WO2018126108A1

WO2018126108A1 - Bacteriophage having modified recognition baseplate protein structural domains

Info

Publication number: WO2018126108A1
Application number: PCT/US2017/068893
Authority: WO
Inventors: Jeffrey A. Radding
Original assignee: Enbiotix, Inc.
Priority date: 2016-12-29
Filing date: 2017-12-29
Publication date: 2018-07-05

Abstract

The present invention relates to engineered Picovirinae bacteriophages, as well as to methods and compositions for the treatment of bacterial infections. Particularly, the present invention provides bacteriophages harboring a modified recognition baseplate protein which allows the bacteriophage to have an altered or broadened infectivity profile.

Description

BACTERIOPHAGE HAVING MODIFIED RECOGNITION BASEPLATE PROTEIN STRUCTURAL DOMAINS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/439,978, filed December 29, 2016, the content of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates, in part, to engineered Picovirinae bacteriophages and recognition baseplate proteins having a broadened or altered scope of infectivity. SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 28, 2017, is named ENB- 004PC_SequenceListing_ST25.txt and is 42,395 bytes in size. BACKGROUND OF THE INVENTION

Bacteriophage are very specific for bacterial surface epitopes, such that, even within a species, bacterial recognition of strains of that species by a specific bacteriophage will be limited to a particular subset of strains from that species. Bacterial surface recognition is the lead event to ultimate infection of the bacteria by the phage. Resistance to phage is often acquired by bacteria through modification or masking of the bacterial surface epitopes recognized by a given phage. This leads to the need for cocktails of natural phages to cover broadly any given bacterial species, and thus introduces both regulatory and manufacturing impediment to phage therapeutics, due to the uncharacterized nature of using multiple unrelated phages and the need to produce each unique phage of the cocktail. Accordingly, there remains a need for bacteriophage with broadened or tailored recognition of bacterial strains, and in particular, bacterial strains that are threats to human or animal health.

SUMMARY OF THE INVENTION In one aspect, the present invention provides engineered recognition baseplate proteins and Picovirinae bacteriophages comprising the same, with a broadened or altered scope of infectivity against bacterial strains. The broadened or altered scope of infectivity is attributable to a modified recognition baseplate protein with modifications in the recognition domain, e.g., the C-terminal domain in some embodiments. For example, the recognition baseplate protein may have a C-terminal domain that has been swapped with the C-terminal domain of another Picovirinae bacteriophage, or an engineered derivative thereof. Alternatively or in addition, the modified recognition baseplate protein may include specific amino acid alterations or a replacement of the recognition domain (or portion thereof) with an Antimicrobial Peptide (AMP) amino acid sequence. The modified bacteriophage can be specific in some embodiments to a bacterial species of the genus Staphylococcus, Streptococcus, Clostridium, Bacillus, Corynebacterium, and Propionibacterium acnes among others. In some embodiments, the modified bacteriophage is specific for Staphylococcus aureus strains.

Accordingly, in one aspect, the present invention provides methods for treating bacterial infections by administering to a subject an engineered bacteriophage comprising the modified recognition baseplate protein. The engineered bacteriophage may recognize a surface determinant on a Gram-positive bacterial species (e.g., Staphylococcus aureus) associated with diseases or conditions including implanted devise-related infections, osteomyelitis, abscess, pneumonia, endocarditis, phlebitis, mastitis, meningitis, metritis, and/or septic shock or toxic shock syndrome and acne vulgaris. The bacteriophage can promote elimination of the bacterial pathogen, and in some embodiments, the bacteriophage can be further engineered to promote clearance of other microbial agents in the infection, and/or promote clearance of a biofilm associated with the infection, and/or block or sequester toxins or enzymes associated with the infection or pathology. Exemplary additional bacteriophage engineering strategies include expression of biofilm-degrading enzymes or lytic enzymes at the infection site, and/or the expression of one or more antibiotic potentiating agents at the infection site, among others.

In various embodiments, the bacteriophage may be provided as a pharmaceutically-acceptable composition suitable for application to subjects afflicted with susceptible bacterial infections.

Other aspects and embodiments of the invention will be apparent from the following detailed description.

DESCRIPTION OF THE DRAWINGS

Figures 1A-D shows a dot-matrix homology analysis of ORF14 (SEQ ID NO: l) from the GRCS bacteriophage and homologous proteins from four other Picovirinae phages (i.e., P68, SI 3', S24-1, and SAP2).

Figures 2A-C shows a BLASTP analysis of the N-terminal and C-terminal domains of ORF14. Figure 2A aligns the N-terminal of GRCS ORF14 (amino acids 1- 151 of SEQ ID NO: l) with one ORF14 homologue in the Picovirinae phage SLPW (amino acids 1-151 of SEQ ID NO: 8). Figure 2B aligns the C-terminal of GRCS ORF14 (amino acids 172-309 of SEQ ID NO: l) with another ORF14 homologue in the Picovirinae phage SLPW (amino acids 9-151 of SEQ ID NO:9). The analysis reveals that the ORF14 homologue in the Picovirinae phage SLPW is split into two separate genes. Figure 3 shows a global protein alignment of ORF14 (SEQ ID NO: 1) with other Staphylococcal phages (SEQ ID NOs: 10, 3, 11, 12, 13, 2, 4, 14, 5, 15, and 16, respectively). Amino acids 140 to 200 of ORF14 (SEQ ID NO: 1) and aligned sequences are highlighted.

DETAILED DESCRIPTION OF THE INVENTION

Picovirinae is a sub-family of viruses in the order Caudovirales, family Podoviridae. There are currently 13 genus in Podoviridae, divided among 2 major subfamilies (Autographiniae and Picovirinae) and a set of diverse genus. The Podoviridae family is characterized by having very short, non-contractile tails. Picovirinae viruses are non-enveloped, with icosahedral and head-tail geometries and further distinguished in having a double stranded (ds) linear genome that utilizes a protein-primed DNA replication mechanism. A further distinguishing characteristic of Picovirinae is the requirement for the covalently-linked terminal protein on the dsDNA to package their dsDNA into the phage head. Picovirinae bacteriophages have been isolated that infect diverse Gram-positive bacterial hosts, including Staphylococcus, Streptocococcus, Clostridium, Propionibacterium, Bacillus and Cornybacterium, among others.

A Picovirinae bacteriophage has been isolated having Staphylococcus aureus as a natural host (GRCS). See generally, Xia and Wolz, Phages of Staphylococcus aureus and their impact on host evolution. Infection, Genetics and Evolution 21 :593- 601 (2014). The GRCS bacteriophage was isolated from raw sewage collected from a treatment plant in India, and its complete genome sequence is known. Swift and Nelson, Complete Genome Sequence of Staphylococcus aureus Phage GRCS. Genome Announc. Vol. 2, Issue 2 (2014). GRCS is a lytic phage.

Infections by Staphylococcus aureus are difficult to treat due to the virulent nature of the bacteria and rapid biofilm production. These microbial organisms secrete numerous enzymes and toxins resulting in pain, inflammation, and other symptoms. Further still, these microbial organisms generate biofilms, which can protect the microorganisms from the host immune system and from antibiotics.

Bacteriophages active against bacterial pathogens (including Staphylococcus aureus) represent a promising alternative to antibiotics in combating bacterial infections. However, bacteriophages are very specific for bacterial surface epitopes. Even within a species, a particular bacteriophage is often limited in its recognition of only a subset of strains from that species. Bacterial surface recognition is a critical event to ultimate infection of the bacteria by the phage. This is evident from the observation that phage resistance acquired by bacteria is often through modification or masking of bacterial surface epitopes recognized by a given phage.

The present invention provides engineered Picovirinae bacteriophages with a broadened or altered profile of infectivity, including against clinical isolates of the host species. As demonstrated herein, the modified recognition baseplate protein of Picovirinae can be modified to alter or broaden the infectivity profile. The engineered bacteriophage has an altered infectivity profile that is distinct from the original (parent) bacteriophage.

In an embodiment, the engineered bacteriophage is a lytic phage. In another embodiment, the engineered bacteriophage is a lytic bacteriophage that is unable to actively propagate the phage infection in natural wild-type bacterial species. In an embodiment, the bacteriophage is a Picovirinae GRCS bacteriophage. Additional exemplary bacteriophages include P68, SAP-2, S13', and S24-1, which can also have alterations in the C-terminal domain of the recognition baseplate protein for altered strain specificity. Corresponding recognition baseplate proteins are provided herein as SEQ ID NOS:2 to 5.

Other Picovirinae bacteriophages can be modified in accordance with the disclosure. Such bacteriophages generally contain a recognition baseplate protein with structural homology to the GRCS bacteriophage recognition baseplate protein (SEQ ID NO: l). Structural homology can be identified by searches of SCOP (structural classification of proteins database) or PDB, for example. In some embodiments, the C- terminal domain has structural homology to GRCS recognition baseplate protein, as well as other phage proteins such as L. lactis phage T901 and phage bIL170 baseplate protein. In some embodiments the bacteriophage is specific for a bacterial host that is gram positive, such as species of Staphylococcus, Streptococcus, Clostridium, Bacillus, Propionobacterium or Corynebacterium.

In various embodiments, the recognition baseplate protein is based on the GRCS bacteriophage protein ORF14. The wild-type ORF14 protein comprises the amino acid sequence of SEQ ID NO: l . In some embodiments, the modified recognition baseplate protein has at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or about 99% identity with SEQ ID NO: l. However, the modified recognition baseplate protein has one or more amino acid mutations relative to SEQ ID NO: l . In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, or deletions. For example, in some embodiments, the modified recognition baseplate protein has from 1 to about 50, or from 1 to about 25, or from 1 to about 15, or from 1 to about 10 amino acid insertions, deletions, and/or substitutions relative to SEQ ID NO: l .

In various embodiments, the modified recognition baseplate protein has one or more amino acids corresponding to positions 140-200 of SEQ ID NO: l modified by amino acid substitution, insertion, and/or deletion. In some embodiments, the positions corresponding to positions 140-200 of SEQ ID NO: l are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the corresponding amino acids of SEQ ID NO:2 (P68), SEQ ID NO:3 (SAP-2), SEQ ID NO:4 (S13'), and SEQ ID NO:5 (S24-1). See Figure 3. In various embodiments, the recognition baseplate protein has a modified C- terminal domain. In an embodiment, the C-terminal domain comprises amino acids corresponding to positions 170-309 of SEQ ID NO: l. Further, positions 140 to 200 (with reference to SEQ ID NO: l) are divergent between Staphylococcus aureus bacteriophages, and thus may represent an opportunity to alter or improve strain specificity. In some embodiments, the C-terminal domain is modified by one or more amino acid substitutions, insertions, and/or deletions, such as from 1 to about 50, or from 1 to about 25, or from 1 to about 15, or from 1 to about 10 amino acid insertions, deletions, and/or substitutions in relation to positions 170 to 309, or in relation to positions 140 to 200 of SEQ ID NO: 1. In some embodiments, the modified recognition baseplate protein has the C- terminal domain swapped with or derived from the C-terminal domain of another Staphylococcus aureus Picovirinae bacteriophage. For example, the C-terminal domain may be swapped with or derived from the Picovirinae bacteriophage recognition baseplate protein of P68 (SEQ ID NO:2), SAP-2 (SEQ ID NO:3), S13' (SEQ ID NO:4), or S24-1 (SEQ ID NO:5), which may further have from 1 to about 50, or from 1 to about 25, or from 1 to about 15, or from 1 to about 10 amino acid insertions, deletions, and/or substitutions.

In some embodiments, the modified recognition baseplate protein has a portion of the recognition domain (e.g., the C-terminal domain) replaced with one or more antimicrobial peptide (AMP) sequences. In some embodiments, the AMP is a helical amphipathic AMP. Without being bound by theory, it is anticipated that helical amphipathic AMPs bind to bacterial membranes in a less specific manner than the wild type recognition baseplate protein, and thus when substituted for or inserted into the recognition domain, can provide a broader or different infectivity profile against S. aureus strains. In some embodiments, the AMP can be selected according to the antimicrobial activity of the AMP for target bacterial species as a free peptide. In some embodiments, the bacteriophage may harbor a modified recognition baseplate protein in which the entire C-terminal domain is replaced with one or more AMP sequences.

Antimicrobial peptides (AMPs) are also called host defense peptides and are produced by species ranging from bacteria, fungi, insects, frogs, and mammals as part of the innate immune response. In some embodiments, the antimicrobial peptide comprises about 10 to about 60 amino acids, or about 12 to about 50 amino acids. In some embodiments, the antimicrobial peptide may include two or more positively charged residues provided by, for example, arginine or lysine, and a large proportion (e.g., greater than 50%) of hydrophobic residues. In some embodiments, the secondary structures of the antimicrobial peptides may be, for example, a-helical, β-stranded (e.g., due to the presence of 2 or more disulfide bonds), β-hairpin or loop (e.g., due to the presence of a single disulfide bond and/or cyclization of the peptide chain), and extended. In an embodiment, the antimicrobial peptide may be an anionic peptide, for example, rich in glutamic and aspartic acids. In another embodiment, the antimicrobial peptide may be a linear cationic α-helical peptide, for example, lacking in cysteine. In a further embodiment, the antimicrobial peptide may be a cationic peptide enriched in specific amino acids. For example, the antimicrobial peptide may be rich in proline, arginine, phenylalanine, glycine, or tryptophan. In another embodiment, the antimicrobial peptide may be an anionic and cationic peptide that contains at least one cysteine and disulfide bond. For example, the antimicrobial peptide may include about 1 to about 3 disulfide bonds. Exemplary antimicrobial peptides include, but are not limited to, Indolicidin, Cecropin PI, Dermaseptin, Ponericin Wl, Ponericin W3, Ponericin W4, Ponericin W5, Ponericin W6, Maximin H5, Dermcidin, Andropin, Moricin, Cerototoxin, Melittin, Megainin, Bombinin, Brevinin, Esculentin, Buforin, CAP 18, LL37, Abaecin, Prophenin, Protegrin, Tachyplesin, Defensin, Drosomycin, or variants thereof. Additional antimicrobial peptides include those described in U.S. Patent Publication No. 2015/0050717, which is hereby incorporated by reference in its entirety.

In some embodiments, the bacteriophage is also engineered to express an antimicrobial peptide in host cells, that is, independent of the recognition baseplate protein. In some embodiments, the antimicrobial peptide is expressed under the control of a bacterial or bacteriophage promoter. In an embodiment, the antimicrobial peptide is expressed under the control of the endogenous bacteriophage promoter. In another embodiment, the antimicrobial peptide is expressed under the control of an exogenous promoter derived from another bacteriophage. In some embodiments, the antimicrobial peptide contains a signal sequence for directing its secretion from bacterial host cells, such as Staphylococcus aureus. In some embodiments, the bacteriophage directs expression of an antimicrobial peptide selected from an Apidaecin and/or Oncocin. Apidaecins (apidaecin-type peptides) are a series of small, proline-rich (Pro-rich), 18- to 20-residue peptides, which are naturally produced by insects. Structurally, Apidaecins consist of two regions, the conserved (constant) region, responsible for the general antibacterial capacity, and the variable region, responsible for the antibacterial spectrum. The small, gene-encoded and unmodified apidaecins are predominantly active against many Gram-negative bacteria by special antibacterial mechanisms.

In various embodiments, the bacteriophage is engineered to encode one or more additional enzymes or polypeptides, which when expressed by the target bacteria, enhance the effectiveness for clearing bacterial infections. In various embodiments, the bacteriophage is engineered to comprise a nucleic acid encoding a biofilm-degrading enzyme, such that the enzyme is expressed and optionally secreted by infected bacteria. Biofilms are polymeric structures secreted by microbial organisms such as bacteria to protect the bacteria from various environmental attacks, such as, host defenses, antibiotics and disinfectants. Biofilms have a regulated lifecycle including attachment, maturation and dispersal phases. For example, initial attachment in Staphylococcal biofilm is generally mediated in part by protein-protein interactions, as Staphylococcus aureus expresses receptors for a number of host plasma proteins including fibrin and fibrinogen. Staphylococcal biofilms are composed of three classes of molecules forming the extracellular polymeric substance: poly-beta- 1,6-N-acetylglucosamine (PNAG), proteins including phenol soluble modulins, Staphylococcus protein A, and others, as well as extracellular DNA of both bacterial and host origin. Further, there are differences between S. aureus and S. epidermidis biofilms. For example, S. epidermidis RP62A biofilm is degraded by DspB enzyme, and not by proteinase K or bovine DNase I, whereas S. aureus biofilms are insensitive to DspB, but degraded by proteinase K and DNase I.

Bacteria in biofilms can be tolerant to antibiotic therapy. Tolerance can be due to the inability of the antibiotic to achieve significant concentrations in the biofilm, coupled with the metabolic quiescence of some biofilm bacteria. Thus, biofilm associated infections are difficult to treat with standard antibiotic therapy.

Biofilms may be found on any surface, including, prosthetic joints and other implantable or indwelling devices. Biofilm-degrading enzymes degrade biofilm matrix polymers by inhibiting biofilm formation, detach established biofilm colonies, and render biofilm-forming cells sensitive to killing by antimicrobial agents. Exemplary enzymes useful for breaking down biofilms include, but are not limited to, dispersin B, alginate lyase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, disaggregatase enzymes, esterase, alpha-galactosidase, beta- galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, polysaccharide depolymerase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, DNase I, or lyase. In some embodiments, the biofilm-degrading enzyme include cellulases, such as the glycosyl hydroxylase family of cellulases (e.g., glycosyl hydroxylase 5 family of enzymes also called cellulase A), polyglucosamine (PGA) depolymerases, and colonic acid depolymerases (e.g., 1,4-L-fucodise hydrolase), depolymerazing alginase, and DNase I. Additional biofilm-degrading enzymes are described, for example, in U.S. Patent No. 8,153,119, which is hereby incorporated by reference in its entirety. In an embodiment, the bacteriophage is engineered to comprise a nucleic acid encoding Dispersin B, an enzyme that hydrolyzes -l,6-N-acetyl-D-glucosamine. Examples of a Dispersin B gene are described, for example, in U.S. Patent No. 8,153,119, which is hereby incorporated by reference in its entirety. In an embodiment, the Dispersin B gene comprises the nucleotide sequence of Dispersin B from Actinobacillus actinomycetemcomitans , as shown for example in SEQ ID NO:6, and/or comprises the amino acid sequence of SEQ ID NO:7, or functional variant thereof.

In various embodiments, the functional variant of the Dispersin B enzyme has an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical with SEQ ID NO:7. Functional variants can be determined by assaying for hydrolysis of -l,6-N-acetyl-D-glucosamine. In various embodiments, the Dispersin B may have one or more amino acid mutations relative to SEQ ID NO: 7. For example, the Dispersin B may have from 1 to about 20, or from 1 to about 15, or from 1 to about 10 amino acid mutations relative to SEQ ID NO:7. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, and/or deletions.

In various embodiments, the bacteriophage is engineered to comprise a nucleic acid encoding at least one antimicrobial polypeptide, such that the antimicrobial polypeptide is expressed and optionally secreted by host bacteria. In some embodiments, the antimicrobial polypeptide is a lytic enzyme, such as an endolysin, a lysozyme, a lysostaphin, or a functional derivative thereof. These enzymes range in size from 50 to several hundreds of amino acids, and are predominantly used by bacteriophages and bacteria in inter- and intraspecies bacteriocidal warfare. In an embodiment, the enzymes induce the lysis of Gram-positive and/or Gram-negative bacteria. For example, the enzymes may effectively lyse one or more of Staphylococcus aureus, coagulase-negative staphylococci, streptococci, enterococci, anaerobes, and Gram-negative bacilli. Exemplary enzymes include, but are not limited to, LysK, lysozyme, lysostaphin or a functional fragment thereof. In an embodiment, the functional fragment of LysK is CHAP 165 as disclosed in U.S. Patent Publication No. 2015/0050717, which is hereby incorporated by reference in its entirety. Additional enzymes are described, for example, in U.S. Patent Publication No. 2015/0050717, which is hereby incorporated by reference in its entirety.

In various embodiments, the bacteriophage is engineered to comprise a nucleic acid encoding an agent that potentiates antibiotic action (i.e., an antibiotic potentiating agent), for example, by inhibiting the expression and/or function of an antibiotic resistance gene or a cell survival repair gene. Exemplary antibiotic resistance genes to target according to these embodiments are those that confer resistance to beta-lactams (e.g., methicillin) or vancomycin.

Exemplary cell survival repair genes include Staphylococcus orthologs of recA, recB, recC, spoT or relA. Additional genes are disclosed, for example, in U.S. Patent Publication No. 2010/0322903, which is hereby incorporated by reference in its entirety. The expression or function of these genes may be targeted, for example, by expression of antisense polynucleotides, or double stranded RNA or other gene silencing techniques that are functional in the targeted host.

In various embodiments, the bacteriophage is engineered to comprise a nucleic acid encoding at least one gene that represses an SOS response gene and/or a non-SOS pathway bacterial defense gene. The SOS response in bacteria is an inducible DNA repair system, which allows bacteria to survive increased DNA damage. In some embodiments, the repressor is the Staphylococcus ortholog of lexA, or modified version thereof such as lexA3. In some embodiments, the gene represses SOS response genes such as marRAB, arcAB and lexO. Additional repressors are disclosed, for example, in U.S. Patent Publication No. 2010/0322903, which is hereby incorporated by reference in its entirety. In some embodiments, a repressor of a non-SOS pathway gene is one or more of soxR, marR, arc, fur, crp, icdA, craA, or ompA, or modified versions thereof. A non-SOS bacterial defense gene refers to genes expressed by a bacteria or a microorganism that serve to protect the bacteria or microorganism from cell death, for example, from being killed or growth suppressed by an antimicrobial agent.

In various embodiments, the bacteriophage is engineered to comprise a nucleic acid encoding an agent that increases the susceptibility of bacteria to an antimicrobial agent. In one embodiment, the agent increases the entry of an antimicrobial agent into a bacterial cell. Exemplary agents that increase the entry of an antimicrobial agent into a bacterial cell include, but are not limited to genes encoding porin or porin-like proteins, such as OmpF, beta barrel porins, or other members of the outer membrane porin (OMP) functional superfamily. In another embodiment, the agent increases iron- sulfur clusters in the bacteria cell and/or increases oxidative stress or hydroxyl radicals in the bacteria. Examples of a susceptibility agent that increases the iron-sulfur clusters include agents that modulate (i.e. increase or decrease) the Fenton reaction to form hydroxyl radicals. Examples of agents that increase iron-sulfur clusters in the bacterial cell include, for example but not limited to genes encoding the proteins or homologues of IscA, IscR, IscS and IscU. Examples of agents which increase iron uptake and utilization include, for example but not limited to genes encoding the proteins or homologues of, EntC, ExbB, ExbD, Feci, FecR, FepB, FepC, Fes, FhuA, FhuB, FhuC, FhuF, NrdH, Nrdl, SodA and TonB. Additional agents that may increase the susceptibility of bacteria to an antimicrobial agent are disclosed, for example, in U.S. Patent Publication No. 2010/0322903, which is hereby incorporated by reference in its entirety.

In various embodiments, the bacteriophage is engineered to comprise a nucleic acid encoding an antibody or antigen-binding molecule against a bacterial toxin or enzyme. Exemplary Staphylococcus toxins include, but are not limited to, TSST-1, enterortoxin type A, enterotoxin type B, or an exfoliatin, alpha toxin, beta toxin, delta toxin, and Panton-Valentine leukocidin (PVL). Exemplary Staphylococcus enzymes include, but are not limited to, coagulase, hyaluronidase, DNase, lipase, staphylokinase, and beta-lactamase. Other toxins of bacterial pathogens that can be targeted are disclosed in US 62/376,960, which is hereby incorporated by reference in its entirety.

Various antibody and antigen-binding platforms are known. 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 molecules 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.

In an embodiment, the bacteriophage is engineered to comprise a nucleic acid encoding a single-chain variable fragment or a single-chain antibody against a Staphylococcus toxin or enzyme. 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. For example, 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. 980250, 2012; I. Benhar, et al, J Mol Biol, vol. 301, pp. 893- 904, Aug 25 2000; A. E. Nixon, et al, MAbs, vol. 6, pp. 73-85, Jan-Feb 2014; A. M. Shukra, et al. Eur J Microbiol Immunol (Bp), vol. 4, pp. 91-8, Jun 2014, all of which are hereby incorporated by reference in their entirety. In various embodiments, the bacteriophage is engineered to comprise a nucleic acid encoding a detectable marker. In an embodiment, the marker is a detectable marker, such as a luminescent or fluorescent protein. Exemplary markers include, for example, luciferase, a modified luciferase protein, blue/UV fluorescent proteins (for example, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, and T-Sapphire), cyan fluorescent proteins (for example, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, and mTFPl), green fluorescent proteins (for example, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, and mWasabi), yellow fluorescent proteins (for example, EYFP, Citrine, Venus, SYFP2, and TagYFP), orange fluorescent proteins (for example, Monomeric Kusabira-Orange, mKOK, mK02, mOrange, and mOrange2), red fluorescent proteins (for example, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, and mRuby), far-red fluorescent proteins (for example, mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP), near-IR fluorescent proteins (for example, TagRFP657, IFP1.4, and iRFP), long stokes-shift proteins (for example, mKeima Red, LSS-mKatel, and LSS-mKate2), photoactivatible fluorescent proteins (for example, PA-GFP, PAmCherryl, and PATagRFP), photoconvertible fluorescent proteins (for example, Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS- CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, and PSmOrange), and photos witchable fluorescent proteins (for example, Dronpa).

In some embodiments, the detectable marker comprises a tag. The tag may be used for the detection or the production of the marker. In some embodiments the tag is an affinity tag used to purify and/or concentrate marker. In some embodiments the tag is a 6xHis tag. In some embodiments, the tag is an epitope specifically recognized by an antibody that is used to purify and/or concentrate marker produced in the sample prior to detection, and/or that is used to detect the marker. In some embodiments, the detectable marker may comprise a unique nucleic acid sequence that may be amplified (e.g., by polymerase chain reaction (PCR)) to detect the presence of or to quantify the gene encoding the specific marker. Thus any nucleic acid sequence contained within the bacteriophage could be used for PCR-based detection or quantification (e.g. , RT- PCR). In various embodiments, the bacteriophage comprises a promoter sequence operatively linked to direct expression of one or more genes disclosed herein (for example, nucleic acids encoding a biofilm-degrading enzyme, an antimicrobial polypeptide, an agent that inhibits an antibiotic resistance gene and/or a cell survival repair gene, an agent that increases the susceptibility of a bacteria cell to an antimicrobial agent, antigen-binding molecule against an enzyme or toxin, and a marker). In some embodiments, the promoter is operatively linked to the nucleic acid. In some embodiments, the promoter is a bacterial promoter. In some embodiments, the promoter is a bacteriophage promoter or a Staphylococcus promoter. Other promoters that may be used are disclosed, for example, in U.S. Patent Publication No. 2010/0322903 and at partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=other_regulator&show=l, which are hereby incorporated by reference in their entireties.

In various embodiments, the bacteriophage delivers the nucleic acids expressing an agent such as, for example, a biofilm-degrading enzyme and an antimicrobial polypeptide, into the infected host bacterial cell. In an embodiment, the agent is released from the host bacterial cell when the host cell is lysed during the lytic cycle of bacteriophage infection. In another embodiment, the agent is secreted from the host cell, for example, via the secretory pathway. In such an embodiment, the agent which is expressed from the bacteriophage-infected host bacterial cell may contain a signal peptide such as a secretory signal sequence. Such a secretory signal sequence allows intracellular transport of the agent to the bacterial cell plasma membrane for its secretion from the bacteria. 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.

In various embodiments, the present invention relates to any of the modified recognition baseplate proteins described herein as well as polynucleotides encoding any of the modified recognition baseplate proteins described herein.

In one aspect, the present invention provides pharmaceutical compositions comprising one or more bacteriophages of the invention. In some embodiments, the pharmaceutical compositions of the invention may additionally include pharmaceutically acceptable excipient or carrier suitable for application to a site of infection.

In various embodiments, the present invention provides methods of treating bacterial infections comprising administering to a subject in need thereof any of the engineered bacteriophage described herein. In some embodiments, the infection involves Staphylococcus. Exemplary infections (which optionally involve S. aureus), include but are not limited to, an implanted or indwelling device-related infection selected from a prosthesis, catheter, cardiovascular device, or artificial heart valve; osteomyelitis; abscess; pneumonia; endocarditis; phlebitis; mastitis; meningitis; metritis; septic shock or toxic shock syndrome. Additional infections include, but are not limited to, MRSA infection, bacteremia, skin infection, cellulitis or folliculitis, or wound infections. In some embodiments, the Staphylococcus aureus-r lated infection is a nosocomial infection. In some embodiments, the subject being treated has an increased risk of developing a Staphylococcus aureus-r iated infection. In some embodiments, the subject is a hospitalized patient such as one in the intensive care unit, an immunocompromised patient, and a patient who has undergone or will undergo a surgical procedure (e.g., a cardiac surgery).

In various embodiments, the Picovirinae bacteriophages can infect at least about 50%, at least about 60%, at least about 70%, at least about 80%, or about at least 90% of clinical isolates from a particular type of infection (e.g., defined by bacterial species and tissue). In some embodiments, the infection is a Staphylococcus aureus- related infections.

In some embodiments, the terms "patient" and "subject" are used interchangeably. In some embodiments, the subject is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In some embodiments, the subject is livestock, such as a cow, sheep, horse, pig or goat. In some embodiments, the subject is a pet (e.g., dog or cat) or zoo animal. For example, methods of the invention may comprise treating bovine mastitis or bovine metritis. In some embodiments, the present invention provides methods of treating prosthetic joint infections comprising administering to the infected area and/or the surface of the prosthetic the engineered bacteriophage and/or pharmaceutical composition as disclosed herein. In some embodiments, the bacteriophage and/or pharmaceutical composition effectively inhibit the growth of and/or kill (or reducing the cell viability) the microorganisms (e.g., Staphylococcus aureus) involved with the prosthetic joint infections. In some embodiments, the bacteriophage and/or pharmaceutical composition is effective in eliminating or reducing the bacterial biofilm produced by the microorganisms (e.g., Staphylococcus aureus) involved with the prosthetic joint infections. In various embodiments, the bacteriophages of the invention can infect at least about 50%, at least about 60%, at least about 70%, at least about 80%, or about at least 90% of clinical isolates from Staphylococcus aureus-r iat d prosthetic joint infections.

In some embodiments, methods of the invention inhibit the growth of and/or kill (or reduce the cell viability) microorganisms in the vicinity of the bacteriophage. In some embodiments, methods of the invention eliminate or reduce bacterial biofilms in the vicinity of the bacteriophage. Without wishing to be bound by theory, it is believed that agents are released into the vicinity from the infected host microbial cell. Accordingly, methods of the invention can target microorganisms involved with prosthetic joint infection even if these microorganisms have not been infected or are resistant to being infected with the bacteriophages of the invention.

In some embodiments, the prosthetic joint infection is a mixed infection involving Staphylococcus aureus and one or more additional microbial species and/or strains. In an embodiment, the additional microbial strain is Gram-positive or Gram- negative. In another embodiment, the additional microbial strain is selected from coagulase-negative staphylococci, streptococci, enterococci, anaerobes, and Gram- negative bacilli. In an embodiment, the additional microbial strain is Staphylococcus epidermidis .

In various embodiments, the bacteriophage or pharmaceutical composition of the invention may be administered in combination with an additional therapeutic agent to a subject in need thereof. In an embodiment, the additional therapeutic agent is an antibiotic or antimicrobial agent, which is administered locally or systemically. In various embodiments, administration of the bacteriophage or pharmaceutical composition of the invention in combination with the additional therapeutic agent produces synergistic effects.

Antibiotics suitable for use in the present invention include, but are not limited to, aminoglycosides, carbapenemes, cephalosporins, cephems, glycopeptides fluoroquinolones/quinolones, oxazolidinones, penicillins, streptogramins, sulfonamides rifamycins and/or tetracyclines. Additional aspects and embodiments of the invention will be apparent from the following examples.

EXAMPLES

Example 1: Analysis of Picovirinae Bacteriophage ORF14 Sequence

The Picovirinae bacteriophage GRCS harbors an ORF14 protein having the amino acid sequence of SEQ ID NO: l. Without wishing to be bound by theory, it is believed that ORF14 is a recognition baseplate protein responsible for the specific recognition of Staphylococcus aureus isolates from, for example, prosthetic joint infections. See PCT/US2016/43815, which is hereby incorporated by reference. It is also believed that Staphylococcus aureus isolates from prosthetic joint infections express a unique cognate epitope that is specifically recognized by ORF14.

BLASTP searches of ORF14 identified only other ORF14 homologues solely and uniquely from other Picovirinae phages infective for Staphylococcus aureus. Dot- matrix comparisons of the GRCS ORF14 sequence against 4 other Picovirinae ORF14 homologues (i.e., P68, SAP-2, S13', and S24-1) showed conserved N-terminal and C- terminal domains with variation in the middle region from about residue 140 to residue 200 (GRCS). There seems to be a series of gene duplications in this region as several phages show off-diagonal homology in this central region (Figures 1A-D). BLASTP searches detected no other proteins from other phages not related to Staphylococcus aureus, meaning there is no primary sequence homology between GRCS ORF14 and any other protein not found in a Staphylococcus aureus specific Picovirinae phage. ORF14 is annotated as a minor tail protein.

As the central region seems to be the most variable, BLASTP was used to search for homologues using the N-terminal domain (residues 1 - 170) and the C- terminal domain (residues 170-309) as queries. One of the sequences detected in both queries was the Staphylococcus aureus Picovirinae phage SLPW. However, it was noted that in the two queries, the homologues recognized were encoded by two separate ORFs. Upon closer examination of the genome structure of phage SLPW, it was confirmed that in this phage, the ORF14 homologue is split into two separate genes, an N-terminal and a C-terminal domain, suggesting that the domains may function independently of each other or may work in concert with each other despite not being covalently linked in a single protein structure (Figures 2A-C).

Figure 3 provides a global alignment of GRCS ORF14 with other

Staphylococcal bacteriophages. Amino acids 140 to 200 of ORF14 and aligned sequences are highlighted.

Further analysis of GRCS ORF14 was performed using a structural prediction method called HHpred (Max Planck Institute). First, using BLASTP with GRCS ORF14 as the query, all the high ranking hits from the other Staphylococcus aureus Picovirinae phages mentioned above were retrieved. These were then aligned using a multiple alignment tool (COBALT in the NCBI tools) and that alignment was used as input into HHpred. HHpred did structural prediction using the multiple alignment and the consensus sequences with their known propensities for certain structural features (using hidden Markov models). The structural prediction was then matched to structural predictions from structural databases such as PDB (crystal and NMR structures), SCOP (structural classification of proteins database) and pfam (protein structural families). Using the entire multiple alignment covering the full sequence of ORF14 as the original seed, HHpred revealed two highly significant structural domains in the sequence. The N-terminal domain belongs to a family of coiled-coiled proteins with a variety of cellular or physiological functions (notably fibrinogen alpha-chain). The C-terminal domain had the most significant hits with L. lactis phage T901 and phage bIL170 baseplate proteins.

EQUIVALENTS

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

Claims

CLAIMS:

1. A modified recognition baseplate protein of a Picovirinae bacteriophage, wherein a Picovirinae bacteriophage comprising the modified recognition baseplate protein has a broadened or altered infectivity profile from that of the original bacteriophage.

2. The modified protein of claim 1, wherein the Picovirinae bacteriophage infects a species of Staphylococcus, Streptococcus, Clostridium, Bacillus, Corynebacterium, Vibrio, enterotoxigenic E. coli, Shigella, and Pseudomonas.

3. The modified protein of claim 1, wherein the Picovirinae bacteriophage infects at least about 60% of clinical isolates from a Staphylococcus aureus-r lated infection selected from:

(a) implanted device-related infection selected from a prosthesis, catheter, cardiovascular device, or artificial heart valve;

(b) osteomyelitis;

(c) abscess;

(d) pneumonia;

(e) endocarditis;

(f) phlebitis;

(g) mastitis;

(h) meningitis;

(i) metritis; and

j) septic shock or toxic shock syndrome.

4. The modified protein of claim 3, wherein the bacteriophage infects at least about 70% of clinical isolates from Staphylococcus aureus-reiated prosthetic joint infections.

5. The modified protein of claim 1, wherein the protein has structural homology the amino acid sequence of SEQ ID NO: 1.

6. The modified protein of any one of claims 1 to 5, wherein the protein has at least about 30% identity to the polypeptide of SEQ ID NO: 1 (ORF14).

7. The modified protein of claim 5, wherein the protein has a C-terminal domain swap from another Staphylococcus aureus Picovirinae bacteriophage, and/or one or more modifications to the C-terminal domain.

8. The modified protein of claim 7, having one or more amino acids substituted, deleted, or inserted at positions corresponding to positions 140 to 200 of SEQ ID NO: 1.

9. The modified protein of claim 7 or 8, wherein the amino acids at positions corresponding to amino acids 140 to 200 of SEQ ID NO: 1 have at least about 50% identity to an amino acid sequence of SEQ ID NO: 2 (P68), SEQ ID NO: 3 (SAP-2), SEQ ID NO:4 (S13'), and/or SEQ ID NO: 5 (S24-1).

10. The modified protein of any one of claims 1 to 8, wherein the protein comprises a replacement of all or a portion of the C-terminal domain with an Antimicrobial Peptide (AMP) amino acid sequence.

11. A polynucleotide encoding the modified recognition baseplate protein of any one of claims 1 to 10.

12. A Picovirinae bacteriophage comprising the modified recognition baseplate protein of any one of claims 1 to 10, or the polynucleotide of claim 11.

13. The Picovirinae bacteriophage of claim 12, wherein the bacteriophage infects at least about 50% of clinical isolates from a bacterial infection selected from:

(b) osteomyelitis;

(c) abscess;

(d) pneumonia; (e) endocarditis;

(f) phlebitis;

(g) mastitis;

(h) meningitis;

(i) metritis; and

(j) septic shock or toxic shock syndrome.

14. The Picovirinae bacteriophage of claim 13, wherein the bacteriophage infects at least about 70% of clinical isolates from Staphylococcus aureus-r iated prosthetic joint infections.

15. The Picovirinae bacteriophage of any one of claims 12 to 14, wherein the bacteriophage is lytic.

16. The Picovirinae bacteriophage of any one of claims 12 to 15, wherein the bacteriophage is a GRCS phage.

17. The Picovirinae bacteriophage of any one of claims 12 to 16, wherein the bacteriophage expresses an antimicrobial peptide (AMP) under the control of a bacterial or bacteriophage promoter.

18. The Picovirinae bacteriophage of claim 17, wherein the AMP contains a signal sequence directing secretion from bacterial host.

19. The Picovirinae bacteriophage of any one of claims 12 to 18, wherein the bacteriophage expresses a biofilm degrading enzyme or lytic enzyme.

20. The Picovirinae bacteriophage of any one of claims 12 to 18, wherein the bacteriophage expresses an antibiotic potentiating gene, such as an inhibitor of an antibiotic resistance gene or inhibitor of a cell survival repair gene.

21. The Picovirinae bacteriophage of any one of claims 12 to 18, wherein the bacteriophage expresses an antibody or antigen-binding molecule against a bacterial toxin or enzyme.

22. The Picovirinae bacteriophage of claim 21 , wherein the antigen-binding molecule is a single chain variable fragment.

23. The Picovirinae bacteriophage of claim 21 or 22, wherein the toxin is TSST-1 , enterortoxin type A, enterotoxin type B, or an exfoliatin, alpha toxin, beta toxin, delta toxin, or Panton-Valentine leukocidin (PVL).

24. The Picovirinae bacteriophage of claim 21 or 22, wherein the enzyme is coagulase, hyaluronidase, DNase, lipase, staphylokinase, or beta-lactamase.

25. The Picovirinae bacteriophage of any one of claims 12 to 24, wherein the bacteriophage further comprises or encodes a detectable marker.

26. A method for treating a bacterial infection, comprising administering the bacteriophage of any one of claims 12 to 25 to a patient in need of treatment.

27. The method of claim 26, wherein the infection involves a species of

Staphylococcus, Streptococcus, Clostridium, Bacillus, Corynebacterium.

28. The method of claim 26, wherein the infection involves Staphylococcus aureus.

29. The method of claim 28, wherein the patient has an implanted device-related infection, osteomyelitis, abscess, pneumonia, endocarditis, phlebitis, mastitis, meningitis, metritis, septic shock, or toxic shock syndrome.

30. The method of any one of claims 26 to 29, wherein the patient is a human or animal patient.

31. The method of claim 29, wherein the patient has a prosthetic j oint infection.

32. The method of claim 29, wherein the infection is bovine mastitis or bovine metritis.