Modified hexa-acylated neisserial LPS

Abstract

The present invention relates to neisserial LPS having a hexa-acylated lipid A moiety, wherein the hexa-acylated lipid A moiety is modified as compared to the lipid A moiety of a wild-type neisserial LPS in that it comprises a palmitoleoyl instead of a lauroyl secondary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety. The invention further relates to mixtures of the hexa-acylated LPS with the corresponding penta-acylated LPS, lacking a secondary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety. The invention also relates to neisserial bacteria that have been genetically modified to reduce expression of the endogenous 1pxL1 gene and to introduce expression of a heterologous thermosensitive 1pxP gene for producing the hexa- and penta-acylated LPS. By selecting the time and/or temperature at which the bacterium is grown, it is feasible to increase or decrease the amount of hexa-acylated lipid A structure relative to the corresponding penta-acylated structure and thereby modulate the TLR4 agonist activity of the neisserial LPS of the invention, to the exact level of activity required for a particular immunotherapeutic approach.

1 . A genetically modified bacterium of the genus  Neisseria , wherein the bacterium comprises:
 a) a genetic modification that reduces or eliminates the activity of a lipid A biosynthesis lauroyl acyltransferase encoded by an endogenous lpxL1 gene; and,   b) a genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyltransferase activity.   
     
     
         2 . The genetically modified bacterium according to  claim 1 , wherein the bacterium is a genetically modified  Neisseria meningitidis, Neisseria gonorrhoeae  or  Neisseria  lactamica. 
     
     
         3 . The genetically modified bacterium according to  claim 1 , wherein the genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyltransferase activity is a genetic modification that introduces the expression of a heterologous lpxP gene. 
     
     
         4 . The genetically modified bacterium according to  claim 3 , wherein the heterologous lpxP gene has a nucleotide sequence that encodes an LpxP lipid A palmitoleoyltransferase that has at least 80% amino acid sequence identity with at least one of SEQ ID NO's: 4-10. 
     
     
         5 . The genetically modified bacterium according to  claim 3 , wherein the nucleotide sequence encodes a LpxP palmitoleoyl acyltransferase having at least 85% amino acid sequence identity with at least one of SEQ ID NO: 4-10. 
     
     
         6 . The genetically modified bacterium according to  claim 3 , wherein the nucleotide sequence encodes a LpxP palmitoleoyl acyltransferase having at least 90% amino acid sequence identity with at least one of SEQ ID NO: 4-10. 
     
     
         7 . The genetically modified bacterium according to  claim 1 , wherein the endogenous lpxL1 gene is a gene encoding an LpxL1 protein having an amino acid sequence with at least 90% sequence identity with at least one of SEQ ID NO's: 1-3. 
     
     
         8 . The genetically modified bacterium according to  claim 1 , wherein the bacterium is further genetically modified to express a heterologous antigen. 
     
     
         9 . The genetically modified bacterium according to  claim 8 , wherein the heterologous antigen is expressed on the extracellular outer membrane surface of the bacterium. 
     
     
         10 . The genetically modified bacterium according to  claim 1 , wherein the bacterium has a genetic modification that reduces or eliminates the expression of at least one of an endogenous lgtB gene and an endogenous galE gene. 
     
     
         11 . The genetically modified bacterium according to  claim 1 , wherein the bacterium is a  Neisseria meningitidis  serogroup B, immunotype L3. 
     
     
         12 . The genetically modified bacterium according to  claim 1 , wherein the bacterium is  Neisseria meningitidis  strain H44/76 or a derivative thereof. 
     
     
         13 . A neisserial LPS having a hexa-acylated lipid A moiety, wherein the hexa-acylated lipid A moiety is modified as compared to the lipid A moiety of a wild-type neisserial LPS in that it has a palmitoleoyl (instead of a lauroyl) as secondary acyl chain bound to the primary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety. 
     
     
         14 . The neisserial LPS according to  claim 13 , wherein the LPS is obtainable from a genetically modified bacterium of the genus  Neisseria , wherein the bacterium comprises:
 a) a genetic modification that reduces or eliminates the activity of a lipid A biosynthesis lauroyl acyltransferase encoded by an endogenous lpxL1 gene; and,   b) a genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyltransferase activity.   
     
     
         15 . The neisserial LPS according to  claim 13 , wherein, except for the hexa-acylated lipid A moiety, the LPS has the structure of LPS of  Neisseria meningitidis, Neisseria gonorrhoeae  or  Neisseria  lactamica. 
     
     
         16 . The neisserial LPS according to  claim 13 , wherein the  Neisseria meningitidis, Neisseria gonorrhoeae  or  Neisseria  lactamica is at least one of lgtB- and galE. 
     
     
         17 . The neisserial LPS according to  claim 13 , wherein the  Neisseria meningitidis  is at least one of serogroup B and immunotype L3. 
     
     
         18 . The neisserial LPS according to  claim 13 , wherein the hexa-acylated lipid A moiety has the structure of formula (I): 
       
         
           
           
               
               
           
         
       
       wherein R 1  and R 2 , independently, are either —P(O)(OH) 2 , —[P(O)(OH)—O] 2 —H, —[P(O)(OH)—O] 2 —CH 2 CH 2 NH 2 , —[P(O)(OH)—O] 3 —CH 2 CH 2 NH 2 , —[P(O)(OH)—O] 3 —H or —P(O)(OH)—O—CH 2 CH 2 NH 2 . 
     
     
         19 . An OMV comprising the neisserial LPS according to  claim 13 . 
     
     
         20 . The OMV according to  claim 19 , wherein the OMV is obtainable from a genetically modified bacterium of the genus  Neisseria , wherein the bacterium comprises:
 a) a genetic modification that reduces or eliminates the activity of a lipid A biosynthesis lauroyl acyltransferase encoded by an endogenous lpxL1 gene; and,   b) a genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyltransferase activity.