Study On Preparation Of O-Specific Polysaccharide,Target Antigen Of Paratyphoid B Polysaccharide Protein Conjugate Vaccine

Paratyphi is a human intestinal infectious disease caused by Paratyphoid pathogens. Salmonella paratyphi belongs to enterobacteriaceae. Salmonella, enterica subspecies. S. paratyphi A, S. paratyphi B and S. paratyphi C are three serotypes of Salmonella subspecies. Clinical manifestations of Paratyphoid A and Paratyphoid B are similar to typhoid fever, however, diseases caused by Paratyphoid A and B are relatively light. Clinical manifestations of Paratyphoid C are more special, manifests as mild typhoid,acute gastroenteritis or sepsis.Vaccination is the main method of preventing typhoid and paratyphoid bacilli infection in humans. These vaccines have been studied for 70 years. Typhoid vaccines were originally crude products, now they are purified vaccines. With the advancement of technology, typhoid and paratyphoid vaccines are gradually divided into two categories, whole cell vaccine and purified subunit vaccine. Typhoid and paratyphoid A & B combined vaccine is whole cell vaccine, Ty21a is an attenuated live vaccine. Vi polysaccharide typhoid vaccine is a purified subunit vaccine. There are currently no commercially available paratyphoid A and paratyphoid B vaccines. At present, the lipopolysaccharide is used as antigen; this is the main research direction at home and abroad. LPS is detoxified and hydrolyzed into O-specific polysaccharide (O-SP),O-SP can be used to develop polysaccharide protein conjugate vaccine. At present, there is no commercial paratyphoid vaccine; most of them are still in clinical reporting stage and clinical research stage. Three companies have received clinical approval documents and clinical studies are still ongoing.In this study, in order to obtain Salmonella paratyphi B wet bacteria that used to hydrolysis and purification, we selected Salmonella paratyphi B CMCC 50074 strain that preserved in the Chinese Food and Drug Biological Products Research Institute. This Salmonella paratyphi B CMCC 50074 strain was recommended by WHO. First, the three-level seed bank system for Salmonella paratyphi B was established, then S. paratyphi B wet cell was prepared according to WHO and China 2015 pharmacopoeia, the paratyphoid vaccine manufacture and verification protocol. Wet bacteria were used to extract lipopolysaccharide. and lipopolysaccharide was hydrolyzed into O-specific polysaccharides. O-specific polysaccharide was further purified. In the lipopolysaccharide extraction. O-specific polysaccharide hydrolysis and purification process, the contents of the study are as follows:1. The effects of dissolution methods, different concentrations of phenol, CaCl2 and alcohol on the extraction of lipopolysaccharide.2. The effects of different glacial acetic acid concentration and hydrolysis temperature on the detoxification of lipopolysaccharide.3. Purification of O-specific polysaccharides using ethanol fractionation and column chromatography and solvent (formaldehyde, ethanol, phenol) removal.4. The contents of protein, nucleic acid, total solid, phenol, formaldehyde, O-acetyl and endotoxin in prepared lipopolysaccharide were detected. The identification experiment was also carried out. Through these studies, the O-specific polysaccharide extraction preparation process was determined.Our research shows that mortar dissolution heating method,95% phenol aqueous solution,5mM CaCl2 concentration and 70% alcohol concentration were suitable for the preparation of LPS.25% glacial acetic acid concentration and 95℃ hydrolysis detoxification temperature met the technical requirements of O-SP preparation.The hydrolyzed O-SP stock solution was ultra filtered with 5 L of water for injection, the membrane size was 1KD.Ultrafiltration concentrated liquid was added CaCl2 and ethanol, the final concentrations were 0.5 mol/L and 25% respectively. Stirred at room temperature for 30 min.2～8℃ standing overnight. The supernatant was collected by centrifugation and then concentrated by ultra filtration using a 1KD filter. The concentrate was dried in vacuo and dissolved in 0.2 mol/L NaCl solution. The macromolecular peak fraction was then collected using an S100 chromatographic column. The macromolecular peak was concentrated by ultra filtration and dried in vacuo, qualified SPB-O-SP was then obtained. The above experimental industrial parameters ensured the effectiveness and safety of the final vaccine. Indicating that preparation process of paratyphoid B polysaccharide protein binding vaccine antigen was successful. This preparation process is suitable for the production of paratyphoid B polysaccharide protein conjugate vaccine.