The Preparation And Immunogenicity Study Of The Conjugate Vaccine Against Streptococcus Pneumoniae Serotype 7F

Streptococcus pneumoniae(pneumococcus)is now the leading cause of several types of serious invasive pneumococcal diseases.Infants under the age of 2 years and the elderly are the main susceptible.Vaccination is the most effective intervention in preventing S.pneumoniae infection.There are two main Pneumococcal vaccine,polysaccharide vaccine and polysaccharide-protein conjugate vaccine.Pneumococcal capsular polysaccharide is T cell-independent antigen and insufficiently immunogenic in infants.Conjugation of PSs to carrier proteins converts it to a T cell-dependent antigen.Pneumococcal conjugate vaccine can induce good immunogenicity and long-term protection against Streptococcus pneumoniae in infants.In this study,we developed a process for the preparation of the conjugate vaccine against one of the serotypes of Streptococcus pneumonia 7F(Pn7F).The main work described in this report is summarized below:(1)The Pn7F polysaccharide was first activated by oxidation using sodium periodate(NaIO4),creating the aldehyde groups.The effect of the activation conditions,including NaIO4 concentrations,reacting time and temperature,on the polysaccharide properties,such as the molecular weight,the degree of oxidation as well as the recovery,was investigated.The results indicated that the degree of oxidation of the Pn7F polysaccharide was reduced with the increase of the concentration of NaIO4.The optimal activation condition was determined as:room temperature for 5 hours,at pH 4.5.The activated polysaccharides were purified and recovered by ultrafiltration using a 100kDa membrane..(2)The conjugate vaccines of the Pn7F polysaccharide conjugated to the nontoxic variant of diphtheria toxin(CRM 197)and pneumococcal surface protein A(PspA)were prepared,in both organic phase and aqueous phase.We studied the effect of different carrier proteins and conjugation processes on the immunogenicity of the resulting vaccine products.The conjugate vaccine preparations using CRM 197 as the carrier were shown to induce better immune response than the conjugates with PspA.When CRM 197 was used as the carrier,the conjugates prepared in the aqueous phase generated better immune response than those prepared in the organic phase.The conjugates when degree of oxidation is 11 were demonstrated to be more immunogenic.(3)The antigenicity of the Pn7F polysaccharide--CRM197 congugates was examined by an indirect competitive inhibition Elisa assay.Our results revealed that the conjugates prepared in the organic phase had additional dominant epitopes,which were missing on the conjugates prepared in the aqueous phase.(4)A method for quantitative determination of free polysaccharides in the Pn7F polysaccharide-CRM197 congugate preparations was developed The Pn7F polysaccharide-CRM197 conjugates could be specifically and efficiently precipitated with Sodium Deoxycholate(NaDOC)at the pH range of 2.5-4.0 and at the protein concentration range of 100-300 mg/L.This assay was fast,accurate and highly reproducible,and could be used for process/quality control in the production of the Pn7F polysaccharide-CRM 197 congugate vaccine.