Perioperative innate immune priming in cancer treatment

Abstract

Provided is a treatment modality that includes the use of a specific repertoire of PRR ligands to improve perioperative immune dysregulation.In fact, innate immune system signaling is induced perioperatively to promote targeted immune response after surgery.Therefore, subject can be treated with immunogenic preparations tailored to specific target tissues to treat subject before and after surgery to remove solid tumors, in this case, target tissue is the site of tumor or the characteristic site of cancer metastasis.

Description

[Technical field]

[0001] In the field of medicine, such as the treatment of cancer, innovations have been disclosed relating to the use of surgery in combination with neoadjuvant and / or adjuvant treatment with tissue-specific preparations containing natural immunogens, such as microbial components. [Background technology]

[0002] In vertebrates, an important aspect of immunological regulation involves the coordinated activity of the innate and adaptive immune systems, which involves metabolic, enzymatic and molecular genetic changes within immune cells and orchestrates an elaborate system of cellular, cytokine and chemokine signaling pathways that mediate the coordinated activity of different components of these complementary systems (see Iwasaki & Madzhitov, 2015, Nature Immunology 16:343-353; WO 0209748; WO 03051305; Turner et al., 2014, BBA-Molecular Cell Research 1843:11 2563-2582). One aspect of this coordinated activity is that ligands of pattern recognition receptors (PRRs) of the innate immune system can be used as vaccine adjuvants to improve adaptive immune responses (see Maisonneuve et al., 2014, PNAS 111(34), 12294-9; WO2007035368), and forms the basis for the recognition that specific repertoires of PRR ligands can be formulated together as site-specific immunomodulatory agents that induce therapeutic immune responses in target tissues (see WO2017185180).

[0003] Immunological memory, such as the recognition of specific antigens by B-cell and T-cell receptors, is a long-recognized central feature of the adaptive immune system and is the basis of vaccine efficacy (see Nature Immunology, Focus on immunological memory: June 2011, Volume 12 No 6 pp461-575). Innate immune memory is a more recently recognized and less well understood feature of the immune system (see Netea et al., 2015, Nature Immunology 16, 675-679, and Bordon, 2014, Nature Reviews Immunology 14, 713).

[0004] The immune system responds in a complex manner to major surgery, and this response is particularly important in the context of primary cancer surgery (see Matzner et al., Nat Rev Clin Oncol. 2020 May;17(5):313-326. doi:10.1038 / s41571-019-0319-9. Epub 2020 Feb 17). Given the potential for surgery-related complications, neoadjuvant and adjuvant cancer treatments have typically been avoided in the immediate perioperative period. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO02 / 009748 [Patent Document 2] WO03 / 051305 [Patent Document 3] WO2007 / 035368 [Patent Document 4] WO2017 / 185180 [Non-patent literature]

[0006] [Non-Patent Document 1] Iwasaki&Madzhitov,2015,Nature Immunology16:343-353 [Non-Patent Document 2] Turner et al.,2014,BBA-Molecular Cell Research 1843:11 2563-2582 [Non-Patent Document 3] Maisonneuve et al.,2014,PNAS 111(34),12294-9 [Non-Patent Document 4] Nature Immunology,Focus on immunological memory:June 2011,Volume 12 No 6 pp461-575 [Non-Patent Document 5] Netea et al.,2015,Nature Immunology 16,675-679 [Non-Patent Document 6] Bordon,2014,Nature Reviews Immunology 14,713 [Non-Patent Document 7] Matzner et al.,Nat Rev Clin Oncol.2020 May;17(5):313-326.doi:10.1038 / s41571-019-0319-9.Epub 2020 Feb 17 Summary of the Invention [Means for solving the problem]

[0007] An embodiment of the therapeutic modality of the present disclosure includes the use of an effective amount of an immunogenic composition to treat perioperative immune dysregulation in a subject. The treatment includes perioperative cancer treatment, in which the immunogenic composition is administered perioperatively before and / or after surgery. Thus, cancer treatment can include neoadjuvant or adjuvant treatment of a mammalian subject whose cancer is characterized by its potential to form tumors in target tissues and / or metastasize to target tissues, and in which surgery is performed to remove the tumor. Perioperative can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more before and / or after the date of surgery, or 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day before and / or after the date of surgery.

[0008] The immunogenic composition may comprise an artificial repertoire of PRR ligands that recapitulate at least a portion of the mammalian pattern recognition receptor (PRR) agonist signature of a microbial mammalian pathogen, such as a pathogen that is pathogenic in a target tissue. The repertoire of mammalian PRR ligands may be formulated together in a therapeutic vehicle for combined presentation after administration to a mammalian subject. The composition may comprise components of a mammalian microbial pathogen that are ligands for multiple mammalian PRRs, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 different mammalian PRRs. The immunogenic composition may be adapted and administered to modulate an immune response in a target tissue, for example, an immune response effective to treat residual disease and thereby treat cancer, for example, by modulating an innate immune response in the target tissue.

[0009] The compositions may be for use by administering an effective amount perioperatively to increase CD69 expression, decrease CD96 expression, increase MHCII expression, increase myelopoiesis, increase neutrophil cell counts, increase CCR2 chemokine receptor expression on immune cells, and / or increase FNγ expression. Thus, the point-of-care test may be used to monitor the perioperative immune response, including any of the aforementioned aspects of the perioperative immune response.

[0010] The therapeutic uses described herein are reflected in the corresponding methods of treatment, and vice versa.

[0011] Embodiments of the invention may include one or more of the following features: A use in which the PRR ligand is a PRR agonist. A use in which the immunogenic composition modulates an innate immune response in a target tissue. A use in which the repertoire of mammalian pattern recognition receptors is an artificial repertoire, and in which the portion of the PRR agonist signature is a distinct portion that is different from any naturally occurring PRR ligand signature of the microbial mammalian pathogen. A use in which the subject is a mouse, a cat, a dog, a horse, a rodent, or a human. A use in which the therapeutic vehicle comprises microbial cells, recombinant microbial cells, a cellular fraction of recombinant microbial cells, a cellular fraction of microbial cells, a bacterial outer membrane fraction, a bacterial inner membrane fraction, a pellet from gradient centrifugation of microbial cell components, microbial chromosomal dna, a microparticle, or a liposome, each of which comprises a component of a microbial mammalian pathogen that provides a PRR agonist that together constitutes a repertoire of PRR agonists. A use in which the recombinant microorganism comprises a recombinant gene encoding at least one component of a PRR agonist. The use wherein the therapeutic vehicle comprises whole killed or attenuated microbial cells or recombinant microbial cells.

[0012] An SSI delivery system is provided, comprising an SSI formulation for use in an amount effective to treat cancer in a mammalian subject, the cancer being characterized by the potential to form tumors in the target tissue and / or metastasize to the target tissue. The delivery system may be used such that the therapeutic composition is deployed in conjunction with surgical removal of the tumor on the day of surgery, when the surgical removal leaves a surgical wound. The therapeutic composition may comprise a PRR ligand of a mammalian microbial pathogen that is pathogenic in the target tissue, or whole killed or attenuated cells of the mammalian microbial pathogen. The delivery system may be adapted for use on the day of surgery to be applied to the surgical wound to mediate the release of the composition, thereby treating residual disease and modulating an immune response in the target tissue effective to treat the cancer. The delivery system may comprise a graded release matrix that encases the mammalian pathogen or the PRR ligand, the graded release matrix may be adapted to release the antigen from the matrix in multiple successive, temporally separated administration steps after the delivery system is applied to the surgical wound on the day of surgery, a therapeutically effective dose of the antigen being released in each administration step during the perioperative period. The sustained release matrix may be composed of, for example, surface eroding polymers such as polyanhydrides and / or poly(orthoesters), or cellulose acetate phthalate complexed with Pluronic F-127. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a data plot showing results from a mouse cancer metastasis model, with the right hand column for each treatment category representing mice that underwent surgery and the total number of lung metastases quantified 3 days after surgery. [Diagram 2] Figure 2 is a data plot showing results from a mouse cancer metastasis model, with the right column representing mice that underwent surgery for each treatment category, and shows NK cell activation in the spleen quantified by cells expressing CD69 3 days after surgery. [Diagram 3]Figure 3 is a data plot showing results from a mouse cancer metastasis model, with the right column for each treatment category representing mice that underwent surgery, and shows that NK cell activation in the spleen was quantified by cells expressing CD96 3 days after surgery. [Figure 4] FIG. 4 is a data plot showing results from a mouse cancer metastasis model, with the right column representing mice that underwent surgery for each treatment category, showing monocyte / macrophage activation in the spleen as quantified by cells expressing MHCII 3 days after surgery. [Diagram 5] FIG. 5 is a data plot showing results from a mouse cancer metastasis model, with the right column representing mice that underwent surgery for each treatment category, and neutrophil cell counts (CD11b+Ly6G+) from the spleen 3 days after surgery quantified as % of live cells. [Figure 6] Figure 6 is a data plot showing results from a mouse cancer metastasis model, with the right column for each treatment category representing mice that underwent surgery and showing splenic monocyte / macrophage CCR2 receptor expression measured 3 days after surgery. [Figure 7] Figure 7 is a data plot showing results from a mouse cancer metastasis model, with the right columns representing mice that underwent surgery for each treatment category. Immune activation was quantified on day 1 after surgery by stimulating whole blood with a cytokine cocktail for 12 hours and measuring IFNγ levels. [Figure 8] FIG. 8 is a data plot showing results from a perioperative mouse liver metastasis model, demonstrating a dramatic reduction in tumor burden (% of liver) with QBECO SSI treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] In the following detailed description, various examples from specific embodiments are presented along with experimental procedures that can be used to perform a wide variety of modifications and variations in the practice of the present invention. For the sake of clarity, various technical terms are used herein in accordance with what is understood to be their commonly understood meanings, as reflected in the definitions set forth below.

[0015] "Immunogen" refers to a molecule, or a composition containing such a molecule, that is capable of eliciting an immune response by the immune system of an organism. "Antigen" refers to a molecule that is capable of binding to the products of an immune response.

[0016] A "pathogenic" agent is an agent, such as a microorganism, such as a bacterium or virus, that is known to cause an infection in a host in nature, and in this sense "pathogenic" is used in the context of the present invention to mean "naturally pathogenic." Although a wide variety of microorganisms may be capable of causing infection under artificial conditions, such as artificial inoculation of the microorganism into a tissue, the range of microorganisms that naturally cause infection is necessarily limited and well established by medical practice.

[0017] "Infection" is a process or condition in which the body or a part thereof is invaded by a pathogenic agent (e.g., a microorganism such as a bacterium) that grows under favorable conditions and produces harmful effects (Taber's Cyclopedic Medical Dictionary, 14th Ed., CL Thomas, Ed., FADAvis Company, PA, USA). Infection is not always clinically evident and may result in only localized cellular damage. Infection may be asymptomatic and may be temporary if the body's defense mechanisms are effective. Infection may spread locally and become clinically evident in the form of acute, subacute, or chronic clinical infection or disease states. Localized infection may also become systemic when the pathogenic agent gains access to lymphatic or vascular systems. Infection is usually accompanied by inflammation, but inflammation can occur without infection.

[0018] "Inflammation" is the characteristic tissue response to injury (characterized by swelling, redness, warmth, and pain) and includes the continuum of changes that occur in living tissues upon injury. Infection and inflammation are distinct conditions, but one can result from the other (Taber's Cyclopedic Medical Dictionary, supra). Thus, inflammation can occur without infection, and infection can occur without inflammation (although inflammation typically results from infection with pathogenic bacteria or viruses). Inflammation is characterized by the following symptoms: rubor, calor, tumour, and dolor. Localized visible inflammation on the skin may be evident from a combination of these symptoms, especially redness at the site of administration.

[0019] According to alternative aspects of the invention, various subjects can be treated or assayed or sampled. As used herein, a "subject" is an animal, e.g., a vertebrate or mammal. Thus, the subject can be a patient, e.g., a human, suffering from a disease or disorder suitable for treatment, such as cancer, a proliferative cell disorder, or an infectious disease, such as persistent viral infection or opportunistic fungal infection, particularly in immunocompromised patients. The subject can also be an experimental animal, e.g., an animal model of immune dysregulation. In some embodiments, the terms "subject" and "patient" may be used interchangeably and may include humans, non-human mammals, non-human primates, rats, mice, cats, or dogs. A healthy subject may be a human not suffering from or suspected of having a disease, such as cancer or immune dysfunction, or not suffering from a chronic disorder or condition. A "healthy subject" may also be a subject that is not immunocompromised. Immunocompromised means any condition in which the immune system functions abnormally or imperfectly. Immunocompromised may be due to a disease, a specific drug, or a condition present at birth. Immunocompromised subjects may be found more frequently among infants, the elderly, and individuals undergoing extensive drug or radiation therapy.

[0020] A "sample" from a subject may include any relevant biological material, such as, for example, a cell, tissue or body fluid sample taken from a patient. For example, a sample may conveniently include a skin, cheek, blood, stool, hair or urine sample. Sample nucleic acid for use in diagnostic and prognostic methods may be obtained, for example, from selected cell types or tissues 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 using a dried sample (e.g., hair or skin).

[0021] Many of the methods described herein can be carried out using a kit that includes, for example, at least one probe or primer nucleic acid, or one or more of the compositions described herein and instructions for use of the kit. The kit can include, for example, at least one probe or primer that can specifically hybridize to or flank a polymorphic region, such that the oligonucleotide is "specific" for the polymorphic region. The kit can also include at least one reagent required to perform a particular assay. The kit can also include positive controls, negative controls, sequencing markers, or antibodies, for example, for determining the genotype or biological marker profile of the subject.

[0022] An "immune response" includes, but is not limited to, one or more of the following responses in a mammal: induction of cellular immune regulators such as cytokines and chemokines, induction or activation 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), for example, by one or more immunogens in the immunogenic composition following administration of the composition. Thus, an immune response to a composition generally includes the development in a host animal of a cellular and / or antibody-mediated response to the composition. In some embodiments, the immune response is such that it also results in a delay or halt in 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.

[0023] "Immune dysregulation" is an inappropriately regulated immune response, e.g., an inappropriately suppressed or inappropriately robust immune response. Immune dysregulation can be associated with, for example, neoplastic diseases such as cancer.

[0024] "Site-specific immunotherapy" (SSI) is an immunomodulatory therapy effective to therapeutically or prophylactically alter an aspect of the immune status or immune system physiology in one or more anatomical sites, such as an organ or tissue. In some instances, for example, an SSI may be tailored to ameliorate immune dysregulation or to treat a condition characterized by immune dysregulation.

[0025] A "cancer" or "neoplasm" is any unwanted growth of cells that does not perform a physiological function. Generally, cancer cells are cells that are free from their normal cell division control, i.e., whose growth is not regulated by the usual biochemical and physical influences in the cellular environment. Thus, "cancer" is a general term for a disease characterized by abnormal and uncontrolled cell proliferation. In most cases, cancer cells proliferate to form malignant clones of cells. A lump or mass of cells, a "neoplasm" or "tumor," generally, can invade and destroy surrounding normal tissues. As used herein, a "malignant tumor" refers to an abnormal growth of any cell type or tissue that has deleterious effects in the organism in which it occurs. The term "malignant tumor" or "cancer" includes cell proliferations that are strictly benign but have the risk of becoming malignant. Cancer cells can spread from the original site to other parts of the body via the lymphatic system or bloodstream, a process known as "metastasis." Many cancers are refractory to treatment and have proven fatal. Examples of cancers or neoplasms include, but are not limited to, transformed and immortalized cells, tumors, carcinomas in various organs and tissues described herein or known to one of skill in the art.

[0026] Most cancers fall into three broad histological categories: carcinomas, which are the predominant cancers and are cancers of epithelial cells or cells lining the external or internal surfaces of organs, glands or other body structures (e.g., skin, uterus, lung, breast, prostate, stomach, intestine) and tend 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 (generally arising in secretory organs or glands such as the breast, lung, colon, prostate or bladder) or squamous cell carcinomas (originating in squamous epithelium and generally occurring in most areas of the body). Sarcomas can be osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelioma or mesothelioma (membrane 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 mesenchymal or mixed mesodermal tumor (mixed connective tissue types). Hematological tumors can be myelomas, which originate in the plasma cells of the bone marrow; leukemias, which are "liquid cancers" and are cancers of the bone marrow, and can be myeloid or granulocytic leukemias (myeloid and granulocytic white blood cells), lymphocytic, lymphocytic or lymphoblastic leukemias (lymphocytic and lymphocytic blood cells) or polycythemia vera or erythrocytemia (various blood cell products, but red blood cells predominate); or lymphomas, which can be solid tumors and arise in the glands or lymph nodes of the lymphatic system, and can be Hodgkin's lymphoma or non-Hodgkin's lymphoma. In addition, there are mixed cancers such as adenosquamous carcinoma, mixed mesodermal tumor, carcinosarcoma or teratocarcinoma.

[0027] Cancers named based on the primary site may be correlated with histological classification. For example, lung cancer is generally small cell or non-small cell lung cancer and may be squamous cell carcinoma, adenocarcinoma or large cell carcinoma, and skin cancer is generally basal cell carcinoma, squamous cell carcinoma or melanoma. Lymphoma may arise in lymph nodes associated with the head, neck and chest, as well as abdominal lymph nodes or axillary or inguinal lymph nodes. Identification and classification of cancer type and stage can be done using information provided, for example, by the Surveillance, Epidemiology, and EndResult (SEER) program of the National Cancer Institute, which is a reliable 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 supplemental registries covering approximately 26% of the U.S. population. The program routinely collects data on patient demographics, primary tumor site, morphology, stage at diagnosis, first course of 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. Information on over 3 million in situ and invasive cancer cases is included in the SEER database, with approximately 170,000 new cases being added annually within the SEER coverage area. Incidence and survival data from the SEER program can be used to access standard survival for specific cancer sites and stages. For example, specific criteria can be selected from the database to ensure optimal comparison groups, such as date of diagnosis and exact stage (e.g., for the lung cancer example herein, the number of years was selected to match the time frame of the retrospective review, and stages 3B and 4 lung cancer was selected, and for the colon cancer example herein, the number of years was also selected to match the time frame of the retrospective review, and stage 4 colon cancer was selected).

[0028] Cancers may also be named based on the organ in which they originate, i.e., "site of origin" (e.g., breast cancer, brain cancer, lung cancer, liver cancer, skin cancer, prostate cancer, testicular cancer, bladder cancer, colon and rectal cancer, cervical cancer, uterine cancer, etc.). This nomenclature remains the same even if the cancer has metastasized to another part of the body different from the site of origin. In the present invention, the treatment is directed to the site of the cancer, not the type of cancer, so that, for example, any type of cancer that is symptomatically or etiologically located in the lung is treated based on this localization in the lung.

[0029] An embodiment of the present invention relates to the use of PRR ligands. PRR ligands may be, for example, commercially available in the form of widely available preparations of attenuated or killed recombinant bacteria, which may be, for example, ligands for TLR2, TLR4 and TLR5. The composition of pathogen-associated molecular patterns (PAMPs) may include PAMPS recognized by PRRs, such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs), including Dectin-1, cytoplasmic dsDNA sensors (CDSs), and NLRs involved in the formation of inflammatory cells.

[0030] Toll-like receptor 2 (TLR2) is involved in the recognition of a wide range 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, namely LPS-binding protein (LBP), CD14, and myeloid differentiation protein 2 (MD-2), inducing a signaling cascade that leads to the activation of NF-κB and the production of proinflammatory cytokines. LPS generally consists of a polysaccharide region anchored to the outer bacterial membrane by a carbohydrate lipid moiety, namely lipid A, which is primarily responsible for the immunostimulatory activity of LPS. Particularly active forms of lipid A contain six fatty acyl groups, as can be found in pathogenic bacteria, for example strains of Escherichia coli or Salmonella spp. Toll-like receptor 5 (TLR5) recognizes flagellins from both gram-positive and gram-negative bacteria. Toll-like receptor 7 (TLR7) and TLR8 recognize single-stranded RNA and synthetic small molecules such as imidazoquinolines and nucleoside analogues. Toll-like receptor 9 (TLR9) recognizes certain unmethylated CpG motifs common to microbes, but not vertebrate genomic DNA.

[0031] NLRs are a family of at least 22 cytoplasmic innate immune sensors, including NOD1 (CARD4) and NOD2 (CARD15), intracellular pattern recognition receptors involved in the recognition of peptidoglycan (PGN). These receptors detect specific motifs within PGN. NOD1 senses diaminopimelic acid (DAP)-containing muropeptides (specifically, d-Glu-meso-DAP dipeptide, "iE-DAP" dipeptide), found primarily in the PGN of Gram-negative bacteria as well as certain Gram-positive bacteria. NOD2 recognizes the muramyl dipeptide (MDP) structure found in almost all bacterial PGN.

[0032] RIG-I-like receptors (RLRs), particularly RIG-I and MDA-5, detect viral RNA species.

[0033] CLR ligands include Dectin-1 and Mincle (macrophage-inducible C-type lectin) agonists. Dectin-1 is a specific receptor 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.

[0034] Cytoplasmic DNA sensors (CDS) bind to intracellular DNA derived from pathogens, and multiple CDSs exist that may exhibit context-dependent preferences for recognition of specific DNA.

[0035] Cyclic dinucleotides (CDNs) and xanthenone derivatives, such as DMXAA, bind to and activate STING (stimulator of interferon genes).

[0036] Inflammasomes are multiprotein complexes involved in the production of mature IL-1β, and in particular the cleavage of pro-IL-1β and pro-IL-18 into active and secreted forms. Inflammasomes can be separated into NLRP1, NLRP3, NLRC4 and AIM2 subtypes that are activated by a wide variety of microbial molecules, danger signals and crystalline substances.

[0037] JPEG2024510510000002.jpg221161

[0038] JPEG2024510510000003.jpg163161

[0039] Thus, aspects of the invention include the use of PRR agonists derived from selected microbial pathogens. For example, peptidoglycan (PGN) can 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 can be obtained from bacteria or bacterial strains that are pathogenic in selected target tissues or organs for use as TLR2 agonists. Similarly, DNA such as double-stranded DNA, particularly repetitive double-stranded DNA, can be obtained from microbial pathogens such as bacteria or bacterial strains that are pathogenic in selected target tissues or organs for use as DAI, LRRFIP1, RIG1, TLR9, AIM2 or cytosolic DNA sensor (CDS) agonists. Beta-glucan peptides can be obtained from fungi or yeasts that are pathogenic in selected target tissues or organs for use as Dectin 1 agonists. Cyclic dinucleotides can be obtained from microbial pathogens that are pathogenic in selected target tissues or organs for use as STING agonists.

[0040] An embodiment of the present invention includes compositions with different PRR agonist signatures, comprising a repertoire of PRR agonists that are collected together in a therapeutic vehicle such that the set of selected PRR agonists is different. In this context, a "therapeutic vehicle" is a formulation that aggregates and holds PRR agonists in a pharma- ceutically acceptable particle or vesicle, such as a recombinant microorganism. For example, the PRR agonist signature can be different from a reference PRR agonist signature, for example, different from the set of PRR agonists that would be present on a non-pathogenic microorganism in a target tissue. The PRR signature can also be different in the sense that it is different from the natural PRR agonist signature of a microbial mammalian pathogen, for example, by recombinant expression of a gene that changes what would otherwise be the wild-type PRR agonist signature of the pathogen. To determine the specificity of a PRR agonist signature, the level or type of PRR agonist may be measured directly, or may be measured, for example, by measuring the activation or inhibition of a signaling pathway in a cell as a result of PRR agonist / receptor binding.

[0041] The various genes and nucleic acid sequences of the present invention may be recombinant sequences. The term "recombinant" means that something has been recombined, and thus when made in reference to a nucleic acid construct, the term refers to a molecule composed of nucleic acid sequences joined or produced by molecular biology techniques. Thus, a nucleic acid "construct" is a recombinant nucleic acid made by aggregating generally interoperable component sequencers. When made in reference to a protein or polypeptide, the term "recombinant" refers to a protein or polypeptide molecule expressed using a recombinant nucleic acid construct made by molecular biology techniques. When made in reference to a genetic composition or an organism or cell, the term "recombinant" refers to a new combination of alleles that did not occur in the parent genome. A recombinant nucleic acid construct may contain nucleotide sequences that are ligated or engineered to be ligated to nucleic acid sequences that are not naturally ligated or that are naturally ligated at a different location. Thus, referring to a nucleic acid construct as "recombinant" indicates that the nucleic acid molecule has been engineered (and thus artificially altered) using genetic engineering, i.e., by human intervention. A recombinant nucleic acid construct may be introduced into a host cell, for example, by transformation. Such recombinant nucleic acid constructs may contain sequences derived from the same host cell species or a different host cell species that have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may be integrated into the host cell genome either as a result of the original transformation of the host cell or as a result of subsequent recombination and / or repair events.

[0042] The recombinant constructs of the invention may include various functional molecules or genomic components necessary to mediate gene expression or repression in, for example, transformed plants. In this context, "DNA regulatory sequences", "control elements" and "regulatory elements" refer to transcriptional and translational control sequences that regulate gene expression, such as promoters, enhancers, polyadenylation signals, terminators and proteolytic signals, as well as epigenetic regulatory signals (e.g., histone methyltransferases or acetyltransferases), which are involved in, for example, methylation or acetylation of histones, resulting in conformational changes in the transcriptional landscape and differences in gene expression. In the context of the present disclosure, "promoter" refers to a sequence sufficient to direct 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 provide for the binding of RNA polymerase and the initiation of transcription. Promoter sequences are typically (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 functionally linked to permit promoter-mediated gene expression. Thus, the term "operably linked" indicates that the DNA segments are positioned to function in concert for their intended purpose, for example, to initiate transcription within a promoter to proceed through the coding segment of a gene to the terminator portion of the gene. In some instances, gene expression can occur when an appropriate molecule (such as a transcriptional activation protein) is bound to the promoter. Expression is the process of converting the information of the coding sequence of a gene into mRNA by transcription and then into a polypeptide (protein) by translation, so that the protein is said to be expressed. As the term is used herein, a gene or nucleic acid is "expressible" if it can be expressed under appropriate conditions in a particular host cell.

[0043] 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% of the cellular or biological components with which it was originally associated. A polynucleotide that has been amplified using PCR such that it is sufficiently distinguishable (e.g., on a gel) from the remainder of the cellular components is thereby "isolated", for example. The polynucleotides of the invention may be "substantially pure", i.e., have a high degree of isolation, such as that achieved using purification techniques.

[0044] In the context of biological molecules, "endogenous" refers to a molecule, such as a nucleic acid, that is naturally found in and / or produced by a given organism or cell. An "endogenous" molecule is sometimes referred to as a "native" molecule. Conversely, in the context of biological molecules, "exogenous" refers to a molecule, e.g., a nucleic acid, that is not normally or naturally found in and / or produced by a given organism or cell in nature.

[0045] The term "heterologous" as used herein to refer to a nucleic acid or amino acid sequence refers to a molecule or a portion of a molecule, e.g., a DNA sequence, that is artificially introduced into a particular host cell, e.g., by transformation. Heterologous DNA sequences can be introduced into a host cell, e.g., by transformation. Such heterologous molecules can include sequences derived from the host cell. Heterologous DNA sequences can be integrated into the host cell genome as a result of the original transformation of the host cell or as a result of a subsequent recombination event.

[0046] Various aspects of the present disclosure encompass nucleic acid or amino acid sequences that are homologous to other sequences. As this term is used herein, an amino acid or nucleic acid sequence is "homologous" to another sequence if the two sequences are substantially identical and the functional activity of the sequence is conserved (as used herein, sequence conservation or identity does not infer evolutionary relatedness). Nucleic acid sequences can also be homologous if they encode substantially identical amino acid sequences even if the nucleic acid sequences themselves are not substantially identical, for example, as a result of the degeneracy of the genetic code.

[0047] PRR ligands for use in alternative aspects of the invention may be derived from microorganisms. More specifically, the microorganism that is the source of the PRR ligand may be the target tissue of interest. Considering a microorganism as a pathogen is a delicate matter, as most animals are colonized to some degree by microorganisms, such as bacteria, that exist in a symbiotic or commensal relationship with the host animal. Thus, many species of normally harmless bacteria are found in healthy animals, usually localized on the surfaces of certain organs and tissues. In many cases, these microbial populations aid in the normal functioning of the body as members of what is called the microbiota. Generally harmless microorganisms, such as E. coli, can cause infections in healthy subjects, with consequences ranging from mild infection 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 the 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 are given, and even the most virulent microorganisms generally require certain circumstances to cause infection. Thus, microbial species that are members of the normal microflora can be pathogenic when they act beyond their normal ecological role in the endogenous microflora. For example, endogenous species may cause infection outside of their ecological niche in anatomically proximate areas, for example, by adjacent transmission. When this happens, these normally harmless endogenous bacteria are pathogenic.

[0048] 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 listed below, but these examples are not limiting in the sense that based on knowledge in the field, as represented, for example, in the following publications: Manual of Clinical Microbiology 8th Edition, Patrick Murray, Ed., 2003, ASM Press American Society for Microbiology, Washington DC, USA, and 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, a person skilled in the art will be able to recognize and identify infectious or pathogenic bacteria that cause or usually cause infections in various organs and tissues of an otherwise healthy organism (and recognize the relative frequency of infection of each bacterial species).

[0049] Skin infections are commonly caused by the following bacterial species: Staphylococcus aureus, Beta-hemolytic Streptococcus groups A, B, C, or G, Corynebacterium diphtheriae, Corynebacterium ulcerans, or Pseudomonas aeruginosa, or viral pathogens: measles, rubella, varicella zoster, echovirus, coxsackievirus, adenovirus, vaccinia, herpes simplex, or parvo B19.

[0050] 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 Clostridium species, or viral pathogens, i.e., influenza or coxsackie viruses.

[0051] Breast infections are commonly caused by the following bacterial species: Staphylococcus aureus or Streptococcus pyogenes.

[0052] 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.

[0053] Infections of the arm / axillary lymph nodes are commonly caused by the following bacterial species, i.e., Staphylococcus aureus or Streptococcus pyogenes, or viral pathogens, i.e., measles, rubella, Epstein-Barr, cytomegalovirus, adenovirus, or varicella zoster.

[0054] Mediastinal lymph node infections are commonly caused by the following bacterial species: Streptococcus viridans, Peptococcus spp., Bacteroides spp., Fusobacterium spp., or Mycobacterium tuberculosis; or by viral pathogens: measles, rubella, Epstein-Barr, cytomegalovirus, varicella zoster, or adenovirus.

[0055] Infections of the hilar lymph nodes are commonly caused by the following bacterial species: Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae, Bordetella pertussis, or Mycobacterium tuberculosis; or by viral pathogens: Haemophilus influenzae, adenovirus, rhinovirus, coronavirus, parainfluenza, respiratory syncytial virus, human metapneumovirus, or coxsackievirus.

[0056] Intraperitoneal lymph node infections are commonly caused by the following bacterial species: Yersinia enterocolitica, Yersinia pseudotuberculosis, Salmonella spp., Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, or Mycobacterium tuberculosis; or viral pathogens: measles, rubella, Epstein-Barr, cytomegalovirus, varicella zoster, adenovirus, influenza, or coxsackievirus.

[0057] 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.

[0058] 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 Streptococcus; or viral pathogens: measles, rubella, varicella zoster, echovirus, coxsackievirus, adenovirus, Epstein-Barr, herpes simplex, or cytomegalovirus.

[0059] Bone infections are commonly caused by the following bacterial species: Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, other Streptococcus species, Escherichia coli, Pseudomonas species, Enterobacter species, Proteus species, or Serratia species; or viral pathogens, i.e., parvovirus B19, rubella, or hepatitis B.

[0060] Joint infections are commonly caused by the following bacterial species: Staphylococcus aureus, coagulase-negative Staphylococcus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, other Streptococcus species, Escherichia coli, Pseudomonas species, Enterobacter species, Proteus species, Serratia species, Neisseria gonorrhoeae, Salmonella species, Mycobacterium tuberculosis, Haemophilus influenzae, or viral pathogens, i.e., Parvovirus B19, Rubella, Hepatitis B, or fungal pathogens, i.e., Scedosporium prolificans.

[0061] Meningeal infections 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.

[0062] Brain infections are commonly caused by the following bacterial species: Streptococcus spp. (including S. anginosus, S. constellatus, and S. intermedia), 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.

[0063] 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.

[0064] Ocular / orbital infections are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Plasmodium falciparum, Escherichia coli, Bacillus cereus, Chlamydia trachomatis, Haemophilus influenzae, Pseudomonas species, Klebsiella species, or Treponema pallidum, or viral pathogens: adenovirus, herpes simplex, varicella zoster, or cytomegalovirus.

[0065] Salivary gland infections are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus viridans (e.g., Streptococcus salivarius, Streptococcus sanguis, Streptococcus mutans), Peptostreptococcus species, or Bacteroides species, or other oral anaerobes, or by viral pathogens, i.e., mumps, influenza, enterovirus, or rabies.

[0066] Oral infections are commonly caused by the following bacterial species: Prevotella melaninogenica, anaerobic streptococci, Streptococcus viridans, Actinomyces spp., Peptostreptococcus spp., or Bacteroides spp., or other oral anaerobes; or by viral pathogens, i.e., herpes simplex, coxsackievirus, or Epstein-Barr.

[0067] Tonsil infections are commonly caused by the following bacterial species: Streptococcus pyogenes, or Streptococcus pyogenes group C or G, or viral pathogens: rhinovirus, influenza, coronavirus, adenovirus, parainfluenza, respiratory syncytial virus, or herpes simplex.

[0068] Sinus infections are commonly caused by the following bacterial species: Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, alpha-Streptococcus, anaerobic bacteria (e.g., Prevotella spp.), or Staphylococcus aureus; or viral pathogens: rhinovirus, influenza, adenovirus, or parainfluenza.

[0069] Nasopharyngeal infections are commonly caused by the following bacterial species: Streptococcus pyogenes or Streptococcus pyogenes of groups C or G, or viral pathogens: rhinovirus, influenza, coronavirus, adenovirus, parainfluenza, respiratory syncytial virus, or herpes simplex.

[0070] Thyroid infections are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, or Streptococcus pneumoniae, or viral pathogens: mumps or influenza.

[0071] Laryngeal infections are commonly caused by the following bacterial species: Mycoplasma pneumoniae, Chlamydia pneumoniae, or Streptococcus pyogenes, or viral pathogens: rhinovirus, influenza, parainfluenza, adenovirus, coronavirus, or human metapneumovirus.

[0072] Infections of the respiratory tract are generally caused by the following bacterial species: Mycoplasma pneumoniae, or viral pathogens: parainfluenza, influenza, respiratory syncytial virus, or adenovirus.

[0073] Bronchial infections are commonly caused by the following bacterial species: Mycoplasma pneumoniae, Chlamydia pneumoniae, Bordetella pertussis, Streptococcus pneumoniae, or Haemophilus influenzae, or viral pathogens: influenza, adenovirus, rhinovirus, coronavirus, parainfluenza, respiratory syncytial virus, human metapneumovirus, or coxsackievirus.

[0074] Pulmonary infections are commonly caused by the following bacterial species: Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, or Haemophilus influenzae; or viral pathogens: influenza, adenovirus, respiratory syncytial virus, or parainfluenza.

[0075] Pleural infections are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Bacteroides fragilis, Prevotella spp., Fusobacterium nucleatum, Peptostreptococcus spp., or Mycobacterium tuberculosis; or by viral pathogens: influenza, adenovirus, respiratory syncytial virus, or parainfluenza.

[0076] 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.

[0077] Cardiac infections are commonly caused by the following bacterial species: Streptococcus spp. (including S. mitior, S. bovis, S. sanguis, S. mutans, S. anginosus), Enterococcus spp., Staphylococcus spp., Corynebacterium diphtheriae, Clostridium perfringens, Neisseria meningitidis, or Salmonella spp., or viral pathogens: enteroviruses, coxsackieviruses, echoviruses, polioviruses, adenoviruses, mumps, measles, or influenza.

[0078] 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.

[0079] Gastric infections are commonly caused by the following bacterial species, namely Streptococcus pyogenes or Helicobacter pylori, or viral pathogens, namely cytomegalovirus, herpes simplex, Epstein-Barr, rotavirus, norovirus, or adenovirus.

[0080] 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 enteritidis, Yersinia enterocolitica, or Shigella; or by viral pathogens: adenovirus, astrovirus, calicivirus, norovirus, rotavirus, or cytomegalovirus.

[0081] Colon / rectal infections are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteritidis, Yersinia enterocolitica, or Shigella; or viral pathogens: adenovirus, astrovirus, calicivirus, norovirus, rotavirus, or cytomegalovirus.

[0082] 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 by viral pathogens, i.e., herpes simplex.

[0083] Perineal infections 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, i.e., herpes simplex.

[0084] Liver infections are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Streptococcus (anginosus group), Enterococcus spp., other viral streptococci, or Bacteroides spp.; or viral pathogens: Hepatitis A, Epstein-Barr, herpes simplex, mumps, rubella, measles, varicella zoster, coxsackievirus, or adenovirus.

[0085] Gallbladder infections are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterobacter spp., Enterococcus faecalis, Bacteroides spp., Fusobacterium spp., Clostridium spp., Salmonella enteritidis, Yersinia enterocolitica, or Shigella spp.

[0086] Biliary tract infections are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterobacter spp., Enterococcus faecalis, Bacteroides spp., Fusobacterium spp., Clostridium spp., Salmonella enteritidis, Yersinia enterocolitica, or Shigella; or viral pathogens: Hepatitis A, Epstein-Barr, herpes simplex, mumps, rubella, measles, varicella zoster, coxsackievirus, or adenovirus.

[0087] Pancreatic infections are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterococcus spp., Pseudomonas spp., Staphylococcus spp., Mycoplasma spp., Salmonella typhimurium, Leptospira spp., or Legionella spp., or viral pathogens: mumps, Coxsackievirus, Hepatitis B, Cytomegalovirus, Herpes simplex 2, or Varicella zoster.

[0088] Splenic infections 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.

[0089] 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 by viral pathogens, i.e., Varicella zoster.

[0090] Kidney infections are commonly caused by the following bacterial species: Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providencia spp., Morganella spp., Enterococcus faecalis, or Pseudomonas aeruginosa; or viral pathogens, namely BK virus or mumps.

[0091] Urinary tract infections are commonly caused by the following bacterial species: Escherichia coli, Proteus mirabilis, Proteus vulva, Providencia spp., Morganella spp., or Enterococcus spp.

[0092] Bladder infections are commonly caused by the following bacterial species: Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providencia spp., Morganella spp., Enterococcus faecalis, or Corynebacterium jeckeum, or by viral pathogens, i.e., adenovirus or cytomegalovirus.

[0093] Peritoneal infections are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Escherichia coli, Klebsiella spp., Proteus spp., Enterococcus faecalis, Bacteroides fragilis, Prevotella melaninogens, Peptococcus spp., Peptostreptococcus spp., Fusobacterium spp., or Clostridium spp.

[0094] Infections of the retroperitoneal region are commonly caused by the following bacterial species: Escherichia coli or Staphylococcus aureus.

[0095] Prostate infections are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus mirabilis, Enterococcus spp., Pseudomonas spp., Corynebacterium spp., or Neisseria gonorrhoeae, or by viral pathogens, i.e., herpes simplex.

[0096] Testicular infections are commonly caused by the following bacterial species: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus species, Streptococcus species, or Salmonella enteritidis; or viral pathogens: mumps, Coxsackievirus, or lymphocytic choriomeningitis virus.

[0097] Penile infections are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Neisseria gonorrhoeae, or Treponema pallidum, or by viral pathogens, such as herpes simplex.

[0098] Ovarian / adnexal infections are commonly caused by the following bacterial species: Neisseria gonorrhoeae, Chlamydia trachomatis, Gardnerella vaginalis, Prevotella spp., Bacteroides spp., Peptococcus spp., Streptococcus spp., or Escherichia coli.

[0099] Uterine infections are commonly caused by the following bacterial species: Neisseria gonorrhoeae, Chlamydia trachomatis, Gardnerella vaginalis, Prevotella spp., Bacteroides spp., Peptococcus spp., Streptococcus spp., or Escherichia coli.

[0100] Cervical infections are commonly caused by the following bacterial species: Neisseria gonorrhoeae, Chlamydia trachomatis, or Treponema pallidum, or by viral pathogens, such as herpes simplex.

[0101] Vaginal infections are commonly caused by the following bacterial species: Gardnerella vaginalis, Prevotella spp., Bacteroides spp., Peptococcus spp., Escherichia coli, Neisseria gonorrhoeae, Chlamydia trachomatis, or Treponema pallidum, or by viral pathogens, i.e., Herpes simplex.

[0102] Vulvar infections are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, or Treponema pallidum, or by viral pathogens, such as herpes simplex.

[0103] Bacterial species are operationally classified as collections of similar strains (which generally refer to groups of putative common ancestry with identifiable physiological but usually no morphological differences, and can be identified using serological techniques against bacterial surface antigens). Thus, each bacterial species (e.g., Streptococcus pneumoniae) has numerous strains (or serotypes) that may differ in their ability to cause infection or in their ability to cause infection in specific organs / sites. For example, there are at least 90 serotypes of Streptococcus pneumoniae, but serotypes 1, 3, 4, 7, 8, and 12 are most often responsible for S. pneumoniae disease in humans.

[0104] Certain strains of E. 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, such as 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), are more likely to cause gastrointestinal infections / diarrhea. Even among subcategories of ExPEC strains, certain virulence factors (e.g., production of type 1 fimbriae) make certain strains more capable of causing infections of the bladder, while other virulence factors (e.g., production of P fimbriae) make other strains more capable of causing infections of the kidney. According to the present invention, for the formulation targeting immune dysregulation in bladder cancer, ExPEC strains that are more likely to cause infection in the bladder can be selected, whereas for the formulation targeting immune dysregulation in kidney cancer, ExPEC strains that are more likely to cause infection in the kidney can be selected.Similarly, for the formulation for treating immune dysregulation in the colon, one or more of the ETEC, EPEC, EHEC, STEC, EAEC, EIEC or DAEC strains of E. coli (i.e., strains that cause colon infection) can be selected.

[0105] Similarly, there may be numerous subtypes of a particular virus. For example, there are three types of influenza virus: influenza A, influenza B, and influenza C, which differ in epidemiology, host range, and clinical characteristics. For example, influenza A is more likely to be associated with viral lung infections, 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 numerous subtypes, which may also differ in epidemiology, host range, and clinical characteristics. According to the present invention, the influenza A subtype most commonly associated with lung infections can be selected to target immune dysregulation in the lung, while the influenza B strain most commonly associated with myositis can be selected to treat immune dysregulation in muscle / soft tissue.

[0106] There are specific microbiota associated with some histopathological conditions, such as the microbiota of specific tumors, e.g., Fusobacterium and Providencia are associated with colorectal cancer.

[0107] The composition of the present invention comprises an immunogen of a pathogenic microbial species (bacteria, viruses or fungi) that is pathogenic in a particular tissue or organ, which is provided in the form of an artificial repertoire of mammalian PRR agonists that reproduces different parts of the PRR agonist signature of a microbial mammalian pathogen that is pathogenic in the target tissue. In selected embodiments, the part of the PRR agonist signature is distinct in the sense that it is both different from the reference PRR agonist signature of a microorganism that is not pathogenic in the target tissue and different from the natural PRR agonist signature of the microbial mammalian pathogen. This different artificial repertoire of mammalian PRR agonists is formulated together in a therapeutic vehicle for combined presentation to innate immune cells present in the target tissue in a mammalian host.

[0108] The compositions of the present invention can be provided in a form suitable for administration to mammals, e.g., humans, 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 carriers ("therapeutic vehicles"). As used herein, "pharma-ceutically acceptable carriers" or "excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Carriers can be suitable for any suitable mode of administration, such as subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intramuscular, sublingual, inhalation, intratumoral, or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. 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 specific bacteria, bacterial antigens, or compositions thereof of the present invention), its use in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0109] Aspects of the invention include the use of nanoparticle (NP) formulations. For example, virus-like particles (VLPs) are essentially empty virus particles with an intact protein shell, and in some embodiments, a membrane envelope. Generally, VLPs lack genetic material. Production of VLPs can be by expression of viral proteins in, for example, mammalian, avian, insect, plant, yeast or bacterial cells. Alternatively, fully synthetic VLPs can be produced. Alternative nanoparticle formulations emulsions, alginate liposomes, chitosan, and polylactide-coglycolide (PLGA) NPs. Examples of NP / TLR ligand preparations that can be adapted for use to induce an immune response are ligands for TLR2 (Pam(3)Cys), TLR9 (poly I:C), TLR4 (3-O-desacyl-4 O-monophosphoryl lipid A (MPL)), TLR7 (9-benzyl-8-hydroxyadenine), TLR7 / 8 (resiquimod, R848) and TLR9 (CpG DNA).

[0110] In addition to the selected co-formulant, a wide variety of adjuvants can be used to enhance the desired immune response (see Levast et al., 2014, Vaccines, 2, 297-322).

[0111] Treatment with PRR ligands according to the present invention can be combined with more traditional existing treatments. For example, in the case of cancer, these can include chemotherapy, radiation therapy, surgery, etc., or therapies 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.

[0112] Conventional pharmaceutical practice can be used to provide suitable formulations or compositions for administering the compounds to a subject. Alternative routes of administration can be used, such as parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ocular, intraventricular, intracapsular, intraspinal, intrathecal, intracapsular, intraperitoneal, intranasal, inhalation, aerosol, topical, intratumoral, sublingual or oral administration. Therapeutic formulations can be in the form of liquid solutions or suspensions; for oral administration, the formulations can be in the form of tablets or capsules; for intranasal formulations, the formulations can be in the form of powders, nose drops or aerosols; for sublingual formulations, the formulations can be in the form of drops, aerosols or tablets.

[0113] Methods well known in the art for making formulations 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 contain, for example, excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, 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 contain excipients, for example, lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholic acid, and deoxycholic acid, or can be oily solutions for administration in the form of nose drops or as gels. In the case of 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 (e.g., compared to a prognosis derived from the SEER database), depending on the disorder.

[0114] Biodegradable polymeric materials can be used for staged delivery of antigenic and immunomodulatory therapeutics such as chitosan, alginate, gelatin, poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly(ε-caprolactone) (PCL), poly(β-amino ester) (PBAE) and poly(methyl methacrylate) (PMMA) (Lu et al., 2021). Similarly, biodegradable systems capable of delivering one or more therapeutics in a predefined time sequence have been developed (Sundararaj et al., 2014). Polyanhydrides and poly(orthoesters) are examples of surface-eroding polymers used in this manner for controlled delivery of therapeutics. Similarly, cellulose acetate phthalate (CAP) and Pluronic F-127 (P), when mixed in an aprotic solvent, associate via hydrogen bonding to provide a matrix capable of mediating the release of therapeutic compositions. The ratio of CAP to Pluronic can be adjusted to modulate the erosion rate and thereby the timing of therapeutic release. Thus, these systems can be provided in a graded release matrix for intermittent graded release encapsulating a therapeutic agent, such as an attenuated or killed mammalian pathogen, adapted to release the mammalian pathogen from the matrix in multiple successive, time-spaced administration steps after the delivery system is applied to a surgical wound, for example on the day of surgery, with a therapeutically effective aliquot of the mammalian pathogen being released in each administration step during the perioperative period, for example within 1, 2, 3, 4, 5 or 6 months of the day of surgery.

[0115] The pharmaceutical composition or formulation can be packaged in a variety of ways depending on the method used to administer the drug. For example, the article of manufacture or package can include a container into which the pharmaceutical formulation in an appropriate form is placed. Suitable containers can include materials such as, for example, bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and vials. The container can have a sterile access port, for example, the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle. The package or container can also include a tamper-evident or multi-use mechanism adapted to control access to the contents of the package or container, for example, a multi-dose vial adapter adapted to a vial contained in the package. The container or package can include a label, for example, a label describing the contents of the container, for example, a drug label identifying the pharmaceutical composition therein and / or designating a mode or route of administration. The label can also include appropriate warnings, for example, designating storage conditions for the container or package, or indicating contraindications or adverse effects of a treatment mode. Thus, the product may take the form of a "kit" that includes the pharmaceutical composition or accessories adapted to facilitate the use of the pharmaceutical composition. The kit may include a label or package insert, where the term "package insert" is used to refer to instructions that are typically included in commercial packaging of therapeutic products and that include information regarding indications, usage, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic products. The kit may further include accessories related to the use of the pharmaceutical composition, such as buffers, diluents, filters, needles, and syringes. The kit may also be adapted for the delivery of a selected dosage form of the pharmaceutical composition, such as, for example, several unit doses. Such a kit may include a memory aid or mechanism that physically or in writing indicates the designated timing of the treatment schedule in which the dosage is to be used.

[0116] 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 examine the applicability of the treatment to a particular patient. A point-of-care companion diagnostic may involve providing a diagnostic composition and / or product in conjunction with providing a pharmaceutical formulation, for example, 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 assay in vitro), providing information related to the safe and effective use of the corresponding drug or biological product. A companion diagnostic may be used with a treatment disclosed herein to provide diagnostic or prognostic information regarding the efficacy of the treatment or evidence of undesirable side effects or risks. The use of a companion diagnostic with a particular therapeutic agent may be specified in instructions, for example, in the labeling of the diagnostic device and / or in the labeling of the corresponding therapeutic product. Types of companion diagnostic tests can include screening and detection, for example in the form of tests that screen for genetic patterns such as genetic SSI response markers; prognostics and theranostics, such as assays for biochemical SSI response markers that help predict the future course of a disease or indicate a patient's response to treatment; monitoring, for example to assess the effectiveness of prescribed treatment and appropriate dosing, or recurrence, including tests to analyze a patient's risk for recurrence of the disease.

[0117] 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 the dosage and time period necessary to achieve a desired therapeutic outcome, such as reducing or eliminating immune dysregulation. A therapeutically effective amount of a composition may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to induce a desired response in the individual. Dosage regimens may be adjusted to provide an optimal therapeutic response. A therapeutically effective amount may also be an amount in which any toxic or harmful effects of the composition are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at the dosage and time period necessary to achieve a desired prophylactic outcome, such as improving immune dysregulation. Typically, a prophylactic dose is used in subjects prior to or at an early stage of cancer, such that the prophylactically effective amount may be less than the therapeutically effective amount.

[0118] 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 need 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 an increase to 0.01-0.02 ml every other day until a sufficient skin reaction is obtained at the injection site (e.g., a delayed reaction of redness visible 1-2 inches in diameter at the injection site). Once this sufficient immune response is obtained, this administration is continued as a maintenance dose. The maintenance dose may be adjusted from time to time to obtain the desired visible skin reaction (inflammation) at the injection site. Administration may be, for example, for a period of administration of at least 1 week, 2 weeks, 2 months, 6 months, 1, 2, 3, 4, or 5 years or more.

[0119] Oral dosages can range, for example, from 4 times a day, daily, or weekly. Administration can be, for example, for at least 1 week, 2 weeks, 2 months, 6 months, 1, 2, 3, 4, or 5 years or more of administration period. In some embodiments, the present invention can include compositions administered sublingually or by inhalation, or administered simultaneously or sequentially to one or more epithelial tissues (i.e., administered to the skin by intradermal or subcutaneous injection, to the pulmonary epithelium by inhalation, to the gastrointestinal mucosa by oral ingestion, to the oral mucosa by sublingual administration). 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, for example, intratumoral administration, intramuscular administration, or intravenous administration.

[0120] For immunogenic formulations, an immunogenic effective amount of the composition of the present invention can be given alone or in combination with other compounds, such as immune 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 expand memory B cells, memory T cells, neutrophils, monocytes, or macrophages of the immune system, including but not limited to these.

[0121] Antigenic compositions comprising killed recombinant bacteria for administration by injection can be made as follows: The bacteria can be grown in a suitable medium and washed with physiological salt solution. The bacteria can then be centrifuged, resuspended in saline and heat killed. The suspension can be standardized by direct microscopic counting, mixed in the required quantities and stored in suitable containers that can be tested for safety, shelf life and sterility by approved methods. 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. Bacterial vaccines can also contain traces of brain heart infusion (beef), peptone, yeast extract, agar, sheep blood, dextrose, sodium phosphate and / or other media components.

[0122] In selected embodiments, the agent may be administered to the administration site in successive doses administered at intervals of 1 hour to 1 month over an administration period of at least 1 week. Optionally, the agent may be administered intradermally or subcutaneously. Optionally, the agent may be administered at a dose such that each dose is effective to cause a visible local inflammatory immune response at the administration site. Optionally, the agent may be administered such that visible local inflammation at the administration site occurs within 1 hour to 48 hours. However, a visible local inflammatory immune response is not always present in all situations, even though an immune response has been initiated. There are other ways in which the initiation 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.

[0123] In another aspect, a method is provided for monitoring the effectiveness of a therapeutic regimen in an individual being treated for an immune dysfunction in a particular organ or tissue, comprising measuring a characteristic of the immune response in a post-treatment immune sample obtained from the particular organ or tissue after the individual has been subjected to the therapeutic regimen for a period of time.

[0124] In some embodiments, the immunogenic composition of the present invention can be formulated using PRR agonists derived from bacteria that are members of the endogenous flora of certain regions of the GIT.In the rows of Table 3, a number of bacterial species are listed, along with the biological regions in which each species can form part of the endogenous flora.For example, Avitrophia species are typically members of the endogenous flora of the mouth.

[0125] JPEG2024510510000004.jpg212161

[0126] JPEG2024510510000005.jpg243161

[0127] JPEG2024510510000006.jpg239161

[0128] JPEG2024510510000007.jpg243161

[0129] JPEG2024510510000008.jpg195161

[0130] Endogenous microflora, such as bacteria, gain access to tissues for pathogenesis either by contiguous transmission or by transmission of bacteremia. Under favorable conditions, endogenous organisms can become pathogenic, invade locally, and spread by contiguous transmission to adjacent tissues and organs. The endogenous flora of the skin, mouth, and colon are species that are understood to be suitable for transmission of bacteremia as well. Thus, bacteria that are members of certain endogenous flora domains can cause infection in tissues or organs where these bacteria can spread. Thus, one aspect of the present invention includes the use of PRR agonists derived from endogenous microbial pathogens to treat immune dysregulation with symptoms localized in the area of ​​the GIT where endogenous bacteria 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 can be symptomatic or pathologically located. Thus, one aspect of the invention includes the use of PRR agonists derived from endogenous microbial pathogens to formulate immunogenic compositions for treating immune dysregulations symptomatically or etiologically located in regions of the GIT where pathogens can spread and cause infection. Thus, in an alternative embodiment, immune dysregulations symptomatic in regions listed in column 1 of Table 2 can be treated with immunogenic compositions comprising an artificial repertoire of mammalian PRR agonists that recapitulate distinct portions of the PRR agonist signatures of microbial mammalian pathogens that are members of the endogenous flora of one or more endogenous flora domains listed in row 1 of Table 2 and indicated by an X or check mark in the appropriate row.

[0131] JPEG2024510510000009.jpg74161

[0132] According to the combined information of Tables 1 and 2, immune dysregulation manifested in specific regions of the GIT shown in column 1 of Table 2 can be treated with an antigen composition comprising an artificial repertoire of mammalian PRR agonists that recapitulates a distinct portion of the PRR agonist signature of a microbial mammalian pathogen that is one of the corresponding bacterial species in Table 1, and thus the column headings of Table 2 are essentially replaced with the bacterial species in Table 1.

[0133] In some embodiments, the PRR agonist may be derived from an exogenous bacterial pathogen. For example, PRR agonists derived from organisms listed in Table 5 may be used in an artificial repertoire of PRR agonists to treat symptomatic immune dysregulation in the region of the GIT listed with the relevant organisms in Table 5. In some embodiments, PRR agonists derived from both endogenous and exogenous microbial species may be used in combination.

[0134] JPEG2024510510000010.jpg150161

[0135] JPEG2024510510000011.jpg151161

[0136] In some embodiments, the PRR agonist for use in the present invention can be derived from a viral pathogen.Table 6 shows an exemplary list of viral pathogens, along with the tissue and organ sites where each virus species is reported to be a pathogen.Therefore, one aspect of the present invention includes utilizing the immunogenic composition of the PRR agonist derived from the designated virus to treat immune dysregulation that is symptomatic in the region of the GIT identified adjacent to the name of the virus in Table 6.

[0137] JPEG2024510510000012.jpg186161

[0138] In some embodiments, the pathogen from which the PRR agonist is derived for use in the immunogenic composition of the invention may be a pathogen that is a common cause of acute infection in the area of ​​the GIT where the immune dysregulation to be treated is symptomatic. Table 7 identifies this type of bacterial and viral pathogen along with the area of ​​the GIT where they commonly cause infection. Thus, in selected embodiments, immune dysregulation that is symptomatic in the area of ​​the GIT identified in the first column of Table 7 can 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.

[0139] JPEG2024510510000013.jpg156161

[0140] Humans are host to a wide range of gastrointestinal parasites, including various protozoa and helminths, which for the purposes of the present invention constitute pathogens of the GIT (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 include PRR agonists for various protozoa, such as Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominus, Isospora belli, Sarcocystis spp., Coccidianoids (Cyclospora spp.), Enterocytozoum biennis, Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmannii, Endolimus nana, Iodoameba butschlii, Dientamoeba hominis, Blastocystis hominis, Cyclospora, Microsporidia, Trypanosoma cruzi, Trichomonas menilii, Trichomonas intestinalis, Balantidium coli, and the like. Similarly, the compositions of the present invention may be used to treat a variety of parasitic helminths, such as tapeworms, Taenia saginata, Taenia solium, Diphyllobothrium spp., Hymenolepis nana, Hymenolepis diminuta, Tapeworm, Roundworms, Ascaris umbellata, Strongyloides umbellata, Ancylostoma ... The antigenic components may include those of the following: Whistling and related species, Angiostrongylus costaricanus, Pinworm, Trematodes (Freakups), Fasciola macrocarpa, Heterophyoma spp., Echinostomum spp., Clonorchis viverrini, Opisthorchis spp., Fasciola spp., Metagonimus yokogawei, Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekogii, Schistosoma intercalatum, Echinosoma spp., and Paragonimus spp.

[0141] In accordance with the above, in various aspects, the present invention provides a method for the preparation of a microbial pathogen, i.e., Acidaminococcus fermentans, Acinetobacter spp., Actinobaculum spp., Actinomyces spp., Aeromonas spp., Anaerorhabdus frucosus, Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus prevotii, Atopobium spp., Bacillus spp., Bacteroides cacca, Bacteroides distasonis, Bacteroides egersii, Bacteroides fragilis, Bacteroides meldae, Bacteroides ketoacilliform ... Bacteroides ovatus, Bacteroides plancnicus, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulata, Bifidobacterium dentium, Bifidobacterium longum, Bilophila wardsworthia, Burkholderia cepacia, Butyrivibrio fibrilsorbens, Campylobacter concisus, Campylobacter curves, Campylobacter gracilis, Campylobacter jejuni, Campylobacter rectus, Campylobacter shoae, Capnocytophaga ochracea, Cideca spp., Citrobacter freundii, Citrobacter koseri, Clostridium spp., Desulfomonas picrum, Dysgononas spp., Eikenella corrodens, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter gergovia, Enterobacter sakazakii, Enterobacter tajarella, Enterococcus spp., Escherichia coli, Escherichia ferritinosa, Gusoni, Escherichia hermannii, Escherichia vanis, Eubacterium spp., Phanegordia magnus, Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium nabiforme, Fusobacterium necroforum, Fusobacterium nucleatum, Fusobacterium lussii, Fusobacterium varium, Gardnerella vaginalis, Gemella morvilloum, Globicatella spp., Hafnia alvei, Helicobacter spp., Klebsiella spp., Lactobacillus acidophilus,Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus salivarius, Leclercsia adecarboxylata, Leminorella spp., Megaphaella elsdenii, Mitsuocera multiacidus, Mobiluncus chrysius, Mobiluncus murielis, Mollerella wissensus, Morganella morganii, Pantoea agglomerans, Pediococcus spp., Peptoniphilus asaccharolyticus, Peptostreptococcus anellobus, Ruminococcus productus, Porphyrocystis spp. Monas asaccharolytica, Proteus mirabilis, Proteus pennellii, Proteus vulgaris, Providencia retogeri, Providencia startii, Pseudomonas aeruginosa, Lethotamonas intestinalis, Ruminococcus productus, Serratia liquefaciens, Serratia marcescens, Serratia odorifera, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus bovis, Streptococcus constellatus, Streptococcus intermedius, Group C + Group G Streptococcus, Succinivibrio dextrinsorbens, Streptomyces spp., Tisserella praeacta, Veillonella spp., Aerobacter spp., Bacillus anthracis, Bacillus cereus, other Bacillus spp., recurrent Borrelia, Brucella spp., Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter spathulum, Clostridium bifermentans, Clostridium botulinum, Clostridium difficile, Clostridium indris, Clostridium spp. Umgenoli, Clostridium perfringens, Clostridium sordellii, Clostridium sporogenes, Clostridium subterminalis, Edwardsiella tada, Francisella tularensis, Listeria monocytogenes, Mycobacterium bovis, Mycobacterium tuberculosis, Pediococcus spp., Plesiomonas shigelloides, Rickettsia rickettsii, Salmonella spp., Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, other Spirillum spp., Streptococcus equi zooepidemicus, Tropherima whipleyi, Vibrio cholerae, Vibrio fulvialis, Vibrio furnisii, Vibrio holisa,The present invention may include the treatment of immune dysregulation by the preparation of artificial repertoires of mammalian PRR agonists that recapitulate distinct portions of the PRR agonist signatures of Vibrio parahaemolyticus, Yersinia parahaemolyticus, Mycobacterium pseudotuberculosis, Herpes simplex virus (1 and 2), Cytomegalovirus, Adenovirus, Orthoreovirus, Rotavirus, Alphavirus, Coronavirus, Torovirus, Human Metapneumovirus, Vesicular Stomatitis Virus, Machupovirus, Juninvirus, Poliovirus, Coxsackievirus, Echovirus, Hepatitis A virus, Norovirus and other Caliciviruses, Astrovirus, Picobirnavirus, or Hepatitis E virus.

[0142] In alternative aspects, the invention may include the treatment of immune dysregulation through the formulation of artificial repertoires of mammalian PRR agonists that recapitulate distinct portions of the PRR agonist signatures of mammalian microbial pathogens that are common small and large intestinal pathogens, such as Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteritidis, Yersinia enterocolitica, Shigella flexneri, Adenovirus, Astrovirus, Calicivirus, Norovirus, Rotavirus, and Cytomegalovirus.

[0143] In selected embodiments, the invention includes a diagnostic step to assess a patient's prior exposure to the organism. For example, the diagnostic step may include taking a medical history of exposure to a selected pathogen and / or assessing the patient's immune response to the selected pathogen. For example, serological tests may be performed to detect antibodies to the selected pathogen in the patient's serum. In connection with 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 based on a diagnostic indication that the patient has been previously exposed to the pathogen one or more times, for example, by the presence of antibodies to an antigenic determinant of the pathogen in the patient's serum.

[0144] In further selected embodiments, the 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, for example, using a serological test to detect antibodies to those immunogenic determinants. In the context of this aspect of the invention, if the evaluation indicates that there is a vigorous immunological response to the immunogenic determinants of the composition, treatment with the selected immunogenic composition may be continued, and the treatment may be discontinued, and if the evaluation indicates that there is not a sufficiently vigorous immunological response to the immunogenic determinants of the immunogenic composition, an alternative treatment with a different immunogenic composition may be initiated.

[0145] In some embodiments, prior exposure of the organism to a microbial pathogen can be used to enhance the effectiveness of a subsequent SSI. For example, in some embodiments, prior exposure to Klebsiella pneumoniae can induce tissue-specific immune memory, e.g., innate immune memory, that promotes tumor cell lysis, particularly in combination with cytotoxic adoptive immune cell therapy.

[0146] The SSI and adoptive cell therapy may be combined with additional components that enhance, for example, the cancer antigen response. The cancer antigen may be mixed, for example, with the SSI. The adoptive cell therapy may then target the antigen mixed with the SSI.

[0147] The microbial component may be formulated as an SSI that includes PRR ligands derived from a microbial fraction, such as the bacterial outer membrane (e.g., from a Gram-negative species), the bacterial inner membrane, the pellet of gradient centrifugation (e.g., obtained from a sucrose gradient), chromosomal DNA, capsular glycoprotein fractions, or peptidoglycan fractions such as peptidoglycan ghosts. In alternative embodiments, engineered or recombinant organisms may be used in SSIs in which genes involved in pathways associated with specific cellular fractions, particularly genes involved in determining the composition of said fractions, have been modified.

[0148] For cell fraction preparation, for example, bacteria can be grown and heat inactivated. Cell fractions can be resuspended, for example, in sterile saline + 0.4% phenol. For example, a two-step sucrose density gradient can be used to recover the inner membrane, as described in Methods in Enzymology, Vol 125: 309-328, 1986. The bacterial pellet obtained after culturing 250 ml of cells can be resuspended in 20% sucrose, 10 mM Tris-HCl (pH 8.0) and 50ug / ml DNase 1. The cells can be incubated at 23°C for 10 minutes. The cells can then be placed on ice and lysed twice through a 15,000 psi French pressure cell, and unbroken cells can be removed by centrifugation at 5,000 x g for 10 minutes at 4°C. The supernatant can be layered on 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. The inner membrane can be collected at the junction between 20% sucrose and 60% sucrose. The sucrose can be diluted to less than 20% with sterile distilled water and the membranes can be pelleted in an ultracentrifuge at 41,000 rpm for 1 hour at 4° C. The inner membrane 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 between the 60% sucrose gradient step and the 70% sucrose gradient step.

[0149] For example, chromosomal DNA of K. pneumoniae can be prepared using the Qiagen Blood and Tissuemidi kit. Cells from 15 or 40 ml broth cultures from each strain can be harvested. The manufacturer's protocol for purification of total DNA can then be followed.

[0150] The SSI may be co-formulated or co-administered with additional therapeutic components. One class of additional therapeutic components includes molecules or compositions for activating or recruiting innate immune cells, including: For example, GMCSF in an amount that synergistically recruits and promotes neutrophil production and enhances SSI-induced innate immune responses. - Vitamin D in an amount effective to, for example, differentiate and activate monocytes and play a role in regulating innate immune function. In alternative embodiments, the vitamin D used with the SSI can be, for example, one or more of vitamin D3, D2, or calcitriol (1,25 dihydroxycholecalciferol). In some embodiments, vitamin D3 and / or D2 can be administered locally, for example, in a dosage effective to provide a locally effective amount of calcitriol to the SSI and vitamin D administration site. For example, vitamin D precursors (D3 and / or D2) can be administered in an amount that is locally effective upon conversion to the calcitriol active form by local monocytes and / or macrophages (expressing CYP27B1) at the administration site. In alternative embodiments, calcitriol can be administered in a locally effective dose at the SSI administration site, which can be, for example, a dose that is less than the dose required for other systemic effects.

[0151] Further classes of therapeutic components for co-formulation or co-administration include the following molecules or compositions that reduce immunosuppression: -Arginase inhibitor NOHA (N(omega)-hydroxy-nor-L-arginine)-arginase breaks down arginine, which is required for immune activation. NOHA can be used in an amount effective to reduce immune suppression, for example, by making free arginine available. For example, alpha-1 antitrypsin in an amount effective to reduce immunosuppression mediated by neutrophil-secreted proteases

[0152] Further classes of therapeutic components for co-formulation or co-administration include the following molecules or compositions that prevent oxidative damage and improve immune function under stress: -Glutathione and other antioxidants

[0153] Further classes of therapeutic components for co-formulation or co-administration include the following costimulatory molecules for innate cytotoxic lymphocytes (e.g., for anti-cancer therapy): - an effective amount of a phosphoantigen (isoprenoid molecule, e.g. isopentenyl pyrophosphate) recognized by human peripheral blood Vy9V52 T cells, which play a central role in anti-cancer responses, to activate and differentiate monocytes, for example working in concert with NK cells to target both solid and liquid cancers. In an exemplary embodiment, it has been found that SSIs in co-formulation or co-administration with zoledronate increase activation markers, e.g. CD25 and CD69, on human peripheral blood Vy9V52 T cells. -Glycolipid molecules recognized by type I NKT cells (e.g., synthetic α-galactosylceramide)

[0154] SSIs can be administered, for example, for systemic distribution. By administering KPN SSI subcutaneously in a mouse model and using cyanine dye (Cy5.5)-labeled total killed KPN cells and optical in vivo dorsal and ventral whole body imaging, it was revealed that the highest concentration of systemic distribution was found at the new injection site and, surprisingly, at the previous injection site. This explains the preferential delivery / retention of SSI at the site of inflammation after systemic dispersion of the locally administered formulation. Distribution of SSI in organs after 24 hours showed preferential accumulation of KPNSSI in the lungs compared to the heart and spleen.

[0155] In selected embodiments, the SSI can be administered directly to the cancer tissue, e.g., at the site of surgical excision of the cancer. For example, the SSI can be applied topically to the site of surgical excision of melanoma in the skin or cutaneous melanoma.

[0156] Clinical data shows the effectiveness of SSIs that act to downregulate PD1 and PDL1 expression in neoplastic disease. Therefore, PD1 and PDL1 can be used as markers of SSI effectiveness. Furthermore, SSIs can be formulated and administered in an effective dosage regimen in target organs or tissues to mediate the increased expression of one or more granzymes or perforins, such as granzyme A, granzyme B, and perforin.

[0157] Various PRR receptors can be used as targets for alternative SSIs. JPEG2024510510000014.jpg241161

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[0161] JPEG2024510510000018.jpg87161Therefore, in selected embodiments, SSI therapy is provided that uses microbial PRR agonists derived from microbial pathogens of target tissues to target selected subsets of PRRs.For example, immunogenic compositions are provided that include microbial agonists for at least a selected number of different PRRs for use in tampering with innate responses in target tissues, where the PRR agonists are microbial components derived from a single species of microorganism that is selectively pathogenic in the target tissue.The number of distinct PRRs that the agonists target can be, for example, 5 to 25, or at least one number within this integer range, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc. The distinct PRRs may be selected, for example, from the PRRs listed in Table 8, Table 9 and / or Table 10.

[0162] Although various embodiments of the present invention are disclosed herein, many adaptations and modifications can be made within the scope of the present invention according to the common general knowledge of those skilled in the art. Such modifications include the substitution of known equivalents for any aspect of the present invention to achieve the same result in substantially the same way. Numerical ranges include the numbers that define the range. The word "including" is used herein as an open-ended term substantially equivalent to the phrase "including, but not limited to," and the word "comprising" has a corresponding meaning. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes two or more such things. The citation of a reference herein is not an admission that such reference is prior art to the present invention. Any priority documents and all publications, including, but not limited to, patents and patent applications cited herein, and all publications cited in such publications and publications, are incorporated herein by reference as if each individual publication was specifically and individually indicated to be incorporated herein by reference and was fully set forth herein. The present invention includes all embodiments and variations substantially as described herein and illustrated with reference to the examples and drawings. In some embodiments, the present invention excludes steps involving medical or surgical treatment.

[0163] JPEG2024510510000019.jpg245161 EXAMPLES

[0164] SSI improves postoperative immunosuppression / metastasis As shown in the Examples below, in murine lung and colon / liver cancer metastasis models, perioperative SSI treatment significantly reduced metastasis and alleviated postoperative immunosuppression. Exemplary data demonstrate therapeutic modulation of multiple immune pathways (MHCII, CD96, CD69, IFNγ) and recruitment of multiple key innate immune cells (NK cells, monocytes, and neutrophils).

[0165] SSI treatment reduces postoperative pulmonary metastases Mice were pretreated with QBKPN or vehicle (placebo) every 2 days for 8 days prior to intravenous tumor injection + / - surgery (laparotomy and nephrectomy) and 1 day later intravenous tumor injection. The total number of lung metastases was quantified 3 days after surgery. As shown in Figure 1, QBKPN SSI treatment substantially reduced lung metastases with or without surgery.

[0166] QBKPN increases NK cell activation Mice were pretreated with QBKPN or vehicle (placebo) every 2 days for 8 days prior to intravenous tumor injection + / - surgery (laparotomy and nephrectomy) and 1 day later intravenous tumor injection. NK cell activation in the spleen was quantified by cells expressing CD69 3 days after surgery. As shown in Figure 2, QBKPN treatment increases NK cell activation as assessed by CD69 expression.

[0167] QBKPN reduces NK cell inhibition Mice were pretreated with QBKPN or vehicle (placebo) every 2 days for 8 days prior to intravenous tumor injection + / - surgery (laparotomy and nephrectomy) and 1 day later intravenous tumor injection. NK cell activation in the spleen was quantified by cells expressing CD96 3 days after surgery. As shown in Figure 3, QBKPN treatment reduces the expression of CD96, an NK cell checkpoint inhibitor, resulting in reduced NK cell inhibition, i.e., NK cell activation.

[0168] QBKPN increases monocyte / macrophage activation Mice were pretreated with QBKPN or vehicle (placebo) every 2 days for 8 days prior to intravenous tumor injection + / - surgery (laparotomy and nephrectomy) and 1 day later intravenous tumor injection. Monocyte / macrophage activation in the spleen was quantified by cells expressing MHCII 3 days after surgery. As shown in Figure 4, QBKPN treatment enhances immune monitoring and M1 monocyte / macrophage polarization (activation) as assessed by increased MHCII expression.

[0169] QBKPN increases neutrophils Mice were pretreated with QBKPN or vehicle (placebo) every 2 days for 8 days prior to intravenous tumor injection + / - surgery (laparotomy and nephrectomy) and 1 day later intravenous tumor injection. Neutrophil cell counts (CD11b+Ly6G+) from the spleen were quantified as % of live cells 3 days after surgery. As shown in Figure 5, QBKPN treatment increases myelopoiesis (neutrophil counts indicative of myelopoiesis that is also associated with increased monocyte counts in SSI therapy).

[0170] QBKPN increases immune cell recruitment Mice were pretreated with QBKPN or vehicle (placebo) every 2 days for 8 days + / - before surgery (laparotomy and nephrectomy) and 1 day after intravenous tumor injection. Splenic monocyte / macrophage CCR2 receptor expression was measured 3 days after surgery. As shown in Figure 6, QBKPN treatment upregulates CCR2 chemokine receptor expression on immune cells. Previous data have shown that QBKPN increases lung-specific release of chemokines, leading to recruitment of activated immune cells expressing CCR2 to the lung.

[0171] QBKPN increases immune activation Mice were pretreated with QBKPN or vehicle (placebo) every 2 days for 8 days prior to intravenous tumor injection + / - surgery (laparotomy and nephrectomy) and 1 day later prior to intravenous tumor injection. Immune activation was quantified 1 day after surgery by stimulating whole blood with a cytokine cocktail for 12 hours and measuring IFNγ levels. As shown in Figure 7, QBKPN treatment increases immune activation as assessed by IFNγ release and reduces postoperative immunosuppression.

[0172] QBECO inhibits perioperative liver metastasis in colon cancer In this mouse model, all animals undergo surgery as an act of tumor seeding. The act of injecting tumor cells via the portal vein involves a laparotomy with significant anatomical manipulation and operative time, with significant associated surgical stress. Female C57Bl / 6 mice were injected subcutaneously every 2 days for 4 times with either vehicle (PBS) or SSI QBECO at a concentration of 10^6 cells / 20uL until the day of MC38-GFP tumor cell implantation via the portal vein, except on the day of implantation. One day after tumor cell implantation, mice continued to receive either vehicle or SSI every 2 days for 7 times. Mice were sacrificed 2 days after the last treatment injection.

[0173] Animals were perfused via the portal vein, then the livers were excised and fixed in 10% formalin for 48 hours before paraffin embedding. Ten sections were taken per liver at 100 um intervals. Each section was cut at a thickness of 4 um and stained with hematoxylin and eosin (H&E).

[0174] Liver images were scanned with a Zeiss AxioScan and processed using FIJI ImageJ software. Tumor burden was calculated as the sum of tumor areas from all sections from each liver (um^2) divided by the sum of total liver areas from all sections (um^2) and expressed as a percentage.

[0175] Statistical significance was determined using the Mann-Whitney test, with an asterisk * indicating p<0.05. Individual data points were plotted in a box and whisker graph, with the whiskers indicating minimum to maximum points. Figure 8 shows the results demonstrating the remarkable efficacy of QBECO in reducing tumor burden (% of liver) in this model of perioperative liver metastasis.

[0176] SSI modulates perioperative NK cell function Female C57Bl / 6 mice were injected subcutaneously six times with vehicle (PBS) or SSI every other day up to and including the day of surgery (laparotomy and nephrectomy). Mice were sacrificed one day after surgery.

[0177] The immunomodulatory effect was assayed by NKVue (IFNg production by NK cells) as follows: Whole blood was collected by cardiac puncture into heparin-coated tubes. Immediately afterwards, blood was incubated with Promoca (proprietary NK stimulating mixture used with the NKVue assay) at 37°C for 12 hours. At the end of the incubation, the supernatant (plasma) was collected and frozen at -80°C until ELISA was performed (stimulated series). A series of unstimulated plasma was also obtained by centrifugation of the blood immediately after removal from the mice. IFNg production by NK cells from both series was measured using the NKVue ELISA kit.

[0178] The immunomodulatory effect was also assayed in a NK cytotoxicity assay as follows: NK cells were isolated from the spleen using the EasySep Mouse NK Cell Isolation Kit (StemCell). Target cells (Yac-1) were labeled with Cell Trace Violet (CTV) and then incubated with NK cells at ratios of 27:1, 9:1, 3:1, and 1:1 (NK cells to Yac-1) to examine the cytotoxicity of NK cells. Cells were cultured at 37°C for 4 hours. Cells were then stained with a viability dye (Zombie NIR from BioLegend) and fixed with 1% PFA for analysis on a BD Fortessa the next day.

[0179] The results of the NK Vue assay showed that in the context of QBSAU treatment in the perioperative period, QBSAU treatment restored NK cell function (assessed by IFN-γ production) in the postoperative period compared to NK cell paralysis (minimal IFN-γ production) in the control group of vehicle-treated mice. This data reflects the efficacy of QBSAU SSI treatment in inducing a therapeutic immunomodulatory effect in common with the therapeutic mechanism of action of other SSIs. The immunomodulatory effect evidenced by the NK cytotoxicity assay was also similar using the various SSIs, QBECO / QBSAU / QBKPN.

[0180] Thus, this data indicates the ability of various SSIs, in addition to QBECO and QBKPN, to have similar perioperative therapeutic effects in modulating immune responses in target tissues in a manner effective to treat residual disease and thereby treat cancer, for example, by reducing perioperative metastases, as demonstrated with QBKPN and QBECO.

[0181] Previous preclinical studies have revealed that NK cells play a key role in the efficacy of SSI and that SSI "trains" NK cells to improve NK cell functionality, e.g., increasing NK cell IFN-γ responses to infectious and non-infectious threats (e.g., cancer). This SSI-induced "training" of NK cell function is believed to play a major role in the SSI-induced reduction of perioperative metastasis, as assessed by IFN-γ production upon NK cell stimulation (e.g., in the NK Vue assay), because without SSI treatment, NK cell function is "paralyzed" in the postoperative period (minimal IFN-γ production in the NK Vue assay), whereas with SSI treatment, NK cell function is therapeutically modulated (as assessed by restoration of IFN-γ production) to allow for NK cell clearance / improvement of metastatic lesions.

[0182] QBECO for treating liver metastases in colon cancer This is an exemplary protocol for treating liver metastases after primary colon cancer surgery, including the use of circulating tumor DNA (ctDNA) as a diagnosis. Surgery to remove the primary colon cancer tumor is often curative. However, in some patients, residual disease progresses in the form of metastases to the liver. In some of these cases, surgery is an option for resection of metastatic colon cancer tumors located in the liver. Due to postoperative immunosuppression, such patients may be at risk for further metastasis or growth of any residual cancer cells during or after surgery. QBECO, an E. coli-based SSI, can be used perioperatively before and after surgery to prevent or remove these residual cancer cells or metastases. A useful adjunct to this SSI treatment is ctDNA monitoring, because if ctDNA is detected in liquid biopsies, specifically for 1, 2, 3 weeks or even 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after liver surgery, it suggests the presence of residual disease (Cescon, DW, Bratman, S., Chan, SM et al. Circulating tumor DNA and liquid biopsy in oncology. Nat Cancer 1, 276-290 (2020) DOI 10.1038 / s4318-020-0043-5, Daniel Andersson, Helena Kristiansson, Mikael Kubista & Anders Stahlberg (2021) Ultrasensitive circulating tumor DNA analysis enables precision medicine: experimental workflow considerations, Expert Review of Molecular Diagnostics, DOI:10.1080 / 14737159.2021.1889371).

[0183] Patients with liver metastases, with or without prior colon cancer surgery, can be treated with QBECO before the planned date of surgery to remove liver tumors.This treatment can be, for example, every other day for 1, 2, 3 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before surgery and / or for 1, 2, 3 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after surgery.During this perioperative period, liquid biopsies can be monitored for ctDNA to provide a prognostic indicator of residual disease.

[0184] Other assays that may be used in perioperative SSI therapy include trained innate immunity immunoassays, the NKVue™ assay, and metabolic assays.

[0185] <References> Lachmann G,von Haefen C,Kurth J,Yuerek F,Spies C.Innate immunity recovers earlier than acquired immunity during severe postoperative immunosuppression.Int J Med Sci 2018;15(1):1-9.doi:10.7150 / ijms.21433)

[0186] Bartal I,Melamed R,Greenfeld K,Atzil S,Glasner A,Domankevich V.et al.Immune perturbations in patients along the perioperative period:alterations in cell surface markers and leukocyte subtypes before and after surgery.Brain,behavior,and immunity.2010;24:376-86

[0187] Goldfarb Y,Sorski L,Benish M,Levi B,Melamed R,Ben-Eliyahu S.Improving postoperative immune status and resistance to cancer metastasis:a combined perioperative approach of immunostimulation and prevention of excessive surgical stress responses.Annals of surgery.2011;253:798-810

[0188] Levi B,Matzner P,Goldfarb Y,Sorski L,Shaashua L,Melamed R,Rosenne E,Page GG,Ben-Eliyahu S.Stress impairs the efficacy of immune stimulation by CpG-C:Potential neuroendocrine mediating mechanisms and significance to tumor metastasis and the perioperative period.Brain Behav Immun.2016 Aug;56:209-220.doi:10.1016 / j.bbi.2016.02.025.Epub 2016 Mar 2

[0189] Benish M,Bartal I,Goldfarb Y,Levi B,Avraham R,Raz A,Ben-Eliyahu S.Perioperative use of beta-blockers and COX-2 inhibitors may improve immune competence and reduce the risk of tumor metastasis.Ann Surg Oncol.2008 Jul;15(7):2042-52.doi:10.1245 / s10434-008-9890-5.Epub 2008 Apr 9

[0190] Matzner P,Sandbank E,Neeman E,Zmora O,Gottumukkala V,Ben-Eliyahu S.Harnessing cancer immunotherapy during the unexploited immediate perioperative period.Nat Rev Clin Oncol.2020 May;17(5):313-326.doi:10.1038 / s41571-019-0319-9.Epub 2020 Feb 17

[0191] Sundararaj SC,Thomas MV,Dziubla TD,Puleo DA.Bioerodible system for sequential release of multiple drugs.Acta Biomater.2014;10(1):115-125

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Claims

1. 1. An immunogenic composition for treating cancer in a mammalian subject, comprising: the cancer is characterized by its potential to form tumors in a target tissue and / or to metastasize to the target tissue; the composition is for use in combination with surgical removal of the tumor on the day of surgery; the composition comprises whole killed or attenuated microbial cells of a microbial mammalian pathogen that is pathogenic in the target tissue, the microbial mammalian pathogen being Escherichia coli, Staphylococcus aureus, or Clubsierra spp.; The immunogenic composition is for perioperative use within one month of the date of surgery to modulate an immune response in the target tissue effective to treat residual disease, thereby treating the cancer.

2. The immunogenic composition of claim 1 , wherein the immunogenic composition modulates an innate immune response in the target tissue.

3. The immunogenic composition of claim 1 , wherein the tumor is removed from the target tissue.

4. The immunogenic composition of claim 1 , wherein the subject is a mouse, a cat, a dog, a horse, a rodent or a human.

5. The immunogenic composition of claim 1 , wherein the immunogenic composition is for administration to an administration site that is not the target tissue.

6. The immunogenic composition of claim 1, wherein the composition is for use by administration of an effective amount during the perioperative period to modulate the immune response to ameliorate postoperative immunosuppression.

7. The immunogenic composition of claim 5 , wherein the administration site is the skin or subcutaneous tissue.

8. 2. The immunogenic composition of claim 1, wherein the immunogenic composition is administered in multiple doses over an administration period, the administration period being at least two weeks, optionally at least one week, optionally before or after the date of surgery.

9. 9. The immunogenic composition of claim 8, wherein the dose is administered subcutaneously every day or every other day around the day of the surgery.

10. 2. The immunogenic composition of claim 1, wherein the immunogenic composition comprises whole killed or attenuated Klebsiella pneumoniae and the target tissue comprises lung, brain, pancreas, prostate, testis or liver.

11. 2. The immunogenic composition of claim 1, wherein the immunogenic composition comprises whole killed or attenuated E. coli and the target tissue comprises colon, intestine, rectum, pancreas, kidney, bladder, prostate, testis, ovary or liver.

12. 2. The immunogenic composition of claim 1, wherein the immunogenic composition comprises whole killed or attenuated Staphylococcus aureus and the target tissue comprises skin, bone, brain or breast.

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