Multiple host range bacteriophage with hybrid tail fibres

Abstract

Modified bacteriophage, uses thereof, and compositions containing the modified bacteriophage are described. The compositions are useful for human treatment and may treat various conditions, including bacterial infections.

1 - 38 . (canceled) 
     
     
         39 . A modified bacteriophage capable of infecting a plurality of different target bacteria, wherein:
 (a) said bacteriophage includes an α/β small acid-soluble spore protein (SASP) gene encoding a SASP which is toxic to the target bacteria;   (b) said bacteriophage expresses a single hybrid host range determinant protein which comprises an amino acid sequence derived from amino acid sequences of range determinant proteins from at least two different bacteriophages, and   (c) said hybrid host range determinant protein is a tail fiber protein,   (d) wherein said hybrid host range determinant protein comprises an N-terminal region wherein said N-terminal region binds to the body of said bacteriophage and a C-terminal region wherein said C-terminal region comprises a receptor binding region for binding to the target bacteria and wherein the amino acid sequence of said N-terminal region of said hybrid host range determinant protein is derived from a different bacteriophage than the amino acid sequence of said C-terminal region.   
     
     
         40 . A modified bacteriophage according to  claim 39  wherein said N-terminal region of said hybrid host range determinant protein comprises amino acids 1 to 628 of said hybrid host range determinant protein based on the amino acid sequence of bacteriophage Phi33 and said C-terminal region comprises amino acids 629 to 964 of the said hybrid host range determinant protein, based on the amino acid sequence of bacteriophage Phi33. 
     
     
         41 . A modified bacteriophage according to  claim 39  wherein said N-terminal region is from any one of bacteriophage Phi33, LBL3, SPM-1, F8, PB1, KPP12, LMA2, SN, 14-1, JG024, NH4, PTP47, PTP92, C36 and PTP93 and/or said C-terminal region is from any one of bacteriophage Phi33, LBL3, SPM-1, F8, PB1, KPP12, LMA2, SN, 14-1, JG024, NH4, PTP47, PTP92, C36 and PTP93. 
     
     
         42 . A modified bacteriophage according to  claim 39 , wherein the percent amino sequence identity of said C-terminal region of said hybrid host range determinant protein to the corresponding C-terminal region of the tail fiber protein of the bacteriophage from which the N-terminal region of said hybrid host range determinant protein is derived is less than 80%, or less than 70%, or less than 60%. 
     
     
         43 . A modified bacteriophage according to  claim 39 , wherein the C-terminal region has no more than 96% amino acid sequence identity with the C-terminal region of bacteriophage Phi33 and/or the N-terminal region has at least 95% amino acid sequence identity with the N-terminal region of bacteriophage Phi33. 
     
     
         44 . A modified bacteriophage according to  claim 39 , wherein the bacterial host specificity of the host range determinant is within the same bacterial genus, optionally wherein said bacterial host genus is  Pseudomonas.    
     
     
         45 . A modified bacteriophage according to  claim 44 , wherein said  Pseudomonas  bacteria comprise  Pseudomonas aeruginosa.    
     
     
         46 . A modified bacteriophage according to  claim 39 , wherein the hybrid host range determinant protein confers a broad host range as defined by more than 50% of a collection of at least 35 or 50 clinical isolates, from a plurality of different infection sites and including a range of antibiotic resistance phenotypes. 
     
     
         47 . A modified bacteriophage according to  claim 39  wherein the bacteriophage is non-lytic. 
     
     
         48 . A modified bacteriophage according to  claim 47 , which comprises an inactivated lysis gene. 
     
     
         49 . A modified bacteriophage according to  claim 49 , wherein the lysis gene is inactivated by insertion of said SASP gene. 
     
     
         50 . A modified bacteriophage according to  claim 39 , wherein the SASP is SASP-C. 
     
     
         51 . A modified bacteriophage according to  claim 51 , wherein the SASP-C is from  Bacillus megaterium.    
     
     
         52 . A modified bacteriophage according to  claim 39 , wherein the SASP gene is under the control of a constitutive promoter which is sufficiently strong to drive production of toxic levels of SASP when the modified bacteriophage is present in multiple copies in the target bacterium and/or wherein the constitutive promoter is selected from pdhA, rpsB, pgi, fda, lasB and promoters having more than 90% sequence identity thereto. 
     
     
         53 . A composition for inhibiting or preventing bacterial cell growth, which comprises at least one modified bacteriophage according to any one of  claim 39 , and a carrier therefor, wherein said composition is formulated for pharmaceutical use, including topical administration, systemic administration, ocular administration, intravenous administration, and/or respiratory administration or is formulated for environmental use, including bacterial decontamination, surface bacterial contamination, land remediation and/or water treatment. 
     
     
         54 . A composition according to  claim 53 , comprising:
 (i) a first modified Phi33 bacteriophage comprising (1) a chimeric tail fiber wherein the N terminal region of the tail fiber is from Phi33 and the C-terminal region of the hybrid tail fiber is from PTP92, (2) a  Pseudomonas aeruginosa  codon-optimized SASP-C gene operably linked to the fda promoter, and (3) an inactivated lysis gene, wherein the lysis gene is inactivated by insertion of said SASP-C gene therein; and   (ii) a second modified Phi33 bacteriophage comprising (1) a chimeric tail fiber wherein the N terminal region of the tail fiber is from Phi33 and the C-terminal region of the hybrid tail fiber is from PTP47, (2) a  Pseudomonas aeruginosa  codon-optimized SASP-C gene operably linked to the fda promoter, and (3) an inactivated lysis gene, wherein the lysis gene is optionally inactivated by insertion of said SASP-C gene therein.   
     
     
         55 . A composition according to  claim 54 , further comprising a third modified Phi33 bacteriophage, wherein the modifications consist of the incorporation of a  Pseudomonas aeruginosa  codon optimized SASP-C gene operably linked to the fda promoter, and (3) an inactivated lysis gene, wherein the lysis gene is inactivated by insertion of said SASP-C gene therein. 
     
     
         56 . A method of treatment of bacterial infection in a subject in need thereof, comprising administration to the subject an effective amount of the composition of  claim 53 . 
     
     
         57 . A method of treatment of bacterial infection in a subject in need thereof, comprising administration to the subject an effective amount of the composition of  claim 54 . 
     
     
         58 . A method of treatment of bacterial infection in a subject in need thereof, comprising administration to the subject an effective amount of the composition of  claim 55 .