METHOD OF PRODUCING MENINGOCOCCAL MENINGITIS VACCINE FOR NEISSERIA MENINGITIDIS SEROTYPES A, C, Y, and W-135

Abstract

Methods for producing quadrivalent meningococcal meningitis polysaccharide and conjugate vaccines for serotypes A, C, Y and W-135 disclosed. Neisseria meningitidis fastidious medium was designed to maximize the yield of capsular polysaccharides and generate minimal cellular biomass and endotoxin in a short duration of fermentation. The crude polysaccharides are isolated, purified, and mechanically depolymerized by sonication. These purified polysaccharides were found in human clinical trials to be safe and immunogenic against meningococcal disease caused by N. meningitidis A, C, Y and W-135 serogroups in sub-Saharan Africa. In the preferred embodiment, the polysaccharides are conjugated to carrier proteins of diphtheria or tetanus toxiod to an average molecular size of 5100 to 9900 Daltons and provide broad spectrum protection to humans of all ages. Accelerated polysaccharide production and the efficacy of the resulting vaccine are demonstrated.

1 . A method of producing a meningococcal meningitis vaccine, the method comprising the steps of:
 1. culturing  Neisseria meningitidis  to produce capsular polysaccharides of serotypes A, C, Y and W-135 in  Neisseria meningitidis  fastidious medium (NMFM);   2. isolating the capsular polysaccharides from the culture;   3. purifying the capsular polysaccharides of any residual cellular biomass; and;   4. depolymerizing the capsular polysaccharide mechanically.   
   
   
       2 . The method according to  claim 1  comprising, producing the maximum amount of polysaccharides with minimal amount of cellular biomass and endotoxins in the minimum amount of time by restricting the fermentation process to about 12 hours. 
   
   
       3 . The method according to  claim 1 , wherein the filter sterilized glucose and amino acids are added to the autoclaved cool media to improve production of polysaccharides the process allowing non degradation of heat sensitive sugars and amino acids and eliminating batch feeding during fermentation process for polysaccharide production. 
   
   
       4 . The method according to  claim 1  comprising, purifying polyanionic capsular polysaccharides using a polycationic compound to specifically collect polyanionic polysaccharides by precipitating slowly with the polycationic compound giving high purity and enhanced production of vaccine polysaccharides. 
   
   
       5 . The method according to  claim 1  comprising, purifying and increasing the rate of production of the capsular polysaccharides from the polysaccharide precipitate obtained from the purification process by slowly adding the polycationic compound and calcium chloride to the polysaccharide precipitate collected in the purification process. 
   
   
       6 . The method according to  claim 1 , wherein mechanical depolymerization of the capsular polysaccharides is carried out using sonication. 
   
   
       7 . The method according to  claim 1  comprising, producing the meningococcal meningitis vaccine without any endotoxin immunizing effectively humans aged above the age group of 5 years against  Neisseria meningitidis  serogroups A, C, Y and W-135 without adverse side effects. 
   
   
       8 . A method of producing a meningococcal meningitis vaccine, the method comprising the steps of:
 1. producing capsular polysaccharides as claimed in  claim 1  and;   2. conjugating the depolymerized capsular polysaccharide to one or more carrier proteins.   
   
   
       9 . The method according to  claim 8  comprising, producing the meningococcal meningitis vaccine to an average molecular weight from 5100 to 9900 Daltons. 
   
   
       10 . The method according to  claim 8 , wherein the carrier protein is diphtheria toxoid. 
   
   
       11 . The method according to  claim 8 , wherein the carrier protein is tetanus toxoid. 
   
   
       12 . The method according to  claim 8  comprising, producing the meningococcal meningitis vaccine immunizing effectively humans of all ages including children below the age group of 2 years against  Neisseria meningitidis  serogroups A,C,Y and W-135. 
   
   
       13 . A method of maintaining the pH from 6.5 to 7.0 during the production of  Neisseria meningitidis  capsular polysaccharides using the NMFM medium composition, the method comprising, using calcium carbonate at a concentration of approximately 0.001 percent per liter of the NMFM medium. 
   
   
       14 . A method of reducing the production of cellular biomass and increasing the production of polysaccharides, using the NMFM medium during the production of  Neisseria meningitidis  capsular polysaccharides, the method comprising, removing the total inorganic phosphate salts from the basal medium and providing it with organic phosphates at a concentration of approximately 0.40 percent. 
   
   
       15 . A method of increasing the yield of  Neisseria meningitidis  serogroup A polysaccharides using the NMFM medium, the method comprising adding Ferric sulphate to the medium during the production of polysaccharides. 
   
   
       16 . A method of increasing the yield of  Neisseria meningitidis  serogroup W-135 polysaccharides using the NMFM medium, the method comprising adding ammonium chloride to the medium during the production of polysaccharides. 
   
   
       17 . A method of increasing the yield of  Neisseria meningitidis  serogroups A, C, Y, and W-135 capsular polysaccharides using the NMFM medium, the method comprising, reducing the availability of oxygen for consumption during the production of capsular polysaccharides. 
   
   
       18 . The method of reducing the yield of  Neisseria meningitidis  serogroups A, C, Y, and W-135 cellular biomass using the NMFM medium, the method comprising reducing the availability of oxygen for consumption during the production of capsular polysaccharides. 
   
   
       19 . A method of reducing the specific rate of endotoxin production using the NMFM medium composition, the method comprising reducing the availability of oxygen for consumption during the production of capsular polysaccharides. 
   
   
       20 . A method of reducing the production of endotoxins using the NMFM medium during the production of  Neisseria meningitidis  capsular polysaccharide vaccine, the method comprising, reducing the amount of organic phosphates available in the NMFM medium by buffering it with morpholinepropanesulfonic acid. 
   
   
       21 . A method of producing increased amount of polysaccharides using the NMFM medium during the production of  Neisseria meningitidis  capsular polysaccharide vaccine, the method comprising reducing the amount of organic phosphates available in the NMFM medium by buffering it with morpholinepropanesulfonic acid. 
   
   
       22 . A method of reducing the production of cellular biomass using the NMFM medium composition during the production of  Neisseria meningitidis  capsular polysaccharide vaccine, the method comprising, reducing the amount of organic phosphates available in the NMFM medium by buffering it with morpholinepropanesulfonic acid. 
   
   
       23 . A method of maintaining the pH of the medium throughout the production of  Neisseria meningitidis  capsular polysaccharides, using the NMFM medium, the method comprising, reducing the amount of organic phosphates available in the NMFM medium by buffering it with morpholinepropanesulfonic acid.