Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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97. WO2004076615 - 10.09.2004
IMMUNOMODULATING PROBIOTIC COMPOUNDS

URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=WO2004076615


Inventor(s): ISRAELSEN HANS (DK); MADSEN SOEREN MICHAEL (DK); GLENTING JACOB (DK); VRANG ASTRID (DK); NOERRELYKKE METTE RINDOM (DK); HANSEN ANNE MARIA (DK); AHRNE SIV ELSA INGEGERD (SE); MOLIN GOERAN (SE); RAVN PETER (DK); BECK HANS CHRISTIAN (DK)
Applicant(s): BIONEER AS (DK); PROBI AB (SE); ISRAELSEN HANS (DK); MADSEN SOEREN MICHAEL (DK); GLENTING JACOB (DK); VRANG ASTRID (DK); NOERRELYKKE METTE RINDOM (DK); HANSEN ANNE MARIA (DK); AHRNE SIV ELSA INGEGERD (SE); MOLIN GOERAN (SE); RAVN PETER (DK); BECK HANS CHRISTIAN (DK)
IP Class 4 Digits: C12N
IP Class: C12N
E Class: C12N1/20; C12N9/02J; C12N9/12B2; C12N9/88; C12N9/90; C12N15/01
Application Number: WO2004DK00138 (20040227)
Priority Number: DK20030000315 (20030227); US20030449840P (20030227); US20030482156P (20030625)
Family: WO2004076615
Cited Document(s): WO0212506
Abstract:

THE PRESENT INVENTION RELATES TO METHODS FOR MODULATING I) AN IMMUNE RESPONSE AND/OR II) THE AMOUNT AND/OR COMPOSITION OF MUCOSAL MUCINS, BY CONTACTING A CELL FORMING PART OF MUCOSALASSOCIATED LYMPHOID TISSUE (MALT), OR AN EPITHELIAL CELL, WITH A MICROBIAL CELL SURFACE POLYPEPTIDE. THE MODULATION OF THE IMMUNE RESPONSE PREFERABLY INVOLVES THE INDUCTION OF ONE OR MORE CYTOKINES. THE MICROBIAL CELL SURFACE POLYPEPTIDE IS PREFERABLY A POLYPEPTIDE OBTAINED FROM PROBIOTIC SPECIES OF LACTOBACILLUS OR BIFIDOBACTERIUM. IT HAS SURPRISINGLY BEEN FOUND THAT INTRACELLULAR ENZYMES ACTING IN METABOLIC PATHWAYS IN LACTOBACILLUS AND BIFIDOBACTERIUM, OR POLYPEPTIDES SUBSTANTIALLY IDENTICAL WITH SUCH INTRACELLULAR ENZYMES, ARE TRANSPORTED TO THE SURFACE OF THE CELL WHERE THEY MAY BECOME AT LEAST PARTIALLY EXPOSED TO THE EXTRACELLULAR MEDIUM. ACCORDINGLY, PREFERRED CELL SURFACE POLYPEPTIDES HAVE INTRACELLULAR (I.E. CYTOPLASM ASSOCIATED) EQUIVALENTS ACTING IN METABOLIC PATHWAYS, SUCH AS E.G. GLYCOLYSIS, IN PROBIOTIC SPECIES OF LACTOBACILLUS AND/OR BIFIDOBACTERIUM. THE SURFACE ASSOCIATED POLYPEPTIDES AND THEIR INTRACELLULAR EQUIVALENTS SHARE AN EXTENDED STRETCH OF CONSECUTIVE AMINO ACID RESIDUES, BUT ARE LOCATED IN DIFFERENT PARTS OF A CELL.Description:

Immunomodulating Probiotic Compounds This application is a non-provisional of U. S. provisional application Serial No.
60/449,840 filed February 27,2003 and of U. S. provisional application Serial No. 60/482,156 filed June 25,2003, which are hereby incorporated by reference in their entirety.
All patent and non-patent references are hereby incorporated by reference in their entirety.
Field of Invention The present invention relates to methods for modulating i) an immune response and/or ii) the amount and/or composition of mucosal mucins, by contacting a cell forming part of mucosal-associated lymphoid tissue (MALT), or an epithelial cell, with a microbial cell surface polypeptide. The modulation of the immune response preferably involves the induction of one or more cytokines.
The microbial cell surface polypeptide is preferably a polypeptide obtained from probiotic species of Lactobacillus or Bifidobacterium. It has surprisingly been found that intracellular enzymes acting in metabolic pathways in Lactobacillus and Bifidobacterium, or polypeptides substantially identical with such intracellular enzymes, are transported to the surface of the cell where they may become at least partially exposed to the extracellular medium.
Accordingly, preferred cell surface polypeptides have intracellular (i. e. cytoplasm associated) equivalents acting in metabolic pathways, such as e. g. glycolysis, in probiotic species of Lactobacillus and/or Bifidobacterium. The surface associated polypeptides and their intracellular equivalents share an extended stretch of consecutive amino acid residues, but are located in different parts of a cell.
The cell surface polypeptide can be administered in isolated form, or associated covalently or non-covalently with the. surface of the cell having produced the polypeptide. When associated with the cell surface the polypeptide can furthermore

be modified, e. g. by post-translational modification, as compared to its intracellular equivalent.


The invention also relates to species of Lactobacillus and Bifidobacterium having an altered expression of at least one cell surface polypeptide. The altered expression can be generated by mutagenising an expression signal directing the expression of a gene encoding a cell surface polypeptide. Alternatively, the altered expression can be generated by fusing a gene encoding a cell surface polypeptide to a heterologous expression signal not natively associated with said gene. The altered expression can be an increased expression or a decreased expression. Preferably, the altered expression is an increased expression.
The invention is useful in the treatment of clinical conditions in an individual which responds to modulation of the mucosal immune system, including modulations involving one or more of e. g. the synthesis and/or secretion of cytokines, the stimulation of IgA antibodies, the inhibition of IgE antibodies, the regulation of the Th1/Th2 response, the stimulation of macrophage function, the stimulation of natural killer cell synthesis, and the general activation of the mucosa-associated lymphoreticular tissue system.
The invention further relates to methods for probiotic strain development and methods for performing a quality control procedure ensuring that a strain have desired probiotic qualities.
Background of Invention Probiotic microorganisms Probiotic microorganisms are defined as microorganisms that are beneficial to animal or human health. This invention pertains to the field of immunomodulatory and mucin modulator compounds produced by probiotic microorganisms.
The Nobel Prize winner Metchnikoff claimed in the beginning of the last century that the consumption of certain microorganisms was beneficial to the health i. e. resulting in a prolonged lifetime. In the following years, research on these so-called"probiotic

microorganisms"has been scattered into several different areas including"im- provement of general health", atherosclerosis, infections, gastrointestinal diseases and cancer. Although the majority of these studies have been performed as clinical trials, many results are difficult to interpret for the reason that non-standardized or non-optimal procedures have been employed regarding the handling, storage, culti- vation and formulation of the probiotic microorganisms. This has led to much speculation, but few clinical results backed by hard experimental facts. Accordingly, scientific data describing the molecular mechanisms behind the alleged positive effects of probiotics is still lacking.


An increasing activity in the research on probiotic microorganisms has emerged in the past five years. For instance, a number of results show that certain probiotic bacterial strains have a positive influence on inflammatory bowel diseases (Hart et al. ; 2003). Also, it has been shown that inactivated probiotic strains cause different cytokines to be produced when the strains are brought into contact with epithelial or immuno-competent mammal cells (massen et al. ; 2000, Christensen et al. ; 2002).
The strains Lactobacillus plantarum 299v (Johansson et al. ; 1993), Lb. rhamnosus 271 (Johansson et al. ; 1993), Lb. paracasei 8700 : 2 and Lb. paracasei 02A (Ahrne et al. ; 1998, Molin et al. ; 1993, Hessle et al. ; 1999, and Antonsson M.; 2001) can be used as standard indicators for probiotic potential as they have been shown to have probiotic properties such as e. g. survival in the gastrointestinal tract, adherence to intestinal mucosa, and induction/modification of cytokine release. Children congeni- tally exposed to human immunodeficiency virus (HIV) have received Lb. plantarum 299v in a fermented oatmeal gruel (freeze-dried) in a pilot study. The results sug- gested that Lb. plantarum 299v elicits specific systemic immune responses after oral supplementation (Cunningham-Rundles et al. ; 2000 and Cunningham-Rundles et al. ; 2002. However, no results have been published concerning the compounds, produced by the probiotic cells, which are responsible for the induction of the cyto- kine production.
A few patents and patent applications speculate that each of a large variety of dif- ferent compounds in or on probiotic strains could be responsible for the immuno- modulator response in the mammalian cells (a. Glenn et al. ; 2002, b. Glenn et al. ; 2002). Genome sequencing and proteome projects have revealed the genetic and protein make-up of selected Lactobacillus strains (Kleerebezem et al. ; 2003,

a. Glenn et al. ; 2002) but the identification and characterisation of one or more com- pounds responsible for the immuno-modulatory effect has not been reported.


Mucosal immune system Mucosal surfaces of the lungs and the Gi tract have several tasks that include ab- sorption, macromolecule transport, barrier and secretory functions. However, the large mucosal surfaces are continuously exposed to millions of more or less harmful antigens from the environment, food and microorganisms. To meet these chal- lenges, the mucosa possesses an immune system that tightly controls the balance between responsiveness and tolerance.
The immune system of the mucosa is part of the entire immune system and, conse- quently, immune responses in the mucosa are reflected in the entire body. It con- sists of an integrated network of tissues, lymphoid and non-lymphoid cells and effector molecules such as antibodies and cytokines. The interaction between anti- gen-presenting cells (APCs), T lymphocytes and cytokines is the key for providing the correct specific immune response.
An incorrect or uncontrolled mucosal T-cell response may lead to immunological diseases such as allergy, inflammation and a number of autoimmune diseases.
Means to control T-cell differentiation and cytokine signaling will be essential for the prevention of or the development of therapeutics against such diseases.
The cytokines tumor necrosis factor-alpha (TNFa) and interleukin () L) 1 (3 have been shown to be essential mediators in stimulating inflammatory responses and the production of these cytokines characterize a pro-inflammatory response. Con- versely, the cytokine IL10 and TGFp are mediators of an anti-inflammatory response (for review see Neurath et al. ; 2002).
The first interaction between microorganisms and the host cells takes place at the so-called mucosa-associated lymphoreticular tissue (MALT), which contains APC, T-lymphocytes and IgA-committed B-cells. In the MALT, specialized epithelial M cells transport antigens and microorganisms to the dome underlying the epithelial

layer, where they encounter antigen presenting cells (APCs) such as dendritic cells (DCs).


Two major outcomes can in principle result from the above-mentioned transport of antigens to the dome underlying the epithelial layer. One outcome results in the development of B-cells capable of producing antigen-specific antibodies. The B-cells can reach the mucosal tissues where they differentiate into plasma cells.
A second outcome of the entry of antigen and antigen presentation by DCs is the activation and differentiation of T cells that subsequently can migrate out of the MALT and reach mucosal or peripheral non-mucosal tissues. The T cells can se- crete cytokines, such as IL10 and TGFp, which are essential for the induction of suppressive T cell responses i. e. an anti-inflammatory response.
Alternatively, mucosal T helper cells, Th1 or Th2, can produce pro-inflammatory cytokines such as TNFoc and IL1i. After the differentiation and migration, CD4+ T cells are termed naive T precursor cells, which are functionally immature.
The activation and further differentiation of naive T precursor cells requires at least two separate signals provided by APCs. The first signal is delivered by the T cell receptor/CD3 complex after the T cell's interaction with antigen/major histocompati- bility complex on APCs. The second signal is produced by a number of co- stimulatory or accessory molecules on the APC that interact with their ligands on T cells.
Cytokines play the most critical role in this so-called Th1/Th2 polarization. IL12 and IL4 are essential in the control of the differentiation of precursor T cells into Th1 and Th2, respectively. Cytokines induce the generation of their own T-helper subset and, simultaneously, inhibit the generation of the other subset.
Besides cytokines such as IL12 and IL4, the cytokine IL18 also favors Th1 devel- opment. Although IL18 alone cannot induce Th1 cell differentiation it strongly en- hances IL12-dependent Th1 cell development. Th1 cells secrete the cytokines interferon-y (INFy), and TNF. The cytokine IL13 plays an important role in the Th2 development and its function is partly overlapping that of IL4.

The cytokine signaling in T cells as carried out by INFy, IL12 and IL4 occurs through the binding to the membrane-located cytokine receptors FN-y R, IL12R and IL4R, respectively. The binding conducts activation of the transcription factors STAT1, STAT4 and STAT6, respectively. STAT1 activates the master transcription factor T- bet for Th1 cells. T-bet induces Th1 cytokine production and IL12R 2 chain expres- sion while it simultaneously suppresses Th2 cytokine production. In contrast, the activation of STAT6 leads to the activation of the master transcription factor GATA-3 for Th2 cells. This activation directs Th2 cytokines production through activation of a number of other activators.


Th1 development eventually leads to a cell mediated immunological response while a Th2 development leads to a humoral response. Some infections require Th1 response while others require Th2. However, uncontrolled responses can result in significant tissue and organ damage, which eventually may result in the death of the host. Examples of diseases resulting from uncontrolled responses include inflam- matory bowel disease, rheumatoid arthritis, multiple sclerosis, arteriosclerosis, al- lergy and diabetes. The inflammatory responses also have an essential role in the protection against growth and development of tumors.
Cvtokine modulation In recent years there has been a growing interest in cytokine therapies and cytokine signaling-directed therapies for T cell mediated diseases using either recombinant cytokines or anti-cytokines strategies.
An attractive approach to prevent and control such diseases would be the therapeu- tic use of compounds that are capable of changing specific cytokine concentrations although knowledge is presently lacking on the characterization on accurate immune responses. For instance, compounds that lower the IL4 levels would be useful for the treatment of allergy and lowering the TNF levels would be useful against Crohn's disease.
The bacterial cell surface is the immediate object for interaction with or binding to eukaryotic host cells. It has been shown that, in general, Gram-negative bacteria induce a pro-inflammatory response while certain Gram-positive induce an anti-

inflammatory response (Maasen et al. ; 2000, Christensen et al. ; 2002). It is speculated that Gram-negative bacteria contains surface located compounds, such as specific lipopolysaccharides or flagellin, which induce the pro-inflammatory response (Liaudet et al. ; 2003). Gram-positive bacteria are generally believed to harbour antiinflammatory inducing compounds although exceptions have been described (Hanage & Cohen; 2002). Various Gram-positive bacteria have been shown to be associated with the induction in DC's of different cytokines in different amounts, but no link have been established between specific cell surface polypeptides and a particular induction profile (Maasen et al. ; 2000, Christensen et al. ; 2002).


Modulation of mucin production It has been shown that specific bacteria can modulate the expression of genes encoding mucins (Mack et al., 1999, Mattar et al. ; 2002). The ability to approach and adhere to the epithelial cells in the intestine changes with differences in the mucin composition. Using a transfection strategy, some strains of E. coli is capable of changing the mucin composition in order to favor its affinity to adhere to the epithelia of the intestinal tract. Some Lactobacillus strains have been shown to remodulate the mucin composition resulting in a disfavorable structure to E. coli adherence (Mack et al. ; 1999).
Mucosal colonization Candida albicans (Gil-Navarro et al. ; 1997) and group A streptococci (Pancholi Fischetti 1992) contain surface located glyceraldehydephosphate dehydrogenase (GAPDH), which is key enzymes of the intracellularly operating glycolysis.
The surface located GAPDH of group A streptococci also functions as an ADPribosylating enzyme, which in the presence of NAD is auto-ADP-ribosylated (Pancholi & Fischetti 1993). Since ADP-ribosylation is involved in signal transduction events, this activity of GAPDH may be involved in the communication between the bacterium and a eukaryotic host cell. This is supported by the finding that the GAPDH of group A streptococci is involved in the activation of protein tyrosine kinase and protein kinase C of human pharyngal cells (Pancholi & Fischetti 1997).
In addition, another key glycolytic enzyme, a-enolase, has been found on the surface of pathogenic streptococci (Pancholi & Fischetti 1997; Pancholi & Fischetti

1998; Bergmann et al. ; 2001) and Candida albicans (Barea et al. ; 1999). Strepto- coccal surface enolase (SEN) displays strong plasmin and plasminogen binding, which could be a virulence factor. Group A streptococci may thus bind to plas- min (ogen) via SEN and subvert the fibrinolytic activity of human plasmin (ogen) to their own advantage for tissue invasion. The presence of enolase on the surface of streptococci and also on a variety of mammalian tissues including the brain provides new insight in the role of SEN-specific antibodies in post-streptococcal autoimmune diseases. Additionally, SEN has been shown to bind to the extracellular matrix, which makes it tempting to speculate if enolase is involved in bacterial signal trig- gering or transduction in eukaryotic host cells.


Most probiotic strains belong to the genera of Lactobacillus and Bifidobacterium.
Several publications, patents and patent applications have described clinical effects related to the ingestion of such strains (Sen et al. ; 2002, for review see Sanders 1999). GAPDH has recently been described on the surface of Lactobacillus rham- nosus (a. Glenn et al. ; 2002).
Only a few publications describe compounds that could be related to the probiotic effects (Adlerberth et al. ; 1996, Grenato et al. ; 1999, Vidal et al. ; 2002,) and specific mechanisms at the molecular level are not described.
Summary of Invention Identification of Lactobacillus and Bifidobacterium compounds capable of either modulating an immune response and/or modulating the amount and/or composition of mucosal mucins would be of great interest in the prevention and treatment of e. g. immuno-dependent diseases and infectious diseases.
It has surprisingly been found that intracellular Lactobacillus enzymes acting in metabolic pathways, or polypeptides being substantially identical with such intracel- lular enzymes as described herein below, are transported to the cell surface and optionally becomes at least partially exposed to the extracellular medium.
In one aspect of the present invention, methods for modulating an immune response and/or modulating the amount and/or composition of mucosal mucins exploit

Lactobacillus and/or Bifidobacterium cell surface polypeptides having substantially identical intracellular equivalents acting in metabolic pathways, such as e. g. the glycolytic pathway, in Lactobacillus and/or Bifidobacterium. The cell surface polypeptides are capable of contacting an animal or human cell forming part of the mucosa-associated lymphoid tissue (MALT) system and/or an animal or human epithelial cell, including MALT cells and epithelial cells of the gastro-intestinal (GI) tract.


This invention in one aspect relates to methods for i) induction of gene expression in an animal or human host cell and, subsequently, ii) increased or decreased produc- tion of compounds such as e. g. cytokines and/or mucins, wherein the production of e. g. cytokines and/or mucins result from the contacting of a microbial cell surface polypeptide and the animal or human host cell.
The increased or decreased production of compounds such as e. g. cytokines and/or mucins is according to one presently preferred hypothesis believed to be the result of the binding of a Lactobacillus or Bifidobacterium surface polypeptide to an epithelial cell, or a cell forming part of mucosa-associated lymphoid tissue (MALT).
The binding of the surface polypeptide can be direct or indirect, i. e. additional binding factors can be involved in order for the Lactobacillus or Bifibobacterium cell surface polypeptide to bind an epithelia cell and/or a cell of the MALT system.
The binding of the Lactobacillus or Bifibobacterium cell surface polypeptide alone can mediate signal transduction, or signal transduction (ultimately resulting in cytokine modulation and/or modulation of the amount and/or composition of mucosal mucins) can require additional factors which may also need to bind the epithelial cell and/or the MALT and/or the surface polypeptide.
Signal transduction can also occur because the binding of a Lactobacillus or Bifidobacterium surface polypeptide to an epithelial cell, or a cell forming part of MALT can prevent a pathogen microbial cell from gaining access to the site of binding of the Lactobacillus or Bifidobacterium surface polypeptide.

According to another presently preferred hypothesis, self-antibodies present in e. g. auto-immune diseases can be titrated by binding to a Lactobacillus or Bifidobacterium surface polypeptide according to the invention, thereby at least alleviating the auto-immune disease.


The present invention in a particularly preferred aspect relates to methods for modulating an immune response, such as a cytokine response, in an animal or human host cell, such as an epithelial cell or a cell of the MALT-system in an animal or human individual, by contacting said cell with a microbial cell surface polypeptide preferably obtained from a probiotic strain of Lactobacillus or Bifidobacterium. The cell surface polypeptide can be in isolated form or associated with the surface of the cell having produced the polypeptide.
The cell surface polypeptide can be modified as compared to a substantially identical intracellular/cytoplasmatically located equivalenvhomolog of the cell surface polypeptide. The intracellular equivalent acts in a metabolic pathway and comprises an enzymatic activity. The cell surface polypeptide can comprise an enzymatic activity, but need not have any enzymatic activity. The modification of the cell surface polypeptide can be any post-translational modification, including ribosylation, phosphorylation, methylation acetylation, alkylation, glycosylation, sulfation, amidation, proteolytic processing, and the cell surface polypeptide can form oligomeric or multimeric complexes with itself or other polypeptides, and attain a different tertiary structure as a result of the cell surface association or the association with e. g. molecular chaperones.
Cell surface polypeptides and their substantially identical cytoplasmic equivalents/homologs share an extensive stretch of consecutive amino acid residues, such as e. g. at least 20 amino acid residues, for example at least 40 amino acid residues, such as e. g. at least 50 amino acid residues, for example at least 60 amino acid residues, such as e. g. at least 70 amino acid residues, for example at least 80 amino acid residues, such as e. g. at least 90 amino acid residues, for example at least 100 amino acid residues, such as e. g. at least 120 amino acid residues, for example at least 140 amino acid residues, such as e. g. at least 160 amino acid residues, for example at least 180 amino acid residues, such as at least 200 amino acid residues.

Cell surface polypeptides and their substantially identical equivalents/homologs preferably comprise amino acid sequences which are e. g. at least 80% identical, such as at least 85% identical, for example at least 90% identical, such as at least 95% identical, for example at least 98% identical, such as completely (100%) identical amino acid sequences. As cell surface polypeptides and their equivalents share extensive stretches of amino acids they are likely also to share some secondary and/or tertiary structure and they can in some embodiments be identified by the same antibody, such as a polyclonal antibody or a monoclonal antibody.

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