KR20070083558A - Bacterial delivery system - Google Patents

Abstract

Helicobacter based preparations comprising a pharmacologically active molecule of interest are disclosed, as well as methods of preparing and using said preparations. In particular, Helicobacter pylori vectors, vector plasmids and recombinant cells that include a sequence encoding a pharmacologically active molecule of interest useful in therapeutic treatments and/or vaccination against disease are provided. Delivery of the pharmacologically active molecules is provided at the mucosal surface, such as the gastric mucosa or nasal membranes, to provide effective and continuous delivery of a pharmacologically active agent. Vectors and shuttle vector constructs are also provided.

Description

Bacterial delivery system {BACTERIAL DELIVERY SYSTEM}

The present invention relates to in vivo delivery of physiologically active substances. More specifically, the present invention relates to the treatment, alleviation or prevention of diseases using bacterial systems that deliver bioactive substances directly to anatomical sites in vivo. In one embodiment of the present invention, a bacterial delivery system comprises a bacterium exhibiting Helicobacter characteristics for delivering heterologous nucleic acid to an animal or animal cell, wherein said heterologous nucleic acid is expressed. In another example of the invention, Helicobacter is engineered to include a DNA vector encoding a heterologous nucleic acid such that when infected, the nucleic acid vector is expressed such that the bioactive material is delivered to the animal's body, particularly the mucosa.

Patients with congenital and acquired diseases require long-term, continuous delivery of drugs and immune agents. In most of these methods, several repeated administrations of the therapeutic compound are carried out on a daily, daily or specific interval. However, it is recognized that long term treatment or prophylaxis has problems such as compliance, side effects and drug resistance. Thus, there is still a need to find a 100% effective, free of side effects and inexpensive treatment.

The type of delivery that has recently been studied for various therapeutic and prophylactic agents is the use of microorganisms. Genes of interest derived from various microorganisms, including bacteria, viruses, parasites, and mammals, are cloned into, for example, various bacteria, viruses and mycobacteria for the purpose of expressing foreign proteins or imparting any desired properties in the microorganism. . These microorganisms are used for vaccination programs, gene replacement therapies, and delivery of therapeutic compositions.

Microorganisms are also used to transform animal (host) cells in vivo. Host cell transformation can include gene transfer vectors (eg, US Pat. No. 5,824,544), naked DNA (eg, US Pat. No. 6,261,834), liposomes, including recombinant expression cassettes, including an incompetent virus. , US Pat. No. 6,271,207. Other methods of delivery of therapeutic compositions based on molecules include methods using replication incompatible recombinant viruses designed to express heterologous surface proteins (eg, US Pat. No. 6,376,236).

Recently, pharmacists have attempted to develop methods for expressing therapeutic compositions in vivo using recombinant organism vectors, inanimate vectors, and naked DNA. Examples of recombinant organism vectors include recombinant bacteria (eg US Pat. No. 5,547,664), alphaviruses (eg US Pat. No. 6,391,632), vaccinia virus (eg US Pat. No. 6,267,965), adenoviruses (eg US Pat. No. 5,698,202) and adenoviruses. Viruses such as adenovirus associated virus (AAV) (eg, US Pat. No. 6,171,597). Inanimate viruses include lipid gene delivery vector constructs, such as DNA / angionic liposome complexes, DNA encapsulated in neutral or anionic liposomes, and liposome-captured, polycation-condensable DNA (LPDI and LPDII) ( Ropert, 1999, Braz J Med Biol Res , 32 (2): 163-9).

Examples of gene transfer by various bacteria include clones of Shigella permeable genes into normally noninvasive E. coli to confer E. coli permeability, thus making them more suitable for use as vaccine strains. The method, or the malaria gene of Plasmodium falciparum , is then cloned into Salmonella typhimurium, which will subsequently express the protein of the malaria, and administration of this bacterium to the specific cytotoxicity of mice against malaria opportunistic infection. There are methods to induce immunity and defense of T cells (eg, Hone et al. , 1991, Vaccine, 9: 810-816; Tacket et al. , 1992, Infect. Immun ., 60: 536-541; Hone et al. , 1992, J. Clin.Invest. , 90: 412-420; Chatfield et al. , 1992, Vaccine , 10: 8-11; Tacket et al. , 1992, Vaccine , 10: 443-446; and Mims et al. , 1993, In : Medical Microbiology, Eds., Mosby-Year Book Europe Ltd., London; Sadoff e t. , 1988, Science , 240: 336-338; Aggrawal et al. , 1990, J. Exp. Med ., 172: 1083-1090).

Shigella of attenuated or low virulence (eg, Noriega et al. , 1994, Infect. Immun ., 62: 5168-5172; US Patent Application No. 20020176848), Salmonella (eg, US Patent 6,531,313; US Patent Application No. 20030170211 ), Listeria (e.g., Schafer et al. , 1992, J. Immunol ., 149: 53-59; US Patent Application No. 20030008839), and other bacteria, orally, to provide a virulent form of these bacteria Immunize against infection As such, attenuated bacteria and mycobacterial organisms, such as Calmetguerein bacilli (BCG) (Lagranderie et al. , 1993, Vaccine , 11: 1283-1290; Flynn, 1994, Cell. Molec. Biol ., 40 ( Suppl. 1): 31-36) are administered parenterally for the purpose of protecting against related organisms such as Mycobacteria tuberculosis ( M. tuberculosis ).

Some other bacterial species used as vectors include Yersinia enterocolitica (van Damme et al. , 1992, Gastroenterol ., 103: 520-531) and Vibrio cholerae (Levine et ). al, 1994, In:.. Vibrio cholerae, Molecular to Global Perspective's, Wachsmuth et al, there is a Eds, ASM Press, Washington, DC , pages 395-414).

Despite numerous studies, the bacterial delivery systems described above have technical difficulties, and therefore there is a need to address these difficulties before use in vivo. Indeed, most bacterial systems require bacteria themselves to produce functional molecules and are dependent on bacteria, which are sufficiently attenuated to be safe for human use but still produce bioactive materials. However, not all previously used attenuated bacterial strains can survive long term in vivo without causing side effects. More importantly, these multiple bacterial species are unable to deliver therapeutic or prophylactic agents to the mucosal epithelial cells of the animal. However, many of the most important therapeutic agents are absorbed through the mucosa, thus requiring a bacterial delivery system capable of delivering bioactive substances directly to the mucosa.

Powell et al., In US Pat. No. 5,877,159, describe a live bacterium capable of invading mucosal cells without developing infection or causing disease, and thus introducing a eukaryotic expression cassette encoding an antigen that can be expressed in animal cells. Is disclosed. This method allows for delivery to the mucosal surface of the DNA vaccine, including easy administration, which is a concern for vaccine delivery in developing countries, but has no effect on providing amplifiable mRNA encoding the gene of interest. In addition, the bacteria disclosed in Powell et al. Can not deliver the drug continuously.

Lactococcus lactis , a non-pathogenic, non-colonising, non-invasive food-grade bacterium, is conventionally used to deliver substances to mucous membranes (eg UK patent GB-2278358B ). However, Lactococcus lactis is non-invasive, cannot establish a chronic infection, and does not deliver therapeutics directly to mucosal epithelial cells.

Therefore, there is a continuing need for a delivery system that can establish a noninvasive chronic infection in proximity to the mucosal epithelial cells so that the bioactive material can be delivered to the mucosal epithelial cells of the seed. There is also a continuing need for delivery mechanisms that are sufficiently improved to elicit useful and beneficial immune responses for delivering pharmacologically active molecules to mucosal surfaces. This mechanism provides an effective in vivo delivery system for pharmacologically active substances, and an effective immunization method, ie, a delivery system that exposes the antigen to mucosal surfaces sufficiently to elicit general humoral and mucosal immune responses.

Summary of the Invention

The present invention is to solve the above problems, some examples of the present invention are described below.

The inventors have surprisingly found that Helicobacter, in particular Helicobacter Philophyll, which can form chronic infections can be used to achieve continuous delivery of the drug. Helicobacter of the present invention can be activated and inactivated at various times, and due to its chronic characteristics, helicobacter can be used to deliver drugs through the gastric mucosa.

Helicobacter pylori is a Gram-negative spherical bacterium found almost exclusively in human gastric mucosa. Human gastric acidity is essentially an effective barrier to colony formation of all bacteria other than Helicobacter spp.

Helicobacter pylori has the unique ability to colonize and survive in human gastric mucosa for years despite mucosal inflammation and immune response. This feature makes Helicobacter pylori an interesting candidate for the delivery of selective substances through the mucosa.

In some aspects, the invention provides compositions, methods, and methods for making and using a Helicobacter construct comprising a Helicobacter sequence having a non-helicobacter sequence encoding a promoter region and a non-Helicobacter pharmacologically active molecule Provide a system. Such constructs are described in some instances as vectors or plasmid vectors, and the promoter sequence may control the expression of the non-helicobacteric pharmacologically active molecule.

Accordingly, a first aspect of the invention comprises administering to a subject an effective amount, preferably a therapeutically or prophylactically effective amount of Helicobacter cells expressing one or more physiologically active substances or bacterial cells having the characteristics of Helicobacter, Provided are methods for delivering one or more bioactive substances to an individual.

A second aspect of the invention provides a noninvasive or non-pathogenic Helicobacter cell expressing one or more heterologous bioactive substances.

Preferably, the Helicobacter is a Helicobacter pylori species.

The physiologically active substance may be heterologous to a cell that is homologous to the genus Helicobacter or is a genus or species of Helicobacter cells used for delivery. Heterogeneous materials may be derived from eukaryotic or prokaryotic sources.

In some aspects, the Helicobacter-based vectors and vector plasmid constructs are bioactive substances, such as antigens, organic or inorganic molecules or substances, pharmacological agents, such as therapeutics such as gene products or gene sequences (isolated nucleic acids). Or prophylactic agents. For example, the substance may be present in or secreted from a bacterial cell for delivery to an immunomodulatory substance, hormone, ligand, enzyme or antisense RNA, catalytic RNA, protein, peptide or animal or animal cell. And other molecules.

In some instances, the physiologically active substance is obtained in a nucleic acid molecule, preferably in an isolated form, and then encoded by a nucleic acid molecule that is inserted into the bacterial delivery vehicle of the present invention. Those skilled in the art will appreciate that the isolated nucleic acid molecules of the invention can be cDNA, genomic DNA, RNA, or hybrid molecules thereof. Preferably, the nucleic acid is cDNA.

For example, the protein and / or peptide of interest may be ghrelin, amylin, insulin, motilin, β-glucosidase, chemical chaperone, or other Goche disease, cell wasting, human immunodeficiency. Sex disorders (AIDS), molecules useful in the treatment of appetite suppression, preparations useful in the treatment of diabetes mellitus.

The isolated nucleic acid is preferably incorporated into an expression vector that can be transformed and maintained in Helicobacter cells.

Accordingly, a third aspect of the invention provides a recombinant vector for delivering a bioactive substance directly to the required anatomical site. The Helicobacter cells of the present invention are preferably adapted to secrete and / or express the bioactive substances on their surfaces.

A fourth aspect of the invention provides a vector or construct for delivering an effective amount of a bioactive substance in vivo, which is

a) a nucleotide sequence encoding a physiologically active substance;

b) a control or regulatory sequence capable of modulating the expression of the nucleotide sequence of (a) such that the bioactive material is produced in vivo when a delivery vehicle comprising the vector, operably linked thereto, is delivered to an individual Include.

The vector may be modified in vivo chemically or by recombinant DNA techniques in a chemical or enzymatic manner to form a modified or variant vector.

In the present invention, such constructs may differ from those disclosed, for example by one or more nucleotide substitutions, deletions or insertions, but the desired physiological activity of the construct, nucleic acid molecule, or product encoded thereby is substantially maintained.

In another embodiment of the invention, a Helicobacter vector or construct is equipped with reporter gene (s) expressed from a constitutive promoter that is cloned into an expression vector and used as a screening tool. Non-limiting examples of reporter genes suitable for use in the present invention include blue fluorescent protein (GFP), β-galactosidase, amylase and chloramphenicol acetyl transferase (CAT).

In another embodiment of the invention, the Helicobacter cells are used to deliver a heterologous gene of interest to an individual in need thereof. The gene of interest may encode a therapeutic product (transgene product), including but not limited to peptide hormones (including but not limited to α-melanosite stimulating hormone (α-MSH), insulin, growth hormone and parathyroid hormone), interferon , Interleukin (IL) -2, IL-4, IL-10, IL-12, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) and erythropoietin (EPO) Same cytokines are included.

In another embodiment, the present invention provides a method for treating, alleviating and preventing a disease in an individual. The method is encouraged by the use of a therapeutic or prophylactic composition. Accordingly, a fifth aspect of the present invention provides a pharmacological composition comprising (a) live non-pathogenic Helicobacter cells expressing and / or secreting a bioactive substance and (b) a therapeutically effective carrier.

A sixth aspect of the invention provides a method of treating, alleviating or preventing a disease comprising administering to a subject in need thereof an effective amount of a Helicobacter cell according to the invention expressing a bioactive substance.

In the present invention, non-limiting examples of individuals may include mammals such as primates, horses, cattle, pigs, sheep and rodents. Also included are fish and algae.

In one embodiment of the invention, the disease to be treated, alleviated or prevented, cancer, immune / hematopoietic diseases or symptoms, reproductive diseases or symptoms, musculoskeletal diseases or symptoms, cardiovascular diseases or symptoms, mixed fetal (mixed fetal) Disease or symptom described, germline disease or symptom, nerve / sensory disease or symptom, endocrine system disease or symptom, respiratory system disease or symptom, digestive system disease or symptom, and connective / epithelial tissue related disease or symptom, or bacteria, virus Or a disease or condition resulting from a parasitic infection.

The present invention provides the patient with a variety of pharmacologically acceptable formulations formulated for gastric, oral or intranasal delivery. In certain embodiments, the composition is suitable for delivery to mucosal surfaces. In certain embodiments, the composition is suitable for delivery to the mucosal surface of the intestine.

For example, the mucous membrane may be a mucous membrane of the stomach, vagina, nose, oral or eye surface, or may be any mucous membrane of the body where a permeable mucosal surface or lining is present. In some instances, the mucosal surface is the mucosal surface of the stomach.

Various delivery forms of the composition take into account specific Helicobacter, Helicobacter constructs and other specific types and ratios of delivery vehicles herein, and formulation techniques known in the formulation art as disclosed in Remington's Pharmaceutical Sciences, 20 ^th edition, Mack Publishing Company. Thus, the present invention is readily prepared for use in the practice of the present invention, which is specifically incorporated herein by reference.

The delivery system can be used for primates, fish and birds including animals, especially humans, horses, cattle, sheep and rodents. Formulations can also be used parenterally or for administration to both infants and adults, and to pregnant or lactating animals. Formulations and methods can be further described as being suitable for both males and females.

Another aspect of the invention provides a method of vaccinating an animal. In some examples, the method comprises administering a vaccine comprising a plasmid vector based on Helicobacter and / or a vaccine comprising a cell transformed with the plasmid vector disclosed herein. In another example, a method is provided for delivering an effective amount of a subject pharmacologically active molecule sufficient to abolish or inhibit an animal's disease or physiological symptom, or to elicit specific immune responses against the subject's pharmacologically active molecule.

For example, a subject non-helicobacter pharmacologically active molecule useful in a vaccine may include a mammalian protein, peptide, enzyme, hormone or any combination thereof. In certain instances, the subject pharmacologically active molecule is further defined as a pharmacologically active molecule of the subject human. In some embodiments, the subject pharmacologically active molecule is a human pathogen molecule / antigen, a human protein antigen, eg, amylin, an analog or derivative thereof, or ghrelin, an analog or derivative thereof.

In certain instances, the vaccine delivers immunity against human pathogens, Ebola virus, HIV virus, Marburg virus, influenza virus, and the like. Jones et al. (2005, Nat. Med . 11: 786-90) discloses a replication competent vaccine based on the attenuated recombinant vesicular stomatitis virus vector, which contains the glycoprotein of Ebola and the glycoprotein of Marburg. Doing. Therefore, constructs using vector systems and plasmid vector systems based on Helicobacter with glycoproteins combined with these or other human pathogens may also be provided in accordance with the present invention in conjunction with the disclosure herein.

Term Definition

Before describing the present invention, definitions of terms to be used hereinafter will be described to help understanding the present invention.

An “antibiotic resistance gene” herein includes heterologous nucleic acid sequences provided in a vector and used as a selection system. The term “antibiotic resistance gene” does not include other mechanisms or genes that confer antibiotic resistance to naturally occurring micro-flora organisms.

The term "attenuated" is used herein to describe bacterial strains, in particular Helicobacter strains such as E. coli or Helicobacter pylori, which are defined as bacterial strains of natural, wild-type type with low virulence and low toxicity (penetrative). .

The term "biologically active" means the ability to perform a biological function and includes, in the context of a polypeptide, the polypeptide adopting a "folded form" that is identical or very similar to its original structure. For example, there are suitable folding units, α-helices, β-sheets, domains, disulfide bonds, etc., and when the folding is done correctly or substantially correctly, the polypeptide must have the ability to perform its original function. In general, the functional unit in a polypeptide is a domain.

The simple ability to bind an antibody or other receptor with or without eliciting an immune response is passive or does not constitute a "biological activity". Any antigen has the ability to bind an antibody but does not necessarily have biological activity.

By "clinical grade vector" is meant herein a plasmid or other expression vector that can be expressed in a non-pathogenic bacterium engineered to have the characteristics of Helicobacter or Helicobacter. Clinical grade vectors of the invention do not use antibiotic resistance markers for selection and / or are modified to prevent replication outside the host, such as suicide vectors.

Examples of suicide systems in Helicobacter pylori can be found in Panthel et al. 2003 ( Infection & Immunity , 71: 109-116). In this system plasmids are introduced into Helicobacter Philo containing PhiX174 cell lysis gene E. In order to remove the strain, it is incubated for 5 hours at 42 ℃. In vivo, this means having the animal drink a beverage at 45-50 ° C. to raise the stomach environmental temperature above 42 ° C.

A second example is the L-Dap screening system, which typically allows for the survival of bacterial mutations in supplemented plates (eg Kirata et al. 1997 ( Infection & Immunity , 65: 4158-4164). In order to survive the DapE deficient Helicobacter pylori mutation, a diet free of a substrate, i.e., diamino-pimelic-acid (DAP), must be provided to the animal. Stop it.

A third possible system concerns the metronidazole susceptibility of Helicobacter pylori due to its rdxA gene. Excessive replication of the rdxA gene is harmful to mammalian cells and E. coli . However, replication may be acceptable to bacteria. Thus, the Helicobacter species of the invention can be engineered to contain two copies of rdxA that prevent normal loss of mutation dependent rdxA. Introducing two or more functional rdxA genes into the Helicobacter genome makes the Helicobacter strain permanently sensitive to metronidazole. Jeong et al. 2000 ( J. Bacteriol ., 182: 5082-5090) demonstrated that nitroreductase produced by the functional rdxA gene converts metronidazole from prodrugs to bactericidal compounds. The mode of action of this active compound is to induce DNA destruction of the Helicobacter genome.

A “detectable immune response” is any one of an antibody (humorous) or cytotoxic (vertical) response that is formed in an animal in response to an antigen that can be measured using general experimental methods herein. Enzyme-linked immunosorbant assay (ELISA), radio-immune assay (RIA), Enzyme-linked ImmunoSPOT (ELISPOT), immunofluorescent assay (IMA), complement fixation assay (CF), western blot (WB) or equivalent methods thereof It includes.

"Subject gene" refers to any nucleic acid sequence that encodes a polypeptide or protein that is suitable for expression herein. The subject gene may or may not include a promoter or other regulatory component. In addition, the gene of interest includes a construct capable of producing antisense RNA.

"Gene therapy" is defined herein as using a recombinant vector to deliver a gene of interest to an animal in need thereof. The gene of interest may be a transgene encoding a therapeutic or prophylactic protein or polypeptide, including, but not limited to, cytokines, anti-inflammatory agents, anti-proliferative agents, antibiotics, metabolic inhibitors / activators and immune active agents and their Contains fragments. Furthermore, "gene therapy" herein also includes gene replacement techniques for both congenital and acquired diseases.

The term Helicobacter includes all bacteria in the genus Helicobacter, including Helicobacter Philoly and Helicobacter Mustelae . The term also includes bacteria that may reside in the gastric mucosa of primates and / or may be chronic and have an ecology similar to Helicobacter pylori in that they are isolated from mucosal infections. The term also encompasses bacteria that have been modified to have the characteristics of Helicobacter pylori such that bacteria can inhabit the gastric mucosa.

A “heterologous” polypeptide is a polypeptide that encodes a nucleic acid for a physiologically active substance that is not inherent to Helicobacter, that is, it is not originally expressed in Helicobacter or prior to introduction into Helicobacter or in its ancestor.

"Host" means a given recipient for a therapeutic composition of the invention herein. Hosts include all animals. In particular, hosts include, but are not limited to, primates (including humans), cattle, horses, dogs, cats, pigs, cattle, rabbits, rodents, birds and fish.

By "immunologically inactive" is meant herein any substance, including a microorganism such as a microflora which does not give rise to a significant immune response in its host. Examples of immunologically inactive materials herein include stainless steel, biocompatible polymers such as poly-L-lactide, medical grade plastics, and organisms of the microflora of the present invention. A "significant immune" response is any immune response that limits or limits the in vivo use of a substance or organism to be used as described herein. A detectable immune response is not necessarily a "significant immune response."

An “isolated nucleic acid” is a nucleic acid structure that is not identical to the structure of a naturally occurring nucleic acid or any fragment structure of a naturally occurring genomic nucleic acid spanning four or more isolated genes. Thus, the term includes, for example, (a) a DNA molecule having some sequence of naturally occurring genomic DNA molecules that are flanked on both sides of a coding sequence flanked by a portion of the molecule in the naturally occurring organism's genome; (b) nucleic acids incorporated into prokaryotic or eukaryotic genomic DNA or vectors in such a way that the molecule produced is not identical to any naturally occurring vector or genomic DNA; (c) isolated molecules such as cDNA, genomic fragments, fragments formed by polymerase chain reaction (PCR), or restriction fragments; And (d) a recombinant nucleotide sequence that is part of a hybrid gene, ie, a gene encoding a fusion protein. In this definition, nucleic acids present in the mixture thereof are particularly excluded, as is made in DNA libraries such as (i) DNA molecules, (ii) transfected cells and (iii) cell clones such as cDNA or genomic DNA libraries.

The "% homology" of two amino acid sequences or two nucleic acids is determined by Karlin and Altschul ( Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993), Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA , 87: 2264-2268, 1990. Such algorithms are described in Altschul et al. ( J. Mol. Biol . 215: 403-410, 1990), incorporated into the NBLAST and XBLAST programs. BLSAT nucleotide searches were performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the reference polypeptide (eg SEQ ID NO: 2). In order to obtain a gapped alignment for the purpose of comparison, Altschul et al. Gapped BLAST is used as disclosed in Nucleic Acids Res . 25: 3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the default parameters of that program (eg XBLAST and NBLAST) are used. These can be found on the World Wide Web at the URL "ncbi.nim.nih.gov".

A "reporter gene" is herein a nucleic acid sequence that provides a transformed vector expressing a subject gene, an identifiable phenotype, integrated into (or adjacent to) a heterologous nucleic acid encoding a gene of interest. Non-limiting examples of reporter genes are GFP, β-galactosidase, amylase and CAT.

By “screening marker” is meant an identification feature (surface) provided to a transformed vector prepared as described herein. In one embodiment of the invention, the screening marker is a reporter gene.

"Selection markers", "selection genes", "reporter genes" and "reporter markers" (hereinafter referred to as "selection markers") are nucleic acids encoding phenotypic properties that allow for the rapid identification and isolation of transformed bacterial vectors. The sequence is shown. In general, bacterial vectors prepared as described herein and considered "clinical grade" are vectors that have a selection marker that does not encode antibiotic resistance.

By "transgene" is meant herein a gene that is inserted into a cell in such a way as to secure its function, replication and transmission as a normal gene using cDNA technology.

By “transgenic nucleic acid sequence” is meant herein a plasmid or other expression cassette containing a nucleic acid sequence encoding a gene of interest. In some examples of the invention, the nucleic acid sequence may encode one or more therapeutic agents. A "transforming nucleic acid sequence" can also be used to refer to a "transgene" according to certain examples of the invention. In another embodiment of the invention, the transgenic nucleic acid sequence comprises a nucleic acid sequence encoding a promoter and / or other regulatory element.

The term "cancer" herein refers to a neoplastic disease (eg leukemia, cancer and "hyperproliferative disease"). Neoplasms include colon, abdomen, bone, rectum, digestive system, liver, interest, prostate, peritoneum, lung, blood (e.g. leukemia), endocrine gland (adrenal gland, parathyroid gland, pituitary gland, testes, ovaries, thymus, thyroid gland) , Uterus, eyes, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest and urogenital tissue.

In one example, the term “cancer” also refers to hyperplasia (eg, endometrial hyperplasia), metaplasia (eg connective tissue metaplasia) and / or dysplasia (eg, endometrial dysplasia and bronchial lungs). Pre-neoplastic conditions selected from the group consisting of dysplasia).

In another example, the term "cancer" also includes a benign dysproliferative disorder selected from the group consisting of benign tumors, fibrocystic symptoms and tissue hypertrophy.

The term “immune / hematopoietic disease or symptom” refers to anemia, thrombocytopenia, leukopenia, thrombocytopenia, leukemia, Hopkins disease, non-Hopkins lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immunodeficiency, inflammatory bowel disease, sepsis, neutropenia, neutropenia, psoriasis, immune response of transplanted organs and tissues, erythematous systemic Refers to a disease or condition selected from the group consisting of lupus, hemophilia, hypercoagulation, diabetes, endocarditis, meningitis, Lyme disease, celiac disease and allergy.

The term "genital disease or symptom" herein refers to latent testicles, prostatitis, inguinal hernia, testicular varicose veins, leydig cell tumors, verrucous carcinoma, prostatitis, scleroderma, Peyronie's disease disease, penile cancer, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner syndrome, mucoculent cervicitis, Sertoli-leydig tumor, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter syndrome, Young's syndrome, premature ejaculation, diabetes, cystic fibrosis, catagener syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, Epididymitis, testicles, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumour, stromal tumour, dysmenorrhea, purple Retroverted uterus, endometriosis, fibroids, adenomyosis, ovulatory bleeding, amenorrhoea, Cushing's syndrome, hydatidiform mole, and Asherman's syndrome syndrome, premature menopause, precocious puberty, uterine polyp, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucous cervicitis ), Cervical dysplasia, cervical polyp, Nabothian cyst, cervical erosion, cervical incompetence, cervical neoplasm, pseudo Refers to a disease or condition selected from the group consisting of pseudohermaphroditism and premenstrual syndrome.

The term “musculoskeletal disease or condition” is used herein to refer to bone cancer (eg, osteochondromas, benign chondromas, chondromas, chondromyxoid fibromas, osteogenic osteomas, giant osteomas). Gant cell tumor, multiple myeloma, osteosarcoma, Paget's disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, rim disease, gout, bursitis, tendonitis , Osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.

The term "cardiovascular disease or condition" is used herein to refer to myxoma, fibroma, rhabdomyoma, cardiovascular disorders (eg, congenital heart defect, cerebral arteriovenous malformation), Septal defects, heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial disease, endocarditis ( endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g. hypertension, coronary artery disease, angina, aneurism, artery Refers to a disease or condition selected from the group consisting of atherosclerosis, peripheral vascular disease, hyponatremia, hypernatremia, hypokalemia and hyperkalemia.

The term “disease or condition described as a mixed fetal” is herein referred to as spina bifida, hydrocephalus, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down syndrome, Patau syndrome, Edward syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crozon syndrome, Relaxation skin disease ( cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Katagener syndrome, McKell- Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith Smith-Lemli-Opitz syndrome, Thrombocytopenia-absent radius syndrome, Tricher Collins syndrome, Williams syndrome, Hirschsprung's disease, Mckel's diverticulum, polycystic kidney disease, Turner syndrome and gonadal dysgenesis, Klippel-Feil syndrome, incomplete osteoblastosis ( Disease or condition selected from the group consisting of Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumour, neuroblastoma and retinoblastoma Means.

The term “germ design disease or condition” is used herein to refer to bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (eg, urinary incontinence, urinary retention, urinary obstruction, urinary tract infections) urinary tract infection, interstitial cystitis, prostatitis, neurogenic bladder, hematuria, kidney disorders (e.g. hydronephrosis, proteinuria, renal failure, pyelonephritis ( refers to a disease or condition selected from the group consisting of pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy.

The term “nerve / sensory system disease or condition” is used herein to refer to brain cancer (eg, brain stem glioma, brain tumor, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral nerves). Cerebral astrocytoma), neurodegenerative (eg Alzheimer's disease, Creutzfeldt-Jakob disease), Parkinson's disease and Idiopathic Presenile Dementia, encephalomyelitis, cerebral malaria ), Meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, capillary dilated coordination (ataxia telangiectasia) and acquired immunity AIDS-Dementia Complex, schizophrenia, attention deficit disorder, hyperactive atten tion deficit disorder, autism and obsessive compulsive disorder.

The term “respiratory disease or condition” is used herein to refer to larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinoma, small cell (Oat cells) respiratory system cancers, such as carcinoma, giant cell carcinoma and adenocarcinoma, allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, invasive lung disease (e.g. pulmonary fibrosis and lymphoid epilepsy) Disease selected from the group consisting of lymphoid interstitial pneumonia, obstructive airway disease (eg, asthma, emphysema, chronic or acute bronchitis), occupational lung (eg, silicosis and asbestosis), pneumonia and pleurisy Or symptoms.

The term “endocrine disease or condition” is used herein to refer to cancer of endocrine tissues and organs (eg, hypothalamic, pituitary, thyroid, parathyroid, interest, adrenal, ovarian and testicular cancers), diabetes (eg, diabetes insipidus, type 1 and 2). Type diabetes mellitus, obesity, pituitary related disorders (e.g., hyperpituitarism, hypopituitarism and pituitary dwarfism), hypothyroidism, hypothyroidism, hypothyroidism, goiter), reproductive disorders (e.g. male and female infertility), adrenal gland disorders (e.g. Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g. high) Phenmephroma, transitional cell cancer and Wilm's tumour, diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis ulonephritis (eg, IgM mesangial proliferative glomerulonephritis and glomerulonephritis due to autoimmune disorders such as Goodpasture's syndrome) and nephropathy .

The term "digestive system disease or symptom" refers herein to ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, digestive system cancer (eg , Cholangiocarcinoma, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, colon tumors (e.g. polyps or cancer) and cirrhosis, pancreatitis, Ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atrophy, benign tumours of the duodenum, overexpansion Irritable bowel syndrome, malabsorption, congenital small bowel disease, bacterial infections, parasitic infections, megacolon, Hirschsprung's disease, congenital megacolon (aganglionic megacolon) AIDS, nodular Inflammation, anorectal disorder (e.g., anal fistula, hemorrhoids), congenital liver disease (e.g. Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia) And alpha 1-antitrypsin deficiency), portal hypertension, cholelithiasis and jaundice.

The term “connective / epithelial disease or condition” is used herein to refer to connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metastasis, mucoepithelial dysplasia, graft versus host disease Polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron macroglobulinemia (Waldenstron's macroglobulinemia) , Crohn's disease, pernicious anaemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes, cystic fibrosis, osteoblastoma, osteoclast Osteocylastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteoarthritis, tooth Disease (periodontal disease), wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis (psoriatic arthritis), disc erythema lupus, systemic lupus erythematosus, sclerodeerma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takaya Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis itis, keloid, Ellus-Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondroisplasia, bullous epidermis Refers to a disease or condition selected from the group consisting of epidermolysis bullosa, Alport syndrome and cutis laxa.

As used herein, the terms "a" and "the" include both one (singular) and two or more (plural).

It is understood herein that a "therapeutically effective amount" of an active substance or combination of substances includes a content sufficient to elicit the desired response but insufficient to cause a toxic response. The desired response can, for example, form sufficient and / or acceptable detectable antibody titer levels in the blood sample. The therapeutic dosage and duration of preparation administered to the patient will be determined by the attending physician of the patient in need of treatment, including age, sex, weight, existing disease state and / or extent of tissue damage in the individual, and as a specific formulation and treatment of Helicobacter. The gene of the product of interest in use will be considered.

The expression “effective level” refers to the desired level of activity of a molecule and does not necessarily limit the number of molecules. For example, the effective level of amylin can be lowered to stimulate ghrelin secretion by using amylin antagonists, without the necessity of reducing the amount of free amylin present in the patient.

The expression “ghrelin related diseases and disorders” means any condition that can be treated, prevented or alleviated through the regulation of ghrelin activity. Such symptoms are symptoms that are enhanced, worsened or stimulated by ghrelin, such as growth hormone secretion, or induced by diet. The physiological action of ghrelin is, for example, stimulation of growth hormone secretion, stimulation of hormone secretion of prolactin and corticotrope, appetite and cardiovascular function, anti-proliferative effect on thyroid and breast tumors And by way of regulation of gastric motility and acid secretion via vagus nerve mediation (see WO 2005021026).

Unless required in context, modifications such as the words "comprise" or "comprises" or "comprising" in the specification encompass the inclusion of the complete or complete group of those mentioned, It will be understood that it does not exclude any other complete or group of completes.

Where the definition of a term differs from the general usage meaning of the term, Applicants are intended to use the definitions set forth herein unless specifically stated otherwise.

Detailed description of the invention

Before describing the present invention in detail, it is to be understood that the invention is not to be limited to the specifically exemplified method, but is to be modified. In addition, the terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. However, it will be limited only by the appended claims.

All publications, patents, and patent applications cited above or below herein are hereby incorporated by reference in their entirety. However, the publications disclosed herein are mentioned for the purpose of describing and disclosing protocols, reactants and vectors that are described in the publications and that may also be used in the present invention. It is not to be understood that the invention is not to be construed to precede such disclosed subject matter by the prior invention.

In addition, in the practice of the present invention, unless otherwise stated, existing immunological and molecular biological techniques and pharmacology within the scope of those skilled in the art are used. Such techniques are well known to those skilled in the art and are fully described in the literature. For example, Coligan et al. "Current protocols in Protein Science." (1999) Volume I and II (John Wiley & Sons Inc.); Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ^nd & 3 ^rd Editions, Cold Spring Harbor Laboratory press (1989) (2001); and Bailey, JE and Ollis, DF, Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986 See.

As used herein and in the appended claims, "a", "an" and "the" include plural instructions unless the context clearly dictates otherwise. Thus, for example, "nucleic acid" includes a plurality of nucleic acids, and "isolated peptide" refers to one or more peptides and the like. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, preferred materials and methods are now described.

Delivery of therapeutic compositions and nucleic acids to specific targets in the body of animals is an ongoing challenge facing drug development companies. The inventors of the present invention have developed a Helicobacter based (Helicobacter) bacterial delivery system capable of transporting a vector encoding a bioactive substance, which is expressed on or secreted from the surface of the bacterium. In one embodiment, the bacterium is a Helicobacter species. More preferably, helicobacter pylori. In some instances, the strains of Helicobacter pylori can be any strain known in the art. In some instances, the Helicobacter strain is a non-pathogenic strain, such as genomic strain 26695.

In another example, genetically modified to characterize Helicobacter or Helicobacter pylori, including the ability to colonize the gastric mucosa or gastrointestinal tract, urinary tract, bronchial epithelial region, or other mucosal organs for a long time without significant toxicity to the host. Bacteria other than Helicobacter are used.

In one example, Helicobacter Philosophy is engineered to remove and / or attenuate some pathogenic features. For example, the baculolytic cell toxins and cag pathogenic islet genes are removed to make Helicobacter phyllori low pathogenic. In one example, Helicobacter pylori is engineered to remove and / or attenuate some pathogenic features. For example, the accululating cytotoxin and cag pathogenic islet genes can be removed so that the Helicobacter Philophyll is low pathogenic. Attenuated mutations can be introduced into Helicobacter using nonspecific mutagenesis, or chemically using either N-methyl-N-nitro-N-nitrosoquianidine or recombinant DNA techniques.

Those skilled in the art will appreciate that the methods of the present invention can be used to deliver a variety of physiologically active substances. Examples of suitable substances include substances capable of local or systemic function, such as substances capable of exerting endocrine activity on local or systemic metabolism, and / or substances capable of modulating the activity of cells belonging to the immune / hematopoietic system, And / or on a substance capable of acting on the survival, proliferation and differentiation of various normal or tumor cells in the body or on the induction or immune regulation of an acute inflammatory response to injury and infection, and / or on its target cell receptor There are substances that can enhance or induce resistance to infection of cells and tissues mediated by acting chemokines or promote the proliferation or wound healing of epithelial cells, and / or substances that regulate the expression or production of substances by cells in the body. .

Specific examples of such bioactive substances include insulin, growth hormone, prolactin, calcitonin, luteinizing hormone, parathyroid hormone, somatostatin, thyroid stimulating hormone, vasoactive intestinal polypeptide, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, GM-CSF, M-CSF, SCF, IFN-γ, Structural group 1 employing antiparallel 4 α helical bundles such as EPO, G-CSF, LIF, OSM, CNTF, GH, PRL or IFN α / β cytokine, structural group 2 cytokines in combination with the cell surface, morphologically symmetric homotrimers, and cytokine of the tumor necrosis factor (TNF) family, Subunits such as TNF α, TNF β, CD40, CD27 or FAS ligands, cytokines of IL-1 Paley, fibroblasts Single-chain α / β molecules produced as macrotransmembrane precursor molecules each comprising a pore growth factor family, platelet derived growth factors, transforming growth factor β and nerve growth factor, and at least one epidermal growth factor (EGF) at the extracellular site. Structural group 3 cytokines, such as EGF family cytokines, having amino acid sequences grouped in a form surrounding a conserved cysteine residue (C--C or C--X--C chemokine subgroup) Structural group 4 cytokines exhibiting a mosaic structure, such as chemokines, or insulin-related cytokines, heregulin or neuregulins consisting of different domains, such as EGF, immunoglobulin-like and kringle domains. ).

Alternatively, the bioactive material may be an antagonist to the receptor or bioactive material as described above.

In some instances, separations contained on the mucosal surface of the host to which the transformed cell preparation has been administered, using vectors and / or vector plasmid constructs based on Helicobacter phyllori (eg, E. coli or Helicobacter cells) Transformed cells are made that allow expression and / or secretion of physiologically active substances from nucleic acid molecules. An isolated nucleic acid contained in a transformed cell (or vector) may comprise one or more nucleic acid constructs in which the nucleic acid encoding a physiologically active substance is under the control of an appropriate expression control sequence in Helicobacter phyllori.

Suitable vectors and shuttle vector sequences comprising nucleic acids for introduction into Helicobacter phyllori can be selected or constructed to include appropriate regulatory sequences, including promoter sequences, terminator fragments, enhancer sequences, marker genes, and other suitable sequences. . The vector may be a plasmid, virus, such as phage or phagemid, if appropriate. More specifically, for example, see Sambrook et al., Supra. Many known nucleic acid engineering techniques and protocols, such as the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells, gene expression, and protein analysis, are described in Short Protocols in Molecular Biology, Second Edition, Ausubel. et al. eds., John Wiley & Sons, 1992. Sambrook et al. supra and Ausubel et al. Is incorporated herein by reference.

In some instances, coding sequences for physiologically active substances are included in operons, ie nucleic acid constructs for multi-cistronic expression. In operons, mRNAs are formed that comprise two or more coding sequences, each having a ribosome binding upstream, each suitably located via transcription from a promoter. Thus, two or more substances can be translated from a single mRNA. The use of operons allows for the expression of coordinated bioactive substances.

Nucleic acid constructs or vectors comprising coding sequences for physiologically active substances of the invention are preferably under the control of expression promoters in Helicobacter phyllori.

In one embodiment, the promoter used according to the present invention is constitutively expressed in Helicobacter pylori. The use of constitutive promoters eliminates the need to provide derivatives or other regulatory signals for expression to occur. Preferably, the promoter causes Helicobacter host cells to be expressed at a level that survives, i.e. maintains some metabolic activity, even if proliferation is inhibited. Advantageously, this expression may be at low levels. For example, when expression products accumulate intracellularly, expression levels can lead to accumulation of expression products at less than about 10% of the cellular proteins, preferably at about 5% or less than 5%, such as about 1-3%. .

The promoter may be homologous to the promoter used in the Helicobacter pylori strain, ie originally found in the strain of Helicobacter pilori. In some instances, the promoter is an arabinose-inducible promoter. Other promoters include the FlaB sigma 54 promoter (Josenhans et al. , 1998, FEMS Microbiol Lett , 161 (2): 263-73), the T7 promoter and the nir B promoter of Salmonella (Chatfield et al. , 1992, Biotechnology , 10 ( 8): 888-92).

In another example, the promoter is inducible. Inducible promoters that can be used in clinical grade vectors include, but are not limited to, lactose inducible promoters (eg, pBluescript ™, Stratagene) as used in E. coli plasmids, pH inducible promoters as disclosed in US Pat. No. 6,242,194 to Kullen et al. , Or the endogenous lactose promoter of Lactobacillus; Aristarkhov et al. , 1999, J. Bacteriology , Vol. As disclosed in 178 (14), 4327-4332, there are, but are not limited to, promoters induced during anaerobic culture, such as alcohol dehydrogenase (adhE) promoters.

In one embodiment, the constructs of the invention also include a virulence gene. Such toxic genes are preferably under the control of an inducible promoter so that, upon completion of treatment, the helicobacter of the present invention can be easily removed by inducing expression of the toxic gene. Non-limiting examples of virulence genes include bacterial autolysin under the control of an inducible promoter. Such self-degradable genes can then be triggered at appropriate times and locations in the gastrointestinal tract by using one or more inducible promoters just described above.

In some instances, the engineered Helicobacter vector and plasmid vector constructs are oxygen sensitive. This oxygen sensitivity is another way to limit the prevalence of clinical grade vectors of the present invention. The human intestinal environment is so low in oxygen that it is suitable for the growth of microorganisms, including Helicobacter, which is anaerobic and microaerophilic. Thus, an effective method of removing the Helicobacter delivery vehicle is a method of manipulating genes that give oxygen sensitivity to transformed microorganisms because the intestinal waste exits the human body when released into an oxygen-rich external environment.

Nucleic acid constructs or constructs of the invention may comprise a secretory signal sequence. Thus, in some instances, a nucleic acid encoding a physiologically active substance, such as a non-Helicobacter polypeptide, is appropriate in the cell membrane by appropriately coupling a nucleic acid sequence encoding a secretory signal sequence with a nucleic acid sequence encoding the molecule (polypeptide) Secretion of the substance can be provided. The ability of Helicobacter with the nucleic acid to secrete a polypeptide can be tested in vitro at culture conditions that maintain the viability of Helicobacter.

Suitable secretion signal sequences include any that have activity in Gram-negative organisms such as Escherichia, Klebsiella and Salmonella. The secretory signal sequence may comprise a secretory leader of staphylokinase enzyme secreted from some Staphylococcus strains, which are known to function in Gram positive and Gram negative hosts ("Gene Expression Using Bacillus", Rapoport (1990)). Curr Opin Biotech 1: 21-27).

Other secretory signal sequences that may be used include, for example, the β-lactamase gene (Talmadge et al. , 1980, Proc. Natl. Acad. Sci. USA 77: 3369-3373) or enteroinvasive E. coli hemonas. Hemolysin A (hlyA) (Su et al. , 1992, Microbial Pathogen , 13: 465-476). Examples of secretory signal sequences are described in Pugsley, 1988, Protein secretion across the outer membrane of gram-negative bacteria. In : Protein Transfer and organelle Biogenesis, RC Dand and PW Robbins (eds), Academic Press, Inc., San Diego, pp 607-652. .

Selection markers provide researchers and technicians with a general means of distinguishing transformed microorganisms from untransformed microorganisms in a mixed population. A method for identifying transformed organisms is to incorporate a selectable marker nucleic acid sequence into a plasmid containing the gene of interest. Selectable marker sequences are generally inserted below the gene of interest and are adjusted from the same promoter. As a result, cells to which the gene of interest has been successfully transformed may also be transformed with selectable marker nucleic acid sequences. When antibiotic resistance is used as a selection marker, only transformed cells will survive and / or proliferate in a medium containing antibiotics.

Thus, antibiotic resistance is a commonly used phenotype when transformants are generated. However, vectors having antibiotic resistance genes as selectable markers are capable of horizontal gene transfer that can confer antibiotic resistance phenotypes to other organisms. This risk is particularly severe when using Helicobacter as part of a therapeutic vector.

In order to use Helicobacter as a gene delivery system for animals, the present invention uses, in some instances, clinical grade vector systems that do not utilize antibiotic selection markers. One alternative to the use of antibiotic resistance genes proposed by the delivery system of the present invention is clinical grade genes with chromosomal deletions or lethal mutations in essential house-keeping genes. Next, a functionally similar house-keeping gene is inserted into the plasmid encoding the gene of interest. Thus, the "house-keeping gene" is a selection marker that allows for the rapid isolation and identification of transformants.

Examples of essential "house-keeping" genes include genes encoding a number of metabolic regulators and / or enzymes, including but not limited to canases, proteases, synthetases, dehydrogenases and the like. It doesn't happen. An alternative to the antibiotic resistance genes provided herein is a clinical grade vector having a reporter gene incorporated into the plasmid containing the gene of interest. Non-limiting examples of reporter genes used in accordance with the present invention include green fluorescent protein (GFP), β-galactosidase and amylase.

In one embodiment, the physiologically active polypeptide preferably has cytokine activity. Cytokines are discussed in "The Cytokine Facts Book", Callard and Gearing (1994), Academic Press. Preferred polypeptides having cytokine activity include interleukins, including interleukin-2 (IL-2) and interleukin-6 (IL-6).

In some instances, the Helicobacter delivery system, vector, or vector plasmid system includes such nucleic acid constructs that are introduced into Helicobacter or other suitable host cell to provide a transformed cell. Accordingly, another aspect of the present invention provides a method comprising introducing a nucleic acid as disclosed in non-pathogenic Helicobacter. Any available technique can be applied to the transformation of host cell cultures such as Helicobacter. For Helicobacter pylori cells, suitable techniques may include calcium chloride transformation, electroshock and transfection with bacteriophage.

Vector plasmids can be introduced into Helicobacter cells, such as by inducing or allowing expression from nucleic acids by culturing Helicobacter pili under conditions in which the gene is expressed. By culturing Helicobacter under conditions for expression of a physiologically active polypeptide, it can be verified that Helicobacter contains a coding nucleic acid and can produce a encoded material.

Another aspect of the invention provides a method of delivering a therapeutic or prophylactic dose of a bioactive substance in vivo, wherein the method comprises administering to the individual an effective amount of a non-pathogenic Helicobacter Philolyri formulation and a vaccine of the invention. Steps.

The methods of the present invention and the use of non-invasive or non-pathogenic Helicobacter as described herein provide a wide range of therapeutic methods by which those skilled in the art can, for example, manipulate an immune response in an individual. Accordingly, one aspect of the invention is a method of modulating survival, proliferation, differentiation, effector function or susceptibility of a cell or tissue, comprising administering to the individual a noninvasive or non-pathogenic Helicobacter as disclosed herein. To provide.

Another aspect of the invention, as disclosed herein, is an immune response to a colony of infection on tumor cells or mucosal surfaces or adjacent or distant tissue, comprising administering to the subject a noninvasive or non-pathogenic Helicobacter. Provides a way to boost.

Another aspect of the invention provides a method of modulating a (antibody to cell mediated) immune response type against a pathogenic infectious agent, comprising administering to a subject a noninvasive or non-pathogenic Helicobacter as disclosed herein. do.

Another aspect of the invention provides a method of controlling infiltration of normal tissue with inflammatory or tumor cells, comprising administering a noninvasive or non-pathogenic Helicobacter to a subject as disclosed herein.

Some aspects provide a method of controlling the rate of proliferation, penetration or survival of tumor cells, comprising administering a non-invasive or non-pathogenic Helicobacter to an individual as disclosed herein.

Another aspect of the invention provides a method of inducing apoptosis in tumor cells, comprising administering to a subject a non-invasive or non-pathogenic Helicobacter as disclosed herein.

Another aspect provides a method of down-regulating an immune response comprising administering to a subject a non-invasive or non-pathogenic bacterium expressing a bioactive substance.

Another aspect of the present invention provides a method of treating an allergic autoimmune or other immunomodulatory dysfunction disease condition comprising administering to a subject a non-invasive or non-pathogenic Helicobacter expressing a bioactive substance.

The subject may be a primate, horse, cow, pig, sheep, rodent, bird or fish. In one embodiment, the subject is a human. Administration can typically be nasal or oral.

In treatment, ie when the biological effect of physiologically active mass transfer to a subject is beneficial to a gas, preferably when administered in a "therapeutically effective amount", this is an amount sufficient to produce a beneficial effect in the subject. This beneficial effect may be to alleviate or reduce at least some of the severity or occurrence of one or more symptoms. As a prophylactic measure, the content may be sufficient to reduce the deleterious effect, for example in subsequent pathogen inoculated individuals by enhancing immune responses. The actual amount administered and the rate and time of administration will depend on the physiological effect desired to be obtained in terms of the purpose of administration, such as the nature and severity of the challenge, and is a topic of general optimization. Therapeutic prescriptions, such as dosage determinations, including prophylactic vaccination, are within the responsibilities of general practitioners and other physicians.

Compositions comprising Helicobacter may be administered simultaneously or sequentially, alone or in combination with other therapeutic agents, in accordance with the present invention.

The present invention also provides a pharmacological composition comprising Helicobacter as disclosed. Such pharmacological compositions are well suited for application to mucous membranes.

Pharmaceutical compositions according to the invention and for use according to the invention may comprise not only helicobacter, but also pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other substances known in the art. Such materials should be nontoxic and should not interfere with the efficacy of the active ingredient. The specific characteristics of the carrier or other material may depend on the route of administration. For oral administration, parenteral acceptable aqueous solutions having suitable pH, isotonicity and stability and free of pyrogen can be used. Those skilled in the art can make suitable solutions. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed. As discussed, the pharmacological composition comprising Helicobacter for administration according to the present invention may comprise one or more nutritional substances, such as energy sources such as glucose, amino acids and the like.

In another aspect, the present invention provides a method of preparing a pharmaceutical formulation comprising formulating Helicobacter as described using a suitable carrier medium suitable for administration to an individual. In one embodiment, the medicament is suitable for application to the mucous membrane of an individual.

In another embodiment, the invention provides non-expressing heterologous physiologically active peptides for pharmacological use, such as in the treatment of a human or animal body by surgery or treatment, including prevention (“vaccination”). Provides pathogenic Helicobacter.

In one embodiment, the methods of the present invention can be used to treat, prevent or alleviate a disease such as cancer. In addition, using the methods and delivery systems, immune / hematopoietic diseases or symptoms, reproductive diseases or symptoms, musculoskeletal diseases or symptoms, cardiovascular diseases or symptoms, diseases or symptoms manifested as a mixed fetus, diseases of the excretory system, symptoms, Treating or treating a neurological or sensory system disease or condition, an endocrine system disease or condition, a respiratory disease or condition, a digestive system disease or condition, and a disease or condition associated with connective / epithelial tissue or a bacterial, viral or parasitic infection or It can be prevented.

In another example, the Helicobacter delivery system can deliver simultaneously or sequentially multiple different nucleic acid molecules encoding products that can treat multiple symptoms or diseases as described herein. In addition, preferred delivery systems deliver compositions that can additionally produce desirable physiological effects, such as suppressing or enhancing appetite.

Examples of suicide systems in Helicobacter pylori are described in Panthel et al. 2003 ( Infection & Immunity , 71: 109-116). This system introduces a plasmid into the Helicobacter Philophyll containing PhiXl74 Lysis gene E. To eradicate the strain, incubate at 42 ° C. for 5 hours. In vivo, this means that the animal ingests a beverage at 45-50 ° C. to raise the gastric environment above 42 ° C.

A second example is the L-Dap screening system, which typically allows for the survival of bacterial mutations in supplemented plates (eg Kirata et al. 1997 ( Infection & Immunity , 65: 4158-4164). In order to survive the DapE deficient Helicobacter pylori mutation, a diet free of a substrate, i.e., diamino-pimelic-acid (DAP), must be provided to the animal. Stop it.

A third possible system concerns the metronidazole susceptibility of Helicobacter pylori due to its rdxA gene. Excessive replication of the rdxA gene is harmful to mammalian cells and E. coli . However, replication may be acceptable to bacteria. Thus, the Helicobacter species of the invention can be engineered to contain two copies of rdxA that prevent normal loss of mutation dependent rdxA. Introducing two or more functional rdxA genes into the Helicobacter genome makes the Helicobacter strain permanently sensitive to metronidazole. Jeong et al. 2000 ( J. Bacteriol ., 182: 5082-5090) demonstrated that nitroreductase produced by the functional rdxA gene converts metronidazole from prodrugs to bactericidal compounds. The mode of action of this active compound is to induce DNA destruction of the Helicobacter genome.

The invention is illustrated in detail by way of the following non-limiting examples. However, it is to be understood that the following examples are illustrative only and are not to be taken in a manner limiting the generality of the invention described herein. In particular, although the invention is described in detail with the use of certain Helicobacter pylori strains, it will be clearly understood that the subject matter of the invention is not limited to such strains.

1 is a schematic of the plasmid construct pHPA1 (2.8 kb).

2 is a schematic of the plasmid construct pHP3 (3.4 kb).

3 is a schematic representation of the plasmid construct pTMI03-8.

4 shows the structure of sulfasalazine (SSN).

5 is a schematic view showing the use of an ion exchange resin (Amberlite XE-96) bonded with a dye (Azure-A).

Brief description of sequence listing

The following nucleic acid and amino acid sequences are referenced in the description of the present invention.

SEQ ID NO: 1-nucleotide sequence of the plasmid pHP1 (2796 nucleotides);

SEQ ID NO: 2-nucleotide sequence of the plasmid pHP1 (2796 nucleotides);

SEQ ID NO: 3-nucleotide sequence of the plasmid pHP3 (3444 nucleotides);

SEQ ID NO: 4-Hepatitis C virus comprises an antigen (HCV) nucleotide sequence;

SEQ ID NO: 5-Immunogenic sequence encoding hepatitis C virus (HCV) core antigen, nucleotide sequence 135 bp (45 amino acids)

SEQ ID NO: 6-Nucleotide sequence corresponding to a loop exposed on the surface of the Hop E gene of Helicobacter pylori (position: nucleotide 504, amino acid 168);

SEQ ID NO: 7-Upstream primers (29);

SEQ ID NO: 8 Downstream primers (28);

SEQ ID NO: 9 Oligonucleotide Primer (15).

Example 1 Vector for stable expression of foreign proteins and

Transgenic Helicobacter Philoly Organisms

The genetic modification of Helicobacter pylori is rare. This example discloses the use of the present invention to provide genetically transformed Helicobacter, in particular transformed Helicobacter pili. Transformed bacteria are prepared using plasmid and plasmid vectors derived from Helicobacter, which have already been engineered in non-Helicobacter organisms such as E. coli .

Several Helicobacter Philophyll plasmids disclosed in the literature have been successfully converted to Helicobacter Philoly / E. coli shuttle vectors. Several E. coli strains have been reported to be inherently suitable for DNA uptake. Resistance markers for streptomycin, rifampin and metronidazole were also successfully transformed with most Helicobacter pylori strains. However, plasmid DNA from E. coli and other organisms can also be introduced into the Helicobacter Philosophy, but these plasmids cannot be stably maintained. In addition, Helicobacter pylori plasmids cannot be transformed into E. coli or Helicobacter spp. Therefore, a Helicobacter pillowy shuttle vector must be constructed.

Two plasmids derived from Helicobacter are shown schematically in FIGS. 1 and 2. Vectors pHPA1 (2.8 kb) (FIG. 1) and pHP3 (3.4 kb) (FIG. 2) were sequenced and confirmed that pHPA1 replicated through plasmid replication theta mode. In contrast to rolling-circle replication plasmids, theta plasmids do not produce single-stranded DNA intermediates during replication, which are more stable vector candidates because they are less prone to undergoing recombination. Furthermore, the pHPA1 replication origin ( ori ) contains a series of direct repeat sequences ("iterons") involved in replication control and stable copy number maintenance. Vector pHP623 shares many of these properties.

The nucleotide sequences of these two vectors are shown below.

(1) Plasmid pHP1 is shown in heterologous strand form (upper strand is SEQ ID NO: 1, lower strand is SEQ ID NO: 2):

(2) Plasmid pHp3 is shown in single stranded form (SEQ ID NO: 3):

Additional cloned nucleotide sequences are set forth in SEQ ID NO: 4, which comprises a 135 bp fragment encoding a peptide consisting of 45 amino acids (SEQ ID NO: 5). This small 45 amino acid peptide is an immunogenic polypeptide of the hepatitis C virus (HCV) core antigen. Nucleic acid sequences encoding 45 amino acid peptides are shown below and 135 nucleotides are underlined (SEQ ID NO: 5).

SEQ ID NO: 4

SEQ ID NO: 5

The nucleic acid of SEQ ID NO: 4 was cloned into Helicobacter Philoly 26695 hop E gene (SEQ ID NO: 6, described below) in SEQ ID NO: 504 (in bold / underlined / corresponding to amino acid residue 168 of the protein product). The expressed product will be located as part of the surface exposed loop of the HopE gene product. This construct, vector pTMI03-8 (FIG. 6), was expressed on the surface of E. coli .

      (SEQ ID NO: 6)

Briefly, one method of performing hopE gene isolation is to amplify from Helicobacter Philoly 22695 using Taq DNA polymerase. The upstream primer is 5'AAGGATCCGATAGGAATGTAAAGGAATGG-3 '(SEQ ID NO: 7) comprising the Bam HI site, and the downstream primer is Eco RI 5'CCGAATTCTAAAGGCATGAACGCTTGCA-3 'comprising the site (SEQ ID NO: 8), these are Perkin-Elmer Applied Biosystems, Inc. model DNA synthesizers such as 332 (ABI; Mississauga, Ontario, Canada). The prepared PCR fragment can be blunt-end cloned into the Eco RV site of pBluescript II KS (+) in the same configuration as the lac promoter.

Then, in order to insert the 135 bp immunogen coding sequence of the hepatitis C virus (HCV) core antigen, a PCR primer for inserting two unique restriction enzyme sites into the hopE gene can be constructed. PCR amplification using Taq DNA polymerase may be performed by a touchdown amplification process as follows. The PCR thermocycler was subjected to the first denaturation step at 96 ° C. for 4 minutes, followed by 65 ° C. (90 seconds) first annealing, which was followed by 0.5 ° C. temperature reduction, 72 ° C. for 6 minutes extension, and 96 ° C. in each successive cycle. It is programmed to perform 18 cycles of 1 minute denaturation. After the first 18 cycles, an additional 14 amplification cycles can be performed with 72 ° C. extension, 96 ° C. denaturation and annealing at a constant 55 ° C. The resulting amplification product is purified by column, precipitated with ethanol and then cleaved with Klenow fragments of DNA polymerase to make blunt. PCR products can be cleaved with restriction enzymes to remove template DNA and relinked to a titration vector such as pTM103.8 under the control of the arabinose inducible promoter and transformed into E. coli JM105.

Recombinant clones can be identified using oligonucleotide primer 5'-AGATCTAAGGACGTC-3 '(SEQ ID NO: 9) and reverse sequencing primer in an amplification reaction. The clones identified can be sequenced to verify that the inserted restriction endonuclease site is inframe and has been mistakenly introduced into the hopE gene. The 135 bp immunogen coding sequence of the hepatitis C virus (HCV) core antigen can then be inserted by standard techniques.

Vector pTMI03-8 was generated, which was transformed into E. coli and then grown at 37 ° C. Cells were then harvested and the expression of HCV inserts verified by Western blot.

Briefly, this process is as follows. Cells were recovered from about 20 plates and resuspended in a 20% sucrose solution containing 50 mg of DNase I (Boehringer Mannheim) in 10 mM Tris-HCl (pH 8.0). Then, high pressure cell crusher (French pressure cells) were destroyed at 15,000 lb / in ² . The destroyed cells were stacked on a sucrose step concentration gradient consisting of 70% 1 ml and 70% 6 ml sucrose in 10 mM Tris-HCl (pH 8.0). The outer membrane fraction was collected and pelleted at 150,000 × g, and the pellet was resuspended in 100 μl of distilled water.

Alternatively, the outer membrane obtained from 500 ml of logarithmic culture was dissolved in 10 mM Tris-HCl (pH 8.0) -3% n -octyl-polyoxyethylene, incubated at 23 ° C. for 1 hour and then at 173,000 × g for 30 minutes. Centrifuged. Pellets are 10 mM Tris-HCl-3% suspended in n -octyl-polyoxyethylene--5mM EDTA (pH8.0), incubated at 23 ° C for 1 hour, and then 173,000 The supernatant was recovered by centrifugation at xg for 30 minutes. Western immunoblot showed HCV / hop E present in the supernatant of the second lysis step. The supernatant containing HCV / hop E was mixed with an equal volume of 0.125 M Tris-HCl, pH 6.8, 4% (wt / vol) sodium dodecyl sulfate (SDS) and 20% (vol / vol) glycerol, SDS-12% polyacrylamide gel electrophoresis (PAGE) was performed. If necessary, HCV / hopE bands were extracted from the unstained site of the gel, and 10 mM Tris-HCl (pH8.0), 1 mM at overnight at 4 ° C. It can be eluted with EDTA (pH 8.0) and 100 mM NaCl. The eluted supernatant can then be electrophoresed on an SDS-PAGE gel to verify purity and Western immunoblot can be performed by standard techniques. For example, the isolated outer membrane can be loaded at a concentration of 15 μg / lane. SDS-PAGE is then performed on a discontinuous 12% polyacrylamide gel. Protein is Coomassie Brilliant Blue (Coomassie) dyed with brilliant blue). For Western immunoblotting, unstained gels were transferred to Immobilon-P membranes (Millipore, Bedford, Mass.) Can be electroblotted. Phosphate-buffered The membrane was blocked for 2 hours at 23 ° C. with 3% bovine serum albumin (BSA; Boehringer Mannheim) -0.1% Tween20 (Sigma) in saline) and then in PBS, anti-HCV rabbit in 1% BSA-0.05% Tween20. The reaction was carried out at 37 ° C. for one hour with an antiserum 1 / 10,000 dilution. The membrane was then washed with PBS and the secondary antibody (Bio-Rad, conjugated with alkaline phosphatase) 1 / 5,000 dilution of Richmond, Calif.) At 37 ℃ for 1 hour. Bound antibodies include 5-bromo-4-chloro-3-indolylphosphate (BCIP, Calbiochem, La Jolla, Calif.) And nitroblue tetrazolium (NBT, Sigma).

Example 2 Expression vector and antigen selection for stable expression

HopE is a natural protein of Helicobacter pylori, and because it is acceptable in this organism, it is possible to construct constructs useful for expressing foreign antigens or other heterologous gene products in Helicobacter pili . Other Helicobacter Philoly / E. Coli shuttle vectors that are readily developed as described above include, for example, vectors comprising two plasmid ori sites and markers that are stable at each host. The marker may include genes for chloramphenicol / kanamycin resistance as well as promoters that can be recognized in both the transcription systems of E. coli and Helicobacter phyllori.

Requirements for E. coli replication can be met using a number of known E. coli plasmids (eg pBR322).

The constructed shuttle vector can be tested for in vitro replication in both E. coli and Helicobacter pylori, and can be compared with the existing shuttle plasmids disclosed in the literature.

The choice of antigen or other heterologous gene product to be expressed is non-toxic to Helicobacter pylori and E. coli , ideally having high immunogenicity (or having other desirable properties) when delivered to the selection of the animal. In the case of an expressing antigen for immunization of a mammal, the site may be a mucosal site.

As disclosed in Example 1, the hopE / HCV core antigen fusion protein can be expressed on the surface of E. coli . The product of pTMI03-8 (FIG. 3) will preferably target the outer membrane of Helicobacter pylori, exposing the HCV core antigen in mucosal environment.

Tetanus toxoid (TT) has been widely studied as an antigen in humans and its immune response is well known. TT elicits good mucosal immune responses upon oral or nasal administration when exposed to the surface of bacterial spores (Duc & Cutting, 2003 , Expert Opinion Biol Ther , 3 (8), 1263-70). Tetanus toxin C fragments can be fused to the hopE gene product as described above, or engineered to contain membrane anchor and cell surface target sequences. The advantage of this system is that there is a very well characterized murine model that can determine the effectiveness of vaccination, but in the case of HCV the known murine model typically relies on immunodeficient mice. GB protein of cytomegalovirus (CMV) has been demonstrated to immunize mice against lethal doses of engineered Vaccinia virus expressing gB antigen. Thus, a shuttle vector comprising a gB antigen instead of an HCV antigen, as described herein, can be constructed using the protocol of Example 1.

Multiple promoters can be used for the shuttle vector. Ideally, the promoters used are inducible by inducing the natural in vivo colonization mechanism of Helicobacter or by using harmless nutrients or compounds. The promoter of Helicobacter Philo histidine kinase HP165, which has been reported to be induced by acidic pH and which may be a toxic factor associated with colonization in the gastric mucosa, is one such promoter. The advantage of these promoters is that they can be prepared in vitro with constructs with foreign antigens that begin to express only when exposed to the acidic environment of the gastric mucosa.

Other promoters include the arabinose inducible promoter used for pTMI03.8, the FlaB sigma 54 promoter ( Josenhans et al. , 1998, FEMS Microbial Lett ., 161 (2), 263-73), many constitutive and inducible E The well-studied T7 promoter used in the coli system, Salmonella's nir B promoter induced in an anaerobic environment (Chatfield et al. , 1992, Biotechnology , 10 (8): 888-92). The ability of these promoters to function in Helicobacter is described by Angelini et al. , Using CAT and GFP reporters as readouts of promoter activity in Helicobacter Philoly plasmid vectors . (2004) ( Plasmid , 51: 101-107).

Expression of the target site will depend on the antigen or other gene product used. Initial research has focused on fusion polypeptides and Hop E proteins that target expressed polypeptides (eg, antigens) to the cell surface of Helicobacter Philophyll.

In addition, plasmid stability is also very important and it is useful to use antibiotic resistance genes in vitro as a selective determinant for plasmid maintenance, but they are less practical in vivo. An alternative is a balanced-lethal system, such as the asd gene used for inactivation in Salmonella. The asd gene, originally found in Helicobacter phyllori, is an aspartate-β-semialdehyde dehydrogenase (biosynthetic pathway enzyme of diaminopimelic acid (DAP), an essential component of the cell wall peptidoglycans of Gram-negative bacteria). Code Without DAP, the asd mutation is Rice. Since DAP is not present in mammalian tissue, the stable- lethal system transports recombinant asd gene containing plasmids using all of the survival requirements of Helicobacter pylori.

To use the asd gene system, genome copies of the asd genes are inactivated with standard gene knockout protocols. Such Helicobacter pylori strains will grow only upon DAP feed or in the presence of plasmids containing the asd gene.

Other systems that can be used for similar purposes include mucosal adjuvant E. coli enterotoxin or cholera toxin (CT). Adjuvants can also be used to boost mucosal immune responses. These two adjuvant are CT and E. coli enterotoxin (LT), and the expressed antigen is fused to LTB and CTB mutations that retain their potent mucosal adjuvant properties but significantly reduce toxicity.

Example 3 Toxic, LD ₅₀

As disclosed in Examples 1 and 2, Helicobacter-based vectors such as pHP3 and pHP1 can provide protection against mammalian infections such as mice or humans. This example demonstrates the utility of using Helicobacter-based systems in providing delivery of pharmacologically active molecules of interest to mammals, including humans, using murine models. The murine model is employed to describe the activity of the transforming strains of Helicobacter pylori which elicit a serological response to surface antigens expressed in vivo.

Mice were infected with the native Helicobacter pylori, and the rest of the mice were inoculated with gavage of the temperature-sensitive Helicobacter pili as in Example 2. Serum was taken from both control and experimental animals to analyze antibodies and gastric tissue of the lethal animals were analyzed according to the scheme described in Table 1. A mouse urea breath test was also performed.

In Table 2, toxicity was reduced by 50% (75% to 40%). Specific antibody titers increased fourfold over baseline, suggesting serological responses. Serum samples were taken at baseline, 12, 24, 48 weeks. At this time, 10 and 20 animals were killed to analyze gastric tissue.

Table 1

In vivo experiment

week Remaining mouse * Used mouse serum group adjustment 0 50 0 40 0 0 12 50 10 40 10 109.2 24 40 10 30 10 218.4 48 30 30 20 20 1310.4 49 0 gun 50 130 40

* C57BL / 6J females, 40 experimental groups and 10 control groups

TABLE 2

POWER CALCULATION TOXICITY STUDIES

Various infection rates of Helicobacter pylori at α = 0.05 and power = 80% Normal = 75% vs TSHP = 50%, mice in each group n = 58 Normal = 75% vs TSHP = 40%, mice in each group n = 30 Number of samples 20 only 37% Power: Difference (75% vs 50%) assay, and 62% power: difference (75% vs 40%) assay. To use sample number 20, TSHP infection rate must be at least 32% or less (80% power and α = 0.05).

Example 4 Comparison of Toxicity and Antigenicity of Temperature-sensitive Helicobacter Strains

To detect major changes in toxicity associated with expression / modification of outer membrane proteins, mice were inoculated with temperature sensitive Helicobacter pylori as in Example 3. Mouse controls of the same size were infected with the native Helicobacter Philoly strain. Noninvasive means were used to confirm the presence or absence of Helicobacter Philophyll. Blood was collected from mice at 6 and 6 months for antibody measurements. After killing, tissue analysis was performed to analyze gastritis and verify colonization. Table 3 shows the serological and histological results of the mice used.

TABLE 3

Toxicity and serology comparison experiment

week Remaining mouse Use mouse Serology histology adjustment 0 180 180 24 180 180 48 180 180 180 180 7862.4 49 0 gun 180 540 180

Example 5 LD to evaluate the efficacy of a temperature sensitive Helicobacter pylori vaccine against pneumococcal antigen ₅₀  Experiment

To confirm the protective effect of Helicobacter-based vaccines from standard pathogens (pneumococcal pneumoniae), mice were inoculated with a temperature sensitive Helicobacter pylori. Controls of the same size were infected with the native Helicobacter pylori strain. The noninvasive method was used to determine the presence or absence of the Helicobacter pylori as in Example 4. Six months after infection, Aaberge et al. (1995, Microb. Pathog. 18: 141-52), mice were inoculated intraperitoneally with 10 times LD ₅₀ (˜20 CFU / mouse) of toxic live pneumococcal diplococci type 4 all.

In Table 4, the control shows 75% mortality, and the power of the experiment to detect a 50% reduction in mortality (75% vs 50%) is 0.8.

Table 4

Pneumococcal Polycocci LD on Immunized Mice ₅₀

week Remaining mouse Use mouse Serology histology adjustment 0 60 0 0 24 60 0 25 60 60 0 0 1365 26 0 gun 60 0 0

Example 6 Determination of Helicobacter Philosophy Status in Mice: Exhalation Testing Methods

In this example, the urea breath test conducted in humans was applied to mice.

Ten mice received an uncooked soy. The mice were then fed 200 μl of scented 3.7 kBq ⁽¹⁴⁾ urea in flavored citrate by feeding and placed in a 2L plastic zipper lock bag filled with air for 20 minutes. The mice were then moved without intra-air exchange. 0.1 mmol of hyamine in ethanol was added and scintillant was added to the hyamine solution and counted until 10 minutes or counted to 1,000 dpm.

Example 7 Human experiment

To confirm toxicity and antibody response to humans, a Helicobacter Philoly-like "Baylor Strain" was used and the following criteria were applied:

1. The infected individual has no symptoms, no damage beyond mild tissue damage, and no evidence of hepatitis virus or HIV infection.

2. Isolates are sensitive to single strains, cagA negative, and metronidazole, clarithromycin, tetracycline and amoxicillin.

3. Candidates who received the challenge were healthy individuals with normal gastric tissue, no history of peptic ulcers, no children at home, no periodic contact with children, and no allergies to antibiotics that may be needed to treat the infection.

Infection consists of 40 mg of famotidine at bedtime and oral administration of Helicobacter Philoly in beef broth in the morning. Subjects were administered daily for 14 days. 13 ^C- UBT was performed after 7 and 14 days, followed by endoscopy, quantitative culture and histological examination after 2 and 3 months. Antibiotics were used to treat the infection.

Example 8 Development of exogenous compound markers for in vivo detection of native and / or TSHP

An example of a compound marker that can be used to detect native or TSHP in vivo is sulfasalazine (SSN), represented by the structure of FIG. 2. In sterile mice and general rat studies, enteric bacteria are the sole cause of diazo-bond reduction, resulting in branching of sulfapipyridine and 5-aminosalicylate and reductive catabolism of SSN. The enzyme (s) that catalyze this reaction is called diazoliductase (s) (synonymous azoriductase (s).) Typical rats administered SSN are 5-aminosalicylate and sulfopyridine in urine and feces. (And associated conjugates), but sterile rats showed no evidence of SSN degradation.

Several bacterial species are known to have diazoliductases (AZR's). Preliminary bioinformatics studies have suggested that Helitobacter pilori may not contain the AZR gene. In addition, the presence of similar analogous sequences results in a negative result. In this situation, the use of such markers could be investigated using the Helicobacter Philoly transforming strain (TSHP) with viable and functional azoductase (azr + TSHP).

Plasmid pTMI03-02 was cleaved with EcoRI and HindIII, linked to azoritactase (AZR) of Bacillus subtilis digested with EcoRI and HindIII, containing HopE 168aa and AZR called pTMI03-azr. Prepare the vector. This plasmid was transformed into E. coli to investigate the expression of AZR in HopE 168aa and Bacillus subtilis. pTMI02 showed HopE :: HCCA delivery to the E. coli outer membrane with Western blot and anti-HopE Abs when treated similarly with full length hepatitis C core antigen (HCCA).

When feeding azr + TSHP to mice (n = 30) and confirming in vivo AZR expression to produce 5-aminosalicylate and sulfapipyridine (and their associated conjugates) in urine and feces, humans You can try

Example 9 Use of Mia Nex blue (DIAGNEX BLUE) as marker

The diagnostic agent "Diagnex Blue" consists of an ion exchange resin (Amberlite XE-96) conjugated with a dye (Azure-A). This test takes advantage of the fact that the resin-dye combination degrades below pH 2.5, and the dye appears in urine after absorption. People without urine dyes are achlorhydric. This principle is shown in FIG. 5.

The same principle can be used to test Helicobacter Philophyll. For example, a dye-resin combination that degrades at pH> 7.0 can detect urease if provided with a urea resin. This forms a pH> 7.0 in the mucosa layer where Helicobacter pillori reside and secrete dye.

Mice (n = 30) were inoculated with the native Helicobacter pylori, and sterile mice (n = 30) were used as a pilot study. After the optimal period of time for the establishment of Helicobacter Philophyll as an active infection, the experimental and control groups were administered a dose of resin-dye complex defined by feeding. Urea solution was then administered (Range 0.01M to 0.5M). Mice were housed in metabolic cages and quantified by monitoring azure dye release. Various ratios of resin and urea concentrations were tested to verify the optimal combination used.

Example 10 Delivery formulations

For administration by aerosol, the present invention may deliver in the form of an aerosol spray presentation using a suitable propellant from a pressurized pack or injector. In the case of a pressurized aerosol, the dosage unit can be measured by providing a valve to deliver a metered amount. The formulation is prepared as a powder for administration by inhalation. Administration by inhalation may also be carried out by an atomizing solution or suspension comprising the composition according to the invention.

The compositions according to the invention can also be formulated in liquid form for oral digestion for administration to a subject as an intravenous preparation.

Various formulations of the invention may be prepared by methods familiar to those skilled in the art, using additional suitable pharmaceutical adjuvants, as appropriate. The composition according to the invention advantageously comprises Helicobacter species alone or in combination with other suitable ingredients.

The single or combination of Helicobacter formulations may be contained in a pharmacological composition comprising a pharmacologically acceptable composition according to the invention. The pharmaceutical compositions herein may be in the form of solids (eg, powders, particles, granules, sachets, tablets, capsules, etc.), semisolids (gels, pastes, etc.), or liquids (solutions, dispersions, suspensions, emulsions, mixtures, etc.). And may be designed, for example, for administration through the gastrointestinal tract and gastric mucosa. Thus, the pharmacological composition may be in powder or particulate form designed to be dispersed in an aqueous medium before use.

The pharmacological composition in liquid form may be in the form of a dispersion comprising an Helicobacter composition and an electrolyte such as a composition adapted to physiological conditions, such as a physiologically acceptable solution.

In addition, the pharmaceutical compositions according to the invention may further comprise other therapeutically, prophylactically and / or diagnostically active substances.

Another aspect of the invention relates to a pharmaceutical kit comprising a first and a second container, wherein the first container comprises a recombinant Helicobacter composition comprising a plasmid and / or plasmid vector according to the invention, the second container being a Helicobacter Including a dispersion medium for the composition, accompanied by instructions for administering and / or administering the Helicobacter composition to the dispersion medium prior to use.

The helicobacter composition according to the present invention contained in the kit may be in powder or particulate form.

The pharmaceutical kit according to the present invention may comprise instructions describing a recommendation for the time interval of the process of dispersing the Helicobacter composition in a dispersion medium and then administering.

Example 11 Immune Regulation with Helicobacter Vaccine Preparation

TH1 response (T-helper cell type 1) is a cell mediated response. Excessive activity of this response is presumed to be the cause of diseases such as rheumatoid arthritis (RA) and lupus. In contrast, TH-2 is a characteristic antibody type serum response of the vaccine. This example provides a technique for obtaining a TH-2 response in an animal when treated with a Helicobacter based vaccine therapeutic preparation in accordance with the present invention.

Herein, Helicobacter vectors and vector plasmid systems can be used to elicit antibody responses in animals. For example, a system using a gene expression cassette in a construct provided for transformation of a bacterium, Clostridium, then secretion of a protein (S-layer protein) from the surface of the transformed clostridium, mucosal vaccination initiation is disclosed in WO0194599 It is incorporated in its entirety by reference. These constructs may also comprise secretory leader sequences selected from ORF1, ORF3, ORF5-7., ORF7 and ORF11.

In some vaccine examples, Helicobacter-based vectors and vector plasmids may comprise sequences encoding bacterial surface layer proteins. Surface layer proteins are defined herein as any molecule of proteinaceous properties and include, for example, lipoproteins that are formed in proteins, glycoproteins or bacterial outer membranes and that can be exposed to bacterial membranes. S-layer proteins can be produced continuously and spontaneously in larger amounts than other protein classes in cells.

A process for the preparation of recombinant cell preparations comprising Gram-negative host cells, Clostridium, with S-layer proteins is also disclosed in WO-97 / 28263. The process can be modified and then incorporate the S-protein as part of the Helicobacter constructs of the invention according to the procedures described herein.

Thus, in some vectors and vector plasmid constructs, a fusion protein is provided comprising a subject non-helicobacterium pharmacologically active molecule and a helicobacter sequence. To enhance the immunogenicity of vaccines employing the Helicobacter constructs of the invention, the Helicobacter sequence of the fusion protein may comprise a sequence encoding an S-layer protein. Bacillus constructs comprising S-layer proteins as part of fusion proteins have been reported to express S-layer proteins on Bacillus surfaces (WO-95 / 19371, Bacillus sphaericus), thus enhancing the immunogenicity of the preparations.

Mucosal immunization has already been provided for some diseases, such as oral polio vaccines and oral (drinking) vaccines (inactivated vaccines) for diarrhea by cholera and E. coli . In some embodiments, it is understood that the vaccines of the present invention may include inactivated vaccines.

The present invention provides a single administration, and because the carrier organism, Helicobacter, continues to produce antigens, ie, subject non-helicobacterium pharmacologically active molecules, the vaccine effect can be sustained for a long time and boosted in vivo. Contains live vaccines. In addition, the vaccine will be administered in combination with adjuvants. Such adjuvants include molecules such as aluminum hydroxide or lipid vesicles that slow the site of vaccination to increase vaccine exposure time. Adjuvants also act by inducing the production of immunoregulatory peptides called cytokines and chemokines (Brewer et al. 1997, J. Cytokines Cell Mol. Ther., 4: 223-246). Thus, the vaccines of the present invention may include cytokine adjuvant to enhance the immune response. When orally administered to mammals such as humans or animals, the transformed Helicobacter or E. coli bacteria will colonize the stomach and the desired polypeptide will be produced and present throughout the gastric wall, the site from which it originally colonized. The gastrointestinal tract is surrounded by a huge immune device that is specialized for increasing various types of immune responses. Thus, gastrointestinal colonization by recombinant Helicobacter vaccines or peptide producing strains can stimulate immunity longer than traditional vaccination. In addition, the antigen may be present preferentially in the barrier or lumen.

Example 12 Helicobacter and its use as an appetite suppressant

This example illustrates the applicability of the present invention as a method of employing Helicobacter in preparations and treatment regimens that provide appetite suppression. In particular, delivering to the intestinal mucosa a construct comprising attenuated Helicobacter and ghrelin (hormone) or a pharmacologically active molecule of a subject non-helicobacter that regulates the level of ghrelin agonists, the intestine modulates appetite and mental It is expected to provide an effective means of providing inhibition of the brain axis.

Studies suggest that ghrelin is an appetite stimulant, ie ghrelin increases food intake in mice (Asakawa et al. 2003, Gut, 52 (7): 947-52). In addition, ghrelin has been reported to reduce fat availability in adipose tissue of rodents (Tschop et al., 2000, Nature, 407: 908-13) and to be involved in the adipogenesis of rats (Choi et al. (2003), Endocrinology, 144 (3): 751-9). In addition, ghrelin has been reported to be a fasting signal that promotes food intake in individuals when nutrient availability is low.

Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), stimulates growth hormone (GH) secretion from pituitary cells cultured in a dose-dependent fashion, and is primarily gastric It is produced and secreted by A-like cells found in the oxygenous gland of fundus. Ghrelin is currently known to work on controlling appetite and body weight as well as GH secretion.

Parenteral and intracerebro-ventricular ghrelin has been shown to stimulate food intake and weight gain in mice and rats even in the absence of GH with free access to food. Appetite and body weight can be independent of GH secretion.

The 28 amino acid peptide, ghrelin, is activated when its third serine residue is acylated by n-octanoic acid, and GHS-R is the first, fourth, or fifth to contain the octanylated serine residues of the whole ghrelin peptide. Responsive to residues. GHS-R has been shown to be present in the pituitary gland, hypothalamus, adrenal gland, thyroid gland, interest, myocardium, spleen and testes. Ghrelin stimulates expression of NPY and AGRP mRNA in the hypothalamus. The gorelin's orexigenic effect is mediated by hypothalamic NPY / AGRP-expressing nerves. Ghrelin has also been reported to inhibit vagal afferent activity. The peripheral appetite-inducing effect of ghrelin may be mediated by at least a portion or more by an inhibitory effect on vagus nerve afferent activity. IL-1β is a proinflammatory cytokine that mediates the cachectic process by stimulating the expression and secretion of leptin and / or by mimicking the effect on the hypothalamus of excessive negative feedback signaling from leptin.

If ghrelin antagonist is given to the intestinal mucosa of the animal, it is suggested to reduce animal food intake and reduce weight gain.

Example 13 Cancer and AIDS with Cell Decline

This example demonstrates the utility of the present invention for use as a preparation for preventing or inhibiting cell decline, in particular cell decline associated with disease states of AIDS and cancer.

Cachexia is a symptom characterized by decline, weakness, weakness, and malnutrition. It has recently been reported that the levels of both ghrelin peptide and ghrelin mRNA are upregulated in the stomach in cancer cachexia mouse models. In cachexic mice with increased plasma levels of IL-1β, ghrelin concentrations also increased with cachexia progression. These results suggest that a close relationship exists between ghrelin kinetics and cachexia processes mediated by IL-1. IL-1β is an appetite suppressant such as CCK, leptin, guestrin related proteins and bombesin and antagonizes the action of ghrelin.

Asakawa et al. Found that parenteral administration of IL-1β inhibits NPY mRNA expression in the hypothalamus and pre-progrelor mRNA expression in the stomach, and intraperitoneal administration of ghrelin inhibits severe IL-1β-induced appetite loss. Reported. Helicobacter pylori infection is known to be a major pathogenic factor in the development of gastritis, gastric ulcer disease and gastric malignancies. When Helicobacter pylori adheres to the gastric mucosa, inflammation is associated with the release of various cytokines, including IL-1β.

Helicobacter pylori eradication is often clinically observed to lead to improvements in some nutritional parameters such as body weight, total cholesterol in serum, total protein and albumin levels. Helicobacter pylori infection is reported to be able to modify plasma and gastric ghrelin dynamics in Mongolian gerbil. In humans, however, the eradication of Helicobacter pylori has been shown to be associated with an increase in plasma ghrelin levels, while Helicobacter pylori infection is not reported to be associated with any change in ghrelin levels in plasma.

It is speculated that by using Helicobacter Philoly as a carrier, it acts to provide ghrelin secretion to the gastric mucosa, thereby providing amylin to patients in need thereof. In some instances, to reduce ghrelin levels, a helicobacter vector comprising an amylin, amylin analogs and / or derivatives thereof, and sequences encoding amylin agents (including calcitonin, calcitonin gene related peptides and analogs thereof) Will build.

Amylin antagonists can increase ghrelin levels. Amylin, amylin agonists, or other similar compounds that reduce the effective level of amylin can be used to adjust the effective level of amylin to inhibit or stimulate antagonist and antibody, ghrelin secretion. Thus, some examples of such methods increase the effective level of amylin or amylin agonist in the body, either by direct or indirect means, or by lowering the effective level of amylin or by using amylin antagonists By inhibiting, it adjusts the endogenous level of ghrelin.

Example 14 Goche disease treatment

This example illustrates the utility of the present invention for use as a therapeutic agent for diseases caused by enzyme deficiencies such as Goche disease. Goche disease is the most common human lysosomal storage disorder and is caused by a deficiency of an enzyme called glucocerebroside (GC) (Nolta et al., (1992), J. Clin. Invest. 90 (2): 342-348).

Enzyme replacement therapy is provided with Helicobacter vaccine constructs comprising sequences encoding chemical chaperones or glucocerebrosides (Sawker et al., (2002), PNAS USA 99 (24): 15428-15433). Thus, enhanced levels of functional β-glycoside (β-Glu, glucocerebroside) can be obtained. In particular, sequences encoding the chemical chaperone deoxynogirimycin can be used in the Helicobacter pili construct and can be administered orally or intragastrically to a patient.

As part of another example, a Helicobacter-based construct is provided that includes a vector having a subject non-helicobacterium pharmacologically active molecule encoding a glucocerebrosidase (GC). Retrovirus-mediated delivery of glucocerebrosidase to cultured Goche bone marrow is described by Nolta et al. (1992) as one method of treating Gosch disease. However, this method is very invasive. Other enzyme replacement therapies that employ constructs based on the Helicobacter of the present invention that include coding sequences for the deficient enzyme, glucocerebrosidase, are a more attractive and cheaper alternative to such therapies.

Example 16

This example uses a vaccination formulation comprising a Helicobacter vector and / or a plasmid vector herein to provide a used formulation suitable for treating and / or inhibiting bacterial carcinoma, such as MALT lymphoma, in particular. The usability of the present invention for the purpose of explanation will be described.

Sutton et al. (2004) (Vaccine, 22 (20): 2541-6) are the result of vaccination / immunization of animals against Helicobacter felis , which are directed against bacterially induced malignancies, particularly primary gastric MALT lymphoma. Reported defense. Thus, using the disclosed Helicobacter Philophyll constructs of the present invention comprising vectors and / or plasmid vectors suitable for providing immunization preparations other than Helicobacter Palace, against bacterially induced malignancies, in particular primary gastric MALT lymphoma Vaccination defenses can be provided.

All documents, patents, journals and other materials cited in this application are incorporated herein by reference.

Although the present invention has been fully described with reference to the accompanying drawings, various modifications and variations will be apparent to those skilled in the art. Such modifications and variations are intended to be included within the scope of the invention as defined by the appended claims unless they depart therefrom.

BIBLIOGRAPHY

The following references are specifically incorporated herein by reference.

                         SEQUENCE LISTING <110> Marshall, Barry   <120> BACTERIAL DELIVERY SYSTEM <130> FP22313 <140> AU 2004904564 <141> 2005-08-12 <150> AU 2004904564 <151> 2004-08-13 <150> US 60 / 602,859 <151> 2004-08-20 <160> 9 <170> PatentIn version 3.3 <210> 1 <211> 2796 <212> DNA <213> Helicobacter pylori <400> 1 gtcatgcgcg ttgtttttaa ttacatttta aacaacttgt tgttgttttt acatgtttta 60 ctcgcatgcg cgcgcgtgag ggattggggg ttgcaacccc ctaaataacg aagctgtagg 120 gtttctcatt tttgtggtga aaatgaataa aacagaactt cttgccaaca ctaacagaac 180 ttcttgccaa cactaacaga acttcttgcc aacactaaca gaacttcttg ccaacactaa 240 cagaacttct ttattttaaa gttatgatta ttaacaattt ttagacataa taacagcgtg 300 tgaagatact tttgtagcgg tatttcctat gtgcggcaaa atttggagca attagcttga 360 cttggttgag ttagtgggtt ggaggataga gagggcgaca cctcgttagg aggtatcaat 420 gtgaaagtat ttgtcgtatt agttctagta ttagtaattc tcgcacaatt gctatattag 480 gcttattcgt ggtctaaccc cttgtttatg ggggttggct cgttataagc atactgatac 540 gatcacactt attatacacc aaaagataag gagtatagag tggaatttga tcaatcagat 600 ttacaaaaag cgttgaaaat attagataca ctcccacaaa ccccacaaat tgagctacaa 660 aaacaagaaa tacaaaaccg catcaacaaa ataacagaga caatcattaa agaattacta 720 tcaaagcatg aaatcaagaa agaagaacta gaacccactc taaccccaaa acccacacca 780 ctcaaagagc cacaaaccac cccaacacca tgcaaagatt tagtggttag cacccctaaa 840 gataaaacct aatatcacct accacaataa cgctaataag gtcaatctag ggaaattgag 900 cgaaagggaa gccaatcttt tattcgctat ttttcaaaaa ctcaaagccc aagggaatac 960 cctcattcgt tttgaaccgc aagatttgaa acgcatgcta aacatagata tttctaatga 1020 gcgcttatca gaagtcgtta ttaagctgtg ggatagcatt aaaaccgctg atttttggaa 1080 aattagcgaa accgaaactt caatcattca agaaaattac atgcttttta gtcggtgtaa 1140 aattgaattg aacaaaccga gtaaagattt gaagtattta gaaatccaac tcaacgataa 1200 ctatcaagac ttactcaaca atctgggcat gggtcaatac acttctttca atctgttaga 1260 atttcaaaga gtgaggggta aatacgctaa aacgctctat cgcttgctca agcaatacaa 1320 aagcacaggg attttgagcg tggaatggac tcaattcagg gagcttttag acattccaaa 1380 agactacaaa atggaaaaca tcgatcaaaa agtcttaacc ccctctctca aagaactcag 1440 aaaaatctac ccttttgaac acttgagcta taaaaaagaa cgcaaaagcc attacaagcg 1500 caaagtaacc cacattgatt tttattttga gcaatttcct taaggcgaaa ataagaaaca 1560 aaacaaagcc gacaagcaac gcgctcaaag ggacatcaag cttgtagcat gggatattca 1620 caaccaaatc gctaaaagaa acgcaaaagc cactatggaa gctaggtttc ttgaattgaa 1680 aactttgatc ggctatcagt tcaggaacaa tgacagtagg aacaaattaa agattgacaa 1740 caccactttt gaaagaatca aatgtattta catgtatctt aaccctaaaa ataagcataa 1800 cccccaaaaa ttccttgtat ccaacaagac attcgcattg gaactactat atatcaatag 1860 atacagccta aaaaaaagac aacttgctag aagaatttaa ccccccaaaa tccaccctat 1920 caccaacgaa cctatcaagg aatttgcaga atacatcggc aaaacgatta acatcaccaa 1980 cttcaatgtg gatcaatgcc atgagggaat cagcaactac ctgacaatca ctaggatcgt 2040 gaactggacg taatcggatc tgtatttggt ccagatgtgg ataagcctgg gacttctcaa 2100 gcctttcatt gctaaagtga gaaaatttgg ggattggttc aagaacacta caggtgaaaa 2160 gacagatgca tgctgactaa actcatagaa aaactgaatc acgaaagaaa gaatgcaagc 2220 agaaaacaaa cacctaaaag aacaaggact agaaaaaatc tacactcaaa aagactacga 2280 gcagttaaaa gaacagcatt tgaaagaaat tgaagcactc aaaaaagaaa tccaaaaaac 2340 caagcaagaa acatacacgc aaccaaaaga atgtagccat ttagcgcatt cttttagccc 2400 taattcattc tttcaatcaa aatccgacta attcatcggc taaacgctaa aaatcgctta 2460 aaacgaaaaa tacaaagcaa aaaacttcat tcccctttta gtcgttaacc atttagccaa 2520 tctaactagt ttagcatcta aaggcgaatc tatcttgtgt tagacatcca accttaccaa 2580 aaccgcagag cgagcttaag agagattcaa gcggttttgc acgattgttt gctgccaaga 2640 aaaccaacaa gcgaagtaag gcgcatagac aaaagcgcat cgcagtttga aagcgtaggc 2700 gtcagaagtg gtttgcgtta gaatcaaaca agatagcgca aacctggcgt taggctaaaa 2760 aacccctaaa aactaaaacc ccaaaatatg tagtgc 2796 <210> 2 <211> 2796 <212> DNA <213> Helicobacter pylori <400> 2 cagtacgcgc aacaaaaatt aatgtaaaat ttgttgaaca acaacaaaaa tgtacaaaat 60 gagcgtacgc gcgcgcactc cctaaccccc aacgttgggg gatttattgc ttcgacatcc 120 caaagagtaa aaacaccact tttacttatt ttgtcttgaa gaacggttgt gattgtcttg 180 aagaacggtt gtgattgtct tgaagaacgg ttgtgattgt cttgaagaac ggttgtgatt 240 gtcttgaaga aataaaattt caatactaat aattgttaaa aatctgtatt attgtcgcac 300 acttctatga aaacatcgcc ataaaggata cacgccgttt taaacctcgt taatcgaact 360 gaaccaactc aatcacccaa cctcctatct ctcccgctgt ggagcaatcc tccatagtta 420 cactttcata aacagcataa tcaagatcat aatcattaag agcgtgttaa cgatataatc 480 cgaataagca ccagattggg gaacaaatac ccccaaccga gcaatattcg tatgactatg 540 ctagtgtgaa taatatgtgg ttttctattc ctcatatctc accttaaact agttagtcta 600 aatgtttttc gcaactttta taatctatgt gagggtgttt ggggtgttta actcgatgtt 660 tttgttcttt atgttttggc gtagttgttt tattgtctct gttagtaatt tcttaatgat 720 agtttcgtac tttagttctt tcttcttgat cttgggtgag attggggttt tgggtgtggt 780 gagtttctcg gtgtttggtg gggttgtggt acgtttctaa atcaccaatc gtggggattt 840 ctattttgga ttatagtgga tggtgttatt gcgattattc cagttagatc cctttaactc 900 gctttccctt cggttagaaa ataagcgata aaaagttttt gagtttcggg ttcccttatg 960 ggagtaagca aaacttggcg ttctaaactt tgcgtacgat ttgtatctat aaagattact 1020 cgcgaatagt cttcagcaat aattcgacac cctatcgtaa ttttggcgac taaaaacctt 1080 ttaatcgctt tggctttgaa gttagtaagt tcttttaatg tacgaaaaat cagccacatt 1140 ttaacttaac ttgtttggct catttctaaa cttcataaat ctttaggttg agttgctatt 1200 gatagttctg aatgagttgt tagacccgta cccagttatg tgaagaaagt tagacaatct 1260 taaagtttct cactccccat ttatgcgatt ttgcgagata gcgaacgagt tcgttatgtt 1320 ttcgtgtccc taaaactcgc accttacctg agttaagtcc ctcgaaaatc tgtaaggttt 1380 tctgatgttt taccttttgt agctagtttt tcagaattgg gggagagagt ttcttgagtc 1440 tttttagatg ggaaaacttg tgaactcgat attttttctt gcgttttcgg taatgttcgc 1500 gtttcattgg gtgtaactaa aaataaaact cgttaaagga attccgcttt tattctttgt 1560 tttgtttcgg ctgttcgttg cgcgagtttc cctgtagttc gaacatcgta ccctataagt 1620 gttggtttag cgattttctt tgcgttttcg gtgatacctt cgatccaaag aacttaactt 1680 ttgaaactag ccgatagtca agtccttgtt actgtcatcc ttgtttaatt tctaactgtt 1740 gtggtgaaaa ctttcttagt ttacataaat gtacatagaa ttgggatttt tattcgtatt 1800 gggggttttt aaggaacata ggttgttctg taagcgtaac cttgatgata tatagttatc 1860 tatgtcggat tttttttctg ttgaacgatc ttcttaaatt ggggggtttt aggtgggata 1920 gtggttgctt ggatagttcc ttaaacgtct tatgtagccg ttttgctaat tgtagtggtt 1980 gaagttacac ctagttacgg tactccctta gtcgttgatg gactgttagt gatcctagca 2040 cttgacctgc attagcctag acataaacca ggtctacacc tattcggacc ctgaagagtt 2100 cggaaagtaa cgatttcact cttttaaacc cctaaccaag ttcttgtgat gtccactttt 2160 ctgtctacgt acgactgatt tgagtatctt tttgacttag tgctttcttt cttacgttcg 2220 tcttttgttt gtggattttc ttgttcctga tcttttttag atgtgagttt ttctgatgct 2280 cgtcaatttt cttgtcgtaa actttcttta acttcgtgag ttttttcttt aggttttttg 2340 gttcgttctt tgtatgtgcg ttggttttct tacatcggta aatcgcgtaa gaaaatcggg 2400 attaagtaag aaagttagtt ttaggctgat taagtagccg atttgcgatt tttagcgaat 2460 tttgcttttt atgtttcgtt ttttgaagta aggggaaaat cagcaattgg taaatcggtt 2520 agattgatca aatcgtagat ttccgcttag atagaacaca atctgtaggt tggaatggtt 2580 ttggcgtctc gctcgaattc tctctaagtt cgccaaaacg tgctaacaaa cgacggttct 2640 tttggttgtt cgcttcattc cgcgtatctg ttttcgcgta gcgtcaaact ttcgcatccg 2700 cagtcttcac caaacgcaat cttagtttgt tctatcgcgt ttggaccgca atccgatttt 2760 ttggggattt ttgattttgg ggttttatac atcacg 2796 <210> 3 <211> 3444 <212> DNA <213> Helicobacter pylori <400> 3 tctacacaat taacaatctt tagctacaat aacagcgtgt gaagatgctt tcacagcggt 60 atttcctatg tgcggcaaaa tttggagcaa ttaacttgac ttggttgggt tagtgggttg 120 gaggatagag agggcgacac ctcgttagga ggtatcaatg tgaaagtatt tgtcgtatta 180 gttctagtat tagtaattct cgcacaattg ctatattagg cttatttgtg gtctaacccc 240 ttgtttatgg gggttagatc cttataagca tactgatacg atcacactta ttatacacca 300 aaagataagg agtatagagt ggaatttgat caattagaat cacaaagatc agacttacaa 360 aaagtgttaa aagaattaga tacactccca aaaaccccac aaattgagct acaaaaacaa 420 gaaatacaaa accgcatcaa caaaataaca gacacaatca ttaaagaatt actatcaaaa 480 catgaaatca aaaaagaaga actagaaccc actctaaccc caaaacccac accaacaaaa 540 gagccacaaa ccacccccac accatgcaaa aatttagtgg ttagcacccc taaagataaa 600 acctatatca cctaccacaa taacgctaat aaggtcaatc tagggaaatt gagcgaaagg 660 gaagccaatc ttttattcgc tatttttcaa aggcttaaag atcaagggaa taccctcatt 720 cgttttgaac cgcaagattt aaaacgcatg atcatggtca aatccaactt aaccaacagg 780 caattattgc aagtcttaaa aaatttgctt gacaacatta gcggtgctaa tttttggatc 840 aattagagag catgttgaaa atggcgaaat ctatgaagat cacactagct acatgctttt 900 caaacaattt gaaatccgca tccataagcc aacacaaact atagaatact tagatgtcca 960 actcaatgat agctatcaat acttgctcaa caatctagga atgggcggtc aatacacttc 1020 tttcaatctc ttagaatttc aaagggtgag gggcaaatag tgagagcgtt aaatttcccc 1080 cccctattcc ccttaaaaag gacccttatc ccagggaatt tttggcccca atacaattag 1140 ggccaaaaac ccggtccctt ccatggctta accaacccaa ttgggggatt ccaatttccc 1200 ctggatggga ataacccaag gctttttttg aaaattccac ctaccatttg gtccaaaatt 1260 ggatggacaa ttccaaattc caaatcttct tttccaagaa tgggggccaa cccttgacaa 1320 actccttaaa ccttttcatt cggctaaaag gttgaaaaac atttggaaga tttggtttaa 1380 ggaaatattt atcgggtgaa aagaccagat gcatggctaa cttaaactcc atagaaaaac 1440 tgaatcacga aagaaagaat gctatcaaaa atggcattta ccacttgatc caaatcaaat 1500 tttcttacaa ctccaatcgc attgaaggaa gcggtttgac ttatgaacaa accgctcata 1560 tttttgacaa atccgttctc ataactgaaa aaaacaccaa tatcaaactt gatgatattt 1620 ttgaaactat caatcatttt gaatgcgtga attacttgct tgaaagctat aaagaacctt 1680 tgagtttaga atactttaag aatttacaca aaatcttgaa aaagaattgt tctgatgaag 1740 ttattggtga ttttaaaaaa cgccctaatt ttgtaggcaa tagcgccaca acaagaccca 1800 aattagttga aagcgaattg acaaatcttg tgaaaaatta tcaacgcaac cttgaagtga 1860 gtttgaaaaa caatatcatg cctttcatca tagaaaacga acacaaagcc ttttactaca 1920 ggggcatcaa agaatatgac aacacaaaag gctacttgaa agacaccatt ttgcaaagtc 1980 aagacaattt caatgaaatg gttagctatt tcttttcttg agtgaaaccg cttatttttg 2040 cttgtgtgct tttgtttttt gcgtttttag ttgtaggtgg taagaaatat cggttttttg 2100 cttttcgttg gttgtaggcg attttagata gcaaaaaaca gctaaaaaat ccaagcaacc 2160 taattgattt caaaccaact tcatttccct tttagtcgtt agccatttag ccaatctaac 2220 tagtttagca tctaaaagcg catataactt gagttagcaa tccaaccaat actaaaaccg 2280 cctagcgaag cgttagcgag caaaataagc ggttttagac cgattgtttg ctgacaagca 2340 aacaccaata agcgagcgtt agcgagcatg gacaaaagcg catcgcagtt tgaaagcgta 2400 ggcgttagcc gaagctgttt tgcgtaagca aatcaaacaa gatagcgcaa gccgaggtgc 2460 agcccaagaa tttgaattaa tccatgcggt gtttagggcg ttttagcgtg atcgctttat 2520 tacatgtttt aaacagcatg ctgtttttta catgttttac tcgcatgcgc gcgcgctagg 2580 tattggtggt tggaatagcc taaataacgc agctgtatgg tttctcattt ttcggtgaca 2640 atgaataagg ggtagttctt gcgagtcata agtgtagttc ttgcgagtca taagtgtagt 2700 tcttgcgagt cataagtgta gttcttgcga gtcataagtg tagttctctt cacaatatct 2760 acacaattca caatctctag ctacaataac agcgtgtgaa gatgctttca cagcggtatt 2820 tcctatgtgc ggcaaaattt ggagcaatta gctttaaaag ctagtgggtt gggagtttgt 2880 agcgggtatg cactccgtta ggaggcacac catgaaagca tttttgatag tagtgatttt 2940 agtggtaatc ttgacacagc cactatatta aaaccttagc gttttaataa cccttataag 3000 tccgccaaga cttcttaagg gtttcactcc tgttattata tcgtcttttg aaaaataagc 3060 attaaaaggc gcttaaatgc ccatgaatac gaattttgaa cagcttagaa aacaagaatt 3120 ggaattacga aaattattag aagaattaga aacgctccca caaaccccac aaattaaact 3180 gcaaaaacaa aaaatacaaa cttacataga caagataaca ccaagtattt tgagcggttt 3240 tgatcaaaaa ttcaaagaaa ttatagaaaa tctatcaaat gaatttgaaa aagaaaaatc 3300 cacaccactc aaagagccac aaaccacccc cacaccatgc aaagatttag tggttagcac 3360 ccctaaagat aacacctata ccacctacca caataacgct aataaggtca atctagggaa 3420 attgagcgaa agggaagcca atct 3444 <210> 4 <211> 580 <212> DNA <213> Hepatitis C virus <400> 4 catgagcacg aatcctaaac ctcaaagaaa aaccaaacgt aacaccaacc gtcgcccaca 60 ggacgtcaag ttcccgggtg gcggtcagat cgttggtgga gtttacttgt tgccgcgcag 120 gggccctaga ttgggtgtgc gcgcgacgag gaagacttcc gagcggtcgc aacctcgagg 180 tagacgtcag cctatcccca aggcacgtcg gcccgagggc aggacctggg ctcagcccgg 240 gtacccttgg cccctctatg gcaatgaggg ttgcgggtgg gcgggatggc tcctgtctcc 300 ccgtggctct cggcctagct ggggccccac agacccccgg cgtaggtcgc gcaatttggg 360 taaggtcatc gataccctta cgtgcggctt cgccgacctc atggggtaca taccgctcgt 420 cggcgcccct cttggaggcg ctgccagggc cctggcgcat ggcgtccggg ttctggaaga 480 cggcgtgaac tatgcaacag ggaaccttcc tggttgctct ttctctatct tccttctggc 540 cctgctctct tgcctgactg tgcccgcttc agcctaccaa 580 <210> 5 <211> 135 <212> DNA <213> Hepatitis C virus <400> 5 aatcctaaac ctcaaagaaa aaccaaacgt aacaccaacc gtcgcccaca ggacgtcaag 60 ttcccgggtg gcggtcagat cgttggtgga gtttacttgt tgccgcgcag gggccctaga 120 ttgggtgtgc gcgcg 135 <210> 6 <211> 1108 <212> DNA <213> Helicobacter pylori <400> 6 atgccatagc atttttatcc ataagattag cggatcctac ctgacgcttt ttatcgcaac 60 tctctactgt ttctccatac ccgttttttg ggctaacagg aggaattaac catggaattt 120 atgaaaaagt ttgtagcttt agggcttcta tccgcagttt taagctcttc gttgttagcc 180 gaaggtgatg gtgtttatat agggactaat tatcagcttg gacaagcccg tttgaatagt 240 aatatttata atacagggga ttgcacaggg agtgttgtag gttgcccccc aggtcttacc 300 gctaataagc ataatccagg aggcaccaat atcaattggc atgctaaata cgctaatggg 360 gctttgaatg gtcttgggtt gaatgtgggt tataagaagt tcttccagtt caagtctttt 420 gatatgacaa gcaagtggtt tggttttaga gtgtatgggc tttttgatta tgggcatgcc 480 actttaggca agcaagttta tgcacctaat aaaatccagt tggatatggt ctcttggggt 540 gtggggagcg atttgttagc tgatattatt gataacgata acgcttcttt tggtattttt 600 ggtggggtcg ctatcggcgg taacacttgg aaaagctcag cggcaaacta ttggaaagag 660 caaatcattg aagctaaggg tcctgatgtt tgtaccccta cttattgtaa ccctaacgct 720 ccttatagca ccaaaacttc aaccgtcgct tttcaggtat ggttgaattt tggggtgaga 780 gccaatattt acaagcataa tggcgtagag tttggcgtga gagtgccgct actcatcaac 840 aagtttttga gtgcgggtcc taacgctact aatctttatt accatttgaa acgggattat 900 tcgctttatt tagggtataa ctacactttt tctcgagatc tgcagctggt acgatatggg 960 aattcgaagc tttctagaac aaaaactcat ctcagaagag gatctgaata gcgccgtcga 1020 ccatcatcat catcattgag tttaacggtc tccagcttgg ctgttttggc ggatgagaga 1080 agattttcag cctgatacag attaaatc 1108 <210> 7 <211> 29 <212> DNA <213> Helicobacter pylori <400> 7 aaggatccga taggaatgta aaggaatgg 29 <210> 8 <211> 28 <212> DNA <213> Helicobacter pylori <400> 8 ccgaattcta aaggcatgaa cgcttgca 28 <210> 9 <211> 15 <212> DNA <213> Helicobacter pylori <400> 9 agatctaagg acgtc 15  

Claims (35)

A composition comprising a Helicobacter molecular structure having a Helicobacter sequence and a non-Helicobacter sequence encoding a non-Helicobacter pharmacologically active molecule. The composition of claim 1, wherein the Helicobacter is Helicobacter pylori . The composition of claim 1 or 2, wherein the pharmacologically active molecule is heterologous to Helicobacter pilori species. The composition of any one of claims 1 to 3, further comprising a promoter sequence. 5. The composition of claim 1, wherein the pharmacologically active molecule is ghrelin, amylin, or an analog thereof. 6. 6. The composition of claim 2, wherein the Helicobacter Philophyll is an attenuated Helicobacter Philophyll. 7. The composition of any one of claims 2 to 6, wherein the pharmacologically active molecule is a protein, peptide or nucleic acid molecule. A vaccine comprising the composition according to claim 4 and a pharmacologically acceptable carrier solution. A vector plasmid comprising the composition according to claim 5. A composition comprising a vaccine, wherein the vaccine is (a) a Helicobacter nucleotide sequence comprising a promoter sequence capable of controlling the expression of a sequence comprising a non-Helicobacter pharmacologically active molecule; And (b) a sequence encoding a non-helicobacterium pharmacologically active molecule; A composition comprising a Helicobacter vector plasmid comprising a. The composition of claim 10 further comprising an adjuvant. The composition according to claim 10 or 11, which is suitable for delivery to mucous membranes. The composition of claim 10, wherein the non-helicobacter pharmacologically active molecule is a protein, peptide or nucleic acid molecule. The composition of claim 10, wherein the non-helicobacter pharmacologically active molecule is ghrelin, amylin or an antagonist or analog thereof. (a) providing a culture comprising Helicobacter pylori cells transformed with a plasmid vector; (b) introducing a plasmid comprising a promoter sequence and a sequence encoding a non-helicobacter immunogen into the culture under appropriate conditions to provide transformed Helicobacter pili cells; And (c) selecting the transformed cells expressing the non-helicobacter immunogen to provide an immunogenic composition; Wherein said promoter sequence is capable of controlling the expression of a sequence comprising said non-helicobacter immunogen, a method for producing an immunogenic composition comprising a non-helicobacter immunogen. The method of claim 15, wherein said promoter sequence is an inducible promoter. The method of claim 15 or 16, further comprising a non-antibiotic resistance gene marker. The method of claim 15, wherein said promoter is constitutively expressed in Helicobacter pylori. 16. The method of claim 15, wherein said promoter is an arabinose inducible promoter. 20. The method according to any one of claims 15 to 19, wherein the Helicobacter pylori is strain 26695. The method of claim 15, wherein the plasmid vector is further defined as comprising an operon construct. (a) providing a composition according to any one of claims 10-14; And (b) administering to said animal an effective amount of said composition; Animal treatment method comprising a. The method of claim 22, wherein said animal is a human. The method of claim 22, wherein the treated animal has an enhanced immune response. 25. The method of any one of claims 22 to 24, wherein the composition is administered to the mucosal surface of the animal. 26. The method of any one of claims 22 to 25, wherein at least one effective amount of said composition is administered to the animal. A composition comprising recombinant cells, said recombinant cells comprising a sequence encoding a non-Helicobacter philotropic pharmacologically active molecule, (a) one or more non-helicobacterium sequences encoding pharmacologically active molecules; And (b) a helicobacter sequence having a promoter sequence capable of controlling the expression of said non-helicobacter sequence, Wherein said recombinant cell is capable of expressing said nucleic acid in a target cell or inducing expression of said nucleic acid molecule. The composition of claim 27, wherein the nucleotide sequence encoding the recombinant cell further comprises a secretory signal polypeptide. 28. The composition of claim 27, wherein said recombinant cell is a recombinant Helicobacter pylori. 30. The composition of claim 29, wherein said recombinant Helicobacter pylori is pTM103-8. (a) a sequence encoding a fusion protein comprising a non-helicobacterium pharmacologically active molecule and a helicobacter sequence; And (b) an inducible promoter sequence of Helicobacter pylori; A composition comprising a recombinant Helicobacter pylori comprising a. A plasmid vector comprising the composition according to claim 31. A pharmaceutical composition comprising the plasmid vector of claim 32 as an active ingredient and a pharmacologically acceptable diluent, carrier, adjuvant or combination thereof. (a) providing a culture of Helicobacter pylori; And (b) providing the plasmid vector according to claim 32 to the culture to provide a recombinant Helicobacter pylori, Wherein said recombinant Helicobacter Philophyll can express or cause expression of said non-Helicobacter pharmacologically active molecule in a target cell. 32. The composition of claim 31, wherein said recombinant Helicobacter further comprises a second nucleic acid molecule encoding an immunoregulatory peptide, said recombinant Helicobacter capable of expressing said second nucleic acid molecule in a target cell.

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