WO1997014802A1 - Novel adherence factors of non pathogenic microorganisms and applications thereof for screening microorganisms for specific probiotic properties; novel pharmaceutical compositions and food additives comprising such microorganisms and adherence factors - Google Patents
Novel adherence factors of non pathogenic microorganisms and applications thereof for screening microorganisms for specific probiotic properties; novel pharmaceutical compositions and food additives comprising such microorganisms and adherence factors Download PDFInfo
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- WO1997014802A1 WO1997014802A1 PCT/NL1996/000409 NL9600409W WO9714802A1 WO 1997014802 A1 WO1997014802 A1 WO 1997014802A1 NL 9600409 W NL9600409 W NL 9600409W WO 9714802 A1 WO9714802 A1 WO 9714802A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- Novel adherence factors of non pathogenic microorganisms and applications thereof for screening microorganisms for specific probiotic properties novel pharmaceutical compositions and food additives comprising such microorganisms and adherence factors
- This invention relates to the screening of bacteria, in particular non pathogenic bacteria for those bacteria that can adhere to specific sites of the mucosa called receptors. More specifically the invention is directed at screening of non pathogenic Gram positive bacteria in particular lactic acid bacterial (LAB) species, more in particular bacteria of the genera Lactobacillus and Bifidobacterium. A preference is expressed for screening bacteria indigenous to farm animals, pets and humans.
- LAB lactic acid bacterial
- the invention comprises a method of screening for a particular group of adherence factors of the non pathogenic bacteria not previously recognised.
- the adherence factors e.g. of Lactobacilli are of interest.
- This novel group of adherence factors of non pathogenic bacteria comprises proteins that are structurally related to virulence factors of certain classes of pathogenic microorganisms.
- the invention also relates to the application of bacteria obtainable via the screening method of the invention, in particular to Lactobacilli producing said adherence factors, application of the adherence factors as such, application of parts of the bacteria and application of parts of an adherence factor from the novel group for various pharmaceutical applications.
- Such application may comprise the treatment or prophylaxis of infections of the gastro-intestinal tract, the respiratory tract, urogenital tract, the oral cavity or any other part of the body in particular any internal part of the body that can be colonised by pathogenic microorganisms.
- Another suitable example of application comprises the targeting of specific compounds to cells of the mucosa, for example with the aim to evoke a specific mucosal immune response against said compound, or to modulate the immune response.
- Novel microorganisms obtainable e.g. through recombinant DNA technology expressing or overexpressing any of the novel adherence factors or effective parts thereof are also included within the invention.
- the nucleic acid sequences encoding the adherence factors and fragments of said sequences encoding mucosa binding expression products are also part of the invention as are the recombinant products resulting from expression of said nucleic acid sequences.
- Novel pharmaceutical compositions comprising the nucleic acid or expression products thereof or microorganisms expressing or overexpressing an adherence factor of the novel type also fall within the scope of the invention.
- Pathogenic viruses and bacteria can adhere to specific sites of the mucosa, called receptors and invade the underlying cells v ia these receptors, resulting in illness or even the death of the host organism.
- receptors specific sites of the mucosa
- v ia these receptors
- the mucosa form the porte d' entree of numerous pathogenic bacteria, for example of Gram negative bacteria of the genera Escherichia, Campylobacter, Haemophi lus, Shige l la, Vibrio, Pasteure l la, Yevsinia, Sa lmone l la, Gram positive bacteria like Mycobacterium, Listeria, C lostridium, Staphylococcus and viruses like rotavirus, poliovirus, measles and many other microorganisms well known to a person skilled in the art of microbial infections.
- Gram negative bacteria of the genera Escherichia, Campylobacter, Haemophi lus, Shige l la, Vibrio, Pasteure l la, Yevsinia, Sa lmone l la, Gram positive bacteria like Mycobacterium, Listeria, C lostridium, Staphylococcus and viruses like rotavirus, poliovirus, measles and many other microorganisms
- Campylobacter Bacteria of the genus Campylobacter for example can cause severe enteritis in humans and animals after oral ingestion.
- C. Jejuni is a major cause of diarrhoea in humans and occasionally in animals. Beside diarrhoea, C . jejuni can occasionally also cause appendicitis, meningitis, abortion and urinary tract infection in humans.
- Campylobacter infections are as common as infections caused by Sa lmone l la , Shige l la or Vibrio cho lerae .
- Mycobacteria such as Mycobacterium tuberculosis and Mycobacterium leprae also cause serious diseases such as tuberculosis and lepra respectively. These bacteria cause the death of many individuals in particular in the less well developed countries. These microorganisms invade the body via the mucosa of the respiratory tract.
- Pathogenic microorganisms can adhere to parts of the body e.g. the gastro-intestinal tract, thereby initiating a disease.
- the studies of the adhesion of pathogenic microorganisms to parts of the body of a host organism have resulted in a wealth of data. From these studies it has become clear that adhesion of pathogenic bacteria can be mediated by proteins. Detailed information is available about proteins from pathogenic bacteria that bind to components of the extra cellular matrix, e.g. collagens, fibronectin or proteoglycans.
- mycobacterial fibronectin-binding proteins the mycobacterial fibronectin-binding proteins, the fibronectin- and collagen-binding proteins of Streptococci and Staphylococci, specific enterobacterial fimbrial types, and surface proteins of Yersinias and the A-protein of Aeromonas (for a review, see Westerlund and Korhonen, Mol. Microbiol. 9:687-6.4 1993).
- Information about the adhesion of Gram-positive, non pathogenic bacteria to surfaces of a host organism is more limited, in particular information regarding specific binding of mucosal receptors by non pathogenic microorganisms is scarce.
- Adhesion of non-pathogenic bacteria may be specific or aspecific. Hydrophobic and electrostatic adhesion mechanisms are involved in non-specific adhesion. Specific adhesion is characterized by a so-called “lock-and key mechanism", in which the adherence factor binds to a specific receptor. Specific adhesion is usually associated with the adhesion of microorganisms to receptors on living tissues. Adherence factors or adhesins are, in general, surface bound molecules. The adhesin can be firmly attached to the surface of the bacterium or loosely bound.
- the receptor is a component or structure on the surface of the cell where the bacterium will bind by an active site of the adhesin (Rutter et al, 1984 Mechanisms of adhesion in "Microbial adhesion and aggregation" Marshall, K.C. ed. pp 5-19. Springer-Verlag, Berlin).
- Lactic acid bacteria particularly Lactobac i l lus and Enterococcus
- Lactobacillus species have been isolated from various regions of the human gastro-intestinal tract (Molin et al, J. Appl. Bacteriol. 74, 314-323 1993).
- LTA lipotheichoic acids
- EP 0 210 579 (with a priority date of November 1984) a preparation is described containing a protein of a Mw of 14 kD claimed to be the responsible compound for the enhancement of bacterial adhesion to squameous epithelium in mice and pigs.
- the preparation containing the 14 kD protein was obtained by cultivating Lactobacillus fermentum in a medium rich in sugars and amino acids. From EP 0 210 579 it is not clear whether the adhesion promoting factor is specific for non-pathogenic bacteria or also may enhance the adhesion of pathogens that normally do not adhere.
- EP 0 210 579 It is also not clear from EP 0 210 579 whether or not the adhesion promoting factor enhances adhesion to specific sites (receptors) or to a-specific sites. Moreover, EP 0 210 579 does not make clear what the origin of the 14 kD protein is. It remains uncertain whether the 14 kD protein is synthesized by L. fermentum as such or is generated from medium components by an activity of L. fermentum. Thus both identity and applicability of the 14 kD protein remain obscure in the publication.
- W0 90/09398 of Conway and Kjelleberg for example describes a fraction derived from L. crispatis 104 of over 30 kD exhibiting anti-pathogenic activity.
- the fraction of over 30 kD maintains it's activity after treatment with pronase or trypsin. It is obtained by growth of L. crispatis in complex medium.
- the application also mentions that the corresponding fraction of 8000-30.000 did not exhibit anti-pathogenic activity.
- Lactobac i l lus fermentum KLD inhibited growth of E. co l i strains, Campi lobacter jejeun i , Sa lmone l la sofia and Streptococcus faec ium in vitro.
- Lactobac i l lus fermentum strain to mouse squamous epithelium said protein being present in a retentate fraction of culture fluid with a MW higher than 250.000 is postulated.
- the publication is silent on the nature of the protein and explicitly states it had not been isolated and that the efficacy had to be verified by further experiments.
- Probiotics are defined as "mono or mixed cultures of living organisms which, applied as dried cells or as a fermented product to humans or animals, beneficially affect the host by improving the properties of the indigenous microflora.”
- Some strains of Lactobac i l lus and B ifidobacter ium strains reportedly, have probiotic properties. The beneficial effects have been attributed to the lowering of the pH, a condition which reduces the proliferation of Gram-negative pathogens like Escherichia co l i .
- many species of lactic acid bacteria produce oligopeptides with antimicrobial properties, called bacteriocines. These compounds are bacteriostatic or bacteriocidal for Gram-positive bacterial pathogens, like Clostridium, Listeria etc.
- Lactobacilli have been suggested as inhibiting adhesion of pathogens in animals and in in vi tro models. These inhibitory effects are usually explained by non-specific steric hindrance of the receptors for pathogens. In contrast, each pathogen has a specific intestinal receptor (Falkow et al. Ann. Rev. Cell. Biol. 8: 333-363 1992).
- Lactobacilli or preparations made with Lactobacilli are thus widely used to treat intestinal and urinary tract disorders (see e.g. WO 9 516 461; RU 2 000 116; W0 9 418 997; EP 0 577 903; GB 2 261 372; W0 9 301 823; wo 0 921 475; US 7 822 505; CA 1 298 556; EP 0 199 535; EP 0 210 579).
- the beneficial effects of such preparations have been attributed to various factors, but the properties and mode of action of such health stimulating compounds have either not been disclosed or are at most mere postulations. Answers regarding the mechanism of probiotics are crucial in order to find novel enhanced probiotics and optimise their use.
- the objective of the invention is to overcome the above mentioned difficulties. It is now proven unequivocally that a 29 kD proteinaceous compound is responsible for specific adhesion of L . fermentum to receptor sites in the mucus of pigs and mice, and thus methods to screen for other microorganisms that synthesize proteins with a similar structure and function are now provided. By demonstrating that the adherence promoting entity of L .
- fermentum is a protein of 29 kD which is structurally related to adherence factors of certain pathogenic bacteria and by demonstrating the nucleotide sequence of the gene encoding the adherence factor
- the present invention provides methods for the rapid screening of microorganisms that contain a gene coding for an adhesin of the novel type and methods for screening of microorganisms that produce such an adherence factor using standard protein and nucleic acid technologies.
- the present invention provides methods to selectively and specifically interfere with the adhesion of pathogens to receptors on the mucosa of the gastro-intestinal tract, of the urogenital tract, of the oral cavity, of the respiratory tract and of the nasal cavity and to screen microorganisms for the capacity to interfere with adhesion of the aforementioned type of pathogens.
- the present invention allows rapidly screening bacteria for the capacity to interfere with the adherence of pathogens to mucosal receptors.
- the present invention provides a method to screen microorganisms for the presence of an adherence factor that enhances the specific adhesion of non-pathogenic Gram positive bacteria, more in particular the adhesion of lactobacilli, to bacterial receptor(s) of the mucosa of the gastro-intestinal tract, the urogenital tract, the respiratory tract and the oral/nasal cavity of humans and animals.
- the microorganisms to be screened will be microorganisms that are non pathogenic in humans and animals. Such microorganisms will preferably be indigenous to humans and/or animals, thus already being able to withstand the environment in which they are to be applied and also obviously not being toxic to the particular species from which they are derived. Examples of suitable non pathogenic microorganisms include bacteria of the genera Lactobac i l lus , Streptococcus, Enterococcus, Bifidobacterium, Clostridium and Bacteroides .
- the screening can occur at protein and/or nucleic acid level using standard technologies known per se for protein detection or nucleic acid detection such as nucleic acid amplification and hybridisation techniques and protein or peptide assays using polypeptide or protein probes and/or antibodies specific for the adherence factor or factors to be detected.
- the present invention thus also provides a method to screen microorganisms for the presence of nucleic acid that encodes proteins with the desired adherence properties. Protein and nucleic acid assays are readily carried out by persons skilled in the art once the relevant amino acid and nucleic acid sequences have been determined as in the instant case.
- probes and primers to be constructed when the nucleic acid sequence and/or the relevant amino acid sequence of the protein has been determined and isolated or synthesized as in the instant case.
- the isolation of the pure protein and/or expression of the pure protein enables production of antibodies in a manner known per se
- bacteria can be screened for the presence of a protein falling within the definition of the novel type of adherence factors of non pathogenic microorganisms as detailed below or the presence of a DNA sequence encoding such a protein or active part thereof.
- Application of the invention thus in particular circumvents the laborious and costly route of screening bacteria for the capacity to adhere to living tissue More in particular, application of the invention circumvents the use of animals and/or human volunteers for screening purposes.
- a method of screening non pathogenic microorganisms for a microorganism capable of specifically binding mucosa comprising detection in a manner known per se of the presence of a particular protein on or in a microorganism on or in a culture of microorganisms, said particular protein being a protein according to any of claims 1-13 falls within the scope of protection.
- a method of screening non pathogenic microorganisms for a microorganism capable of specifically binding mucosa said method comprising detection in a manner known per se of the presence of a particular gene on or in a microorganism on or in a culture of microorganisms, said particular gene encoding a protein according to any of claims 1-13 also falls within the scope of the invention.
- the invention also covers a kit suitable for detection of a non pathogenic microorganism capable of specifically binding mucosa, said kit comprising a component capable of specifically binding to a protein according to any of claims 1-13 such as an antibody.
- the invention comprises a kit suitable for detection of a non pathogenic microorganism capable of specifically binding mucosa, said kit comprising a component capable of specifically binding to a part of a nucleic acid sequence encoding a protein according to any of claims 1-13 such as a nucleic acid probe or primer.
- the invention offers a method to efficiently and specifically interfere with the adhesion of certain classes of pathogens to bacterial receptors of the mucosa and to screen for microorganisms capable of interfering with adhesion of certain classes of pathogens.
- Pathogens that may now be combatted comprise both Gram positive and Gram negative microorganisms in particular those that specifically bind mucosa receptors.
- pathogens to be combatted comprise strains of the genera Escherichia, Campy lobacter, Haemophi lus, Shige lla, Vibrio, Pasteure l la, Yers inia, Salmone lla, Mycobacterium, L isteria, C lostridium, Staphy lococcus and viruses like rotavirus, poliovirus and measles.
- the invention exploits the conclusion that the infectivity of pathogens that adhere to a mucosal receptor through an adherence factor similar to that of the adhesion protein of L . fermentum 104R, will be reduced by probiotic bacteria harbouring an adhesion protein with a structure like that of the adhesion protein of L . fermentum 104R, by specific interaction with the receptor, rather than by the more general mechanism of steric hindrance.
- a strategy can be devised to specifically inhibit adherence of certain pathogens (those that adhere by means of an adherence factor that is stucturally related to the adhesion protein of L . fermentum ) , by administering e.g. in food or feed or as pharmaceutical composition such adhesion proteins or, microorganisms that produce such adhesion proteins.
- the application can be topical, oral or intravenous in any dosage form normally applied for pharmaceutical compositions and/or feed additives.
- the dosage form selected will depend on the type of infectious pathogen to be combatted.
- the dosage form may be solid or liquid. Certain standards with regard to purity and hygiene i.e. sterility normally applicable for such compositions must be adhered to. Such circumstances are well known to a person skilled in the art.
- composition comprising a component selected from the group of components comprising
- a composition comprising the abovementioned components in a form suitable for use as food additive is also envisaged to fall within the scope of the invention.
- a non pathogenic microorganism capable of expressing a protein or peptide according to any of claims 1-21 or a part of said microorganism, said part expressing mucosa binding promoting activity as pharmaceutically active component in a pharmaceutical composition for prophylaxis and/or treatment of disease or illness associated with a mucosa colonising pathogenic microorganism also falls within the scope of the invention.
- a method for improving food products comprising addition of a product according to any of claims 1-21 or 24-27 and/or a non pathogenic microorganism capable of expressing a protein or peptide according to any of claims 1-21 or a part of said microorganism, said part expressing mucosa binding promoting activity to the food product forms an embodiment of the invention.
- a method comprises addition of a product according to any of claims 1-21 or 24-27 to the food product.
- a food product comprising a product according to any of claims 1-21 or 24-27 and/or a non pathogenic microorganism capable of expressing a protein or peptide according to any of claims 1-21 or a part of said microorganism, said part expressing mucosa binding promoting activity as additive is also covered.
- a food product comprising a product according to any of claims 1-21 or 24-27 as additive is a particularly suitable embodiment.
- the inhibiting effect may also be obtained by addition of parts of the adherence protein, e.g. peptides derived from the 29 kD adherence protein of L. fermentum that are found to specifically bind to mucus and mucin.
- the active peptides can either be synthesized chemically or made micro-biologically by a genetically engineered microorganism.
- the protein can be produced by a non recombinant or recombinant microorganism and subsequently e.g. via proteolytic digestion and optionally separation of the proteolytic fragments the desired polypeptide can be obtained.
- the 29 kD protein according to the invention comprises three such sequences. They are more or less evenly distributed over the protein molecule at positions 47-52 (KKMGLK), 173-178 (KKNSTK) and 223-238
- KKLSEK of the mature protein.
- the numbering corresponds to amino acids 54-59. I80-185 and 230-235 of sequence id no. 2. of the sequence listing, in which the mature protein commences with Ala at position 8.
- the presence of at least one of the KKXXXK sequences, preferably two of these sequences in a protein or peptide according to the invention is preferred. Most desirably three such sequences are present.
- the consensus sequence will be one of the natively occurring amino acid sequences present in the 29 kD protein disclosed above.
- sequences corresponding to those present in their native environment will be used, such sequences can however be arrived at through genetic engineering or synthetic means generally known in the art such as through DNA synthesizers, Merrifield synthesis and cloning technology as mentioned above.
- sequences will also be present in a sequence such that the tertiary structure mimics that of the native protein. This can be ascertained using computer technology in a manner known per se. Such sequences are involved in binding to negatively charged intestinal receptors.
- Microorganisms that have the GRAS status like Aspergi l lus , Lactobac i l lus and Lactococcus are well suited for such purposes. A person skilled in the art will realise that other microorganisms can also be used for production of adherence factors or peptides derived thereof. However it will be preferred for applications to humans to employ GRAS organisms. Lists of GRAS organisms are readily available to a person skilled in the field of foodstuffs and/or pharmaceuticals and are incorporated herein by reference. The US FDA for example maintains a list of such organisms.
- fermentum 104R might be that adherence of such bacteria to a specific receptor will also limit the adherence of pathogens with adherence factors other than the L . fermentum-like adhesion factor, by a general mechanism of steric hindrance.
- the pathogenic microorganisms that can be combatted do not only comprise microorganisms that bind the mucosal receptor specifically bound by the adherence factor from the non pathogenic organism.
- the microorganism will be a GRAS organism such as a lactic acid bacterium. It is also possible to incorporate the encoding sequences or sequences such that they are operably linked to regulating sequences that enable higher expression than with the regulating sequence normally associated with the encoding sequence.
- GRAS microorganisms such as lactic acid bacteria.
- Recombinant microorganisms capable of expressing or overexpressing the polypeptide or protein capable of promoting the binding of mucosa of the novel group of adherence factors from non pathogenic microorganisms or recombinant expression vectors comprising the appropriate nucleic acid also fall within the scope of the invention.
- the microorganism that is genetically engineered may already express the adherence factor but the microorganism may also be selected from a group that does not natively express an adherence protein of the novel group.
- the microorganism may simply be used as production plant for the protein or polypeptide which may subsequently be isolated and applied as pharmaceutical or as food/feed additive, or the microorganism itself may be used as pharmaceutical or as food/feed additive.
- the protein or polypeptide producing microorganism will be non pathogenic.
- GRAS microorganisms are preferred in order to enable applications of the expression product and/or microorganisms as active component of a pharmaceutical composition or food/feed additive.
- the nucleic acid sequences may be incorporated onto a plasmid vector or integrated into the chromosome in any embodiment known per se in the recombinant DNA technology field.
- a large number of transformation and expression vectors and technologies are known in the state of the art and are currently also commercially available. Preferred are food-grade transformation and expression vectors and methods of transformation suitable for GRAS microorganisms.
- microorganisms to be selected and/or transformed have the following characteristics:
- a recombinant microorganism comprising a nucleic acid sequence according to claim 23 and/or an expression vector according to claim 24 or 25, said nucleic acid sequence and/or expression vector being absent or in the alternative being present in a lower copy number or being expressed to a lower degree in the corresponding non recombinant microorganism.
- the invention comprises a recombinant microorganism as just defined (according to claim 26), said microorganism being a non pathogenic microorganism, preferably indigenous to the microflora of a human or animal, more preferably to the microflora of a human.
- the invention also encompasses a nucleic acid sequence encoding any of the proteins or peptides according to any of claims 1-21 and an expression vector comprising such a nucleic acid sequence, operably linked to an expression regulating sequence, said expression vector being capable of expressing the nucleic acid in a non pathogenic microorganism such as a GRAS microorganism and said expression vector preferably comprising nucleic acid derived from a GRAS microorganism.
- the expression vector according to the invention is a vector, wherein the expression regulating sequences are not naturally associated with the gene encoding the adherence factor from which the nucleic acid sequence is derived.
- amino acid sequence and nucleic acid sequence of an adherence protein have now been ascertained and the similarity between other protein groups has been determined it lies within reach of a person skilled in the art to design a protein or polypeptide exhibiting improved binding characteristics and thus improved results in pharmaceutical applications or as food/feed additive.
- the invention thus also covers mutant polypeptides and proteins exhibiting better mucosa binding than the protein with amino acid sequences of figure 3 and better mucosa binding activity than any of polypeptides I-V as defined in the experimental part of the subject description.
- the invention also comprises equivalent sequences as available in nature and as mutants i.e.
- the microorganism may be selected for already having this particular characteristic or may be genetically engineered so that it subsequently produces the desired drug, immunomodulator or antigen.
- the invention covers such novel microorganisms and molecules and applications thereof as pharmaceutical compositions.
- the invention thus also covers a method for targeting a bacterium that expresses a gene of interest, for example a gene encoding an antigen of a pathogenic organism, to specific receptors of the mucosa, thereby evoking a specific immune response against the antigen and/or modulating an immune reponse.
- the invention covers a fusion protein or peptide comprising a protein or peptide according to any of the claims 1-21 attached to a drug, immunomodulator or antigen of choice.
- non pathogenic microorganism capable of expressing a protein or peptide according to any of claims 1-21 or a part of said microorganism, said part expressing mucosa binding promoting activity
- targeting component in a pharmaceutical composition for targeting an additional pharmaceutically active component to mucosa, said additional pharmaceutical component being physically linked to the targeting component falls within the scope of the invention.
- lactobacilli to receptors of the mucosa, according to the invention, providing the opportunity to specifically target bacteria carrying compounds of interest, for example lactobacilli expressing an antigen of a pathogenic organism or a human protein, to the cells of the mucosa, thereby modulating the immune response against the antigen/human protein is a preferred embodiment of the invention.
- the adhesion capacity of probiotic strains may be modulated by altering the properties of the adhesion protein.
- properties may involve interaction of the adhesion protein with the mucosal receptor or interaction with other (accessory) proteins.
- the novel protein has adhesion promoting activities.
- the adhesion promoting activity comprises exhibiting binding to mucosa or mucin.
- the adhesion protein is present on the surface and is also shed off into the culture medium by L. fermentum 104R.
- the invention more in particular exploits a special property of the adhesion promoting protein, namely that it is structurally similar to virulence proteins of several pathogenic bacteria, e.g. to adherence factors from Campy lobacter jejuni , Pasteure l la haemo lytica and Mycobacterium .
- adhesion promoting protein namely that it is structurally similar to virulence proteins of several pathogenic bacteria, e.g. to adherence factors from Campy lobacter jejuni , Pasteure l la haemo lytica and Mycobacterium .
- the adhesion promoting protein from L . fermentum 104R belongs to a class of proteins, called Class III solute transporters, of which the histidine transporter (HisJ), glutamine transporter (GlnH) and the lysine, arginine and ornithine transporter (LAO) of Enterobacter iaceae are the prototypes.
- HisJ histidine transporter
- GlnH glutamine transporter
- LAO arginine and ornithine transporter
- the amino acid sequence of the adhesion promoting protein of L. fermentum 104R shows a striking similarity with on the one hand adherence proteins of pathogens, Peb1 of C. jejun i and LapT of P.
- a protein belonging to the group of novel proteins as defined according to the invention is defined as a protein obtainable from a non pathogenic microorganism, said protein having mucosa binding promoting activity and a molecular weight of 20-40 kD. Preferably the weight lies between 20-30 kD.
- a protein according to the invention comprises one or more of the following properties:
- d has a 3D structure with 2 lobes like LAO or HisJ
- e comprises one or more amino acid sequences that are 90% or more similar to the following amino acid sequences
- IAGTGTNNA preferably of the amino acid sequences II-V.
- a specific embodiment is formed by the group of proteins further characterised in that the protein exhibits the consensus sequence illustrated in figures 4 and 5.
- the proteins claimed as such do not comprise virulence factors of pathogenic microorganisms or Class III transporters, neither does the class of recombinant proteins comprise recombinant virulence factors or recombinant Class III reporters that could perhaps form state of the art at the filing date of the subject patent application.
- a protein belonging to the group of proteins suitable for application according to the invention will exhibit binding promoting activity for mucosal receptors used by any of C jejuni , P . haemo lyt ica or Mycobacterium higher or equal to that exhibited by the 29 kD protein of L. fermentum 104 with the amino acid sequence of figure 3 as can be determined by the mucosa binding assay illustrated in the Example.
- nucleotide structure of the adhesion promoting protein is known, non pathogenic microorganisms can also be screened for the presence of DNA sequences encoding proteins with a structure similar to that of the adhesion protein of L . fermentum 104R.
- a nucleic acid sequence is a nucleic acid sequence encoding the amino acid sequence of figure 2 corresponding to that of the 29 kD protein of L. fermentum 104R.
- a nucleic acid sequence encoding the consensus amino acid sequence of the figures 4 and 5 as such also falls within the scope of the invention.
- nucleic acid sequence encoding a protein of 20-40 kD comprising the amino acid consensus sequence and further corresponding to the sequence of the 29 kD sequence, the only difference being in the presence of one or more mutations resulting in substitution of amino acids by other similar amino acids such that the hydropathy profiles remain similar and no serious conformation change can be expected of the resulting protein or polypeptide falls within the scope of the invention.
- sequences are known as those wherein conservative exchange of amino acids has occurred in comparison to the sequence according to sequence id. no 2.
- the invention comprises any nucleic acid sequence capable of hybridising under stringent hybridisation conditions when carrying out a blot assay in a manner known per se.
- sequences thus comprise sequences encoded by nucleic acid sequences derivable from other non pathogenic microorganisms through cross hybridisation technology using oligonucleotide probes encoding parts of the amino acid sequence according to sequence id no 2, preferably probes in which the preferred codon usage of the microorganism to be screened has been taken into account in a manner known per se.
- Stringent hybridisation conditions as described for example in Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory New York Maniatis, T. Fritsch, E.F. and Sambrook, J. (1982) can be suitably applied to obtain such equivalent sequences.
- the cited reference also provides information regarding a number of other standard technologies mentioned elsewhere in the description and is incorporated herein by reference.
- sequences from non pathogenic microorganisms belonging to the genera mentioned previously in the description are preferred.
- at least one consensus sequence according to sequence id no 30 will be present.
- one of the sequences of polypeptides I-V will be present.
- a protein or polypeptide with the amino acid sequence of the mature protein of sequence id no 2 in which amino acids have been mutated, outside the consensus sequences, having at least the mucosa binding activity of the mature protein of sequence id no 2 is also comprised within the invention.
- any sequence combining any of the above definitions is also included within the scope of the invention and forms a preferred embodiment.
- an equivalent sequence is not derivable as such from a microorganism but can be produced in an alternative manner e.g. recombinant DNA technology, PCR etc.
- the above embodiments are also valid for such alternative (mutant) sequences and fall within the scope of the invention.
- a protein or polypeptide according to the invention will be free of cell extract and other contaminating proteins.
- Substantial purity is preferable i.e. more than 80% pure. The purity being sufficient for application as pharmaceutical and food additive and for achieving the activity required in the applications according to the invention.
- bacteria may also be screened for the presence of proteins like the L . fermentum 104R adhesion protein, that can adhere to non-living surfaces like plastics or metal surfaces, such screening can occur as described above using oligo probes based on the amino acid sequence of the 29 kD adhesion protein.
- a probe will encode a part of a consensus sequence of the figures 4 and 5.
- a suitable probe will comprise a sequence encoding the consensus sequence of sequence id no 30.
- the consensus sequence or the part thereof will be at least 5 contiguous amino acids long and preferably the probe will be comprised completely of consensus sequence.
- the adhesion promoting protein of L. fermentum 104R IS produced by cultivating bacteria in MRS broth or LDM medium (Conway and Kjelleberg, J. Gen. Microbiol. 135:1175-1186 1989) for l4 to 24 hours.
- the 29 kD adhesion protein is purified from the medium to apparent homogeneity by ammonium sulphate precipitation, gel-filtration and affinity chromatography.
- the adhesion promoting activity is detected in the fractions by adhesion inhibition and dot blot assays, and visualized by PAGE, SDS-PAGE and western blots using horse radish peroxidase labelled mucus or mucin.
- the purified protein has an estimated Mw of 29 kD, under non-denaturing conditions as well as under reducing and denaturing conditions (non gradient denaturing SDS-PAGE, using a calibration curve obtained with standard proteins, and gel-filtration chromatography, relative to the standard curve) and is sensitive to pronase, and therefore, differs from the adhesion proteins described and/or implied in EP 0 210 579 and WO 90/09398, as well as those described by Conway and Kjellenberg (J. Gen. Microbiol. 135. 1175-1186), Blomberg et al (Appl. Environm. Microbiol. 59, 34-39 1993) and Aleljung et al (Current Microbiology vol 28 (1994) p.
- the adhesion promoting protein could be extracted from the cell surface of L fermentum by treatment of the bacteria with 1 M LiCl and low concentrations of lysozyme.
- the adhesion promoting protein which had an affinity for both small intestine mucus and gastric mucin from pigs or mice, was released into the culture supernatant fluid after 24h of growth . ii) Screening of microorganisms for the presence of a L . fermentum-like adhesion protein
- lactobacilli are screened for the presence of an adhesion promoting protein with properties similar to those of the adhesion promoting protein from L . fermentum, by separating proteins from the culture medium of an overnight culture by SDS-PAGE, and Western blotting using polyclonal antibodies raised in rabbits against purified adhesion protein of L. fermentum 104R. iii) Screening of microorganisms for the presence of a L . fermentum-like adhesion protein encoding gene
- DNA is isolated from microorganisms to be screened and subjected to PCR analysis, using sets of primers that are based on the nucleotide sequences of the L. fermentum 104R adhesion protein encoding gene.
- the products formed are analysed by standard molecular biological techniques as are described in handbooks (e.g. as cited elsewhere in this description) or commercially available kits.
- the gene encoding the adhesion protein from L . fermentum 104R or from another selected strain, isolated by the aforementioned procedure is cloned behind a strong, preferably inducible promoter and secretion signal encoding sequence, in a GRAS production organism like Aspergi l lus niger , Lactobac i l lus etc.
- the culture medium is either used as such and used as food/feed additive or pharmaceutical composition, or the adhesion promoting protein is first purified (by standard techniques) and then added to food/ feed preparations or pharmaceutical compositions.
- the nucleic acid sequence may be adjusted such that it encodes the identical amino acid sequence of the 29 kD L .
- peptides derived from the L . fermentum 104R adhesion protein that show adhesion promoting properties are synthesized chemically and used as food/feed additive.
- DNA sequences, coding for such peptides are cloned behind a strong, preferably inducible promoter in a GRAS production organism like A . n iger or Lactobac i l lus etc.
- the medium can be used as food/feed additive.
- the entire organisms, or extracts made from such organisms can be used as food/feed additive.
- the desired proteins or polypeptides may be isolated e.g. using chromotagraphy in a manner known per se for isolating protein or polypeptide e.g. in combination with antibodies specific for the protein or polypeptide to be isolated.
- An antibody or antibody fragment capable of binding an epitope or protein or peptide according to any of claims 1-20 falls within the scope of the invention.
- Such an antibody may be a polyclonal antibody (see Example) or a monoclonal antibody.
- An antibody specifically disclosed in any of the above cited references is excluded from the scope of protection for antibody claims as such. vi) Targeting of an antigen or human protein to mucosa
- adhesion protein in another embodiment, is exploited to target an antigen of a pathogen to the mucosa to enhance a mucosal immune response against the antigen.
- microorganisms are constructed that are capable of synthesizing the adhesion protein and the antigen of interest.
- microorganisms carrying a gene encoding a human protein are genetically engineered in such a way that they synthesize an adhesion protein with properties similar to those of the L. fermentum adhesion protein.
- Spent culture fluids from 14 or 24 hour cultures were collected by centrifuging at 6000g for 20 min and dialysing at 4 ⁇ C against ultra pure water. The retenate was concentrated by ultra filtration through a 14 KDa molecular weight cut off membrane. The high molecular weight fraction was freeze dried and stored at 4 ⁇ C. Spent culture fluid was also concentrated 10 times by hollow fibre ultrafilter and ammonium sulphate was dissolved in the concentrate (40, 60 and 100% of saturation at 4 ⁇ C). The precipitates were collected by centrifugation (l8000xg/30 min), dissolved in ultra pure water and dialysed against 0.01M ammonium bicarbonate. The solutions were freeze dried and kept at 4 ⁇ C.
- the freeze dried preparation from 24 hours spent culture fluid concentrated by ultra filtration was dissolved in HEPES-Hanks and filtered (0.22 ⁇ m) to remove insoluble particles.
- a 4 ml aliquot of the solution (2.1 mg of protein) was applied to a Sephadex G 200 in XK-26 column (Pharmacia-LKB, Uppsala Sweden) for gel filtration chromatography.
- HEPES-Hanks buffer was used to equilibrate the column and elute the sample.
- the fractions in each 280 nm-absorbing peak were assayed for the capacity to bind HRP-mucin and HRP-crude mucus by dot blot assay and in the inhibition of lactobacilli binding to crude mucus in microtiter plates adhesion inhibition assay.
- the active fractions in each 280 nm absorbing peak were pooled, dialysed and freeze dried for SDS-PAGE and western blot analysis.
- Mucin was covalently coupled to Activated CH-sepharose 4B according to the instructions of the manufacturer (Pharmacia-LKB. Biotechnology).
- a column C10/40 (30 ml bed volume) was packed with this adsorbent and equilibrated with HEPES-Hanks.
- L fermentum spent culture fluid, cell extracts, or active fractions from Gel filtration chromatography were loaded throw the column.
- Column was washed with two bed volumes of equilibrating buffer, then successively washed with different solutions (0.1 M glycine pH 3. 0.1M tris pH 8 and 0-2 M gradient of sodium chloride) at flow rates of 6 ml h -1 .
- the adhesion promoting activity was detected in the fractions by adhesion inhibition and dot blot assays, and was visualized by PAGE, SDS- PAGE and western blots using horse radish peroxidase labelled mucus or mucin.
- the adhesion promoting protein could be extracted from the cell surface of L . fermentum by treatment of the bacteria with 1 M LiCl and low concentrations of lysozyme.
- the adhesion promoting protein which had an affinity for both small intestine mucus and gastric mucin, was released into the culture supernatant fluid after 24h of growth.
- the active fraction was characterized by assessing the presence of carbohydrates in (periodic-acid Schiff stain procedure, SIGMA, and DIG glycan detection kit, Boehringer Mannheim, Germany) and the heat sensitivity of the active region of the adhesion promoting protein.
- the adhesion promoting activity lacked carbohydrates and remained completely biologially active, when LiCl cell extracts from L. fermentum were heated for 5 min at 100 ⁇ C and tested by dot blot adhesion assay.
- the purified protein has an estimated Mw of 29 kD, under non-denaturing conditions as well as under reducing and denaturing conditions (SDS-PAGE, using a calibration curve obtained with standard proteins, and gel-filtration chromatography, relative to the standard curve; Figure 1).
- the adhesion promoting protein was further characterized by determination of the N-terminal amino acid sequence, showing the following sequence:
- the adhesion promoting protein gene was cloned from a genomic bank of L . fermentum 104R.
- oligonucleotide primers were synthesized, based on the aminoacid sequence data of the sequenced peptides of the adhesion promoting protein. These oligonucleotides were used in various combinations in PCR reaction. Oligonucleotides 42 (sense; 5'-CTI.GCI.GTI.AAC/T.TCI.GAG/A.TTG/A.GT-3') and 105 (antisense;
- FIG. 1 SDS-PAGE and Western blot of the adhesion promoting protein (APP) using HRP labelled mucus for blotting.
- Figure 2 Nucleotide sequence of the adhesion promoting protein of L. fermentum 104R. The open reading frame starts at nucleotide 1 and ends at nucleotide 734.
- Figure 3 Amino acid sequence of the adhesion promoting protein of L. fermentum 104R.
- FIG. 4 Comparison of the amino acid sequences of the adhesion promoting protein of L. fermentum 104R, Peb1 from C. jejuni and LapT from P. haemolytica. A consensus sequence is given below the sequences. Bold letters indicate identical aminoacids or conserved substitutions.
- FIG. 5 Comparison of the aminoacid sequences of the adhesion promoting protein of L. fermentum 104R and Class III solute transport proteins (Atunop, nopaline of Agrobacter tumefaciens; Atuoct, octopine Agrobacter tumefaciens; GlnH, glutamine binding protein of E. coli; HisJ, histidine binding protein, LAO, lysine, arginine, ornithine binding protein of Salmonella thyphimurium. A consensus sequence is given below the sequences. Aminoacids in adhesion promoting protein that also occur in other proteins are indicated in bold capital letters; colons indicate a conserved substitution and asterics a less conserved substitution.
- FIG. 6 Comparison of the aminoacid sequences of the adhesion promoting protein of L. fermentum 104R and proteins of the 85K complex of Mycobacterium. A consensus sequence is given below the sequences. Aminoacids that are identical in adhesin and in one or more Mycobacterium proteins are indicated in bold capital letters. conserveed substitutions are indicated with a colon, and less conserced substitutions with an asterisc.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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JP9515715A JP2000505642A (en) | 1995-10-20 | 1996-10-21 | Novel adhesion factors of non-pathogenic microorganisms and their application for screening microorganisms for specific symbiotic properties; novel pharmaceutical compositions and food additives containing such microorganisms and adhesion factors |
AU72294/96A AU727676B2 (en) | 1995-10-20 | 1996-10-21 | Novel adherence factors of non pathogenic microorganisms and applications thereof for screening microorganisms for specific probiotic properties; novel pharmaceutical compositions and food additives comprising such microorganisms and adherence factors |
EP96933659A EP0857213A1 (en) | 1995-10-20 | 1996-10-21 | Novel adherence factors of non pathogenic microorganisms and applications thereof for screening miroorganisms for specific probiotic properties; novel pharmaceutical compositions and food additives comprising such microorganisms and adherence factors |
US09/051,755 US6506389B2 (en) | 1995-10-20 | 1996-10-21 | Adherence factors of non pathogenic microorganisms and applications thereof for screening microorganisms for specific probiotic properties; novel pharmaceutical compositions and food additives comprising such microorganisms and adherence factors |
US10/231,055 US20030095978A1 (en) | 1995-10-20 | 2002-08-30 | Novel adherence factors of non pathogenic microorganisms and applications thereof for screening microorganisms for specific probiotic properties; novel pharmaceutical compositions and food additives comprising such microorganisms and adherence factors |
Applications Claiming Priority (2)
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NLPCT/NL95/00367 | 1995-10-20 |
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US09/051,755 A-371-Of-International US6506389B2 (en) | 1995-10-20 | 1996-10-21 | Adherence factors of non pathogenic microorganisms and applications thereof for screening microorganisms for specific probiotic properties; novel pharmaceutical compositions and food additives comprising such microorganisms and adherence factors |
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EP (1) | EP0857213A1 (en) |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2778187A1 (en) * | 1998-04-30 | 1999-11-05 | Sanofi Elf | METHOD FOR SELECTING BACTERIAL STRAINS |
WO2000071139A2 (en) * | 1999-05-25 | 2000-11-30 | Gregor Reid | Lactobacillus compositions and methods for preventing wound infections and biofilm formation on implantable surgical devices |
US6682744B1 (en) * | 1999-08-09 | 2004-01-27 | University Of Maryland | Pro-gut maturation and anti-inflammatory effects of lactobacillus and lactobacillus secreted proteins, carbohydrates and lipids |
US7011826B1 (en) * | 1999-07-02 | 2006-03-14 | The University Of New England | Control of acidosis |
US7060687B2 (en) | 2001-02-07 | 2006-06-13 | Genmont Biotechnology Co. | Live vaccines for allergy treatment |
US9631185B2 (en) | 2011-06-09 | 2017-04-25 | Novozymes A/S | Fusion of bioactive molecules |
WO2018015380A1 (en) * | 2016-07-18 | 2018-01-25 | Philippe Ulsemer | Natural microorganisms which are naturally capable of binding toxins and/or toxin receptors |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990009398A1 (en) * | 1989-02-17 | 1990-08-23 | Bioinvent International Ab | Products for inhibiting the adhesion, growth and/or survival of pathogens |
-
1996
- 1996-10-21 WO PCT/NL1996/000409 patent/WO1997014802A1/en not_active Application Discontinuation
- 1996-10-21 JP JP9515715A patent/JP2000505642A/en not_active Ceased
- 1996-10-21 EP EP96933659A patent/EP0857213A1/en not_active Ceased
- 1996-10-21 AU AU72294/96A patent/AU727676B2/en not_active Ceased
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990009398A1 (en) * | 1989-02-17 | 1990-08-23 | Bioinvent International Ab | Products for inhibiting the adhesion, growth and/or survival of pathogens |
Non-Patent Citations (8)
Title |
---|
ALELJUNG P ET AL: "Purification of collagen-binding proteins of Lactobacillus reuteri NCIB 11951", CURRENT MICROBIOLOGY, 28 (4). 1994. 231-236., XP000573194 * |
GREENE JD ET AL: "Factors involved in adherence of lactobacilli to human Caco-2 cells.", APPL ENVIRON MICROBIOL, DEC 1994, 60 (12) P4487-94, UNITED STATES, XP000614693 * |
HARTY DW ET AL: "Pathogenic potential of lactobacilli.", INT J FOOD MICROBIOL, DEC 1994, 24 (1-2) P179-89, NETHERLANDS, XP000573171 * |
HENRIKSSON A ET AL: "ADHESION TO PORCINE SQUAMOUS EPITHELIUM OF SACCHARIDE AND PROTEIN MOIETIES OF LACTOBACILLUS-FERMENTUM STRAIN 104-S", J GEN MICROBIOL, 138 (12). 1992. 2657-2661., XP000614690 * |
OH BH ET AL: "The bacterial periplasmic histidine-binding protein. structure/function analysis of the ligand-binding site and comparison with related proteins.", J BIOL CHEM, FEB 11 1994, 269 (6) P4135-43, UNITED STATES, XP002023710 * |
RODRIGUEZ F ET AL.: "An operon encoding a novel ABC-type transport system in Bacillus subtilis", MICROBIOLGY, vol. 141, no. 7, July 1995 (1995-07-01), pages 1781 - 1784, XP000573094 * |
TOBA T ET AL: "A collagen-binding S-layer protein in Lactobacillus crispatus", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 61 (7). 1995. 2467-2471., XP000573172 * |
WOLF A ET AL: "Structure/function analysis of the periplasmic histidine-binding protein. Mutations decreasing ligand binding alter the properties of the conformational change and of the closed form.", J BIOL CHEM, JUL 7 1995, 270 (27) P16097-106, UNITED STATES, XP002023711 * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2778187A1 (en) * | 1998-04-30 | 1999-11-05 | Sanofi Elf | METHOD FOR SELECTING BACTERIAL STRAINS |
EP0955545A1 (en) * | 1998-04-30 | 1999-11-10 | Sanofi Sante Nutrition Animale | Process to select bacterial strains |
US6346422B1 (en) | 1998-04-30 | 2002-02-12 | Ceva Sante Animale | Method of selecting bacterial strains |
WO2000071139A2 (en) * | 1999-05-25 | 2000-11-30 | Gregor Reid | Lactobacillus compositions and methods for preventing wound infections and biofilm formation on implantable surgical devices |
WO2000071139A3 (en) * | 1999-05-25 | 2001-03-08 | Gregor Reid | Lactobacillus compositions and methods for preventing wound infections and biofilm formation on implantable surgical devices |
US7011826B1 (en) * | 1999-07-02 | 2006-03-14 | The University Of New England | Control of acidosis |
US6682744B1 (en) * | 1999-08-09 | 2004-01-27 | University Of Maryland | Pro-gut maturation and anti-inflammatory effects of lactobacillus and lactobacillus secreted proteins, carbohydrates and lipids |
US7060687B2 (en) | 2001-02-07 | 2006-06-13 | Genmont Biotechnology Co. | Live vaccines for allergy treatment |
US9631185B2 (en) | 2011-06-09 | 2017-04-25 | Novozymes A/S | Fusion of bioactive molecules |
WO2018015380A1 (en) * | 2016-07-18 | 2018-01-25 | Philippe Ulsemer | Natural microorganisms which are naturally capable of binding toxins and/or toxin receptors |
Also Published As
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EP0857213A1 (en) | 1998-08-12 |
AU7229496A (en) | 1997-05-07 |
AU727676B2 (en) | 2000-12-21 |
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