Preparation Of Meningococcal Conjugate Vaccine Using Rfhbp Of Neisseria Meningitidis Serogroup B As A Protein Carrier And Research On Its Immunogenicity

Neisseria meningitidis is a Gram-negative diplococcus and an obligate human pathogen. Meningococcal strain can be divided into13sero-groups(A、B、C、D、H、I、K、L、X、Y、Z、29E、W135) based on the chemically and antigenically distinct polysaccharide capsule expressed. Five serogroups (A、B、C、Y and W135) account for virtually all the cases of meningococcal disease. The capsule polysaccharide of serogroup A, C, Y and W135can be the vaccine target immunogen even if their poor immunogenicity, it will be the efficient vaccines when conjugate them with protein carrier (change capsule polysaccharide from TI-2Ag to TD Ag). The meningococcal serogroup B polysaccharide capsule is composed of a homolinear polymer of a2-8N-acetylneuraminic acid (sialic acid). The unique chemical structure and antigenic properties of the serogroup B capsule render it poor immunogenic in human. Conventional wild-type OMP vaccines are the only formulations against serogroup B meningococcal disease. These vaccines are suitable for epidemic control and to be used in localized, clonal outbreaks. The most important limitation of this approach is the strain-specific. Following the complete sequencing of the meningococcal genome, the term ’reverse vaccinology’ was launched. Five vaccine targets were found, they are efficient and preserved in all meningococcal. The most promising protein is fHBP. The vaccine that contains recombinant fHBP from E. coli is in clinical trial now.This study was to express and purify the meningococcal outer membrance protein factor H binding protein(fHBP), conjugated this recombinant protein with group C meningococcal polysaccharide(GCMP) using chemical method. Mice were immunized with this conjugate, and then the IgG and serum bactericidal antibody (SB A) against GCMP(GCMP-IgG) and recombinant fHBP(rfHBP-IgG) were detected to discuss its application as a protein carrier in conjugates vaccine. The gene encoding for the fHBP was amplified from the genomic DNA of meningococcal using PCR. The PCR product was then cloned into the prokaryotic expression vector pET30a(+) and the recombinant was transformed into host cell E.coli BL21(DE3). The rfHBP was induced by IPTG and purified by Ni-NTA. The rfHBP was successfully conjugated with GCMP. Mice were immunized subcutaneously with the conjugates and the immune responses against GCMP and rfHBP were detected by ELISA. The SBA was detected with the TTC method. The inserted fhbp gene sequence was confirmed by DNA sequencing and the rfHBP was successfully induced and conjugated with GCMP. High level of rfHBP-IgG and GCMP-IgG were significantly boosted in the mice immunized with conjugate vaccine in comparison with those immunized with GCMP alone(P<0.05). There was no statistical significance in rfHBP-IgG and SBA between the conjugate vaccine group and the rfHBP group(P>0.05).There was no statistical significance between the SBA against ATCC700111and CMCC29361(P>0.05). The mice immunized with the conjugate vaccine were detected high level of antibody and SBA, this suggest that the rfHBP is a promising protein carrier, at the same time, high level of antibody and SBA against meningococcal group B were also detected, show that this kind of conjugate vaccine may provide universal protection against meningococcal.