WO2007093849A2 - P100 bactériophage p100 anti monocytogènes de listéria - Google Patents

P100 bactériophage p100 anti monocytogènes de listéria Download PDF

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WO2007093849A2
WO2007093849A2 PCT/IB2006/004168 IB2006004168W WO2007093849A2 WO 2007093849 A2 WO2007093849 A2 WO 2007093849A2 IB 2006004168 W IB2006004168 W IB 2006004168W WO 2007093849 A2 WO2007093849 A2 WO 2007093849A2
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seq
nucleotides
nucleic acid
phage
ploo
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PCT/IB2006/004168
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WO2007093849A3 (fr
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Martin J. Loessner
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Ebi Food Safety, B.V.
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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/097—Preservation
    • A23C19/10—Addition of preservatives
    • 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
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3571—Microorganisms; Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • Listeria monocytogenes is a bacterial pathogen that contaminates many food products, the list of which includes but is not limited to soft cheeses, pates, ice cream, smoked and cured fish, frozen seafood, salads, and processed meats. When ingested, these bacteria can produce a disease termed listeriosis, characterized by a variety of symptoms and conditions, including diarrhea, abortion, and encephalitis. Collectively, in the industrialized countries, hundreds of deaths occur each year as a result of Listeria monocytogenes food contamination.
  • the food processing industry has not been sufficiently successful in eradicating Listeria monocytogenes bacteria from the environment of the processing plants. As a result, even foods that have been pasteurized at temperatures high enough to kill these bacteria nevertheless become contaminated, post-pasteurization.
  • the bacteria gain access to the foodstuffs through one or more routes, including (i) from the raw materials (e.g. raw milk, and/or milk that has been pasteurized at low temperatures); (ii) from the processing machinery (in and on which the bacteria can grow as biofilms that are difficult to eradicate ); and (iii) from airborne bacteria present in the plant environment which can settle onto the surface of the foodstuffs during curing, packaging, and so on.
  • the methods currently in use to control Listeria in the food industry include: (i) pasteurization of primary ingredients (e.g. milk) and heat treatment of the products, which is often unsuccessful because recontamination frequently occurs and many products cannot undergo a final (listeriocidal) heat treatment; (ii) application of physicochemical agents such as
  • compositions and methods related to pi 00, a phage which can be used in the treatment of listeria caused problems. IV. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 shows the effect of phage PlOO on growth of L. monocytogenes on surface- ripened, soft cheese with a washed rind. All tested cheese were contaminated with L. monocytogenes on day 1 after cheese making.
  • PlOO was repeatedly applied to the cheese surface at different concentrations during all rind smearings until day 13. The data point for repeated low dose application on day 16 was not measured.
  • B A single high dose of PlOO was added to the brine during first smearing of the cheese rind. The control cheeses received no phage. AU cheeses were packaged on day 16 after cheese making (indicated by a star).
  • Figure 2 shows the effect of phage PlOO on the growth of L. monocytogenes in cooked chicken fillet (vacuum, 7°C).
  • Figure 3 shows the evolution of the phage titer on the cooked ham during storage.
  • Figure 4 shows the effect of PlOO on the growth of the L. monocytogenes cocktail (LISl, LIS2 and LIS3) in the cooked ham (vacuum packaged and stored at 7°C).
  • Figure 5 shows the effect of PlOO on the evolution of the total aerobic psychrotrophic count in the cooked ham (vacuum packaged and stored at 7°C).
  • Figure 6 shows the effect of PlOO on the evolution of the number of lactic acid bacteria in the cooked ham (vacuum packaged and stored at 7°C).
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of vames disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • Primer are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur.
  • a primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
  • Probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. 22. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. 23.
  • These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular vector is disclosed and discussed and a number of vector components including the promoters are discussed, each and every combination and permutation of promoters and other vector components and the modifications that are possible unless specifically indicated to the contrary.
  • compositions and methods of treating and preventing Listeriosis can involve controlling Listeria monocytogenes contamination, such as for example, in a food product, on food processing equipment or on food storage containers.
  • the compositions and methods can also involve treating an animal infected with Listeria monocytogenes.
  • the compositions and methods can also involve delivering a nucleic acid to a Listeria monocytogenes bacterium.
  • the compositions and methods can also involve detecting the presence of Listeria monocytogenes.
  • Listeriosis 25 Listeriosis is an infection resulting from the ingestion of foods contaminated by
  • Listeria monocytogenes and is characterized by a variety of symptoms, from diarrhea to abortion and infections of the brain and central nervous system. Because of its high mortality rate of approximately 25-30% (Vasquez-Boland et al., 2001), the disease ranks among the most severe food-borne illnesses. It was estimated that approximately 2000 hospitalizations and 500 deaths occur annually in the United States alone, as a result of the consumption of foods contaminated with L. monocytogenes (Mead et al., 1999). Listeria does not belong to the normal flora of healthy animals or man, but is an environmental bacterium and usually contaminates foods during fermentation, processing, storage, or even packaging of foods. This includes most ready- to-eat products such as milk and cheeses (mostly soft cheese), cold cuts (different types of meats), hot dogs, smoked fish, seafoods, and various delicatessen items.
  • compositions comprising isolated, purified, or recombinant bacteriophages and bacteriophage-derived proteins, such as endolysins, for treating listeriosis.
  • Bacteriophages can be regarded as natural enemies of bacteria, and therefore are candidates to evaluate as agents for the control of foodborne bacterial pathogens, such as
  • phages include the following: (i) they are designed to kill live bacterial target cells, (ii) they generally do not cross species or genus boundaries, and will therefore not affect (a) desired bacteria in foods (e.g., starter cultures), (b) commensals in the gastrointestinal ⁇ tract, or (c) accompanying bacterial flora in the environment. Moreover, (iii) since phages are generally composed entirely of proteins and nucleic acids, their eventual breakdown products consist exclusively of amino acids and nucleic acids. Thus, they are not xenobiotics, and, unlike antibiotics and antiseptic agents, their introduction into and distribution within a given environment may be seen as a natural process.
  • phages are the most abundant self-replicating units in our environment, and are present in significant numbers in water and foods of various origins, in particular fermented foods (reviewed by Sulakvelidze and Barrow, 2005). On fresh and processed meat and meat products, more than 108 viable phage per gram are often present (Kennedy and Bitton, 1987). It is a fact that phages are routinely consumed with food, in quite significant numbers. Moreover, phages are also normal commensals of humans and animals, and are especially abundant in the gastrointestinal tract (Furuse, 1987; Breitbart et al, 2003).
  • phages for controlling foodborne pathogens is reflected in recent studies dealing with Salmonella (Goode et al., 2003;Leverentz et al., 2001; Whichard et al., 2003), Campylobacter (Atterbury et al., 2003; Goode et al., 2003), E. coli (HuV et al., 2005; Toro et al., 2005), and L. monocytogenes (Dykes and Moorhead, 2002; Leverentz et al., 2003, 2004).
  • Salmonella Goode et al., 2003;Leverentz et al., 2001; Whichard et al., 2003), Campylobacter (Atterbury et al., 2003; Goode et al., 2003), E. coli (HuV et al., 2005; Toro et al., 2005), and L. monocytogenes (Dykes and Moorhead, 2002; Leverentz e
  • Solutions to circumvent this problem can include (i) careful selection and pooling of different phages with different lysis ranges, and/or (ii) the use of single broad host range phages which are able to infect all (or a majority of) the targeted organisms.
  • the latter solution permits a precise definition of the agent, and use of a single phage (rather than a pooled mixture) can be expected to facilitate the process of obtaining regulatory approval. 29. Almost all of the phages infecting organisms of the genus Listeria are temperate and feature a very narrow host range (Loessner and Rees, 2005).
  • PlOO represents one of the few virulent phages for this genus, which is strictly lytic and therefore invariably lethal to a bacterial cell once an infection has been established.
  • PlOO features an unusually broad host range within the genus Listeria, similar to phage A511 (Loessner, 1991; Loessner and Busse, 1990; van der Mee-Marquet et al., 1997).
  • more than 95% of approximately 250 different foodborne Listeria isolates belonging to serovar groups 1/2, 4 (L. monocytogenes), and 5 (L. ivanovii) were infected and killed by PlOO.
  • compositions can be used for treating and preventing Listeriosis, such as by controlling Listeria contamination in a food product, on food processing equipment or on food storage containers.
  • the provided composition can comprise recombinant PlOO phage or recombinant PlOO polypeptides.
  • methods for decontaminating an object which has a particular pathogen, such as a Listeria bacterium by incubating the object with or admninstering to the object, one or more of the compositions disclosed herein.
  • decontaminating means that the number of bacteria on an object have been reduced. It is not required that the bateria be completely eradicated or removed, although in certain embodiments this may occur. 31.
  • recombinant proteins can be used as disclosed herein, but can at least be used in conjunction with whole phage to reduce Listeria bacteria. Also disclosed are variants of the PlOO phage which are capable of being made and used based on the genetic information. Also disclosed herein are specific conditions for use of the PlOO phage as well as its variants.
  • composition comprising recombinant PlOO phage.
  • the phage are do not include the PlOO phage designated as ATCC PTA-4383.
  • embodiments of the proteins produced by PlOO which are recombinant.
  • recombinant is meant proteins which are produced within an environment which has been manipulated in some way by recombinant biotechnology.
  • the herein provided recombinant PlOO phage can comprise a nucleic acid having mutations, substitutions, or deletions to the sequence set forth in SEQ JD NO:1 (PlOO genome).
  • the folly annotated PlOO genome sequence (SEQ ID NO:1) has been deposited in GenBank under Accession No. DQ004855.
  • the PlOO genome comprises 174 open reading frames (ORFs), shown in Table 3.
  • ORFs open reading frames
  • the provided recombinant PlOO phage can be produced from a nucleic acid vector comprising all of these ORFs if at least one ORF comprises a mutation, substitution, and/or deletion to the sequence of the phage deposited in ATCC PTA- 4383.
  • the provided recombinant PlOO phage can also be produced from a nucleic acid vector comprising less than all of these ORFs.
  • the provided recombinant PlOO phage can also be produced from a vector comprising a mixture (chimera) of PlOO ORFs and ORFs from other phages (e.g. A511). It is understood that one of skill in the art using standard methods can routinely select ORFs for deletion, substitution or mutation for use in the herein provided methods. For example, substitution of the PlOO capsid protein encoding nucleic acids (gplO; nucleotides 5,472 to 6,497 of SEQ ID NO:1) with nucleic acids encoding other phage capsid proteins of a different phage family then P 100 can alter the specificity of the resulting phage.
  • PlOO capsid protein encoding nucleic acids (gplO; nucleotides 5,472 to 6,497 of SEQ ID NO:1)
  • P 100 can alter the specificity of the resulting phage.
  • composition comprising PlOO phage and one or more of A511, P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971 C, 907515, 12981, 11711 A, 00241, 13441, A500, A640.
  • the recombinant PlOO phage that can be used herein can be defined based on homology to Pl 00.
  • the herein disclosed recombinant PlOO phage can be 65%, 70%, 75%, 80%, 85%, 90%, 95% homologous to the PlOO phage as encoded by SEQ ED NO:1.
  • the disclosed recombinant PlOO phage is not A511, P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971 A, 02971C, 907515, 12981, 11711 A, 00241, 13441, A500, A640, B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425 A, 2425, or 3551 phage.
  • composition comprising a recombinant phage PlOO polypeptide.
  • the provided polypeptide can be a large terminase, endolysin, portal protein, capsid protein, tail sheath protein, baseplate protein, tail protein, helicase, replicase, primase, exonuclease, dUTPase, ribonucleoside-diphosphate reductase alpha subunit, ribonucleoside-diphosphate reductase beta subunit, ribose-phosphage pyrophosphokinase, nicotinamid phosphoribosyl transferase, DNA polymerase, recombinase, sigma factor, alanyl- tRNA synthetase, ATPase, ligase, pyrphosphathydrolase, or repressor.
  • a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 8,189 to 9,832 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 11,790 to 13,196 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 17,130 to 18,818 of SEQ ID NO: 1, or a fragment or conservative variant thereof.
  • a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 29,606 to 30,316 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 30,330 to 31,376 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 41,690 to 43,435 of SEQ ID NO: 1, or a fragment or conservative variant thereof.
  • a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 43,450 to 45,090 of SEQ ID NO: 1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 45,108 to 46,571 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 47,733 to 48,113 of SEQ ID NO: 1 , or a fragment or conservative variant thereof.
  • a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 50,227 to 51,288 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 51,335 to 51,982 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 53,678 to 55,027 of SEQ ID NO: 1, or a fragment or conservative variant thereof.
  • a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 55,238 to 56,269 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 56,445 to 57,476 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 60,267 to 61,181 of SEQ TD NO:1, or a fragment or conservative variant thereof.
  • a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 61,192 to 62,985 of SEQ E) NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 66,415 to 66,729 of SEQ ID NO:1, or a fragment or conservative variant thereof.
  • a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 66,812 to 67,648 of SEQ TD NO: 1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 67,983 to 70,091 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 72,029 to 73,273 of SEQ ID NO:1, or a fragment or conservative variant thereof.
  • a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 73,712 to 74,350 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 80,225 to 80,707 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 83,439 to 84,455 of SEQ TD NO:1, or a fragment or conservative variant thereof.
  • a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 117,468 to 116,527 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 120,547 to 120,344 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 120,209 to 120,985 of SEQ E) NO:1, or a fragment or conservative variant thereof.
  • a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions to a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO: 1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 8,189 to 9,832 of SEQ ID NO:1.
  • nucleic acid that can hybridize under stringent conditions to a nucleic acid having the sequence set forth in nucleotides 120,209 to 120,985 of SEQ E) NO:1.
  • Phage-encoded lysins or endolysins are highly active enzymes which hydrolyze bacterial cell walls. These phage encoded cell wall lytic enzymes are synthesized late during virus multiplication and mediate the release of progeny virions. Endolysins can be used to lyse Listeria cells to recover nucleic acids or cellular protein for detection or differentiation. The endolysin can also be used to treat animals (including humans) which are infected with Listeria and to treat surfaces which may be contaminated with Listeria.
  • Endolysin (ply) genes from Listeria phages (Al 18, A500 and A511) have been cloned and purified (Loessner, et al., Applied and Environmental Microbiology, August 1996, p. 3057-3060).
  • PlOO endolysin polypeptide (Ply 100).
  • the polypeptide can be isolated and purified from the herein disclosed recombinant PlOO phages.
  • PlylOO can be isolated by techniques known in the art including but not limited to lysis, chromatography, filtration, and centrifugation.
  • the endolysin can be isolated from Listeria which have been incubated with Pl 00.
  • PlylOO can also be recombinantly expressed in a host bacteria (e.g. E. coli, L. lactis, S. aureus, and B. cereus) or other cell or other in vitro system as understood.
  • a host bacteria e.g. E. coli, L. lactis, S. aureus, and B. cereus
  • the provided endolysin polypeptide can be produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO:1, or a fragment or conservative variant thereof.
  • the provided endolysin polypeptide can also be produced by a cell comprising a nucleic acid that can hybridize under stringent conditions to a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO:1, or a fragment or conservative variant thereof.
  • the endolysin can be isolated from the host bacteria or the host bacteria containing the endolysin can be directly applied or administered without isolation of the endolysin.
  • a host bacteria which produces the endolysin can be administered to an animal or applied to a surface where the endolysin would be secreted into the food, onto the surface or into the animal's gut.
  • the endolysin can then attack Listeria cells present in this environment.
  • One unit of endolysin activity is defined as the amount of endolysin necessary to decrease the optical density at 600 nm by 0.01/min, at pH 8.0 and 25 0 C. in a volume of 1 ml, when heat-killed, washed cells of Listeria monocytogenes are used as a substrate.
  • PlOO Capsids 45 PlOO particles can be used for the delivery of a composition to ⁇ Listeria cell.
  • PlOO capsid polypeptide and phage particles produced therefrom The PlOO capsid polypeptides can be recombinantly expressed in a host bacteria (e.g. E. coli, L. lactis, S. aureus, and B. cereus).
  • a host bacteria e.g. E. coli, L. lactis, S. aureus, and B. cereus.
  • the provided capsid polypeptide can be produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 11,790 to 13,196 of SEQ TD NO: 1, or a fragment or conservative variant thereof.
  • nucleic acid encoding a bacteriophage Pl 00-specific polypeptide.
  • a nucleic acid comprising a fragment or conservative variant of the sequence set forth in SEQ ID NO: 1.
  • a nucleic acid comprising a sequence set forth in SEQID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1.
  • the provided nucleic acid can also comprise a nucleic acid that can hybridize under stringent conditions to a sequence set forth in SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO: 1.
  • a sequence set forth in SEQ ID NO: 1 is meant any portion or unique fragment of the PlOO genome such that the nucleic acid is at least 10, 20, 100 nucleotides in length.
  • nucleic acid having the sequence set forth in nucleotides 11,790 to 13,196 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO: 1. Also provided is a nucleic acid having the sequence set forth in nucleotides 17,130 to 18,818 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 29,606 to 30,316 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1.
  • nucleic acid having the sequence set forth in nucleotides 30,330 to 31,376 of SEQ ID NO:1, wherein thenucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 41,690 to 43,435 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 43,450 to 45,090 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1.
  • nucleic acid having the sequence set forth in nucleotides 45,108 to 46,571 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 47,733 to 48,113 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 50,227 to 51,288 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO: 1. Also provided is a nucleic acid having the sequence set forth in nucleotides
  • nucleic acid having the sequence set forth in nucleotides 53,678 to 55,027 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 55,238 to 56,269 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1.
  • nucleic acid having the sequence set forth in nucleotides 56,445 to 57,476 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 60,267 to 61,181 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 61,192 to 62,985 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ TD NO:1.
  • nucleic acid having the sequence set forth in nucleotides 66,415 to 66,729 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 66,812 to 67,648 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 7,983 to 70,091 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1.
  • nucleic acid having the sequence set forth in nucleotides 72,029 to 73,273 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 73,712 to 74,350 of SEQ E) NO:1, wherein the nucleic acid does not consist of SEQ TD NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 80,225 to 80,707 of SEQ TD NO:1, wherein the nucleic acid does not consist of SEQ E) NO:1.
  • nucleic acid having the sequence set forth in nucleotides 83,439 to 84,455 of SEQ TD NO: 1 wherein the nucleic acid does not consist of SEQ TD NO: 1.
  • nucleic acid vector comprising a nucleic acid encoding a bacteriophage PlOO-specific polypeptide, wherein the nucleic acid does not consist of SEQ TD NO:1.
  • cell comprising a nucleic acid encoding a bacteriophage P100-specific polypeptide, wherein the nucleic acid does not consist of SEQ TD NO: 1.
  • compositions include combinations of the compositions.
  • Effective concentrations/ dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • concentrations/ dosages ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are effected.
  • the herein provided compositions can be used for controlling Listeria contamination in a food product, on food processing equipment or on food storage containers.
  • the provided compositions can be applied on or into food products, and/or into various physical sites within the food processing plants, by a number of means including, but not limited to, admixing the compositions into the food products, spraying the compositions onto the foodstuffs, spraying the compositions onto the plant equipment, and/or directly applying the compositions to the plant equipment. Said applications significantly reduce the numbers of Listeria monocytogenes bacteria that would otherwise be present.
  • the concentration of PlOO phage for administration on or into food products, and/or into various physical sites within the food processing plants is contemplated to be in the range of about 10 3 to 10 ° pfu (plaque forming units) per mL, including about 10 6 to 10 9 pfu/ mL.
  • the PlOO phage can be administered to the surface of the food products and/or physical sites within the food processing plants with a final surface concentration of about 10 5 to 10 8 pf ⁇ /crn 2 , including 10 6 to 10 7 pfu/cm 2 .
  • the concentration of PlylOO for administration on or into food products, and/or into various physical sites within the food processing plants is contemplated to be in the range of about 2-2000 ng/ ml, such as about 20-200 ng/ ml.
  • the compositions are administered until successful reduction or elimination of the Listeria monocytogenes is achieved or until the amount of Listeria monocytogenes is substantially reduced. 52.
  • the herein provided compositions can also be used to treat animals, including humans, infected with Listeria monocytogenes. When administered to a subject, the dosages should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosages will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • Any suitable route of administration can be used to administer the phage including but not limited to: oral, aerosol or other device for delivery to the lungs, nasal spray, intravenous, intramuscular, intraperitoneal, intrathecal, vaginal, rectal, topical, lumbar puncture, intrathecal, and direct application to the brain and/or meninges.
  • Excipients which can be used as a vehicle for the delivery of the phage, endolysin and/or host bacteria containing the endolysin will be apparent to those skilled in the art.
  • the compositions could be in lyophilized form and be dissolved just prior to administration by IV injection.
  • the dosage of administration for PlOO phage is contemplated to be in the range of about 10 3 to about 10 13 pfu/per kg/per day, such as about 10 12 pfu/per kg/per day.
  • the dosage of administration for the PlylOO is contemplated to be in the range of about 2-2000 ng/per g/per day, such as about 20-200 ng/per g/per day.
  • the compositions are administered until successful elimination of the Listeria monocytogenes is achieved or until the amount of Listeria monocytogenes is substantially reduced. 54.
  • Also provided is a method for detecting the presence of Listeria monocytogenes comprising obtaining a sample suspected to contain Listeria monocytogenes, incubating said sample with recombinant PlOO phage or recombinant PlylOO, and detecting any change in said sample caused by PlOO, as an indication of the presence of Listeria monocytogenes.
  • said change in said sample is due to lysis by PlOO or a detectable label or signal.
  • the method can comprise recombinantly inserting a gene construct into the genome of PlOO before incubation with said sample, wherein expression of said gene construct results in a detectable signal in the presence of Listeria monocytogenes.
  • the gene construct can, fore example, encode a bio-luminescent protein, such as one selected from the group consisting of luciferase and a fluorescent protein.
  • Luciferase can be from bacteria or insects.
  • the fluorescent protein can be green fluorescent protein or a variant thereof.
  • the method can comprise immobilizing said Listeria monocytogenes on a solid support, such as a test strip, and detecting any change on said solid support.
  • Listeria monocytogenes can be immobilized using ⁇ tnti-Listejia antibodies. Samples that can be tested using the provided method includes those obtained from patients suspected of being infected with Listeria monocytogenes or those obtained from a food product, food processing equipment or food storage 1 containers.
  • recombinant phage PlOO or a nucleic acid or polypeptide derived therefrom, for the production of a pharmaceutical composition for the treatment of an animal infected with Listeria monocytogenes.
  • a nucleic acid encoding a PlOO polypeptide for the production of a pharmaceutical composition for the treatment of an animal infected with Listeria monocytogenes.
  • a recombinant PlOO polypeptide for the production of a pharmaceutical composition for the treatment of an animal infected with Listeria monocytogenes. 4.
  • compositions in combination with other anti-Listerial agents known in the art.
  • anti-Listerial agents include but are not limited to endolysins, surface disinfectants, antimicrobial agents, enzymes, surfactants, and other phage.
  • the provided compositions can be combined with listeriolysins which are enzymes which have been shown to selectively control Listeria in food and the environment.
  • the herein provided compositions can be administered in combination with, for example, Plyl 18, Ply 500 orPly511 endolysins.
  • the provided compositions can be combined with known surface disinfectants such as (i) preservatives of various kinds, such as but not limited to benzoic acid and BHT; and (ii) various disinfectants with which the phages are compatible, such as but not limited to quaternary ammonium compounds.
  • compositions can be combined with known antimicrobial agents (including antibiotics and chemotherapeutic agents) including but not limited to vancomycin, nisin, danofloxacin and neomycin.
  • the provided compositions can be combined with enzymes to aid in breaking up biofilms (e.g. biofilms found in food processing equipment). Such enzymes are known in the art and include but are not limited to polysaccharide depolymerase enzymes, and protease. 61.
  • the provided compositions can be combined with known surfactants when used to treat food processing equipment. The surfactant helps to wet the surface so that the phage are properly distributed over the various surfaces, and to solubilize and remove dirt so that the Listeria are accessible to the phage. Suitable surfactants include but are not limited to Tween 80, 20 and 81 and Dobanols.
  • compositions can be combined with phage specific for Listeria monocytogenes and/or phage specific for other bacteria known to contaminate food processing equipment and food products.
  • bacteria include but are not limited to E. coli, and bacterial species from the genera Salmonella, Bacillus, Staphylococcus, Streptococcus, Clostridium, and Pseudomonas.
  • the herein provided phage can be applied in a liquid or a powdered form to food products and food processing equipment. If applied as a liquid, the phage are applied at a concentration of about 10 3 to 10 10 pfu/ mL, including about 10 6 to 10 9 pfu/ mL. If applied as a dry powder the phage are applied at a concentration of about 10 3 to 10 10 pfu/ mg, including about 10 6 to 10 9 pfu/ mg.
  • the phage can be suspended in a suitable carrier prior to application or drying, including but not limited to protein solutions containing BSA, casein, whey protein, soy bean protein, etc and sugar based carriers containing sugars such as mannitol.
  • the phage can be lyophilized or cryopreserved by vitrification and either suspended in a solution prior to application or applied directly as a dry powder.
  • Suitable amounts of phage for use in the present invention can be obtained by techniques known in the art, including but not limited to a batch technique where a culture of host bacteria is grown and then seeded with phage. After an amount of time suitable to allow maximal phage propagation and bacterial lysis, the culture is further lysed by physical or chemical means and the lysate spun down.
  • the phage containing supernatant can be used as is or further purified using techniques such as ultrafiltration, chromatography and centrifugation.
  • the endolysin can be applied in a liquid or a powdered form to food products and food processing equipment.
  • the endolysin is applied in a concentration between 2 to 2000 ng endolysin per ml or per gram of carrier, and preferably between 20 to 200 ng endolysin per ml or per gram of carrier.
  • Food products to which the herein provided compositions can be administered include, but are not limited to dairy products, meat products, fish products, unpasteurized food products, and salads.
  • dairy product is intended to include any food product made using milk or milk products, including but not limited to milk, yogurt, ice cream, cheese, butter, and cream.
  • dairy product is intended to include any food product which contains animal tissue, including but not limited to beef, pork, and poultry.
  • ready to eat meat product in intended to include any meat product which does not require cooking prior to consumption, including but not limited to pates, hot dogs, bologna, salami, and cold cuts.
  • fish product is intended to include any food product which contains tissue from an aquatic animal including but not limited to lobster, crab, fresh water and saltwater fish and other seafoods.
  • unpasteurized food product is intended to include any food product which is prepared using unpasteurized primary ingredients and which does not undergo a final (listeriocidal) heat treatment.
  • siad is intended to include any food product which contains mixtures of vegetables or fruits, and particularly such mixtures as are presented for consumers to choose from in a display commonly referred to as a "salad bar”. 6.
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions maybe administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • stealth and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either Tecycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses ana toxins, dissociation and degradation of ligand, and receptor-level regulation.
  • the pharmaceutically-acceptable carrier examples include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders maybe desirable..
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • primers are designed based upon conserved sequences found in multiple members of a protein family. Since sequence conservation is rarely perfect (especially at the nucleotide level), primers are said to be "degenerate,” meaning that more than one nucleotide is permitted at certain positions.
  • Primer mixtures are used to amplify DNA samples (cDNAs or genomic DNAs) thought to contain sequences encoding unknown members of the protein family. There are two major roles for this method: 1) clone novel members of known families and 2) clone homologues in different species.
  • Homology PCR can be used when target novel sequence must have at least two regions with sequences very similar to known proteins. (One primer is modeled after each of these two regions.) These regions may be small, perhaps as short as 12 nucleotides (4 amino acids). However, more reliable results are obtained with somewhat larger areas (18-24 nt, 6-8 aa). Ideally, regions should be separated by 150-500 nt. Avoid excessive codon degeneracy. Find an area where there is as much identity as possible between sequences. Avoid regions containing many leucine, serine, and arginine residues, since each can be encoded by six different codons. The ideal amino acids are tryptophan and methionine (one codon each).
  • deoxyinosine in the primer sequence
  • primer sequence it may be useful to use deoxyinosine ("I" in the primer sequence) instead of mixing all four nucleotides at • completely degenerate positions. This can substantially reduce overall primer degeneracy and should reduce false priming.
  • I deoxyinosine
  • Other methods can be used to clone products made using primers without restriction sites (e.g., blunt-end or TA cloning).
  • nucleic acids can be used to design degenerate primers for the cloning and sequencing of A511, P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971C, 907515, 12981, 11711 A, 00241, 13441, A500, A640, B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425A, 2425, or 3551 phage.
  • degenerate primers can be used to clone the homologous genes for the indicated phage or other phage. 8. Sequence similarities
  • homology and identity mean the same thing as similarity.
  • the use of the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences.
  • Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.
  • variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • Another.way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.
  • a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).
  • hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
  • Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide.
  • the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.
  • selective hybridization conditions can be defined as stringent hybridization conditions.
  • stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps.
  • the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5 0 C to 20°C below the Tm.
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA- RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids).
  • a preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C.
  • Stringency of hybridization and washing if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.
  • stringency of hybridization and washing if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.
  • Another way to define selective hybridization is by looking at the amount
  • selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid.
  • the non-limiting primer is in for example, 10 or 100 or 1000 fold excess.
  • This type of assay can he performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k ⁇ j, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k d . 90.
  • Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation.
  • selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended.
  • Preferred conditions also include those suggested
  • Nucleic acids 93 There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example, Plyl 00, as well as any other proteins disclosed herein, as well as various functional nucleic acids.
  • the disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, that the expressed rrJRNA will typically be made up of A, C, G, and U.
  • an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment.
  • a nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage.
  • the base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).
  • the sugar moiety of a nucleotide is a ribose or a deoxyribose.
  • the phosphate moiety of a nucleotide is pentavalent phosphate.
  • An non-limiting example of a nucleotide would be 3'-AMP (3'- adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).
  • a nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties.
  • Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid. 97. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake.
  • conjugates to nucleotides or nucleotide analogs to enhance for example, cellular uptake.
  • Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86, 6553-6556),
  • a Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute.
  • the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
  • a Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA.
  • the Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.
  • compositions including primers and probes which are capable of interacting with the genes disclosed herein, hi certain embodiments the primers are used to support DNA amplification reactions.
  • the primers will be capable of being extended in a sequence specific manner.
  • Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer.
  • Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred.
  • the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Typically the disclosed primers hybridize with the nucleic acid or region of the nucleic acid or they hybridize with the complement of the nucleic acid or complement of a region of the nucleic acid. d) Functional Nucleic Acids
  • Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction.
  • Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting.
  • functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences.
  • the functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.
  • Functional nucleic acid molecules can interact with any macromolecule, such as DNA 5 RNA, polypeptides, or carbohydrate chains.
  • functional nucleic acids can interact with the mRNA of PlOO or the genomic DNA of PlOO or they can interact with a polypeptide PlOO.
  • functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.
  • Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC.
  • antisense molecules bind the target molecule with a dissociation constant (k d )less than or equal to 10 "6 , 10 "8 , lO "10 , or 10 "12 .
  • k d dissociation constant
  • a representative sample of methods and techniques which aid in the design and use of antisense molecules can be found in the following non-limiting list of United States patents: 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.
  • Aptamers are molecules that interact with a target molecule, preferably in a specific way.
  • aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
  • Aptamers can bind small molecules, such as ATP (United States patent 5,631,146) and theophiline (United States patent 5,580,737), as well as large molecules, such as reverse transcriptase (United States patent 5,786,462) and thrombin (United States patent 5,543,293).
  • Aptamers can bind very tightly with k d S from the target molecule of less than 10 "12 M.
  • the aptamers bind the target molecule with a k d less than 10 "6 , 10 "8 , 10 "10 , or 10 "12 .
  • Aptamers can bind the target molecule with a very high degree of specificity.
  • aptamers have been isolated that have greater than a 10000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (United States patent 5,543,293). It is preferred that the aptamer have a k d with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the k d with a background binding molecule.
  • the background molecule be a different polypeptide.
  • the background composition could be A511.
  • Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,476,766, 5,503,978, 5,631,146, 5,731,424 , 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660 , 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698.
  • Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions.
  • ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (for example, but not limited to the following United States patents: 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but not limited to the following United States patents: 5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,3
  • ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited to the following United States patents: 5,580,967, 5,688,670, 5,807,718, and 5,910,408).
  • Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates.
  • Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions.
  • Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a k d less than 10 " , 10 "8 , 10 '10 , or 10 "12 .
  • EGSs External guide sequences
  • RNase P RNase P
  • RNAse P aids in processing transfer RNA (tRNA) within a cell.
  • Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altaian, Science 5 238:407-409 (1990)).
  • RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukarotic cells.
  • the disclosed nucleic acids can be in the form of naked DNA or RNA, or the nucleic acids can be in a vector for delivering the nucleic acids to the cells, whereby the antibody-encoding DNA fragment is under the transcriptional regulation of a promoter, as would be well understood by one of ordinary skill in the art.
  • the vector can be a commercially available preparation, such as an 0 adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms.
  • delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other 5 liposomes developed according to procedures standard in the art.
  • LIPOFECTIN LIPOFECTAMINE
  • SUPERFECT Qiagen, Inc. Hilden, Germany
  • TRANSFECTAM Promega Biotec, Inc., Madison, WI
  • the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Arlington, AZ).
  • vector delivery can be via a viral system, such as a retroviral 0 vector system which can package a recombinant retroviral genome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486, 1988; Miller et al., MoI. Cell. Biol. 6:2895, 1986).
  • the recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding a broadly neutralizing antibody (or active fragment thereof).
  • the exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use ot retroviral vectors.
  • adenoviral vectors Mitsubishi et al., Hum. Gene Ther. 5:941-948, 1994
  • adeno-associated viral (AAV) vectors Goodman et al., Blood 84:1492-1500, 1994
  • lentiviral vectors Non-deficiency virus vectors
  • pseudotyped retroviral vectors Agrawal et al., Exper. Hematol. 24:738-747, 1996.
  • Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood 87:472-478, 1996).
  • compositions and methods can be used in conjunction with any of these or other commonly used gene transfer methods.
  • the dosage for administration of adenovirus to humans can range from about 10 7 to 10 9 plaque forming units (pfu) per injection but can be as high as 10 12 pfu per injection (Crystal, Hum. Gene Ther. 8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997).
  • a subject can receive a single injection, or, if additional injections are necessary, they can be repeated at six month intervals (or other appropriate time intervals, as determined by the skilled practitioner) for an indefinite period and/or until the efficacy of the treatment has been established.
  • Parenteral administration of the nucleic acid or vector, if used, is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained.
  • suitable formulations and various routes of administration of therapeutic compounds see, e.g., Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • the nucleic acids that are delivered to cells typically contain expression controlling systems.
  • the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
  • a promoter contains core elements required for basic interaction of KNA polymerase and transcription factors, and may contain upstream elements and response elements.
  • Preferred promoters controlling transcription from vectors in mammalian host cells maybe obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
  • viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)).
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as aHindin E restriction fragment (Greenway, PJ. et al., Gene 18: 355-360 (1982)).
  • promoters from the host cell or related species also are useful herein.
  • Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., MoI. Cell Bio. 4: 1293 (1984)).
  • Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression.
  • Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the promo tor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function.
  • Systems can be regulated by reagents such as tetracycline and dexamethasone.
  • reagents such as tetracycline and dexamethasone.
  • the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed, hi certain constructs the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time.
  • a preferred promoter of this type is the CMV promoter (650 bases).
  • Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral
  • GFAP glial fibrillary acetic protein
  • Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contain a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA.
  • the identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs.
  • the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct. b) Markers
  • the viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed.
  • Preferred marker genes are the E. CoIi lacZ gene, which encodes ⁇ -galactosidase, and green fluorescent protein.
  • the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydro folate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin.
  • the transformed mammalian host cell can survive if placed under selective pressure.
  • selective regimes There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are: CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.
  • the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1 : 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., MoI. Cell. Biol. 5: 410-413 (1985)).
  • the three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin. 13. Peptides a) Protein variants
  • Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications.
  • amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants.
  • Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • Immunogenic fusion protein derivatives are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion.
  • Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule.
  • These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • substitution mutations at predetermined sites in DNA having a known hcquciiuc aie wen Known, for example Ml 3 primer mutagenesis and PCR mutagenesis.
  • Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues.
  • Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
  • substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 2 and 3 and are referred to as conservative substitutions.
  • Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 3, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • an electropositive side chain e.g., lysyl, arginyl, or histidyl
  • an electronegative residue e.g., glutamyl or aspartyl
  • substitutions include combinations such as, for example, GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.
  • Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).
  • Deletions of cysteine or other labile residues also may be desirable.
  • Deletions or substitutions of potential proteolysis sites, e.g. Arg is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
  • Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post- translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.
  • variants and derivatives of the disclosed proteins herein are through defining the variants and derivatives in terms of homology/identity to specific known sequences.
  • SEQ JD NO:1 sets forth the PlOO genome encoding PlOO proteins. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the encoded proteins.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level. 131. Another way of calculating homology can be performed by published algorithms.
  • Optimal alignment of sequences for comparison maybe conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.
  • nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence.
  • a particularly preferred non-peptide linkage is -CH 2 NH-. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, and the like.
  • Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such.
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type e.g., D-lysine in place of L- lysine
  • Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations.
  • antibodies specific for the PlOO polypeptides disclosed herein.
  • the term "antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with a PlOO polypeptide.
  • the antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods. 140.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chai ⁇ (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. ScL USA, 81:6851-6855 (1984)).
  • the disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies.
  • disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro, e.g., using the HIV Env-CD4-co-receptor complexes described herein.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.).
  • DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains- of murine antibodies).
  • Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.
  • In vitro methods are also suitable for preparing monovalent antibodies.
  • Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566.
  • Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
  • the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • antibody can also refer to a human antibody and/or a humanized antibody.
  • Many non-human antibodies e.g., those derived from mice, rats, or rabbits
  • are naturally antigenic in humans and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
  • nucleic acids and proteins can be represented as a sequence consisting of the nucleotides of amino acids.
  • nucleotide guanosine can be represented by G or g.
  • amino acid valine can be represented by VaI or V.
  • Those of skill in the art understand how to display and express any nucleic acid or protein sequence in any of the variety of ways that exist, each of which is considered herein disclosed. Specifically contemplated herein is the display of these sequences on computer readable mediums, such as, commercially available floppy disks, tapes, chips, hard drives, compact disks, and video disks, or other computer readable mediums.
  • kits 148 Disclosed Jtierem are kits that are drawn to reagents that can be used in practicing the methods disclosed herein.
  • the kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice, of the, disclosed methods.
  • the kits could include recombinant phage, as well as the buffers and enzymes required to use the phage as intended.
  • C Methods of making the compositions
  • compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted. 1. Nucleic acid synthesis
  • the nucleic acids such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.
  • Peptide synthesis 151 One method of producing the disclosed proteins, such as SEQ TD NO:23, is to link two or more peptides or polypeptides together by protein chemistry techniques.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA).
  • Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry Applied Biosystems, Inc., Foster City, CA.
  • a peptide or polypeptide corresponding to the disclosed proteins for example, can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
  • peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof.
  • the peptide or polypeptide is independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions. 152. For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments.
  • This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
  • the first step is the chemoselective reaction of an unprotected synthetic peptide—thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product.
  • this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J.Biol. Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
  • unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)).
  • This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry TV. .Academic Press, New York, pp. 257-267 (1992)).
  • compositions 154 Disclosed are processes for making the compositions as well as making the intermediates leading to the compositions. For example, disclosed are nucleic acids in SEQ DD O: 1. There are a variety of methods that can be used for making these compositions, such as synthetic chemical methods and standard molecular biology methods. It is understood that the methods of making these and the other disclosed compositions are specifically disclosed.
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid comprising the sequence set forth in SEQ ID NO:1 and a sequence controlling the expression of the nucleic acid.
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence having 80% identity to a sequence set forth in SEQ ID NO:1, and a sequence controlling the expression of the nucleic acid.
  • nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence that hybridizes under stringent hybridization conditions to a sequence set forth SEQ ID NO: land a sequence controlling the expression of the nucleic acid.
  • compositions can be used in a variety of ways as research tools.
  • the disclosed compositions such as SEQ ID NO:1 can be used to study the interactions between bacteriophage and Listeria, by for example acting as inhibitors of binding.
  • compositions can also be used diagnostic tools related to diseases, such as listeriosis.
  • compositions can be used in any known method of screening assays, related to chip/micro arrays.
  • compositions can also be used in any known way of using me computer rea ⁇ aoie emDodiments of the disclosed compositions, for example, to study relatedness or to perform molecular modeling analysis related to the disclosed compositions.
  • Example 1 Materials and methods
  • Phage PlOO was first isolated from a sewage effluent sample taken irom a dairy plant in southern Germany. Liquid samples were centrifuged, filter-sterilized, and tested for presence of Listeria phages by spotting small drops on preformed lawns of a selection of different Listeria indicator strains as previously described (Loessner and Busse, 1990). One particular phage which formed large, clear plaques on most tested strains was isolated, purified, and designated as PlOO. A stock lysate of PlOO, containing approximately 3xlO 9 pfu/ml (plaque forming units), was then prepared using L. monocytogenes WSLC 1001 as a host, and stored at 4°C.
  • Propagation of PlOO was performed using either L. monocytogenes WSLC 1001 or the non-pathogenic host L. innocua WSLC 2096 or WSLC 2321. Purification of virions by polyethylene-glycol precipitation and CsCl density gradient centrifugation, and extraction of the DNA molecules was performed as previously described (Loessner et al., 1994; Loessner and Scherer, 1995). The sequence of the PlOO double-stranded DNA genome was determined using a "shotgun" cloning strategy (Loessner et al., 2000; Zimmer et al., 2003), with some modifications.
  • ⁇ o oligonucleotide primers complementary to vector sequences flanking the inserts in an automated nucleotide sequencer (ABI 3700; Applied Biosystems). After approximately 50 contigs of various lengths could be assembled, gaps were closed by using phage DNA directly as template in the sequencing reaction, employing oligonucleotide primers complementary to the ends of the contigs (primer walking). Regions of low redundancy or showing sequence ambiguities were checked again by primer walking, or by sequencing a PCR amplification product designed to encompass the region of interest.
  • nucleotide position 1 (left end of the genome) was set directly upstream of the putative terminase subunit genes.
  • the information encoded by the PlOO genome was then analyzed using Vector NTI software (version 8; InforMax), and the annotated genome and all predicted open reading frames (ORF), gene products (gp) and secondary structures were again confirmed by visual inspection.
  • the basic prerequisites for an ORF were the presence of one of the three potential start codons ATG, TTG or GTG, a suitable ribosomal binding site (Loessner and Scherer, 1995; Loessner et al., 2000), and a length of at least 40 encoded amino acids.
  • Nucleotide and amino acid sequence alignment searches (BlastN, BlastX, and BlastP) using the ORFs and deduced gene products, respectively, were performed with Vector NTIs integrated BLAST engine which used the non-redundant database (available through the NCBI web sites http://www.ncbi.nlm.nih.gov/). Searches for specific protein domains and conserved motifs with known function were performed using the PFAM tools (available online at http://pfam.wustl.edu/hmmsearch.shtml). Transmembrane domains were predicted by using the hidden Markov model (TMHMM) (available at http://www.cbs.dtu.dk/services/TMHMM/).
  • TSHMM hidden Markov model
  • Helix-turnhelix-Scans were performed using SeqWeb Version 2.1.0 (GCG package) (accessed via the biocomputing services of the University of Zurich online at http://www.bio.unizh.ch/bioc/).
  • Potential tRNA genes were identified using the bioinformatics tool provided by http://www.genetics.wustl.edu/eddy/tRNAscan-SE (Lowe and Eddy, 1997).
  • Loops and hairpins were identified using H ⁇ BIO software (Hitachi) and VectorNTI, and a preliminary graphical genetic map of PlOO was constructed using VectorNTI.
  • a total of 10 healthy male and 10 healthy female Wistar albino rats (Ace Animals, Boyertown, USA) of about 8 weeks of age were used, with a pre-test body weight range of 202-231 g per male, and 193-214 gper female. Animals were randomly selected and assigned to two groups of five males and five females per group, and individually identified by ear tags. The rats were housed 1 per cage in stainless steel wire bottom cages, in a temperature controlled animal room, with a 12 h light/dark cycle. Fresh rodent chow diet was provided ad libitum, except for the fasting period of one day prior to sacrifice. Fresh water was available ad libitum. 170.
  • AU animals were examined for gross pathology. The esophagus, stomach, duodenum, jejunum, ileum, cecum, and colon were preserved in 10% neutral buffered formalin. Histopathologic preparation (cross-sections and longitudinal sections) and microscopical analysis were performed according to standardized procedures. All results were evaluated based on the relationship between the dose levels and incidents or severity of responses (if any). Appropriate statistical evaluations were performed using Instat Statistics Version 2.0 software.
  • PlOO lysates were purified by tangential-flow ultrafiltration (30 kDa cut-off), and adjusted to approximately 1x10 10 pfu/ml, in MOPS buffer (1OmM 3-(7V-morpholino) propanesulfonic acid, pH 7.3).
  • the cheese rinds (65 cm 2 , corresponding to approximately 30-40 g) were homogenized with buffer (5OmM trisodium-citrate, pH 7.3; added to 250ml) using a Stomacher laboratory blender. The homogenate and decimal dilutions prepared thereof were . surface plated on Listeria selective Oxford agar plates (Oxoid), in triplicate. The plates were incubated at 37 0 C for 48 h, until typical Listeria colonies could be enumerated and viable counts calculated. The lower limit of detection was approximately 5 cfu/cm 2 of cheese.
  • Necropsy results were normal in all animals except one of the animals of the PlOO test group which showed a small red area in the mucosa at the junction of jejunum and ileum. Multiple thin sections from this area of the gastrointestinal tract were then examined, and all were within normal histological limits with no microscopic change to correlate with the gross observation.
  • Fig. 1 demonstrate the effect of PlOO on Z. monocytogenes contamination on a surface-ripened Munster-type soft cheese.
  • the manufacturing process used was indistinguishable from that employed in commercial production of this type of cheese, including the specific parameters of inoculation with a standardized bacterial/ yeast ripening flora, ripening conditions (temperature and duration), washing of the rind, and time point of packaging.
  • Example 2 a) Experimental setup 191. The test product was industrially prepared, cooked chicken fillet that was sliced into slices of approximately 17.5 g and approximately 1 dm 2 or 100 cm 2 and a thickness of approximately 1.5 mm.
  • the product was treated in three different ways: (1) Blank: not inoculated product (2) LIS: product inoculated with a mixture of three Listeria monocytogenes (LISl, LIS2 and LIS3) strains at a realistic contamination level of 17 cfu/g (Table 6)
  • LIS + PHA product inoculated with phages (at 7x10 7 pfu/g or 1x10 9 pfu/dm 2 or 1x10 7 pfu/cm 2 ) and a mixture of three Listeria monocytogenes
  • Vacuum packaging was performed using a Multivac A300/42 (Hagenm ⁇ ller, Wolfertschr, Germany) gas packaging machine in a high barrier film (NX90, Euralpak, Wommelgem, Belgium) of 90 ⁇ m thickness with an oxygen transmission rate of 5.2 ml/m 2 24h atm at 23 0 C and 85% of relative humidity.
  • Table 7 presents the results of the determination of the total aerobic psychrotrophic count, the number of lactic acid bacteria and the number of Z. monocytogenes, ha Figure 2, the effect of phage PlOO on the growth of L. monocytogenes in cooked chicken fillet (vacuum, 7°C) is presented. Table 7. Total aerobic psychrotrophic count, lactic acid bacteria and L. monocytogenes
  • Lactic acid bacteria (loglO cfu/g)
  • the test product was 'Meesterlyck' cooked ham, an industrially prepared cured cooked ham that meets the criteria of Table 8.
  • the product was produced by Brackenier NV (Oosterzele, Belgium) and after production, the cooked hams were sliced and vacuum packaged per 50Og in the same company. The shelf-life at 7°C was estimated at ⁇ 2 weeks. Table 8. Criteria for 'Meesterlyck' cooked ham
  • K-nitrate max. 30 mg per kg
  • Na-nitrite max. 30 mg per kg
  • Na-nitrite + K-nitrate max. 50 mg per kg
  • Phosphate (P2O5)/protein max. 2,2
  • Glutamic acid max. 300 mg per kg
  • Citrate max. 100 mg per kg
  • Lactobacilli max. 5.000 per gram
  • Salmonella and Listeria monocytogenes absent in 25 gram
  • Escherichia coli max. 10 per gram
  • test product was first characterized by determining the pH, water activity (aw), salt level, dry matter, lactate level and nitrite level.
  • LIS product inoculated with a mixture of three Listeria monocytogenes (LISl, LIS2 and LIS3) strains (at a level of 10 cfu/g)
  • LIS + PHA-I product inoculated with phages (IxIO 7 pfu/cm 2 ) and a mixture of three Listeria monocytogenes (LISl, LIS2 andLIS3) strains (at a level of lO cfu/g)
  • LIS +PHA-2 product inoculated with phages (5x10 6 pfu/cm 2 ) and a mixture of three Listeria monocytogenes (LISl, LIS2 and LIS3) strains (at a level of 10 cfu/g)
  • LIS + PHA-I and LIS + PHA-2 more or less 30-60 seconds after inoculation with the L.monocytogenes mixture, the product was inoculated with the phage solution. From the appropriate dilution of the phage solution containing 2xl0 10 pfu/ml, 150 ⁇ l was divided over and spread on the surface of ⁇ 150 g of product with a spatula to reach the desired inoculation levels (PHA-I: ⁇ 1x10 7 pfu/cm 2 and PHA-2: ⁇ 5x10 6 pfu/cm 2 ).
  • NaCl is determined according to the method of Mohr (titrimetric determination of chloride ions)
  • Phage titer as a function of time of three replicate samples (Tl, T2 and T3) of the cooked ham and for both treatment levels (PHA-I and PHA-2)
  • Lactic acid bacteria (loglO cfu/g)
  • Lactic acid bacteria (loglO cfu/g)
  • Table 15 Total aerobic psychrotrophic count, lactic acid bacteria and number of L. monocytogenes as a function of time of three replicate samples of the Listeria-inoculated cooked ham that was treated with Listex at 5x10 6 pfu/cm 2
  • Lactic acid bacteria (loglO cfu/g)
  • Lactobacillus plantarum bacteriophage LP65 a new member of the SPOl- like genus of the family Myoviridae. J. Bacterid. 186, 7069-7083.
  • Gerba, C.P. Bitton, G. (Eds.), Phage Ecology. John Wiley & Sons, New York, pp. 289-316. 233. Leverentz, B., Conway, W.S., Alavidze, Z., Janisiewicz, WJ., Fuchs, Y., Camp,
  • Listeria phage A511 late gene region comprising the major capsid and tail sheath protein genes cps and tsh. J. Bacterid. 177, 6601- 6609.
  • Genome and proteome of Listeria monocytogenes phage PSA an unusual case for programmed +1 translational frameshifting in structural protein synthesis. MoI. Microbiol. 50, 303-317.

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Abstract

L'invention porte sur des compositions et procédés de traitement et prévention de la listériose utilisant des bactériophages de recombinaison ou des endolysines en dérivant.
PCT/IB2006/004168 2005-09-16 2006-09-15 P100 bactériophage p100 anti monocytogènes de listéria WO2007093849A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010010192A1 (fr) * 2008-07-25 2010-01-28 Profos Ag Nouvelle endolysine plyp40
US20120128652A1 (en) * 2008-07-25 2012-05-24 Martin Loessner Endolysin plyp40
WO2012159773A1 (fr) * 2011-05-26 2012-11-29 Dsm Ip Assets B.V. Endolysines pour le contrôle de listeria dans du fromage à pâte filée et les produits alimentaires connexes
JP2015525061A (ja) * 2012-05-07 2015-09-03 マイクレオス ビー．ブイ． サルモネラの生物的防除のための及び食品の製造若しくは加工におけるバクテリオファージ
CN113136371A (zh) * 2021-05-29 2021-07-20 甘肃农业大学 基于单增李斯特菌噬菌体的分离及筛选方法
EP3978514A1 (fr) * 2020-10-01 2022-04-06 Micreos Food Safety B.V. Nouveau phage pour la listeria, y compris la listeria monocytogenes

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2004004495A1 (fr) * 2002-07-08 2004-01-15 Exponential Biotherapies, Inc. Phages virulents destines a la lutte contre la listeria monocytogenes dans des produits alimentaires et des usines de transformation des aliments

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WO2004004495A1 (fr) * 2002-07-08 2004-01-15 Exponential Biotherapies, Inc. Phages virulents destines a la lutte contre la listeria monocytogenes dans des produits alimentaires et des usines de transformation des aliments

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CARLTON ET AL: "Bacteriophage P100 for control of Listeria monocytogenes in foods: Genome sequence, bioinformatic analyses, oral toxicity study, and application" REGULATORY TOXICOLOGY AND PHARMACOLOGY, ACADEMIC PRESS,NEW YORK, NY,, US, vol. 43, no. 3, December 2005 (2005-12), pages 301-312, XP005153177 ISSN: 0273-2300 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010010192A1 (fr) * 2008-07-25 2010-01-28 Profos Ag Nouvelle endolysine plyp40
JP2011528898A (ja) * 2008-07-25 2011-12-01 バイオメリュー エス．エー． エンドリシンＰｌｙＰ４０
US20120128652A1 (en) * 2008-07-25 2012-05-24 Martin Loessner Endolysin plyp40
WO2012159773A1 (fr) * 2011-05-26 2012-11-29 Dsm Ip Assets B.V. Endolysines pour le contrôle de listeria dans du fromage à pâte filée et les produits alimentaires connexes
JP2015525061A (ja) * 2012-05-07 2015-09-03 マイクレオス ビー．ブイ． サルモネラの生物的防除のための及び食品の製造若しくは加工におけるバクテリオファージ
EP3978514A1 (fr) * 2020-10-01 2022-04-06 Micreos Food Safety B.V. Nouveau phage pour la listeria, y compris la listeria monocytogenes
WO2022069607A1 (fr) * 2020-10-01 2022-04-07 Micreos Food Safety B.V. Nouveau phage pour listeria, y compris listeria monocytogenes
CN113136371A (zh) * 2021-05-29 2021-07-20 甘肃农业大学 基于单增李斯特菌噬菌体的分离及筛选方法

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