WO2018035407A1 - Bactériophages pour neutraliser des toxines et leurs procédés d'utilisation - Google Patents

Bactériophages pour neutraliser des toxines et leurs procédés d'utilisation Download PDF

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WO2018035407A1
WO2018035407A1 PCT/US2017/047503 US2017047503W WO2018035407A1 WO 2018035407 A1 WO2018035407 A1 WO 2018035407A1 US 2017047503 W US2017047503 W US 2017047503W WO 2018035407 A1 WO2018035407 A1 WO 2018035407A1
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toxin
bacteriophage
phage
binding protein
infection
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PCT/US2017/047503
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Jeffrey A. Radding
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Enbiotix, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C12N2795/10211—Podoviridae
    • C12N2795/10241—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to methods and compositions for neutralizing toxins.
  • the present invention provides bacteriophages for neutralizing bacterial toxins, and their use for treating, preventing, and controlling bacterial toxicosis in humans and animals.
  • Bacterial infections are often associated with the release of bacterial toxins that are responsible for the manifestation of the disease ("toxicoses"). While many bacteria release virulence factors that enhance the ability of the bacteria to survive in the infected host, for example, by subverting or avoiding the immune system, obtaining nutrients from the host, and inducing dormancy to survive host defenses and antibiotic treatment; for bacterial toxicoses, the disease can be recapitulated by the toxin itself in the absence of any bacteria.
  • Bacterial toxicoses include intestinal diseases caused by bacterial contamination of water sources across the world. These include cholera caused by Vibrio spp. and cholera toxin, as well as E. coli and Shigella diarrheal disease (e.g., shiga toxin). In addition, childhood immunizations against diphtheria and tetanus toxoids are part of routine immunizations in most of the world. Bacterial toxicoses are also relevant to the food industry. For example, necrotic enteritis in poultry is largely caused by Clostridium perfringens toxin. C. perfringens is a commensal bacteria found in about 75% of chickens.
  • the present disclosure provides bacteriophages that encode toxin- binding proteins for expression in host bacterial cells.
  • Host cells include toxin- producing bacteria, or commensal bacteria that do not themselves produce the target toxin.
  • the bacteriophage provides for the neutralization of toxin, including those produced by bacterial pathogens in humans and animals, such as Clostridium difficile (C. difficile), Clostridium perfringens (C. perfringens), Corynebacterium diphtheriae, Clostridium tetani, Clostridium botulinum, Vibrio cholerae, Enterotoxigenic E.
  • the toxin is produced by a bacterial pathogen, including a commensal bacterial species that becomes a pathogen due to pre-disposing conditions, for example, in the gastrointestinal tract, upper respiratory system, or nasopharyngeal tract.
  • the toxin-binding protein may take a variety of forms, including antigen- binding portions of antibody molecules, or engineered antibody platforms or antibody mimics known in the art.
  • An exemplary toxin-binding molecule is a single-chain variable fragment antibody (scFv).
  • the toxin-binding protein in some embodiments is directed against or neutralizes toxin A or toxin B from C. difficile.
  • the toxin is a C. perfringens toxin, such as beta-like toxin B.
  • C. perfringens is a significant pathogen of poultry.
  • the toxin-binding protein neutralizes pneumolysin, a toxin produced by S. pneumoniae during pneumococcal pneumonia.
  • the bacteriophage may be engineered from a lysogenic or lytic bacteriophage.
  • the bacteriophage is a lytic phage that targets/infects the toxin-producing bacteria.
  • the bacteriophage is a lytic phage that targets/infects a commensal bacteria, which may or may not produce the toxin.
  • the bacteriophage is a lysogenic phage that targets a commensal bacteria that does not itself produce the target toxin.
  • Exemplary bacteriophage include those of the order Caudovirales, such as Siphoviridae, Myoviridae and Podoviridae.
  • the toxin-binding protein may be released upon lysis of infected bacteria, or in addition or alternatively, release of the toxin-binding protein is directed by an encoded secretory signal.
  • the toxin-binding protein may be fused with a phage structural protein or other protein.
  • the present disclosure provides compositions suitable for use as therapeutics for treating, preventing, or controlling bacterial toxicosis, including C. difficile associated disease in humans, or necrotic enteritis in poultry, among others.
  • the compositions are suitable for use in treating or controlling pneumococcal pneumonia.
  • the compositions may comprise a carrier for delivering the bacteriophage to the site of infection, such as the GI tract or nasopharyngeal tract or middle ear.
  • bacteriophage may be mixed or applied to feed or drinking water, providing an economical means for treating large flocks or herds.
  • the present disclosure is related to methods of preventing, ameliorating, treating, or controlling a toxin-associated infection, comprising administering to the subject a bacteriophage described herein.
  • the subject may be a human or animal subject, and may have been exposed, or may be exposed, to toxin producing bacteria or spores of such bacteria, such as C. difficile or C. perfringens in some embodiments.
  • Figure 1 shows phage plaques resulting from transformation of genomic DNA isolated from natural GRCS phage, or engineered phage DNA assembled in a bacterial artificial chromosome vector, on Sau strain RN4220 (left panel), and amplification of DNA isolated from those phage plaques by PCR (right panel).
  • FIG 2 is a schematic showing sites of GFP gene insertion into the phage genome, assembled using a bacterial artificial chromosome vector.
  • Figure 3 shows that insertion of GFP gene at locus 1 results in only natural phage.
  • Figure 4 shows that insertion of GFP at locus 2 shows some GFP gene insert in recovery lysate.
  • Figure 5 shows that 0.5M sucrose stabilizes phage carrying the GFP gene in recovery lysate at locus 2.
  • the present disclosure provides bacteriophages that encode toxin-binding proteins for expression in host bacterial cells, as well as methods for treating, preventing, or controlling bacterial toxicoses.
  • Bacteriophages phage
  • the bacteriophages according to this disclosure express and optionally secrete anti-toxin molecules, including but not limited to single-chain antibodies, which will bind, sequester, and/or neutralize target toxins.
  • the toxin-binding protein binds to a toxin from a bacterial species, which in some embodiments is a human or animal pathogen, or a commensal bacteria of poultry, livestock or humans.
  • the pathogen is of the genus Clostridium, Vibrio, Escherichia, Streptococcus, Staphylococcus, or Shigella.
  • the toxin-producing bacterium is selected from Clostridium difficile (C. difficile), Clostridium perfringens (C.
  • the toxin-binding protein binds to a toxin from bacterium including, but not limited to, those disclosed in U.S. Patent Application No. 2009/0087478, which is hereby incorporated by reference in its entirety.
  • the target toxin is a Clostridium toxin.
  • Clostridium difficile toxin such as toxin A or toxin B
  • Clostridium perfringens toxin such as toxin types A, B, C, D, or E, or combination thereof, or beta- like toxin B.
  • C. perfringens toxins include C. perfringens alpha toxin (CPA), C. perfringens beta toxin (CPB or CPB2), epsilon toxin (ETX), iota toxin (ITX), perfringolysin O (PFO), C.
  • CPE perfringens enterotoxin
  • TpeL toxin perfringens large
  • NetB Necrotic enteritis toxin B-like
  • the toxin is pneumolysin, for example, as produced by
  • the toxin-binding protein may be an antibody or antigen-binding portion thereof.
  • Various antibody variants and antigen-binding platforms are known, including those for preparing small, compact, target-binding molecules. Examples include a single-domain antibody, a heavy-chain-only antibody (VHH), a single-chain variable fragment antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin, Adnectin, Tetranectin, Affibody; Transbody, Anticalin, Affilin, Microbody, peptide aptamers, phylomer, stradobody, maxibody, evibody, fynomer, an armadillo repeat protein, a Kunitz domain, avimer, atrimer, probody, immunobody, triomab, troybody, pepbody, UniBody, and DuoBody.
  • Exemplary antigen-binding formats are described in US Patent Nos. or Patent Publication Nos. US 7,417, 130, US 2004/132094, US 5,831,012, US 2004/023334, US 7,250,297, US 6,818,418, US 2004/209243, US 7,838,629, US 7, 186,524, US 6,004,746, US 5,475,096, US 2004/146938, US 2004/157209, US 6,994,982, US 6,794, 144, US 2010/239633, US 7,803,907, US 2010/1 19446, and/or US 7, 166,697, the contents of which are hereby incorporated by reference in their entireties.
  • the toxin-binding protein is a single-chain antibody.
  • a single-chain antibody is a modified variable heavy and/or light chains of human, camelid, or avian antibodies, which can be expressed as single domain proteins recognizing specific antigens. These single-chain antibodies generally lack immune effector function, as they do not contain the effector function domains of typical IgG from mammalian systems.
  • single-chain variable fragment antibodies (scFv) binding to the desired toxin target can be identified based on phage display, where the scFv is covalently attached to the phage particle, and screened for binding against specific antigens. See Z. A. Ahmad, et al. Clin Dev Immunol, vol. 2012, p.
  • scFv can also be screened in functional assays for inhibition of target toxin function.
  • the toxin-binding protein has a molecular weight of less than about 30 kDa, or less than about 20 kDa, or less than about 15 kDa, or less than about 10 kDa, or less than about 5 kDa. In various embodiments, the toxin-binding protein binds to its target with an affinity (KD) of better (less) than about 100 mM, or less than about 10 mM, or less than about 1 mM, or less than about 100 M, or less than about 10 ⁇ , or less than about 1 ⁇ , or less than about 100 nM, or less than about 10 nM, or less than about 1 nM in various embodiments.
  • KD affinity
  • the toxin-binding protein can comprise one or several (2, 3, or 4) VH and VL hypervariable region chains (the portion of each chain that together form the antigen-binding epitope) linked together in head to head or head to tail configurations by short peptide linkers.
  • a library of scFv antibodies (or other binding-protein format described above) is provided using a phage, yeast, ribosome or other display technology, which is useful for screening for binding proteins to a selected bacterial toxin antigen.
  • the target may be toxin B (tcdB), or alternatively toxin A (tcdA).
  • tcdB toxin B
  • tcdA toxin A
  • the target may be toxin A (pic) and/or ⁇ -like toxin (netB).
  • the gene encoding the scFv antibody is then inserted into a phage genome.
  • the phage may be a lytic phage of the family Podoviridae, such as phi24R, phiZP2, phiCPV4 or phiCP7R for C. perfringens, or similar phage.
  • the phage is a lytic phage of the P68 genus of the family Podoviridae such as GRCS, P68, 44AHJD, SAP-2, S24-1, 66 phage which targets Staphylococcus aureus.
  • the phage is Cp-1 or SOCP, or other member of the Picovirinae family that infects Streptococcal pneumoniae.
  • the phages can have broad spectrum activity against target bacterial field or clinical isolates, which may be tested prior to the initiation of genome engineering. For example, in the case of C. difficile, since it lacks identified lytic phages, temperate phages could be converted to lytic phages by engineering the phage to interfere with mechanisms of genomic integration. Alternatively, phage that infect common commensal organisms may be engineered in similar fashion.
  • lytic phages to C. difficile may be obtained by screening (e.g., screening of stool samples).
  • the phage is a lytic phage that infects commensal bacteria, which may or may not produce the toxin. In these embodiments, the phage may be engineered to be weakly lytic.
  • phage lysis is controlled by the expression and concentration-dependent oligomerization of holin to form a pore, which allows lysin to exit the cytoplasm and access the cell wall for degradation and lysis.
  • the oligomerization rate can be reduced and lysis delayed.
  • the holin and/or lysin genes are mutated to delay lysis.
  • the bacteriophage engineered to encode a toxin-binding protein may be produced according to techniques known in the art (See, US Patent Nos. 8,182,804, 9,056,899, and 8,153, 119; and U.S. Patent Application Publication No. 2015/0050717, all of which are incorporated herein by reference in their entirety).
  • the bacteriophage is produced by inserting into the phage genome the nucleic acid sequence encoding the toxin-binding protein.
  • the selected scFv or other toxin-binding protein may be either covalently attached (e.g., by translational fusion) to a phage protein, designed for secretion from the bacteria during phage infection, or released from phage-infected bacteria upon lysis.
  • a phage protein designed for secretion from the bacteria during phage infection, or released from phage-infected bacteria upon lysis.
  • the phage genome may be assembled in vitro, or by homologous recombination in yeast vectors, into a workhorse plasmid-based construct, such as a bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC).
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • Transformation of the assembled genome in the BAC or YAC may then be transformed into the target bacterial species and phage production assessed by plaque formation in a standard overlay assay.
  • the nucleic acid sequence encoding toxin-binding protein may be inserted into the phage genome in several different loci to determine expression levels using a combination of PCR detection of recovered engineered phage (e.g. for stability of the inserted gene) and immunodetection of epitope -tagged versions of the scFv genes to determine protein expression.
  • the nucleic acid sequence encoding toxin-binding protein described herein may contain a secretion sequence to promote secretion of the toxin-binding protein during phage infection; it may be expressed independently of secretion and released upon lysis of the phage-infected cell; or it may be fused to a capsid protein or other structural phage protein, and released as a phage/anti-toxin particle that sequesters the targeted toxin.
  • the phage is engineered for stability of the transgene, which can include positive selection in the presence of serially decreasing osmotic stabilizer (such as sucrose), and/or negative selection of phage without the transgene (e.g., by targeting CRISPR/Cas9 endonuclease to the wild type locus).
  • serially decreasing osmotic stabilizer such as sucrose
  • negative selection of phage without the transgene e.g., by targeting CRISPR/Cas9 endonuclease to the wild type locus.
  • the engineered phage is tested for anti-toxin activity in lysed culture supernatants of engineered phage, e.g., by ELISA and/or western blot against the target toxin.
  • the lytic activity of the engineered phage against a number of pathogen clinical or field isolates may compared to the spectrum of the original natural phage chosen for engineering. The spectrum may be enhanced by identifying the recognition baseplate proteins in the chosen phage genomes, and modification of these baseplate proteins by either baseplate swaps with other phage baseplates, or modification such as with other possible recognition elements (e.g. linear amphipathic cationic antimicrobial membrane-binding peptides).
  • the bacteriophage is a virulent phage.
  • the bacteriophage may be identified or engineered from a lysogenic bacteriophage.
  • a virulent phage may be created from the lysogenic bacteriophage by deletion of genes in the phage genome that are required for lysogeny.
  • the phage is of the family Podoviridae and subfamily Picovirinae. Examples include GRCS phage, which infects Staphylococcus aureus.
  • the phage (e.g., Cp- 1 or SOCP phage) infects Streptococcus pneumoniae.
  • Other families of phage which may be engineered in accordance with this disclosure, include Myoviridae and Siphoviridae.
  • the bacteriophage is a Podoviridae that contains a deletion of all or part of the minor tail protein. Phage genomes are small, and tightly packed, with only around 20 to 22 open reading frames. While most of the genes are not believed to be dispensable, some variants of Podoviridae, for example, exhibit truncated forms of the minor tail protein, suggesting that this open reading frame might be partially or completely deleted to make room for the gene encoding the toxin- binding protein. In some embodiments, this will reduce or alter the host range of the phage.
  • the toxin is a Clostridium toxin, and is optionally a Clostridium difficile toxin.
  • the bacteriophage may also infect C. difficile.
  • C. difficile is a gram-positive, motile, obligate anaerobic, spore- forming bacterium. It is the causative agent of CDAD ⁇ Clostridium difficile associated disease) in humans, usually predisposed by antibiotic therapy in hospital and long-term care facilities. It may cause severe colitis, sepsis and death.
  • the bacterium is acquired through ingestion of spores, usually by the fecal-oral route, and can be transmitted person to person.
  • the target toxin to be neutralized is toxin A or toxin B from C. difficile.
  • Toxin A ⁇ tcdA) and toxin B ⁇ tcdB) are chromosomally encoded at the PaLoc (pathogencity locus) that encodes a number of genes besides the toxin genes, and which contribute to virulence and pathogenicity.
  • PaLoc pathogencity locus
  • tcdA and tcdB are generally highly homologous to each other (M. Rupnik and S. Janezic, J Clin Microbiol, vol. 54, pp. 13- 8, Jan 2016).
  • CDT binary toxin
  • Toxin A and toxin B mechanism of action is to glucosylate the Rho- family of GTPases, inducing actin depolymerization (I. Just, et al., Nature, vol. 375, pp.
  • toxin-binding proteins can be engineered to contain antigen-binding sequences of actoxumab or bezlotoxumab.
  • the toxin-binding protein comprises an antigen-binding sequence of actoxumab or bezlotoxumab, optionally from 1 to 10 (e.g., 1 to 5) amino acid substitutions, insertions, or deletions.
  • the toxin-binding protein is designed to bind to a competing epitope to actoxumab or bezlotoxumab.
  • the bacteriophage infecting C. difficile may be identified or engineered from a lysogenic bacteriophage.
  • a virulent phage against C. difficile may be created from the lysogenic bacteriophage by deletion of genes in the phage genome that are required for lysogeny.
  • the lysogenic phages that infect Clostridium difficile have been described (J. Y. Nale, et al, Antimicrob Agents Chemother, vol. 60, pp. 968-81, Feb 2016; K. R. Hargreaves and M. R. Clokie, Front Microbiol, vol. 5, p. 184, 2014; US Patent Application Publication No.
  • the lysogenic bacteriophage include NCTC 12081404, NCTC 12081405, NCTC 12081406, NCTC 12081407, NCTC 12081408, NCTC 12081409, and NCTC 12081410.
  • the bacteriophage may be engineered from a phage selected from Caudovirales, Siphoviridae, Myoviridae and Podoviridae, for example.
  • the bacteriophage is against a commensal bacterium other than C. difficile.
  • the nucleotide sequence encoding the toxin-binding protein may be inserted into a lytic phage that infects a commensal bacterium.
  • the commensal bacterium may be of a species of Clostridiales, Lachnospiraceae, Ruminococcaceae, Veillonellaceae, Bacteroidales, Lactobaci Hales, Clostridium, Bacteroides, Fusobacterium, Streptococcus, Staphylococcus, Enterococcus, Parabacteroides, or Salmonella.
  • the commensal bacterium may be Escherichia coli or Lactobacillus spp. , commonly found in CDAD stool samples (J. Y. Chang, et al, J Infect Dis, vol. 197, pp. 435-8, Feb 1 2008; M. J. Hopkins and G. T. Macfarlane, J Med Microbiol, vol. 51, pp. 448-54, May 2002; M. J. Hopkins, R. Sharp, and G. T. Macfarlane, Gut, vol. 48, pp. 198-205, Feb 2001; A. Khoruts, et al., J Clin Gastroenterol, vol. 44, pp. 354-60, May-Jun 2010).
  • the commensal bacterium is Streptococcus (e.g., Streptococcus pneumoniae), which is commonly found in the upper respiratory system (e.g., nasopharyngeal tract), but can become pathogenic.
  • the bacteriophage is a phage that is deficient in lysis, but does not integrate its genome into the genome of the host bacteria. The phage may accumulate in the cytoplasm of the infected commensal bacteria, while expressing the anti-toxin and transporting it through a secretion pathway. The secreted anti-toxin may then be available to sequester toxin produced by C. difficile.
  • the antitoxin molecule is not secreted, but sequesters toxin inside the pathogenic bacteria (e.g., S. pneumoniae).
  • the target toxin is produced by Clostridium perfringens (C. perfringens).
  • C. perfringens Clostridium perfringens
  • the bacteriophage may infect C. perfringens, or may target another commensal bacteria.
  • the bacteriophage may be engineered from a phage selected from Caudovirales, Siphoviridae, Myoviridae and Podoviridae, for example.
  • C. perfringens is a commensal gram-positive facultative anaerobic spore- forming bacterium, found in the intestinal tract of poultry (e.g., in about 75 to 80% of chickens).
  • C. perfringens consists of 5 toxinotypes (A-E), each toxinotype secreting a different combination of toxins.
  • Type A is the most common cause of necrotic enteritis in poultry, producing primarily toxin A (pic), with type C less common, but producing both toxin A and toxin B (F. Van Immerseel, et al., Avian Pathol, vol. 33, pp. 537-49, Dec 2004).
  • beta-like toxin B (netB) has been shown to be a primary toxin causing the necrotic enteritis in poultry (A. L. Keyburn, et al, PLoS Pathog, vol. 4, p. e26, Feb 8 2008; A. L. Keyburn, et al., Toxins (Basel), vol. 2, pp. 1913-27, Jul 2010; K. A. Hassan, et al., Res Microbiol, vol. 166, pp. 255-63, May 2015; A. L. Keyburn, et al., Infect Immun, vol. 74, pp. 6496-500, Nov 2006; F. A.
  • Toxin A may be primarily involved in maintaining sub-clinical infection and in myonecrosis in poultry. Predisposing conditions that initiate overgrowth of the organism and induce necrotic enteritis include: stress on the animals and intestinal mucosal damage caused by parasitic infection (e.g., apicomplexan Eimeria spp. parasites). Intestinal damage caused by parasitic infection may release nutrients into the gut, prompting overgrowth of C. perfringens.
  • parasitic infection e.g., apicomplexan Eimeria spp. parasites
  • diets consisting of high indigestible, water-soluble non- starch polysaccharides, such as rye, wheat and barley, predispose chickens to necrotic enteritis. This may be due, in part, to the presence of fungal mycotoxins, such as fumonisin B, that have been shown to induce intestinal epithelial damage and are common contaminants in poultry feed (G. Antonissen, et al, Vet Res, vol. 46, p. 98, 2015).
  • fungal mycotoxins such as fumonisin B
  • the toxin to be neutralized is toxin beta-like toxin B
  • NetB from C. perfringens.
  • NetB is a beta-like pore-forming toxin similar to the Staphylococcus aureus a-hemolysin, ⁇ -toxin and leukocidin toxin. These toxins self- assemble and target host epithelial membranes, forming beta-barrel pore complexes in the membrane leading to cytosolic content leakage and loss of membrane polarity (A. L. Keyburn, et al., Toxins (Basel), vol. 2, pp. 1913-27, Jul 2010).
  • NetB is plasmid encoded and therefore represents a mobile genetic element for toxin transfer by conjugation.
  • the NetB pore complex consists of six monomers forming a ⁇ -barrel pore divided into three ultrastructural domains, the ⁇ -sandwich, rim, and stem (C. G. Sawa, et al., J Biol Chem, vol. 288, pp. 3512-22, Feb 1 2013).
  • the rim domain mediates binding of the complex to phospholipid membranes.
  • cholesterol has been shown to play a role in monomer oligimerization to form the pore structure, and thus monomer produced by the bacteria should be available for sequestration and inhibition of oligomer formation.
  • the bacteriophages may be lytic bacteriophages against C. perfringens (US Patent Nos. 9,320,795, 7,625,740, and 7,625,739, U.S. Patent Application Publication No. 2016/0076003, and 2016/0076004, C. A. Morales, et al., Arch Virol, vol. 157, pp. 769-72, Apr 2012, B. B. Oakley, et al, BMC Genomics, vol. 12, p. 282, 2011, B. S. Seal, Poult Sci, vol. 92, pp. 526-33, Feb 2013, B. S. Seal, et al , Arch Virol, vol. 156, pp. 25-35, Jan 2011, N. V.
  • the phages cover three families of Caudovirales, the Siphoviridae, Myoviridae and Podoviridae. These families are characterized by related ultrastructure of the virion particles, and not necessarily by genomic sequences. Phylogeny based upon genome relatedness is progressing as more phages are characterized and sub-families are being characterized through genome sequencing.
  • Podoviridae sequences for virulent phages to Clostridium perfringens have been deposited in the NCBI database (C. A. Morales, et al, Arch Virol, vol. 157, pp. 769-72, Apr 2012; N. V. Volozhantsev, et al., PLoS One, vol. 7, p. e38283, 2012).
  • the Podoviridae phages infecting C. perfringens are lytic phages with small genomes and are similar in structure and organization to the S. aureus phage GRCS.
  • the toxin-binding protein gene, selected by phage, yeast or other display technology, would be engineered into the C. perfringens phage genome according to these embodiments.
  • the encoded toxin-binding protein further comprises an
  • N-terminal secretory signal directing secretion of the toxin-binding protein from the host bacteria into the gut environment.
  • Exemplary secretory signal sequences are disclosed, for example, in U.S. Patent Publication No. 2015/0050717, which is hereby incorporated by reference in its entirety.
  • the toxin-binding protein is released into the gut environment upon lysis of the engineered phage-infected bacterium.
  • the toxin-binding protein is fused to a structural phage protein (e.g. capsid protein) or a lytic enzyme.
  • the bacteriophage comprises a promoter sequence operatively linked to direct expression of the anti-toxin protein gene disclosed herein.
  • the promoter is a bacteriophage promoter.
  • compositions may be included as part of a composition for delivering the phage to its intended site of action, such as the intestinal tract.
  • Compositions in some embodiments may contain pH resistant coatings, for delivering phage through acidic portions of the GI, including the stomach.
  • Compositions may be therapeutic compositions formulated for oral administration. In the case of poultry and livestock, compositions may be delivered mixed or applied to animal feed or drinking water.
  • the bacteriophage may be included as part of a composition for delivering the phage to the upper respiratory system, including the nasopharyngeal tract.
  • Compositions include those suitable for delivery to the throat, nasal passages, sinuses, and ear (including ear canal or middle ear).
  • the present disclosure is also related to methods of preventing, ameliorating, treating, controlling, or preventing recurrence of a toxin-associated infection.
  • the method comprises administering the bacteriophage described herein to a subject exhibiting symptoms of a bacterial toxicosis, or at risk of developing a bacterial toxicosis.
  • a subject can be at risk of developing a bacterial toxicosis when exposed to other subjects having the condition, or exposed to spores that place the subject at risk, such as Clostridium spores.
  • the patient is hospitalized, and/or is immunocompromised, placing the patient at risk of nosocomial infection.
  • toxin-associated infection or toxicosis is associated with C. difficile infection (e.g., CD AD) or C. perfringens infection (e.g., necrotic enteritis).
  • the present disclosure provides methods for treating Streptococcus pneumoniae infection, including pneumonia, otitis media, sinusitis, meningitis, and bacteremia.
  • the invention comprises administering the engineered phage against Streptococcus pneumoniae to a subject.
  • the subject may or may not receive antibiotic treatment concurrently.
  • the infection is antibiotic resistant.
  • the engineered phage persists in vivo for at least several days (e.g., about 2 to 5 days, such as about 3, about 4, or about 5 days), or in some embodiments from 1 to 2 weeks.
  • the persistence of the phage allows treatment or prevention of acute infection and toxicosis, without long term impact on the subject or microbiome of the subject.
  • C. difficile infections For C. difficile infections, the current standard of care is treatment with gram- positive antibiotics. Approximately 20% of patients treated and cured of the acute infection experience recurrent infections, and treatment with antibiotics becomes less effective. The recurrence is thought to arise through the germination of spores of the bacterium in the gut, and this may be triggered by release of the toxins during sporulation, preparing the micronutrient environment in the gut to support germination by degrading the intestinal epithelium. Sequestration of the toxin prevents germination most likely (precise mechanisms remain unknown) by maintaining a nutrient poor environment for germination and allowing the gut epithelium to repair itself. Spores are eventually passed through the gut and eliminated in the feces.
  • the anti-toxin engineered bacteriophage described herein has distinct advantages over passive immunization.
  • a lytic phage engineered against the pathogen itself would generate anti-toxin, thus reducing the likelihood of recurrence, and also kill the bacteria directly through its lytic activity.
  • the alternative strategy of infecting commensal bacteria provides anti-toxin activity, but will not kill the target (toxin-producing) bacterium.
  • This type of therapeutic approach may require concurrent use of standard-of-care antibiotics. In either case, targeted at C.
  • the bacteriophage may be delivered to flocks through feed or drinking water.
  • the dose of the bacteriophage could be relatively low per bird, and thus could have significant economic and logistical advantages over flock vaccination.
  • Example 1 Engineering of GRCS Phage against Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus is a Gram-positive, catalase-positive, coagulase-positive, facultative aerobe that is an opportunistic pathogen with a high prevalence of antibiotic resistance genes and the ability to form biofilms on abiotic surfaces, rendering Sau infections often recalcitrant to standard antibiotic therapy. The development of phage therapy to treat Sau infections is therefore gaining prominence as an alternative to traditional antibiotic treatment.
  • SAgs Superantigens
  • SEB entertoxin B
  • SEB entertoxin B
  • aching feeling head and muscles
  • enteric dysfunction i.e., nausea, vomiting, and diarrhea
  • Nanogram levels of inhaled SEB are incapacitating, while microgram levels can be fatal.
  • SEB staphylococcal enterotoxin A
  • SEB is carried on a chromosomal Sau pathogenicity island (SaPI), a mobile genetic element that under stress conditions can excise from the chromosome, package into virus like particles and be transmitted to other Sau strains.
  • SEB induces inflammatory cytokines, including TNF-a, interleukin (IL)-2, IL-6, IL-10, and interferon- ⁇ , and chemokines, including monocyte chemoattractant protein 1, and normal T-cell expressed and secreted proteins.
  • T cell-mediated toxicity is initially mediated by TNF-a induced inflammation, resulting in SEB-induced septic shock.
  • SEB in vitro studies showed that SAgs activate TNF transcriptionally in T cells and monocytes, the major cause of shock syndrome.
  • SEB alone can induce fatal disease, as demonstrated by intrapulmonary instillation of purified SEB inducing hemorrhagic lung tissue, respiratory distress, and lethal toxic shock syndrome in rabbits.
  • the three-dimensional structure of staphylococcal enterotoxin B has been determined to a resolution of 2.5 A.
  • the structure contains an unusual two domain, main chain fold, a general motif adopted by most staphylococcal enterotoxins.
  • a shallow cavity formed by both domains forms the T-cell receptor binding site, while the MHCII molecule binds to an adjacent site.
  • a strain, isolated from a human prosthetic joint infection, known to carry the SEB gene was tested in both planktonic and biofilm cultures for SEB expression and mitogenic activity. Transcription of SEB was significant and mitogenic activity was similarly high, suggesting that this strain produces biologically active SEB.
  • This strain is also readily infected by the lytic dsDNA phage GRCS, as detected by the rapid increase in the major capsid protein gene expression by PCR upon infection by phage and rapid lysis in short term planktonic culture.
  • Bacteriophage GRCS is a member of the Picovirinae subfamily of the Podoviridae family of viruses and has shown a high rate of infectivity toward Sau isolates that have been recovered from prosthetic joint infections.
  • the natural GRCS phage genome was introduced into a bacterial artificial chromosome using PCR fragments from isolated phage DNA and reconstructed in the BAC vector using Gibson assembly. Transformation of the assembly mixture into E. coli amplified the construct, which was then isolated and used for transformation in the GRCS Sau host strain.
  • the GRCS/BAC transformation was allowed to recover for up to 3 hours and the recovery supernatant was tested for phage production by overlay plating on Sau strain RN4220. Plaque formation by natural phage transduction and the transformation supernatant were similar, illustrating efficient transduction by both natural phage and reconstructed engineered phage ( Figure 1, left panel). Phage DNA from the respective plates was then prepared and amplified by PCR using primers used to prime the Gibson assembly fragment, and resulted in identical banding pattern on gel electrophoresis ( Figure 1, right panel), demonstrating that plaques formed by the GRCS/BAC construct were GRCS phage.
  • a reporter gene green fluorescent protein
  • the GFP gene is approximately 700 bp and encodes the 238 amino acid (26.9kDa) GFP protein. This is approximately the same size as a single-chain fragment variant antibody (scFv), with a molecular mass of approximately 27 kDa.
  • the purpose of inserting GFP was to examine the different loci for gene expression from the phage genome for placement of an anti-toxin scFv or VHH antibody.
  • the GFP construct at loci 1 yielded transducing phage.
  • Individual isolated plaques were tested for the presence of the GFP gene using the set of primers pictured schematically on the upper right ( Figure 3).
  • the loss of the GFP insert is clear, as the lane containing the BAC-GRCS-GFP1 control shows the distinct band at 1.3 kb, corresponding to the inserted GFP 1 gene.
  • the plaques however, showed no presence whatsoever of the GFP1 gene.
  • Detection of the phage DNA was confirmed using a set of primers (D and E) to another site in the GRCS genome. This suggested a loss of the GFP 1 gene upon transformation and recovery, and that insertion in this locus is unstable.
  • the insertion of the trans-genes will be stabilized by initially providing osmotic support to counteract the imbalance in head pressure.
  • the phage head will be subjected to negative selective pressure for reversion to the natural phage by targeting a CRISPR- Cas endonuclease to natural phage revertant sequences at the site of trans-gene insertion, thus cutting the natural phage genome, while maintaining the engineered GFP phage genome integrity.
  • the engineered phage containing the GFP trans-gene will be passaged for several generations under conditions of progressively decreasing osmotic support, in the presence of CRISPR-Cas negative selection for the natural phage.
  • the insertion of a 700 bp reporter gene will produce an intact phage particle that can accommodate an anti-toxin scFV or VHH antibody of approximately the same size.
  • the evolved genome of the engineered phage will be used as a new scaffold for insertion of trans-genes of similar size for either measurement of expression using GFP at different loci, or insertion of the anti-toxin protein.
  • the target for the anti-toxin protein would be SEB, and it would be expected that phage infection of an SEB carrying strain by the engineered anti-toxin phage would kill the bacteria by lysis, but also neutralize the cyto- and immune- toxic actions of SEB.
  • Example 2 Engineering phage targeting Streptococcus pneumoniae for production of anti-pneumolysin antibody
  • Streptococcus pneumoniae is a Gram-positive lancet shaped diplococci, and a common commensal organism found in the nasopharyngeal tract in human. Upon viral infection or other insults to the upper respiratory system, Spn can become pathogenic and cause pneumonia, otitis media, sinusitis, meningitis and bacteremia [1] . In the United States there are approximately 900,000 cases per year of pneumococcal pneumonia, of which 400,000 cases require hospitalization [2]. This is despite the availability of both polyvalent polysaccharide vaccine (PP23) and polyvalent conjugate vaccine (PCV 13, Prevnar) [3-6].
  • PP23 polyvalent polysaccharide vaccine
  • PCV 13, Prevnar polyvalent conjugate vaccine
  • Phage Cp- 1 and SOCP are virulent members of the Picovirinae subfamily of the Podoviridae family of viruses that specifically infect Spn [9]. These phages have small genomes, ⁇ 19kB, with 345 bp inverted terminal repeats and covalently linked terminal proteins, required for DNA replication and packaging. These phage genomes have no significant sequence homology to the Sau phage GRCS (Example 1), but are organized in similar functional clusters, ordered differently, and transcribed in a single direction, left to right. The phage engineering methodology utilized for constructing the Sau phage GRCS would be amenable to engineering the Spn phages CP- 1 and SOCP.
  • Pneumolysin is a 52.7 kDa cholesterol-binding protein that oligomerizes in the eukaryotic membrane, forming a very large circular pore consisting of 42 membrane -inserted monomers, and roughly 350-450 A in diameter [10, 11].
  • pneumolysin also directly induces specific inflammatory responses at sub-lytic concentration [12-14].
  • pneumolysin's role in infection has been demonstrated by the protective effect of antibodies to pneumolysin in attenuating aspects of pneumococcal pneumonia, including cytokine activation, inflammation, lung injury and infection [15, 16]. Inactive forms of pneumolysin have been used to generate protective antibody response and are broad spectrum, serotype independent, vaccine candidates [17, 18]. In addition, it has been shown that lytic antibiotics, such as ⁇ -lactams, can release significant amounts of pneumolysin from lysing pneumococcal bacteria, exacerbating potential lung injury in the treatment of pneumonia [19].
  • Neutralizing pneumolysin as a payload would be advantageous in phage therapy. Natural phages with their lytic action would release pneumolysin from Spn, and thus may exacerbate lung injury in treating pneumococcal pneumonia, similar to the effect of lytic ⁇ -lactam antibiotics. The delivery of an anti-toxin molecule, via phage infection and bacterial expression, would neutralize the effect of pneumolysin from Spn, minimizing its inflammatory and cytotoxic impact on the lung epithelium.
  • an engineered anti-toxin phage would provide superior efficacy in treatment of pneumococcal pneumonia, and other Spn related infections, by clearing the infection by Spn, but also ameliorating or preventing significant epithelial cell damage and inflammation due to pneumolysin.
  • NCEZID National Center for Emerging and Zoonotic Infectious Diseases
  • DHQP Healthcare Quality Promotion

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Abstract

La présente invention concerne des bactériophages qui codent pour des protéines de liaison à la toxine pour l'expression dans des cellules bactériennes hôtes, ainsi que des procédés de traitement, de prévention ou de lutte contre les toxicoses bactériennes. Les bactériophages selon la présente invention expriment et sécrètent éventuellement des molécules anti-toxines, comprenant, mais sans y être limitées, des anticorps à chaîne unique, qui se lient, séquestrent, et/ou neutralisent des toxines cibles.
PCT/US2017/047503 2016-08-19 2017-08-18 Bactériophages pour neutraliser des toxines et leurs procédés d'utilisation WO2018035407A1 (fr)

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CN112703201A (zh) * 2018-07-05 2021-04-23 诺沃班德畜牧业治疗公司 针对家禽致病原的抗体和其用途

Citations (2)

* Cited by examiner, † Cited by third party
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US20140120614A1 (en) * 2004-02-06 2014-05-01 Medarex, L.L.C. Antibodies against clostridium difficile toxins and uses thereof
US20150050717A1 (en) * 2009-03-05 2015-02-19 Massachusetts Institute Of Technology Bacteriophages expressing antimicrobial peptides and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140120614A1 (en) * 2004-02-06 2014-05-01 Medarex, L.L.C. Antibodies against clostridium difficile toxins and uses thereof
US20150050717A1 (en) * 2009-03-05 2015-02-19 Massachusetts Institute Of Technology Bacteriophages expressing antimicrobial peptides and uses thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112703201A (zh) * 2018-07-05 2021-04-23 诺沃班德畜牧业治疗公司 针对家禽致病原的抗体和其用途

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