 rs6426833 A 1.10 rs566416 C 1.09 rs941823 G 1.09 rs10896794 A 1.09 rs2930047 G 1.08 rs1847472 T 1.07 rs1748195 G 1.06 rs477515 A 1.06 rs7758080 A 1.06 rs1 1054935 A 1 .05
rs2227564 G 1 .05
rs2227551 T 1 .05
rs3766606 T 1 .05
rs7240004 A 1 .02
rs9491697 G 1 .00
[00160] Within the foregoing subset of 1 12 SNPs, a number were individually associated with particular markers of clinical efficacy, and these SNPs are in turn spatially associated with genes, so that alternative markers, such as SNPs, associated with these genes may also serve as markers of SSI efficacy, as set out in Table 14.
Table 14: Select SNPs and associated Genes
Figure imgf000085_0001
CD - rs17391694 7.21 E-03 See CD - response
Cal protecti n: rs2413583 1.09E-02 MAP3K7IP1 , PDGFB, RPL3, Comparing SYNGR1 , SNORD43, SNORD83A, drop in fecal SNORD83B, FLJ23865, TAB1 , calprotectin ATF4
after 8 weeks rs 1 1209026 1.62E-02 IL12RB2,IL23R
of treatment rs2472649 2.85E-02 CXCL3, PF4, PPBP, CXCL5, for all CD PPBPL2, IL8, CXCL1 , CXCL6, subjects CXCL2
CD - CRP: rs2641348 2.20E-03 NOTCH2, ADAM30, REG4, NBPF7 Comparing
drop in CRP
after 8 weeks
of treatment
for all CD
subjects
UC - Mayo: rs17085007 1.81 E-02 GPR12
Comparing rs2024092 2.50E-02 CNN2, GPX4, POLR2E, STK1 1 , drop in Mayo ABCA7, SBN02, HMHA1 score after 16 rs6017342 2.50E-02 HNF4A, SERINC3, PKIG, TTPAL, weeks of R3HDM
treatment for rs2188962 3.65E-02 IRF1 , SLC22A4, SLC22A5, C5orf56 all UC rs941823 3.71 E-02 LOC646982, FOX01
subjects
UC-CRP: rs17085007 1.95E-02 see UC-Mayo
Comparing rs2024092 1.95E-02 see UC-Mayo
drop in CRP rs6017342 3.81 E-02 see UC-Mayo
after 16 rs17229285 3.96E-02
weeks of
treatment for all UC
subjects
[00161 ] The foregoing IBD associated SNPs are spatially associated with genes (Liu et al., Nature Genetics. 47.9 (Sept. 2015): p979), so that alternative markers, such as SNPs, associated with these genes may also serve as markers of SSI efficacy, as set out in Table 25:
Table 15: Additional SNPs and associated Genes
SNP Candidate Gene GRAIL gene
rs1748195 USP1
rs34856868 BTBD8
rs1 1583043 SLC30A, EDG1 EDG1
rs6025 SELP, SELE, SELL SELP, SELE, SELL
NA (rs10798069) PTGS2, PLA2G4A
NA (rs7555082) PTPRC
rs1 1681525 —
rs4664304 MARCH7, LY75, PLA2R1 LY75
rs31 16494 ICOS, CD28, CTLA4 ICOS, CD28, CTLA4 rs7556897,rs1 1 1781203 CCL20 CCL20
rs35320439 PDCD1 , ATG4B PDCD1 ,ATG4B rs1 13010081 FLJ78302, LTF, CCR1/2/3/5 FLJ78302, LTF, CCR1 ,
CCR3, CCR5 rs616597 NFKBIZ NFKBIZ
rs724016 —
rs2073505 HGFAC
rs4692386 —
rs6856616 —
rs2189234 —
rs395157 OSMR, FYB, LIFR OSMR, FYB
rs4703855 — rs564349 C5orf4, DUSP1 DUSP1
rs7773324 IRF4, DUSP22 IRF4, DUSP22 rs13204048 —
rs7758080 MAP3K7IP2 MAP3K7IP2 rs1077773 AHR AHR
rs2538470 CNTNAP2
rs17057051 PTK2B, TRIM35,EPHX2 PTK2B
rs701 1507 —
rs3740415 NFKB2, TRIM8, TMEM180 NFKB2
rs7954567 CD27, TNFRSF1A, LTBR CD27, TNFRSF1A,
LTBR
rs653178 SH2B3, ALDH2, ATXN2 SH2B3
rs1 1064881 PRKAB1
rs9525625 AKAP1 , TNFSF1 1 TNFSF1 1 rs3853824 —
rs17736589 —
rs9319943 —
rs7236492 NFATC1 , TST NFATC1 rs727563 TEF, NHP2L1 , PMM1 ,
L3MBTL2, CHADL
[00162] The foregoing IBD associated SNPs are spatially associated with genes (Jostins, et ai, Nature. 2012; 491 : 1 19-124), so that alternative markers, such as SNPs, associated with these genes may also serve as markers of SSI efficacy, as set out in Table 16:
Table 16: Further SNPs and associated Genes
SNP IC_SNP Key Genes (N)
rs17391694 rs17391694 (5)
rs6679677 rs6679677 PTPN22,(8) rs3897478 rs2641348 ADAM30 ,(6)
rs9286879 rs7517810 TNFSF18.FASLG
rs1728918 rs 1260326 UCN,(22)
rs10865331 rs 10865331 (3)
rs6716753 rs6716753 SP140,(5)
rs12994997 rs12994997 ATG16L1 ,(8)
rs6837335 rs7438704 TEC,TXK,SLC10A4,(3)
rs13126505 rs13126505 (1)
rs10065637 rs 10065637 IL6ST,IL31 RA,(2)
rs7702331 rs10061469 (4)
rs17695092 rs 17695092 CPEB4,(2)
rs12663356 rs 12663356 (2)
rs9264942 rs9264942 HLA-C,PS0RS1 C1 ,(1 )
rs9491697 rs2503322 (3)
rs13204742 rs13204742 (2)
rs212388 rs212388 (6)
rs10486483 rs 10486483 (2)
rs864745 rs864745 CREB5.JAZF1
rs7015630 rs7015630 RIPK2,(4)
rs6651252 rs6651252 (0)
rs3764147 rs3764147 LACC1 ,FLJ38725,(2)
rs16967103 rs16967103 RASGRP1 ,SPRED1 ,(2)
rs2066847** rs5743289 N0D2, (?)
rs2945412 rs2945412 LGALS9,NOS2,(4)
rs2024092 rs2024092 APC2,GPX4,(21 )
rs4802307 rs4802307 (11 )
rs516246 rs516246 DBPJZUM01 ,FUT2,SPHK2,(22) rs2284553 rs2284553 IFNGR2, IFNAR1 , IL10RB, TMEM50B,
IFNAR2, GART,(7) [00163] The foregoing IBD associated SNPs are spatially associated with genes (Jostins, et ai, Nature. 2012; 491 : 1 19-124), so that alternative markers, such as SNPs, associated with these genes may also serve as markers of SSI efficacy, as set out in Table 17:
Table 17: Further Select SNPs and associated Genes
Figure imgf000090_0001
PS0RS1C3, HCG27, HLA-C, HLA-B, MICA,
HCP5, HCG26, MICB, MCCD1, DDX39B, SN0RD117, SNORD84, ATP6V1G2, NFKBIL1 rs9491697 rs2503322 RSP03, RNF146, ECHDC1
rs13204742 rs13204742 THEMIS, PTPRK
rs212388 rs212388 EZR, 0STCP1, C6orf99, RSPH3, TAGAP,
FNDC1
rs10486483 rs 10486483 C7orf71 , SKAP2
rs864745 rs864745 JAZF1, L0C100128081, CREB5
rs7015630 rs7015630 RIPK2, 0SGIN2, NBN, DECR1, CALB1 rs6651252 rs6651252
rs3764147 rs3764147 EN0X1, CCDC122, LACC1, LINC00284 rs16967103 rs 16967103 SPRED1, FAM98B, RASGRP1, C15orf53 rs2066847** rs5743289 ADCY7, BRD7, NKD1, SNX20, N0D2, CYLD rs2945412 rs2945412 WSB1, LOC440419, KSR1, LGALS9, N0S2 rs2024092 rs2024092 MED16, R3HDM4, KISS1R, ARID3A, WDR18,
GRIN3B, C19orf6, CNN2, ABCA7, HMHA1, P0LR2E, GPX4, SBN02, STK11, C19orf26, ATP5D, MIDN, CIRBP-AS1, CIRBP, C19orf24, EFNA2, MUM1
rs4802307 rs4802307 IGFL3, IGFL2, DKFZp434J0226, IGFL1, HIF3A,
PPP5C, CCDC8, PNMAL1, PNMAL2
rs516246 rs516246 GRWD1, KCNJ14, CYTH2, LMTK3, SULT2B1,
FAM83E, SPACA4, RPL18, SPHK2, DBP, CA11, SEC1, NTN5, FUT2, MAMSTR, RASIP1, IZUM01, FUT1, FGF21, BCAT2, HSD17B14, PLEKHA4, PPP1R15A, TULP2, NUCB1, DHDH rs2284553 rs2284553 C21orf54, IFNAR2, IL10RB, IFNAR1, IFNGR2,
TMEM50B, DNAJC28, GART, SON, DONSON, CRYZL1, ITSN1 [00164] The correlation coefficient between pairs of loci may be reflected by the term r-squared (r2), which may be used a measure of the degree to which alternative genetic markers provide similar diagnostic or prognostic information. The value of r2 ranges between 0 and 1 (1 when two markers provide identical information, and 0 when they are in perfect equilibrium). Conventionally, markers with r2 > 0.8 may be considered to be in high linkage disequilibrium, so that they may provide similar diagnostic or prognostic information. Accordingly, an aspect of the assays described herein involves the use of makers that are in linkage disequilibrium with the markers identified above, having for example r2 > 0.7, r2 > 0.8, r2 > 0.9 or r2 > 0.95. In addition, markers that provide related information may be characterized by physical proximity in the genome, for example being within 1 Mbp of each other, for example within 50Kb, 60Kb, 70Kb, 80Kb, 90Kb, 100Kb, 200Kb, 300Kb, 400Kb or 500Kb of each other.
[00165] In accordance with the foregoing, a "genetic SSI response marker" means a genetic biomarker, the presence of which is correlated with the probability of response to a treatment with an SSI. Exemplary genetic SSI response markers are disclosed in this Example, evidencing a correlation with response to an SSI in IBD patients. Genetic SSI response markers may be detected by a wide range of genomic assays, and may also be detected by assays that interrogate the transcription or translation products of a genome, for example protein isoforms associated with a particular genomic allele. Similarly, "biochemical SSI response markers" are disclosed herein that provide a biochemical indication of response to an SSI therapy, these for example include temporal or special changes in cellular populations or in the abundance or concentration of biologically relevant molecules. Biochemical and genetic SSI response markers may be used as diagnostic or prognostic indicators in the context of an SSI treatment, for example for IBD in general, or for specific forms of IBD such as Crohn's Disease and ulcerative colitis. Exemplary genetic SSI response markers are set out in Table 18, as well as
Tables 13 to 17
Table 18: Genetic SSI Response Markers SNP Response SNP related allele (or isoform) I BD Allele
rs9286879 A TNFSF18, TNFSF4, FASLG Crohn's rs7517810 G TNFSF18, TNFSF4, FASLG Crohn's rs17391694 G DNAJB4 (HSP-40 family member), GIPC2, Crohn's
NEXN, FUBP1 , MGC27382
rs17085007 A GPR12 UC rs2024092 A CNN2, GPX4, POLR2E, STK1 1 , ABCA7, UC
SBN02, HMHA1
rs6017342 T HNF4A, SERI NC3, PKIG, TTPAL, R3HDM UC
[00166] Figure 6 includes bar graphs illustrating the frequency of selected SNP genotypes in responders and non-responders, for the SNPs rs9286879, rs7517810 and rs17391694, where "response" is indicative of the last recorded response for CD patients undergoing a QBECO SSI therapy. Figure 7 shows similar data for 4 SNPs when response is measured by a drop in Crohn's Disease Activity Index (CDAI , Best et al. , 1976, Gastroenterology 70 (3): 439-444), after 8 weeks of QBECO SSI treatment, for the SNPs: rs1734907, rs7517810, rs9286879 and rs4836519. Figure 8 shows similar data for 4 SNPs when response is measured by a drop in fecal calprotectin after 8 weeks of QBECO SSI treatment, for the SNPs: rs17391694, rs2413583, rs1 1209026 and rs2472649. Figure 9 shows similar data for 1 SNPs when response is measured by a drop in CRP (serum levels of C-reactive protein) after 8 weeks of QBECO SSI treatment, for the SNP: rs2641348.
[00167] Figure 10 includes bar graphs illustrating the frequency of selected SNP genotypes in UC responders and non-responders, for the SNPs rs17085007, rs2024092, rs6017342, rs2188962 and rs941823, where "response" is indicative of a drop in Mayo score after 16 weeks of treatment for UC patients undergoing a QBECO SSI therapy. Figure 11 shows similar data for 4 SNPs when response is measured in UC patients by a drop in CRP (serum levels of C-reactive protein) after 16 weeks of QBECO SSI treatment, for the SNPs: rs17085007, rs2024092, rs6017342 and rs17229285. Figure 12 shows similar data for 3 SNPs when response is measured in UC patients by last recorded histological score (indicative of histological healing), for the SNPs: rs17085007, rs6017342, rs2024092. Figure 13 shows similar data for 4 SNPs when response is measured in UC patients by a drop in histology score after 16 weeks of QBECO SSI treatment, for the SNPs: rs10065637, rs1 1739663, rs6017342 and rs2651244. Figure 14 shows similar data for 4 SNPs when response is measured in UC patients by a drop in continuous score after 16 weeks of QBECO SSI treatment, for the SNPs: rs10065637, rs1 1739663, rs1 1083840 and rs2651244. Figure 15 shows similar data for 5 SNPs when response is measured in UC patients by a drop in last recorded continuous score, for the SNPs: rs6017342, rs13277237, rs4802307, rs9847710 and rs2188962.
[00168] In this context, it is relevant that SNP rs17085007 is associated with the protein ubiquitin specific peptidase 12 (USP12) which is an important protein for macrophage mediated inflammatory response to bacterial endotoxin (LPS) and may mediate a key response to bacteria, and bacterial derived products (Nayak et al. , Biochem Biophys Res Commun 2017;483:69-74.). Similarly, SNP rs2188962 is located in the IBD genetic locus of susceptibility 5, which includes many important inflammatory genes for immune activation and modulation, including IL-13, II-4, I L- 5, and 11-13, among others
[00169] Figure 16 shows similar data for 6 SNPs when response is measured by the last recorded response for CD and UC patients undergoing a QBECO SSI therapy, for the SNPs: rs7517810, rs9286879, rs2413583, rs10758669,
rs10865331 and rs2024092.
[00170] In instances where homozygosity was observed in the responder genotype and heterozygosity in the non-responder genotype, one allele may have dominance over the other with respect to response to an SSI treatment.
Accordingly, some embodiments involve assays to distinguish homo- or
heterozygosity in the SNP alleles identified herein. Example 7: QBECO PRR Receptor Targets
[00171 ] This Example provides an analysis of the PRR receptors that are the targets for QBECO SSIs.
Table 28: List of PRRs stimulated by QBECO SSI. Where a PRR is Optional", this indicates that some embodiments may be designed to include agonists for the specified PRR.
Figure imgf000095_0001
Macrophage a- or -/V-acetylgalactosamine Optional galactose-type (GalNAc, Tn) residues of N- lectin (MGL) and O-glycans carried by
glycoproteins and/or
glycosphingolipids (PMID 15802303)
DC-SIGN (CD- High-mannose-containing Optional 209) glycoproteins
Langerin (CD207) Similar to CD-209 Optional
Mannose Binding Mannose and N- Optional Lectin (MBL) acetylucosamine
Myeloid DAP12- Unclear, dengue viral particles Optional associating lectin (PMC3204838)
(MDL-1/CLEC5A)
Dectin1/CLEC7A B glucans on fungi, Optional mycobacteria
DNGR1/CLEC9A Actin filaments (no microbial Optional ligands identified)
SIGNR3 Mycobacterium tuberculosis Optional
CLEC4B1 Not Determined Optional
CLEC4B2 Not Determined Optional
CLEC2 Endogenous (prodoplanin), Optional snake venom, HIV
CLEC12B Not Determined Optional
CLEC12A Not Determined Optional
DCI R/CLEC4A HIV-1 Optional
Dectin 2/CLEC6A Mannose-type carbohydrates Optional
CLEC4C Unclear Optional
CLEC4E (Mincle) Fungal a-mannose and others Optional
NLR (Nod-Like Receptors) NOD1 diaminopimelatic acid (DAP)- Optional containing muropeptide
NOD2 muramyl dipeptide (MDP) Yes moieties universal to all bacterial peptidoglycan
NLRC3 (NOD3) Cytosolic DNA, cyclic di-GMP, Optional
DNA viruses (PMID 24560620)
This is an inhibitory PRR.
NLRC4 (NOD4) Flagellin, components of the Optional type three secretion system,
others
NLRC6 Unclear Optional
NLRX1 (NOD5) Unclear Optional
NALP1 -14 Pathway unclear (Anthrax and Optional muramyldipeptide for NALP1 )
NAIP Unclear Optional
CI ITA (NLRA) Unclear (does not directly bind Optional
DNA)
RLR (Rig-1 Like Receptors)
RIG-1 dsRNA (viral), maybe bacterial Optional
MDA5 dsRNA (viral) Optional
LGP2 dsRNA (viral) Optional
Others
DAI (DNA- DNA Optional dependent
activator of IRFs)
(PMID 20098460)
AIM2 (PMI D dsDNA Optional 20098460)
Caspase 1 1 LPS Optional (PMID 25145754) LBP LPS Optional
(Lipopolysacchari
de Binding
Protein
CD14 LPS Optional
Scavenger LPS Optional
Receptors
Beta2 Integrins LPS Optional
Peptidoglycan Peptidoglycan Minor
receptor proteins
(4 different
receptors)
Table 29: PRR agonists in fractionated QBECO SSIs, particularly in the DNA fractions Exemplified herein.
Figure imgf000098_0001
Table 30: PRR agonists in fractionated QBECO SSIs, particularly in the outer membrane fractions as exemplified herein.
Figure imgf000099_0001
[00172] Accordingly, in select embodiments, SSI therapies are provided that target a select subset of PRRs, using microbial PRR agonists derived from microbial pathogens of a target tissue. For example, an immunogenic composition is provided that comprises microbial agonists for at least a select number of distinct PRRs, for use so as to illicit an innate response in a target tissue, wherein the PRR agonists are microbial components from a single species of microbe that is selectively pathogenic in the target tissue. The number of distinct PRRs targeted by the agonists may for example be a number from 5 to 25, or at least a number within that range of integers, for example at least 5, 6, etc. The distinct PRRs may for example be selected from the PRRs set out in Tables 28, 29 and/or 30.
Example 8: Cytokine Markers of SSI Therapy in CD
[00173] This Example provides an indication of cytokine markers indicative of various facets of SSI therapies. This data reflects the analysis of 42
cytokines/chemokines from a cohort of Crohn's Disease patients undergoing SSI therapy with QBECO, at baseline, week 4, week 8, week 16, and week 24, of a randomized placebo-controlled trial involving 68 patients.
Cytokines changes with QBECO exposure
[00174] QBECO exposure increased I L-18 and I P-10 at both the 8 week and 16 week time points. Serum levels of IL-18 showed the most significant differences between patients treated with QBECO vs. Placebo at week 8 (median change 24 pg/mL, adjusted p=0.0256) (Figure 74). This increase in IL-18 was evident at the week 16 time point as well. The second serum biomarker to show significant differences was IFNv-inducible protein 10 (IP-10, also known as CXCL10) which showed greater increases in QBECO exposed patients at both week 8 (median change 7 pg/mL, adjusted p=0.036) and week 16 (median change 22 pg/mL, adjusted p=0.0151 ).
[00175] Vascular endothelial growth factor A (VEGF-A) showed some increase in the QBECO group at the week 8 mark (median change 14 pg/mL, adjusted p=0.0483), but this difference was lost at the end of the week 16 treatment point. A number of other immune factors showed strong trends in being increased from baseline to 8 weeks of QBECO exposure; these included: granulocyte colony stimulating factor (GCSF), I FNv, I L-17A, IL-6, IL-7, and transforming growth factor- α (TGFa).
[00176] None of the serum immune factors remained elevated after patients were taken off all treatment after week 16 and evaluated again at week 24, illustrating that these biomarkers are most helpful to assess the immune
responsiveness to QBECO while on treatment.
Serum biomarker cytokine concentration changes that associate with clinical response
[00177] A sub-analysis was performed in patients exposed to QBECO (N=42, including those initially randomized to QBECO and those who were switched from placebo at week 8) to assess whether any of the immune changes over time associated with clinical outcome. 11-18 increased less among those with clinical response and remission, compared to non-responders. I P-10 increased less among those with clinical response and remission, compared to non-responders. I FNv, IL- 12p70, IL-17A and TGFa showed a significant difference in increase over time for responders compared to non-responders. In particular, IFNv, IL-12p70, IL-17A and TGFa, had greater increases over-time (adjusted p=0.0344) in patients who experienced a clinical response to QBECO in comparison to non-responders at week 8 (Figure 75).
Baseline serum immune factors that associate with clinical response
[00178] Lower Eotaxin 1 was a predictive biomarker for remission in response to QBECO treatment. In particular, baseline serum levels of Eotaxin-1 (C-C chemokine 1 1 ) had the strongest link to clinical remission (adjusted p=0.0016), with patients who had higher levels at baseline being less likely to go into clinical remission by week 8 with QBECO treatment (Figure 76). Although not reaching statistical significance after correcting for multiple comparisons, patients with higher baseline IL-10 and IL-12p40 were also less likely to have a clinical response to QBECO treatment by week 8 (Figure 76).
[00179] Trial results indicate that patients who had been previously exposed to TNFa inhibitors, such as Remicade™ or Humera™, were less likely to experience clinical remission or response after 8 weeks of QBECO treatment. This more difficult to treat group may have more severe immune dysfunction, due to their exposure to these immunosuppressive drugs and/or by virtue of the nature of their condition. Stratifying the mean baseline serum levels of the immune factors that associated with clinical outcome by previous TNFa inhibitor exposure provides evidence to support this. The baseline serum immune factors that inversely associated with patient response to QBECO, Eoxtaxin-1 , IL-10 and I L-12p40, were higher in patients previously exposed to anti-TNFa therapy relative to unexposed patients (Table 31 ).
Table 31. Mean baseline serum levels of Eotaxin-1 , IL-10 and I L-12p40 stratified by
Previous 95%
Mean
anti- Mean ± Confidence
N* Difference
TNFa SD Interval of the
± SD
therapy Difference
Eotaxin-1 No 39 84 ± 43
-17 ± 12 (-41 , 7)
Yes 26 100 ± 54
IL-10 No 28 6 ± 14
-5 ± 5 (-14, 4)
Yes 17 1 1 ± 17
IL-12p40 No 36 19 ± 67
-52 ± 36 (-123, 20)
Yes 24 70 ± 198
*20 reads from the IL-10 assay and 5 reads from the IL-12p40 were out of range of the assay or unreliable
High Response and Remission Rates in Anti-TNFa Naive Patients
[00180] In anti-TNFa naive patients, treatment with QBECO SSI for 8 weeks resulted in a statistically significant response rate of 64% compared to 27% in the placebo control (p=0.041 ). Clinical remission rates after 8 weeks of treatment were also impressive at 50%, more than double the placebo rate of 23% (p=0.16).
Clinical response and remission rates were assessed using the standard Crohn's Disease Activity Index (CDAI), defined as a decrease in CDAI of >70 points (response) and CDAI score <150 points (remission). Anti-TNFa naive patients include, for example, patients who have not been treated with the
immunosuppressive drugs Remicade®, Humira®, Cimzia® and Simponi®. In patients previously been treated with TNFa inhibitors who completed 16 weeks of SSI treatment, 40% were in remission, indicating that this more challenging patient group may respond to QBECO SSI with longer treatment.
Building a composite prediction model to assess likelihood of patient response to QBECO by 8 weeks of therapy
[00181 ] Using a Regularized Logistic Regression modelling approach, which simultaneously selects variables with the strongest association with response and optimally weights them to generate a prediction score, a composite prediction model was built including both the baseline biomarker measures (i.e. the 42 immune factors including cytokines, chemokines and growth factors) and baseline clinical and demographic characteristics. The variables available for the latter included enrollment year, age at randomization, age at diagnosis, time from diagnosis to randomization, sex, race (Caucasian or not), site (Vancouver or not), prior anti-TNFa therapy, baseline Crohn's Disease Activity Index (CDAI) score, baseline fecal calprotectin levels, and baseline C-reactive protein levels.
[00182] An "optimism-adjusted" area-under the receiver operating curve (AUROC) was made to correct for the potential over-estimation of the model fit. This "optimism-adjusted" AUROC can thus be more readily reliably applied to future independent data.
[00183] As shown in the analysis in this Example, high baseline serum
Eotaxin-1 was the strongest negative biomarker predictor for clinical response after 8 weeks of QBECO treatment and was included in all models generated. Of the clinical/demographic variables - sex (females were more likely to respond to QBECO treatment) and previous anti-TNFa therapy (those previously exposed less likely to respond to QBECO) were the strongest predictors. Table 32 summarizes the different models generated. Typical commercial biomarker standards require an AUROC > 7 for commercial viability of a prediction model. After optimism- adjustment, the composite model generated from this data achieved this level of predictive value with the inclusion of the following variables: sex, prior TNFa therapy, and baseline levels of Eotaxin-1 , GROa (also called CXCL1 - a neutrophil chemokine), IL-10, PDGF AA and RANTES (also called CCL5 - a chemokine for activated T cells, eosinophils, basophils). Alternatively, a predictive model may also be developed using Eotaxin, GROa, IL10, PDGF AA, RANTES, Sex and prior aTNFa, predicting response with high confidence.
Table 32. Performance of four prediction models for clinical response and clinical remission following 8 weeks of QBECO treatment
Figure imgf000104_0001
5 * - significant at 0.05 level.
Analysis
[00184] Cytokine change with QBECO exposure: I L-18 (adjusted p = 0.01 1 0 @ 8 weeks and 0.067 @ 16 weeks) and I P-10 (adjusted p = 0.036 @ 8 weeks and 0.015 @ 16 Weeks) demonstrated a substantial and statistically significant increase with exposure to QBECO. These two cytokines also demonstrated significantly different trajectories for Clinical Responders vs Non-Responders (adjusted p = 0.0328 for both) and those in and not in Clinical Remission (adjusted p = 0.0368 for both) at week 8. Further, IL-18 demonstrated significantly different trajectory for those randomized to QBECO vs Placebo (adjusted p = 0.0256).
[00185] Cytokine Association with Outcome: Baseline Eotaxin-1 concentration was most strongly associated with clinical outcome among QBECO exposed subjects; those with higher Eotaxin-1 concentration at baseline were more likely to achieve Clinical Remission (adjusted p = 0.0016) following 8 weeks of QBECO exposure.
[00186] Composite Biomarker: Baseline concentration of Eotaxin-1 , GRO-a, IL-10, PDGF AA and RANTES, combined with clinical variables Sex, and Prior anti- TNFAa exposure provided predictions of 8-week clinical outcomes that were significantly better than chance (optimism-adjusted AUROC = 0.70, 95% CI [0.59, 0.81 ] for Response and 0.71 , 95% CI [0.60,0.82] for Remission). This model had some observable predictive ability for subjects in the Placebo group (AUROC = 0.67 95% CI [0.45, 0.89] for Response and 0.70 [0.47, 0.93] for Remission. Summary
[00187] QBECO SSI therapy provokes a biological response by increasing certain cytokines (IL-18 and IP-10) over time. Surprisingly, although both cytokines are increased after QBECO treatment, patients who were responders increased less. Treatment protocols, such as dosing, may accordingly be adjusted to achieve this result.
[00188] IFNgamma, I L-12P70, IL-17A and TGFalpha increased more in responders than non-responders. Treatment protocols, such as dosing, may accordingly be adjusted to achieve this result. TGFalpha may for example be used as a marker of mucosal healing.
[00189] Lower Eotaxin 1 levels may be used as an indicator of patients more amenable to SSI treatment.
[00190] In conclusion: • an increase in serum IL-18 from baseline to week 8 and 16 of treatment was the best biomarker (of the 42 assessed) for QBECO exposure/activity;
• a subsequent rise in serum levels of I FNy, IL-12p70, IL-17A and TGFa after 8 weeks of QBECO treatment associated with clinical response;
· Crohn's patients with higher baseline levels of Eotaxin-1 (and to a lesser extent, IL-10 and IL-12p40) were less likely to experience a clinical response or remission to QBECO after 8 weeks of treatment; previous anti-TNFa therapy may predispose to having higher levels of these factors, and anti-TNFa naive patients represent a distinct Crohn's patient population amenable to QBECO SSI therapy;
• a composite model that includes baseline serum biomarkers and
clinical/demographic data would be able to predict, after optimism-adjustment (AUROC >7), a patient's likelihood to respond to 8 weeks of QBECO treatment; the variables in the final model includes sex, previous anti-TNFa therapy and baseline serum levels of Eotaxin-1 , GROa, IL-10, PDGF AA and RANTES.
[00191 ] This biomarker analysis illustrates the formulation of a viable predictive composite model that can provide personalized treatment for Crohn's disease. This biomarker analysis maybe useful alone, or in combination with the genetic analysis exemplified herein, which showed significant stratification between responders and non-responders based on a derivation of a gene score.
Example 9: Cytokine Markers of SSI Therapy in UC
[00192] This Example illustrates the availability of cytokine markers of efficacious SSI therapy in ulcerative colitis, demonstrated in an open label, dose ranging, therapeutic intervention using a QBECO SSI for the treatment of subjects with moderate to severe ulcerative colitis. Blood samples were collected from 1 1 patients, and serum immune factors, that included cytokines, chemokines and growth factors, were measured by multiplex technology (Eve Technologies, Calgary, AB, Canada) using the Human Cytokine/Chemokine Array 42-Plex with IL- 18 (HD42; Millipore). [00193] Four cytokines were identified as significantly increased with QBECO treatment, from baseline to 16 Weeks of QBECO treatment, including interferon inducible protein 10 (I P10, also known as CXCL10), MIP-1 a, IL-1 a and IL-2.
CXCL10 is a chemokine that is produced upon IFNv production and bacterial stimulation and is a chemoattractant for monocytes/macrophages, NK cells, T cells and others. Importantly, CXCL10 was also increased in QBECO treated patients with Crohn's Disease (QBECO-01 ) IL-18, which was also shown to be increased with QBECO treatment in patients with Crohn's Disease (QBECO-01 ), showed a trend to increase in this Example in ulcerative colitis patients.
[00194] Serum immune factors were also identified that were associated with clinical response, these serum immune factor changes were different for Week 16 responders compared to Week 16 non-responders, including FGF2, IFNv, IL-17A, IL-6, CXCL10, MDC, MIP 1 a, MIP 1 β, TNFa, Fir 3L, IL-12p70, IL-1 a and I L-2. Two serum immune factors, CXCL10 and I L-1 a, were changed in patients with histological healing when compared to patients without histological healing. The serum immune factors that were significant were increased in responders when compared to non-responders. These results indicate that patients who are able to induce an immune response to QBECO over time, as indicated by increase in inflammatory cytokines like I L-6, IFNv and others, are more likely to respond to QBECO treatment.
[00195] In the foregoing analysis of QBECO use in patients with Crohn's Disease, four serum immune factors where increased in responders vs non- responders over time with QBECO treatment. These included I FNv, IL-12p70, IL- 17A, and TGFa. Importantly, IFNv, IL-12p70 and IL-17A were shown to increase in both CD and UC patient undergoing treatment with QBECO, identifying these serum immune factors as important markers for response to QBECO.
[00196] Changes in cytokine levels in UC QBECO responders vs. non- responders are illustrated in Figure 23. In conclusion, this exemplary analysis of serum cytokines, chemokines and growth factors in relation to QBECO treatment for Ulcerative Colitis shows:
• Increased serum CXCL10, MIP-1 a, IL-1 a and IL-2 from baseline to Week 16. • Increased serum levels of FGF2, IFNv, I L-17A, IL-6, CXCL10, MDC, MIP 1 a, MIP 1 β, TNF a, Fir 3L, IL-12p70, IL-1 a and IL-2 from baseline to Week 16 that was correlated to response to QBECO
• Increased serum levels of CXCL10 and IL-1 a from baseline to week 16 that was correlated to histological healing
• Limited correlation between baseline serum immune factors and week 16 response or histological healing.
Example 10: Histological Healing in UC
[00197] In this example of histological healing in UC patients, with a QBECO SSI treatment, biopsy samples were collected from inflamed areas of the colon for histology. Samples were collected at Week 0 (all 1 1 patients), Week 8 (5 patients) and Week 16 (10 patients). Biopsy tissue samples were scored by a clinical pathologist, blinded to time-point and treatment, using the Geboes' scale. The Geboes' classifies histological changes on ordinal scale as grade 0 (structural change only), grade 1 (chronic inflammation), grade 2 (2a, lamina propria neutrophils; 2b, lamina propria eosinophils), grade 3 (neutrophils in the epithelium), grade 4 (crypt destruction), or grade 5 (erosion or ulcers), and generates a score from 0 to 5, with higher scores indicating greater inflammation. The mean histological score for all patients improved from 16.3 (Extended Geboe's scale) at baseline to 12.1 at Week 16 (p=0.06). A reduction in histology score to 13 or less (Extended Geboe's scale) is considered to be a good response in UC.
[00198] Of the 8 patients who were deemed responders to QBECO (i.e., decrease in Mayo score of >3 points) at Week 8 and/or Week 16, 6 had
improvement in histology grade (circles). The 3 non-responders as measured by Mayo score had no histological improvement (squares). None of the patients had an increase in histological score in this study.
Example 11 : DSS Colitis Model
[00199] This Example illustrates results from a mouse model of chemically induced colitis, used to assess the efficacy of QBECO SSI therapy. Mice were given dextran sodium sulfate (DSS) in drinking water to induce colitis that mimics human ulcerative colitis. In the disease model, one cohort of mice was exposed to DSS for 7 days, a second cohort was exposed to DSS for 7 days followed by 3 days of water. Mice were given SSI injections every other day during a 10 day period prior to DSS exposure. The SSI injections continued every other day during DSS exposure. The results, as illustrated, indicate that SSI treatment with QBECO ameliorates disease severity by limiting weight loss (Figure 77), lowering disease severity (Figure 78) and maintaining mucosal barrier function (Figure 79). The pharmacodynamics of QBECO SSI treatment in this model are illustrated by the blood neutrophil (Figure 80) and blood cytokine (Figure 81 ) levels in disease-free mice treated with QBECO or placebo, with the pharmacokinetics of QBKPN (Figure 82) used to model the pharmacokinetics of SSIs in general, including QBECO (QBKPN SSI was fluorescently labelled and subcutaneously injected into disease- free mice, mice were bled at different time points over 48 hours).
Example 12: SNPs and Response to SSIs
[00200] Tables 33, 34 and 35 are set out below, in which each Table includes a SNP I D, Odds ratios (ORs) and "risk" allele. In these Tables, the OR reflects the "risk" of responding to the treatment. For example, a SNP with a G allele and OR of 5 would be predictive of response to SSI (QBECO) while a SNP with a G allele and OR of 0.1 would be predictive of NOT responding to SSI (QBECO). The first three Tables are, respectively, response to SSI (QBECO) in UC patients, response to SSI (QBECO) in CD and UC patients, and response to SSI (QBECO) in CD patients.
Figure imgf000109_0001
rs3197999 5 G 0.677083333 rs724016 4.488677506 G 0.427083333 rs2155219 4.250590881 C 0.479166667 rs6088765 4.250590881 C 0.458333333 rs1734907 4 G 0.833333333 rs2651244 4 G 0.666666667 rs35320439 4 G 0.302083333 rs7236492 4 G 0.90625 rs2227551 3.861917278 C 0.28125 rs1077773 3.689845905 G 0.458333333 rs6920220 2.800257315 G 0.739583333 rs7134599 2.800257315 G 0.666666667 rs10896794 2.571641737 G 0.270833333 rs2836878 2.5 G 0.71875 rs3742130 2.5 G 0.8125 rs568617 2.106256354 G 0.822916667 rs13204048 2.103002238 G 0.40625 rs9264942 2.103002238 G 0.447916667 rs1893217 1 .820393751 G 0.145833333 rs3764147 1 .820393751 G 0.21875 rs2231884 1 .75 G 0.84375 rs6426833 1 .75 G 0.489583333 rs7097656 1 .75 G 0.8125 rs2930047 1 .739800613 G 0.416666667 rs38904 1 .629678753 G 0.395833333 rs17780256 1 .6 C 0.1875 rs7210086 1 .6 C 0.1875 rs10781499 1 .595544408 G 0.510416667 rs1801274 1 .595544408 G 0.59375 rs12778642 1 .433765451 C 0.541666667 rs6667605 1 .374131521 G 0.458333333 rs1 1672983 1 .303984992 G 0.645833333 rs1847472 1 .303984992 C 0.708333333 rs7282490 1 .243266737 G 0.416666667 rs1 1083840 1 .212219242 C 0.46875 rs1042058 1 G 0.604166667 rs13277237 1 G 0.520833333 rs1517352 1 C 0.583333333 rs17229285 1 G 0.458333333 rs564349 1 G 0.333333333 rs566416 1 C 0.260416667 rs7517810 1 G 0.760416667 rs9286879 1 G 0.239583333 rs9491697 1 G 0.395833333 rs12654812 1 G 0.71875 rs4976646 0.828852697 G 0.260416667 rs4728142 0.824933284 G 0.614583333 rs483905 0.804332626 G 0.645833333 rs1654644 0.766879992 C 0.322916667 rs7758080 0.766879992 G 0.25 rs4802307 0.762040442 C 0.59375 rs921720 0.762040442 G 0.604166667 rs917997 0.748379567 G 0.729166667 rs1 1150589 0.727732379 G 0.583333333 rs26528 0.727732379 G 0.510416667 rs516246 0.727732379 G 0.614583333 rs211 1485 0.626745326 G 0.489583333 rs7240004 0.626745326 G 0.395833333 rs1569723 0.625 C 0.404255319 rs477515 0.625 G 0.760416667 rs6074022 0.625 G 0.375 rs1016883 0.549331703 G 0.864583333 rs1728918 0.549331703 G 0.666666667 rs3766606 0.549331703 C 0.875 rs212388 0.530808718 G 0.302083333 rs1292053 0.469040769 G 0.489583333 rs9319943 0.4 G 0.15625 rs2227564 0.4 G 0.739583333 rs4409764 0.373881338 C 0.375 rs9847710 0.373881338 G 0.427083333 rs1 1168249 0.3571 10039 G 0.40625 rs2823286 0.265869932 G 0.760416667 rs1 1583043 0.258938742 G 0.71875 rs1260326 0.258938742 G 0.489583333 rs4243971 0.258938742 C 0.666666667 rs4692386 0.258938742 G 0.583333333 rs171 19 0.253238771 G 0.15625 rs1 1054935 0.25 G 0.21875 rs4836519 0.25 G 0.229166667 rs925255 0.2 G 0.572916667 rs2189234 0.164479021 C 0.53125 rs395157 0.149412587 G 0.427083333
Table 34: Odds ratios for IBD SNPs associated with response to SSI (QBECO) at last recorded timepoint in both CD and UC patients
rsID OR RiskAllele AlleleFrequency rs2413583 8.125 G 0.916666667 rs7517810 5.624999996 G 0.760416667 rs1734907 3.75 G 0.833333333 rs17391694 3.703703704 G 0.927083333 rs13204048 3.398089721 G 0.40625 rs1 1739663 3.233594035 G 0.270833333 rs10865331 3.228774214 G 0.635416667 rs1801274 2.731309451 G 0.59375 rs3742130 2.698184378 G 0.8125 rs194749 2.558917835 G 0.229166667 rs1 1209026 2.5 G 0.9375 rs864745 2.382624526 G 0.614583333 rs7702331 2.37052769 G 0.427083333 rs559928 2.304093059 G 0.875 rs1 1672983 2.221837057 G 0.645833333 rs686341 1 2.162217745 T 0.670212766 rs10781499 2.125176575 G 0.510416667 rs2155219 2.043031813 C 0.479166667 rs3764147 1 .922252178 G 0.21875 rs212388 1 .828457192 G 0.302083333 rs1042058 1 .771333171 G 0.604166667 rs35320439 1 .741978904 G 0.302083333 rs2472649 1 .722168091 G 0.864583333 rs4802307 1 .683716549 C 0.59375 rs9847710 1 .590751653 G 0.427083333 rs1 1742570 1 .58769402 G 0.614583333 rs516246 1 .568074844 G 0.614583333 rs26528 1 .546842723 G 0.510416667 rs724016 1 .540335421 G 0.427083333 rs12654812 1 .504755786 G 0.71875 rs4243971 1 .46058754 C 0.666666667 rs568617 1 .426961626 G 0.822916667 rs9264942 1 .349312494 G 0.447916667 rs483905 1 .328155321 G 0.645833333 rs2231884 1 .319444444 G 0.84375 rs564349 1 .319444444 G 0.333333333 rs4728142 1 .319064408 G 0.614583333 rs2930047 1 .285406559 G 0.416666667 rs2227551 1 .279806235 C 0.28125 rs38904 1 .243044484 G 0.395833333 rs10896794 1 .243032385 G 0.270833333 rs1 1083840 1 .238870685 C 0.46875 rs566416 1 .166666667 C 0.260416667 rs1748195 1 .165851737 G 0.322916667 rs1893217 1 .146589948 G 0.145833333 rs4836519 1 .140327047 G 0.229166667 rs9297145 1 .138528723 C 0.260416667 rs7495132 1 .090909091 G 0.916666667 rs1 1150589 1 .066197308 G 0.583333333 rs1847472 1 .019186167 C 0.708333333 rs1260326 1 G 0.489583333 rs1292053 1 G 0.489583333 rs2189234 0.988074793 C 0.53125 rs1077773 0.952817489 G 0.458333333 rs9491697 0.944323796 G 0.395833333 rs7240004 0.937912704 G 0.395833333 rs2227564 0.918923858 G 0.739583333 rs6426833 0.917329468 G 0.489583333 rs1517352 0.9101 1894 C 0.583333333 rs17780256 0.907601791 C 0.1875 rs7210086 0.907601791 C 0.1875 rs477515 0.906484996 G 0.760416667 rs7758080 0.893817031 G 0.25 rs7097656 0.858257102 G 0.8125 rs941823 0.858257102 G 0.8125 rs13277237 0.856714576 G 0.520833333 rs925255 0.832324192 G 0.572916667 rs1 182188 0.828923213 G 0.385416667 rs171 19 0.826778583 G 0.15625 rs2651244 0.820644837 G 0.666666667 rs1016883 0.7921 12329 G 0.864583333 rs1 1054935 0.792029259 G 0.21875 rs7282490 0.790192932 G 0.416666667 rs4409764 0.782545328 C 0.375 rs3197999 0.779901 195 G 0.677083333 rs2836878 0.777500421 G 0.71875 rs12778642 0.743556063 C 0.541666667 rs4976646 0.72977943 G 0.260416667 rs3766606 0.727548988 C 0.875 rs211 1485 0.717123548 G 0.489583333 rs921720 0.713134705 G 0.604166667 rs4692386 0.701555735 G 0.583333333 rs6920220 0.697933763 G 0.739583333 rs9319943 0.697515307 G 0.15625 rs2188962 0.693763628 G 0.489583333 rs7134599 0.689814297 G 0.666666667 rs616597 0.686868687 C 0.770833333 rs6017342 0.685686984 C 0.5625 rs6651252 0.68236335 G 0.145833333 rs1569723 0.663893014 C 0.404255319 rs12199775 0.66 G 0.0625 rs6074022 0.638942383 G 0.375 rs3024505 0.633333333 G 0.864583333 rs17229285 0.627512244 G 0.458333333 rs1728918 0.619332216 G 0.666666667 rs7236492 0.61560618 G 0.90625 rs1 1583043 0.613137022 G 0.71875 rs10521318 0.611449573 G 0.947916667 rs1654644 0.604847933 C 0.322916667 rs6667605 0.583267612 G 0.458333333 rs1 1168249 0.551544601 G 0.40625 rs395157 0.537660042 G 0.427083333 rs2823286 0.525718 G 0.760416667 rs6088765 0.509340798 C 0.458333333 rs917997 0.502168027 G 0.729166667 rs17085007 0.466666667 G 0.21875 rs2641348 0.4 G 0.0625 rs2816958 0.383838384 G 0.854166667 rs10065637 0.346153846 G 0.833333333 rs10758669 0.340429623 C 0.364583333 rs2024092 0.239497706 G 0.75
rs9286879 0.177777778 G 0.239583333
Table 35: Odds ratios for IBD SNPS associated with response to an SSI (QBECO) at last recorded timepoint in CD patients
rsID OR RiskAllele AlleleFrequency rs7517810 19.83333333 G 0.760416667 rs17391694 12 G 0.927083333 rs2413583 5.666666667 G 0.916666667 rs13204048 3.835420339 G 0.40625 rs1 1209026 3.642857143 G 0.9375 rs1734907 3.555555556 G 0.833333333 rs212388 3.191301966 G 0.302083333 rs1 1739663 3.191227916 G 0.270833333 rs3742130 3 G 0.8125 rs1 1672983 2.992922607 G 0.645833333 rs1801274 2.935766197 G 0.59375 rs559928 2.679566565 G 0.875 rs1042058 2.67880675 G 0.604166667 rs9847710 2.656301029 G 0.427083333 rs4802307 2.488650666 C 0.59375 rs4836519 2.3749731 G 0.229166667 rs194749 2.307390547 G 0.229166667 rs4243971 2.281284057 C 0.666666667 rs10781499 2.256027345 G 0.510416667 rs26528 1 .989154969 G 0.510416667 rs864745 1 .90038981 1 G 0.614583333 rs516246 1 .873128962 G 0.614583333 rs2472649 1 .867362024 G 0.864583333 rs12654812 1 .850316563 G 0.71875 rs3764147 1 .846153846 G 0.21875 rs2155219 1 .788828406 C 0.479166667 rs12199775 1 .6875 G 0.0625 rs4728142 1 .628424226 G 0.614583333 rs1 182188 1 .6272403 G 0.385416667 rs171 19 1 .598419229 G 0.15625 rs2189234 1 .594050132 C 0.53125 rs483905 1 .580715707 G 0.645833333 rs925255 1 .489864535 G 0.572916667 rs7702331 1 .475664741 G 0.427083333 rs564349 1 .444444444 G 0.333333333 rs35320439 1 .426791 163 G 0.302083333 rs10865331 1 .374262891 G 0.635416667 rs1016883 1 .333333333 G 0.864583333 rs7495132 1 .333333333 G 0.916666667 rs1292053 1 .294723494 G 0.489583333 rs1260326 1 .273385051 G 0.489583333 rs724016 1 .25 G 0.427083333 rs9264942 1 .228491033 G 0.447916667 rs1 1742570 1 .205765561 G 0.614583333 rs3024505 1 .2 G 0.864583333 rs1 1083840 1 .19616662 C 0.46875 rs686341 1 1 .177923008 T 0.670212766 rs1 1150589 1 .157962356 G 0.583333333 rs2188962 1 .152803395 G 0.489583333 rs38904 1 .149706388 G 0.395833333 rs2231884 1 .142857143 G 0.84375 rs568617 1 .142857143 G 0.822916667 rs566416 1 .090909091 C 0.260416667 rs941823 1 .085709812 G 0.8125 rs2930047 1 .078849391 G 0.416666667 rs1748195 1 .062701801 G 0.322916667 rs2227564 1 .047793286 G 0.739583333 rs9491697 1 G 0.395833333 rs7240004 0.976371643 G 0.395833333 rs3766606 0.955484658 C 0.875 rs2227551 0.954386722 C 0.28125 rs1 1054935 0.951451784 G 0.21875 rs7758080 0.946314303 G 0.25 rs477515 0.943664067 G 0.760416667 rs1847472 0.930768562 C 0.708333333 rs10896794 0.921056427 G 0.270833333 rs6426833 0.905495049 G 0.489583333 rs1893217 0.898335775 G 0.145833333 rs4409764 0.88785958 C 0.375 rs13277237 0.860720579 G 0.520833333 rs6017342 0.824817035 C 0.5625 rs1517352 0.823155868 C 0.583333333 rs1 1583043 0.811912223 G 0.71875 rs4692386 0.793716938 G 0.583333333 rs2823286 0.787256899 G 0.760416667 rs211 1485 0.766410064 G 0.489583333 rs395157 0.759855802 G 0.427083333 rs17780256 0.756557265 C 0.1875 rs7210086 0.756557265 C 0.1875 rs921720 0.751440036 G 0.604166667 rs616597 0.740740741 C 0.770833333 rs10521318 0.713348967 G 0.947916667 rs9319943 0.710706963 G 0.15625 rs7282490 0.704565134 G 0.416666667 rs1569723 0.688930197 C 0.404255319 rs4976646 0.683412252 G 0.260416667 rs9297145 0.674858124 C 0.260416667 rs6074022 0.665512098 G 0.375 rs7097656 0.663968714 G 0.8125 rs1077773 0.655248509 G 0.458333333 rs1 1168249 0.632624826 G 0.40625 rs10758669 0.624387584 C 0.364583333 rs1728918 0.6075707 G 0.666666667 rs2651244 0.5856617 G 0.666666667 rs12778642 0.576750853 C 0.541666667
rs17229285 0.559025112 G 0.458333333
rs2836878 0.556147005 G 0.71875
rs6667605 0.502816839 G 0.458333333
rs1654644 0.484848485 C 0.322916667
rs10065637 0.476190476 G 0.833333333
rs2641348 0.4375 G 0.0625
rs2816958 0.428571429 G 0.854166667
rs7134599 0.407260929 G 0.666666667
rs6651252 0.396529179 G 0.145833333
rs917997 0.378766997 G 0.729166667
rs6088765 0.376373488 C 0.458333333
rs2024092 0.375087927 G 0.75
rs3197999 0.335887072 G 0.677083333
rs7236492 0.324639362 G 0.90625
rs17085007 0.238095238 G 0.21875
rs6920220 0.205188582 G 0.739583333
rs9286879 0.050420168 G 0.239583333
[00201] Tables 36, 37 and 38 reflect SNP associations, respectively, with Mayo score drop in the UC population, CDAI score drop in the CD population, and Histological drop in the UC population. These three categories of analysis (Mayo score drop in the UC population, CDAI score drop in the CD population, and Histological drop in the UC population) are continuous values, and therefore are reflected in epsilon squared values to reflect the "effect size". These epsilon squared values range from 0 to 1 for each SNP, with 1 being a large effect size. They are not linked to a specific allele (i.e. G, C, T or A), but to the effect size of a SNP overall. Table 36: IBD SNPs CDAI drop after 8 weeks of SSI
(QBECO) treatment in CD patients
rsID epsilon_squared rs4836519 0.447142857 rs1734907 0.428571429 rs4802307 0.418231293 rs7517810 0.396825397 rs9286879 0.396825397 rs7134599 0.368630952 rs35320439 0.3375 rs171 19 0.321071429 rs559928 0.317694805 rs477515 0.313214286 rs1 182188 0.31
rs3742130 0.277232143 rs10896794 0.274285714 rs2227551 0.255089286 rs2227564 0.255089286 rs2930047 0.253988095 rs686341 1 0.247278912 rs7240004 0.244897959 rs2823286 0.238636364 rs10865331 0.233333333 rs724016 0.232857143 rs2188962 0.225 rs1 1583043 0.218962585 rs2651244 0.215242347 rs6920220 0.21
rs4409764 0.203877551 rs1 1054935 0.196825397 rs212388 0.196309524 rs3024505 0.194444444 rs516246 0.175892857 rs7702331 0.175324675 rs13204048 0.169642857 rs1569723 0.166815476 rs6074022 0.166815476 rs6651252 0.165535714 rs4243971 0.164732143 rs925255 0.159970238 rs921720 0.158660714 rs6017342 0.154897959 rs2024092 0.1541 12554 rs941823 0.153061224 rs9319943 0.148917749 rs2413583 0.147321429 rs7236492 0.147252747 rs211 1485 0.146825397 rs10758669 0.143928571 rs7282490 0.138571429 rs1260326 0.135034014 rs1042058 0.1 10544218 rs26528 0.10952381 rs4692386 0.108163265 rs13277237 0.099206349 rs4976646 0.099206349 rs17391694 0.09862013 rs10521318 0.095663265 rs1 1742570 0.089540816 rs1517352 0.083333333 rs3197999 0.082589286 rs2231884 0.080357143 rs568617 0.080357143 rs6667605 0.080357143 rs1 1739663 0.08015873 rs1 1150589 0.078571429 rs7097656 0.077487245 rs7758080 0.077487245 rs1728918 0.07577381 rs2641348 0.068571429 rs9847710 0.068367347 rs3764147 0.059659091 rs1893217 0.0581 16883 rs9491697 0.0581 16883 rs1016883 0.053571429 rs1292053 0.052517007 rs395157 0.050595238 rs10781499 0.047261905 rs1 1209026 0.043831 169 rs1847472 0.040816327 rs2472649 0.038690476 rs1801274 0.037202381 rs10065637 0.035714286 rs194749 0.035714286 rs3766606 0.034821429 rs9297145 0.034438776 rs17229285 0.032397959 rs4728142 0.024489796 rs1 1083840 0.024064626 rs12654812 0.023915816 rs2189234 0.023313492 rs38904 0.023051948 rs6426833 0.02244898 rs483905 0.01875 rs7495132 0.015306122 rs17085007 0.015306122 rs17780256 0.013392857 rs7210086 0.013392857 rs1 1168249 0.011020408 rs6088765 0.010595238 rs1077773 0.009642857 rs1748195 0.008928571 rs616597 0.008928571 rs864745 0.008928571 rs1 1672983 0.008609694 rs9264942 0.005952381 rs2816958 0.00487013 rs12778642 0.004345238 rs564349 0.004285714 rs1654644 0.003968254 rs2155219 0.002380952 rs6856616 0.00206044 rs917997 0.001071429 rs2836878 0.000956633 rs566416 0.000956633 rs12199775 0 Table 37: IBD SNPs
Histological score at last recorded response after SSI
(QBECO) treatment in UC patients
rsID epsilon_squared rs10521318 NA
rs17391694 NA
rs2641348 NA
rs559928 NA
rs7240004 NA
rs9847710 0.648076923 rs17085007 0.646367521 rs6017342 0.646367521 rs2188962 0.61965812 rs1801274 0.5531 13553 rs724016 0.547008547 rs10758669 0.519230769 rs1292053 0.512820513 rs4836519 0.465384615 rs2024092 0.462820513 rs564349 0.455357143 rs1 1672983 0.452724359 rs1654644 0.452724359 rs4409764 0.41025641 rs2823286 0.394230769 rs4802307 0.38034188 rs921720 0.370726496 rs6920220 0.367628205 rs17780256 0.359134615 rs7210086 0.359134615 rs1893217 0.358287546 rs211 1485 0.352564103 rs13277237 0.346153846 rs194749 0.346153846 rs2189234 0.346153846 rs7702331 0.346153846 rs9264942 0.346153846 rs3197999 0.33349359 rs2930047 0.329166667 rs17229285 0.296703297 rs395157 0.292200855 rs2836878 0.289903846 rs2472649 0.288461538 rs7495132 0.288461538 rs6088765 0.282451923 rs686341 1 0.268055556 rs1077773 0.264957265 rs1042058 0.262820513 rs864745 0.262820513 rs2651244 0.257936508 rs171 19 0.243990385 rs7097656 0.242387821 rs1 1742570 0.234432234 rs212388 0.230929487 rs1 182188 0.228098291 rs568617 0.228098291 rs9319943 0.216666667 rs12778642 0.200320513 rs941823 0.200320513 rs13204048 0.192307692 rs1748195 0.192307692 rs7282490 0.173076923 rs10896794 0.166826923 rs7758080 0.166826923 rs1569723 0.16159188 rs6074022 0.16159188 rs9491697 0.15 rs1 1083840 0.140384615 rs10065637 0.133547009 rs1 1168249 0.125 rs2227551 0.1 10576923 rs516246 0.109615385 rs9297145 0.103846154 rs1517352 0.101388889 rs3764147 0.098214286 rs2231884 0.097680098 rs35320439 0.097680098 rs477515 0.097680098 rs10781499 0.090659341 rs1728918 0.090659341 rs4692386 0.089903846 rs10865331 0.083760684 rs4243971 0.082051282 rs1 1739663 0.074786325 rs3024505 0.074786325 rs7134599 0.072222222 rs925255 0.072222222 rs2155219 0.0721 15385 rs2413583 0.0721 15385 rs6856616 0.0721 15385 rs4728142 0.06971 1538 rs6667605 0.065438034 rs1260326 0.063461538 rs1847472 0.051442308 rs483905 0.051442308 rs6426833 0.048076923 rs4976646 0.040064103 rs12654812 0.040064103 rs38904 0.038461538 rs26528 0.037774725 rs1 1150589 0.034615385 rs1016883 0.033653846 rs1 1583043 0.033386752 rs3742130 0.033386752 rs2227564 0.033386752 rs3766606 0.03221 1538 rs1 1209026 0.032051282 rs616597 0.032051282 rs6651252 0.032051282 rs7236492 0.032051282 rs12199775 0.032051282 rs1 1054935 0.024420024 rs1734907 0.006105006 rs7517810 0.006105006 rs9286879 0.006105006 rs566416 0.00133547 rs2816958 0
rs917997 0 Table 38: IBD SNPs Mayo score drop after 16 weeks of SSI (QBECO) treatment in UC patients rsID epsilon_squared rs10521318 NA
rs17391694 NA
rs2472649 NA
rs2641348 NA
rs559928 NA
rs7240004 NA
rs2188962 0.827586207 rs17085007 0.698275862 rs10865331 0.667241379 rs1517352 0.629885057 rs2024092 0.628448276 rs6017342 0.628448276 rs2930047 0.55316092 rs941823 0.543103448 rs1 1672983 0.525862069 rs1654644 0.525862069 rs864745 0.491954023 rs10758669 0.465517241 rs12778642 0.458333333 rs724016 0.434482759 rs171 19 0.433497537 rs1801274 0.433497537 rs1292053 0.433497537 rs4976646 0.423850575 rs12654812 0.423850575 rs1 1083840 0.422413793 rs1 1739663 0.422413793 rs212388 0.387931034 rs10065637 0.380172414 rs3742130 0.380172414 rs1 1742570 0.356321839 rs395157 0.356321839 rs3197999 0.344827586 rs568617 0.331034483 rs9847710 0.331034483 rs1 182188 0.32183908 rs1893217 0.32183908 rs4802307 0.32183908 rs6088765 0.315270936 rs3024505 0.310344828 rs4836519 0.310344828 rs686341 1 0.301724138 rs921720 0.296551724 rs1077773 0.281034483 rs9319943 0.279310345 rs211 1485 0.218390805 rs7517810 0.215517241 rs9286879 0.215517241 rs10896794 0.193965517 rs17780256 0.193965517 rs3766606 0.193965517 rs7210086 0.193965517 rs9297145 0.193965517 rs2155219 0.192528736 rs1042058 0.183908046 rs13277237 0.183908046 rs194749 0.183908046 rs2823286 0.182471264 rs17229285 0.177339901 rs6651252 0.177339901 rs7097656 0.177339901 rs516246 0.174712644 rs1 1209026 0.174568966 rs2413583 0.174568966 rs7236492 0.174568966 rs12199775 0.174568966 rs7758080 0.155172414 rs13204048 0.149425287 rs1728918 0.149425287 rs4409764 0.147126437 rs2651244 0.137931034 rs35320439 0.137931034 rs925255 0.1 15517241 rs38904 0.105747126 rs1016883 0.103448276 rs1260326 0.086206897 rs2189234 0.082758621 rs2227551 0.082758621 rs7134599 0.082758621 rs7702331 0.082758621 rs4728142 0.079022989 rs1847472 0.077586207 rs4692386 0.077586207 rs616597 0.077586207 rs6426833 0.077586207 rs6667605 0.077586207 rs917997 0.077586207 rs9491697 0.057471264 rs6920220 0.048275862 rs1748195 0.04597701 1 rs26528 0.04597701 1 rs3764147 0.04597701 1 rs1 1054935 0.044334975 rs1734907 0.044334975 rs564349 0.044334975 rs7282490 0.040229885 rs483905 0.036781609 rs1 1150589 0.034482759 rs477515 0.034482759 rs566416 0.034482759 rs1 1583043 0.031034483 rs1569723 0.031034483 rs2836878 0.031034483 rs6074022 0.031034483 rs1 1168249 0.012931034 rs2231884 0.00862069 rs4243971 0.007758621 rs10781499 0
rs2816958 0
rs6856616 0
rs7495132 0
rs9264942 0
rs2227564 0 [00202] Tables 39-44 are set out below, reflecting a P value summary: Tables 39-41 reflect the P value of the SNP associations of the three analysis reflected in Tables 33, 34 and 35; and, Tables 42-44 reflect P values for Mayo score drop in the UC population, CDAI score drop in the CD population, and Histological drop in the UC population (the analysis reflected in Tables 36, 37 and 38.
Table 39: IBD SNPS associated Table 40: IBD SNPS associated with a with a Mayo score drop with 16 CDAI score drop with 8 weeks of weeks SSI (QBECO) treatment in QBECO treatment in CD
UC patients
rsID pvalue FDRpvalue rsID pvalue FDRpvalue rs17085007 0.018102 0.640126 rs 1734907 0.014306 0.6939 rs2024092 0.024947 0.640126 rs7517810 0.018422 0.6939 rs6017342 0.024947 0.640126 rs9286879 0.018422 0.6939 rs2188962 0.036504 0.640126 rs4836519 0.043718 0.750178 rs941823 0.037122 0.640126 rs 10896794 0.050044 0.750178 rs1 1739663 0.06602 0.640126 rs4802307 0.053524 0.750178 rs10865331 0.069324 0.640126 rs2823286 0.067577 0.750178 rs1517352 0.080497 0.640126 rs7134599 0.075742 0.750178 rs10065637 0.081 167 0.640126 rs2651244 0.082579 0.750178 rs3742130 0.081 167 0.640126 rs6920220 0.08641 1 0.750178 rs2930047 0.10941 1 0.640126 rs35320439 0.094184 0.750178 rs3024505 0.1 151 0.640126 rs3024505 0.09896 0.750178 rs1 1672983 0.122035 0.640126 rs171 19 0.105663 0.750178 rs1654644 0.122035 0.640126 rs559928 0.10819 0.750178 rs9319943 0.134962 0.640126 rs477515 0.1 1 1637 0.750178 rs864745 0.139762 0.640126 rs1 182188 0.1 14178 0.750178 rs10758669 0.155351 0.640126 rs7702331 0.1 17185 0.750178 rs12778642 0.15988 0.640126 rs3742130 0.143614 0.750178 rs724016 0.175884 0.640126 rs2413583 0.150962 0.750178 rs171 19 0.176578 0.640126 rs2227551 0.167692 0.750178 rs1801274 0.176578 0.640126 rs2227564 0.167692 0.750178 rs1292053 0.176578 0.640126 rs2930047 0.16899 0.750178 rs4976646 0.183525 0.640126 rs686341 1 0.1771 16 0.750178 rs12654812 0.183525 0.640126 rs7240004 0.180092 0.750178 rs1 1083840 0.184583 0.640126 rs 10865331 0.195278 0.750178 rs7517810 0.189161 0.640126 rs724016 0.19593 0.750178 rs9286879 0.189161 0.640126 rs2188962 0.207008 0.750178 rs212388 0.21 1882 0.640126 rs1 1583043 0.215944 0.750178 rs17780256 0.212881 0.640126 rs4976646 0.238593 0.750178 rs7210086 0.212881 0.640126 rs17391694 0.239985 0.750178 rs9297145 0.212881 0.640126 rs4409764 0.239994 0.750178 rs6651252 0.233614 0.640126 rs10521318 0.24716 0.750178 rs7097656 0.233614 0.640126 rs1 1054935 0.252138 0.750178 rs1 1209026 0.237302 0.640126 rs212388 0.25305 0.750178 rs2413583 0.237302 0.640126 rs2231884 0.288844 0.750178 rs7236492 0.237302 0.640126 rs568617 0.288844 0.750178 rs12199775 0.237302 0.640126 rs516246 0.291927 0.750178 rs1 1742570 0.240439 0.640126 rs7097656 0.297621 0.750178 rs395157 0.240439 0.640126 rs7758080 0.297621 0.750178 rs3197999 0.251752 0.640126 rs 13204048 0.304983 0.750178 rs568617 0.266032 0.640126 rs 1569723 0.31 1079 0.750178 rs9847710 0.266032 0.640126 rs6074022 0.31 1079 0.750178 rs1 182188 0.275999 0.640126 rs6651252 0.313878 0.750178 rs1893217 0.275999 0.640126 rs4243971 0.315649 0.750178 rs4802307 0.275999 0.640126 rs925255 0.326348 0.750178 rs6088765 0.283347 0.640126 rs2641348 0.327187 0.750178 rs4836519 0.288985 0.640126 rs921720 0.329353 0.750178 rs2651244 0.29351 1 0.640126 rs6017342 0.338143 0.750178 rs35320439 0.29351 1 0.640126 rs2024092 0.340007 0.750178 rs686341 1 0.299124 0.640126 rs941823 0.342519 0.750178 rs921720 0.305377 0.640694 rs9319943 0.352599 0.750178 rs1077773 0.324932 0.66861 1 rs7236492 0.356732 0.750178 rs211 1485 0.417461 0.805784 rs21 1 1485 0.357801 0.750178 rs616597 0.430791 0.805784 rs3764147 0.360765 0.750178 rs6426833 0.430791 0.805784 rs 10758669 0.365131 0.750178 rs6667605 0.430791 0.805784 rs7282490 0.379083 0.757075 rs917997 0.430791 0.805784 rs1016883 0.386476 0.757075 rs10896794 0.460307 0.805784 rs 1260326 0.388587 0.757075 rs3766606 0.460307 0.805784 rs1 1209026 0.433422 0.8301 13 rs2155219 0.46296 0.805784 rs 1042058 0.461253 0.854799 rs1042058 0.479202 0.805784 rs26528 0.464559 0.854799 rs13277237 0.479202 0.805784 rs4692386 0.469005 0.854799 rs194749 0.479202 0.805784 rs 10065637 0.4795 0.860056 rs2823286 0.481964 0.805784 rs9297145 0.487453 0.86066 rs17229285 0.491959 0.805996 rs13277237 0.499352 0.868104 rs516246 0.497156 0.805996 rs1 1742570 0.534306 0.905491 rs7758080 0.537574 0.813722 rs1517352 0.558035 0.905491 rs13204048 0.550075 0.813722 rs3197999 0.560949 0.905491 rs1728918 0.550075 0.813722 rs12654812 0.562834 0.905491 rs1 1054935 0.551476 0.813722 rs6667605 0.569783 0.905491 rs1734907 0.551476 0.813722 rs1 1739663 0.570575 0.905491 rs564349 0.551476 0.813722 rs1 1 150589 0.57695 0.905491 rs4409764 0.555156 0.813722 rs1728918 0.58836 0.910749 rs477515 0.599426 0.837433 rs9847710 0.619668 0.940393 rs566416 0.599426 0.837433 rs7495132 0.643429 0.940393 rs1 1583043 0.618292 0.837433 rs17085007 0.643429 0.940393 rs1569723 0.618292 0.837433 rs17780256 0.665006 0.940393 rs2836878 0.618292 0.837433 rs7210086 0.665006 0.940393 rs6074022 0.618292 0.837433 rs1893217 0.665765 0.940393 rs925255 0.629979 0.842597 rs9491697 0.665765 0.940393 rs38904 0.655086 0.862704 rs1292053 0.692381 0.957155 rs1016883 0.661 138 0.862704 rs395157 0.701758 0.957155 rs1260326 0.708342 0.871686 rs10781499 0.718325 0.957155 rs2189234 0.71818 0.871686 rs616597 0.723674 0.957155 rs2227551 0.71818 0.871686 rs864745 0.723674 0.957155 rs7134599 0.71818 0.871686 rs1 1672983 0.728454 0.957155 rs7702331 0.71818 0.871686 rs 1847472 0.751477 0.965678 rs4728142 0.728992 0.871686 rs2472649 0.762744 0.965678 rs1847472 0.733194 0.871686 rs1801274 0.77073 0.965678 rs4692386 0.733194 0.871686 rs 194749 0.778801 0.965678 rs2231884 0.792849 0.917787 rs3766606 0.783684 0.965678 rs9491697 0.794625 0.917787 rs2816958 0.794003 0.965678 rs4243971 0.803255 0.917787 rs17229285 0.797092 0.965678 rs6920220 0.824397 0.917787 rs564349 0.806496 0.965678 rs1748195 0.832012 0.917787 rs 1654644 0.813664 0.965678 rs26528 0.832012 0.917787 rs4728142 0.84246 0.965678 rs3764147 0.832012 0.917787 rs1 1083840 0.844971 0.965678 rs7282490 0.851361 0.929547 rs2189234 0.849426 0.965678 rs483905 0.863185 0.93214 rs38904 0.850983 0.965678 rs1 1150589 0.871 159 0.93214 rs6426833 0.854582 0.965678 rs1 1168249 0.949591 1 rs6856616 0.865135 0.967923 rs10781499 1 1 rs483905 0.876998 0.971577 rs2816958 1 1 rs917997 0.902523 0.973887 rs6856616 1 1 rs2836878 0.907869 0.973887 rs7495132 1 1 rs566416 0.907869 0.973887 rs9264942 1 1 rs1 1 168249 0.925758 0.973887 rs2227564 1 1 rs6088765 0.928517 0.973887 rs10521318 NA NA rs 1077773 0.934728 0.973887 rs17391694 NA NA rs1748195 0.939413 0.973887 rs2472649 NA NA rs9264942 0.959189 0.985349 rs2641348 NA NA rs12778642 0.970041 0.987519 rs559928 NA NA rs2155219 0.983471 0.992252 rs7240004 NA NA rs12199775 1 1
Table 41 : IBD SNPS associated Table 42: IBD SNPS associated with with a decrease in histological score a response to SSI (QBECO) at the to SSI (QBECO) at the last recorded last recorded timepoint in CD patients timepoint
rsID pvalue FDRpvalue rsID pvalue FDRpvalue rs17085007 0.015869 0.665032 rs9286879 0.00332 0.187606 rs6017342 0.015869 0.665032 rs7517810 0.00332 0.187606 rs2024092 0.041258 0.665032 rs17391694 0.040563 0.885879 rs9847710 0.054131 0.665032 rs 13204048 0.073646 0.885879 rs2188962 0.061516 0.665032 rs6088765 0.077979 0.885879 rs1801274 0.082992 0.665032 rs2413583 0.079044 0.885879 rs724016 0.085304 0.665032 rs1801274 0.082457 0.885879 rs10758669 0.096662 0.665032 rs17085007 0.083625 0.885879 rs1292053 0.099491 0.665032 rs212388 0.097082 0.885879 rs2472649 0.107123 0.665032 rs1 1739663 0.1 1 1048 0.885879 rs7495132 0.107123 0.665032 rs 1042058 0.1 15727 0.885879 rs4836519 0.123165 0.665032 rs4802307 0.13173 0.885879 rs2651244 0.127603 0.665032 rs9847710 0.134241 0.885879 rs564349 0.12885 0.665032 rs3742130 0.134939 0.885879 rs1 1672983 0.130386 0.665032 rs3197999 0.141844 0.885879 rs1654644 0.130386 0.665032 rs7134599 0.144341 0.885879 rs7097656 0.139679 0.665032 rs1 1672983 0.148994 0.885879 rs4409764 0.157843 0.665032 rs6920220 0.155264 0.885879 rs9319943 0.162587 0.665032 rs 1734907 0.159342 0.885879 rs2823286 0.169646 0.665032 rs559928 0.160592 0.885879 rs941823 0.179364 0.665032 rs4243971 0.176306 0.885879 rs4802307 0.180588 0.665032 rs10781499 0.184193 0.885879 rs921720 0.188573 0.665032 rs2024092 0.186009 0.885879 rs6920220 0.191221 0.665032 rs6667605 0.189722 0.885879 rs17780256 0.198671 0.665032 rs917997 0.200242 0.885879 rs7210086 0.198671 0.665032 rs 1 1209026 0.203831 0.885879 rs1893217 0.19943 0.665032 rs 194749 0.22378 0.936561 rs211 1485 0.204633 0.665032 rs6651252 0.240484 0.965674 rs13277237 0.210622 0.665032 rs516246 0.265362 0.965674 rs194749 0.210622 0.665032 rs12654812 0.300291 0.965674 rs2189234 0.210622 0.665032 rs7236492 0.300358 0.965674 rs7702331 0.210622 0.665032 rs864745 0.30594 0.965674 rs9264942 0.210622 0.665032 rs4836519 0.31031 1 0.965674 rs3197999 0.222969 0.665032 rs4728142 0.320293 0.965674 rs2930047 0.227353 0.665032 rs2472649 0.326866 0.965674 rs1569723 0.227835 0.665032 rs2651244 0.327197 0.965674 rs6074022 0.227835 0.665032 rs17229285 0.339862 0.965674 rs17229285 0.2631 15 0.70353 rs2155219 0.339862 0.965674 rs395157 0.2685 0.70353 rs26528 0.353137 0.965674 rs2836878 0.27129 0.70353 rs2816958 0.358419 0.965674 rs10065637 0.272938 0.70353 rs 1654644 0.36272 0.965674 rs6088765 0.280541 0.70353 rs 12778642 0.365771 0.965674 rs686341 1 0.299318 0.70353 rs 10065637 0.370819 0.965674 rs1077773 0.30352 0.70353 rs2641348 0.376015 0.965674 rs1042058 0.306453 0.70353 rs1728918 0.397277 0.966101 rs864745 0.306453 0.70353 rs 1077773 0.404018 0.966101 rs171 19 0.333552 0.70353 rs 10758669 0.419283 0.966101 rs9297145 0.333666 0.70353 rs2836878 0.421062 0.966101 rs1 1742570 0.348212 0.70353 rs1 1 168249 0.432777 0.966101 rs2231884 0.348443 0.70353 rs483905 0.432777 0.966101 rs35320439 0.348443 0.70353 rs2189234 0.436028 0.966101 rs477515 0.348443 0.70353 rs6074022 0.450515 0.97029 rs212388 0.353744 0.70353 rs7282490 0.489627 0.97029 rs1 182188 0.358279 0.70353 rs 1569723 0.495864 0.97029 rs568617 0.358279 0.70353 rs925255 0.509166 0.97029 rs4243971 0.390154 0.733163 rs4976646 0.510103 0.97029 rs12778642 0.405984 0.733163 rs3764147 0.510634 0.97029 rs1 1739663 0.41 1981 0.733163 rs1 182188 0.512464 0.97029 rs3024505 0.41 1981 0.733163 rs7702331 0.552396 0.97029 rs2413583 0.420457 0.733163 rs7097656 0.568644 0.97029 rs13204048 0.42089 0.733163 rs9297145 0.568719 0.97029 rs1748195 0.42089 0.733163 rs171 19 0.571843 0.97029 rs6667605 0.442828 0.759134 rs35320439 0.58669 0.97029 rs7282490 0.458935 0.772406 rs395157 0.595429 0.97029 rs10896794 0.472026 0.772406 rs921720 0.618005 0.97029 rs7758080 0.472026 0.772406 rs 10865331 0.633601 0.97029 rs9491697 0.509156 0.81 1065 rs4692386 0.639269 0.97029 rs6426833 0.510671 0.81 1065 rs9319943 0.650755 0.97029 rs1 1083840 0.531671 0.818596 rs564349 0.650873 0.97029 rs1 1168249 0.569783 0.818596 rs724016 0.651874 0.97029 rs1 1583043 0.583581 0.818596 rs12199775 0.669929 0.97029 rs3742130 0.583581 0.818596 rs1292053 0.674363 0.97029 rs2227564 0.583581 0.818596 rs21 1 1485 0.678043 0.97029 rs1 1209026 0.591208 0.818596 rs 17780256 0.692467 0.97029 rs616597 0.591208 0.818596 rs7210086 0.692467 0.97029 rs6651252 0.591208 0.818596 rs 1260326 0.700157 0.97029 rs7236492 0.591208 0.818596 rs616597 0.704862 0.97029 rs12199775 0.591208 0.818596 rs1 1742570 0.705638 0.97029 rs2227551 0.60799 0.824346 rs6017342 0.716059 0.97029 rs516246 0.610627 0.824346 rs1517352 0.72203 0.97029 rs1517352 0.633655 0.836379 rs9264942 0.722223 0.97029 rs1 1054935 0.639207 0.836379 rs10521318 0.735245 0.97029 rs3764147 0.642773 0.836379 rs1 1083840 0.740049 0.97029 rs10781499 0.665001 0.837961 rs1 1583043 0.745157 0.97029 rs1728918 0.665001 0.837961 rs2823286 0.747668 0.97029 rs4692386 0.667265 0.837961 rs2188962 0.767362 0.97029 rs10865331 0.685969 0.851548 rs686341 1 0.774614 0.97029 rs7134599 0.722527 0.857821 rs7495132 0.77585 0.97029 rs925255 0.722527 0.857821 rs1016883 0.77585 0.97029 rs2155219 0.722875 0.857821 rs1 1 150589 0.813698 0.97029 rs6856616 0.722875 0.857821 rs4409764 0.81681 0.97029 rs4728142 0.730737 0.857821 rs3024505 0.832995 0.97029 rs1260326 0.751581 0.872803 rs38904 0.835414 0.97029 rs1847472 0.79335 0.88978 rs13277237 0.838896 0.97029 rs483905 0.79335 0.88978 rs6426833 0.860846 0.97029 rs1734907 0.814672 0.88978 rs2231884 0.86773 0.97029 rs7517810 0.814672 0.88978 rs568617 0.86773 0.97029 rs9286879 0.814672 0.88978 rs 1893217 0.871206 0.97029 rs4976646 0.835029 0.88978 rs2930047 0.885155 0.97029 rs12654812 0.835029 0.88978 rs1748195 0.8971 14 0.97029 rs38904 0.841073 0.88978 rs 1847472 0.903288 0.97029 rs26528 0.843676 0.88978 rs941823 0.90504 0.97029 rs1 1150589 0.855757 0.88978 rs 10896794 0.90504 0.97029 rs1016883 0.859467 0.88978 rs566416 0.912885 0.97029 rs3766606 0.865064 0.88978 rs477515 0.920609 0.97029 rs566416 0.912701 0.929922 rs1 1054935 0.925984 0.97029 rs2816958 1 1 rs7758080 0.930394 0.97029 rs917997 1 1 rs2227551 0.935943 0.97029 rs10521318 NA NA rs2227564 0.935943 0.97029 rs17391694 NA NA rs3766606 0.955499 0.981558 rs2641348 NA NA rs7240004 0.967599 0.985033 rs559928 NA NA rs6856616 0.994028 1 rs7240004 NA NA rs9491697 1 1
Table 43: IBD SNPS associated with Table 44: IBD SNPS associated with a response to SSI (QBECO) at the response to QBECO at the last last recorded timepoint in both UC recorded timepoint in UC patients and CD patients
rsID pvalue FDRpvalue rsID pvalue FDRpvalu e rs7517810 0.018325 0.817225 rs7702331 0.097349 1 rs9286879 0.018325 0.817225 rs395157 0.124909 1 rs2413583 0.024164 0.817225 rs2189234 0.176843 1 rs10758669 0.040681 0.817225 rs3197999 0.1867 1 rs10865331 0.04345 0.817225 rs925255 0.1867 1 rs2024092 0.044268 0.817225 rs864745 0.1867 1 rs13204048 0.050625 0.817225 rs171 19 0.230159 1 rs1801274 0.075683 0.941 123 rs4836519 0.231919 1 rs1 1739663 0.088943 0.941 123 rs1077773 0.248748 1 rs1734907 0.092344 0.941 123 rs724016 0.273916 1 rs864745 0.1 1 19 0.941 123 rs2823286 0.27729 1 rs3742130 0.1 1576 0.941 123 rs7134599 0.294598 1 rs686341 1 0.125775 0.941 123 rs6920220 0.294598 1 rs17391694 0.126558 0.941 123 rs1 1 168249 0.294598 1 rs7702331 0.141397 0.941 123 rs1 1583043 0.303916 1 rs194749 0.144892 0.941 123 rs4243971 0.303916 1 rs10065637 0.162503 0.941 123 rs 1260326 0.303916 1 rs395157 0.16797 0.941 123 rs2227551 0.303916 1 rs6088765 0.172201 0.941 123 rs4692386 0.303916 1 rs1 1672983 0.17886 0.941 123 rs2651244 0.326959 1 rs10781499 0.179602 0.941 123 rs35320439 0.326959 1 rs559928 0.188506 0.941123 rs2155219 0.327799 1 rs2155219 0.210396 0.941123 rs6088765 0.327799 1 rs11168249 0.225452 0.941123 rs11054935 0.392197 1 rs1042058 0.239263 0.941123 rs 1734907 0.392197 1 rs212388 0.239888 0.941123 rs7236492 0.392197 1 rs6667605 0.246313 0.941123 rs4409764 0.417783 1 rs917997 0.265894 0.941123 rs9847710 0.417783 1 rs2816958 0.270352 0.941123 rs 10896794 0.447427 1 rs17085007 0.273557 0.941123 rs568617 0.44976 1 rs2823286 0.288266 0.941123 rs2930047 0.473875 1 rs4802307 0.289372 0.941123 rs212388 0.496481 1 rs11209026 0.307633 0.941123 rs9319943 0.511715 1 rs2641348 0.307633 0.941123 rs2227564 0.511715 1 rs11742570 0.319756 0.941123 rs2836878 0.511715 1 rs724016 0.352481 0.941123 rs3742130 0.511715 1 rs1728918 0.356508 0.941123 rs 13204048 0.551288 1 rs35320439 0.358195 0.941123 rs9264942 0.551288 1 rs2472649 0.35902 0.941123 rs1292053 0.553972 1 rs3764147 0.362512 0.941123 rs1728918 0.611198 1 rs516246 0.365203 0.941123 rs3766606 0.611198 1 rs6074022 0.37114 0.941123 rs3764147 0.611198 1 rs9847710 0.378984 0.941123 rs1016883 0.611198 1 rs17229285 0.383956 0.941123 rs1893217 0.611198 1 rs11583043 0.390211 0.941123 rs38904 0.630689 1 rs12654812 0.396596 0.941123 rs 12778642 0.678743 1 rs2188962 0.402445 0.941123 rs2231884 0.702246 1 rs1654644 0.413383 0.941123 rs7097656 0.702246 1 rs1569723 0.419077 0.941123 rs6426833 0.702246 1 rs7134599 0.421037 0.941123 rs17780256 0.736436 1 rs6017342 0.42616 0.941123 rs7210086 0.736436 1 rs4692386 0.435533 0.941123 rs477515 0.736436 1 rs4243971 0.454404 0.941123 rs1569723 0.736436 1 rs26528 0.463162 0.941123 rs6074022 0.736436 1 rs921720 0.48871 0.941123 rs2111485 0.736816 1 rs6920220 0.502638 0.941123 rs1801274 0.736816 1 rs4976646 0.5057 0.941123 rs10781499 0.736816 1 rs4728142 0.508738 0.941123 rs7240004 0.736816 1 rs12778642 0.522151 0.941123 rs6667605 0.780048 1 rs7236492 0.546309 0.941123 rs11150589 0.780048 1 rs3024505 0.554443 0.941123 rs516246 0.780048 1 rs2930047 0.555192 0.941123 rs26528 0.780048 1 rs483905 0.557278 0.941123 rs921720 0.795114 1 rs568617 0.563031 0.941123 rs4802307 0.795114 1 rs2111485 0.567283 0.941123 rs7758080 0.79518 1 rs6651252 0.567463 0.941123 rs 1847472 0.79518 1 rs9319943 0.569384 0.941123 rs 1654644 0.79518 1 rs9264942 0.569944 0.941123 rs11672983 0.79518 1 rs7282490 0.592114 0.941123 rs917997 0.795565 1 rs4409764 0.598755 0.941123 rs483905 0.817746 1 rs616597 0.599216 0.941123 rs7282490 0.817746 1 rs10521318 0.625697 0.941123 rs11083840 0.826535 1 rs3766606 0.626648 0.941123 rs4728142 0.826535 1 rs2227551 0.637285 0.941123 rs4976646 0.82661 1 rs11083840 0.637448 0.941123 rs6856616 0.996024 1 rs11054935 0.637708 0.941123 rs2413583 0.996154 1 rs3197999 0.652116 0.941123 rs12199775 0.996154 1 rs12199775 0.672186 0.941123 rs17085007 0.996213 1 rs2651244 0.680175 0.941123 rs6651252 0.996213 1 rs2836878 0.686341 0.941123 rs11739663 0.996213 1 rs38904 0.691141 0.941123 rs941823 0.996213 1 rs2231884 0.691718 0.941 123 rs6017342 0.996444 1 rs564349 0.691718 0.941 123 rs10521318 0.996655 1 rs925255 0.706641 0.941 123 rs7495132 0.996655 1 rs10896794 0.708126 0.941 123 rs2472649 0.996655 1 rs1748195 0.716253 0.941 123 rs559928 0.996655 1 rs1016883 0.741501 0.960292 rs1 1209026 0.996655 1 rs171 19 0.751778 0.960292 rs9297145 0.996802 1 rs1 182188 0.762896 0.960292 rs1 182188 0.996827 1 rs13277237 0.800314 0.960292 rs1 1742570 0.996827 1 rs7097656 0.80846 0.960292 rs2816958 0.996935 1 rs941823 0.80846 0.960292 rs 17391694 0.996935 1 rs1893217 0.809145 0.960292 rs616597 0.996935 1 rs566416 0.818722 0.960292 rs1748195 0.997 1 rs4836519 0.82248 0.960292 rs 194749 0.997 1 rs7758080 0.833766 0.960292 rs3024505 0.997172 1 rs9297145 0.833966 0.960292 rs 10065637 0.997172 1 rs1517352 0.843628 0.960292 rs 10865331 0.997184 1 rs477515 0.855593 0.960292 rs 10758669 0.997216 1 rs6426833 0.868683 0.960292 rs686341 1 0.99726 1 rs2227564 0.875464 0.960292 rs2024092 0.997349 1 rs17780256 0.877273 0.960292 rs2188962 0.999569 1 rs7210086 0.877273 0.960292 rs13277237 1 1 rs9491697 0.901308 0.960292 rs1517352 1 1 rs1 1150589 0.905561 0.960292 rs566416 1 1 rs7240004 0.905561 0.960292 rs 12654812 1 1 rs1077773 0.909303 0.960292 rs564349 1 1 rs7495132 0.926206 0.969086 rs1042058 1 1 rs1847472 0.970233 1 rs17229285 1 1 rs2189234 0.980702 1 rs9491697 1 1 rs6856616 0.994267 1 rs9286879 1 1 rs1292053 1 1 rs7517810 1 1 rs1260326 1 1 rs2641348 NA NA
[00203] As reflected in this Example, there are SNPs that will be predictive of SSI (QBECO) response in CD. Further, there are SNPs that are predictive of SSI (QBECO) response in UC. In addition, there are SNPs that are predictive of SSI (QBECO) response in both CD and UC. In effect, certain SNPs are disease specific for SSI (QBECO) response - and other SNPs are SSI (QBECO) response specific, but are not disease specific. This utility of the alternative SNPs is reflected in uses that are disease specific tests, and alternative uses that are SSI response tests in general.

Claims

A method of predicting the degree to which a mammalian subject has an increased likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists, the method comprising screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the subject has an increased likelihood of benefiting from said treatment if the diagnostic SNP comprises a response allele, and wherein the diagnostic SNP and the corresponding response allele are one or more of:
SNP rsID Response Allele
rs9286879 A
rs7517810 G
rs17391694 G
rs2413583 G
rs6920220 A
rs17085007 A
rs13204048 G
rs1 1209026 G
rs1734907 G
rs212388 G
rs1 1739663 G
rs7236492 A
rs3742130 G
rs1 1672983 G
rs3197999 A
rs1801274 G
rs559928 G
rs1042058 G
rs2024092 A rs6088765 T rs9847710 G rs917997 A rs6651252 A rs4802307 C rs7134599 A rs4836519 G rs2816958 A rs194749 G rs2641348 A rs4243971 C rs10781499 G rs10065637 A rs1654644 T rs26528 G rs6667605 A rs864745 G rs516246 G rs2472649 G rs12654812 G rs3764147 G rs2836878 A rs17229285 A rs2155219 C rs12778642 T rs2651244 A rs12199775 G rs1728918 A rs4728142 G rs1 182188 G rs10758669 T rs171 19 G rs2189234 C rs483905 G rs1 1 168249 A rs1077773 A rs7097656 A rs6074022 A rs925255 G rs9297145 T rs7702331 G rs4976646 A rs1569723 T rs564349 G rs35320439 G rs7282490 A rs9319943 A rs10521318 A rs10865331 G rs616597 T rs7495132 G rs1016883 G rs921720 A rs17780256 T rs7210086 T rs395157 A rs21 1 1485 A rs1292053 G rs 1260326 G rs2823286 A rs4692386 A rs724016 G rs1 1583043 A rs9264942 G rs1517352 T rs6017342 T rs1 1742570 G rs3024505 G rs1 1083840 C rs686341 1 T rs13277237 A rs1 1 150589 G rs2188962 G rs38904 G rs2231884 G rs568617 G rs4409764 T rs1893217 A rs6426833 A rs566416 C rs941823 G rs10896794 A rs2930047 G rs1847472 T rs1748195 G rs477515 A rs7758080 A rs1 1054935 A rs2227564 G rs2227551 T rs3766606 T
rs7240004 A
rs9491697 G
2. The method of claim 1 , wherein the subject is a human patient.
3. The method of claim 2, wherein the patient has a disease characterized by immune dysregulation.
4. The method of claim 2, wherein the patient has an IBD.
5. The method of claim 4, wherein the IBD is Crohn's Disease.
6. The method of claim 4, wherein the IBD in ulcerative colitis.
7. The method of claim 4, wherein the IBD is collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome or indeterminate colitis.
8. The method of any one of claims 1 to 7, wherein the treatment with an antigenic formulation comprising PRR agonists is a site specific immunotherapy (SSI).
9. The method of claim 8, wherein the SSI comprises use of a preparation of a killed or attenuated microbe of one or more pathogenic microbial species; or a cell wall extract, a cell membrane extract, a whole cell extract, or a PRR agonist formulation comprising components of the microbial species; for use in treating an immune dysfunction in a specific organ or tissue in the subject, wherein the preparation, cell wall extract, cell membrane extract, whole cell extract or PRR agonist formulation elicits an immune response in the subject, and wherein the pathogenic microbial species is pathogenic in the corresponding specific organ or tissue in a healthy subject.
10. The method of claim 9, wherein the immune dysfunction is an IBD, or Crohn's diseases or ulcerative colitis, and the pathogenic microbial species is an E. coli. 1 1 . The method of any one of claims 1 to 8, wherein the antigenic formulation comprises an artificial repertoire of mammalian pattern recognition receptor (PRR) agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is
pathogenic in a target tissue, the distinct portion of the PRR agonist signature being different from any native PRR agonist signature of the microbial mammalian pathogen, wherein the artificial repertoire of mammalian PRR agonists are formulated together in a therapeutic vehicle for combined presentation following administration to a
mammalian host, and the composition comprises components of the microbial mammalian pathogen that are agonists for at least 5 distinct mammalian PRRs, and wherein the composition is for use so as to modulate an innate immune response in the target tissue.
12. The method of any one of claims 1 to 1 1 , wherein the genomic polymorphism is within 1 Mbp of the diagnostic SNP, or within 500Kbp, or within 10Okbp.
13. The method of any one of claims 1 to 12, wherein the subject is homozygous for the response allele. 14. The method of any one of claims 1 to 12, wherein the patient is heterozygous for the response allele.
15. The method of any one of claims 1 to 14, wherein the genomic polymorphism is in linkage disequilibrium with the diagnostic SNP, with an r-squared value of greater than 0.8, or greater than 0.9. The use of a genetic marker linked to one or more SNP that is associated with an IBD, for assessing the susceptibility of an IBD patient to a therapy with an immunogenic formulation comprising PRR receptor agonists.
The use according to claim 16, wherein the SNP is one or more of: rs1748195 rs34856868, rs1 1583043, rs6025, rs10798069, rs7555082, rs1 1681525 rs4664304, rs31 16494, rs7556897, rs1 1 1781203, rs35320439, rs1 13010081 rs616597, rs724016, rs2073505, rs4692386, rs6856616, rs2189234 rs395157, rs4703855, rs564349, rs7773324, rs13204048, rs7758080 rs1077773, rs2538470, rs17057051 , rs701 1507, rs3740415, rs7954567 rs653178, rs1 1064881 , rs9525625, rs3853824, rs17736589, rs9319943 rs7236492, rs727563, rs17391694, rs6679677, rs3897478, rs9286879 rs1728918, rs10865331 , rs6716753, rs12994997, rs6837335, rs13126505 rs10065637, rs7702331 , rs17695092, rs12663356, rs9264942, rs9491697 rs13204742, rs212388, rs10486483, rs864745, rs7015630, rs6651252 rs3764147, rs16967103, rs2066847, rs2945412, rs2024092, rs4802307 rs516246, rs2284553, rs10797432, rs6426833, rs2816958, rs1016883 rs17229285, rs9847710, rs3774959, rs1 1739663, rs254560, rs6927022 rs798502, rs4722672, rs4380874, rs4728142, rs483905, rs561722 rs28374715, rs1 1 150589, rs1728785, rs7210086, rs1 126510, rs6088765 rs6017342, rs12103, rs35675666, rs12568930, rs1 1209026, rs2651244 rs4845604, rs670523, rs4656958, rs1801274, rs2488389, rs755451 1 rs3024505, rs6545800, rs925255, rs10495903, rs7608910, rs6740462 rs917997, rs21 1 1485, rs1517352, rs2382817, rs3749171 , rs4256159 rs3197999, rs2472649, rs7657746, rs2930047, rs1 1742570, rs1363907 rs4836519, rs2188962, rs686341 1 , rs1 1741861 , rs6871626, rs12654812 rs171 19, rs9358372, rs1847472, rs6568421 , rs3851228, rs6920220 rs12199775, rs1819333, rs1456896, rs9297145, rs1734907, rs38904 rs921720, rs1991866, rs10758669, rs4743820, rs4246905, rs10781499 rs12722515, rs1042058, rs1 1010067, rs2790216, rs10761659, rs2227564 rs1250546, rs6586030, rs791 1264, rs4409764, rs90761 1 , rs10896794 rs1 1230563, rs4246215, rs559928, rs2231884, rs2155219, rs6592362 rs630923, rs1 1612508, rs1 1564258, rs1 1 168249, rs7134599, rs17085007, rs941823, rs9557195, rs194749, rs4899554, rs8005161 , rs17293632, rs7495132, rs529866, rs7404095, rs26528, rs10521318, rs3091316, rs12946510, rs12942547, rs1292053, rs1893217, rs7240004, rs727088, rs1 1879191 , rs17694108, rs1 1672983, rs6142618, rs491 1259, rs1569723, rs913678, rs259964, rs6062504, rs2823286, rs2836878, rs7282490, rs2266959, rs2412970, rs2413583, rs2641348, rs7517810, rs1260326, rs7438704, rs10061469, rs2503322, rs5743289, rs6667605, rs1440088, rs3774937, rs477515, rs1 182188, rs17780256, rs1 1083840, rs3766606, rs13407913, rs6708413, rs2457996, rs10051722, rs4976646, rs7746082, rs3891 1 , rs13277237, rs2227551 , rs7097656, rs12778642, rs1 1229555, rs174537, rs568617, rs2226628, rs566416, rs1 1054935, rs3742130, rs1569328, rs2361755, rs3091315, rs1654644, rs4243971 , rs6087990, rs6074022, rs5763767.
A method of treating a subject having an immune dysfunction, comprising carrying out, or requesting the results of, the method of any one of claims 1 to 15, and administering an effective amount of the antigenic formulation if the subject has an increased likelihood of benefiting from said treatment
The method of claim 18, wherein the patient is an anti-TNFa naive patient.
A method of diagnostic or insurance coverage screening, comprising requesting the results of a diagnostic assay that comprises the method of any one of claims 1 to 15.
Use of the antigenic formulation comprising PRR agonists for treatment of the subject, wherein prior to treatment the subject is found to have an increased likelihood of benefiting from said treatment by the method of any one of claims 1 to 17.
A method of identifying a subject for treatment, or excluding a subject from treatment, with an antigenic formulation comprising PRR agonists, comprising determining if the subject has an increased likelihood of benefiting from said treatment by the method of any one of claims 1 to 17.
23. A kit for use in predicting the degree to which a mammalian subject has an increased likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists, the kit comprising an agent for screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the subject has an increased likelihood of benefiting from said treatment if the diagnostic SNP comprises a response allele, and wherein the diagnostic SNP and the corresponding response allele are as set out in claim 41 .
24. The kit of claim 23, wherein the agent for screening is a probe or primer specific for the genomic polymorphism.
25. The method of claim 8, wherein the SSI comprises a formulation of PRR agonists derived from an E. coli.
26. The method of any one of claims 1 to 15, wherein wherein the diagnostic SNP, the corresponding response allele, and the genomic location of the genomic polymorphism are one or more of:
Figure imgf000154_0001
rs6017342 T HNF4A, SERINC3, PKIG, TTPAL,
R3HDM
The method of any one of claims 1 to 15, wherein wherein the subject has Crohn's disease, and the diagnostic SNP, the corresponding response allele, and the genomic location of the genomic polymorphism are one or more of:
Figure imgf000155_0001
The method of any one of claims 1 to 15, wherein wherein the subject has ulcerative colitis, and the diagnostic SNP, the corresponding response allele, and the genomic location of the genomic polymorphism are one or more of:
Figure imgf000155_0002
An in vitro method for determining efficacy of an SSI treatment of a human patient having an inflammatory bowel disease, comprising determining in vitro a level of one or more of IL-18, IP-10, I FNgamma, IL-12P70, IL-17A and TGFalpha, in a sample from the patient undergoing treatment.
30. The method of claim 29, wherein the SSI comprises treatment with a formulation comprising PRR agonists derived from an E. coli.
31 . A method of predicting the degree to which a mammalian subject has an increased likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists, the method comprising screening a sample isolated from said patient for levels of one or more of Eotaxin 1 , GROa, IL-10, PDGF AA and RANTES.
32. The method of claim 31 , wherein the subject has an increased likelihood of benefiting from the treatment if the level of Eotaxin 1 is below a predetermined threshold level.
33. The method of claim 31 or 32, wherein the antigenic formulation comprises an artificial repertoire of mammalian pattern recognition receptor (PRR) agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is pathogenic in the target tissue, the distinct portion of the PRR agonist signature being different from any native PRR agonist signature of the microbial mammalian pathogen, wherein the artificial repertoire of mammalian PRR agonists are formulated together in a therapeutic vehicle for combined presentation following administration to a mammalian host, and the composition comprises components of the microbial mammalian pathogen that are agonists for at least 5 distinct mammalian PRRs, and wherein the composition is for use so as to modulate an innate immune response in the target tissue.
34. The method of claim 33, wherein the therapeutic vehicle comprises a recombinant microbe, a cellular fraction of the recombinant microbe, a cellular fraction of a microbial cell, a microparticle or a liposome, each comprising components of the microbial mammalian pathogen that provide the PRR agonists that together make up the artificial repertoire of PRR agonists.
35. The method of claim 34, wherein the recombinant microbe comprises a recombinant gene encoding a component of at least one of the PRR agonists.
36. The method of claim 34 or 35, wherein the therapeutic vehicle comprises a whole killed or attenuated cell of the recombinant microbe.
37. The method of claim 34, wherein the cellular fraction of the microbial cell comprises an isolated cellular fraction of the microbial mammalian pathogen. 38. The method of claim 37, wherein the isolated cellular fraction is one or more of: a bacterial outer membrane fraction; a bacterial inner membrane fraction; a pellet from a gradient centrifugation of microbial cell components; or chromosomal DNA. 39. The method of any one of claims 33 to 38, wherein the PRRs and the corresponding PRR agonists are selected from the group consisting of:
Figure imgf000157_0001
NOD1 iE-DAP, Acylated iE-DAP, D-gamma- Glu-mDAP, L-Ala-gamma-D-Glu-mDAP
NOD2 MDP (MurNAc-L-Ala-D-isoGIn,
muramyl dipeptide), N-glycolylated muramyldipeptide, N-Acetyl-muramyl- L- Alanyl-D-Glutamin-n-butyl-ester, MurNAc-Ala-D-isoGln-Lys, N- Acetylmuramyl-L-Alanyl-D- Isoglutamine (L-D isoform), 6-0- stearoyl-N-Acetyl-muramyl-L-alanyl-D- isoglutamine, Pam2C-Aca-Benzyl- Murabutide,
TLR2/NOD2 Pam2C-conjugated murabutide
NOD1 /NOD2 PGN, Pam2C-conjugated murabutide
RIG1/MDA5 5' triphosphate double stranded RNA
(18-20mer),
polyriboinosinic:polyribocytidylic acid
DAI, LRRFI P1 , AIM2, dsDNA, poly(dA-dT poly(dT-dA) RIG1
Dectin-1 Beta-glucan peptide, fungal cell wall preparations
Mincle damaged microbial cells, fungus, yeast and mycobacteria, Trehalose-6,6- dibehenate, trehalose-6,6-dimycolate
STING Cyclic dinucleotides (c-di-nucleotides), xanthenone derivatives, 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, 2'2'-cGAMP, c-di-AMP (cyclic di-adenylate monophosphate), c-di-GMP, c-di-IMP, c-di-UMP, c-di-AMP
RIG-I PPP-ssRNA (PPP-ssRNA, ssRNA with a 5'-tri phosphate group), RNA with
base pairing and polyl:C
MDA5 Long dsRNA
LGP2 dsRNA
DDX41 B-form DNA and CDNs (cyclic
dinucleotides)
DHX9 DNA, RNA, CpG-A
oligodeoxynucleotids and CpG-B
ODNs
DDX3 Viral RNA
DHX36 DNA, RNA, CpG-A
oligodeoxynucleotids and CpG-B oligodeoxynucleotids
DDX1 -DDX21 -DDX36 RNA and polyl :C
DDX60 ssRNA, dsRNA and dsDNA
KU70 DNA
cGAS DNA
STING CDNs (c-di-GMP and c-di-AMP)
NOD2 ssRNA
NLRP3 ssRNA, dsRNA, bacterial mRNA and oxidized mitochondrial DNA
AI M2 DNA
IFI 16 dsDNA
LRRFI P1 B-form DNA, Z-form DNA and dsRNA
DAI DNA
IFIT1 .2.3 and 5 PPP-ssRNA
A method for evaluating a patient with an IBD, comprising:
(a) measuring in a biological sample from the patient levels of one or more biological markers selected from the group consisting of Eotaxin 1 , GROa, IL-10, PDGF AA, RANTES, IL-18, IP-10, IFNgamma, IL-12P70, I L- 17A and TGFalpha;
(b) determining a genomic polymorphism status of the patient;
(c) comparing the level of one or more biological markers from step (a) to a threshold level of the biological marker derived from a cohort of marker test individuals;
(d) comparing the genomic polymorphism status of the patient to a reference genomic polymporphism status of a cohort of genomic test individuals; and,
(e) identifying the patient as having or not having a likelihood of benefiting from treatment with an antigenic formulation comprising mammalian pattern recognition receptor (PRR) agonists based on:
the comparison of the measurements in step (a) to the threshold level derived from the cohort of marker test individuals; and,
the comparison of the genomic polymorphism status of the patient to the corresponding reference genomic polymorphism status of the cohort of genomic test individuals.
The method of claim 40, wherein the genomic polymorphism status of the patient comprises the allelic identity of a genetic marker linked to one or more SNP that is positively or negartively associated with a degree risk for suffering from an I BD.
The method of claim 41 , wherein the SNP is one or more of: rs1748195, rs34856868, rs1 1583043, rs6025, rs10798069, rs7555082, rs1 1681525, rs4664304, rs31 16494, rs7556897, rs1 1 1781203, rs35320439, rs1 13010081 , rs616597, rs724016, rs2073505, rs4692386, rs6856616, rs2189234, rs395157, rs4703855, rs564349, rs7773324, rs13204048, rs7758080, rs1077773, rs2538470, rs17057051 , rs701 1507, rs3740415, rs7954567, rs653178, rs1 1064881 , rs9525625, rs3853824, rs17736589, rs9319943, rs7236492, rs727563, rs17391694, rs6679677, rs3897478, rs9286879, rs1728918, rs10865331 , rs6716753, rs12994997, rs6837335, rs13126505 rs10065637, rs7702331 , rs17695092, rs12663356, rs9264942, rs9491697 rs13204742, rs212388, rs10486483, rs864745, rs7015630, rs6651252 rs3764147, rs16967103, rs2066847, rs2945412, rs2024092, rs4802307 rs516246, rs2284553, rs10797432, rs6426833, rs2816958, rs1016883 rs17229285, rs9847710, rs3774959, rs1 1739663, rs254560, rs6927022 rs798502, rs4722672, rs4380874, rs4728142, rs483905, rs561722 rs28374715, rs1 1 150589, rs1728785, rs7210086, rs1 126510, rs6088765 rs6017342, rs12103, rs35675666, rs12568930, rs1 1209026, rs2651244 rs4845604, rs670523, rs4656958, rs1801274, rs2488389, rs755451 1 rs3024505, rs6545800, rs925255, rs10495903, rs7608910, rs6740462 rs917997, rs21 1 1485, rs1517352, rs2382817, rs3749171 , rs4256159 rs3197999, rs2472649, rs7657746, rs2930047, rs1 1742570, rs1363907 rs4836519, rs2188962, rs686341 1 , rs1 1741861 , rs6871626, rs12654812 rs171 19, rs9358372, rs1847472, rs6568421 , rs3851228, rs6920220 rs12199775, rs1819333, rs1456896, rs9297145, rs1734907, rs38904 rs921720, rs1991866, rs10758669, rs4743820, rs4246905, rs10781499 rs12722515, rs1042058, rs1 1010067, rs2790216, rs10761659, rs2227564 rs1250546, rs6586030, rs791 1264, rs4409764, rs90761 1 , rs10896794 rs1 1230563, rs4246215, rs559928, rs2231884, rs2155219, rs6592362 rs630923, rs1 1612508, rs1 1564258, rs1 1 168249, rs7134599, rs17085007 rs941823, rs9557195, rs194749, rs4899554, rs8005161 , rs17293632 rs7495132, rs529866, rs7404095, rs26528, rs10521318, rs3091316 rs12946510, rs12942547, rs1292053, rs1893217, rs7240004, rs727088 rs1 1879191 , rs17694108, rs1 1672983, rs6142618, rs491 1259, rs1569723 rs913678, rs259964, rs6062504, rs2823286, rs2836878, rs7282490 rs2266959, rs2412970, rs2413583, rs2641348, rs7517810, rs1260326 rs7438704, rs10061469, rs2503322, rs5743289, rs6667605, rs1440088 rs3774937, rs477515, rs1 182188, rs17780256, rs1 1083840, rs3766606 rs13407913, rs6708413, rs2457996, rs10051722, rs4976646, rs7746082 rs3891 1 , rs13277237, rs2227551 , rs7097656, rs12778642, rs1 1229555 rs174537, rs568617, rs2226628, rs566416, rs1 1054935, rs3742130 rs1569328, rs2361755, rs3091315, rs1654644, rs4243971 , rs6087990, rs6074022, rs5763767.
The method of claim 40, wherein determining the genomic polymorphism status of the patient comprises screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the subject has an increased likelihood of benefiting from said treatment if the diagnostic SNP comprises a response allele, and wherein the diagnostic SNP and the corresponding response allele are one or more of:
Figure imgf000162_0001
rs6088765 T rs9847710 G rs917997 A rs6651252 A rs4802307 C rs7134599 A rs4836519 G rs2816958 A rs194749 G rs2641348 A rs4243971 C rs10781499 G rs10065637 A rs1654644 T rs26528 G rs6667605 A rs864745 G rs516246 G rs2472649 G rs12654812 G rs3764147 G rs2836878 A rs17229285 A rs2155219 C rs12778642 T rs2651244 A rs12199775 G rs1728918 A rs4728142 G rs1 182188 G rs10758669 T rs171 19 G rs2189234 C rs483905 G rs1 1 168249 A rs1077773 A rs7097656 A rs6074022 A rs925255 G rs9297145 T rs7702331 G rs4976646 A rs1569723 T rs564349 G rs35320439 G rs7282490 A rs9319943 A rs10521318 A rs10865331 G rs616597 T rs7495132 G rs1016883 G rs921720 A rs17780256 T rs7210086 T rs395157 A rs21 1 1485 A rs1292053 G rs 1260326 G rs2823286 A rs4692386 A rs724016 G rs1 1583043 A rs9264942 G rs1517352 T rs6017342 T rs1 1742570 G rs3024505 G rs1 1083840 C rs686341 1 T rs13277237 A rs1 1 150589 G rs2188962 G rs38904 G rs2231884 G rs568617 G rs4409764 T rs1893217 A rs6426833 A rs566416 C rs941823 G rs10896794 A rs2930047 G rs1847472 T rs1748195 G rs477515 A rs7758080 A rs1 1054935 A rs2227564 G rs2227551 T rs3766606 T
rs7240004 A
rs9491697 G
The method of claim 40, wherein the cohort of marker and/or genomic test individuals comprises at least 200 individuals.
The method of any one of claims 40 to 44, wherein the antigenic formulation comprises an artificial repertoire of PRR agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is pathogenic in the target tissue, the distinct portion of the PRR agonist signature being different from any native PRR agonist signature of the microbial mammalian pathogen, wherein the artificial repertoire of mammalian PRR agonists are formulated together in a therapeutic vehicle for combined presentation following administration to a mammalian host, and the composition comprises components of the microbial mammalian pathogen that are agonists for at least 5 distinct mammalian PRRs, and wherein the composition is for use so as to modulate an innate immune response in the target tissue.
The method of claim 45, wherein the therapeutic vehicle comprises a recombinant microbe, a cellular fraction of the recombinant microbe, a cellular fraction of a microbial cell, a microparticle or a liposome, each comprising components of the microbial mammalian pathogen that provide the PRR agonists that together make up the artificial repertoire of PRR agonists.
The method of claim 46, wherein the recombinant microbe comprises a recombinant gene encoding a component of at least one of the PRR agonists.
The method of claim 46 or 47, wherein the therapeutic vehicle comprises a whole killed or attenuated cell of the recombinant microbe.
49. The method of claim 46, wherein the cellular fraction of the microbial cell comprises an isolated cellular fraction of the microbial mammalian pathogen.
50. The method of claim 49, wherein the isolated cellular fraction is one or more of: a bacterial outer membrane fraction; a bacterial inner membrane fraction; a pellet from a gradient centrifugation of microbial cell components; or chromosomal DNA.
51 . The method of any one of claims 45 to 50, wherein the PRRs and the corresponding PRR agonists are selected from the group consisting of:
Figure imgf000167_0001
Acetylmuramyl-L-Alanyl-D- Isoglutamine (L-D isoform), 6-0- stearoyl-N-Acetyl-muramyl-L-alanyl-D- isoglutamine, Pam2C-Aca-Benzyl- Murabutide,
TLR2/NOD2 Pam2C-conjugated murabutide
NOD1 /NOD2 PGN, Pam2C-conjugated murabutide
RIG1/MDA5 5' triphosphate double stranded RNA
(18-20mer),
polyriboinosinic:polyribocytidylic acid
DAI, LRRFI P1 , AIM2, dsDNA, poly(dA-dT poly(dT-dA) RIG1
Dectin-1 Beta-glucan peptide, fungal cell wall preparations
Mincle damaged microbial cells, fungus, yeast and mycobacteria, Trehalose-6,6- dibehenate, trehalose-6,6-dimycolate
STING Cyclic dinucleotides (c-di-nucleotides), xanthenone derivatives, 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, 2'2'-cGAMP, c-di-AMP (cyclic di-adenylate monophosphate), c-di-GMP, c-di-IMP, c-di-UMP, c-di-AMP
RIG-I PPP-ssRNA (PPP-ssRNA, ssRNA with a 5'-triphosphate group), RNA with base pairing and polyl:C
MDA5 Long dsRNA
LGP2 dsRNA
DDX41 B-form DNA and CDNs (cyclic
dinucleotides)
DHX9 DNA, RNA, CpG-A oligodeoxynucleotids and CpG-B
ODNs
DDX3 Viral RNA
DHX36 DNA, RNA, CpG-A
oligodeoxynucleotids and CpG-B oligodeoxynucleotids
DDX1 -DDX21 -DDX36 RNA and polyl :C
DDX60 ssRNA, dsRNA and dsDNA
KU70 DNA
cGAS DNA
STING CDNs (c-di-GMP and c-di-AMP)
NOD2 ssRNA
NLRP3 ssRNA, dsRNA, bacterial mRNA and oxidized mitochondrial DNA
AI M2 DNA
IFI 16 dsDNA
LRRFI P1 B-form DNA, Z-form DNA and dsRNA
DAI DNA
IFIT1 .2.3 and 5 PPP-ssRNA
52. The method of any one of claims 40 to 51 , wherein the I BD is Crohn's Disease. 53. The method of any one of claims 40 to 51 , wherein the IBD in ulcerative colitis.
54. The method of any one of claims 40 to 51 , wherein the IBD is collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome or indeterminate colitis.
55. The method of any one of claims 40 to 54, wherein the treatment with an antigenic formulation comprising PRR agonists is a site specific immunotherapy (SSI).
56. The method of claim 55, wherein the SSI comprises use of a preparation of a killed or attenuated microbe of one or more pathogenic microbial species; or a cell wall extract, a cell membrane extract, a whole cell extract, or a PRR agonist formulation comprising components of the microbial species; for use in treating an immune dysfunction in a specific organ or tissue in the subject, wherein the preparation, cell wall extract, cell membrane extract, whole cell extract or PRR agonist formulation elicits an immune response in the subject, and wherein the pathogenic microbial species is pathogenic in the corresponding specific organ or tissue in a healthy subject.
57. The method of claim 56, wherein the pathogenic microbial species is an E. coli.
58. A method of predicting the degree to which a mammalian subject has a likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists, the method comprising screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the diagnostic SNP is one or more SNP listed in any one of Tables 33 to 44.
59. The method of claim 58, wherein the subject is a human patient.
60. The method of claim 59, wherein the patient has a disease characterized by immune dysregulation.
61 . The method of claim 59, wherein the patient has an I BD.
62. The method of claim 61 , wherein the I BD is Crohn's Disease. 63. The method of claim 61 , wherein the I BD in ulcerative colitis.
64. The method of claim 61 , wherein the I BD is collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome or indeterminate colitis.
65. A method of predicting the degree to which a mammalian subject has a likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists, the method comprising screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the diagnostic SNP is one or more SNP listed in Table 33, and wherein the subject is a human patient suffing from ulcerative colitis and the treatment is with an SSI comprising E. coli PRR agonists.
66. A method of predicting the degree to which a mammalian subject has a likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists, the method comprising screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the diagnostic SNP is one or more SNP listed in Table 34, and wherein the subject is a human patient suffing from Crohn's disease or ulcerative colitis and the treatment is with an SSI comprising E. coli PRR agonists. 67. A method of predicting the degree to which a mammalian subject has a likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists, the method comprising screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the diagnostic SNP is one or more SNP listed in Table 35, and wherein the subject is a human patient suffing from Crohn's disease and the treatment is with an SSI comprising E. coli PRR agonists.
68. A method of predicting the degree to which a mammalian subject has a likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists, the method comprising screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the diagnostic SNP is one or more SNP listed in Table 36, and wherein the subject is a human patient suiting from Crohn's disease and the treatment is with an SSI comprising E. coli PRR agonists.
69 A method of predicting the degree to which a mammalian subject has a likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists, the method comprising screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the diagnostic SNP is one or more SNP listed in Table 37, and wherein the subject is a human patient suffing from ulcerative colitis and the treatment is with an SSI comprising E. coli PRR agonists. 70 A method of predicting the degree to which a mammalian subject has a likelihood of benefiting from treatment with an antigenic formulation comprising PRR agonists, the method comprising screening a sample isolated from said patient for a genomic polymorphism that is a diagnostic SNP, or is in genetic linkage disequilibrium with the diagnostic SNP, wherein the diagnostic SNP is one or more SNP listed in Table 38, and wherein the subject is a human patient suffing from ulcerative colitis and the treatment is with an SSI comprising E. coli PRR agonists.
Figure imgf000172_0001
The method of any one of claims 58 to 70, wherein the treatment with an antigenic formulation comprising PRR agonists is a site specific immunotherapy (SSI). 72 The method of claim 71 , wherein the SSI comprises use of a preparation of a killed or attenuated microbe of one or more pathogenic microbial species; or a cell wall extract, a cell membrane extract, a whole cell extract, or a PRR agonist formulation comprising components of the microbial species; for use in treating an immune dysfunction in a specific organ or tissue in the subject, wherein the preparation, cell wall extract, cell membrane extract, whole cell extract or PRR agonist formulation elicits an immune response in the subject, and wherein the pathogenic microbial species is pathogenic in the corresponding specific organ or tissue in a healthy subject.
73. The method of claim 72, wherein the immune dysfunction is an IBD, or Crohn's diseases or ulcerative colitis, and the pathogenic microbial species is an E. coli.
74. The method of any one of claims 58 to 73, wherein the genomic polymorphism is within 1 Mbp of the diagnostic SNP, or within 500Kbp, or within 10Okbp.
75. The method of any one of claims 58 to 74, wherein the genomic polymorphism is in linkage disequilibrium with the diagnostic SNP, with an r-squared value of greater than 0.8, or greater than 0.9.
76. The method of any one of claims 58 to 75, wherein the antigenic formulation comprises an artificial repertoire of mammalian pattern recognition receptor (PRR) agonists that recapitulates a distinct portion of a PRR agonist signature of a microbial mammalian pathogen that is pathogenic in the target tissue, the distinct portion of the PRR agonist signature being different from any native PRR agonist signature of the microbial mammalian pathogen, wherein the artificial repertoire of mammalian PRR agonists are formulated together in a therapeutic vehicle for combined presentation following administration to a mammalian host, and the composition comprises components of the microbial mammalian pathogen that are agonists for at least 5 distinct mammalian PRRs, and wherein the composition is for use so as to modulate an innate immune response in the target tissue.
77. The method of claim 76, wherein the therapeutic vehicle comprises a recombinant microbe, a cellular fraction of the recombinant microbe, a cellular fraction of a microbial cell, a microparticle or a liposome, each comprising components of the microbial mammalian pathogen that provide the PRR agonists that together make up the artificial repertoire of PRR agonists.
78. The method of claim 77, wherein the recombinant microbe comprises a recombinant gene encoding a component of at least one of the PRR agonists.
79. The method of claim 77 or 78, wherein the therapeutic vehicle comprises a whole killed or attenuated cell of the recombinant microbe. 80. The method of claim 77, wherein the cellular fraction of the microbial cell comprises an isolated cellular fraction of the microbial mammalian pathogen.
81 . The method of claim 80, wherein the isolated cellular fraction is one or more of: a bacterial outer membrane fraction; a bacterial inner membrane fraction; a pellet from a gradient centrifugation of microbial cell components; or chromosomal DNA.
82. The method of any one of claims 76 to 81 , wherein the PRRs and the corresponding PRR agonists are selected from the group consisting of:
Figure imgf000174_0001
TLR9 unmethylated CpG DNA motifs
NOD1 iE-DAP, Acylated iE-DAP, D-gamma- Glu-mDAP, L-Ala-gamma-D-Glu-mDAP
NOD2 MDP (MurNAc-L-Ala-D-isoGIn,
muramyl dipeptide), N-glycolylated muramyldipeptide, N-Acetyl-muramyl- L- Alanyl-D-Glutamin-n-butyl-ester, MurNAc-Ala-D-isoGln-Lys, N- Acetylmuramyl-L-Alanyl-D- Isoglutamine (L-D isoform), 6-0- stearoyl-N-Acetyl-muramyl-L-alanyl-D- isoglutamine, Pam2C-Aca-Benzyl- Murabutide,
TLR2/NOD2 Pam2C-conjugated murabutide
NOD1 /NOD2 PGN, Pam2C-conjugated murabutide
RIG1/MDA5 5' triphosphate double stranded RNA
(18-20mer),
polyriboinosinic:polyribocytidylic acid
DAI, LRRFI P1 , AIM2, dsDNA, poly(dA-dT poly(dT-dA) RIG1
Dectin-1 Beta-glucan peptide, fungal cell wall preparations
Mincle damaged microbial cells, fungus, yeast and mycobacteria, Trehalose-6,6- dibehenate, trehalose-6,6-dimycolate
STING Cyclic dinucleotides (c-di-nucleotides), xanthenone derivatives, 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, 2'2'-cGAMP, c-di-AMP (cyclic di-adenylate monophosphate), c-di-GMP, c-di-IMP, c-di-UMP, c-di-AMP RIG-I PPP-ssRNA (PPP-ssRNA, ssRNA with a 5'-triphosphate group), RNA with base pairing and polyl:C
MDA5 Long dsRNA
LGP2 dsRNA
DDX41 B-form DNA and CDNs (cyclic
dinucleotides)
DHX9 DNA, RNA, CpG-A
oligodeoxynucleotids and CpG-B ODNs
DDX3 Viral RNA
DHX36 DNA, RNA, CpG-A
oligodeoxynucleotids and CpG-B oligodeoxynucleotids
DDX1 -DDX21 -DDX36 RNA and polyl :C
DDX60 ssRNA, dsRNA and dsDNA
KU70 DNA
cGAS DNA
STING CDNs (c-di-GMP and c-di-AMP)
NOD2 ssRNA
NLRP3 ssRNA, dsRNA, bacterial mRNA and oxidized mitochondrial DNA
AI M2 DNA
IFI 16 dsDNA
LRRFI P1 B-form DNA, Z-form DNA and dsRNA
DAI DNA
IFIT1 .2.3 and 5 PPP-ssRNA
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