Francisella glycoconjugate vaccines

Abstract

The disclosure relates to a glycoconjugate vaccine conferring protection against Francisella tularensis infections and a method to manufacture a glycoconjugate antigen.

1 . A vaccine or immunogenic composition comprising a carrier polypeptide, wherein the carrier polypeptide comprises:
 one or more T-cell dependent epitopes;   one or more amino acid sequences having at least one amino acid motif comprising the amino acid sequence D/E-X-N-X-S/T, wherein X is any amino acid except proline; and   an O-antigen antigenic polysaccharide isolated from  Francisella  crosslinked to said carrier polypeptide.   
     
     
         2 . The vaccine or immunogenic composition according to  claim 1  wherein said O-antigen comprises:
 2-acetamido-2-deoxy-O-D-galact-uronamide, 4, 6-dideoxy-4-formamido-D-glucose and/or 2-acetamido-2, 6-dideoxy-O-D-glucose and/or N-acetyl glucosamine, 
 or comprises or consists of 
 4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-QuiNAc-(1-2)-β-D-Qui4NFm-(1-), wherein GalNAcAN is 2-acetamido-2-deoxy-O-D-galacturonamide, Qui4NFm is 4,6-dideoxy-4-formamido-D-glucose and the reducing end group QuiNAc is 2-acetamido-2,6-dideoxy-O-D-glucose, 
 or comprises or consist of 
 4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-GlcNAc-(1-2)-β-D-Qui4NFm-(1-), wherein GalNacAN is 2-acetomido-2-deoxy-O-D-galacturonamide, Qui4NFm is 4, 6-dideoxy-4-formamido-D-glucose and the reducing end group GlcNAc is N-acetyl glucosamine. 
 
     
     
         3 . The vaccine or immunogenic composition according to  claim 1 , wherein said O-antigen polysaccharide is a tetrasaccharide. 
     
     
         4 . The vaccine or immunogenic composition of  claim 1 , wherein said carrier polypeptide,
 comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7,   comprises a polymorphic sequence variant of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7,   comprises a polypeptide fragment of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or   comprises one or more glycosylation motifs of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.   
     
     
         5 .- 10 . (canceled) 
     
     
         11 . The vaccine or immunogenic composition according to  claim 1 , wherein said composition includes an adjuvant. 
     
     
         12 . The vaccine or immunogenic composition according to  claim 11  wherein said adjuvant is an aluminium based adjuvant suitable for use in a human subject. 
     
     
         13 . A method of treating or preventing a  Francisella  infection comprising:
 administering the vaccine or immunogenic composition of  claim 1  to a subject, thereby treating or preventing the  Francisella  infection in the subject.   
     
     
         14 . An antigenic polypeptide comprising: a carrier polypeptide comprising one or more T-cell dependent epitopes and one or more amino acid sequences having at least one amino acid motif comprising the amino acid sequence D/E-X-N-X-S/T wherein X is any amino acid except proline and crosslinked to said carrier polypeptide is an antigenic polysaccharide wherein the polysaccharide is isolated from  Francisella  and is an O-antigen. 
     
     
         15 . The antigenic polypeptide according to  claim 14  wherein said carrier polypeptide,
 comprises the amino acid sequence as set forth in of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, 
 comprises a polymorphic sequence variant of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, 
 comprises a polypeptide fragment of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or 
 comprises one or more glycosylation motifs of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. 
 
     
     
         16 .- 21 . (canceled) 
     
     
         22 . The antigenic polypeptide according to  claim 14 , wherein said O-antigen comprises:
 2-acetamido-2-deoxy-O-D-galact-uronamide, 4, 6-dideoxy-4-formamido-D-glucose and/or 2-acetamido-2, 6-dideoxy-O-D-glucose and/or N-acetyl glucosamine,   or comprises or consist of:   4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-QuiNAc-(1-2)-β-D-Qui4NFm-(1-), wherein GalNAcAN is 2-acetamido-2-deoxy-O-D-galacturonamide, Qui4NFm is 4,6-dideoxy-4-formamido-D-glucose and the reducing end group QuiNAc is 2-acetamido-2,6-dideoxy-O-D-glucose,   or comprises or consists of:   4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-GlcNAc-(1-2)-β-D-Qui4NFm-(1-), wherein GalNacAN is 2-acetomido-2-deoxy-O-D-galacturonamide, Qui4NFm is 4, 6-dideoxy-4-formamido-D-glucose and the reducing end group GlcNAc is N-acetyl glucosamine.   
     
     
         23 . A bacterial cell genetically modified to include:
 i) a nucleic acid molecule comprising the nucleotide sequence of the  Francisella  O-antigen biosynthetic polysaccharide locus [SEQ ID NO: 8];   ii) a nucleic acid molecule comprising a nucleotide sequence of an oligosaccharyltransferase; and/or   iii) a nucleic acid molecule comprising a nucleotide sequence of carrier polypeptide wherein the a carrier polypeptide comprising one or more T-cell dependent epitopes and one or more amino acid sequences having at least one amino acid motif comprising the amino acid sequence D/E-X-N-X-S/T wherein X is any amino acid except proline, wherein said bacterial cell is adapted for expression of each nucleic acid molecule and synthesizes an antigenic polypeptide according to  claim 14 .   
     
     
         24 . The bacterial cell according to  claim 23  wherein said nucleic acid molecule encoding an oligosaccharyltransferase comprises the nucleotide sequence of SEQ ID NO: 9, 23, 25 or 26 or a polymorphic sequence variant thereof, wherein said variant comprises a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence of SEQ ID NO: 9, 23, 25 or 26 and wherein said nucleic acid molecule encodes an oligosaccharyltransferase; 
     
     
         25 . The bacterial cell according to  claim 23 , wherein said carrier polypeptide is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 10, 11, 13, 14, 15, 16 or 29, or a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence of SEQ ID NO: 10, 11, 13, 14, 15, 16 or 29. 
     
     
         26 . The bacterial cell according to  claim 23 , wherein said bacterial cell expresses a glycosyltransferase encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO 17, or a sequence variant thereof wherein said variant comprises a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence set forth in SEQ ID NO: 17 and wherein said nucleic acid molecule encodes an glycosyltransferase. 
     
     
         27 . The bacterial cell according to  claim 23 , wherein said glycosyltransferase encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO 17 is modified wherein said modification is the addition, substitution or deletion of at least one nucleic acid base wherein said modified nucleic acid sequence encodes a glycosyltransferase polypeptide which has reduced or undetectable enzyme activity when compared to an unmodified glycosyltransferase polypeptide. 
     
     
         28 . The bacterial cell according to  claim 27  wherein said cell is deleted for all or part of the nucleic acid sequence of SEQ ID NO: 17 to provide a non-functional glycosyltransferase polypeptide. 
     
     
         29 . The bacterial cell according to  claim 23 , wherein at least the oligosaccharyltransferase is integrated into the bacterial genome to provide a stably transfected and expressing oligosaccharyltransferase. 
     
     
         30 . The bacterial cell according to  claim 23 , wherein one or more nucleic acid molecules encoding carrier polypeptides are also integrated into the bacterial genome. 
     
     
         31 . A bacterial cell culture comprising a genetically modified bacterial cell according to  claim 23 . 
     
     
         32 . A process for producing one or more glycoconjugates, comprising:
 i) providing the bacterial cell culture according to  claim 31 ;   ii) providing cell culture conditions; and   iii) isolating one or more glyconjugates from the bacterial cell or cell culture medium.   
     
     
         33 . A cell culture vessel comprising the bacterial cell culture according to  claim 31 . 
     
     
         34 . The cell culture vessel according to  claim 33 , wherein said cell culture vessel is a fermentor.