Therapeutic induction of innate immune responses in target tissues

Abstract

To provide therapeutic compositions that present an artificial repertoire of mammalian pattern recognition receptor (PRR) agonists so that the pattern of PRR agonists recapitulates a distinct portion of a PRR agonist signature of a mammalian pathogen.SOLUTION: The artificial repertoire of PRR agonists may be formulated together in a therapeutic vehicle for combined presentation to an innate immune cell resident in a target tissue in a mammalian host, and such a vehicle is adapted to deliver the PRR agonists to the target tissue so as to modulate an immune response.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] Innovations have arisen with respect to preparations that include immunogens, such as microbial components, that have been disclosed in the medical and veterinary sciences, that have been formulated for medical purposes, and that provide methods for using such preparations in therapy. [Background technology]

[0002] Immunological dysregulation, a state of imbalance between immune response and immune tolerance, is increasingly being recognized not only as a major contributing factor in allergic and autoimmune diseases, but also as playing a fundamental mechanistic role in a wide variety of pathologies, including cancer (see Mills et al., 2016, Cancer Res; 76(3); 1-4), metabolic diseases (obesity, diabetes), degenerative diseases (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, osteoporosis), respiratory and cardiovascular diseases (see Immune Rebalancing, 1st Edition: The Future of Immunosuppression, 2016, Boraschi and Penton-Rol Eds, Academic Press).

[0003] In vertebrates, the activities of the innate and adaptive immune systems are coordinated in key aspects of immunological regulation, involving metabolic, enzymatic, and molecular genetic changes within immune cells that orchestrate an elaborate system of cellular, cytokine, and chemokine communication pathways that mediate the coordinated activities of the different components of the complement system (see Iwasaki & Madzhitov, 2015, Nature Immunology 16:343-353; WO0209748; WO03051305; Turner et al., 2014, BBA-Molecular Cell Research 1843:11 2563-2582). This aspect of coordinated activity is the basis for the recognition that the use of ligands of pattern recognition receptors (PRRs) of the innate immune system as vaccine adjuvants can improve adaptive immune responses (see Maisonneuve et al., 2014, PNAS 111(34), 12294-9; WO2007035368).

[0004] Immunological memory involves the recognition of specific antigens by B and T cell receptors and is a long-standing key feature of the adaptive immune system and the basis of vaccine efficacy (see Nature Immunology, Focus on immunological memory: June 2011, Volume 12 No 6 pp461-575). Innate immune memory has been recognized more recently and is less well understood in the immune system (see Netea et al., 2015, Nature Immunology 16, 675-679, and Bordon, 2014, Nature Reviews Immunology 14, 713).

[0005] Non-lymphoid tissues are understood to be resident to a wide variety of innate and adaptive immune cells, which have diverse roles in tissue homeostasis (see Nature Immunology, Focus on tissue-resident leukocytes, October 2013, Volume 14 No 10 pp977-1100). The complexity of this homeostasis can be seen in the finding that the development of tissue-resident immune cells can even differ from the development of similar immune cells that are not tissue-resident (Italiani and Boraschi, Frontiers in Immunology, October 2014, Vol 5, article 514). Summary of the Invention [Means for solving the problem]

[0006] Provided is an immunomodulatory or immunogenic composition that is an artificial repertoire of mammalian pattern recognition receptor (PRR) agonists. The PRR agonist repertoire is selected so that it effectively reproduces different parts of the PRR agonist signature of a microbial pathogen, more specifically, a pathogen that is pathogenic in a selected target tissue. The PRR agonist signature is different in the sense that it is different from the PRR agonist signature of a microorganism that is not pathogenic in the target tissue, and is also different in the sense that it is different from the natural PRR agonist signature of a wild-type pathogen. And this different artificial repertoire of PRR agonists can be formulated so that the PRR agonists are presented together in a therapeutic medium, for example, the PRR agonist repertoire can be presented in combination. The therapeutic medium can be, for example, a recombinant microorganism, 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 different mammalian PRRs, the number of different mammalian PRRs being, for example, at least 5, as described in further detail herein. The vehicle may then be delivered, for example, systemically, to present the PRR agonist repertoire to innate immune cells resident in the target tissue of a host, such as a mammalian host. The therapeutic vehicle may, for example, assemble an artificial repertoire of PRR agonists such that the proximity of multiple PRR agonists is maintained during systemic distribution in the host. This type of composition may be used to treat a wide variety of diseases characterized by immune dysregulation, including neoplastic and autoimmune diseases.

[0007] Aspects of this innovation include the use of an immunogenic composition in a method for treating immune dysregulation in a target tissue of a mammalian host, the composition comprising the aforementioned artificial repertoire of mammalian PRRs. The artificial repertoire of PRR agonists can be formulated together in a therapeutic vehicle to be presented in combination after administration to a mammalian host. The composition can, for example, comprise components of a microbial mammalian pathogen that are agonists for a selected number of different mammalian PRRs, the number of different mammalian PRRs being discussed in more detail below, for example, at least 5. The composition can, for example, be suitable for use to modulate an innate immune response in a target tissue. The therapeutic vehicle can, for example, comprise a recombinant microorganism, a cellular fraction of a recombinant microorganism, a cellular fraction of a microbial cell, a microparticle, or a liposome, each of which comprises a component of a microbial mammalian pathogen that provides a PRR agonist that together constitutes an artificial repertoire of PRR agonists. The recombinant microorganism can, for example, comprise a recombinant gene encoding at least one component of a PRR agonist. In alternative embodiments, the therapeutic medium may comprise, for example, whole killed or whole attenuated cells of recombinant microorganisms. Alternatively, cellular fractions of microbial mammalian pathogens may be used, such as bacterial outer membrane fractions, bacterial inner membrane fractions, pellets obtained from gradient centrifugation of microbial cell components, or chromosomal DNA. The therapeutic medium may be formulated, for example, for use in delivering PRR agonists to target tissues.

[0008] In alternative embodiments, the PRR and corresponding PRR agonist may be selected from the group consisting of, for example, those set forth in the following table:

[0009] [Table 1]

[0010] [Table 2]

[0011] The therapeutic medium may include additional therapeutic moieties, such as, for example, one or more of GMCSF, vitamin D, NOHA, alpha 1 antitrypsin, glutathione, isoprenoids, or alpha-galactosylceramide. In alternative embodiments, the therapeutic medium further includes an antigen, such as a cancer antigen. Alternatively, the therapeutic medium may further include a heterologous PRR agonist, such as a PRR agonist that is not a component of a microbial mammalian pathogen.

[0012] The subject of treatment, such as a mammalian host or human patient, may be one suffering from a disease or condition characterized by immune dysregulation in a target tissue, such as, for example, cancer or an inflammatory disorder.

[0013] The composition may be suitable for use in an amount effective to modulate biomarkers such as, for example, one or more of PD1, PDL1, IP-10, MIG, RANTES, neutrophils, Ly6C monocytes, and NKG2D. In alternative embodiments, the composition may be suitable for use in an amount effective to down-regulate expression of PD1 and / or PDL1 in cells present in a target tissue. Thus, the composition may be suitable for use to modulate an adaptive immune response in a host, for example, as a co-agent to modulate an innate immune response.

[0014] The therapeutic vehicle is intended for administration to an administration site that is not a target tissue, and such site may be, for example, the skin, subcutaneous tissue, or airway. Administration may be enteral or non-enteral. The therapeutic vehicle may be formulated so that the PRR agonist is distributed systemically after administration to a local administration site. The therapeutic vehicle may be administered in multiple doses over an administration period, which may be, for example, at least two weeks, or any of the various other administration regimens disclosed herein or known in the art.

[0015] In alternative embodiments, the human patient treated according to the present invention may be, for example, immunosuppressed or immunocompromised, or may be a geriatric or pediatric patient.

[0016] The therapeutic applications described herein are reflected in the corresponding methods of treatment, and vice versa. [Brief description of the drawings]

[0017] [Figure 1] Schematic timeline of site-specific immunotherapy (SSI) according to one embodiment of the present invention showing intratracheal (IT) instillation of K. pneumoniae (KPN) whole killed cell SSI on day -31 and subcutaneous (SQ) injections of SSI or saline (placebo) every other day starting on day -10 (Lewis Lung Carcinoma (LLC) is administered intravenously (IV) on day 0), followed by sacrifice (sac) on day 18. [Diagram 2] 1 is a graph showing the therapeutic efficacy of alternative SSI formulations in a mouse cancer model. [Diagram 3] Schematic timeline showing a mouse pre-infection model of SSI-mediated anti-tumor efficacy. [Figure 4] 1 is a graph showing the anti-cancer efficacy of SSI following pre-infection in a mouse Lewis Lung Carcinoma (LLC) cancer model. [Diagram 5] 1 is a line graph showing tumor volume over time for alternative SSI treatments in a mouse B16 skin cancer model. Herein, 10X QBSAU and 1X QBSAU are designated as QBSAUR and QBSAU, respectively. [Figure 6] 1 is a bar graph showing tumor volumes at day 7 for alternative SSI treatments in a mouse B16 skin cancer model. [Figure 7] 1 is a bar graph showing tumor volumes at day 8 for alternative SSI treatments in a mouse B16 skin cancer model. [Figure 8] 1 is a bar graph showing tumor volumes at day 10 for alternative SSI treatments in a mouse B16 skin cancer model. [Figure 9] 1 is a bar graph showing tumor volumes at day 12 for alternative SSI treatments in a mouse B16 skin cancer model. [Figure 10]1 is a bar graph showing tumor volumes at day 14 for alternative SSI treatments in a mouse B16 skin cancer model. [Figure 11] Schematic diagram showing the administration schedule of SSI (upper panel) and bar graph showing the therapeutic efficacy of SSI in a mouse cancer model (lower panel). [Figure 12] 1 is a chart showing the efficacy of various SSI combinations in mouse cancer models. [Figure 13] FIG. 13 is an alternative bar graph diagram showing the efficacy of various SSI combinations in mouse cancer models. [Figure 14-1] FIG. 1 is a series of graphs showing the efficacy of SSI treatment in a surrogate animal model of colitis. The logarithmic scale of the Y-axis shows the relative expression levels of IFN-gamma (A) and IL-17A (B). [Figure 14-2] A series of graphs showing the efficacy of SSI treatment in surrogate animal models of colitis, showing cumulative IL-17A expression data (C) and evidence that the efficacy of QBECO to increase IL-18 gene expression compared to QBKPN (D) is site-specific in colon tissue. [Figure 15-1] FIG. 1 is a series of graphs showing efficacy of SSI treatment in a surrogate animal model of colitis; the microbiome (A) is shown. [Figure 15-2] A series of graphs showing the efficacy of SSI treatment in surrogate animal models of colitis, showing the microbiome (B) as well as histology (C). [Figure 16] 1 is a bar graph showing the efficacy of SSIs in a mouse asthma / allergy model. [Figure 17] FIG. 1 includes two bar graphs showing efficacy of SSI in a mouse asthma / allergy model: A) eosinophil counts; B) lymphocyte counts. [Figure 18] FIG. 1 includes two bar graphs showing efficacy of SSIs in a mouse asthma / allergy model, showing A) IL-4 concentrations and B) IL-5 concentrations. [Figure 19]1 is a bar graph showing the results of ex vivo imaging of Cy5.5-labeled KPN SSI (QBKPN) measured in organs (heart, lung, and spleen) 24 hours after the third subcutaneous injection of SSI. [Figure 20] 1 is a bar graph showing HDM-specific IgE responses following exposure to saline or house dust mites (HDM) and treatment with either placebo or QBKPN. [Figure 21] A series of bar graphs (A-E) depicting the profile of anti-inflammatory SSI treatment for asthma derived from animal models, specifically, BAL cell numbers and differences in saline- or HDM-exposed mice treated with placebo or QBKPN SSI. [Figure 22] FIG. 1 includes two bar graphs showing the profile of anti-inflammatory SSI treatment for asthma derived from animal models, specifically, serum (A) and BAL (B) mediators associated with eosinophilia. [Diagram 23] FIG. 1 is a series of bar graphs showing the profile of anti-inflammatory SSI treatment for asthma derived from an animal model, specifically showing Th1 (A) and Th2 (B and C) gene expression in the lungs following HDM exposure and QBPKN treatment. [Figure 24] FIG. 1 is a series of bar graphs depicting the profile of anti-inflammatory SSI treatment for asthma derived from animal models, specifically the effect of HDM exposure and QBKPN treatment on levels of Th1-mediated (A-C) and Th2-mediated (D-F) cytokines in BAL fluid. [Diagram 25] FIG. 1 is a graph showing the profile of anti-inflammatory SSI treatment for asthma derived from an animal model, specifically, a principal component analysis (PCA) of BAL cytokines showing a partially normalized total cytokine profile. [Figure 26] FIG. 1 is a bar graph showing the profile of anti-inflammatory SSI treatment on asthma derived from an animal model, specifically quantification of airway goblet cells following HDM exposure and QBPKN treatment. [Figure 27-1]FIG. 27A is a bar graph showing the profile of anti-inflammatory SSI treatment for COPD from an animal model, specifically showing differences in BAL cells. [Figure 27-2] Figure 27B reflects this data and shows that the increase in macrophages and lymphocytes induced in the lung by cigarette smoke exposure was attenuated by KPN SSI intervention treatment, but not the increase in total cells or neutrophils. Figure 27B shows the number and difference in BAL cells after treatment with placebo and KPN SSI in the filtered air or cigarette smoke exposed groups: (a) BAL total cells, (b) lymphocytes, (c) macrophages, and (d) neutrophils. *p<0.05 compared to these groups and controls, #p<0.05 compared to the KB group and placebo control. Data are mean ± SD (9-10 mice per group). [Figure 28-1] Figure 28A shows data indicating that the increase in cigarette smoke exposure-induced Th1-biased lung inflammatory response (listed below) was attenuated by KPN SSI intervention treatment. BAL supernatants were analyzed after placebo and KB treatment in filtered air or cigarette smoke exposed groups: (a) IFNγ, (b) CXCL9, (c) CXCL10, (d) CCL5, (e) IL-6, (f) G-CSF, (g) CXCL1, (h) IL-17. *p<0.05 compared to these groups and controls, #p<0.05 compared to KPN SSI group and placebo control. Data are mean ± SD (10 mice per group). [Figure 28-2] Figure 28B shows data indicating that tobacco smoke exposure-induced changes in serum immune mediators, including: (a) VEGF, (b) IL-1β, (c) CCL2, (d) CXCL9, (e) CXCL10, and (f) CCL5, were analyzed after treatment with placebo and KPN in the filtered air or tobacco smoke exposed groups. *p<0.05 compared to these groups and controls, #p<0.05 compared to the KB group and placebo control. Data are mean ± SD (9-10 mice per group). [Figure 28-3] Figure 28C shows data showing that blood and lung Ly6CHI monocytes and neutrophils (listed below) were increased by KPN SSI intervention treatment. Ly6CHI monocytes and neutrophils in blood (a-b) and lungs (c-d) were analyzed by flow cytometry after placebo and KB treatment in filtered air or tobacco smoke exposed groups. *p<0.05 compared to these groups and controls. #p<0.05 compared to KB group and placebo control. Data are mean ± SD (10 mice per group). [Figure 28-4] FIG. 28D is a series of bar graphs (A-C) depicting the profile of anti-inflammatory SSI treatment for COPD derived from animal models, specifically the expression profile of selected genes in the lung. [Figure 29] FIG. 1 is a series of bar graphs (A-G) depicting the profile of anti-inflammatory SSI treatment for COPD derived from animal models, specifically the expression profile of select BAL cytokines. [Diagram 30] FIG. 1 is a series of bar graphs (A-C) showing the profile of anti-inflammatory SSI treatment for COPD derived from animal models, specifically, serum cytokine expression profiles. [Diagram 31] 1 is a bar graph showing that use of Klebsiella variicola SSI reduces tumor burden in a B16 melanoma model of lung metastasis. [Diagram 32] 1 is a bar graph showing the efficacy of QBKPN SSI in reducing lung nodules in the absence of CD25 positive cells. [Figure 33-1] Figure 33A is a bar graph showing delta Ct (cycle threshold) values ​​associated with administered KPN SSI formulations (QBKPN) in a B16 melanoma model of lung metastasis, where the KPN SSI was administered in serial dilutions (10x, 100x, and 1000x). The delta Ct values ​​are inversely proportional to the amount of target nucleic acid in the sample. As shown, tumor burden increased with increasing dilution of the SSI. [Figure 33-2]Figure 33B is a bar graph showing the dose-dependent similar effect of KPN SSI, as measured by the number of B16 tumor nodules in the lungs, and Figure 33C is a bar graph showing the therapeutic effect of various dosing regimens, all of which had injection intervals ranging from 1 to 7 days. [Diagram 34] FIG. 1 contains two bar graphs showing that the percentage of cells expressing Rae-1 inversely correlates with tumor burden in a B16 melanoma model of lung metastasis (A) and that this is dependent on NKG2D expression (B). [Diagram 35] 1 is a bar graph showing a significantly enhanced effect of QBKPN SSI in reducing lung tumor nodules in the Lewis Lung Carcinoma (LLC)-RFP model. [Diagram 36] 1 is a bar graph showing a concomitant decrease in the number of LLC-RFP cells in the lungs 15 days after LLC inoculation. [Figure 37] 1 is a line graph showing a survival advantage obtained from QBECO SSI compared to that obtained from either QBKPN or QBSAU in an MC38 colon cancer model. [Figure 38] 1 is a bar graph showing that mice treated with QBKPN SSI, but not 10× QBSAU, showed increased lung-specific Rae-1 expression in skin and lung tumor-bearing models. [Figure 39] 1 is a bar graph showing that PD-1 expression was decreased in the lungs of mice treated with QBKPN compared to placebo in a skin and lung tumor model. [Diagram 40] 1 is a bar graph showing that treatment of mice with 10× QBSAU, but not QBKPN, reduced skin tumor burden compared to placebo controls in a skin and lung B16 tumor model. [Diagram 41]1 is a bar graph showing that both intravenous (IV) and subcutaneous (SQ) SSI treatments provide therapeutic benefit in the B16 lung metastasis model. [Diagram 42] 1 is a schematic timeline showing as an example a study design based on the efficacy of QBKPN in treating and preventing cancer in a B16 lung cancer model. [Diagram 43] It contains four bar graphs showing the efficacy of QBKPN in treating and preventing cancer in the B16 lung cancer model. [Diagram 44] 1 is a bar graph showing the efficacy profile of QBKPN in treating and preventing cancer in a B16 lung cancer model. [Diagram 45] 1 is a bar graph showing profile of how quickly treatment of an SSI produces a detectable therapeutic effect involving myeloid cell populations, specifically neutrophils. [Figure 46] 1 is a bar graph showing profile of how quickly SSI treatment produces a detectable therapeutic effect involving myeloid cell populations, specifically Ly6C monocytes. [Figure 47-1]

[0036] Figure 47A(i) is a series of graphs showing the efficacy of alternative cell fractions in the lung of a B16 melanoma model, including dose-dependent and site-specific efficacy. Figure 47A(i) shows that both 1X KPN outer membrane fraction and 0.01X KPN outer membrane fraction were effective in a dose-dependent manner (the 1X fraction had equivalent efficacy to the whole killed cell formulation). [Figure 47-2]

[0036] Figure 47A(ii) is a series of graphs showing the efficacy of alternative cell fractions in the lung of a B16 melanoma model, including dose-dependent and site-specific efficacy. Figure 47A(ii) shows that both 1X and 0.01X KPN outer membrane fractions were effective in a dose-dependent manner (1X fraction was as effective as the whole killed cell formulation), which was also true for 1X and 4X DNA fractions. [Figure 47-3]A series of graphs showing the efficacy of alternative cell fractions in the lungs of the B16 melanoma model, including dose-dependent and site-specific efficacy. Figure 47A(iii) shows that the intimal fraction showed a dose-dependent trend, but this trend did not have strong statistical significance. Figure 47B is a bar graph showing the results after 10 injections of adventitial SSI, showing that the adventitial fraction increased Rae-1 expression in a dose-dependent manner. [Figure 47-4] Figure 47C is a series of graphs showing the efficacy of alternative cell fractions in the lungs of a B16 melanoma model, including dose-dependent and site-specific efficacy. Figure 47C includes two bar graphs showing OM dose-dependent increases in blood neutrophil and monocyte counts in blood collected 2 days prior to tumor implantation after four injections of QBKPN SSI, placebo, or various concentrations of OM fractions (0.01X, 1X, 10X, or 20X). [Figure 47-5] Figure 47A is a series of graphs showing the efficacy of alternative cell fractions in the lung of a B16 melanoma model, including dose-dependent and site-specific efficacy. Figure 47D is a column scatter plot showing that the KPN fraction has site-specific efficacy compared to the E. coli fraction in a B16 lung cancer model. [Figure 48] 1 is a bar graph showing that high doses (1 / 20 dilution, 1 / 200 dilution) of QBKPN SSI increase the death of NCI-H358 cancer cells in a 24 hour killing assay. [Figure 49] 1 is a bar graph showing increased γδ T cell mediated killing of NCI-H358 cancer cells by alternative doses (1 / 20 dilution, 1 / 200 dilution) of KPN SSI in a 24 hour cell killing assay. [Figure 50] 1 is a bar graph showing that KPN SSI (QBKPN) at 1 / 200 and 1 / 2000 dilutions enhanced the effect of zoledronate in inducing γδ T cell-mediated cancer cell lysis in a 24 hour cell killing assay. [Figure 51] 1 is a line graph showing the therapeutic efficacy of QBECO SSI in an MC-38 colon cancer model. [Figure 52] 1 is a line graph showing that NKG2D expression correlates with QBECO efficacy in the MC-38 colon cancer model using NKG2D knockout mice. [Figure 53] FIG. 1 is a schematic diagram of a treatment profile in a neutropenic model system. [Figure 54] FIG. 1 is a set of four graphs showing flow cytometry results in a neutropenic model, gating on viable CD45+CD11b+ cells and enumerating specific cell populations from lung samples. [Figure 55] Two column scatter plots are included showing the percentage (A) and number (B) of neutrophils in lung samples from a neutropenic model. [Figure 56] Two figures are included, showing the percentage (A) and number (B) of neutrophils in spleen samples from a neutropenic model. [Figure 57] 1 is a bar graph showing the percentage of cells with the indicated characteristics in blood samples from lung cancer patients separated into neoplastic and pre-neoplastic patient populations, showing increased expression of PDL1 and PD1 in the neoplastic patient population compared to pre-neoplastic patients. [Figure 58] 1 is a bar graph showing the relative numbers of cells with the indicated characteristics in blood samples from lung cancer patients separated into neoplastic and pre-neoplastic patient populations, showing increased expression of PDL1 and PD1 in the neoplastic patient population compared to pre-neoplastic patients. [Figure 59] Contains two bar graphs showing SSI-mediated decrease in PD-L1 expression in neoplastic lung cancer in two patients (Patient 01-001 (Panel A) and Patient 01-002 (Panel B)) at week 1 day 4 (W1D4), week 1 day 5 (W1D5), week 2 (W2), week 4 (W4), week 12 (W12), and week 16 (W16). [Figure 60] It contains two bar graphs showing SSI-mediated decrease in PD-1 expression in two neoplastic lung cancer patients, patient 01-001 (Panel A) and patient 01-002 (Panel B). [Figure 61]It contains two bar graphs showing that the proportion of M1 macrophages was increased in two neoplastic lung cancer patients, patient 01-001 (Panel A) and patient 01-002 (Panel B). [Figure 62] Two bar graphs are included, showing RT-qPCR fold changes of (A) GzmA, GzmB, Prf1, and (B) Tyr in the lungs of B16-vaccinated mice receiving various doses of QBKPN. Data points are mean + / - SD. Significance was calculated using one-way Tukey multiple comparison ANOVA test. **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 63] Contains three bar graphs showing activation of pattern recognition receptors in HEK cells following stimulation with QBECO or QBKPN, respectively showing A) activation of Toll-like receptors (TLRs) (measured by activation of NK-κB), B) NOD2 and C-type lectin receptors (CTLs) (measured by activation of NK-κB), and C) RLRs (Rig-1-like receptors) (measured by activation of IRF3). [Figure 64] 1 is a bar graph of PRR repertoire fingerprints, in which PRR fingerprints for QBECO SSI and QBKPN SSI were constructed from 1 / 10 dilution data after subtraction of negative control data. Bars represent, in order, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1, NOD2, Dectin1a, Dectin1b, and Mincle. RIG-1 and MDA5 are not shown. Positive controls are specific for each PRR (i.e., LPS for TLR4). [Figure 65] 1 is a radar graph of PRR fingerprints, in which PRR fingerprints for QBECO SSI and QBKPN SSI were constructed from 1 / 10 dilution data after subtraction of negative control data and plotted on a radar graph. [Figure 66]Bar graph showing neutrophil levels in blood on day 7 after treatment with placebo, QBKPN, or rabies vaccine. Neutrophil levels were measured by flow cytometry and assessed as the percentage of neutrophils (Ly6G+) among total CD45+ cells. N=4–5 mice per group. * indicates P<0.05 compared to placebo and was assessed by Student's t-test. Mean values ​​± standard deviation are shown. QBKPN is a bacterial SSI from Klebsiella. Rabies is Imrab 3TF rabies vaccine, which contains killed rabies virus. [Figure 67] Bar graph showing Ly6CHI monocyte levels in blood on day 7 after treatment with placebo, QBKPN, or rabies vaccine. Ly6CHI monocyte levels were measured by flow cytometry and assessed as the percentage of Ly6CHI monocytes (Ly6CHILy6G-) among total CD45+ cells. N=4–5 mice per group. * indicates P<0.05 compared to placebo and was assessed by Student's t-test. Mean ± standard deviation is shown. QBKPN, bacterial SSI from Klebsiella. Rabies, Imrab 3TF rabies vaccine, which contains killed rabies virus. Fel-O-Vac, feline rhinotracheitis-calicivirus infection-panleukopenia vaccine, which contains the three killed viruses. Nobivac is a canine influenza H3H8 vaccine, which contains killed influenza H3H8. [Figure 68] Column scatter plot showing that boosting cancer antigens with QBKPN enhances the effect of melanoma-associated antigen gp100. Antitumor efficacy of QBKPN SSI in combination with gp100 is compared to an irrelevant control antigen, OVA (SIINFEKL), including OVA adjuvanted with CpG. [Figure 69]Column scatter plot showing surface metastasis-like tumor nodules in mice burdened with B16 melanoma, providing evidence that augmenting the microbial SSI QBKPN with an additional PRR agonist, the STING agonist 2'2'-cGMAP, enhances its efficacy. [Figure 70] Column scatter plot showing treatment-induced IFN-γ levels in plasma of B16 melanoma-burdened mice, providing evidence that augmenting the microbial SSI QBKPN with STING agonists enhances IFN-γ levels. [Figure 71] Figure 71A is a violin plot showing the logarithmic distribution of risk scores, comparing the last recorded response for all CD subjects using a risk score based on 112 IBD SNPs (P value 2.430E-05). Figure 71B is a violin plot showing the logarithmic distribution of risk scores, comparing the last recorded response for all CD subjects using a risk score based on 3 IBD SNPs (P value: 1.385E-04). [Figure 72] Violin plot showing logarithmic distribution of risk scores, comparing last recorded response for all UC subjects using a risk score based on 84 IBD SNPs (P value: 1.255E-02). [Figure 73] Violin plot showing the logarithmic distribution of risk scores, comparing last recorded response for all CD and UC subjects using a risk score based on 112 IBD SNPs (P value: 8.184E-07). [Figure 74] 1 is a graph showing the change in serum IL-18 levels in patients treated with QBECO compared to patients treated with placebo. [Figure 75] FIG. 1 is a set of four graphs showing that changes in serum immune cytokines with QBECO treatment correlate with clinical response. [Figure 76] FIG. 1 is a series of three graphs showing baseline levels of Eotaxin-1, IL-10, and IL-12p40 by patient response to QBECO. [Figure 77]Fig. 1 is a graph showing changes in body weight over time in a mouse DSS colitis model. [Figure 78] 1 is a graph showing changes in disease activity index over time in a mouse DSS colitis model. [Figure 79] Fig. 1 is a graph showing the time course of FITC-dextran assay in a mouse DSS colitis model. [Figure 80] FIG. 1 is a graph showing blood neutrophil levels over time in disease-free mice with or without initial QBECO SSI treatment (mean + / - SEM, n=10 mice per group). [Figure 81] A set of three graphs showing blood cytokine levels over time in disease-free mice with or without initial QBECO SSI treatment (mean + / - SEM, n=10 mice per group). [Figure 82] FIG. 2 shows the pharmacokinetics of QBKPN. QBKPN SSI was fluorescently labeled and injected subcutaneously into disease-free mice. Mice were bled at different time points over 48 hours and blood cell counts were quantified. [Figure 83] Contains six bar graphs, showing gene expression of CXCL10 (IP-10), CCL2 (MCP-1), and CCR2 in lung tissue. Mice were treated with placebo, QBKPN, or QBECO every other day for 10 days and then implanted with B16F10 tumors in the lungs via tail vein injection. Treatment continued every other day after tumor implantation. Mice were euthanized on day 5 (A, C, E) or day 17 (B, D, F). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In the following detailed description, various examples are given based on specific embodiments along with experimental procedures that can be used to effect various modifications and variations in the practice of the invention. For purposes of clarity, various terms are used herein consistent with what is understood to have commonly understood meanings, which are reflected in the definitions set forth below.

[0019] General definition "Immunogen" refers to a molecule capable of eliciting an immune response by the immune system of an organism, or a composition containing such a molecule. "Antigen" refers to a molecule capable of binding to the products of an immune response.

[0020] A "pathogen" entity is an entity, such as a microorganism (such as a bacterium or virus), that is known to cause infection in the natural host, and in this sense "pathogenic" is used in the context of the present invention to mean "naturally pathogenic." Although a wide variety of microorganisms may have the ability to cause infection under artificial conditions, such as artificial inoculation of tissue with the microorganism, the range of microorganisms that naturally cause infection is necessarily limited and well established by medical science.

[0021] An "infection" is a state or condition in which the body or a part thereof is invaded by a pathogen (e.g., a microorganism such as a bacterium) which, under favorable conditions, multiplies and exerts a damaging effect (Taber's Cyclopedic Medical Dictionary, 14th Ed., CL Thomas, Ed., FADAvis Company, PA, USA). An infection may not always be clinically evident and may only result in localized cell damage. An infection may remain asymptomatic and may be temporary if the body's defense mechanisms are effective. An infection may spread locally and become clinically evident as an acute, subacute, or chronic clinical infection or condition. A local infection may become systemic if the pathogen gains access to lymph or blood vessels. An infection is usually accompanied by inflammation, but inflammation can occur without infection.

[0022] "Inflammation" is the characteristic tissue response to injury (characterized by swelling, redness, heat, and pain) and involves a continuum of changes that occur in living tissues when they are injured. Infection and inflammation are distinct states, although one may arise from the other (Taber's Cyclopedic Medical Dictionary, supra). Thus, inflammation can occur without infection (although inflammation is typically due to infection with a pathogenic bacteria or virus), and infection can occur without inflammation. Inflammation is characterized by the following symptoms: redness (rubor), heat (calor), swelling (tumor), and pain (painful). A combination of these symptoms may make inflammation visible locally on the skin, specifically redness at the site of administration.

[0023] Various subjects may be treated or assayed or sampled according to alternative aspects of the invention. A "subject" as used herein is an animal, e.g., a vertebrate or a mammal. Thus, 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, a mouse, or a dog. A healthy subject may be a human not suffering from a disease such as cancer or immune dysfunction, or a human not suspected of having such a disease, or a human not suffering from a chronic disorder or condition. A "healthy subject" may also be a subject that is not immunocompromised. Immunocompromised is intended to be any condition in which the immune system functions in an abnormal or defective manner. Immunocompromised may result from a disease, a certain medication, or a condition present at birth. Immunocompromised subjects may be more frequently found among infants, the elderly, and individuals undergoing extensive drug or radiation therapy.

[0024] A "sample" from a subject may include any relevant biological material, including, for example, a cell, tissue, or body fluid sample taken from a patient. For example, a sample may conveniently include a skin, cheek, blood, feces, hair, or urine sample. Sample nucleic acid for use in diagnostic and prognostic methods may be obtained, for example, from a selected cell type or tissue of a subject. For example, a subject's body fluid (e.g., blood) may be obtained by known techniques. Alternatively, nucleic acid testing may be performed on a dry sample (e.g., hair or skin).

[0025] The term "polymorphism" refers to a position in a biological sequence, such as a genomic sequence, and such positions vary within a population. A polymorphism is composed of different "alleles." The term "genotype" refers to a particular allele in the genome, for example, of a cell, tissue sample, or an individual. The position of a polymorphism can be identified, for example, by its position in a genome, or by its position in a sequence (such as a protein) that reflects a genomic locus. This can be given, for example, in a form that characterizes the different amino acids or bases found at a specified position. For a diploid genome, a genotype is typically composed of at least two alleles, and such alleles can be identical (homozygous) or different (heterozygous). Each polymorphism is typically assigned an identifier (such as a "reference SNP," "refSNP," or "rs number") specific to the art, for example, in the Single Nucleotide Polymorphism Database of Nucleotide Sequence Variations (dbSNP) available at the NCBI website.

[0026] Characterization of polymorphisms, alleles, or genotypes can be performed by any of a wide variety of methods. Such methods can include, for example, hybridization, labeling, cloning, sequencing, and / or amplification of nucleic acids (such as genomic DNA) in various forms, for example, PCR, LCR, xMAP, Invader assay, mass spectrometry, pyrosequencing, selective oligonucleotide hybridization, selective amplification, selective primer extension, or using probes. In this context, the term "probe" includes naturally occurring or recombinant single-stranded or double-stranded nucleic acids, or chemically synthesized nucleic acids. Probes can be, for example, polynucleotides of a length suitable for selective hybridization to nucleic acids containing polymorphic regions. Labeled probes can also be used in conjunction with amplification of polymorphisms. DNA microarray technology (sometimes referred to as DNA chips or gene chips) can be used, for example, for genome characterization, for example, to characterize point mutations, single nucleotide polymorphisms (SNPs), and / or short tandem repeats (STRs). For example, several probes capable of specifically hybridizing to allelic variants can be attached to a solid support by various processes, including lithography. Additional methods include laser capture microdissection (LCM), comparative genomic hybridization (CGH), and chromatin immunoprecipitation (ChiP). Allele-specific hybridization can utilize, for example, about 5, or 10, or 20, or 25, or 30 probes that overlap the polymorphic site and include the surrounding nucleotides of the polymorphic region. Alternatively, the presence of a particular allele in DNA from a subject can be characterized, in some cases, by restriction enzyme analysis. Similarly, a technique that can be described as a "mismatch cleavage" assay can be used to detect mismatched bases in RNA / RNA, DNA / DNA, or RNA / DNA heteroduplexes by noting their protection from cleavage agents (such as nucleases, hydroxylamine, or osmium tetroxide).Alterations in electrophoretic mobility can be used to characterize allelic variants and, for example, single-stranded conformation polymorphisms can be detected.

[0027] Many of the methods described herein can be carried out using, for example, a kit that includes at least one probe or primer nucleic acid or one or more compositions described herein, and instructions for using the kit. The kit can include, for example, at least one probe or primer that has the ability to specifically hybridize to the polymorphic region or the adjacent region of the polymorphic region, so that the oligonucleotide is "specific" for the polymorphic region. The kit can also include at least one reagent required for carrying out a particular assay. The kit can also include, for example, a positive control, a negative control, a sequencing marker, or an antibody for determining the genotype or biological marker profile of the subject.

[0028] An "immune response" includes, but is not limited to, one or more of the following responses in a mammal: induction or activation (such as that caused by one or more immunogens in an immunogenic composition following administration of the composition) of antibodies, neutrophils, monocytes, macrophages (including both M1-like 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). Thus, an immune response to a composition generally results in a cellular and / or antibody-mediated response to the composition in a host animal. In some embodiments, the immune response will also result in a slowing or halting of the progression of immune dysregulation or a disease characterized by immune dysregulation. Thus, the immune response may include one or both of a cellular immune response and / or a humoral immune response, and may be an adaptive or innate immune response.

[0029] "Immune dysregulation" refers to an immune response that is inappropriately regulated, such as an inappropriately restricted or robust immune response. Immune dysregulation may be associated with, for example, autoimmune, inflammatory, or degenerative diseases (such as rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, multiple sclerosis, neurodegenerative diseases, or allergies), or neoplastic diseases such as cancer, or host defense against pathogens. Inflammatory bowel disease (IBD) is the name frequently given to a group of inflammatory conditions of the colon and small intestine that are generally characterized by similar symptoms of immune dysregulation and uncertain etiology. The major subtypes of IBD are clinically recognized as Crohn's disease and ulcerative colitis. In addition to Crohn's disease and ulcerative colitis, IBD may also include conditions that are recognized as any one of the following: collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome, or unclassified colitis. The differences between these conditions are primarily related to the location and nature of the inflammatory changes in the gastrointestinal tract (GIT). For example, Crohn's disease is generally recognized as potentially affecting any part of the gastrointestinal tract from the mouth to the anus, and is characterized by a relapsing-remitting granulomatous inflammation of the gastrointestinal tract, most often in the terminal ileum and colon. In contrast, ulcerative colitis is generally recognized as being limited to the colon and rectum. The various areas of the digestive tract where such inflammatory conditions may be symptomatic include the bowel or intestine, which includes the small intestine (which has three parts: the duodenum, jejunum, and 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.

[0030] "Site-specific immunotherapy" (SSI) is an immunomodulatory therapy effective in therapeutically or prophylactically altering aspects of the immune status or immune system physiology in one or more anatomical sites, such as an organ or tissue. In some cases, for example, SSIs may be suitable for ameliorating immune dysregulation or treating a condition characterized by immune dysregulation.

[0031] A "cancer" or "neoplasm" is any unwanted cell proliferation that serves no physiological function. Generally, cancer cells are cells that are freed from their normal cell division controls, i.e., cells whose proliferation is not regulated by the usual biochemical and physical influences in the cellular environment. Thus, "cancer" is a general term for diseases characterized by abnormal and uncontrolled cell proliferation. In most cases, cancer cells form malignant clonal cells as a result of proliferation. A mass or cell mass that is a "neoplasm" or "tumor" generally has the ability to invade or destroy surrounding normal tissues. As used herein, "malignant tumor" refers to the abnormal growth of any cell type or tissue that has deleterious effects on the organism that has the abnormal growth. The term "malignant tumor" or "cancer" includes cell proliferation that is technically benign but carries the risk of becoming malignant. Cancer cells can spread from their original site to other parts of the body via the lymphatic system or bloodstream in a process known as "metastasis". Many cancers are resistant to treatment and prove fatal. Examples of cancers or neoplasms include, but are not limited to, cancerous and immortalized cells, tumors, carcinomas in various organs and tissues, as described herein or known to one of skill in the art.

[0032] Most cancers fall into three broad histological categories: predominantly cancers of the epithelial cells or cells lining the external or internal surfaces of organs, glands, or other structures of the body (e.g., skin, uterus, lung, breast, prostate, stomach, intestine) and which have a tendency to metastasize, carcinomas originating in connective or supporting tissues (e.g., bone, cartilage, tendons, ligaments, fat, muscle), and hematological tumors originating in bone marrow and lymphatic tissue. Carcinomas can be adenocarcinomas (which generally arise in organs or glands with secretory capacity, such as the breast, lung, colon, prostate, or bladder) or squamous cell carcinomas (which originate from squamous epithelium and generally arise in most areas of the body). Sarcomas can be osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesosarcoma or mesothelioma (lining of body cavities), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessels), liposarcoma (fatty tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), or a mixed mesenchymal or mesodermal tumor (mixed connective tissue type). Hematological tumors can be myeloma (originating from the plasma cells of the bone marrow), leukemia ("liquid cancer" - cancer of the bone marrow, which can be myeloid or granulocytic leukemia (myeloid and granulocytic white blood cells), lymphocytic, lymphocytic, or lymphoblastic leukemia (lymphatic and lymphocytic blood cells), or polycythemia vera or erythrocyte (various blood cell products exist, but red blood cells predominate)), or lymphoma (which can be a solid tumor, arising in the glands or nodes of the lymphatic system, which can be Hodgkin's lymphoma or non-Hodgkin's lymphoma). In addition, there are mixed types of cancers, such as adenosquamous carcinoma, mixed mesodermal tumor, carcinosarcoma, or teratocarcinoma.

[0033] Cancers named based on the primary site may be associated with a histological classification. For example, lung cancer is generally small cell lung cancer or non-small cell lung cancer (which may be squamous cell carcinoma, adenocarcinoma, or large cell carcinoma), and skin cancer is generally basal cell carcinoma, squamous cell carcinoma, or melanoma. Lymphomas may arise in lymph nodes associated with the head, neck, and chest, as well as in abdominal, axillary, or inguinal lymph nodes. Identification and classification of cancer type and stage may be performed, for example, by using information provided by the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute, which is an authoritative source of information on cancer incidence and survival in the United States and is recognized worldwide. The SEER program currently collects and publishes cancer incidence and survival data from 14 population-based cancer registries and three additional registries, which cover approximately 26 percent of the U.S. population. The program routinely collects data on patient demographics, primary tumor site, morphology, stage at diagnosis, initial treatment, and vital status follow-up, and is the only comprehensive source of population-based information in the United States, including cancer stage at diagnosis and survival within each stage. The SEER database contains more than 3 million cases of in situ and invasive cancer, with approximately 170,000 new cases added annually within the SEER coverage area. Incidence and survival data from the SEER program can be used to obtain standardized survival rates for specific cancer sites and stages. For example, to ensure optimal comparison groups, specific criteria can be selected from the database, including date of diagnosis and exact stage (e.g., in the case of lung cancer exemplified herein, the number of years was selected to match the period of retrospective review, and the stage of lung cancer was selected as 3B and 4; similarly, in the case of colon cancer exemplified herein, the number of years was selected to match the period of retrospective review, and the stage of colon cancer was selected as 4).

[0034] Cancers may also be named based on their organ of origin, i.e., "primary site," such as cancers of the breast, brain, lung, liver, skin, prostate, testes, bladder, colon and rectum, cervix, uterus, etc. This nomenclature does not change even if the cancer has metastasized to another part of the body different from the primary site. In the context of the present invention, the treatment is directed to the site of the cancer, not the type of cancer, so that any type of cancer that is symptomatic in the lung or etiologically located in the lung will be treated based on this localization in, for example, the lung.

[0035] PRR Ligands An embodiment of the present invention relates to the use of PRR ligands. PRR ligands may be, for example, commercially available and widely available, for example, as preparations of attenuated or killed recombinant bacteria, and may be, for example, ligands for TLR2, TLR4, and TLR5. The composition of pathogen-associated molecular patterns (PAMPs) may include PAMPs recognized by PRRs, including Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) (including Dectin-1), cytosolic dsDNA sensors (CDSs), and NLRs involved in the formation of inflammasomes.

[0036] Toll-like receptor 2 (TLR2) is involved in the recognition of a variety of microbial molecules representing a broad group of species, including Gram-positive and Gram-negative bacteria, as well as mycoplasmas and yeast. TLR2 recognizes cell wall components such as peptidoglycan, lipoteichoic acid, and lipoproteins from Gram-positive bacteria, lipoarabinomannan from mycobacteria, and zymosan from yeast cell walls. 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), inducing a signaling cascade that results in the activation of NF-κB and the production of proinflammatory cytokines. LPS generally consists of a polysaccharide region anchored to the bacterial outer membrane by a glycolipid moiety, lipid A, which is responsible for most of the immunostimulatory activity of LPS. Particularly active forms of lipid A contain six fatty acyl groups and can be found in pathogenic bacteria, for example, strains of Escherichia coli or Salmonella species. 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 RNA, synthetic small molecules (such as imidazoquinolines), and nucleoside analogues. Toll-like receptor 9 (TLR9) recognizes specific unmethylated CpG motifs that are widespread in microorganisms but absent from vertebrate genomic DNA.

[0037] NLRs are a family of at least 22 cytoplasmic innate immune sensors, including the intracellular pattern recognition receptors NOD1 (CARD4) and NOD2 (CARD15) involved in the recognition of peptidoglycan (PGN). These receptors detect specific motifs in PGN. NOD1 senses diaminopimelic acid (DAP)-containing muropeptides (specifically, d-Glu-meso-DAP dipeptide ("iE-DAP" dipeptide)) that are found primarily in the PGN of Gram-negative bacteria, as well as in certain Gram-positive bacteria. NOD2 recognizes the muramyl dipeptide (MDP) structure found in almost all bacterial PGN.

[0038] RIG-I-like receptors (RLRs), specifically RIG-I and MDA-5, detect viral RNA species.

[0039] CLR ligands include agonists of Dectin-1 and Mincle (macrophage-inducible C-type lectin). Dectin-1 is a receptor specific for β-glucan, a glucose polymer found in fungal cell walls. Mincle is a multitasking danger signal receptor that recognizes a wide variety of ligands, including damaged cells, fungal, yeast, and mycobacterial components.

[0040] Cytosolic DNA sensors (CDSs) bind to intracellular DNA derived from pathogens, and several CDSs exist that may exhibit context-specific selectivity in recognizing specific DNA.

[0041] Cyclic dinucleotides (CDNs) and xanthenone derivatives (such as DMXAA) bind to and activate STING (stimulator of interferon genes).

[0042] Inflammasomes are multiprotein complexes involved in the production of mature IL-1β, specifically by cleavage of pro-IL-1β and pro-IL-18 to active, secretable forms. Inflammasomes can be divided into subtypes, NLRP1, NLRP3, NLRC4, and AIM2, which are activated by a wide variety of microbial molecules, danger signals, and crystalline substances.

[0043] [Table 3]

[0044] [Table 4]

[0045] Thus, embodiments of the present invention include the use of PRR agonists derived from selected microbial pathogens. For example, peptidoglycan (PGN) may be obtained from bacteria or bacterial strains that are pathogenic in selected target tissues or organs for use as NOD1 / NOD2 agonists. Similarly, cell wall components may be obtained from bacteria or bacterial strains that are pathogenic in selected target tissues or organs for use as TLR2 agonists. Similarly, DNA, including double-stranded DNA, particularly repetitive double-stranded DNA, may be obtained from microbial pathogens such as bacteria or bacterial strains that are pathogenic in selected target tissues or organs for use as agonists of DAI, LRRFIP1, RIG1, TLR9, AIM2, or cytosolic DNA sensor (CDS). Beta-glucan peptides may be obtained from fungi or yeasts that are pathogenic in selected target tissues or organs for use as Dectin-1 agonists. Cyclic dinucleotides may be obtained from microbial pathogens that are pathogenic in a selected target tissue or organ for use as STING agonists.

[0046] Aspects of the present invention include compositions with different PRR agonist signatures, which imply a repertoire of PRR agonists collected together in a therapeutic medium such that the selected collection of PRR agonists is different.A "therapeutic medium" in this context is, for example, a formulation that integrates and holds a PRR agonist in a medicamentously acceptable particle or vesicle, such as a recombinant microorganism.For example, such a PRR agonist signature can be different from a reference PRR agonist signature, for example, different from the collection of PRR agonists assumed to be present in a non-pathogenic microorganism in a target tissue.Such a PRR signature can also be different in the sense that it is different from the natural PRR agonist signature of a microbial mammalian pathogen, which is modified, for example, by recombinantly expressing a gene that modifies what would normally be the wild-type PRR agonist signature of the pathogen. For purposes of determining the uniqueness of a PRR agonist signature, the level or type of PRR agonist can be measured directly or, for example, by determining the activation or inhibition of a cellular signaling pathway as a result of PRR agonist / receptor binding.

[0047] Recombinant embodiments The various genes and nucleic acid sequences of the present invention may be recombinant sequences. The term "recombinant" means that something has been recombined, so that when it is performed with respect to a nucleic acid construct, the term refers to a molecule composed of nucleic acid sequences that have been co-linked or produced by molecular biology techniques. Thus, a nucleic acid "construct" is a recombinant nucleic acid, and such recombinant nucleic acids are generally prepared by integrating interoperable component sequencers. The term "recombinant", when used with respect to a protein or polypeptide, refers to a protein or polypeptide molecule that is expressed using a recombinant nucleic acid construct created by molecular biology techniques. The term "recombinant", when used with respect to a genetic composition or an organism or cell, refers to a new combination of alleles that did not occur in the parent genome. A recombinant nucleic acid construct may include a nucleic acid sequence to which it is not naturally linked, or a nucleotide sequence that is linked to a nucleic acid sequence to which it is naturally linked in a different location, or a nucleotide sequence that has been engineered to be linked to such a nucleic acid sequence. Thus, reference to a nucleic acid construct as "recombinant" indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e., by human intervention (resulting in the nucleic acid molecule being of human origin). For example, a recombinant nucleic acid construct can be introduced into a host cell by transformation. Such recombinant nucleic acid constructs can contain sequences derived from the same or a different host cell species, which sequences have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences can become integrated into the host cell genome as a result of the original transformation of the host cell or as a result of subsequent recombination and / or repair events.

[0048] The recombinant constructs of the invention may include various functional molecular or genomic elements, as appropriate, for example, to mediate gene expression or repression in transformed plants. 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 proteolytic signals that regulate gene expression, as well as epigenetic regulatory signals that, for example, involve histone methylation or acetylation (e.g., histone methyltransferases or histone acetyltransferases) to cause conformational changes that result in differential transcriptional status and gene expression. In the context of this disclosure, a "promoter" refers to a sequence sufficient to induce transcription of a gene when the promoter is operably linked to the gene. Thus, a promoter is a portion of a gene that contains DNA sequences that cause binding of RNA polymerase and initiation of transcription. Promoter sequences are usually, but not universally, located in the 5' non-coding region of a gene. A promoter and a gene are "operably linked" when such sequences are operably linked to allow gene expression mediated by the promoter. Thus, the term "operably linked" indicates that DNA segments are arranged so that they function in concert for the purpose intended thereby, such as transcription initiating at the promoter and proceeding through the coding segment of the gene to reach the terminator portion of the gene. Gene expression can occur, in some cases, when an appropriate molecule (such as a transcriptional activator protein) binds to the promoter. Expression is the process by which the information of the coding sequence of a gene is converted into mRNA by transcription and then into a polypeptide (protein) by translation, such that the protein is said to be expressed. If a gene or nucleic acid has the ability to be expressed under appropriate conditions in a particular host cell, it is "expressible" as the term is used herein.

[0049] As used herein, an "isolated" nucleic acid or polynucleotide refers to a component that has been removed from its original environment (e.g., its natural environment if it occurs in nature). 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% with which it is originally associated. Using PCR, a polynucleotide that has been amplified such that it is sufficiently distinguishable (e.g., on a gel) from the remaining cellular components is, for example, thereby "isolated". The polynucleotides of the invention may be "substantially pure", i.e., a high degree of isolation has been achieved using purification techniques.

[0050] In the context of biological molecules, "endogenous" refers to a molecule, such as a nucleic acid, that is found in nature and / or produced by a given organism or cell. An "endogenous" molecule can also refer to a "natural" 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 and / or produced by a given organism or cell in nature.

[0051] The term "heterologous" as used herein to describe a nucleic acid sequence or an amino acid sequence refers to a molecule or a portion of a molecule, such as a DNA sequence, that is artificially introduced into a particular host cell, for example, by transformation. Heterologous DNA sequences can be introduced into a host cell, for example, by transformation. Such heterologous molecules can include sequences derived from the host cell. Heterologous DNA sequences can become integrated into the host cell genome as a result of the original transformation of the host cell or as a result of subsequent recombination events.

[0052] Various aspects of the present disclosure encompass nucleic acid sequences or amino acid sequences that are homologous to other sequences. An amino acid sequence or nucleic acid sequence is "homologous" to another sequence, as the term is used herein, if the two sequences are substantially identical and the functional activity of the sequences is preserved (as used herein, no evolutionary relatedness is inferred from sequence conservation or identity). Nucleic acid sequences can also be homologous, even if the nucleic acid sequences themselves are not substantially identical, for example, as a result of the degeneracy of the genetic code, if they code for substantially identical amino acid sequences.

[0053] "Substantial homology" or "substantial identity" with respect to biological sequences is intended to mean, in alternative terms, homology of more than 70%, more than 71%, more than 72%, more than 73%, more than 74%, more than 75%, more than 76%, more than 77%, more than 78%, more than 79%, more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or 99% to 100% sequence identity. Homology may refer to nucleic acid or amino acid sequences, depending on the context. In alternative embodiments, sequence identity may be, for example, at least 75%, at least 90%, or at least 95%. Optimal sequence alignment for identity comparison can be performed using various 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 computer implementations of such algorithms (such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., USA). Sequence identity can also be determined using the BLAST algorithm, which is described in Altschul et al. (1990), J. Mol. Biol. 215:403-10 (can be determined using the published default settings). Software for performing BLAST analyses may be available through the National Center for Biotechnology Information (NCBI) at its internet site.In the BLAST algorithm, high scoring sequence pairs (HSPs) are first identified by identifying short strings of length W in a query sequence that match or meet some positive threshold score T when aligned with a string of the same length in a database sequence. T is referred to as the contiguous string score threshold. The initial contiguous string hits act as seeds to initiate searches to find longer HSPs. The string hits are extended in both directions along each sequence for as long as the cumulative alignment score can be increased. String hit extension in each direction is stopped when the following parameters are met: the cumulative alignment score falls by an amount X from its maximum achieved value, the cumulative score reaches zero or below due to the accumulation of one or more negatively scoring residue alignments, or the end of either sequence is reached. The sensitivity and speed of the alignment are determined by the BLAST algorithm parameters W, T, and X. In the BLAST program, a word length (W) of 11, a BLOSUM62 scoring matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919), alignment (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands may be used as defaults. One measure of statistical similarity between two sequences using the BLAST algorithm is the minimum sum probability (P(N)), which is an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments, nucleotide or amino acid sequences are considered to be substantially identical if the minimum sum probability in the comparison of test sequences is less than about 1, less than about 0.1, less than about 0.01, or less than about 0.001.

[0054] An alternative indication that two amino acid sequences are substantially identical is that one peptide specifically and immunologically reacts with an antibody that also has specific immunoreactivity to the other peptide. If an antibody binds preferentially to a peptide and does not bind in significant amounts to other proteins present in a sample, the antibody has specific immunoreactivity to the peptide, such that the antibody's preferential binding to the peptide is detectable in an immunoassay and distinguishable from non-specific binding to other peptides. The specific immunoreactivity of an antibody to a peptide can be assessed using a variety of immunoassay formats, such as solid-phase ELISA immunoassays to select monoclonal antibodies that have specific immunoreactivity with a protein (see Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York).

[0055] 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. Moderately stringent hybridization to filter-bound sequences can be achieved, for example, by 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., with washing in 0.2×SSC / 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 under stringent conditions to filter-bound sequences can be performed in, for example, 0.5 M NaHPO 4Hybridization may be performed at 65° C. in 0.1×SSC / 0.1% SDS, 7% SDS, 1 mM EDTA, and washing may be performed at 68° C. in 0.1×SSC / 0.1% SDS (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified according to 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, NY). In general, stringent conditions are selected to be about 5° C. below the thermal melting point for the specific sequence at a defined ionic strength and pH. The term "polynucleotides that hybridize under stringent (low, medium) conditions" is intended to include both single-stranded and double-stranded polynucleotides, although only one strand will hybridize to the complementary strand of another polynucleotide. Washing in a particular solution can be performed for a period ranging from a few minutes to a few days, and a person skilled in the art will easily select an appropriate washing time to distinguish between different levels of homology in the binding sequence.

[0056] It is well known in the art that some modifications and changes can be made to the structure of a polypeptide to obtain a biologically equivalent polypeptide without substantially altering the biological function of the peptide. The term "conservative amino acid substitution" as used herein refers to the replacement of one amino acid with another amino acid at a given position in a peptide, which can be made without any appreciable loss or gain of function, resulting in a biologically equivalent polypeptide. When making such modifications, the replacement of similar amino acid residues can be made based on the relative similarity of the side chain substituents (e.g., their size, charge, hydrophobicity, hydrophilicity, and the like), and the effect of such replacement on the function of the peptide can be assayed by routine testing. Conversely, the term "non-conservative amino acid substitution" as used herein refers to the replacement of one amino acid with another amino acid at a given position in a peptide, which can result in a non-biologically equivalent polypeptide by causing the peptide to lose or gain any appreciable function.

[0057] In some embodiments, conservative amino acid substitutions may be made such that an amino acid residue is replaced with another amino acid having a similar hydrophilicity value (e.g., within a value of ±2.0), with the following hydrophilicity values ​​being assigned to amino acid residues (as described in detail 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), Gln (+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), Ile (-1.8), Tyr (-2.3), Phe (-2.5), and Trp (-3.4). Non-conservative amino acid substitutions can be made so that the hydrophilicity values ​​of the residues differ significantly, for example, so that the hydrophilicity values ​​of the residues differ by more than 2.0.

[0058] In alternative embodiments, conservative amino acid substitutions may be made such that an amino acid residue is replaced with another amino acid having a similar hydropathic index (e.g., within a value of ±2.0). In such embodiments, a hydropathic index may be assigned to each amino acid residue based on its hydrophobicity and charge characteristics, as follows: Ile (+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), Gln (-3.5), Asp (-3.5), Asn (-3.5), Lys (-3.9), and Arg (-4.5). Non-conservative amino acid substitutions can be made so that the hydropathic indexes of the residues differ significantly, for example, so that the hydropathic indexes of the residues differ by more than 2.0.

[0059] In alternative embodiments, conservative amino acid substitutions may be made such that an amino acid residue is replaced with another amino acid of the same class, which are grouped into non-polar, acidic, basic and neutral classes as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met, acidic: Asp, Glu, basic: Lys, Arg, His, neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr. Non-conservative amino acid substitutions may be made such that residues do not belong to the same class, for example, a basic amino acid is replaced with a neutral or non-polar amino acid.

[0060] microorganisms Most animals are colonized to some degree by microorganisms, such as bacteria, that exist in a mutualistic or commensal relationship with the host animal. Thus, normally harmless bacterial species are abundant in healthy animals, and these species are usually localized on the surfaces of certain organs and tissues. These microbial communities often serve the normal functioning of the body as members of what is called the microbiome. Generally harmless microorganisms, such as Escherichia coli, can cause infections in healthy subjects, with outcomes ranging from mild infections to death. Whether a microorganism is pathogenic (i.e., causes an infection) depends on factors such as the route of entry and access to a particular host cell, tissue, or organ, the intrinsic pathogenicity of the microorganism, the amount of microorganism present at the site of potential infection, or the health of the host animal. Thus, normally harmless microorganisms can become pathogenic when conditions favorable for infection arise, and even most pathogenic microorganisms generally require certain circumstances to cause an infection. Thus, a microbial species that is a member of the normal flora can become a pathogen when it acts beyond its normal ecological role in the endogenous flora. For example, endogenous species can cause infections outside their ecological niche in anatomically proximal areas, e.g., by contiguous spread, and when this occurs, these normally harmless endogenous bacteria become pathogenic.

[0061] Certain microbial species are known to cause infections in specific cells, tissues, or organs of otherwise healthy subjects. Examples of bacteria and viruses that commonly cause infections in specific organs and tissues of the body are set forth below, and these examples are not intended to be limiting in the sense that a person skilled in the art would be able to recognize and identify infectious or pathogenic bacteria that cause or commonly cause infections in various organs and tissues of an otherwise healthy organism (and recognize the relative frequency of infection for each bacterial species) based on knowledge in the field 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, GLMandell, JE Bennett, R. Dolin, Eds., 2000, Churchill Livingstone, Philadelphia, PA, USA (all of which are incorporated herein by reference).

[0062] Skin infections are commonly caused by the following bacterial species: Staphylococcus aureus, beta-hemolytic streptococci groups A, B, C, or G, Corynebacterium diptheriae, Corynebacterium ulcerans, or Pseudomonas aeruginosa, or viral pathogens: measles, rubella, varicella zoster, echovirus, coxsackievirus, adenovirus, vaccinia, herpes simplex, or parvo B19.

[0063] Infections of soft tissues (e.g., fat and muscle) are commonly caused by the following bacterial species: Streptococcus pyogenes, Staphylococcus aureus, Clostridium perfringens, or other species of the genus Clostridium, or viral pathogens: influenza, or coxsackievirus.

[0064] Breast infections are commonly caused by the following bacterial species: Staphylococcus aureus, or Streptococcus pyogenes.

[0065] Infections of the lymph nodes of the head and neck are commonly caused by the following bacterial species: Staphylococcus aureus, or Streptococcus pyogenes, or viral pathogens: Epstein-Barr, cytomegalovirus, adenovirus, measles, rubella, herpes simplex, coxsackievirus, or varicella zoster.

[0066] Infection of the lymph nodes in the arm / axilla is commonly caused by the following bacterial species: Staphylococcus aureus, or Streptococcus pyogenes, or viral pathogens: measles, rubella, Epstein-Barr, cytomegalovirus, adenovirus, or varicella zoster.

[0067] Mediastinal lymph node infections are commonly caused by the following bacterial species: Streptococcus viridans, Peptococcus spp., Peptostreptococcus spp., Bacteroides spp., Fusobacterium spp., or Mycobacterium tuberculosis, or viral pathogens: measles, rubella, Epstein-Barr, cytomegalovirus, varicella zoster, or adenovirus.

[0068] Infection of the hilar lymph nodes is commonly caused by the following bacterial species: Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenza, Chlamydophila pneumoniae, Bordetella pertussis, or Mycobacterium tuberculosis, or by viral pathogens: influenza, adenovirus, rhinovirus, coronavirus, parainfluenza, respiratory syncytial virus, human metapneumovirus, or coxsackievirus.

[0069] Intraperitoneal lymph node infections are commonly caused by the following bacterial species: Yersinia enterocolitica, Yersinia pseudotuberculosis, Salmonella species, Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, or Mycobacterium tuberculosis, or viral pathogens: measles, rubella, Epstein-Barr, cytomegalovirus, varicella zoster, adenovirus, influenza, or coxsackievirus.

[0070] Infections of the lymph nodes in the legs / groin are commonly caused by the following bacterial species: Staphylococcus aureus, or Streptococcus pyogenes, or viral pathogens: measles, rubella, Epstein-Barr, cytomegalovirus, or herpes simplex.

[0071] Infections of the blood (i.e., sepsis) are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, coagulase-negative staphylococci, Enterococcus spp., Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, Bacteroides fragilis, Streptococcus pneumoniae, or group B streptococci, or viral pathogens: measles, rubella, varicella zoster, echovirus, coxsackievirus, adenovirus, Epstein-Barr, herpes simplex, or cytomegalovirus.

[0072] Bone infections are commonly caused by the following bacterial species: Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, other species of streptococci, Escherichia coli, Pseudomonas species, Enterobacter species, Proteus species, or Serratia species, or viral pathogens: parvovirus B19, rubella, or hepatitis B.

[0073] Joint infections are commonly caused by the following bacterial species: Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, other species of streptococci, Escherichia coli, Pseudomonas spp., Enterobacter spp., Proteus spp., Serratia spp., Neisseria gonorrhea, salmonella spp., Mycobacterium tuberculosis, Hemophilus influenza, or viral pathogens: parvovirus B19, rubella, hepatitis B, or fungal pathogens: Scedosporium prolificans.

[0074] Infections of the meninges are commonly caused by the following bacterial species: Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, or Listeria monocytogenes, or viral pathogens: echoviruses, coxsackieviruses, other enteroviruses, or mumps.

[0075] Brain infections are commonly caused by the following bacterial species: Streptococcus spp. (including S. anginosus, S. constellatus, S. intermedius), Staphylococcus aureus, Bacteroides spp., Prevotella spp., Proteus spp., Escherichia coli, Klebsiella spp., Pseudomonas spp., Enterobacter spp., or Borrelia burgdorferi, or viral pathogens: coxsackievirus, echovirus, poliovirus, other enteroviruses, mumps, herpes simplex, varicella zoster, flavivirus, or bunyavirus.

[0076] Spinal infections are commonly caused by the following bacterial species: Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, Listeria monocytogenes, or Borrelia burgdorferi, or viral pathogens: coxsackievirus, echovirus, poliovirus, other enteroviruses, mumps, herpes simplex, varicella zoster, flavivirus, or bunyavirus.

[0077] Ocular / orbital infections are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus milleri, Escherichia coli, Bacillus cereus, Chlamydia trachomatis, Haemophilus influenza, Pseudomonas species, Klebsiella species, or Treponema pallidum, or viral pathogens: adenovirus, herpes simplex, varicella zoster, or cytomegalovirus.

[0078] Infections of the salivary glands are commonly caused by the following bacterial species: Staphylococcus aureus, viridans streptococci (e.g., Streptococcus salivarius, Streptococcus sanguis, Streptococcus mutans), Peptostreptococcus species, or Bacteroides species, or other oral anaerobes, or viral pathogens: mumps, influenza, enterovirus, or rabies.

[0079] Oral infections are commonly caused by the following bacterial species: Prevotella melaninogenicus, anaerobic streptococci, viridans streptococci, Actinomyces species, Peptostreptococcus species, or Bacteroides species, or other oral anaerobes, or viral pathogens: herpes simplex, coxsackievirus, or Epstein-Barr.

[0080] Infections of the tonsils are generally caused by the following bacterial species: Streptococcus pyogenes, or B-hemolytic streptococci of groups C or G, or viral pathogens: rhinovirus, influenza, coronavirus, adenovirus, parainfluenza, respiratory syncytial virus, or herpes simplex.

[0081] Sinus infections are commonly caused by the following bacterial species: Streptococcus pneumoniae, Haemophilus influenza, Moraxella catarrhalis, alpha-streptococci, anaerobic bacteria (e.g., Prevotella species), or Staphylococcus aureus, or viral pathogens: rhinovirus, influenza, adenovirus, or parainfluenza.

[0082] Infections of the nasopharynx are generally caused by the following bacterial species: Streptococcus pyogenes, or B-hemolytic streptococci of groups C or G, or viral pathogens: rhinovirus, influenza, coronavirus, adenovirus, parainfluenza, respiratory syncytial virus, or herpes simplex.

[0083] Infections of the thyroid gland are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, or Streptococcus pneumoniae, or viral pathogens: mumps, or influenza.

[0084] Laryngeal infections are commonly caused by the following bacterial species: Mycoplasma pneumoniae, Chlamydophila pneumoniae, or Streptococcus pyogenes, or viral pathogens: rhinovirus, influenza, parainfluenza, adenovirus, coronavirus, or human metapneumovirus.

[0085] Infections of the respiratory tract are commonly caused by the following bacterial species: Mycoplasma pneumoniae, or viral pathogens: parainfluenza, influenza, respiratory syncytial virus, or adenovirus.

[0086] Infections of the bronchus are commonly caused by the following bacterial species: Mycoplasma pneumoniae, Chlamydophila pneumoniae, Bordetella pertussis, Streptococcus pneumoniae, or Haemophilus influenzae, or viral pathogens: influenza, adenovirus, rhinovirus, coronavirus, parainfluenza, respiratory syncytial virus, human metapneumovirus, or coxsackievirus.

[0087] Pulmonary infections are commonly caused by the following bacterial species: Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, or Haemophilus influenza, or viral pathogens: influenza, adenovirus, respiratory syncytial virus, or parainfluenza.

[0088] Infections of the pleura are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Bacteroides fragilis, Prevotella species, Fusobacterium nucleatum, peptostreptococcus species, or Mycobacterium tuberculosis, or viral pathogens: influenza, adenovirus, respiratory syncytial virus, or parainfluenza.

[0089] Mediastinal infections are commonly caused by the following bacterial species: Streptococcus viridans, Peptococcus spp., Peptostreptococcus spp., Bacteroides spp., Fusobacterium spp., or Mycobacterium tuberculosis, or viral pathogens: measles, rubella, Epstein-Barr, or cytomegalovirus.

[0090] Infections of the heart are commonly caused by the following bacterial species: Streptococcus species (including S. mitior, S. bovis, S. sanguis, S. mutans, S. anginosus), Enterococcus species, Staphylococcus species, Corynebacterium diptheriae, Clostridium perfringens, Neisseria meningitidis, or Salmonella species, or viral pathogens: enterovirus, coxsackievirus, echovirus, poliovirus, adenovirus, mumps, measles, or influenza.

[0091] Infections of the esophagus are commonly caused by the following bacterial species: Actinomyces spp., Mycobacterium avium, Mycobacterium tuberculosis, or Streptococcus spp., or viral pathogens: cytomegalovirus, herpes simplex, or varicella zoster.

[0092] Gastric infections are commonly caused by the following bacterial species: Streptococcus pyogenes or Helicobacter pylori, or viral pathogens: cytomegalovirus, herpes simplex, Epstein-Barr, rotavirus, norovirus, or adenovirus.

[0093] Infections of the small intestine 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: adenovirus, astrovirus, calicivirus, norovirus, rotavirus, or cytomegalovirus.

[0094] Colon / rectal infections 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: adenovirus, astrovirus, calicivirus, norovirus, rotavirus, or cytomegalovirus.

[0095] Anal infections 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 a viral pathogen: herpes simplex.

[0096] 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 by viral pathogens: herpes simplex.

[0097] Liver infections are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Streptococcus (anginosus group), Enterococcus spp., other viridans streptococci, or Bacteroides spp., or viral pathogens: Hepatitis A, Epstein-Barr, herpes simplex, mumps, rubella, measles, varicella zoster, coxsackievirus, or adenovirus.

[0098] Gallbladder infections are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterobacter spp., Enterococci, Bacteroides spp., Fusobacterium spp., Clostridium spp., Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri.

[0099] Infections of the biliary tract are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterobacter spp., Enterococci, Bacteroides spp., Fusobacterium spp., Clostridium spp., Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri, or viral pathogens: Hepatitis A, Epstein-Barr, herpes simplex, mumps, rubella, measles, varicella zoster, coxsackievirus, or adenovirus.

[0100] Pancreatic infections are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterococcus spp., Pseudomonas spp., Staphylococcal spp., Mycoplasma spp., Salmonella typhi, Leptospirosis spp., or Legionella spp., or viral pathogens: mumps, coxsackievirus, hepatitis B, cytomegalovirus, herpes simplex type 2, or varicella zoster.

[0101] Infections of the spleen are commonly caused by the following bacterial species: Streptococcus spp., Staphylococcus spp., Salmonella spp., Pseudomonas spp., Escherichia coli, or Enterococcus spp., or viral pathogens: Epstein-Barr, cytomegalovirus, adenovirus, measles, rubella, coxsackievirus, or varicella zoster.

[0102] Infections of the adrenal glands are commonly caused by the following bacterial species: Streptococcus spp., Staphylococcus spp., Salmonella spp., Pseudomonas spp., Escherichia coli, or Enterococcus spp., or the viral pathogen: Varicella zoster.

[0103] Kidney infections are commonly caused by the following bacterial species: Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providentia species, Morganella species, Enterococcus faecalis, or Pseudomonas aeruginosa, or viral pathogens: BK virus, or mumps.

[0104] Urinary tract infections are commonly caused by the following bacterial species: Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providentia species, Morganella species, or Enterococcus species.

[0105] Bladder infections are commonly caused by the following bacterial species: Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providentia species, Morganella species, Enterococcus faecalis, or Corynebacterium jekeum, or viral pathogens: adenovirus, or cytomegalovirus.

[0106] Infections of the peritoneum are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Escherichia coli, Klebsiella spp., Proteus spp., Enterococcus, Bacteroides fragilis, Prevotella melaninogenica, Peptococcus spp., Peptostreptococcus spp., Fusobacterium spp., or Clostridium spp.

[0107] Infections of the retroperitoneal region are generally caused by the following bacterial species: Escherichia coli, or Staphylococcus aureus.

[0108] Infections of the prostate are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus mirabilis, enterococci spp., Pseudomonas spp., Corynebacterium spp., or Neisseria gonorrhoeae, or a viral pathogen: herpes simplex.

[0109] Infection of the testes is commonly caused by the following bacterial species: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus species, Streptococcus species, or Salmonella enteriditis, or viral pathogens: mumps, coxsackievirus, or lymphocytic choriomeningitis virus.

[0110] Infections of the penis are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Neisseria gonorrhoeae, or Treponema pallidum, or a viral pathogen: herpes simplex.

[0111] Ovarian / adnexal infections are commonly caused by the following bacterial species: Neisseria gonorrhoeae, Chlamydia trachomatis, Gardenerella vaginalis, Prevotella spp., Bacteroides spp., Peptococcus spp., Streptococcus spp., or Escherichia coli.

[0112] Infections of the uterus are commonly caused by the following bacterial species: Neisseria gonorrhoeae, Chlamydia trachomatis, Gardenerella vaginalis, Prevotella spp., Bacteroides spp., Peptococcus spp., Streptococcus spp., or Escherichia coli.

[0113] Cervical infections are commonly caused by the following bacterial species: Neisseria gonorrhoeae, Chlamydia trachomatis, or Treponema pallidum, or a viral pathogen: herpes simplex.

[0114] Vaginal infections are commonly caused by the following bacterial species: Gardenerella vaginalis, Prevotella spp., Bacteroides spp., peptococci spp., Escherichia coli, Neisseria gonorrhoeae, Chlamydia Trachomatis, or Treponema pallidum, or a viral pathogen: herpes simplex.

[0115] Vulvar infections are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, or Treponema pallidum, or a viral pathogen: herpes simplex.

[0116] Bacterial species are classified in classification practice as a group of similar strains, which generally have identifiable physiological differences, but usually no morphological differences, and are presumed to have a common ancestor, and can be identified using serological techniques against bacterial surface antigens. Thus, each bacterial species (e.g., Streptococcus pneumoniae) has a large number of strains (or serotypes), which may differ in their ability to cause infection or in their ability to cause infection in a particular organ / site. For example, there are at least 90 serotypes of Streptococcus pneumoniae, but serotypes 1, 3, 4, 7, 8, and 12 are the most frequent causes of pneumococcal disease in humans.

[0117] Certain strains of Escherichia coli, termed extraintestinal pathogenic E. coli (ExPEC), are more likely to cause urinary tract infections or other extraintestinal infections such as neonatal meningitis, while other strains are more likely to cause gastrointestinal infections / diarrhea, 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 diffusely adherent E. coli (DAEC). Even within subcategories of ExPEC strains, certain virulence factors (e.g., production of type 1 pili) allow certain strains to have an increased ability to induce bladder infections, while other virulence factors (e.g., production of P pili) allow other strains to have an increased ability to induce kidney infections. According to the present invention, for the preparation targeting immune dysregulation in bladder cancer, ExPEC strain(s) that are more likely to cause infection in bladder can be selected, while for the preparation targeting immune dysregulation in renal cancer, ExPEC strain(s) that are more likely to cause infection in kidney can be selected.Similarly, for the preparation treating immune dysregulation in colon, one or more of E.coli ETEC strain, EPEC strain, EHEC strain, STEC strain, EAEC strain, EIEC strain or DAEC strain (i.e., strain that causes colon infection) can be selected.

[0118] Similarly, a particular virus may have many subtypes. For example, influenza virus has three types, influenza A, influenza B, and influenza C, which have different epidemiology, host range, and clinical characteristics. For example, influenza A is more likely to be associated with viral lung infection, while influenza B is more likely to be associated with myositis (i.e., muscle infection). Furthermore, each of these three types of influenza virus has many subtypes, which may also have different epidemiology, host range, and clinical characteristics. According to the present invention, to target immune dysregulation in the lung, influenza A subtypes that are most commonly associated with lung infection may be selected, while influenza B strains that are most commonly associated with myositis may be selected to treat immune dysregulation in muscle / soft tissue.

[0119] There are specific microbiota associated with some pathological tissue conditions, such as the microbiota of certain tumors, for example Fusobacterium and Providencia are associated with colorectal cancer.

[0120] The compositions of the present invention comprise immunogens of pathogenic microbial species (bacteria, viruses or fungi) that are pathogenic in a particular tissue or organ, and in these compositions, the immunogens are provided in the form of artificial repertoires of mammalian PRR agonists that reproduce different parts of the PRR agonist signature of microbial mammalian pathogens that are pathogenic in target tissue.In alternative embodiments, the parts of the PRR agonist signature are different in the sense that they are different from the reference PRR agonist signature of a microorganism that is not pathogenic in the target tissue, and they are different from the natural PRR agonist signature of microbial mammalian pathogens.These different artificial repertoires of mammalian PRR agonists are formulated together in a therapeutic medium so that they are presented in combination to the innate immune cells that reside in the target tissue of the mammalian host.

[0121] Preparations and therapeutic vehicles The compositions of the present invention may be provided alone or in combination with other compounds (e.g., nucleic acid molecules, small molecules, peptides, or peptide analogs) in the presence of liposomes, adjuvants, or any pharma- ceutically acceptable carrier ("therapeutic vehicle") in a form suitable for administration to a mammal, such as a human. As used herein, a "pharma-ceutically acceptable carrier" or "excipient" includes any or all of physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The carrier may be suitable for any suitable mode of administration, including subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intramuscular, sublingual, inhalation, intratumoral, or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or suspensions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or suspensions. The use of such media and agents for pharma-ceutically 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 particular bacteria, bacterial antigen, or composition thereof of the present invention), its use in the pharmaceutical compositions of the present invention is contemplated. Additional active compounds may also be incorporated into the compositions.

[0122] Aspects of the invention include the use of nanoparticle (NP) formulations. For example, virus-like particles (VLPs) are a form of essential empty virus particles that contain an intact protein coat and, in some embodiments, a membrane envelope. Generally, VLPs lack genetic material. VLPs can be produced, for example, by expressing viral proteins in mammalian, avian, insect, plant, yeast, or bacterial cells. Alternatively, fully synthetic VLPs can be produced. Alternative nanoparticle formulations are emulsions, liposomes, alginate, chitosan, and polylactide-glycolide copolymer (PLGA) NPs. Examples of NP / TLR ligand preparations that may be suitable for use to induce an immune response are a ligand for TLR2 (Pam(3)Cys), a ligand for TLR9 (poly I:C), a ligand for TLR4 (3-O-desacyl-40-monophosphoryl lipid A (MPL)), a ligand for TLR7 (9-benzyl-8-hydroxyadenine), a ligand for TLR7 / 8 (resiquimod, R848), and a ligand for TLR9 (CpGDNA).

[0123] In addition to the selected combination, a wide variety of adjuvants can be used to enhance the desired immune response (see Levast et al., 2014, Vaccines, 2, 297-322).

[0124] Treatment with PRR ligands according to the present invention can be combined with more traditional and existing treatments. For example, for cancer, such treatments can include chemotherapy, radiation therapy, surgery, etc., or such treatments can be combined with treatments that stimulate the immune system, reduce inflammation, or otherwise benefit the subject, such as nutrients, vitamins, and supplements. For example, vitamin A, vitamin D, vitamin E, vitamin C, vitamin B complex, selenium, zinc, coenzyme 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]) can also be administered to the subject.

[0125] Usual pharmaceutical practice may be employed to obtain suitable formulations or compositions for administering the compound to a subject. Alternative routes of administration may be employed, such as parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intraarticular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, inhalation, aerosol, topical, intratumoral, sublingual, or oral administration. Therapeutic formulations may be in the form of a solution or suspension; for oral administration, the formulation may be in the form of a tablet or capsule; for intranasal formulations, the formulation may be in the form of a powder, nasal spray, or aerosol; and for sublingual formulations, the formulation may be in the form of a drop, aerosol, or tablet.

[0126] Methods for preparing formulations are well known in the art, and such methods can be found, for example, in "Remington's Pharmaceutical Sciences" (20th edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, PA. Formulations for parenteral administration can include, for example, excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible and biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation can include, for example, excipients such as lactose, or can be aqueous solutions including, for example, polyoxyethylene-9-lauryl ether, glycolate, and deoxycholate, or can be oily solutions for administration in the form of nasal drops or as a gel. For therapeutic or prophylactic compositions, the pathogenic bacterial species is administered to an individual in an amount effective to stop or slow the progression or metastasis of cancer, or to increase the subject's survival rate in response to the disorder (e.g., compared to a prognosis obtained from the SEER database).

[0127] The pharmaceutical composition or formulation may be packaged in a variety of ways depending on the method used to administer the drug. For example, the article of manufacture or packaging may include a container in which the pharmaceutical formulation is received in an appropriate form. Suitable containers may include materials such as, for example, 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 with a stopper that can be pierced by a hypodermic needle. The package or container may also include a tamper-evident or multi-purpose mechanism suitable for controlling access to the contents of the package or container, for example, a multi-dose vial adapter that fits the 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 that identifies the pharmaceutical composition present therein and / or a drug label that identifies the mode or route of administration. The label may also include appropriate warnings, for example, warnings that identify the storage conditions of the container or package, or warnings that indicate contraindications or adverse effects of the treatment mode. Thus, the article of manufacture may take the form of a "kit" that includes the pharmaceutical composition or accessories suitable for facilitating the use of the pharmaceutical composition. The kit may include a label or package insert, the term "package insert" being used to refer to instructions that are customarily included in commercial packages of therapeutic products and that include information about directions, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. The kit may further include accessories associated with the use of the pharmaceutical composition, including buffers, diluents, filters, needles, and syringes. The kit may also be suitable for delivery of the pharmaceutical composition in a selected dosage form, including, for example, a number of unit doses. Such a kit may include a memory aid or memory mechanism in the form of a physical or written indication of the intended timing of the treatment schedule in which the dose is to be used.

[0128] A "companion diagnostic" may be associated with a pharmaceutical treatment or composition. A companion diagnostic is an assay that facilitates an associated treatment by providing diagnostic or prognostic information, typically in the form of a diagnostic test, to determine the applicability of the treatment to a particular patient. A point-of-care companion diagnostic may include, for example, providing a diagnostic composition and / or article of manufacture in conjunction with the provision of a pharmaceutical formulation, such as provided as part of a kit. Alternatively, a companion diagnostic may be provided separately, as an assay for monitoring the treatment of a subject or for predicting the therapeutic efficacy of an intended treatment. A companion diagnostic may take the form of, for example, a medical device, such as an imaging tool, or a process performed by such a device (e.g., for performing an in vitro assay), which provides information related to the safety and effective use of the corresponding drug or biological product. A companion diagnostic may be used in conjunction with a treatment disclosed herein to provide diagnostic or prognostic information about the efficacy of the treatment, or evidence of undesirable side effects or risks. The use of a companion diagnostic with a particular treatment may be specified, for example, in instructions present on the label of the diagnostic device and / or the label of the corresponding treatment product. Types of companion diagnostic tests may include screening and detection in the form of tests that screen for genetic patterns, such as genetic SSI response markers, predictive and theranostics, such as assays for biochemical SSI response markers that help predict the future course of the disease or indicate the patient's response to treatment, monitoring, for example, to evaluate the effectiveness and appropriate dosing of prescribed treatment, or recurrence tests to analyze the patient's risk of disease recurrence.

[0129] An "effective amount" of a composition according to the present invention includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic result, such as reduction or elimination of immune dysregulation. A therapeutically effective amount of a composition may vary according to factors such as the individual's condition, age, sex, and weight, and the ability of the compound to induce a desired response in the individual. Dosage regimens may be adjusted to obtain an optimal therapeutic response. A therapeutically effective amount may also be one in which any toxic or adverse effects of the composition are outweighed by therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired prophylactic result, such as remission of immune dysregulation. Typically, a prophylactic dose is used in a subject before or at an early stage of cancer, such that the prophylactically effective amount may be less than the therapeutically effective amount.

[0130] For any particular subject, the timing and dosage of treatment may be adjusted over time (e.g., timing may be daily, every other day, weekly, monthly) according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition. For example, in the context of subcutaneous or intradermal administration, the composition may be administered every other day. An initial dose of about 0.05 ml may be administered subcutaneously, followed by increments of 0.01 to 0.02 ml every other day until an appropriate skin reaction is achieved at the injection site (e.g., a delayed type reaction with a diameter of 1 inch to 2 inches resulting in a visible reddening at the injection site). Once this appropriate immune response is achieved, this dosing is continued as a maintenance dose. The maintenance dose may be adjusted from time to time to achieve a desired skin reaction (inflammation) visible at the injection site. Dosing may be administered over a period of administration, such as, for example, at least 1 week, 2 weeks, 2 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years or more.

[0131] Oral dosing can range from, for example, 4 times per day, daily, or weekly. Dosing can be performed over a period of administration, such as, for example, at least 1 week, 2 weeks, 2 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years or more. In some embodiments, the present invention can include compositions administered sublingually or by inhalation, or administered to one or more epithelial tissues (i.e., administered to the skin by intradermal or subcutaneous injection, administered to the pulmonary epithelium by inhalation, administered to the gastrointestinal mucosa by oral ingestion, administered to the oral mucosa by sublingual administration), simultaneously or sequentially. Thus, in some embodiments, the compositions of the present invention are administered to induce an immune response in epithelial tissues. In some embodiments, one or more epithelial administration routes can be combined with one or more additional administration routes, such as intratumoral administration, intramuscular administration, or intravenous administration.

[0132] In the case of immunogenic formulations, an immunogenically effective amount of the composition of the present invention can be provided alone or in combination with other compounds, such as, for example, immunological adjuvants. The composition can include, for example, a compound linked to 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 induce a desired immune response. An immunogenic composition can select, activate, or proliferate memory B cells, memory T cells, neutrophils, monocytes, or macrophages of the immune system, including, but not limited to,.

[0133] Antigenic compositions comprising killed recombinant bacteria for administration by injection can be prepared as follows: The bacteria can be grown in a suitable medium and washed with saline. The bacteria can then be centrifuged and resuspended in saline before heat killing. The suspension can be standardized by direct counting under a microscope, mixed in the required quantities, stored in a suitable container, and its safety, shelf life, and sterility can be tested in an approved manner. In addition to the pathogenic bacterial species and / or its antigens, killed bacterial vaccines suitable for administration to humans can contain 0.4% phenol preservative and / or 0.9% sodium chloride. The bacterial vaccine can also contain traces of brain heart infusion (bovine), peptone, yeast extract, agar, sheep blood, dextrose, sodium phosphate, and / or other media components.

[0134] In an alternative embodiment, the drug may be administered to the administration site over a dosing period of at least one week, with successive doses given at dosing intervals between one hour and one month. Optionally, the drug may be administered intradermally or subcutaneously. Optionally, the drug may be administered in a dose such that each dose is effective in eliciting a visible local inflammatory immune response at the administration site. Optionally, the drug may be administered such that a visible local inflammation occurs at the administration site within 1 to 48 hours. However, a visible local inflammatory immune response may not necessarily be present in all situations, even if an immune response has been initiated. There are other ways in which the onset of an immune response can be monitored. For example, the profile (and relative changes in characterization) of immune cells from a subject undergoing an immune response can be compared to that from a subject not undergoing an immune response.

[0135] In another aspect, there is provided a method for monitoring the efficacy of a therapeutic regimen in an individual undergoing treatment for immune dysfunction in a particular organ or tissue, in which the individual is subjected to a period of the therapeutic regimen, and then a characteristic of the immune response is measured in a post-treatment immune sample obtained from the particular organ or tissue.

[0136] In some embodiments, PRR agonists derived from bacteria that are members of the endogenous flora of a particular region of the GIT can be used to formulate the immunogenic composition of the present invention.Many bacterial species are listed in the rows of Table 3, along with the biological region in which each species may form part of the endogenous flora.For example, species of the genus Abiotrophia are typically members of the endogenous flora of the oral cavity.

[0137] [Table 5]

[0138] [Table 6]

[0139] [Table 7]

[0140] [Table 8]

[0141] [Table 9]

[0142] [Table 10]

[0143] [Table 11]

[0144] [Table 12]

[0145] [Table 13]

[0146] Endogenous microbial flora, such as bacteria, can access tissues for pathogenesis either through proximal or bacteremic spread. Under favorable conditions, endogenous organisms can become pathogenic, invade locally, and spread to adjacent tissues and organs by proximal spread. The endogenous bacterial flora of the skin, oral cavity, and colon are species that are also known to be prone to bacteremic spread. Therefore, bacteria that are members of certain endogenous flora domains can cause infections in tissues or organs to which they can spread. Thus, one aspect of the present invention includes the use of PRR agonists derived from endogenous bacteria to treat immune dysregulation with symptoms localized in the area of ​​the GIT where endogenous microbial pathogens can spread and cause infection. The columns of Table 2 list the domains of the endogenous flora. The rows of Table 4 list the areas of the GIT where immune dysregulation is symptomatic or may be localized pathogenetically. Thus, one aspect of the invention involves the use of PRR agonists derived from endogenous microbial pathogens to formulate immunogenic compositions for treating immune dysregulation that is symptomatic or etiologically localized in regions of the GIT where the pathogens can spread and cause infection. Thus, in an alternative embodiment, immune dysregulation that is symptomatic in the regions listed in the first column of Table 2 may be treated with an immunogenic composition comprising an artificial repertoire of mammalian PRR agonists that recapitulate different portions of the PRR agonist signature of a microbial mammalian pathogen that is a member of one or more endogenous flora of the endogenous flora domains listed in the first row of Table 2 and indicated by an X or check mark in that row.

[0147] [Table 14]

[0148] According to the combined information of Tables 1 and 2, immune dysregulation manifested in specific regions of the GIT as shown in column 1 of Table 2 may be treated with an antigenic composition comprising an artificial repertoire of mammalian PRR agonists that recapitulate different portions of the PRR agonist signature of a microbial mammalian pathogen that is one of the corresponding bacterial species in Table 1, and consequently the column headings of Table 2 are valid when substituted with the bacterial species of Table 1.

[0149] In some embodiments, PRR agonists can be derived from foreign bacterial pathogens. For example, PRR agonists derived from organisms listed in Table 5 can be used in the artificial repertoire of PRR agonists to treat symptomatic immune dysregulation in the region of GIT listed with the organism in Table 5. In some embodiments, PRR agonists derived from both endogenous and foreign microbial species can be used together.

[0150] [Table 15]

[0151] [Table 16]

[0152] In some embodiments, the PRR agonist for use in the present invention can be derived from viral pathogens.Table 6 illustrates a list of viral pathogens, with the tissue and organ sites where each virus species is reported to be a pathogen for that site.Therefore, one aspect of the present invention includes utilizing the immunogenic composition of the PRR agonist derived from the named virus to treat symptomatic immune dysregulation in the region of GIT shown next to the name of the virus in Table 6.

[0153] [Table 17]

[0154] In some embodiments, the pathogen from which the PRR agonist for use in the immunogenic composition of the invention originates may be a common cause of acute infections in the area of ​​the GIT where the immune dysregulation to be treated is symptomatic. Table 7 shows this type of bacterial and viral pathogens, along with the area of ​​the GIT where they commonly cause infection. Thus, in an alternative embodiment, immune dysregulation symptomatic in the area of ​​the GIT shown in the first column of Table 7 may be treated with an immunogenic composition comprising an artificial repertoire of mammalian PRR agonists that reproduce different parts of the PRR agonist signature of the pathogenic organisms listed in the second column of Table 7.

[0155] [Table 18]

[0156] Humans are host to a wide range of gastrointestinal parasites, including various protozoa and helminths, which constitute GIT pathogens for the purposes of the present invention (Schafer, TW, Skopic, A. Parasites of the small intestine. Curr Gastroenterol Reports 2006; 8: 312-20; Jernigan, J., Guerrant, RL, Pearson, RD Parasitic infections of the small intestine. Gut 1994; 35: 289-93; Sleisenger & Fordtran's Gastrointestinal and liver disease. 8th ed. 2006; Garcia, LS Diagnostic medical parasitology. 5th ed. 2007). Thus, the compositions of the present invention may include PRR agonists from a variety of protozoans, including, for example, Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominus, Isospora belli, Sarcocystis spp., Coccidian-like bodies (Cyclospora spp.), Enterocytozoon bieneusi, Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Endolimax nana, Iodamoeba butschlii, Dientameoba fragilis, Blastocystis hominus, Cyclospora cayetanensis, Microsporidia, Trypanosoma cruzi, Chilomastix mesnili, Pentatrichomonas hominis, and Balantidium coli.Similarly, the compositions of the invention may contain antigenic components from a variety of helminths, including, for example, Cestodes (tapeworms), Taenia saginata, Taenia solium, Diphyllobothrium spp., Hymenolepis nana, Hymenolepis diminuta, Dipylidium caninum, Nematodes (roundworms), Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus, Ancylostoma duodenale, Ancylostoma caninum, Tichuris trichiura, Capillaria philippinensis, Trichostrongylus spp., Trichinella spp., Necator americanus, Anisakis and related species, Angiostrongylus costaricensis, Enterobius vermicularis, Trematodes (flukes), Fasciolopsis buski, Heterophyes species, Echinostoma species, Clonorchis sinensis, Opisthorchis species, Fasciola species, Metagonimus yokogawi, Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, Schistosoma intercalatum, Echinostoma species, and Paragonimus species.

[0157] In accordance with the foregoing, in various aspects, the invention may comprise treating immune dysregulation with the formulation of an artificial repertoire of mammalian PRR agonists that recapitulate distinct portions of the PRR agonist signature of a microbial pathogen, such as 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 species, Citrobacter freundii, Citrobacter koseri, Clostridium species, Desulfomonaspigra, Dysgonomonas species, Eikenella corrodens, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter gergoviae, Enterobacter sakazakii, Enterobacter taylorae, Enterococcus species, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Eubacterium species, Finegoldia magnus, Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium necrophorum, Fusobacterium nucleatum, Fusobacterium russii, Fusobacterium varium, Gardnerella vaginalis, Gemella morbillorum, Globicatella species, Hafnia alvei, Helicobacter species, Klebsiella species, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus salivarius, Leclercia adecarboxylata, Leminorella species, Megasphaera elsdenii, Mitsuokella multiacidus, Mobiluncus curisii, Mobiluncus mulieris, Moellerella wisconsensis, Morganella morganii, Pantoea agglomerans, Pediococcus species, Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobus, Peptostreptococcus productus, Porphyromonas asaccharolytica, ProteusMirabilis, 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 + Group G streptococci, Succinivibrio dextrinosolvens, species of the genus Sutterella, Tissierella praeacuta, species of the genus Veillonella, species of the genus Aerobacter, Bacillus anthracis, Bacillus cereus, other species of the genus Bacillus, Borrelia recurrentis, species of the genus Brucella, 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, Edwardsiella tarda, Francisella tularensis, Listeria monocytogenes, Mycobacterium bovis, Mycobacterium tuberculosis, species of the genus Pediococcus, Plesiomonas shigelloides, Rickettsiarickettsiae, species of the genus Salmonella, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, other species of the genus Spirillum, Streptococcus zooepidemicus, Tropheryma whipplei, Vibrio cholerae, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio parahaemolyticus, Yersinia enterocolitica, Yersinia pseudotuberculosis, herpes simplex virus (types 1 and 2), cytomegalovirus, adenovirus, orthoreovirus, rotavirus, alphavirus, coronavirus, torovirus, human metapneumovirus, vesicular stomatitis virus, Machupo virus, Junin virus, poliovirus, coxsackievirus, echovirus, hepatitis A virus, norovirus and other caliciviruses, astrovirus, picobirnavirus, or hepatitis E virus.

[0158] In alternative aspects, the invention may include treating immune dysregulation with the formulation of artificial repertoires of mammalian PRR agonists that recapitulate distinct portions of the PRR agonist signatures of microbial mammalian pathogens that are common small intestinal and large intestinal pathogens, such as Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri, adenovirus, astrovirus, calicivirus, norovirus, rotavirus, and cytomegalovirus.

[0159] In an alternative embodiment, the invention includes a diagnostic step for evaluating a patient's exposure history to an organism. For example, the diagnostic step may include obtaining a history of exposure to a selected pathogen and / or evaluating a patient's immune response to the selected pathogen. For example, a serological test may be performed to detect antibodies to the selected pathogen in the patient's serum. In the context of this aspect of the invention, an antigenic determinant of a selected pathogen may be selected for use in an immunogenic composition for a selected patient, the selection being based on a diagnostic indication that the patient has had one or more exposure(s) to the pathogen, for example because antibodies to an antigenic determinant of the pathogen are present in the patient's serum.

[0160] In an additional alternative embodiment, the present invention includes a diagnostic step for evaluating the patient's immunological response to treatment with the selected immunogenic composition. For example, the diagnostic step may include evaluating the patient's immune response to the immunological determinants of the immunogenic composition, which may be performed, for example, using a serological test to detect antibodies to such immunogenic determinants. In the context of this aspect of the invention, treatment with the selected immunogenic composition may be continued if the evaluation indicates that there is an active immunological response to the immunogenic determinants of the composition, and treatment may be discontinued and 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.

[0161] The immunomodulatory properties of the formulations of the present invention can be harnessed for use in the treatment of a variety of diseases characterized by pathological immune dysregulation, for example, by using PRR agonists derived from endogenous or exogenous pathogens that are pathogenic in tissues or organs where the immune dysregulation is symptomatic or manifest, including bacterial, viral, and fungal pathogens. Table 8 describes diseases characterized by immune dysregulation that can be treated according to alternative embodiments of the present invention.

[0162]

Table 19

[0163]

Table 20

[0164]

Table 21

[0165]

Table 22

[0166]

Table 23

[0167]

Table 24

[0168] As shown in the table above, arthritis is a chronic inflammatory disease. Specifically, arthritis is understood to describe inflammation of one or more joints. There are many types of arthritis, or conditions with arthritic symptoms, including (but not limited to): ankylosing spondylitis, Behcet's disease, Ehlers-Danlos syndrome, familial Mediterranean fever, fibromyalgia, fifth disease, giant cell arteritis, gout, hemochromatosis, Henoch-Schonlein purpura, hyperglobulinemia with periodic fever, inflammatory bowel disease arthritis, and inflammatory bowel disease. arthritis (including Crohn's disease and ulcerative colitis), juvenile rheumatoid arthritis, juvenile spondyloarthropathy, Lyme disease, Marfan syndrome, osteoarthritis, pseudogout, psoriatic arthritis, reactive arthritis (Reiter's syndrome), rheumatoid arthritis, sarcoidosis, scleroderma, SEA syndrome (seronegative, enthesopathy, arthropathy), Sjogren's syndrome, Still's disease, systemic lupus erythematosus (SLE), TNF receptor-associated periodic syndrome, and Wegener's granulomatosis (and other vasculitic syndromes).

[0169] screening Patients may be advantageously screened for disorders of innate immunity, such as genetic disorders, for example, by primary sequence analysis or analysis of epigenetic changes. For example, various genetic disorders associated with gene products involved in innate immunity, such as genes for TLRs (TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9), genes for signaling proteins (MyD88, Mal, IRAK1, IRAK4, NEMO, IκBα, IRF5), genes for NLRs (NOD2, NALP1, NALP3), and others (CD14, UNC93B), have been identified (see Mogensen T., 2009, Clinical Microbiology Reviews, Vol. 22, No. 2, p. 240-273). Patients identified as having primary Mendelian immune deficiencies associated with impaired TLR signaling or NF-κB activation, for example, may not benefit from, or may require approaches appropriate to their condition, depending on the embodiment. Patients with polymorphisms in genes encoding components of innate signaling pathways, for example, mutations in the gene encoding TIR domain-containing adaptor-inducing interferon beta (TRIF), can also be identified prior to treatment with an SSI.

[0170] Microorganisms have various strategies to evade the innate immune system (Mogensen T., 2009, Clinical Microbiology Reviews, Vol. 22, No. 2, p. 240-273), and therefore embodiments of the present invention may be suitable for avoiding the suppressive effect of such strategies on the innate response elicited. In an alternative embodiment, recombinant microorganisms are provided that lack virulence factors that interfere with TLR signaling, such as recombinant E. coli that lack TIR domain-containing proteins (Cirl, C. et al., 2008, Nat. Med. 14: 399-406). Gram-negative bacterial preparations may advantageously contain LPS that is recognized by TLRs, such as TLR4, rather than forms of LPS that are not recognized by TLRs (Hornef, MW et al., 2002, Nat. Immunol. 3: 1033-1040). Similarly, bacterial preparations may advantageously include flagellins of the class that activate TLRs, such as TLR5, rather than those that do 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 elicited innate response, such as the amastigotes-specific cysteine ​​peptidase of Leishmania mexicana, which proteolytically degrades IκB and NF-κB (Cameron, P. et al., 2004, J. Immunol. 173:3297-3304). In alternative embodiments, such undesirable components may be removed from the preparation by appropriate manufacturing steps, for example, to wash or fractionate the microbial preparation so that the components are removed.

[0171] Patients can be genotyped, for example, by identifying polymorphisms in PRR genes (see WO2009003905). Genes associated with inflammation and immunity related diseases and disorders may be screened for, for example, including AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFN gamma, CXCL12, SDF1), autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, FAS, CD95, ALPS1A), combined immunodeficiency (IL2RG, SCIDX1, SCIDX, IMD4), HIV-1 (CCL5, SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5(CCR5)), immunodeficiencies (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNF SF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI), inflammation (IL-10, IL-1 (IL-1a, IL-1 b), IL-13, IL-17 (IL-17a(CTLA8), IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3cr1, ptpn22, TNFa, IBD These include NOD2 / CARD15, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1), and severe combined immunodeficiency (SCID) (JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4).Alternatively, genes involved in selected signaling pathways can, for example, be screened to identify patients who are more or less sensitive to, for example, SSI treatment, such as GM-CSF signaling (LYN, ELK1, MAPK1, PTPN11, AKT2, PIK3CA, CAMK2A, STAT5B, PIK3CB, PIK3C3, GNB2L1, BCL2L1, MAPK3, ETS1, KRAS, RUNX1, PIM1, PIK3C2A, RAF1, MAP2K2, AKT1, JAK2, PIK3R1, STAT3, MAP2K1, CCND1, AKT3, STAT1), IL-1 0 signaling (TRAF6, CCR1, ELK1, IKBKB, SP1, FOS, NFKB2, MAP3K14, MAPK8, MAPK13, RELA, MAPK14, TNF, IKBKG, RELB, MAP3K7, JAK1, CHUK, STAT3, NFKB1, JUN, IL1R1, IL6), Toll-like receptor signaling (IRAK1, EIF2AK2, MYD88, TRAF6, PPARA, ELK1, IKBKB, FOS, NFKB2, MAP3K14, MAPK8, MAPK13, RELA, TLR4, MAPK14, IKBKG, RELB, MAP3K7, CHUK, NFKB1, TLR2, JUN), etc.

[0172] Additionally, patients can be genotyped for SNPs located in non-coding regions of the genome that are associated with inflammatory disorders, such as SNPs identified via publicly available GWAS datasets, e.g., SNPs in genomic regions associated with sequences that play a regulatory role in immune function-related gene expression.

[0173] Alternative Embodiments Although various embodiments of the present invention are disclosed herein, many adaptations and modifications may be made within the scope of the present invention according to the ordinary general knowledge of one skilled in the art. Such modifications include the replacement of any of the aspects of the present invention with known equivalents to achieve the same result in substantially the same way. Numerical ranges are inclusive of the numbers defining the range, including all numbers and fractions contained within each range. The term "comprising" is used herein as an open-ended term and is substantially equivalent to the phrase "including, but not limited to," and the term "comprises" has a similar meaning. Terms such as "consisting essentially of" and "consists essentially of" allow for elements not expressly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics 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, a reference to "one" includes a plurality of such things. Citation of a reference herein is not an admission that such reference is prior art to the present invention. Any priority document(s) and all publications cited herein, including but not limited to patents and patent applications, are incorporated herein by reference as if each individual publication was specifically and individually indicated to be incorporated herein by reference and as if fully set forth herein. The present invention includes all of the embodiments and modifications substantially as described in the preceding portion of this specification and in conjunction with the examples and drawings. No commitment to any particular utility for all embodiments is intended herein.

[0174] The terms "about" or "approximately" as used herein, when referring to measurable values ​​such as parameters, amounts, time periods, and the like, are intended to encompass a variation within + / -20% of the particular value, preferably a variation within + / -10% of the particular value, more preferably a variation within + / -5% of the particular value, and even more preferably a variation within + / -1% of the particular value, provided that such variations are appropriate for the practice of the disclosed invention. A value modified by "about" or "approximately" is to be understood as being itself disclosed as specific and preferred.

[0175] All references cited herein are incorporated by reference in their entirety. Specifically, the teachings of all references specifically mentioned herein are incorporated by reference, along with all documents cited in the documents cited herein.

[0176] Standard references illustrating 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, NY, 1989; Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (periodically updated) ("Ausubel et al. 1992"); the Methods in Enzymology series (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); Antibodies, a Laboratory Manual (Harlow and Lane, eds. (1988)); and Animal Cell Culture (RI Freshney, ed. (1987). General principles of microbiology are presented, for example, in Davis, BD et al., Microbiology, 3rd edition, Harper & Row, publishers, Philadelphia, Pa. (1980).

[0177] Throughout this specification, a reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one of the embodiments of the invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, although they may. Moreover, as will be apparent to one of ordinary skill in the art from this disclosure, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Moreover, as will be apparent to one of ordinary skill in the art, while some of the embodiments described herein include some features included in other embodiments but not other features included therein, combining features of different embodiments is intended to be within the scope of the invention and forms different embodiments. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0178] In this description of the invention, reference is made to the accompanying drawings which form a part hereof, and which show by way of illustration only 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. Therefore, the description is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims.

[0179] Preferred descriptive statements (features) and embodiments may be combined with any other feature or embodiment, unless expressly stated to the contrary. Specifically, any feature indicated as being preferred or advantageous may be combined with any other feature or features or descriptive statements indicated as being preferred or advantageous.

[0180] In some embodiments, the present invention excludes steps involving medical or surgical treatment. Similarly, in some embodiments, the present invention disclaims claims to embodiments of natural origin, and as a result, aspects of the present invention relate only to compositions of artificial origin. Furthermore, in alternative aspects of the present invention, claims to known in the art products, processes for preparing the products, or methods of using the products are disclaimed herein.

[0181] Common symbols and abbreviations: SSI: Site-specific immunomodulators MC-38: Mouse colon adenocarcinoma cell line PD1: Programmed cell death 1 OD: optical density IP: intraperitoneal SC: Subcutaneous SOP: Standard Operating Protocol RPM: Revolutions per minute EDTA: Ethylenediaminetetraacetic acid ANOVA: Analysis of variance Ly6G: lymphocyte antigen 6 complex, G locus (Lymphocyte antigen 6 complex, locus G) Ly6C: lymphocyte antigen 6 complex, C locus (Lymphocyte antigen 6 complex, locus C) CD45: Surface antigen classification 45 SD: standard deviation NA: No value, not available, not present Rae1: ribonucleic acid transporter 1 CD3: Surface antigen classification 3 CD11b: Surface antigen classification molecule 11B KO: Knockout PBS: Phosphate buffered saline NKG2D: Natural killer group 2, member D G: grams μM: micrometer μL: microliter hr :hour min : minute QBECO: Escherishia coli whole cell SSI QBKPN: Klebsiella pneumoniae phylogroup III (also known as K. variicola) whole-cell SSI QBSAU: Staphylococcus aureus whole-cell SSI EXAMPLES

[0182] Example 1: Recombinant Microorganisms A family of virulence factors from Escherichia coli and Brucella melitensis, termed TIR domain-containing proteins, disrupt TLR signaling through direct binding to MyD88, thereby suppressing innate immunity and increasing bacterial virulence. Thus, aspects of the invention provide recombinant bacteria lacking expression of TIR domain-containing proteins or other virulence factors that disrupt the host innate immune response to pathogens.

[0183] Staphylococcus aureus In alternative embodiments, compositions may be prepared from recombinant S. aureus strains, such as those of sequence type ST-291, having the following alleles or homologous sequences with at least 99% identity thereto: arcc-3, aroe-37, glpf-19, gmk-2, pta-20, tpi-26, and yqil-32 (Larsen et al., 2012, OJ Clin. Microbiol 50(4):1355-1361). The strains may completely lack resistance genes to the following classes of antibiotics: aminoglycosides, beta-lactams, fluoroquinolones, fosfomycin, fusidic acid, MLS-macrolides, lincosamides and streptogramins B, nitroimidazoles, oxazolidinones, phenicols, rifampicin, sulfonamides, tetracyclines, trimethoprim, and glycopeptides. Alternatively, the strain may have one or more resistance genes, such as the blaZ beta-lactam resistance gene (Accession No. AP004832). Similarly, the strain may or may not include one or more virulence factor genes (Cosentino et al., 2013, PLoS ONE 8(10):e77302), which have, for example, at least 90%, 95%, 99%, or 100% identity to selected database sequences (identified by accession numbers in the table below). [Recall strains that lack leukotoxins, specifically those that target the innate immune system]

[0184] [Table 25]

[0185] [Table 26]

[0186] [Table 27]

[0187] [Table 28]

[0188] [Table 29]

[0189] The recombinant strain may contain one or more plasmids (Carattoli et al., 2014, Antimoclobic Agents and Chemotherapy 58(7):3895-3903), such plasmids having, for example, 90%, 95%, 99%, or 100% identity to plasmid rep5 (accession number NC005011) or plasmid rep16 (accession number CP002115.1).

[0190] Klebsiella species In alternative embodiments, compositions may be prepared from recombinant Klebsiella strains, such as K. pneumoniae or K. variicola (previously identified as K. pneumoniae), such as strains of sequence type having the following alleles or homologous sequences with at least 99% identity thereto: gapa16, infb24, mdh30, pgi40, phoe92, rpob17, tonb67. Strains may completely lack resistance genes to the following classes of antibiotics: aminoglycosides, beta-lactams, fluoroquinolones, fosfomycin, fusidic acid, MLS-macrolides, lincosamides and streptogramins B, nitroimidazoles, oxazolidinones, phenicols, rifampicin, sulfonamides, tetracyclines, trimethoprim, and glycopeptides. Alternatively, the strain may have one or more resistance genes, such as the blaLEN24 beta-lactam resistance gene (accession number AM850914).Similarly, the strain may or may not contain one or more of the virulence factors shown in the table below (see Leticia et al., 2014, BMC Biology 12:41).

[0191] [Table 30]

[0192] Escherichia coli.(prostate) In an alternative embodiment, the composition may be prepared from a recombinant E. coli strain that is particularly suitable for the treatment of immune dysfunction of the prostate, such as a strain of sequence type that has the following alleles or has a homologous sequence with at least 99% identity thereto: adk-37, fumc-38, gyrb-19, icd-37, mdh-151, pura-11, reca-26 (sequence type 1231). The strain may completely lack resistance genes to the following classes of antibiotics: aminoglycosides, beta-lactams, fluoroquinolones, fosfomycin, fusidic acid, MLS-macrolides, lincosamides and streptogramins B, nitroimidazoles, oxazolidinones, phenicols, rifampicin, sulfonamides, tetracyclines, trimethoprim, and glycopeptides. Similarly, the strain may or may not contain one or more virulence factor genes that have, for example, at least 90%, 95%, 99%, or 100% identity to selected database sequences (identified by accession numbers in the table below). The strain may also lack the stx holotoxin virulence factor.

[0193] [Table 31]

[0194] The serotype of the E. coli strain can be, for example, O18ac:H7, e.g., in which the H serotype gene fliC (accession number AF228492) and the O serotype genes wzx (accession number GU299793) and wzy (accession number GU299793) are present.

[0195] The recombinant strain may comprise one or more plasmids, such plasmids having, for example, 90%, 95%, 99%, or 100% identity to plasmid IncFIB (accession number AP001918), and / or plasmid IncFII (29) (accession number CP003035), and / or plasmid ColRNAI (accession number DQ298019), and / or plasmid Col156 (accession number NC009781).

[0196] The recombinant E. coli can be, for example, 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 USA 103:5977-82), or derived therefrom.

[0197] Escherichia coli. (colon) In an alternative embodiment, the composition may be prepared from a recombinant E. coli strain that is particularly suitable for the treatment of immune dysfunction of the colon, for example a strain of sequence type that has the following alleles or has a homologous sequence with at least 99% identity thereto: adk-76, fumc-43, gyrb-9, icd-36, mdh-404, pura-14, reca-10 (sequence type ST-5292). The strain may completely lack resistance genes to the following classes of antibiotics: aminoglycosides, beta-lactams, fluoroquinolones, fosfomycin, fusidic acid, MLS-macrolides, lincosamides and streptogramins B, nitroimidazoles, oxazolidinones, phenicols, rifampicin, sulfonamides, tetracyclines, trimethoprim, and glycopeptides. Alternatively, the strain may have one or more resistance genes, such as the strB or strA aminoglycoside resistance genes (accession number M96392 or accession number AF321551), and / or the sul1 sulfonamide resistance gene (accession number AY224185), and / or the sul2 sulfonamide resistance gene (accession number GQ421466), and / or the dfrA5 trimethoprim resistance (accession number X12868). Similarly, the strain may or may not include one or more virulence factor genes, such as those having at least 90%, 95%, 99%, or 100% identity to selected database sequences (identified by accession number in the table below). The strain may also have a gene that is at least 95% or 99% or 100% identical to the stx holotoxin virulence factor gene stx1 (accession number M19437).

[0198] [Table 32]

[0199] The serotype of the E. coli strain can be, for example, O117:H7, eg, in which the H serotype gene fliC (accession number AF228492) and the O serotype gene wzx (accession number EU694096) are present.

[0200] The recombinant strain may contain one or more of the plasmids shown in the table below.

[0201] [Table 33]

[0202] The recombinant E. coli (coli) can be, or be derived from, for example, E. coli SE15 or any E. coli strain having at least 80%, 90%, or 95% sequence identity to E. coli of the O117:H7 serotype.

[0203] Example 2: Minimal SSI formulation In this example, the modest efficacy of a minimal SSI formulation containing a TLR agonist and a microbial antigen is shown. In this example, the TLR agonist is a diacylated lipoprotein (InVivogen Pam2CSK4), a TLR2 / 6 agonist, and a TLR4 agonist (LPS, Sigma L1519). The microbial antigen used was recombinant outer membrane protein A (ompA) from Klebsiella pneumoniae (CUSABIO CSB-EP340587KBG). These components were formulated together in a liposomal vehicle and administered to a mouse model of SSI treatment according to the treatment timeline shown in Figure 1. As shown in Figure 2, the activity of TLR-only liposomes was not as high as the TLR+Ag liposomal construct, indicating that SSI action may be enhanced by engaging alternative immunogenic pathways. In the context of this data, it is important to note that the antigen components of these minimal formulations were recombinantly expressed in E. coli and were provided by the manufacturer with a purity of >90% (SDS-PAGE), thus providing the concomitant characteristic that these antigen preparations contain additional bacterial components that act as additional natural immunogens, including additional PRR agonists.

[0204] Example 3: SSI mediates antitumor activity in MyD88- / - mice Pathogen recognition and inflammatory signaling in innate immune defense involve many pathways, including MyD88-dependent and MyD88-independent pathways (Mogensen, 2009, Clinical Microbiology Reviews, 22(2):240). In this example, using a commercially available strain of MyD88- / - mice in a B16 melanoma model, we observed enhanced B16 tumor burden in MyD88 mice compared to B6 mice (genetically matched except for the MyD88 knockout). This is consistent with literature reports to the effect that MyD88 knockouts have an inherently poorer ability to control disease. In a model of SSI treatment, using Klebsiella SSI ("KPN SSI"), we observed a significant reduction in tumor burden in both wild-type B6 and MyD88-knockout mice after treatment with KPN SSI. This is evidence that SSIs can act at least partially in a MyD88-independent manner, indicating that alternative PRR signaling pathways beyond what is generally considered to be classical TLR signaling are involved in this SSI-mediated innate immune response. This is consistent with another observation that NOD-like receptor NLRP3 is upregulated in cultured cells treated with KPN SSI and ECO SSI. Alternatively, in some embodiments, SSIs may mediate classical TLR signaling through adaptors other than MyD88 (such as TRIF) downstream of TLR. Furthermore, additional studies have shown that SSI treatment can engage the MyD88 signaling pathway. Taken together, these results provide evidence that SSI treatment can engage robust and diverse PRR signaling pathways.

[0205] Example 4: Pre-exposure to related strains enhances the efficacy of SSI This example demonstrates that in some embodiments, pre-exposure of an organism to a microbial pathogen enhances the efficacy of a subsequent SSI.

[0206] In an animal model of SSI treatment treated according to the treatment timeline shown in Figure 3, pre-infection with Klebsiella pneumoniae enhanced the efficacy of KPN SSI using a different strain of Klebsiella genus, whereas pre-infection with S. pneumoniae failed to induce KPN SSI efficacy (in a model of lung cancer). As shown in Figure 4, the results indicate that the SSI response requires or may benefit from pre-exposure of the organism to at least a closely related pathogen. This is consistent with another observation that mice from colonies that test negative for K. pneumoniae but may harbor K. oxytoca, a strain commonly found in laboratory animal colonies, exhibit KPN SSI activity, indicating that in some cases pre-exposure to K. oxytoca is suitable for inducing responsiveness to KPN SSI. Comparing mice obtained from Jackson Labs (JAX) and Taconic (TAC), differences in pre-exposure of these mice are evident in Figure 4, indicating that even in the absence of pre-infection, S. pneumoniae (SPN) did not induce the same therapeutic efficacy as QBKPN did in mice of JAX origin. In mice of TAC origin, pre-infection was required for QBKPN to be effective.

[0207] Example 5: Treatment with killed Klebsiella pneumoniae improves mouse erythrocyte Reduce tumor burden in rheumatoid arthritis models. This example shows that the use of heat-killed Klebsiella pneumoniae SSI results in Klebsiella-mediated anti-cancer efficacy in metastatic B16 melanoma. Subcutaneous injection of KPN SSI significantly reduced tumor burden. Furthermore, subcutaneous treatment with heat-killed E. coli (ECO SSI) or Staph. aureus (SAU SSI) had no therapeutic effect in the lung tumor model, indicating that subcutaneous immune induction using a lung-specific pathogen activates a lung-specific anti-tumor response. To demonstrate the effect of pre-exposure to K. pneumoniae, mice were exposed to live K. pneumoniae via intratracheal infection before subcutaneous injection of KPN SSI. Pre-exposure to K. pneumoniae significantly enhanced KPN SSI-induced anti-tumor immunity and control of metastatic B16 melanoma in the lung. Antitumor efficacy in challenged mice correlated with monocyte and neutrophil influx into the lungs, but not with T cell influx into the lungs. Collectively, these data indicate that pre-exposure to K. pneumoniae can, in some embodiments, induce tissue-specific immune memory (e.g., innate immune memory) that mediates tumor cell lysis.

[0208] Example 6: Site specificity in skin cancer models This example demonstrates the site specificity of S. aureus SSI in a mouse skin cancer model. In this example, concentrated S. aureus SSI (10X QBSAU (herein designated "QBSAUR"), as well as a Klebsiella species SSI, E. coli SSI, and placebo were used in a B16 skin tumor model in which approximately 100,000 B16 melanoma cells were injected into the right flank of C57BL / 6 mice on day 0 in a volume of 100 ul. SSI treatment was initiated on day -10 and continued through day +12. Tumor volume monitoring began on day 7 with the endpoint being reached on day 14. The tumor volume results shown in Figures 5-10 very clearly demonstrate the site specificity of the S. aureus SSI formulation.

[0209] Example 7: Use of SSI enhances cancer antigen responses This example demonstrates the effect of combining a model cancer antigen expressed by transformed cancer cells, in this case OVA (or chicken ovalbumin), with an SSI, such as a Klebsiella species SSI (abbreviated as "KPN SSI"), in this case in a B16 melanoma animal model. OVA does not occur naturally in mammals or bacteria, is known to have CD4- and CD8-associated epitopes, and is immunogenic in C57Bl / 6 mice. In this example, B16 cells were transfected to express cytoplasmic OVA protein, which presented the OVA epitope in the tumor MHC, thereby enabling OVA-specific T cell recognition of melanoma. Some studies used the whole protein, which was found to be processed by the host immune system into the relevant antigen. Other studies used purified OVA antigen (SIINFELK (Class I-restricted CD8-specific antigen)). Some studies also used OT1 cells (transgenic CD8 cells that specifically recognize SIINFEKL via their T cell receptor) as a readout system.

[0210] The data provided provide evidence of enhanced efficacy when model cancer antigens are combined with SSI. One result was a reduction in the number of tumor nodules associated with treatment with Klebsiella species SSI+OVA compared to PBS+OVA (as assessed by photographic and quantitative PCR for the melanoma-specific gene Tyrp1). Consistent with this, the proportion of OVA-specific CD8 T cells in the lung was increased by KPN SSI+OVA. Similarly, expression of genes associated with effective antitumor immunity (granzyme B and IFN-gamma) was increased by KPN SSI+OVA compared to PBS+OVA. The same study presented survival data in a lung cancer model, showing improved survival with Klebsiella species SSI+OVA compared to control, and a significant improvement in survival with E. coli SSI+OVA.

[0211] In an alternative study, it was observed that the process of epitope spreading was induced or enhanced by SSI (see Figure 11; for background, see Vanderlugt & Miller, 2002, Nature Reviews Immunology 2, 85-95). In this study, mice were treated with SSI of a species of Klebsiella and then challenged with B16 melanoma (intravenous injection). Five days later, mice were adoptively transferred with TCR-transfected cells (Pmel T cells) specific for a natural cancer antigen in melanoma. To prevent the release of T cells from lymph nodes, mice were also treated with FTY-720 (fingolimod) to allow the cells to be harvested from the tumor-draining lymph. The activity status of the harvested pmel T cells was evaluated and it was found that the proportion of activated (i.e., antigen-exposed) T cells in the draining lymph nodes was enhanced by SSI. This provides evidence that SSIs may be suitable for enhancing the processing and presentation of immunogenic cancer antigens, including self-antigens and exogenously administered cancer antigens.

[0212] Example 8: Component Formulation This example relates to fractionation of microbial preparations for the formulation of alternative SSIs. In alternative embodiments, fractions can be prepared from, for example, bacterial outer membranes (e.g., from gram-negative species), bacterial inner membranes, gradient centrifugation pellets (e.g., from sucrose gradients), chromosomal DNA, capsule glycoprotein fractions, or peptidoglycan fractions such as peptidoglycan ghosts. In alternative embodiments, engineered or recombinant organisms can be used in SSIs, in which genes involved in pathways associated with specific cellular fractions are modified, and in particular genes involved in determining the composition of the aforementioned fractions are modified.

[0213] For cell fraction preparation, bacteria can be, for example, heat inactivated after growth. Cell fractions can be, for example, resuspended in sterile saline + 0.4% phenol. Intima can be, for example, collected using a two-step sucrose density gradient, such collection methods are described, for example, in Methods in Enzymology, Vol 125:309-328, 1986. Bacterial pellets obtained after culturing 250 ml of cells can be resuspended in 20% sucrose, 10 mM Tris-HCI (pH 8.0), and 50 ug / ml DNase 1. Cells can be incubated at 23°C for 10 minutes. Afterwards, cells can be placed on ice and solubilized twice by using a French pressure cell at 15,000 psi. Unbroken cells can be removed by centrifugation at 5,000xg for 10 minutes at 4°C. The supernatant can be layered on top of a two-step sucrose gradient (60% and 70%) and centrifuged in an SW28 swinging bucket rotor at 23,000 rpm for 18 hours at 4° C. Inner membranes can be collected from the junction between 20% and 60% sucrose. Sucrose can be diluted to less than 20% with sterile distilled water and membranes can be pelleted by ultracentrifugation at 41,000 rpm for 1 hour at 4° C. Inner membranes can be washed once with sterile water and then resuspended in sterile saline + 0.4% phenol. A crude outer membrane preparation can also be collected from the junction of the 60% and 70% sucrose gradient steps.

[0214] Chromosomal DNA (e.g., of Klebsiella pneumoniae) can be prepared using the Qiagen Blood and Tissue midi kit. Cells can be harvested from 15 ml or 40 ml broth cultures from each strain. Total DNA can then be purified according to the manufacturer's protocol.

[0215] Efficacy of component formulations was demonstrated using the outer membrane fraction of Klebsiella pneumoniae (KPN) in a B16 lung cancer model, where injections of the SSI were started every other day 10 days before inoculation of tumor cells by intravenous injection. Three SSIs were compared: total killed cell KPN (QBKPN), 1X outer membrane (OM) fraction (containing an outer membrane concentrate roughly equivalent to that of the whole cell formulation), and a 0.01X dilution of the outer membrane fraction. As shown in FIG. 47A, both 1× and 0.01× KPN outer membrane fractions (i) were effective in a dose-dependent manner in the pulmonary B16 melanoma model (the 1× fraction was as potent as the whole-cell formulation), as was the case for the 1× and 4× DNA fractions (ii), whereas the inner membrane fraction (iii) showed a dose-dependent trend, although this trend did not have strong statistical significance.

[0216] As shown in FIG. 47B, after 10 injections of adventitial SSI, 1× adventitial membrane fraction increased Rae-1 expression in a dose-dependent manner.

[0217] Of note, higher concentrations of membrane fractions caused pathology in animals prior to tumor cell inoculation. Specifically, 10X and 20X outer membrane fractions induced strong toxicity in mice, as evidenced by highly elevated mobilization of innate cells (monocytes and neutrophils) into the blood accompanied by poorer health (e.g., dramatic weight loss, gait, hunched posture, eye pathology). Similarly, in some embodiments, enriched whole cell preparations did not induce toxicity. Figure 47C shows elevated blood neutrophil and monocyte counts in blood collected 2 days prior to tumor implantation after 4 injections of SSI or placebo.

[0218] As shown in Figure 47D, in the B16 lung cancer model, the KPN fraction shows site-specific and preferential activity in the lung compared to the E. coli fraction. Specifically, compared to the placebo control, the whole QBKPN was effective in reducing lung tumor burden. The whole killed E. coli formulation (QBECO) was not as effective as QBKPN. The QBKPN fractions (OM alone or DNA alone) were effective. In combination (OM+DNA), the QBKPN fraction was almost as effective as the whole QBKPN. The QBECO fractions (OM, DNA, or OM+DNA) did not show the same potency as the QBKPN fraction. Taken together, this indicates that site specificity occurs in association with the QBKPN fraction, specifically the combined DNA (4x) and OM (1x) fraction.

[0219] Example 9: Combinations and Simultaneous Administration This example illustrates an embodiment in which an SSI is combined or co-administered with an additional therapeutic component.

[0220] One class of additional therapeutic components includes molecules or compositions for activating or recruiting innate immune cells, such as: - GMCSF (specifically cancer-directed) (e.g., in amounts that synergistically recruit and promote neutrophil production and enhance SSI-induced innate immune responses) - Vitamin D (targeting inflammatory diseases such as IBD, and cancer) (e.g. in amounts that are beneficial for monocyte differentiation and activation, and play a role in regulating innate immune function) Includes: In an alternative embodiment, the vitamin D used in combination with the SSI is, for example, vitamin D 3 , Vitamin D 2 In some embodiments, the vitamin D may be one or more of: 3 and / or Vitamin D 2For example, calcitriol may be administered locally in an amount effective to provide an effective amount of calcitriol locally to the site of administration of the SSI and vitamin D. For example, vitamin D precursor (D 3 and / or D. 2 ) may be administered in an amount that is locally effective upon conversion to the calcitriol active form by monocytes and / or macrophages (which express CYP27B1) localized at the site of administration. In an alternative embodiment, calcitriol may be administered in a locally effective dose at the site of administration of the SSI, which may be a small dose, for example, compared to the dose required for other systemic effects.

[0221] Additional classes of therapeutic components for combination or co-administration include molecules or compositions that reduce immunosuppression: - NOHA (N(omega)-hydroxy-nor-L-arginine) (arginase inhibitor) - Arginase breaks down arginine, which is necessary for immune activation. NOHA can be used in an amount effective to reduce immune suppression, for example, by making free arginine available. - alpha 1 antitrypsin - for example, in an amount effective to reduce immunosuppression mediated by protease-secreting neutrophils.

[0222] Additional classes of therapeutic components for combination or co-administration include molecules or compositions that block oxidative damage and improve immune function under stress: - Glutathione and other antioxidants, specifically targeting fibrotic diseases (such as IBD).

[0223] Additional classes of therapeutic components for combination or co-administration include costimulatory molecules for natural cytotoxic lymphocytes (e.g., directed to anti-cancer therapy): - phosphoantigens (isoprenoid molecules such as isopentenyl pyrophosphate) - recognized by human peripheral blood Vy9V52 T cells that play a central role in anti-cancer responses, e.g., in an amount effective to activate and differentiate monocytes that work in concert with NK cells to target both solid and liquid tumors. In an exemplary embodiment, SSIs, in combination or co-administration with zoledronate, have been shown to increase activation markers (e.g., CD25 and CD69) present on human peripheral blood Vy9V52 T cells. - Glycolipid molecules recognized by type I NKT cells (e.g., synthetic α-galactosylceramide)

[0224] As shown in Table 18 and Figures 12 and 13, the SSI combination was demonstrated to improve anti-cancer activity in vivo using the LLC model, where the combination with GMCSF and vitamin D (D 3 ) has been shown to have the highest potency, followed by NOHA (an arginase inhibitor) and then alpha-1-antitrypsin.

[0225] [Table 34]

[0226] Example 10: Colitis animal model, anti-inflammatory efficacy This example shows results from a mouse spontaneous colitis model (Muc2 knockout "KO" mice) that mimics the underlying immune system abnormalities and chronic bacterial infections associated with Crohn's disease and ulcerative colitis. IBD patients typically exhibit structural and / or functional abnormalities in their normally protective colonic mucosal barrier. The mucus barrier is highly dependent on the release of goblet cell-derived mucin (Muc2), which prevents microorganisms and luminal antigens from contacting epithelial surfaces in the gastrointestinal tract. Muc2 knockout mice are healthy immediately after weaning (1 month of age), but develop progressive diarrhea and isolated rectal prolapse with age. Histological analysis of colonic tissue reveals crypt hyperplasia, crypt abscesses, infiltration of inflammatory cells, and submucosal edema. Thus, Muc2 knockout mice have an abnormal gastrointestinal mucosal barrier and after some time spontaneously develop colitis that resembles ulcerative colitis in humans. In this model, young (2 month old) Muc2 knockout mice develop less severe colitis, whereas aged (3 month old) Muc2 knockout mice develop more severe colitis.

[0227] Results from this animal model are shown in Figures 14A, 14B, and 14C, showing that pro-inflammatory markers in the colon are reduced by E. coli SSI (QBECO) (using qPCR gene expression data). Figure 14D shows that QBECO has activity in site-specifically increasing gene expression of IL-18 in the colon compared to QBKPN. IFN-gamma expression data specifically shows how the efficacy of SSI can be affected by the stage of colitis (comparison of expression data from old vs. young mice). IL17A data is for a cytokine produced by activated T cells (inflammatory marker of IBD) and shows that this inflammatory marker is significantly reduced after E. coli SSI treatment. Thus, QBECO treatment substantially improved all components of the histopathology score, including infiltration, integrity, hyperplasia, and edema. Infiltration of T lymphocytes in colonic tissue is a hallmark of IBD in patients and mouse models, and QBECO treatment significantly reduced this infiltration. Thus, this example shows that an SSI such as QBECO can be used to significantly reduce disease severity in IBD models, including to substantially attenuate the hyperresponsiveness of the adaptive immune system.

[0228] QBECO was also shown to have a positive effect on the gastrointestinal microbiome. Alterations in the bacterial species in the gut microbiome can be either harmful ("unhealthy" bacteria) or therapeutic ("healthy" bacteria) in IBD patients (and mouse models). In IBD, some bacteria (e.g., Lactobacillus species) can promote a healthy immune environment and improve symptoms, while others (e.g., γ-proteobacteria) can have adverse effects. We analyzed the gut microbiome before and after QBECO SSI treatment. As shown in Figures 15A and 15B, QBECO SSI improved the gut dysbiosis imbalance in the colon of Muc2 mice by increasing the relative proportion of Lactobacillus (a healthy bacterial species) and decreasing the relative proportion of Gamma-proteobacteria (an unhealthy bacterial species). As shown in Figure 15C, all aspects of histological inflammation / injury scores (infiltration, integrity, hyperplasia, and edema) in the colon of MUC2 spontaneously colitis mice were also reduced by QBECO SSI. These results indicate that SSI treatment using a GI pathogen-derived formulation, such as QBECO, has a therapeutic effect on the gastrointestinal microbiome. Thus, embodiments of the present invention include the use of SSIs, such as E. coli-derived SSIs, to treat gut dysbiosis imbalance in IBD.

[0229] In summary, QBECO treatment significantly improved the overall histological score and reduced T cell infiltration in colonic tissue. Furthermore, a reduction in pro-inflammatory mediators (IL-17A) in the colon and in serum (KC) was observed. QBECO treatment did not affect the expression of regulatory T cell markers (FoxP3) and anti-inflammatory growth factor (TGF-β) in affected tissue. Furthermore, it was demonstrated that the levels of antimicrobial lectins RegIII-β and RegIII-γ were decreased in mice treated with SSI. The changes in antimicrobial lectins brought about by QBECO allowed the modulation of the gut microbiome, resulting in a decrease in Gamma-Proteobacteria and a significant increase in Lactobacillus.

[0230] Example 11: Efficacy of SSI in Asthma / Allergy In this example, animal model data is presented showing the efficacy of SSI therapy (KPN SSI) in treating asthma / allergy. As shown in Figure 16, KPN SSI reduces BAL total cell counts in asthmatic mice. As shown in Figure 17, KPN SSI reduces eosinophil and lymphocyte counts in BAL: A) eosinophils, B) lymphocytes. As shown in Figure 18, KPN SSI reduces TH2 cytokines in BAL supernatants: A) IL-4, B) IL-5.

[0231] Example 12: Systemic distribution of subcutaneously administered SSI In this example, the systemic distribution of KPN SSI administered subcutaneously in a mouse model is shown using total dead KPN cells labeled with a cyanine dye (Cy5.5) and in vivo dorsal and ventral optical whole body imaging. After the first injection, imaging (1, 3, 6, 24, and 47 hours) revealed that the SSI was distributed systemically, with the highest concentration at the injection site. After the first injection, the SSI was cleared from the circulation within approximately 24 hours. Subsequent injections were performed at a different injection site, and imaging (1, 3, 6, and 24 hours) revealed that systemic distribution occurred, with the highest concentration at the previous injection site in addition to the new injection site, surprisingly. This indicates that the SSI is preferentially delivered / retained at the site of inflammation after systemic dispersion of the locally administered formulation. Microscopic evaluation of blood samples confirmed that the Cy5.5 fluorescence detected in the blood was not free dye. As shown in FIG. 19, distribution of SSI in organs after 24 hours demonstrated preferential accumulation of KPN SSI in the lung compared to the heart and spleen.

[0232] Example 13: Surgical Wound Treatment In this example, a topical formulation of an SSI is formulated for administration to a wound, such as a surgical wound, a partial-thickness burn, a laceration, a chronic wound, or a vascular ulcer. The topical SSI formulation may include a PPR agonist derived from a microorganism that is, for example, a skin pathogen, and may be formulated into an ointment or gel.

[0233] Example 14: Durability of Treatment Response in IBD This example shows that treatment of Crohn's disease can be effective over a long period of time with regular dosing of SSI. Specifically, in a randomized, placebo-controlled, double-blind Phase 1 / 2 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) decreased on average by week 16 of treatment, and decreased more significantly at week 16 of treatment compared to week 8. More specifically, the mean reduction in CDAI score for patients treated with SSI was about 80 points by week 8, and the mean reduction in CDAI score by week 16 was about 120 points. This indicates that clinical improvement continued throughout the 16 weeks of SSI treatment.

[0234] In this example, a whole killed E. coli SSI preparation was used administered by subcutaneous injection every other day. Doses were individualized for each patient by titrating the dose so that each dose was effective in eliciting a visible local inflammatory immune response at the injection site (a delayed reaction 1"-2" in diameter resulting in visible redness at the injection site).

[0235] Thus, aspects of the invention include the use of SSIs over an extended period of time with appropriate dosing intervals and durations to improve therapeutic efficacy over the entire dosing period, such as a progressive reduction in CDAI scores in patients with Crohn's disease over a period of at least 16 weeks.

[0236] Example 15: Pulmonary Inflammation - Asthma This example shows the therapeutic efficacy of SSI (KPN SSI) in a house dust mite (HDM)-induced asthma model in mice, illustrating the basic mechanisms underlying the use of SSI in the treatment of asthmatic inflammation. In this example, BALB / c mice were exposed intranasally to HDM for 2 weeks. Mice were subcutaneously treated with either KPN SSI or placebo 1 week prior to HDM exposure and throughout the 2-week exposure period. Twenty-four hours after the final exposure, lung inflammatory cell infiltrates, gene expression, cytokine levels, and goblet cell metaplasia were analyzed, as well as serum allergen-specific serum IgE levels.

[0237] method animal Six to eight week old female mice (BALB / c) were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were used at 10 per group. Mice were housed under specific pathogen-free and environmentally controlled conditions with a 12:12 h light / dark cycle for the duration of the study.

[0238] Allergen Exposure Protocol Under isoflurane anesthesia, mice were exposed intranasally to saline (35 μL) or house dust mites (HDM, Dermatophagoides pteronyssimus, Greer Laboratories, Lenoir, NC) (25 μg in 35 μL saline). Nasal exposure to HDM or saline was performed for 5 consecutive days in week 1 and 4 consecutive days in week 2 (experimental days: 1–5, 8–11, Figure 1). Mice were euthanized 24 h after the last exposure.

[0239] Klebsiella interventional therapy strategy KPN SSI is derived from Klebsiella isolated from a patient infection and has a final OD 600Total heat-killed cells were suspended in saline containing 0.4% phenol as a preservative to a pH of 5.0. The placebo was saline containing 0.4% phenol. KB or placebo was administered prophylactically on study days -7, -5, and -3, and treatment continued on study days 1, 3, 5, 8, and 10. 30 μL of placebo or KB was injected subcutaneously into alternating adjacent sites of the skin on the lower right and left abdomen and the upper right and lower left chest.

[0240] Blood collection, bronchoalveolar lavage (BAL), and cytospin analysis of BAL cell differentials BAL cell differential counts were performed by examining cytospins for cell morphology and Wright-Giemsa staining. A total of 100 cells per mouse were identified by a blinded observer.

[0241] Quantification of HDM-specific immunoglobulins by ELISA 96-well plates were coated with HDM (2.5ug / well) and incubated overnight at 4°C. After blocking with PBS containing 5% FBS, undiluted serum was added and incubated overnight at 4°C. After washing, biotin anti-mouse IgE (BD Bioscience-San Jose, CA, USA) was added and incubated for 1 hour at 37°C. Levels were visualized using streptavidin-HRP / TMB substrate and absorbance at 450nm was recorded.

[0242] Gene expression Right lung tissue was solubilized by homogenization with a TissueLyser LT (Qiagen-Toronto, Ontario, Canada), and RNA isolation was performed using the PureLink RNA Mini kit (Life Technologies-Carlsbad, CA, USA). For cDNA synthesis, the iScript cDNA Synthesis Kit-170-8891 (Biorad) was used. Gene expression was examined by quantitative RT-PCR using TaqMan Fast Advanced Master Mix (Applied Biosystems) with Taqman probes for IL-4 (Mn00445259_m1), IL-13 (Mn00434204_m1), and IFN-γ (Mn01168134_m1) on a StepOnePLus RT-PCR instrument (Applied Biosystems-Foster City, CA, USA).

[0243] Cytokine and chemokine analysis of BAL and serum samples Thirty-one cytokine / chemokine / growth factor biomarkers were simultaneously quantified using the Milliplex Mouse Cytokine / Chemokine Kit (Millipore, St. Charles, MO, USA) according to the manufacturer's protocol. This multiplex quantification was performed by using the Bio-Plex™ 200 System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The 31-plex consisted of eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, and VEGF. The assay sensitivity range for these markers is 0.1–33.3 pg / mL. As L-13 levels in this multiplex quantification were mostly below the detection limit, IL-13 protein levels in BAL fluid were measured by ELISA (eBioscience San Diego, CA, USA).

[0244] histology Lungs were dissected and inflated with 5 mL of 10% formalin. Tissues were paraffin embedded and sectioned at 3 μm. Sections were stained with Periodic Acid-Schiff to quantify mucus-containing goblet cells. Stained sections were scanned at 60X magnification using Aperio Slidescanner (Vista, CA) (version 11.1.2.760). Positively stained pixels were identified by color segmentation in a cross-sectional fashion of the lung airways using Aperio Image Scope software to show the number of strongly positive pixels (Periodic Acid-Schiff) normalized to basement membrane length (μM).

[0245] 4. Data Analysis Data were analyzed using GraphPad Prism and are presented as mean ± SD. Multiple group comparisons were performed by one-way ANOVA followed by Sidak post-hoc tests. Four combinations of experimental groups were compared: saline-placebo vs. saline-Klebsiella, saline-placebo vs. HDM-placebo, saline-Klebsiella vs. HDM-Klebsiella, and HDM-placebo vs. HDM-Klebsiella. For statistical analysis purposes, any values ​​below the minimum standard were recorded as half the minimum standard. Principal component analysis (PCA) of the multiplexed BAL data was performed with or without the IL-13 ELISA data. PCA was completed in R (version 3.2.4) using the prcomp command (R Core Team (2016) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https: / / www.R-project.org / ).

[0246] result FIG. 20 is a graph showing HDM-specific IgE responses following exposure to saline or HDM, and treatment with either placebo or QBKPN. * P<0.05 compared with HDM-treated mice versus their appropriate controls (HDM placebo vs. saline placebo, and HDM QBKPN vs. saline QBKPN).

[0247] FIG. 21 is a series of graphs showing the numbers and differences of BAL cells in placebo- or QBKPN-treated mice exposed to saline or HDM: A) BAL total cells, B) BAL neutrophils, C) BAL lymphocytes, D) BAL macrophages, panel (E) BAL eosinophils. Data are shown as mean±SEM (10 mice per group) ( *= p<0.05). Data are means ± SD (10 mice per group). * P<0.05 compared with HDM-treated mice vs. their appropriate controls. #P<0.05 compared with HDM-treated QBKPN-treated mice vs. HDM-treated placebo-treated mice.

[0248] FIG. 22 includes two graphs showing BAL and serum mediators associated with eosinophilia: IL-5 in serum (A) and eotaxin in BAL (B). Data are shown as mean ± SD (10 mice per group) ( * = p < 0.05) (comparison between HDM-treated mice and their appropriate controls) # P < 0.05 (comparison between HDM-treated QBKPN-treated mice and HDM-treated placebo-treated mice).

[0249] FIG. 23 is a series of graphs showing Th1 and Th2 gene expression in the lungs following HDM exposure and QBPKN treatment: A) Th-1 mediated response (gene expression of IFN-γ cytokine), B) Th-2 mediated response (gene expression of IL-4 cytokine), and C) IL-13 cytokine gene expression (data are mean ± SD (10 mice per group). * P<0.05 compared with HDM-treated mice and their appropriate controls).

[0250] FIG. 24 is a series of bar graphs showing the effect of HDM exposure and QBKPN treatment on Th1- and Th2-mediated cytokine levels in BAL fluid: A) IFN-γ cytokine levels, B) IL-2 cytokine levels, C) TNF-α cytokine levels, D) IL-4 cytokine levels, E) IL-5 cytokine levels, F) IL-13 cytokine levels (data are mean ± SD (10 mice per group). * P<0.05 compared with HDM-treated mice and their appropriate controls).

[0251] FIG. 25 is a graph showing principal component analysis (PCA) of BAL cytokines showing partially normalized total cytokine profiles. This shows an example of group variation of total BAL cytokine profiles using principal component analysis (PCA) based on all multiplex data. Based on the first principal component (PC1), the different experimental groups were clustered separately. Among the placebo-treated mice, saline-exposed mice were clustered separately from HDM-exposed mice. Separation of HDM-exposed mice from saline controls was minimized by KB treatment. Among the saline control group, KB- and placebo-treated mice were similar, as shown by their clustering together. Since PC1 appeared to best separate the mice into different groups, the cytokines with the largest contribution to PC1 were identified. The top five cytokines defining PC1 were LIF (leukemia inhibitory factor, 8.2%), IL-5 (8.2%), eotaxin (8.1%), IL-4 (7.5%), and CXCL10 (7.3%). Completing the principal components analysis with the addition of an asthma marker measured by ELISA (IL-13) yielded a similar clustering, with the top five cytokines defining PC1 being IL-5 (7.3%), eotaxin (7.2%), LIF (leukemia inhibitory factor, 7.2%), IL-4 (6.8%), and IL-13 (6.7%).

[0252] Figure 26 is a bar graph showing quantification of airway goblet cells after HDM exposure and QBPKN treatment. Quantification of goblet cells was shown as the number of strongly positive pixels / basement membrane length. Data are mean ± SD (10 mice per group). * P<0.05 compared with HDM-treated mice vs. their appropriate controls. #P<0.05 compared with HDM-treated QBKPN-treated mice vs. HDM-treated placebo-treated mice.

[0253] [Table 35]

[0254] [Table 36]

[0255] [Table 37]

[0256] [Table 38]

[0257] As shown in this Example, asthma QBKPN SSI results in decreased BAL total cells, neutrophils, lymphocytes, macrophages, and eosinophils, decreased mediators of eosinophilia including serum IL-5 and BAL eotaxin, decreased Th2 cytokines (IL-4 and IL-5) in the BAL, and decreased goblet cell hyperplasia. Specifically, in summary, this Example shows that HDM-exposed mice have decreased goblet cell hyperplasia, elevated allergen-specific serum IgE, airway eosinophilia, and T H HDM-mediated airway eosinophilia, BAL total cell count, bronchoalveolar lavage (BAL) T cell count, and IL-13 / IL-5 T cell count were shown to induce classical symptoms of experimental allergic asthma, including concomitant increases in IL-4, IL-13, and IL-5 cytokines. H 2 cytokine production and goblet cell metaplasia were attenuated by treatment with KPN SSI. H This study demonstrates that HDM induction of a biased airway inflammatory response toward T 2 and associated goblet cell metaplasia was attenuated by treatment with KPN SSI. Thus, in embodiments of the present invention, e.g., allergen-induced T HTreatment (e.g., subcutaneous treatment) with microbial biologics (e.g., compositions derived from bacterial lung pathogens) is provided as a treatment for allergic airway disease to attenuate the 2-biased airway inflammatory response and / or associated goblet cell metaplasia. Thus, these results demonstrate that SSI treatment with biologics has the ability to simultaneously target two of the key molecular components that drive airway eosinophilia in a process independent of allergen-specific IgE regulation. Specifically, HDM-induced T H A KPN SSI may be administered at a dosage and for a duration effective to attenuate 2-biased allergic airway inflammation, and / or airway eosinophilia, and / or mucus content of goblet cells, which may be independent of, for example, modulating levels of allergen-specific IgE.

[0258] Example 16: Pulmonary Inflammation - COPD This example demonstrates anti-inflammatory efficacy in a mouse model of COPD (short-term (3 weeks) smoking model). In this model, mice are pre-treated (with placebo or KPN SSI) every other day for three times (Monday, Wednesday, and Friday of week 1). Then, mice are exposed to smoke (exposure to air or cigarette smoke for 5 consecutive weekdays for the first 2 weeks and 4 consecutive weekdays for week 3) from days 8 to 25 (no treatment or exposure on weekends) while continuing treatment (placebo or KPN SSI) every other day of the week (Monday, Wednesday, and Friday). On day 26, mice were euthanized 24 hours after the last air / cigarette smoke exposure and samples were collected.

[0259] Briefly, tobacco smoke exposure (Kentucky research grade cigarettes) was performed by placing mice in a nose-only exposure plexiglass chamber, ensuring that the nose was within reach of the main chamber. The cigarette was placed in the smoking apparatus, lit with a lighter, and evacuated in a fume hood. A 20 cc syringe in the smoking apparatus was filled with smoke, and the automatic valve was turned before injecting the smoke into the nose-only exposure chamber. Each inhale-exhale cycle lasted 1.5 minutes. Each mouse smoked 3 cigarettes per day for a total exposure of 45 minutes. Control air-exposed mice were restrained for a similar period of time without exposure to smoke. Animals were monitored throughout the smoke exposure procedure and for 30 minutes after smoke exposure.

[0260] Heat-killed Klebsiella strains (originating from patient infections) were administered as follows: KPN SSI or placebo vehicle (0.4% phenol in saline) was administered prophylactically three times every other day, and this regimen was continued therapeutically throughout the smoking period. Thirty microliters of placebo or KPN SSI was injected subcutaneously into the right lower quadrant, left lower quadrant, right upper thorax, and left upper thorax, with the subcutaneous injections being performed in a clockwise circular fashion around the injection sites for each injection.

[0261] Cytospins were performed and evaluated based on morphology and Wright-Giemsa staining, and then the prepared cytospin slides were used to count BAL cell differentials, where 100 cells per mouse were counted in a blinded fashion.

[0262] Immune mediator profiling of BAL and serum samples was performed as follows: Analysis of soluble mediators in BAL and serum was performed using the Bio-Ple™ 200 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using a 31 cytokine / chemokine / growth factor multiplex kit according to the manufacturer's protocol (Eve Technologies Corp, Calgary, AB, Canada). The 31-plex assays performed included the following mediators: eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, IP-10(CXCL10), KC(CXCL1), LIF, MCP-1(CCL2), M-CSF, MIG(CXCL9), MIP-1α(CCL3), MIP-1β(CCL4), MIP-2(CXCL2), RANTES(CCL5), TNFα, and VEGF. The assay sensitivity range for these markers is 0.1-33.3 pg / mL.

[0263] Flow cytometry analysis of blood Ly6CHI monocytes / macrophages and neutrophils was performed as follows. Blood was collected in EDTA-coated tubes (BD Microtainer) to prevent clotting and kept on ice until staining. Blood was stained with CD11b-FITC, Ly6G-PE, CD11c-PerCPCy5.5, and Ly6C-APC before erythrocyte lysis (BD lysis buffer). Flow cytometry was performed on a FACSCalibur (BD Bioscience). Analysis was completed with FlowJo V10.1. Neutrophils were stained with Ly6G + CD11b + Ly6CHI monocytes / macrophages were defined as Ly6C HI Ly6G - CD11b + was defined as a cell.

[0264] Data analysis was performed as follows. Statistical analysis of the results was performed using GraphPad Prism 6 software (GraphPad Software, San Diego, CA). Data are presented as mean ± SD. When comparing selected groups, one-way ANOVA analysis was followed by multiple comparisons using Sidak post-hoc test. Four combinations of experimental groups were compared: air-placebo vs. air-KB, air-placebo vs. tobacco smoke-placebo, air-KB vs. tobacco smoke-KB, and tobacco smoke-placebo vs. tobacco smoke-KB.

[0265] Body weight and clinical scores (e.g., hunched posture, interaction with other animals, activity level) were used to monitor the general health of mice exposed to filtered air or cigarette smoke with or without KPN treatment. Body weight was normalized to the starting weight of each animal. No changes in body weight were recorded in the placebo- or KPN-treated air-exposed groups (p>0.05). However, mice exposed to cigarette smoke experienced a significant weight loss (p<0.05), and this adverse effect was not reversed by KPN treatment (p>0.05). No changes in clinical scores were observed for any group.

[0266] To assess airway pulmonary inflammation, BAL total cell counts and cell differentials were analyzed. Figure 27A is a bar graph showing BAL cell differentials, which shows that QBKPN reduces BAL total cell counts after smoke exposure by decreasing lymphocyte and macrophage populations. In placebo-treated animals, cigarette smoke exposure induced an increase in total cell counts in the BAL, which was not attenuated by KPN intervention treatment (Figure 27B(a), p<0.05). The increase in BAL total cells induced by cigarette smoke exposure was attributable to lymphocytes, macrophages, and neutrophils (Figure 27B(b)-(d), p<0.05), but not eosinophils (p>0.05, data not shown). The increase in lymphocytes and macrophages in the cigarette smoke-exposed group was attenuated by KPN treatment (p<0.05), but macrophages remained elevated compared to air+KPN (Figure 27B(b)-(c)). The cigarette smoke-induced increase in neutrophils was not affected by PN treatment (Figure 27B(d)).

[0267] Previous reports have shown that a TH1-biased inflammatory response occurs in the mouse cigarette smoke exposure model. This example shows that the cigarette smoke exposure-induced TH1-biased lung inflammatory response was attenuated by KPN SSI intervention treatment, as evidenced by multiplex analysis of 31 cytokines, chemokines, and growth factors that induced TH1 and non-TH1 mediators. Fifteen of the 31 mediators measured in BAL fluid (46.7%) were induced by cigarette smoke exposure, and the 15 induced mediators included IFNγ, CXCL9, CXCL10, CCL5, IL-6, IL-17, G-CSF, CXCL1, LIF, CCL2, CCL3, CCL4, TNFα, eotaxin, and VEGF (p<0.05). IL-17 was elevated with cigarette smoke exposure in 4 of 10 samples, approaching the detection level of this mediator (0.64 pg / ml). The cigarette smoke-induced increases in IFNγ, CXCL9, CXCL10, CCL5, IL-6, G-CSF, and IL-17 were attenuated by KPN intervention (Figure 28A), all of which are associated with a TH1-biased inflammatory response. KPN SSI intervention had no effect on air-exposed animals for any of the mediators measured.

[0268] Protein expression profiles of serum immune mediators were minimally affected by cigarette smoke exposure but enhanced by KPN intervention treatment, as evidenced by applying the same multiplex assay of 31 mediators to sera from the four experimental groups. Cigarette smoke exposure induced an increase in only VEGF, and the increased levels of VEGF were elevated compared to air-exposed controls (Figure 28B(a), p<0.05). The cigarette smoke exposure-induced increase in serum VEGF was not reversed by KPN SSI intervention treatment. Only one mediator (IL12p40) was decreased by KPN intervention treatment in air-exposed animals compared to air+placebo, whereas IL-1β, CCL2, CXCL9, and CXCL10 levels (Figure 28B(b)-(d) p<0.05) were increased. In smoke-exposed mice, KPN intervention treatment increased the levels of CXCL9, CXCL10, and CCL5 compared to the cigarette smoke+placebo group. Collectively, these serum data indicate that SSI interventions, which can be implemented to be effective in locally suppressing tobacco smoke-induced lung inflammation, induced systemic effects independent of tobacco smoke exposure.

[0269] To demonstrate the systemic cellular immune response in this COPD model, flow cytometry was used to measure the Ly6C in blood after cigarette smoke exposure and KPN intervention. HI The levels of monocytes / macrophages, inflammatory monocyte populations, and neutrophils were assessed. Cigarette smoke exposure increased the levels of Ly6C in the blood. HI No increase in monocytes / macrophages or neutrophils was induced (Figure 28C(a)-(b)). Surprisingly, the KPN SSI intervention treatment did not induce an increase in blood Ly6C in the cigarette smoke-exposed group. HI Monocytes / macrophages and neutrophils were increased (p<0.05), with neutrophils increased in air-exposed animals. HIThis increase in monocytes / macrophages and neutrophils correlated with a similar pattern of increase locally in lung tissue (Figure 28C(c)-(d)), and when KB induced an increase in these cell types, this increase was further amplified by cigarette smoke exposure (p<0.05).

[0270] FIG. 28D contains three bar graphs of expression profiles of selected genes in the lung, showing that QBKPN reduces the expression of three key inflammatory cytokine genes (IL-6, IL-1 beta, and IL-17A) in lung tissue after smoke exposure.

[0271] FIG. 29 shows the expression profile of selected BAL cytokines with six bar graphs indicating that QBKPN significantly reduced G-CSF, IL-6, and IP-10 in the COPD model, and produced a downward trend in IL-4, KC, MIG, and TNF alpha.

[0272] Figure 30 shows the serum cytokine expression profile, and many markers of SSI efficacy have been identified, in particular elevated serum levels of IP-10, MIG, and RANTES. Thus, such serum markers can be used as biomarkers for SSI efficacy, for example to identify responders or non-responders to a particular SSI, or as markers of effective dosing in dose titration protocols.

[0273] This example shows that QBKPN SSI reduced many markers of the inflammatory environment in a COPD model, specifically, reductions in BAL total cells, lymphocytes, and macrophages, gene expression of cytokines normally elevated in COPD, including IL-6, IL-1beta, and IL-17A, and levels of key cytokines in COPD BAL, including IL-6, IP-10, and G-CSF. More specifically, these results show that cigarette smoke-induced TH1-biased lung inflammation and BAL cell types were attenuated by KPN treatment. In air-exposed control and cigarette smoke-exposed experimental animals, systemic immune responses, including production of immune mediators, and monocyte and neutrophil influx were induced by KPN SSI, which was associated with increased expression of Ly6C HI The results mimicked the local lung environment with increased monocytes / macrophages and neutrophils. Thus, this example shows that intervention with microbial components that enhance certain aspects of the immune response, rather than broadly suppressing the immune response, could be used to alter the course of COPD pathogenesis associated with tobacco smoke exposure.

[0274] COPD shares many underlying pathways associated with other inflammatory diseases, including asthma and inflammatory bowel disease (IBD). IBD and COPD share common findings, including altered microbiome, immune dysfunction, altered epithelial cell function, and chronic inflammation. There is also significant overlap between asthma and IBD, including altered respiratory microbiome and immune dysfunction. Overall, the similarities between COPD and other inflammatory diseases that benefit from SSIs indicate that enhancing aspects of the immune response with a repertoire of PRR agonists, such as microbial products or synthetic preparations, as demonstrated herein, may be used as a therapeutic approach for COPD.

[0275] In this example of cigarette smoke exposure-induced acute inflammation, we observed that IFN-γ, CXCL9, CXCL10, CCL-5, IL-6, G-CSF, and IL-17 were elevated in the BAL, and that this elevation was attenuated by KPN treatment. This reduction in TH1-biased inflammatory mediators was associated with a reduction in BAL lymphocyte and macrophage abundance with KPN treatment, as well as a reduction in macrophage and lymphocyte recruitment to the lung. Interestingly, there was a significant increase in T cells in both air- and cigarette smoke-exposed animals. H The systemic induction of a 1-biased chemokine signature (CXCL9, CXCL10, CCL-5) by KPN is similar to that seen during infection. H 1 In parallel with the attenuation of inflammation, Ly6C HI Systemic immune activation with increases in monocytes / macrophages and neutrophils was induced by KPN treatment, thus this example shows that KPN SSI actively stimulates aspects of the immune response that may be suitable for generating mobilization and recruitment of TH1-biased immune cells systemically but reducing locally in the BAL.

[0276] This example illustrates the expression of pro-inflammatory cytokines (e.g., IL-1β) and inflammatory monocytes (Ly6C) in the blood. HI These results show that administration of KPN SSI increases inflammatory monocytes and neutrophils systemically, similar to the response seen in acute infections. We further identified increased inflammatory monocytes and neutrophils in lung tissue by flow cytometry. In the lung inflammation examples (Examples 15 and 16), serum cytokine levels are increased by QBKPN SSI in control mice (air-exposed in the COPD study and saline-exposed in the asthma study). These can therefore be used as biomarkers for efficacy, specifically IP-10 (CXCL10), which was increased in both asthma and COPD cases in control mice treated with QBKPN SSI.

[0277] Example 17: Klebsiella variicola SSI In a murine B16 melanoma model of lung metastasis, an SSI formulated with whole killed Klebsiella lineage III (K. varicolla) cells was effective in reducing tumor burden, as shown in FIG. 31 (K. varicolla is designated as "QBKPN" in the figure).

[0278] Example 18: CD25 depletion CD25 can be used on activated T cells, activated B cells, and T cells. reg and resting memory T cells (cells involved in adaptive immunity). In this example, an anti-CD25 antibody was utilized to demonstrate efficacy of QBKPN SSI in reducing lung nodules in the absence of CD25 positive cells, as shown in FIG. 32. This demonstrates that the efficacy aspect of the SSI is independent of CD25 positive adaptive immune cells in the B16 melanoma model (mice challenged with B16 melanoma cells were given prophylactic intravenous injections of SSI and tumor foci were counted 18 days after B16 injection). Thus, an aspect of the present invention is the use of CD25 positive adaptive immune cells to inhibit the proliferation and proliferation of CD25 positive cells. + Involved in regulating cell-independent immune responses (eg, innate immune responses).

[0279] Example 19: Dose dependency and expression of Rae-1 In a murine B16 melanoma model of lung metastasis, the potency of KPN SSI decreased progressively with dilution (tumor burden was measured by quantifying Trp-1 expression by QPCR). Figure 33A shows the C10-fold increase associated with the KPN SSI formulation (QBKPN) and serial dilutions of KPN SSI (10x, 100x, and 1000x). t (cycle threshold) values ​​(5 days after B16 loading) are shown. Therefore, C t The values ​​indicate the number of PCR cycles required for the fluorescent signal to exceed the threshold (i.e., above background level). Delta C t Values ​​are the C of housekeeping genes tThe dose-dependence factored in the amount of target nucleic acid in the sample, and therefore the level is inversely proportional to the amount of target nucleic acid in the sample. As shown, tumor burden increased with increasing dilution of SSI. This dose-dependence is also reflected in the assay of the number of B16 tumor nodules in the lung, as shown in Figure 33B. Figure 33C is a bar graph showing that various dosing regimens, with injection intervals ranging from 1 to 7 days, all of which produced therapeutic effects.

[0280] Further analysis shown in Figure 34 showed that the ratio of cells expressing Rae-1 was inversely correlated with tumor burden, proving the fact that SSI increases the expression of Rae-1 in target tissues in a dose-dependent manner. It is assumed that increased Rae-1 signaling promotes immune stimulation via the NKG2D (see below) receptor present on innate lymphoid cells such as NK cells, which increases the killing of cancer cells and leads to the reduction in tumor burden demonstrated in this example. Indeed, high doses of SSI induce Rae-1 expression, which reduces cancer burden.

[0281] As shown in Figure 34(B), NKG2D (natural killer group 2, member D) knockout mice abrogated the therapeutic efficacy of QBKPN in the B16 lung metastasis model, indicating a critical role for NKG2D signaling in various aspects of the therapeutic SSI response, strengthening the significance of the evidence that Rae-1 expression is increased in target tissues.

[0282] Example 20: Site specificity lung The efficacy of KPN SSI (QBKPN) was compared with E. coli (QBECO) SSI and Staphylococcus aureus (QBSAU) SSI in mouse Lewis lung carcinoma expressing red fluorescent protein (LLC-RFP) (KPN is a pulmonary pathogen in mice, whereas ECO and SAU are not). As shown in Figure 35, a significantly enhanced effect of reducing tumor nodules in the lung was obtained from QBKPN. As shown in Figure 36, the number of LLC-RFP cells in the lung was simultaneously reduced at 15 days after LLC inoculation.

[0283] Alternative data show that while immune infiltrates in QBKPN and QBECO can be comparable at early time points in some systems, there are enhanced neutrophil levels at day 7 in QBKPN compared to QBECO (flow data). Also, gene array analysis has demonstrated longer persistence of spontaneous infiltrates in QBKPN compared to QBECO (approximately 72 hours). These data further indicate that ongoing immune responses in the lung differ as QBKPN is compared to QBECO.

[0284] colon In the MC38 colon cancer model, QBECO conferred a survival advantage over that obtained from either QBKPN or 10× concentrated QBSAU (QBSAUR), as shown in FIG.

[0285] skin In the B16 melanoma model, 100,000 B16 melanoma cells were injected into the right flank of C57BL / 6 mice in a volume of 100 μl on day 0. SSI treatment began on day -10 and continued until day +12. Tumor volume was monitored starting on day 7 with the endpoint reached on day 14. As shown in FIG. 5, 10× concentrated QBSAU or QBSAUR were much more effective in reducing B16 tumor volume in the skin compared to either QBKPN or QBECO.

[0286] Skin and lungs A B16 melanoma model was used in which lung tumors were implanted intravenously and skin tumors were implanted subcutaneously on the dorsal side, so that each animal had both skin- and lung-located cancer. In this study, placebo (30 μl), QBKPN (30 μl (OD 600 value 4.91), or 10X QBSAU (30 μl (OD 600 value 8.6) was subcutaneously injected into the experimental group of mice (N=5 / group), followed by B16 melanoma cells (1x10 5 Four control groups of mice (N=5 / group) were subcutaneously injected with either QBKPN or 10X QBSAU on days -8, -6, -4, and -2, and two of these control groups were inoculated with B16 tumors on day 0 either intravenously (QBKPN single cancer positive control) or subcutaneously (10X QUSAU single cancer positive control) to serve as single cancer positive controls, while two of these control groups did not receive any tumor inoculation to serve as negative controls. Mice were given either SSI treatment or placebo control subcutaneously every other day consecutively until the experiment was terminated on day 5 after tumor implantation. Tumor burden in lungs and skin was counted on day 5.

[0287] Mice treated with QBKPN, but not 10X QBSAU, showed increased lung-specific Rae-1 expression (Figure 38) and recruitment of monocytes and neutrophils to the lungs. PD-1 expression was also reduced in the lungs of mice treated with QBKPN compared to placebo-treated mice in the same model (Figure 39). In contrast, there was no difference between groups in PD-L1 expression in the lungs. In a model of B16 tumors in the skin and lungs, treatment of mice with 10X QBSAU, but not QBKPN, reduced skin tumor burden compared to placebo controls (Figure 40).

[0288] Thus, QBKPN demonstrated site specificity in the lungs by increasing Rae-1 expression and recruiting monocytes and neutrophils in animals bearing both skin and lung tumors. Similarly, 10X QBSAU demonstrated site specific efficacy by reducing skin tumors in these animals.

[0289] Example 21: Routes and Schedules of Dosage Comparison of intravenous and subcutaneous SSIs In this example, KPN SSI (QBKPN) was administered either intravenously or subcutaneously in a B16 lung metastasis model. B16 cells were administered intravenously on day 0 to implant tumors. KPN SSI was administered (intravenously or subcutaneously) on days 1, 3, 5, and 7. End point was reached on day 9, and tumor counts were measured. As shown in Figure 41, both routes of administration provided therapeutic efficacy.

[0290] Comparison of prophylaxis and treatment schedules In this example, we compared scheduling SSI treatment either before (prevention) or after (treatment) cancer cell challenge. This example also shows immune-related factors associated with efficacy, specifically the M1 / M2 ratio of macrophages. Figure 42 is a schematic diagram of the study design based on comparing the efficacy of QBKPN between treatment and prevention regimens in a B16 lung cancer model. As shown in Figure 43, the treatment regimen showed a very strong efficacy trend by day 17, while the prevention regimen achieved a treatment effect earlier. The efficacy of both the prevention and treatment regimens is reflected in the common efficacy-related factor of the lung M1 / M2 macrophage ratio, as shown by the surrogate M2 marker CD206, at day 10 for the treatment group and day 17 for the prevention group in Figure 44.

[0291] Early blood analysis This example illustrates how quickly SSI treatment produces a detectable therapeutic effect involving myeloid cell populations and provides an example of a therapeutic biomarker. As shown in Figure 45, there is an increase in neutrophils at all time points for the QBKPN SSI and the QBECO SSI, with a significant increase even within 3 hours after injection. As shown in Figure 46, Ly6C HI Ly6G + CD11b + There was a significant increase in the number of cells (Ly6C monocytes) with a trend toward a decrease back to placebo levels by approximately 24 hours. Thus, the cellular immune response induced by SSI treatment can be characterized as occurring rapidly within hours and then dissipating within days. This pattern of cellular response supports a dosing schedule with repeated administration at intervals of several days, such as once every 1, 2, 3, 4, 5, 6, or 7 days.

[0292] Example 22: Cytotoxicity of SSI This example shows that high doses of SSI (QBKPN) can directly induce increased cancer cell death. NCI-H358 cells (human lung cell line) were incubated in vitro with serially diluted QBKPN for 24 hours. Potency was assessed using a carboxyfluorescein succinimidyl ester (CFSE) labeling assay (a green fluorescent cell staining dye to label target cells) along with 7-AAD (7-aminoactinomycin D), a red live / dead viability dye used to identify dead / dead cells present in cytotoxicity assay samples. Figure 48 shows that high doses (1 / 20 dilution, 1 / 200 dilution) of QBKPN increase the death of NCI-H358 cancer cells in this 24-hour killing assay. Using the same assay, it was also shown that similar doses (1 / 20 and 1 / 200 dilutions) of KPN SSI increased γδ T cell-mediated killing of NCI-H358 cancer cells in a 24 hour killing assay, as shown in Figure 49. Furthermore, 1 / 200 and 1 / 2000 dilutions of KPN SSI (QBKPN) enhanced the effect of zoledronate in inducing γδ T cell-mediated cancer cell lysis, as shown in Figure 50.

[0293] As shown in this example, in select embodiments, the SSI can be administered directly to the cancerous tissue, for example, directly to the site of surgical removal of the cancer. For example, an SSI such as QBSAU can be applied topically to a cutaneous melanoma or to the site of surgical removal of a cutaneous melanoma.

[0294] Example 23: NKG2D knockout mice in MC-38 intraperitoneal injection model This example shows that NKG2D receptors are involved in mediating therapeutic responses to SSI. This was shown in a survival study of mice after intraperitoneal injection of MC-38 cells (a mouse adenocarcinoma cell line derived from a primary mouse colon cancer). Tumor cells were injected intraperitoneally to engraft the intestine, creating an MC-38 cell colon cancer model. QBECO treatment was compared to placebo in wild-type mice (C57BL / 6 mice) and NKG2D knockout mice (with a C57BL / 6 background). Wild-type and NKG2D mice (10 per group) were treated with either QBECO or placebo every other day for 10 days before injection with MC-38. Every other day treatment was continued throughout the survival study.

[0295] As shown in Figure 51, this study confirms the therapeutic efficacy of QBECO in the MC-38 colon cancer model, showing a statistically significant increase in survival in wild-type mice with QBECO treatment compared to placebo treatment. As shown in Figure 52, NKG2D expression correlates with QBECO efficacy, as there is no statistical difference in survival between NKG2D knockout mice treated with either QBECO or placebo. Immunophenotyping confirmed that the levels of NKG2D-positive cells were reduced in NKG2D knockout mice. Interestingly, NKG2D was reduced by QBECO at each time point in wild-type mice (indicating the use of NKG2D expression as a biomarker for SSI efficacy). Immunophenotyping also showed a characteristic increase in neutrophils by day -9 and monocytes by day -1 in both wild-type and NKG2D knockout mice treated with QBECO compared to placebo. PDL1+ was also increased after treatment with QBECO, which caused an increase in blood PDL1+ cells throughout the experiment, which occurred with significance in wild-type mice on days -9 and -1, and a similar phenomenon with a weakened pattern in NKG2D knockout mice. In wild-type mice, QBECO increased blood PD1+ cells in non-tumor-bearing mice (days -9 and -1), while QBECO induced an early increase in PD1+ cells in tumor-bearing mice, but this increase weakened by day 11, with a continued trend of QBECO decreasing blood PD1+ cells during survival. NKG2D knockout mice followed this trend, but to a lesser extent and without significance. Taken together, this data indicates that PD1 and PDL1 can be used as biomarkers of SSI efficacy.

[0296] Example 24: Treatment of Neutropenia In this example, the use of SSI to treat neutropenia in a mouse model is shown. As shown in the treatment outline in Figure 53, the response of neutrophil populations to SSI treatment (QBKPN) and neutrophil-depleting (anti-Ly6G) monoclonal antibody was evaluated in the spleen (representative of the circulation in mice) and lungs. In the indicated mice, SSI treatment was performed every other day from day -10 to +10. SSI (QBKPN) was injected subcutaneously at alternating sites as a 0.03 ml solution containing a dose of OD5.0. In the indicated mice, anti-Ly6G treatment was performed every other day from day -1 to +11. Antibody (Bio-X-Cel clone 1A8) was injected intraperitoneally at a dose of 200 μg / mouse. All mice were injected intravenously with B16 melanoma (200,000 cells / mouse) on day 0. Mice were sacrificed on day +12. Single cell suspensions were prepared from spleen and lung. Neutrophil populations (CD45+CD11b+Ly6G-hi Ly6C-moderate) were assessed by flow cytometry using monoclonal antibodies (BioLegend) and a Miltenyi MacsQuant cytometer and analyzed using FlowJo software. Representative staining data from lung is shown in Figure 54, which was obtained from a lung sample and gated on viable CD45+CD11b+ cells. The percentage of neutrophils in lung (Figure 55A, % of viable CD45+CD11b+ cells shown) and spleen (Figure 56A) was calculated from the primary cytometry data. The number of neutrophils (CD45+CD11b+Ly6G+Ly6C+ cells) in lung (Figure 55B) and spleen (Figure 56B) was calculated by multiplying the percentage of cells by the total cell type.

[0297] QBKPN treatment significantly increased the proportion of neutrophils in both the lung and spleen (Figure 55A and Figure 56A). QBKPN treatment significantly increased the number of neutrophils in the spleen (Figure 56B). These data indicate that QBKPN SSI induces an increase in the proportion and number of circulating neutrophils.

[0298] A parallel cohort of mice was treated with anti-Ly6G monoclonal antibody. In the absence of QBKPN treatment, anti-Ly6G completely depleted neutrophils in the lungs and spleen, both in terms of percentage and number (Figures 55 and 56). However, in QBKPN-treated mice, the neutrophil population remained at high levels in the lungs (Figure 55) and spleen (Figure 56), despite treatment with anti-Ly6G monoclonal. As neutrophils were detected using fluorescently labeled anti-Ly6G antibody (indicating antigen expression), the data indicate that QBKPN treatment did not render neutrophils resistant to anti-Ly6G-mediated depletion. Thus, these data indicate that QBKPN SSI induces proliferation of the neutrophil compartment in a model of neutropenia.

[0299] Many common treatments, including chemotherapy drugs used to treat cancer, suppress bone marrow function and reduce neutrophil counts, resulting in neutropenia. Therefore, as shown herein, SSIs can be administered to restore neutrophil counts. This type of SSI treatment has additional useful therapeutic effects. In addition to treating neutropenia, in the context of treating underlying diseases, selecting targeted SSIs with PRR agonist signatures that recapitulate different parts of the PRR agonist signatures of pathogenic microbial pathogens in the target tissue will restore innate immune function in a site-specific manner in the target tissue. This can involve, for example, anti-cancer immune responses, or anti-inflammatory immune responses mediated by SSIs (which occur in addition to the therapeutic effect of neutropenia).

[0300] Chemotherapy commonly causes myelosuppression, the most clinically relevant of which is neutropenia, which occurs between days 2 and 10 after chemotherapy. This has significant clinical implications as it interferes with the ability to maintain chemotherapy doses and schedules and creates the risk of neutropenic sepsis. Thus, SSIs can be administered to patients undergoing myelosuppressive chemotherapy as prophylaxis or treatment for neutropenia, for example, every other day between chemotherapy cycles.

[0301] Example 25: PD1 and PDL1 markers in patients In this example, clinical data is presented that demonstrates the efficacy of SSIs that act to downregulate PD1 and PDL1 expression in neoplastic disease. It also demonstrates the use of PD1 and PDL1 as markers of SSI efficacy, reinforcing the NKG2D mouse model data in Example 23. This is significant given the knowledge that PD1 (expressed on T cells) and its ligand PD-L1 play a role in blocking T cell activation and mediating pathological immune suppression.

[0302] The KPN SSI lung cancer clinical trial included six patients with two different disease groups, pre-neoplastic and neoplastic (two of the six patients were neoplastic patients). In this example, data obtained from blood samples analyzed by flow cytometry are presented.

[0303] As shown in Table 21, two neoplastic patients had elevated expression of PDL1 and PD1 compared to preneoplastic patients. This is shown in the bar graphs in Figure 57 as percentages of different cell populations and in Figure 58 as relative numbers of cells with the indicated characteristics. Neoplastic patients (01-001 and 01-002) expressed higher levels of PD1 and PDL1 and lower levels in M1 macrophages compared to preneoplastic patients.

[0304] [Table 39]

[0305] After SSI treatment, the expression of PD1 and PDL1 was significantly decreased in neoplastic patients, and the expression of M1 (CD45 + CD14 + HLA-DR + CD86 +) macrophage cells significantly increased. Figure 59 shows that PD-L1 expression decreased in patients 01-001 (Panel A) and 01-002 (Panel B) at week 1 day 4 (W1D4), week 1 day 5 (W1D5), week 2 (W2), week 4 (W4), week 12 (W12), and week 16 (W16) during the course of every other day SSI treatment. Figure 60 shows that PD-1 expression decreased in these patients at these time points. Figure 61 shows that the proportion of M1 macrophages increased in these patients at these time points. As shown, SSI treatment decreased PDL1 expression on CD45+ cells (all white blood cells), decreased PD1 expression on CD3+ cells (lymphocytes), and increased the proportion of M1 macrophages (CD45 + CD14 + HLA-DR + CD86 + In these patients, SSI treatment was discontinued at week 12 (W12), and assays showed that the relative M2 macrophage population (CD14+CD163+) generally declined until treatment was discontinued, after which it rebounded.

[0306] Example 26: Expression of Granzymes and Perforin This example shows the relationship between SSI dose, tumor burden, and expression of granzyme A, granzyme B, and perforin in a B16 melanoma mouse lung model, and provides evidence that effective SSI treatment increases granzyme and perforin levels accompanied by a decrease in tumor burden.

[0307] A mouse lung cancer model was obtained by intravenously injecting mice with B16 melanoma cells. Five groups of mice (n=5) were subcutaneously injected with: placebo (saline), 1xQBKPN, 1 / 50xQBKPN, 1 / 500xQBKPN, or 1 / 5000xQBKPN (serial dilutions of 1xKPN SSI). SSI was administered prophylactically every other day for 10 days prior to tumor inoculation. Mice were continuously injected with SSI every other day until mice were euthanized 14 days after tumor injection. The right lung post-caval lobe was removed and stored in RNAlater®.

[0308] Mouse right lung inferior vena cava lobes were homogenized in lysis buffer using a mini bead mill (Qiagen, Cat. No. 85600). RNA was extracted from all homogenates using the PureLink® RNA Mini Kit (ThermoFisher Scientific, Cat. No. 12183018A). RNA concentration and purity were quantified using a Nanodrop™ spectrophotometer. One microgram of RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad, Cat. No. 170-8891). For quantitative PCR, 50 nanograms of cDNA was loaded into each well of the reaction plate. In addition to the cDNA, TaqMan® Fast Advanced Master Mix (ThermoFisher Scientific, Cat. No. 4444554) and TaqMan® gene expression assay probes were included in the wells. Samples were quantified for Granzyme A (ThermoFisher Scientific, Mm01304452_m1), Granzyme B (ThermoFisher Scientific, Mm00442837_m1), Perforin (ThermoFisher Scientific, Mm00812512_m1), Tyrosinase (ThermoFisher Scientific, Mm00495817_m1), and GAPDH (ThermoFisher Scientific, Mm99999915_g1) (as housekeeping genes). For genes of interest (GOI), two technical replicates were added to the plate, and for housekeeping genes, a single replicate was added.

[0309] Calculation of fold changes in gene expression was performed using ddC t The dCt values ​​were calculated using the ROUT method. For GOI, technical replicates were averaged, and for biological replicates, outlier testing was performed using the ROUT method in GraphPad Prism 7.00 with 95% confidence intervals. For GzmB:1 / 500x-3 and Prf1:1 / 500x-3, technical outliers were excluded from further analysis. t ,GOI to Ct , calculated by subtracting GAPDH. t The value is the dC t The fold change was calculated by subtracting the mean dCt of the placebo group from the mean dCt of the placebo group. The fold change was calculated by taking the negative exponential of the ddCt to base 2 (2-ddC t The mean fold change for each treatment group was analyzed for significance using one-way Tukey's multiple comparison ANOVA test with 95% confidence intervals.

[0310] Figure 62 shows the results of the aforementioned assays, showing RT-qPCR fold changes of (A) GzmA, GzmB, Prf1, and (B) Tyr in the lungs of B16-inoculated mice administered various doses of QBKPN and euthanized on day 14. Taqman® gene expression assays were performed on 50 ng of cDNA isolated from the right pulmonary inferior vena cava lobe of mice. Values ​​were normalized to GAPDH and compared to gene expression in the placebo group, which were mice injected with saline. Data points are mean + / - SD. All data points are n=5, except for GzmB-1 / 500X and Prf1-1 / 500X, which showed significantly higher dC t Outliers were excluded, resulting in an n = 4. Significance was calculated using one-way Tukey's multiple comparison ANOVA test. ** p<0.01, *** p<0.001, and **** p<0.0001.

[0311] According to one aspect of this embodiment, the SSI can be formulated and administered in an effective dosing regimen in a target organ or tissue to mediate increased expression of one or more granzymes or perforins, such as granzyme A, granzyme B, and perforin.

[0312] Example 27: Different SSIs stimulate different PRRs In this example, it is shown that both the QBECO SSI and the QBKPN SSI activate multiple PRRs, and that QBECO and QBKPN each activate different PRRs, and distinct PRR repertoire fingerprints are identified for each SSI.

[0313] The data in this example were obtained by assaying PRR activation by QBKPN and QBECO in cell lines with a single PRR: HEK293 cell lines expressing a single human Toll-like receptor (TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9), NOD-like receptors (NOD1 and NOD2), C-type lectins (Dectin1a, Dectin1b, and Mincle), or Rig-1-like receptors (RIG-1 and MDA5).

[0314] As shown in Figure 63, two TLRs (TLR2 and TLR4) were highly activated by both QBECO and QBKPN. One TLR (TLR5) was highly activated by QBKPN only. One PRR (NOD2) was moderately activated by both QBECO and QBKPN. Four (TLR3, TLR7, TLR8, TLR9) were moderately activated by QBECO only, while two (Dectin1a, Dectin1b) were moderately activated by QBKPN only. NOD1, Mincle, RIG-1, and MDA5 were not activated by either QBECO or QBKPN.

[0315] TLR2 and TLR4 are localized in the cell membrane and mainly recognize lipoproteins and LPS, respectively. TLR5 is a cell membrane receptor that responds to flagellin. Among the TLRs that recognize RNA / DNA, TLR3 was only slightly activated by QBECO (no activation occurred with QBKPN). TLR3 is mainly a dsRNA receptor for viral RNA. TLR7 and TLR8 are located in the endolysosome, and these TLRs also recognize RNA (bacterial and viral), and were activated only by QBECO. Finally, TLR9 recognizes CpG-DNA and is located in the endolysosome, and this TLR was also activated only by QBECO. In light of this situation, the absence of knowledge that HEK cells internalize bacteria to a high degree in the endolysosome is relevant. Therefore, the failure of QBKPN to activate TLR7, TLR8, and TLR9 may be due to the lack of interaction of these receptors with DNA / RNA. Nod-like receptors (NLRs) are cytoplasmic receptors. NOD1 was not activated by either QBECO or QBKPN, whereas NOD2 (which recognizes muramyl dipeptide (MDP)) was activated by both QBECO and QBKPN. Other cytoplasmic receptors, RIG-1 and MDA5, which recognize short and long dsRNA, respectively, were not activated. C-type lectin receptors (CLRs) are located in the cell membrane and primarily recognize carbohydrates. Mincle was not increased by either QBKPN or QBECO. Dectin-1a and Dectin-1b are primarily fungal receptors for beta-glucan, but can also discriminate bacterial carbohydrates.

[0316] When plotted as either a bar graph (Figure 64) or a radar graph (Figure 65), a global PRR repertoire fingerprint emerges. All these results are obtained from 1 / 10 dilutions of the relevant SSIs and negative controls have been subtracted from the absorbance values.

[0317] Example 28: Viral SSI In this example, it was shown that viral SSI-induced immune changes are similar to those of bacterial SSI, which was demonstrated by comparing immune-related factors in blood after 7 days of viral SSI treatment with those of QBKPN SSI treatment. Mice were treated with placebo, QBKPN, or three viral SSI models (rabies vaccine, feline rhinotracheitis-calicivirus infection-panleukopenia vaccine, and canine influenza vaccine). Thirty microliters of treatment was injected subcutaneously every other day. The endpoint was 24 hours after the fourth SSI injection (day 7). Blood was collected at the endpoint and stained for flow cytometry to detect CD45 + Neutrophils and Ly6C in blood as a percentage of cells HI Monocyte numbers were determined.

[0318] As shown in Figure 66, compared to placebo, rabies vaccine (killed rabies virus) increased neutrophil levels to levels similar to those seen with QBKPN treatment. As shown in Figure 67, rabies vaccine, Fel-O-Vac (feline rhinotracheitis-calicivirus infection-panleukopenia virus), and Novivac (canine influenza H3H8 virus) treatment all increased Ly6C levels similar to those seen with QBKPN treatment. HI An increase in monocytes occurred as well.

[0319] This data indicates that SSIs derived from viral compositions induce immune responses similar to those derived from bacterial compositions. Viral SSIs induce responses equivalent to those generated by QBKPN in blood neutrophils and Ly6C. HI It has been demonstrated to occur at the level of monocytes.

[0320] Example 29: Enhancement by cancer antigen This example shows that SSI combined with cancer antigen enhances immune response. As shown in more detail below, lung-targeted SSI QBKPN mediated tumor burden reduction and further reduced tumor burden when used in combination therapy with melanoma-associated antigen (gp100). This effect was specific to the use of cancer antigen, as evidenced by the fact that unrelated (immunogenic but not tumor-associated) antigens did not affect tumor burden. SSI-cancer antigen combination therapy was effective both as a combined composition and as separate injections of SSI and antigen. This demonstrates the use of SSI as an adjuvant to induce immune response to immunogenic cancer antigens.

[0321] The antitumor efficacy of QBKPN SSI in combination with the melanoma-specific antigen, gp100, was compared to an irrelevant control antigen, OVA (also called SIINFEKL), in C57Bl / 6 mice obtained from Jackson Laboratories. At approximately day -31, mice were infected with K. pneumoniae (2.5x10 5 Mice were infected by oral instillation after anesthetization with isoflurane (1 cell / mouse) and then allowed to rest. Within 5-7 days, all mice fully recovered from the K. pneumoniae challenge. On day -10, some mice were injected subcutaneously with QBKPN SSI, and mice were injected every other day from day -10 to day +12. Injections were performed with a 0.03 ml dose of a suspension with an OD of 5.0 (following the typical protocol described in the Examples herein) circulating around the injection site.

[0322] On days -10, -6, and -4, some mice were also treated with the indicated antigens and / or adjuvants, either co-mixed with the SSI or injected subcutaneously at a distant site (nuchal area). 25-33 (KVPRNQDWL, 100 μg / mouse), or an immunogenic control antigen derived from OVA (OVA 257-264The adjuvant used consisted of commercially available CpG (ODN 1585 VacciGrade, 30 μg / mouse (subcutaneous injection)). On day 0, B16 melanoma (3x10 5 Mice were challenged with 1000 cells / mouse (intravenous injection). On day +14, mice were sacrificed, the number of surface metastases counted, and spleens and blood were collected. Splenocytes were pooled within each group. gp100 25-33 , OVA 257-264 , or with a control peptide (Influenza NP366-374) (all peptides at 10 μg / ml) in culture with splenocytes (1 × 10 6 After 5 days of culture of 1000 cells / well (1000 cells / well), supernatants were assessed for IFN-γ (by specific ELISA) as a readout for immunogenicity. Blood was collected into heparin-containing tubes and then centrifuged to remove cells. ELISA (RND Systems DuoSetDY485, detection limit 31.2 pg / ml) was performed according to the manufacturer's protocol. Cytokine values ​​for each culture condition (restimulated splenocytes) or animal (serum analysis) were obtained by performing ELISA on technical replicates (n=3) for all samples. Cytokine data are reported as group mean + / - standard deviation. Statistical differences were evaluated by unpaired T-test (GraphPad PRISM).

[0323] As shown in Figure 68, QBKPN treatment of mice pre-exposed to K. pneumoniae significantly (p<0.0001) reduced metastatic-like B16 melanoma in the lungs. Consistent with the relative lack of response to the vaccine without adjuvant in the mouse system, administration of cancer antigen (gp100) without adjuvant had no significant effect. Administration of cancer antigen with adjuvant (CpG) reduced tumor burden (p=0.0023), but to a lesser extent compared to SSI alone. Combining QBKPN SSI with gp100 (tumor-associated antigen) significantly (p<0.05) enhanced antitumor efficacy compared to QBKPN alone. There was no statistical difference in antitumor efficacy between SSI / antigen combinations used as combined agents and those used as separate injections. Combining SSI with a non-specific antigen (OVA) did not enhance antitumor efficacy above the level of QBKPN SSI alone.

[0324] These data indicate that QBKPN SSI can act as an adjuvant to induce / enhance the efficacy of cancer vaccines, and this adjuvant effect can be utilized either as a combined drug or administered separately. These results demonstrate that SSI (alone or combined with antigen) was superior to antigen with adjuvant (gp100+CpG) in anticancer activity. Consistent with this, SSI treatment, with or without antigen, enhanced circulating levels of IFN-γ.

[0325] Example 30: STING agonists and SSIs In this example, it is shown that augmenting microbial SSI with an additional PRR agonist (in this case a STING agonist) enhances its efficacy. These formulations constitute a class of artificial PRR agonist repertoires in which a microbial PRR agonist repertoire is augmented with one or more additional heterologous PRR agonists.

[0326] The antitumor efficacy of QBKPN SSI in combination with STING agonist 2'2'-cGMAP (inVivoGen) was demonstrated in C57Bl / 6 mice as follows. On approximately day -31, after anesthesia with isoflurane, K. pneumoniae (2.5x10 5 Mice were infected with 1000 cells / mouse and then allowed to rest. Within 5-7 days, all mice fully recovered from K. pneumoniae challenge. On day -10, some mice were injected subcutaneously with QBKPN SSI, and mice were injected every other day from day -10 to day +12. Injections were performed with a 0.03 ml dose of a suspension with an OD of 5.0, circulating around the injection site. On days -10, -6, and -4, some mice were also treated with STING agonist (10 μg / mouse subcutaneously in 20 μl saline), either co-mixed with the SSI or injected subcutaneously at a distant site (nuchal region). On day 0, tumor cells (2.0x10 5 Mice were challenged with B16 melanoma by tail vein (i.v.) injection of a single cell suspension of 1000 cells / mouse. On day +14, plasma was collected for ELISA, after which mice were sacrificed and the number of visible metastases was counted and recorded. Serum was collected in heparin-containing tubes and then centrifuged to remove cells. ELISA (RND Systems DuoSet DY485, detection limit 31.2 pg / ml) was performed according to the manufacturer's protocol. Cytokine values ​​for each animal were obtained by performing ELISA on technical replicates (n=3) for all plasma samples. Cytokine data are reported as group mean + / - standard deviation (mice (n=7) / group). Statistical differences were evaluated by unpaired T-test (GraphPad PRISM).

[0327] As shown in Figure 69, QBKPN treatment of mice pre-exposed to K. pneumoniae significantly (p<0.0001) reduced metastasis-like B16 melanoma in the lungs. Administration of STING agonist alone also reduced tumor burden (p=0.0038). Combining SSI with STING agonist further reduced tumor burden, with a significant reduction in the number of tumor nodules after co-injection of SSI and agonist compared to no treatment (p<0.0001), SSI alone (p=0.0206), or STING agonist alone (p=0.0006) (Figure 1). Similarly, co-treatment with SSI and STING agonist (separate injection sites) reduced tumor burden compared to no treatment (p<0.0001) or agonist alone (p=0016).

[0328] As shown in Figure 70, both SSI and STING agonist treatments enhanced circulating levels of IFN-γ (p<0.0001 and p=0.0038, respectively). Combining SSI and STING agonist further increased cytokine levels, even though STING agonist treatment was not administered for 18 days. Cytokine levels after combination treatment were statistically higher compared to single agent treatment. There was a significant inverse correlation between tumor burden and plasma IFN-γ levels. Taken together, this data indicates that combination therapy using STING agonist and SSI is effective.

[0329] Example 31: Genetic markers for SSI treatment response This example shows the genetic analysis of IBD subjects treated with SSI therapy and the use of genetic markers associated with IBD to identify patient populations suitable for SSI therapy. In this example, 48 subjects with IBD and approximately 2.4 million single nucleotide polymorphisms (SNPs) were analyzed according to genotyping on the Infinium Omni2.5-8 bead chip. The endpoints used in these genetic analyses were varied, including those using targeted markers of disease activity in addition to clinical response. A total of 1,271,655 SNPs were available for analysis according to standard quality control methods (including call frequency, minor allele frequency, and Hardy-Weinberg equilibrium test). 113 known IBD loci were represented on the chip and passed quality control. The study subjects included 31 Crohn's disease (CD) cases and 12 ulcerative colitis (UC) cases.

[0330] Many IBD-associated SNPs are associated with the outcome of SSI treatment in IBD cases using p=0.05 as nominal significance, such associations include, for example: CD phenotypes and IBD-associated SNPs Last recorded response in CD (response vs. no response) – SNPs associated with FASLG and TNFSF18 genes were the most highly associated (p=0.0033). The same FASLG and TNFSF18 loci were also associated with reduced CDAI in CD cases (p=0.018). Reduced calprotectin levels in CD were associated with multiple SNPs associated with the following four gene loci: ○NEXN, FUBP1, DNAJB4, GIPC2, MGC27382, ○ATF4, TAB1, ○IL23R, ○IL8, CXCL1, CXCL6, CXCL3, PF4, CXCL5, CXCL2 (all p<0.05). The 8-week reduction in CRP was associated with a concomitant SNP in NOTCH2 (p=0.002). UC phenotypes and IBD-associated SNPs A decrease in Mayo score at 16 weeks was associated with SNPs associated with: ○HNF4A ○IRFI ○GPR12 ○nd FOXO1 (all p<0.05) · HNF4A and GPR12 are also associated with lower CRP in UC after 16 weeks of treatment. IBD phenotypes and IBD-associated SNPs When the final response in all IBD cases was combined, an association was observed with SNPs associated with the FASLG and TNFSF18 loci (p=0.02), as well as with SNPs associated with the JAK2 locus (p=0.04).

[0331] A cross-chip analysis of all SNPs for the above phenotypes revealed numerous associations, summarized in Table 22:

[0332] [Table 40]

[0333] Using a cumulative Gene-Risk Score (GRS; see Jostins et al., (2013) PLoS ONE 8(10):e76328) based on all SNPs known to be associated with IBD, we identified a highly significant association (p=2.43x10) in CD responders to SSI treatment compared to non-responders, with a higher GRS from 112 IBD-associated SNPs (listed below), as shown in Figure 71A. -5). Using only three of these SNPs (rs9286879, rs7517810, rs17391694) with raw p-values ​​below 0.05, a significant association with CD responders (P-value: 1.385E-04) was also demonstrated, as shown in Figure 71B. Similarly, an association was observed with higher GRS in UC responders compared to non-responders (p=0.012), as shown in Figure 72, providing an independent validation of the CD phenotype and GRS findings summarized above. Given that the vast majority of IBD-associated loci are shared between CD and UC, it is reasonable to combine these data as a cumulative GRS for all IBD cases. Although the number of cases is small, a highly significant association exists between GRS and response at last follow-up, as shown in Figure 73 (p=8.18x10 - 7).

[0334] The significant association between cumulative GRS and final reported response in CD, UC and combined IBD patient populations indicates that IBD individuals with genetic enrichment of IBD-associated genetic markers are more likely to respond to SSI. Furthermore, since most of these genetic mutations are associated with other immune-mediated diseases, this indicates that this approach can be extended to other patient cohorts other than IBD when treated with SSI. These findings indicate that it is possible to identify subjects, such as IBD subjects, who are more likely to respond to SSI treatment. Thus, an aspect of the present invention includes the provision of a companion diagnostic genetic test assay associated with SSI treatment. SNPs and loci that can be used in such assays are as follows:

[0335] · List of IBD susceptibility SNPs of 243: rs1748195, rs34856868, rs11583043, rs6025, rs10798069, rs7555082, rs11681525, rs4664304, rs3116494, rs7556897, rs111781203, rs35320439, rs113010081, rs616597, rs724016, rs2073505, rs4692386, rs6856616, rs2189234, rs395157, rs4703855, rs564349, rs7773324, rs13204048, rs7758080, rs1077773, rs2538470, rs17057051, rs7011507, rs3740415, rs7954567, rs653178, rs11064881, 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, rs11739663, rs254560, rs6927022, rs798502, rs4722672, rs4380874, rs4728142, rs483905, rs561722, rs28374715, rs11150589, rs1728785, rs7210086, rs1126510, rs6088765, rs6017342, rs12103, rs35675666, rs12568930, rs11209026,rs2651244、rs4845604、rs670523、rs4656958、rs1801274、rs2488389、rs7554511、rs3024505、rs6545800、rs925255、rs10495903、rs7608910、rs6740462、rs917997、rs2111485、rs1517352、rs2382817、rs3749171、rs4256159、rs3197999、rs2472649、rs7657746、rs2930047、rs11742570、rs1363907、rs4836519、rs2188962、rs6863411、rs11741861、rs6871626、rs12654812、rs17119、rs9358372、rs1847472、rs6568421、rs3851228、rs6920220、rs12199775、rs1819333、rs1456896、rs9297145、rs1734907、rs38904、rs921720、rs1991866、rs10758669、rs4743820、rs4246905、rs10781499、rs12722515、rs1042058、rs11010067、rs2790216、rs10761659、rs2227564、rs1250546、rs6586030、rs7911264、rs4409764、rs907611、rs10896794、rs11230563、rs4246215、rs559928、rs2231884、rs2155219、rs6592362、rs630923、rs11612508、rs11564258、rs11168249、rs7134599、rs17085007、rs941823、rs9557195、rs194749、rs4899554、rs8005161、rs17293632、rs7495132、rs529866、rs7404095、rs26528、rs10521318、rs3091316、rs12946510、rs12942547、rs1292053、rs1893217、rs7240004、rs727088、rs11879191、rs17694108、rs11672983、rs6142618、rs4911259、rs1569723、rs913678、rs259964、rs6062504、rs2823286、rs2836878、rs7282490、rs2266959、rs2412970、rs2413583、rs2641348、rs7517810、rs1260326、rs7438704、rs10061469、rs2503322、rs5743289、rs6667605、rs1440088、rs3774937、rs477515、rs1182188、rs17780256、rs11083840、rs3766606、rs13407913、rs6708413、rs2457996、rs10051722、rs4976646、rs7746082、rs38911、rs13277237、rs2227551、rs7097656、rs12778642、rs11229555、rs174537、rs568617、rs2226628、rs566416、rs11054935、rs3742130、rs1569328、rs2361755、rs3091315、rs1654644、rs4243971、rs6087990、rs6074022、rs5763767。、

[0336] A subset of 112 SNPs that together generated the GRS in Figure 71A: rs10065637, rs1016883, rs1042058, rs10521318, rs10758669, rs1077773, rs10781499, rs10865331, rs10896794, rs11054935, rs11083840, rs11150589, rs11168249, rs11209026, rs11583043, rs11672983, rs11739663, rs11742570, rs1182188, rs1260326, rs11706 ... 2778642, rs13204048, rs13277237, rs1517352, rs1569723, rs1654644, rs17085007, rs17119, rs17229285, rs1728918, rs1734907, rs17391694, rs1 748195, rs17780256, rs1801274, rs1847472, rs1893217, rs194749, rs2024092, rs2111485, rs212388, rs2155219, rs2188962, rs2189234, rs2227551 , rs2231884, rs2413583, rs2472649, rs2641348, rs2651244, rs26528, rs2816958, rs2823286, rs2836878, rs2930047, rs3024505, rs35320439, rs37 42130, rs3764147, rs3766606, rs38904, rs395157, rs4243971, rs4409764, rs4692386, rs4728142, rs477515, rs4802307, rs4836519, rs483905, rs49 76646, rs516246, rs559928, rs564349, rs566416, rs568617, rs6017342, rs6088765, rs616597, rs6426833, rs6651252, rs6667605, rs6856616, rs68 63411, rs6920220, rs7097656, rs7134599, rs7210086, rs7236492, rs7240004, rs724016, rs7282490, rs7495132, rs7517810, rs7702331, rs7758080,rs864745, rs917997, rs921720, rs925255, rs9264942, rs9286879, rs9297145, rs9319943, rs94182 3, rs9491697, rs9847710, rs12199775, rs12654812, rs1292053, rs2227564, rs3197999, rs6074022. ,

[0337] A subset of the aforementioned 112 SNPs show varying degrees of association with response to SSI treatment, as shown in Tables 23A and 23B, which reveal associated alleles for each SNP and odds ratios reflecting the association of the alleles with SSI response. In Table 23A, odds ratios greater than 1 indicate that the indicated allele is positively associated with response to SSI treatment, odds ratios less than 1 indicate that the alternative allele is positively associated with response to SSI treatment, and the alleles shown in this table are negatively associated with response to SSI treatment. In Table 23B, the negative odds ratios in Table 23A are converted to positive odds ratios for the alternative allele, so that all odds ratios are greater than 1 and the response alleles are the alleles associated with response to SSI treatment.

[0338] [Table 41]

[0339] [Table 42]

[0340] [Table 43]

[0341] [Table 44]

[0342] [Table 45]

[0343] [Table 46]

[0344] Of the subset of 112 SNPs mentioned above, many are individually associated with specific clinical efficacy markers, and these SNPs are spatially associated with genes, such that surrogate markers (such as SNPs) associated with these genes may also serve as markers of SSI efficacy, as shown in Table 24.

[0345] [Table 47]

[0346] The aforementioned IBD-associated SNPs are spatially associated with genes (Liu et al., Nature Genetics. 47.9 (Sept. 2015): p 979), and as a result, surrogate markers (such as SNPs) associated with these genes may also serve as markers of SSI efficacy, as shown in Table 25:

[0347] [Table 48]

[0348] [Table 49]

[0349] The aforementioned IBD-associated SNPs are spatially associated with genes (Jostins, et al., Nature. 2012;491:119-124), and as a result, surrogate markers (such as SNPs) associated with these genes may also serve as markers of SSI efficacy, as shown in Table 26:

[0350] [Table 50]

[0351] The aforementioned IBD-associated SNPs are spatially associated with genes (Jostins, et al., Nature. 2012;491:119-124), and as a result, surrogate markers (such as SNPs) associated with these genes may also serve as markers of SSI efficacy, as shown in Table 27a:

[0352] [Table 51]

[0353] [Table 52]

[0354] The correlation coefficient between pairs of loci is r-squared (r 2 ), which can be used as a measure of the extent to which alternative genetic markers provide similar diagnostic or prognostic information. 2 The value of r ranges from 0 to 1 (1 when the two markers provide identical information and 0 when they are in perfect equilibrium). By convention, r 2 Markers with a r >0.8 may be considered to be in high linkage disequilibrium, such that such markers may provide similar diagnostic or prognostic information. Thus, embodiments of the assays described herein are in linkage disequilibrium with the above markers, e.g., r 2 >0.7, r 2 >0.8, r 2>0.9, or r 2 >0.95. Additionally, informative markers can be characterized by their physical proximity in the genome, such as being within 1 Mbp of each other (e.g., within 50 Kb, 60 Kb, 70 Kb, 80 Kb, 90 Kb, 100 Kb, 200 Kb, 300 Kb, 400 Kb, or 500 Kb of each other).

[0355] In accordance with the foregoing, a "genetic SSI response marker" refers to a genetic biomarker, the presence of which correlates with the probability of responding to treatment with SSI. Examples of genetic SSI response markers are disclosed in this Example, which demonstrate a correlation with response to SSI in IBD patients. Genetic SSI response markers can be detected by a wide range of genomic assays, including assays that examine transcriptional or translational products of the genome, such as protein isoforms associated with specific genomic alleles. Similarly, "biochemical SSI response markers" are disclosed herein that provide a biochemical indication of response to SSI treatment, including, for example, temporal or specific changes in cell populations, or abundance or concentration of biologically relevant molecules. Biochemical and genetic SSI response markers can be used in the context of SSI treatment, for example, as diagnostic or prognostic indicators for IBD in general, or specific forms of IBD, such as Crohn's disease and ulcerative colitis. Examples of genetic SSI response markers are shown in Table 27b, as well as Tables 23-26.

[0356] [Table 53]

[0357] Example 32: PRR Receptor Targeting In this example, an analysis of PRR receptors that are targets of alternative SSIs is presented. Table 28: List of PRRs stimulated by selected SSIs, including QBKPN, QBECO, and QBSAU. When a PRR is "optional," this indicates that some embodiments may be designed to include an agonist for that PRR.

[0358] [Table 54]

[0359] [Table 55]

[0360] [Table 56]

[0361] [Table 57]

[0362] [Table 58]

[0363] Thus, in selected embodiments, SSI treatment is provided using microbial PRR agonists derived from microbial pathogens of target tissues to target a select subset of PRRs. For example, immunogenic compositions are provided that include microbial agonists for at least a select number of different PRRs for use in eliciting an innate response in a target tissue, where the PRR agonists are microbial components derived from a single type of microorganism that is selectively pathogenic in the target tissue. The number of different PRRs targeted by the agonist can be, for example, a number between 5 and 25, or at least an integer within that range, such as at least 5, at least 6, etc. The different PRRs can be selected, for example, from the PRRs shown in Table 28, Table 29, and / or Table 30.

[0364] Example 33: Cytokine markers for SSI treatment This example provides a cytokine marker index of various aspects of SSI treatment. The data reflect an analysis of 42 cytokines / chemokines at baseline, 4 weeks, 8 weeks, 16 weeks, and 24 weeks from a cohort of Crohn's disease patients undergoing SSI treatment with QBECO in a randomized, placebo-controlled study involving 68 patients.

[0365] Cytokine changes following exposure to QBECO QBECO exposure increased IL-18 and IP-10 at both 8 and 16 weeks. Serum levels of IL-18 showed the most significant difference between QBECO-treated and placebo-treated patients at week 8 (median change 24 pg / mL, adjusted p=0.0256) (Figure 74). This increase in IL-18 was also evident at week 16. The second serum biomarker showing a significant difference was IFNγ-inducible protein 10 (IP-10 (also known as CXCL10)), which was increased more in patients exposed to QBECO 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).

[0366] Vascular endothelial growth factor A (VEGF-A) showed a modest increase in the QBECO group at week 8 (median change 14 pg / mL, adjusted p=0.0483), but this difference was lost by the end of treatment at week 16. Many other immune factors showed strong trends of increase from baseline to 8 weeks of QBECO exposure, including granulocyte colony-stimulating factor (GCSF), IFNγ, IL-17A, IL-6, IL-7, and transforming growth factor-α (TGFα).

[0367] Patients were removed from all treatment after week 16 and re-evaluated at week 24, and no serum immune factors remained elevated, indicating that these biomarkers are most useful for assessing immune responsiveness to QBECO during treatment.

[0368] Changes in serum biomarker cytokine concentrations associated with clinical response A secondary analysis was performed in QBECO-exposed patients (N=42, including patients initially randomized to QBECO and those who switched from placebo at week 8) to assess whether any of the immune changes over time were associated with clinical outcomes. Compared to non-responders, there was a smaller increase in Il-18 among patients who had clinical responses and remissions. Compared to non-responders, there was a smaller increase in IP-10 among patients who had clinical responses and remissions. Compared to non-responders, responders showed a significant increase in IFNγ, IL-12p70, IL-17A, and TGFα over time. Specifically, at week 8, patients who developed a clinical response to QBECO showed a greater increase in IFNγ, IL-12p70, IL-17A, and TGFα over time compared to non-responders (adjusted p=0.0344) (Figure 75).

[0369] Baseline serum immune factors associated with clinical response Low eotaxin-1 was a biomarker predictive of remission in response to QBECO treatment. Specifically, baseline serum levels of eotaxin-1 (CC chemokine 11) had the strongest association with clinical remission (adjusted p=0.0016), with patients with higher baseline levels being less likely to achieve clinical remission by week 8 with QBECO treatment (Figure 76). Patients with higher baseline IL-10 and IL-12p40 were also less likely to experience a clinical response to QBECO treatment by week 8, although this did not reach statistical significance after correcting for multiple comparisons (Figure 76).

[0370] Results show that patients with a history of exposure to TNFα inhibitors, such as Remicade™ or Humera™, were less likely to achieve clinical remission or clinical response after 8 weeks of QBECO treatment. This makes it more difficult to treat a group that may experience severe immune dysfunction, which may be due to their history of exposure to these immunosuppressants and / or the nature of their condition. Evidence supporting this is found when mean baseline serum levels of immune factors associated with clinical outcomes are stratified by history of exposure to TNFα inhibitors. Baseline serum immune factors (eotaxin-1, IL-10, and IL-12p40) that are inversely correlated with patient response to QBECO were higher in patients with a history of exposure to anti-TNFα treatment compared to those without (Table 31).

[0371] [Table 59]

[0372] High response and remission rates in anti-TNFα naive patients In anti-TNFα naïve patients, treatment with QBECO SSI for 8 weeks resulted in a statistically significant response rate of 64% compared to 27% for placebo controls (p=0.041). Clinical remission rates after 8 weeks of treatment were also excellent at 50%, more than double the placebo remission rate of 23% (p=0.16). Clinical response and remission rates were assessed using the standard Crohn's Disease Activity Index (CDAI) and were defined as a reduction in CDAI score of 70 points or more (response) and a reduction in CDAI score of 150 points or less (remission). Anti-TNFα naïve patients include, for example, patients who have not been previously treated with the immunosuppressants Remicade®, Humira®, Cimzia®, and Simponi®. Forty percent of patients previously treated with TNFα inhibitors who completed 16 weeks of SSI treatment achieved remission, indicating that this more challenging patient population may respond to QBECO SSI with extended treatment.

[0373] Building a composite predictive model to assess the likelihood of patients responding to QBECO after 8 weeks of treatment Using a regularized logistic regression modeling approach, which simultaneously selects the variables most strongly associated with response and optimally weights them to obtain a predictive score, a composite predictive model was constructed that included both baseline biomarker measures (i.e., 42 immune factors, including cytokines, chemokines, and growth factors) and baseline clinical and demographic characteristics, including year of enrollment, age at randomization, age at diagnosis, time from diagnosis to randomization, sex, race (Caucasian or not), location (Vancouver or not), prior anti-TNFα treatment, baseline Crohn's Disease Activity Index (CDAI) score, baseline fecal calprotectin level, and baseline C-reactive protein level.

[0374] To correct for potential overestimation in model fitting, we created an "optimism-adjusted" area under the receiver-operating characteristic curve (AUROC), which can then be more easily and reliably applied to future, unique data.

[0375] As shown in the analysis of this example, high baseline serum eotaxin-1 was the strongest negative biomarker predictor of clinical response after 8 weeks of QBECO treatment and was included in all models created. Among the clinical / demographic variables, gender (women were more likely to respond to QBECO treatment) and anti-TNFα treatment history (those with a history of exposure were less likely to respond to QBECO) were the strongest predictors. Table 32 summarizes the different models created. A typical commercial biomarker standard requires an AUROC>7 for a predictive model to be practical. After optimism correction, the combined model generated from this data achieved this level of predictive value including the following variables: gender, TNFα treatment history, and baseline levels of eotaxin-1, GROα (also known as CXCL1 (neutrophil chemokine)), IL-10, PDGF AA, and RANTES (also known as CCL5 (chemokine for activated T cells, eosinophils, and basophils)). Alternatively, predictive models may be developed using eotaxin, GROα, IL10, PDGF AA, RANTES, gender, and anti-TNFα history to reliably predict response.

[0376] [Table 60]

[0377] analysis Cytokine changes with QBECO exposure: IL-18 (adjusted p=0.011 at 8 weeks and 0.067 at 16 weeks) and IP-10 (adjusted p=0.036 at 8 weeks and 0.015 at 16 weeks) demonstrated substantial and statistically significant increases with exposure to QBECO. These two cytokines also demonstrated significantly different courses at week 8 when comparing clinical responders with non-responders (adjusted p=0.0328 for both) and those who achieved clinical remission with those who did not (adjusted p=0.0368 for both). Additionally, IL-18 demonstrated significantly different courses when comparing those randomized to QBECO with those randomized to placebo (adjusted p=0.0256).

[0378] Cytokine associations with outcome: Baseline eotaxin-1 concentrations were most strongly associated with clinical outcome among subjects exposed to QBECO, with subjects with higher baseline eotaxin-1 concentrations being more likely to achieve clinical remission after 8 weeks of QBECO exposure (adjusted p = 0.0016).

[0379] Composite biomarkers: Baseline concentrations of eotaxin-1, GRO-α, IL-10, PDGF AA, and RANTES, combined with the clinical variables of sex and prior anti-TNFAα exposure, provided significantly better prediction of 8-week clinical outcomes (optimistically corrected AUROC=0.70, 95% CI [0.59, 0.81] for response and AUROC=0.71, 95% CI [0.60, 0.82] for remission). The model had some observable predictive ability for placebo subjects (AUROC=0.67, 95% CI [0.45, 0.89] for response and AUROC=0.70 [0.47, 0.93] for remission).

[0380] overview QBECO SSI treatment induces a biological response by increasing certain cytokines (IL-18 and IP-10) over time. Surprisingly, both cytokines increased after QBECO treatment, but the increase was smaller in responder patients. Therefore, treatment protocols such as dosing can be adjusted to achieve this result.

[0381] IFNg, IL-12P70, IL-17A, and TGFa were increased in responders compared to non-responders. Thus, treatment protocols such as dosing can be adjusted to achieve this result. TGFa can be used, for example, as a marker of mucosal healing.

[0382] A low level of Eotaxin 1 may be used as an indicator that a patient is more suitable for SSI treatment.

[0383] in conclusion: Increase in serum IL-18 from baseline to weeks 8 and 16 of treatment was the best biomarker (among 42 biomarkers evaluated) for QBECO exposure / activity. Increased serum levels of IFNγ, IL-12p70, IL-17A, and TGFα following 8 weeks of QBECO treatment were associated with clinical response. Crohn's disease patients with higher baseline levels of eotaxin-1 (and to a lesser extent IL-10 and IL-12p40) are less likely to experience a clinical response or remission to QBECO after 8 weeks of treatment; prior anti-TNFα treatment may predispose to higher levels of these factors, and anti-TNFα naïve patients represent a different Crohn's disease patient population suitable for QBECO SSI treatment. A combined model including baseline serum biomarkers and clinical / demographic data was assumed to be able to predict a patient's likelihood of response to 8 weeks of QBECO treatment after optimism correction (AUROC ≥ 7). Variables in the final model included sex, prior anti-TNFα treatment, and baseline serum levels of Eotaxin-1, GROα, IL-10, PDGF AA, and RANTES.

[0384] This biomarker analysis demonstrates the creation of a viable predictive composite model that can provide personalized treatment for Crohn's disease. This biomarker analysis may be useful alone or in combination with the genetic analysis exemplified herein, which has been shown to significantly stratify responders and non-responders based on the derivation of genetic scores.

[0385] Example 34: DSS colitis model In this example, results are presented from a mouse model of chemically induced colitis, which was used to evaluate the efficacy of QBECO SSI treatment. Mice were administered drinking water containing dextran sulfate sodium (DSS) to induce colitis that mimics human ulcerative colitis. In this disease model, one cohort of mice was exposed to DSS for 7 days, and a second cohort was exposed to water for 3 days after exposure to DSS for 7 days. Mice received SSI injections every other day for 10 days prior to DSS exposure. SSI injections were continued during DSS exposure. As shown, the results show that SSI treatment with QBECO ameliorated disease severity by limiting weight loss (Figure 77), reducing disease severity (Figure 78), and maintaining mucosal barrier function (Figure 79). The pharmacodynamics of QBECO SSI treatment in this model are illustrated by blood neutrophil (Figure 80) and blood cytokine (Figure 81) levels in disease-free mice treated with QBECO or placebo, and the pharmacokinetics of QBKPN (Figure 82) were used to model the pharmacokinetics of SSIs in general, including QBECO (QBKPN SSI was fluorescently labeled and then injected subcutaneously into disease-free mice, and mice were bled at different time points over 48 hours).

[0386] Example 35: Tissue Biomarkers In this example, results from a mouse cancer model are presented, showing tissue-specific biomarker responses to SSI treatment. As shown in Figure 83, gene expression in lung tissue demonstrates that tissue-specific SSI responses occur for CXCL10 (IP-10), CCL2 (MCP-1), and CCR2. In this example, mice were treated with placebo, QBKPN, or QBECO every other day for 10 days, and then implanted with B16F10 tumors in the lungs via tail vein injection. Treatment continued every other day after tumor implantation. Mice were euthanized on day 5 (A, C, E) or day 17 (B, D, F). Thus, in alternative embodiments, CXCL10 (IP-10), CCL2 (MCP-1), and / or CCR2 may be used as biochemical SSI response markers, for example, in biopsy tissue sample assays.

Claims

1. 1. An in vitro method for determining efficacy of site-directed immunotherapy (SSI) treatment in a human patient having Crohn's disease and having low baseline levels of eotaxin-1 and IL-10, comprising: determining in vitro the levels of IL-12P70, IL-17A, Eotaxin 1, GROα, IL-10, PDGF AA, RANTES, IFN-gamma and TGF-alpha in a sample from said patient receiving treatment; If the SSI results in increased levels of IL-12P70, IL-17A, IFN-gamma, and TGF-alpha, assess that the patient is likely to benefit from continuing the SSI. The method, wherein the SSI comprises treatment with a formulation comprising whole killed or whole attenuated E. coli cells.

2. 2. The method of claim 1, wherein if the level of eotaxin 1 is below a predetermined threshold level in the sample, the patient is likely to benefit from the SSI treatment.

3. 3. The method of claim 1 or 2, wherein the E. coli is recombinant E. coli.

4. 4. The method of claim 3, wherein the recombinant E. coli comprises a recombinant gene encoding at least one component of an E. coli derived mammalian pattern recognition receptor (PRR) agonist.

5. The method according to any one of claims 1 to 4, wherein the patient is an anti-TNFα naive patient.

6. The method according to any one of claims 1 to 5, The method further comprises screening a sample isolated from the patient for a genomic polymorphism that is a diagnostic SNP or is in genetic linkage disequilibrium with the diagnostic SNP; If the diagnostic SNP contains a response allele, then the patient is likely to benefit from the treatment; The method, wherein the diagnostic SNPs and the corresponding response alleles are one or more of those set forth in the table below. 【Table 1】 【Table 2】 【Table 3】

7. 7. The method of claim 6, wherein the patient is homozygous for the response allele or heterozygous for the response allele.

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