Non-toxic mutants of pathogenic gram-negative bacteria

Abstract

A method is provided for identifying, isolating, and producing htrB mutants of gram-negative bacterial pathogens. The method comprises mutating the htrB gene of a gram-negative bacterial pathogen so that there is a lack of a functional htrB protein, resulting in a mutant that lacks one or more secondary acyl chains contained in the wild type gram-negative bacterial pathogen, and displays substantially reduced toxicity as compared to the wild type strain. Also, the present invention provides methods for using a vaccine formulation containing the htrB mutant, the endotoxin isolated therefrom, or the endotoxin isolated therefrom which is then conjugated to a carrier protein, to immunize an individual against infections caused by gram-negative bacterial pathogens by administering a prophylactically effective amount of the vaccine formulation.

1 . A vaccine formulation comprising an active ingredient selected from the group consisting of an htrB mutant of a gram-negative bacterial pathogen, endotoxin isolated from the htrB mutant of said gram-negative bacterial pathogen, endotoxin isolated from the htrB mutant of said gram-negative bacterial pathogen said endotoxin conjugated to a carrier protein, and an htrB mutant of said gram-negative bacterial pathogen which has been genetically engineered to express at least one heterologous vaccine antigen; wherein said htrB mutant lacks one or more secondary acyl chains of lipid A contained in the gram-negative bacterial pathogen resulting in substantially reduced toxicity when compared to lipid A of the gram-negative bacterial pathogen. 
     
     
         2 . The vaccine formulation of  claim 1 , wherein the active ingredient consists essentially of an htrB mutant of said gram-negative bacterial pathogen. 
     
     
         3 . The vaccine formulation of  claim 1 , wherein the active ingredient consists essentially of endotoxin isolated from the htrB mutant of said gram-negative bacterial pathogen. 
     
     
         4 . The vaccine formulation of  claim 1 , wherein the active ingredient consists essentially of endotoxin isolated from the htrB mutant of said gram-negative bacterial pathogen, wherein the isolated endotoxin is conjugated to a carrier protein. 
     
     
         5 . The vaccine formulation of  claim 1 , wherein the active ingredient consists essentially of an htrB mutant of said gram-negative bacterial pathogen which has been genetically engineered to express at least one heterologous antigen from a microbial pathogen. 
     
     
         6 . The vaccine formulation of  claim 1 , further comprising a physiological carrier and an adjuvant. 
     
     
         7 . The vaccine formulation of  claim 1 , wherein the gram-negative bacterial pathogen is a  Neisseria, Haemophilus, Moraxella, Campylobacter, Salmonella, Shigella , or  Pseudomonas  gram-negative bacterial pathogen. 
     
     
         8 . The vaccine formulation of  claim 7 , wherein the gram-negative bacterial pathogen is  Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Haemophilus ducreyi, Moraxella catarrhalis, Campylobacter jejuni, Salmonella typhimurium, Shigella dysentariae , or  Pseudomonas aeruginosa.    
     
     
         9 . The vaccine formulation of  claim 8 , wherein the gram-negative bacterial pathogen is  Haemophilus influenzae.    
     
     
         10 . The vaccine formulation of  claim 9 , wherein the gram-negative bacterial pathogen is non-typeable  Haemophilus influenzae.    
     
     
         11 . The vaccine formulation of  claim 10 , wherein the endotoxin of the htrB mutant contains a decreased phosphoethanolamine content and an increased hexose content in the mutant endotoxin's inner core, and a pentaacylated or tetraacylated lipid A lacking one or two secondary acyl chains compared to the corresponding wild-type non-typeable  Haemophilus influenzae  hexaacylated endotoxin. 
     
     
         12 . A method for immunizing an individual to prevent disease caused by a gram-negative bacterial pathogen, the method comprising vaccinating the individual with a prophylactically effective amount of the vaccine formulation of  claim 1 . 
     
     
         13 . The method of  claim 12 , wherein the individual is a human. 
     
     
         14 . The method of  claim 12 , wherein the individual is not a human. 
     
     
         15 . The method of  claim 12 , wherein the vaccine formulation is introduced by a route of administration selected from the group consisting of intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, ocular, intranasal, and oral administration. 
     
     
         16 . The method of  claim 12 , wherein the vaccine formulation comprises an active ingredient consisting essentially of an htrB mutant of said gram-negative bacterial pathogen. 
     
     
         17 . The method of  claim 12 , wherein the vaccine formulation comprises an active ingredient consisting essentially of endotoxin isolated from the htrB mutant of said gram-negative bacterial pathogen. 
     
     
         18 . The method of  claim 12 , wherein the vaccine formulation comprises an active ingredient consisting essentially of endotoxin isolated from the htrB mutant of said gram-negative bacterial pathogen, wherein the isolated endotoxin is conjugated to a carrier protein. 
     
     
         19 . The method of  claim 12 , wherein the vaccine formulation comprises an active ingredient consisting essentially of an htrB mutant of said gram-negative bacterial pathogen which has been genetically engineered to express at least one heterologous antigen from a microbial pathogen. 
     
     
         20 . The method of  claim 12 , wherein the vaccine formulation further comprises a physiological carrier and an adjuvant. 
     
     
         21 . The method of  claim 15 , wherein the vaccine formulation is administered orally as an additive to the individual's feed. 
     
     
         22 . The method of  claim 12 , wherein the gram-negative bacterial pathogen is a  Neisseria, Haemophilus, Moraxella, Campylobacter, Salmonella, Shigella , or  Pseudomonas  gram-negative bacterial pathogen. 
     
     
         23 . The method of  claim 22 , wherein the gram-negative bacterial pathogen is  Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Haemophilus ducreyi, Moraxella catarrhalis, Campylobacter jejuni, Salmonella typhimurium, Shigella dysentariae , or  Pseudomonas aeruginosa.    
     
     
         24 . The method of  claim 23 , wherein the gram-negative bacterial pathogen is  Haemophilus influenzae.    
     
     
         25 . The method of  claim 24 , wherein the gram-negative bacterial pathogen is non-typeable  Haemophilus influenzae.    
     
     
         26 . The method of  claim 24 , wherein the endotoxin of the htrB mutant contains a decreased phosphoethanolamine content and an increased hexose content in the mutant endotoxin's inner core, and a pentaacylated or tetraacylated lipid A lacking one or two secondary acyl chains compared to the corresponding wild-type non-typeable  Haemophilus influenzae  hexaacylated endotoxin.