Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5047—Cells of the immune system
  • G01N33/505—Cells of the immune system involving T-cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
  • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/101—Bovine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/02—Animal zootechnically ameliorated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K2035/11—Medicinal preparations comprising living procariotic cells
  • A61K2035/115—Probiotics

Definitions

• the present disclosure relates to methods and systems for identifying immunomodulatory substance and in particular immunomodulatory microorganisms and compounds.
• Bacteroides fragilis is a human commensal microorganism that has been shown to have immunomodulatory properties.
• immunomodulatory properties of Bacteroides fragilis have been associated to Polysaccharide A (PSA) production by the bacterium.
• PSA Polysaccharide A
• identification of the mechanism and/or molecular triggers by which B. fragilis (or any commensal bacteria) delivers beneficial microbial molecules to the immune system and performs the immunomodulatory properties has been challenging.
• kits for screening microorganisms and related substances having immunomodulatory and, in particular, anti inflammatory properties are provided herein.
• methods and systems that in several embodiments allow identification of bacterial substances that are able to induce in an individual an immunomodulatory response comparable to the one of Bacteroides fragilis.
• a method for identifying a bacterial substance having immunomodulatory ability.
• the method comprises contacting a candidate bacterial substance with a T cell alone or in presence of an antigen presenting cell, and detecting expression of at least one of one or more anti inflammatory biomarkers selected from the group consisting of IL-IO, Foxp3, TGF ⁇ l,TGF ⁇ 2 Perform and Granzyme B, and one or more inflammatory biomarkers selected from the group consisting of IFN ⁇ , IFN ⁇ , IFN ⁇ IL- l ⁇ , IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17 or TNF ⁇ .
• the method further comprises determining an anti inflammatory ability of the candidate bacterial substance through detection of an increase of the expression of the one or more anti-inflammatory biomarkers or a decrease of the expression of the one or more inflammatory biomarkers following the contacting.
• a method for identifying a bacterial substance having immunomodulatory ability.
• the method comprises contacting a candidate bacterial substance with an antigen presenting cell, and incubating the antigen presenting cell with a T cell following the contacting.
• the method further comprises detecting expression of at least one of one or more anti inflammatory biomarker selected from the group consisting of IL-10, Foxp3, TGF ⁇ l, TGF ⁇ 2, Perforin and Granzyme B, and one or more inflammatory biomarker selected from the group consisting of IFN ⁇ , IFN ⁇ , IFN ⁇ IL-l ⁇ , IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17 or TNF ⁇ .
• the method further comprises determining an anti inflammatory ability of the candidate bacterial substance through detection of an increase of the expression of the one or more anti-inflammatory biomarkers or a decrease of the expression of the one or more inflammatory biomarkers following the incubating.
• a method for identifying a bacterial substance having immunomodulatory ability in animals.
• the method comprises treating a transgenic marker non-human animal with a candidate bacterial substance, the transgenic marker non- human animal genetically modified to express at least one of one or more labeled inflammatory biomarkers selected from the group consisting of IL-IO, Foxp3, TGF ⁇ l, TGF ⁇ 2, Perform and Granzyme B, and one or more labeled inflammatory biomarker selected from the group consisting of IFN ⁇ , IFN ⁇ , IFN ⁇ IL-l ⁇ , IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL- 17 or TNF ⁇ .
• the method further comprises detecting expression in the transgenic marker non-human animal of at least one of the one or more anti-inflammatory biomarkers or at least one of the inflammatory biomarkers following the treating and determining an anti inflammatory ability of the candidate bacterial substance through detection of an increase of the expression of the one or more anti-inflammatory biomarkers or a decrease of the expression of the one or more inflammatory biomarkers following the treating.
• a system for screening a bacterial substance comprises at least two of a T cell, an antigen presenting cell and reagents for detection of at least one of one or more anti-inflammatory biomarkers selected from the group consisting of IL-IO, Foxp3, TGF ⁇ l, TGF ⁇ 2, Perforin and Granzyme B and one or more labeled inflammatory biomarker selected from the group consisting of IFN ⁇ , IFN ⁇ , IFN ⁇ IL-l ⁇ , IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17 or TNF ⁇ , for simultaneous, combined or sequential use in a method to identify an anti-inflammatory bacterial substance herein described.
• Figure 1 shows that outer membrane vesicles from Bacteroides fragilis contain PSA.
• Figure Ia shows OMV produced wild type B. fragilis (WT-OMV) and B. fragilis ⁇ PSA ( ⁇ PSA- OMV) detected by transmission electron microscopy of EDL (electron dense layer) enriched B. fragilis.
• Figure Ib shows an immunoblot analysis of whole cell (WC) and outer membrane vesicles (OMV) extracts from wild-type and PSA-mutant bacteria.
• Figure Ic shows immunogold labeling of purified OMVs, stained with anti-PSA and anti-IgG-colloidal gold conjugate (5nm), analyzed by electron microscopy.
• Figure Id shows a glycoprotein staining of capsular polysaccharide preparations from whole cells and OMVs.
• Figure 2 illustrates exemplary results showing that OMVs protect animals from experimental colitis and intestinal inflammation in a PSA-dependent manner.
• Figure 2a shows a diagram reporting weight loss in animal groups following the induction of TNBS colitis (day 0) measured as reduction from initial weight until day of sacrifice (day 4). All groups contained at least 4 animals, with error bars indicating standard error of the mean (SEM). Results are representative of 3 independent trials, p values determined by one-way ANOVA: * /? ⁇ 0.05; *** / ⁇ .001.
• Results are shown from 3 combined experiments performed independently, p values determined by one-way ANOVA: *** / ⁇ .001.
• NS not significant.
• Figure 2c shows images from hematoxylin and eosin (H & E) stained colon sections representative of each treatment group.
• Figure 2d shows colitis scores from animals assigned by a blinded pathologist (G.W.L) according to a standard scoring system (ONLINE METHODS) (Scheiffele and Fuss. (2001) Induction of TNBS colitis in mice. Current Protocols in Immunology . 1.19.1-15.19.14) Each symbol represents an individual animal. Results are shown from 3 combined experiments performed independently. *** /? ⁇ 0.001. NS: not significant.
• Figures 2e and f show diagrams illustrating cytokine transcript analysis by qRT-PCR from RNA recovered from whole colons (Figure 2e) or purified CD4 + T cell from mesenteric lymph nodes (Figure 2f). Error bars indicate SEM from 4 animals/group. Results are representative of 3 independent trials.
• FIG. 3 shows results indicating that PSA containing OMVs induce IL-IO production and Foxp3 expression from T cells co-cultured with treated DCs.
• Figure 3a shows flow cytometry (FC) analysis of OMV internalization by DCs. OMVs were labeled with FITC (Fluorescein isothiocyanate) and incubated with cultured DCs for various times (as indicated). Cells were stained with anti-CD l ie. Percentages show CDI lC + OMV + populations.
• Figure 3b shows FC plots of DCs incubated with WT-OMVs and ⁇ PSA-OMVs for various times (as indicated) and stained with anti-CD l ie and anti-MHCII.
• FIG. 3c shows ELISA analysis for IL-IO of culture supernatants from DCs or DC-T cell co-cultures, where DCs were pulsed with OMVs for 18 hours, washed and incubated with primary CD4 + T cells or not. Supernatants were collected at day 4 of culture. Media samples indicate DCs that were not pulsed with OMVs, but otherwise treated identically. Anti-CD3 was added to some samples to augment T cell responses. Error bars indicate SEM from triplicate samples. Results are representative of over 5 independent trials. * /? ⁇ 0.05; ** p ⁇ 0.0l.
• Figure 3d shows ELISA analysis similar to Figure 3c, but also including DCs differentiated from IL-IO "7" animals. Error bars indicate SEM from triplicate samples. Results are representative of 3 independent trials. * p ⁇ 0.05. NS: not significant.
• Figure 3d (right panel) shows ELISA analysis similar to Figure 3c, but also including DCs differentiated from TLR2 "7" animals. SEA: staphylococcal enterotoxin A. Error bars indicate SEM from triplicate samples. Results are representative of 3 independent trials. * p ⁇ 0.05. NS: not significant.
• Figure 3e shows transcript levels of IL-IO (left) and Foxp3 (right) as determined by qRT-PCR of RNA received from purified T cell subsets following in vitro culture with DCs.
• Co-cultures were set up as in (c-e); on day 4, CD4 + CD25 + and CD4 + CD25 " T cell were purified by magnetic bead separation (>95% purity) and RNA extracted with RNeasy mini kit. Relative values were normalized to ⁇ -actin. Error bars indicate SEM from triplicate samples. Results are representative of 3 independent trials. * /? ⁇ 0.05; *** /? ⁇ 0.001. NS: not significant.
• Figure 3f shows co-cultures were set up as in (c-e),, but using CD4 + T cells from Foxp3-GFP mice. Following 4 days of culture with OMV pulsed DCs, cells were stained with anti-CD4 and Foxp3 detected by GFP expression using FC. Results are representative of 2 independent trials.
• Figure 4 shows exemplary results indicating in vitro generation of Treg suppressive function by PSA-containing OMVs.
• Figure 4a shows FC histograms of IL-IO expression by CD4 + T cell subsets following 4 day co-culture with DCs treated with OMVs.
• Splenic CD4 + T cell were purified from IL-IO-GFP mice, stained with anti-CD4 and anti-CD25 following co- culture, and IL-IO expression measured by GFP expression. Percentages show IL-IO + populations among CD4 + CD25 + and CD4 + CD25 " subsets. Results are representative of 3 independent trials.
• Figure 4b shows in vitro suppression of na ⁇ ve responder cells by purified CD4 + CD25 + T cells following co-culture with DCs treated with media (control), WT-OMVs and ⁇ PSA-OMVs.
• Cell proliferation was measured by FC of CFSE dilution.
• Treg:Teff ratios are indicated, and percentages show total proliferating cells.
• No Treg: CD4 + CD25 " cells only. Results are representative of 2 independent trials.
• Figure 4c shows the quantification of percentage of CD4 + T cells in each proliferating peak (as is labeled as 1, 2, 3, 4, 5 in Fig4b). Results are shown from 3 combined experiments performed independently. * p ⁇ 0.05. NS: not si 'g6n' ificant.
• Figure 5 shows wild-type B. fragilis and PSA deletional mutant B. fragilis produce similar amount of OMVs during in vitro culture. The amount of total protein recovered from each OMVs preparation normalized by OD OOO of the culture at the time of harvest. Error bars indicate SEM. Result is shown from >10 combined experiments preformed independently, p value determined by Student's t-test. NS: not significant.
• FIG. 6 shows Majority OMVs purified from wild-type B. fragilis contain PSA Immunogold labeling of PSA on purified OMVs shows -60% of WT-OMVs observe among random sampling of 10 areas (l ⁇ m x l ⁇ m) of sample are associated with PSA, but none of ⁇ PSA-OMVs observed are stained positively for PSA. (Immunogold labeling of WT-OMvs with anti-IgG-gold only confirms the specificity of the labeling).
• Figure 7 shows OMVs from wild-type or PSA deletional mutant B. fragilis show no significant difference in protein composition.
• Proteome mass spectrometry shows 100% overlap of the identified proteins (>1 unique peptide identified for each protein) between WT-OMVs and ⁇ PS A-OMVs.
• we semi-quantitatively compared the amount of those relatively abundant proteins according to the number of unique peptides identified for each of them. Majority of them show no difference performed independently. Errors indicate SEM.
• FIG. 8 shows actin polymerization is required for OMV uptake by DCs.
• OMVs were labeled with FITC (Fluorescein isothiocyanate) and incubated with cultured DCs for various times (as indicated).
• Cells were stained with anti-CD l ie. Percentages show CDI lC + OMV + populations.
• FIG. 9 shows WT-OMVs or ⁇ PSA-OMVs are internalized and localized in the cytoplasm of DCs.
• OMVs were labeled with FITC (gray arrow) and incubated with cultured DCs for 2hrs. Cells were fixed and cell membrane was stained with Wheat Germ Agglutinin (WGA)- tetramethylrhodamine (black arrow). Scale bar: 7.5 ⁇ m.
• Figure 10 shows WT-OMVs and ⁇ PSA-OMVs upregulate co-stimulatory molecule for DC activation.
• FIG 11 shows PSA induced expression of biomarkers in an embodiment herein described.
• the data illustrated in each diagram are representative of three independent experiments. Light bars indicated cells derived from PBS treated mice and dark bars from PSA-treated mice.
• immunomodulatory indicates the ability to promote a state associated with absence of an inflammatory response.
• immunomodulatory properties comprise anti inflammatory properties, wherein the term anti-inflammatory refers to the property of a substance or treatment that prevents or reduces inflammation.
• inflammation indicates the complex biological response of vascular tissues of an individual to harmful stimuli, such as pathogens, damaged cells, or irritants, and includes secretion of cytokines and more particularly of pro-inflammatory cytokine, i.e. cytokines which are produced predominantly by activated immune cells such as microglia and are involved in the amplification of inflammatory reactions.
• pro-inflammatory cytokine include but are not limited to IL-I, IL-6, TNF-a, IL-17, IL21, and IL23.
• inflammations include acute inflammation and chronic inflammation.
• acute inflammation indicates a short- term process characterized by the classic signs of inflammation (swelling, redness, pain, heat, and loss of function) due to the infiltration of the tissues by plasma and leukocytes.
• An acute inflammation typically occurs as long as the injurious stimulus is present and ceases once the stimulus has been removed, broken down, or walled off by scarring (fibrosis).
• chronic inflammation indicates a condition characterized by concurrent active inflammation, tissue destruction, and attempts at repair. Chronic inflammation is not characterized by the classic signs of acute inflammation listed above. Instead, chronically inflamed tissue is characterized by the infiltration of mononuclear immune cells (monocytes, macrophages, lymphocytes, and plasma cells), tissue destruction, and attempts at healing, which include angiogenesis and fibrosis.
• the term "substance” as used herein indicates a matter of particular or definite chemical constitution.
• the term "bacterial substance” indicate matter of bacterial origin such as, a live bacteria, dead bacteria, and in particular heat- killed bacteria, bacterial extracts, purified molecules from bacteria, a combination of molecules purified from bacteria, or vesicles containing one molecule or a combination of molecules purified from bacteria or purified vesicles from bacteria.
• bacterial substance can be formed by outer membrane vesicles (OMVs) which are vesicles released from the surface of bacteria and deliver a suite of molecular cargo to distant target cells.
• Bacterial substance comprises substance derived from a same bacteria and substance derived from two or more different bacteria.
• bacteria indicates large group of unicellular, prokaryote, microorganisms, typically of a few micrometers in length, and having a wide range of shapes.
• bacteria in the sense of the present disclosure comprise bacteria of the human flora, i.e. the assemblage of microorganisms that reside on the surface and in human tissues and bodily fluids such as deep layers of skin, in saliva, oral or vaginal mucosa, and in the gastrointestinal tracts.
• Bacterial flora comprises gut flora (bacteria detectable in the digestive tract of humans) vaginal flora (bacteria detectable in the fibromuscular tubular tract leading from the uterus to the exterior of the body in female humans) and skin flora (bacteria detectable in human skin). More specifically, bacteria in the sense of the present disclosure can be formed by one or more bacteroidetes of the gut flora and in particular one or more bacteroides, a genus of Gram- negative, non endospore forming anaerobes bacillus bacteria, symbiotic with humans and identifiable by a skilled person. A representative bacteroides is B. fragilis.
• the immunomodulatory ability of a bacterial substance can be determined by detection of inflammatory and/or anti-inflammatory biomarkers.
• biomarker indicates a substance or characteristic used as an indicator of a biological state, such as a phase of cellular cycle, a biological process, as well as health and disease state. The presence, absence, reduction, upregulation of the biomarker is associated with and is indicative of a particular state.
• the term "inflammatory biomarker” as used herein indicates a biomarker that is indicative of presence of an inflammatory state and comprise an IFN ⁇ , IFN ⁇ , IFN ⁇ IL-I ⁇ , IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17, TNF ⁇ and additional cell markers and cytokines identifiable by a skilled person.
• the term antiinflammatory biomarker as used herein indicates a biomarker that is indicative of absence of an inflammatory state and comprises Foxp3, IL-IO, TGF ⁇ l, TGF ⁇ 2 Perforin and Granzyme B and additional cell markers and cytokines identifiable by a skilled person.
• transgenic marker non-human animal refers to an animal that contains non-native, genetic material that has been transferred naturally or by any of a number of genetic engineering techniques. Such non-native, genetic material (or transgene) may act as a "biomarker” (as defined herein) and/or retain the ability to produce RNA or protein in the non-human animal.
• identification of an immunomodulatory bacterial substance is performed by detecting expression of inflammatory or anti-inflammatory biomarkers by a T cells alone or in presence of an antigen presenting cell.
• T cell indicates a indicates a sub-group of lymphocytes (a type of white blood cell or leukocyte) including different cell types identifiable by a skilled person.
• T-helper cell according to the present disclosure and include effector T h cells (such as ThI, Th2 and Thl7)- i.e. Th cells that secrete cytokines, proteins or peptides that stimulate or interact with other leukocytes, including T h cells - and suppressor Th cells (such as Treg) i.e. Th cells that suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-anti 'g6'ens.
• antigen presenting cell indicates a cell that displays foreign antigen complex with major histocompatibility complex (MHC) on its surface.
• antigen presenting cell comprise dendritic cell, macrophage, B cells and additional cells identifiable by a skilled person.
• methods herein described comprise contacting a candidate bacterial substance with a T cell alone or in presence of an antigen presenting cell. In an embodiment, methods here described comprise contacting a candidate bacterial substance with an antigen presenting cell and incubating the antigen presenting cell with a Tcell following the contacting.
• contacting indicates actions directed to creation of a spatial relationship between two items provided for a time and under condition such that at least one of the reciprocal or non reciprocal action or influence between the two items can be exerted.
• incubation can be performed between a bacterial substance and a cell and can result in a direct contact and/or interaction between the bacterial substance and the cell or can result in a modification of the cell following an indirect action of the bacterial substance (e.g. following activation or modification of another substance which directly interacts with the cell).
• Incubation can also be performed between a first cell and a second cell following contacting of the first cell with a bacterial substance and can result in a direct contact and/or interaction between the first cell and the second cell or can result in a modification of the second cell following an indirect action of the first cell (e.g. following secretion of cytokines or other molecules which directly interact with the second cell).
• Exemplary contacting a bacterial substance with a T cell or an antigen presenting cell can be performed in vitro by bathing a whole sample comprising one or more types of cells, in a solution containing the bacterial substance under suitable conditions which depend on the specific cells and the specific bacterial substance and are identifiable by a skilled person upon reading of the present disclosure.
• exemplary contacting between the bacterial substance and a T cell or antigen presenting cell can be performed in vitro by introducing the bacterial substance to a cell culture of purified cells under suitable conditions, and in vivo by treating an individual (e.g. a transgenic animal, other than a human, genetically modified to express a labeled inflammatory or an anti inflammatory biomarker) with the bacterial substance.
• Systemic administration includes enteral administration (e.g. oral administration, administration by gastric feeding tube, administration by duodenal feeding tube, gastrostomy, enteral nutrition, and rectal administration) and parenteral administration (e.g. intravenous administration, intra- arterial administration, intramuscular administration, subcutaneous administration, intradermal, administration, intraperitoneal administration, and intravesical infusion.
• Topical administration include but is not limited to epicutaneous administration, inhalational administration (e.g. in asthma medications), enema, eye drops (E.G. onto the conjunctiva), ear drops, intranasal route (e.g. decongestant nasal sprays), and vaginal administration.
• Exemplary incubation of an antigen presenting cell with a T cell can be performed in vitro by mixing a cell culture comprising the antigen presenting cell with a cell culture comprising the T cell, by adding a purified antigen presenting cell to a culture of T cells, or by adding a purified T cell to a culture of antigen presenting cells. Additional exemplary incubation between a T cell and an antigen presenting cell in vivo comprise transplanting antigen presenting cell in a into a tissue of an individual, the tissue comprising T cell, or transplanting T cell into a tissue of an individual the tissue comprising antigen presenting cell.
• the individual is a transgenic animal other than humans genetically modified to express a labeled inflammatory or an anti inflammatory biomarker.
• Suitable conditions for performing the contacting or incubation are identifiable by a skilled person and comprise providing a suitable environment for cell culture in vitro or suitable environment for e.g. by use of a robotic incubator which controls and monitors the temperature and optionally also the atmospheric CO 2 , N 2 and/or O 2 content, relative humidity, nutrient amounts and other conditions suitable to provide the cells with a suitable growth environment. Also additional procedures and techniques suitable for performing contacting between a substance and a cell in vitro or in vivo can be identified by a skilled person upon reading of the present disclosure.
• the amount of substance given to in vitro cultures or in vivo to animals, as well as the time frame of incubation can be determined by the skilled person experimentally in view of the specific bacterial substance, the specific T cell, antigen presenting cell, animal treated, related suitable conditions and the experimental design.
• detection of the expression of an inflammatory or an anti-inflammatory biomarker can be performed in vitro and in vivo by techniques identifiable by a skilled person which comprise use of labeled molecules, including labeled biomarkers or labeled molecule specific for the biomarker or molecule associated thereto.
• detect indicates the determination of the existence, presence or fact of an analyte or related signal in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate.
• a detection is "quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the analyte or related signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the analyte or related signal.
• a detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the analyte or related signal in terms of relative abundance to another analyte or related signal, which is not quantified.
• label and "labeled molecule” or as used herein as a component of a complex or molecule referring to a molecule capable of detection, including but not limited to radioactive isotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (such as biotin, avidin, streptavidin or haptens) and the like.
• fluorophore refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable image.
• the wording "signal” or “labeling signal” as used herein indicates the signal emitted from the label that allows detection of the label, including but not limited to radioactivity, fluorescence, chemiluminescence, production of a compound in outcome of an enzymatic reaction and the like.
• Detection can be performed by detecting levels of expression of the biomarker, a precursor or analog thereof, and/or of an analyte associated thereto.
• the wording "associated to" as used herein with reference to two items indicates a relation between the two items such that the occurrence of a first item is accompanied by the occurrence of the second item, which includes but is not limited to a cause-effect relation and sign/symptoms-disease relation.
• the detection can be performed qualitatively or quantitatively and can involve detection of molecules such as RNA, protein, their precursors, differing types (i.e. mRNA, tRNA, and rRNA) and/or degradation products, and/or detection or measurable properties associated thereof. Techniques and procedures to perform detection are identifiable by a skilled person upon reading of the present disclosure.
• Exemplary methods for detection of a biomarker expression comprise methods known to a skilled person including but not limited to ELISA, Q-PCR and intracellular cytokine staining detected by FACs.
• expression of a biomarker can be detected via fluorescent based readouts on a cell culture performed using an antibody specific for the biomarker or molecule associated thereto, labeled with fluorophore, which includes, but not exhaustively, small molecular dyes, protein chromophores, quantum dots, and gold nanoparticles.
• expression of a biomarker can be detected by detecting expression of a label under the transcriptional control of a biomarker promoter in vivo (e.g., in an animal tissue) or in vitro (e.g. in a cell culture).
• a biomarker promoter in vivo (e.g., in an animal tissue) or in vitro (e.g. in a cell culture).
• the biomarker can be in particular IL-IO or Foxp3. Additional techniques are identifiable by a skilled person upon reading of the present disclosure and will not be further discussed in detail.
• an anti inflammatory ability of the candidate bacterial substance can be determined through detection of an increase of expression of the one or more anti-inflammatory biomarkers or a decrease of the expression of the one or more inflammatory biomarkers following the contacting and/or the incubating. Determining increase and/or decrease of a biomarker expression can be performed by comparing the detected expression of the biomarker following the contacting and/or incubating, with a predetermined detected expression of the same biomarker in absence of contacting and/or incubating. Determining increase and/or decrease of a biomarker expression can be performed by comparing the detected expression marker with the detected expression of the same biomarker in a control cell in absence of contacting and/or incubating.
• detection of an increase in expression of the anti-inflammatory or detection of a decrease in expression of an inflammatory biomarker indicates the ability of a substance to induce anti-inflammatory molecules such as IL-IO, Foxp3, TGF ⁇ l or TGF- ⁇ 2, perforin and granzyme B or a combination thereof, or suppress inflammatory cytokines such as IFN ⁇ , IFN ⁇ , IFN ⁇ IL-I ⁇ , IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17 or TNF ⁇ or a combination thereof.
• anti-inflammatory molecules such as IL-IO, Foxp3, TGF ⁇ l or TGF- ⁇ 2, perforin and granzyme B or a combination thereof, or suppress inflammatory cytokines such as IFN ⁇ , IFN ⁇ , IFN ⁇ IL-I ⁇ , IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22,
• the method comprises contacting a candidate bacterial substance in vitro and/or in vivo with a T cell alone or in presence of an antigen presenting cell, and detecting expression of at least one anti inflammatory biomarker selected from the group consisting of IL- 10, Foxp3, TGF ⁇ l, TGF ⁇ 2, perforin and granzyme B, wherein an increase of the expression of the anti-inflammatory biomarker following the contacting indicates an anti inflammatory ability of the candidate bacterial substance.
• expression of IL-IO, TGF ⁇ l, TGF ⁇ 2, perforin and/or granzyme B can be performed on Foxp3 expressing T cell, and more particularly Foxp3 expressing Treg cell.
• the method comprises contacting a candidate bacterial substance with an antigen presenting cell, and incubating the antigen presenting cell with a T cell following the contacting.
• the method further comprises detecting the expression of at least one antiinflammatory biomarker selected from the group consisting of Foxp3, IL-10, TGF ⁇ l, TGF ⁇ 2, TGF ⁇ 2, Perforin and Granzyme B, wherein detection of an increase of the biomarker expression following the incubating indicates an anti inflammatory ability of the candidate substance.
• expression of IL-10, perforin and/or granzyme B can be performed on Foxp3 expressing T cell, and more particularly Foxp3 expressing Treg cell.
• the method comprises contacting a candidate bacterial substance with a T cell alone or in presence of an antigen presenting cell, and detecting expression of at least one inflammatory biomarker selected from the group consisting of IL 17 and TNF ⁇ , wherein a decrease of the expression of the inflammatory biomarker following the contacting indicates an anti-inflammatory ability of the candidate bacterial substance.
• expression of IL17 and TNF ⁇ can be performed on Foxp3 expressing T cell, and more particularly Foxp3 expressing Treg cell.
• the method comprises contacting a candidate bacterial substance with an antigen presenting cell, and incubating the antigen presenting cell with a T cell following the contacting.
• the method further comprises detecting the expression of at least one antiinflammatory biomarker selected from the group consisting of consisting of IL 17 and TNF ⁇ , wherein a decrease of the expression of the inflammatory biomarker following the contacting indicates an anti-inflammatory ability of the candidate bacterial substance.
• expression of IL17 and TNF ⁇ can be performed on Foxp3 expressing T cell, and more particularly Foxp3 expressing Treg cell.
• the candidate bacterial substance comprises bacteria in a pure pool. In other embodiments, the candidate bacterial substance comprises bacteria in a mixed pool.
• the bacteria can be live bacteria, dead bacteria, extracts or products isolated from bacteria, or combinations of each.
• the bacteria may also be from laboratory strains, isolates from repositories such as ATCC, isolates from animals, isolates from humans, or combinations of each.
• the isolate from animals is isolated from feces.
• the isolate can also come from intestinal contents or combinations of feces and other intestinal contents.
• the isolate from humans is isolated from feces.
• the isolate can also come from intestinal contents or combinations of feces and other intestinal contents.
• the method comprises treating a transgenic marker non-human animal and in particular a transgenic marker mammal such as a mouse or a rat, with a candidate substance, the transgenic non-human marker animal genetically modified to express a labeled anti-inflammatory or inflammatory biomarker selected from the group consisting of IL-IO and Foxp3 but also perforin and granzyme B, IL17 and TNF ⁇ ; and detecting biomarker expression in the transgenic marker animal following the treating.
• the aforementioned transgenic non-human animal models can be treated either orally or intravenously with the substance, or permanently colonized with live bacteria by oral gavage and the amount of biomarker expression Foxp3 or ILlO expression will be monitored in the various compartments of the mouse including the spleen, mesenteric lymph nodes, small and large intestine, lungs, pancreas, and bone marrow as a measure of the immunomodulatory ability of the substance.
• dendritic cells, T cells, T regulatory, B cells or macrophages can be purified or differentiated from mice where expression of IL-IO (or Foxp3 in the case of T cell analysis) is marked by the fluorophore Green fluorescent protein (GFP).
• the candidate bacterial substance is contacted with one of the aforementioned cell types and the amount of GFP expression will be determined indicating the immunomodulatory ability of the substance being tested.
• dendritic cells or other antigen presenting cells can be incubated with the substance, the substance can be washed off and the dendritic cells can be subsequently incubated with T cells to determine the ability of the dendritic cells to elicit immunomodulatory activity from the T cell.
• GFP expression can be determined using fluorescent cell sorting (FACS) or a microplate reader.
• a T cell, antigen presenting cell, and reagents for detection of at least one of one or more anti-inflammatory biomarkers selected from the group consisting of IL- 10, Foxp3, TGF ⁇ 2, Perforin and Granzyme B and one or more labeled inflammatory biomarker selected from the group consisting of IL 17 and TNF ⁇ an be comprised in a system for identifying a bacterial substance having immunomodulatory ability according to methods herein described.
• the system comprises: dendritic cell, T cell, T regulatory, B cells and/or macrophages that, in some embodiments, can be purified or differentiated from mice where expression of IL-10 (or Foxp3 in the case of T cell analysis) is marked by the fluorophore Green fluorescent protein (GFP).
• GFP fluorophore Green fluorescent protein
• kits of parts can be provided in the form of kits of parts.
• the multi- ligand capture agent and other reagents to perform the method can be comprised in the kit independently.
• the antigen presenting cell, the T cell and the reagents can be included in one or more compositions, and each cell and reagent can be in a composition together with a suitable vehicle.
• Additional components can include labeled molecules and in particular, labeled capture agents specific for an anti-inflammatory or an inflammatory biomarker or a molecule associated to the expression thereof, a microfluidic chip, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure.
• the term "capture agent” as used herein indicates a compound that can specifically bind to a target.
• the wording "specific” “specifically” or “specificity” as used herein with reference to the binding of a first molecule to second molecule refers to the recognition, contact and formation of a stable complex between the first molecule and the second molecule, together with substantially less to no recognition, contact and formation of a stable complex between each of the first molecule and the second molecule with other molecules that may be present.
• Exemplary specific bindings are antibody-antigen interaction, cellular receptor-ligand interactions, polynucleotide hybridization, enzyme substrate interactions etc.
• stable complex is meant a complex that is detectable and does not require any arbitrary level of stability, although greater stability is generally preferred.
• the kit can comprise labeled polynucleotides or labeled antibodies.
• kits can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here described.
• the kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, for carrying out the assay, will usually be included in the kit.
• the kit can also contain, depending on the particular method used, other packaged reagents and materials (i.e. wash buffers and the like).
• biomarker such asILlO, Foxp3, TGF ⁇ l, TGF ⁇ 2, perforin, granzyme B, TNF ⁇ IL17A to other biomarkers herein described.
• Bacterial strains and culture conditions and mice B. fragilis strain NCTC 9343 was obtained from the American Type Culture Collection, its isogenic PSA deletion mutant and mpi44 mutant (produces only PSA but not other polysaccharides) has been described (MJ. Coyne, A.O. Tzianabos, B.C. Mallory, VJ. Carey, D. L. Kasper and L.E. Comstock, (2001) Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis, Infect. Immun.
• Bacteria were grown either in a rich medium containing 37g BHI (BD #237200), 0.5 ⁇ g/ml Hemin (Sigma H5533), and 0.5 ⁇ g/ml Vitamin K (Sigma V3501) in IL ddH 2 O or a customized minimum medium (MM), which contained 8g Glucose, 1% FBS, 0.5 ⁇ g/ml Hemin, and 0.5 ⁇ g/ml Vitamin K in IL of RPMI (Invitrogen SKU#11835-030).
• SPF mice of strains C57BL/6 and Balb/c were purchased from Taconic Farms (Germantown, NY).
• TLR2 "7" and IL-10 knockout mice were purchased from Jacksom laboratories IL-10GFP mice were procured from the laboratory of Christopher Karp from Cinncinati Childrens medical hospital, and, Foxp3GFP mice were a given by the laboratory of Talal Chatila from the University of California Los Angeles.
• OMV purification and labeling This method is adapted from a previously described protocol for the preparation of OMVs from E. coli (Amanda L. Horstman and Meta J. Kuehn. (2000) Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles. / Biol Chem. 275: 12489-12496.) Briefly, EDL-enriched B. fragilis was grown in customized MM. OMVs were recovered from the bacteria-free supernatant of the culture by centrifugation at 40,00Og for 2hrs at 4°C and further washed twice with PBS and filtered through 0.45 ⁇ m spin columns (Millipore #20-218).
• OMVs Total protein concentration of the purified OMVs was determined by Bradford assay (Biorad #500-0205).
• FITC-labeled OMVs were prepared as previously described (Nicole C. Kesty and Meta J. Keuhn. (2004) Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles. / Biol Chem. 279: 2069-2076). Briefly, OMVs were incubated in the staining buffer (lmg/ml FITC (Thermo Scientific #46424), 10OmM NaCl, 5OmM Na 2 CO 3 , pH 9.2) for lhr at RT. Labeled OMVs were collected by centrifugation at 40,00Og for 30min at 4°C and washed twice with PBS+200mM NaCl.
• Microb Pathog_. 20(4): 191-202. Briefly, a tiny drop of purified OMV was applied to formvar/carbon coated gold grids (EMS #FF200-Au) and air-dried. Immunogold labeling was performed at RT by floating these grids with "OMVs"-side down on a series of small drops of antibody and wash solutions. Particularly, samples were blocked in 10% FBS for lOmin after 5min incubation in 0.12% glycine. After blocking, samples were further incubated with antibody against PSA diluted in 10% FBS for 20min, followed by 5 washes x 3min with PBS. Subsequently, secondary antibody- IgG conjugated with 5nm gold (kind gift from Dr.
• TNBS Chemical (TNBS)-induced experimental colitis. This protocol is adapted from a previously described method (Scheiffele and Fuss. (2001) Induction of TNBS colitis in mice. Current Protocols in Immunology.15.19.1-15.19.14.) Briefly, wild-type (Balb/c) male mice were orally treated with PBS, WT-OMV (5 ⁇ g) or ⁇ PSA-OMV (5 ⁇ g) every other day for a week before TNBS administration. The treated mice were anesthetized with isofluorene and rectal administration of 2% TNBS (in 50% EtOH, Sigma P2297) was applied through a 3.5F catheter (Instech Solomon; SIL-C35).
• Tissue pathology analysis Mouse colons were fixed in neutral buffered 10% formalin (ScyTek Laboratories CAS#50-00-0) and processed by Pacific Pathology for H & E staining. Colitis scores for each colon section were evaluated in a blinded fashion by a pathologist (Dr. Gregory Lawson, David Geffen School of Medicine, UCLA, Los Angeles). Histology images were taken using light microscopy (Zeiss) at 2Ox magnification.
• Quantitative real-time PCR RNA was collected either from mouse tissues using Trizol (Invitrogen #15596-018) or from purified cells using RNeasy Mini Kit (Qiagen #74104).
• iSCRIPT cDNA synthesis kit BioRad #170-8890 was used for conversion of cDNA and IQ SYBR Green supermix (BioRad #172-8882) was used for real-time PCR.
• BMDCs from OMV uptake assay or activation assay were collected and blocked in 5% mouse serum for 30min on ice. After blocking, cells were stained with anti-CD 1 Ic-APC, anti-MHCII-FITC or anti-CD86-PE (ebioscience) for 30min on ice and washed 2x with FACS buffer (HBSS (no Ca 2+ Mg 2+ ), 1% FBS, 2mM EDTA, 1OmM HEPES) at 4 0 C before flow cytometry analysis.
• FACS buffer HBSS (no Ca 2+ Mg 2+ )
• FBS 1% FBS
• 2mM EDTA 1OmM HEPES
• CD4 + splenic T cells were isolated by magnetic microbead purification (Miltenyi Biotec #130-090-860) (Cell Purity is > 95%.)
• OMV- pulsed BMDCs (lO ⁇ l/ml OMVs, 100,000 cells/ml, 12hrs-24hrs) were washed with HBSS and then incubated with CD4 + T cells (1,000,000 cells/ml) in a round bottom 96 well plate with addition of O.Ol ⁇ l/ml anti-CD3 (day 0, Fig 3c as indicated, 3d, e, f , Fig 4a), 2ng/ml TGFb (day 0, Fig 3c, d & f , Fig4a), and 5ng/ml IL-2 (day 1 and day 3, all in vitro DC-T cell co-culture assays). After total 4 days of culture, supernatants were collected for ELISA (ebioscience #88- 7104-77) or cells were harvested for
• CD4 + CD25 + cells purified from BMDC (pulsed with WT- OMV or ⁇ PSA-OMV)-T cell co-culture were used as a source of Tregs (Miltenyi Biotec, #130- 091-041).
• CD4 depleted mouse splenocytes treated with Mitomycin C (Sigma M4278) were used as APCs (100,000 cells/ml).
• CD4 + CD25 " T cells directly purified from mouse spleen were pulsed with CFSE for lOmin at 37 0 C, followed by first wash with PBS and a second wash with culture media, and used immediately (500,000 cells/ml) as responder cells (Teff).
• This assay was conducted in a round bottom 96 well plate with an addition of 5 ⁇ g/ml of anti-CD3 (ebioscience #16-0031-86) in 200 ⁇ l volume. Teff:Treg ratio was titrated and cells were collected after 2-3 days of culture for FACS analysis.
• fragilis APSA illustrate no defect in OMV synthesis, and the size, shape and abundance of OMVs produced were indistinguishable from wild-type bacteria ( Figure Ia and Figure 5).
• Figure Ia the results illustrated in Figure Ia reveal that vesicles are actively budding from the surface of bacteria.
• PSA is a heterogeneous polymer of repeating subunits. Size separation of PSA recovered from whole cell extracts by chromatography was performed as well as an immunoblot analysis with anti-PSA of capsular polysaccharide preparations from whole cells and purified OMVs as indicated in material and methods.
• Example 2 OMVs protect animals from experimental colitis and intestinal inflammation in a PSA-dependent manner.
• mice were treated by gavage with OMVs during the induction of TNBS (2,4,6-trinitrobenzene sulfonic acid) colitis.
• TNBS 2,4,6-trinitrobenzene sulfonic acid
• Example 3 PSA containing OMVs inhibits TNF- ⁇ /IL-17, enhances IL-IO expression.
• the production of canonical pro- and anti-inflammatory cytokines associated with colitis was measured in mice treated as exemplified in Example 2.
• cytokine transcript analysis was performed by qRT-PCR from RNA recovered from whole colons or purified CD4 + T cell from mesenteric lymph nodes.
• Example 4 PSA containing OMVs from B. fragilis induce dendritic cell responses.
• DCs Dendritic cells
• MLNs Dendritic cells
• PSA administered orally to animals is associated with CDl Ic + DCs in the MLN. Accordingly, Applicants sought to test whether OMV containing PSA can also be taken up by DCs.
• PSA mediated induction in the DCs of T cell activation markers was also investigated.
• the results illustrated in Figure 3b indicated that expression of T cell activation markers (MHCII, CD86) was elevated equally following internalization of both WT-OMVs and ⁇ PSA- OMVs ( Figure 3b and Figure 10).
• Increased expression of MHC and co-stimulatory molecules indicate that PSA containing OMVs from B. fragilis can influence T cell responses.
• Example 5 PSA induce IL-IO expression in CD4 + T cell through DCs IL-IO expression.
• Example 6 PSA programs DCs to direct Foxp3 Treg development and/or expansion.
• Microbial ligands are sensed by several classes of pattern recognition receptors, and PSA has been shown to signal through toll-like receptor 2 (TLR2) to elicit ThI cytokine production.
• TLR2 toll-like receptor 2
• a series of experiments were therefore performed to test whether TLR2 is required for induction of IL-IO by PSA, as recent reports have shown that Treg function and IL-IO expression are influenced by TLR2.
• CD4 + CD25 + and CD4 + CD25 " T cells were purified following co-culture with DCs and the expression of IL-IO and Foxp3 were measured by qRT-PCR.
• Example 7 PSA-containing OMVs induce ILlO mediated Foxp3 Treg suppressive function
• the use of Tregs as cellular therapies has been proposed for IBD, autoimmunity and allergies.
• IL-IO expression by CD4 + T cell subsets following 4 day co-culture with DCs treated with OMVs was investigated.
• Splenic CD4 + T cell were purified from IL-IO-GFP mice, stained with anti-CD4 and anti-CD25 following co-culture, and IL-IO expression measured by GFP expression.
• CD4 + CD25 " responder cells effector cells; Teff) were purified from spleens of wild- type mice, pulsed with intracellular dye CFSE (Carboxyfluorescein succinimidyl ester), incubated with Tregs and stimulated with anti-CD3 for 3 days. Cell proliferation was measured by FC of CFSE dilution.
• CFSE Carboxyfluorescein succinimidyl ester
• Example 8 PSA induce various biomarkers combination in Foxp3 Treg
• Foxp3-GFP mice were orally treated with purified PSA every other day for 6 days.
• MLNs were extracted and CD4+Foxp3+ or the CD4+Foxp3- T cells were purified by FACS based on ⁇ GFP expression (purity >99%).
• RNA was extracted and used for q-PCR.
• the above results provide a molecular mechanism for the probiotic activity of B. fragilis, and reveal a seminal example for a microbial ligand that links innate immune receptor signaling to regulatory T cell development.
• PSA is delivered to the host by outer membrane vesicles (OMVs), secretion structures that target bacterial molecules to host cells.
• OMVs containing PSA are internalized by dendritic cells of the host immune system.
• PSA programs dendritic cells to induce the differentiation of regulatory T cells (Treg) that express Foxp3 and the anti-inflammatory cytokine interleukin-10 (IL-IO).
• Treg development by OMVs requires toll-like receptor 2 (TLR2) expression and IL-IO production by dendritic cells.
• TLR2 toll-like receptor 2
• purified OMVs direct the in vitro differentiation of functional Tregs with potent suppressive activity in a PSA dependent manner.
• Treatment of animals with OMVs containing PSA prevents experimental colitis and suppresses pro-inflammatory cytokine responses in the gut.
• fractions of bacterial products can be purified and the same assays performed as above until a pure compound is found which mimics that outcome when whole bacteria are used. Also, the approaches above could be used to screen a mutant library of a microorganism of interest to identify immunomodulatory molecules that have been deleted in a respective clone of a strain which possesses this activity.

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