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 sero types A, C, Y and W-135 disclosed. Neisseria meningitidis fastidious medium was designed to maximize the yield of capsular polysaccharides and generate minimal cellular bio mass 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 sero groups in sub-Saharan Africa. In the preferred embodiment, the polysaccharides are conjugated to carrier proteins of diphtheria or tetanus toxoid 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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of U.S. Utility application Ser. No. 11 / 761,667 which was published as 20080020002 claiming the priority date of Jul. 19, 2006, being the Non Provisional Application of U.S. Provisional Application No. 60 / 831,682 filed on Jul. 19, 2006.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates generally to the field of medical microbiology, immunology, vaccines and the prevention of infection by a bacterial pathogen by immunization.[0004]2. Description of the Related Art[0005]Meningococcal meningitis is an infection of the meninges, the thin lining that surrounds the brain and the spinal cord. The causative agent, Neisseria meningitidis (the meningococcus), was identified in 1887. Meningococcal disease was first reported in 1805 when an outbreak swept through Geneva, Switzerland.[0006]Twelve subtypes or serogroups of N. meningitidis have been identified and fo...

AI Technical Summary

Benefits of technology

[0050]Methods for producing quadrivalent meningococcal meningitis polysaccharide vaccine for serotypes A, C, Y and W-135 by mechanical means: The methods employ Neisseria meningitidis fastidious medium specially designed to maximize the yield of capsular polysaccharides and minimize yield of the cellular biomass and endotoxins. The crude polysaccharides are isolated and purified by ultra-filtration and gently treated with a polycationic compound that precipitates the polyanionic capsular polysaccharides and to maximize the yield of precipitated polysaccharides from liquid cultures. The polysaccharides are then mechanically depolymerized, preferably by sonication. The pure polysaccharides were found in human clinical trials to be highly effective against meningitis caused by N. meningitidis A, C, Y and W-135 serogroups. In the most preferred embodiment the pure polysaccharides are conjugated to carrier proteins of diphtheria or tetanus toxoid to provide broad spectrum protection to humans of all age groups.

[0051]The present invention is directed to a method of producing meningococcal meningitis vaccine in the Neisseria meningitidis fastidious medium with composition of medium comprising DI water, NaCl, K2SO4, KCl, Trisodium citrate.2H2O, MgSO4.7H2O, MnSO4.H2O, MnCl2.6H2O, Vitamin B12 (from a Plant source, for example, Saccharomyces cerevisiae), NAD (Nicotinamide adenine dinucleotide), Thiamine HCL, Soy peptone, D-Glucose, L-Glutamic acid, L-Arginine, L-Serine, L-Cysteine, Glycine, Morpholinepropanesulphonic acid [MOPS], CaCO3 with the PH maintained at 6.5 to 7.0 (Fe2 (SO4)3 for serogroup A and NH4Cl for serogroup W-135). The specific formulation used in the experiments conducted is given below.Neisseria Meningitidis Fastidious Medium (NMFM) for serogroups A, C, Y and W-135: (grams per Liter)Components: with the PH maintained atQuantityConcentration6.5 to 7.0(g / L)(mM)DI water900mLNaCl0.35gK2SO40.20gKCl0.20gTrisodium citrate•2H2O0.70gMgSO4•7H2O0.60gMnSO4•H2O1.00mgMnCl2•6H2O40mgVitamin B12 (source: Saccharomyces10.0gcerevisiae)NAD (Nicotinamide adenine dinucleotide)0.25gThiamine HCLSoy peptone15gD-Glucose10gL-Glutamic acid5.10L-Arginine0.237L-Serine0.476L-Cysteine0.254Glycine1.998Morpholinepropanesulphonic acid10[MOPS]CaCO30.25* Fe2(SO4)3 = 0.5 g / L for seogroup A* NH4Cl = 1.25 g / L for serogroup W-135* The addition of Ferric Sulphate to the NMFM medium was found to increase the production of Serogroup A and the addition of Ammonium Chloride to the NMFM medium was found to increase the production of Serogroup W-135 poysaccharides, while their absence leads to reduced production of the respective serogroups.

[0052]Filter sterilized glucose and amino acids were added to the autoclaved cool medium, which improved production of polysaccharides by 25%. This type of process allowed non-degradation of heat sensitive sugars and amino acids and eliminated batch feeding during the fermentation process for polysaccharide production. The above medium is specially designed to increase the production of capsular polysaccharide and decrease the production of cellular biomass. One more special feature of the medium is that the pH is maintained from about 6.5 to about 7.0 during the fermentation process without using buffers and pH probes. Here in this invention the phenol extraction step is replaced by activated carbon filtration to avoid any phenol interaction in purification processes. The isolated polyanionic polysaccharides are then precipitated with a polycationic compound. The precipitated polysaccharides are then subjected to ultra-filtration for the isolation of pure polysaccharides. The isolated pure polysaccharides are depolymerized by sonication. These low molecular weight polysaccharides proved very effective when compared with other inventions. The human trials for pure polysaccharides of serotypes A, C, Y and W-135 in this invention indicated very mild adverse side effects, none of which were severe, and also proved to be very effective for humans above the age of 13 years and may provide effective protection against meningococcal meningitis for humans above the age of 5 years.

[0053]In another preferred embodiment the pure low molecular weight polysaccharides were conjugated to carrier proteins of diphtheria or tetanus toxoids to produce quadrivalent meningococcal meningitis conjugated vaccine for the serotypes A, C, Y and W-135. This conjugated vaccine proved effective for all ages. The vaccine proved to be non-toxic and immunogenic in animal trials using neonatal mice and mice of 7-8 weeks, when compared with the known state of the art. The use of mice models in animal trials may show that the conjugated quadrivalent polysaccharide vaccine A, C, Y and W-135 may also be effective for at risk age groups of the children below 2 years and can immunize effectively humans of all ages.

Problems solved by technology

Bacterial meningitis may result in brain damage, hearing loss, or learning disability in 10 to 20% of survivors.

During the dry season, between December and June, because of dust winds and upper respiratory tract infections due to cold nights, the local immunity is diminished, increasing the risk of meningitis.

Polysaccharides themselves are poor at stimulating an effective antibody response in the highest risk age groups (infants).

The problem with the invention disclosed in this patent is that the phenol contaminants may interfere with the pure polysaccharide production process.

The problem with the invention disclosed in this patent is that the phenol contaminants may interfere with the pure polysaccharide production process.

The problem with that invention is the chemical process for depolymerization may interfere with the purity in polysaccharide vaccines production.

The problem with the invention art is that large scale biomass production reduces the production of capsular polysaccharides.

The problem with this invention is that the chemical residues tend to induce adverse side effects during routine immunization.

The average size of purified capsular polysaccharides is about 8,000 to 35,000 Daltons, which may not provide efficient immune response in humans.

The problem with this medium is that it requires pH adjustment during the fermentation process.

Glucose utilization is higher in this medium, resulting in excessive cellular biomass.

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