Characterization of the target antigen of mAb 54-07 and evaluation of a multivalent vaccine against the meningococcal infection
| Autor: | Chi-Jui Lin, 林祺叡 |
|---|---|
| Rok vydání: | 2013 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 Neisseria meningitidis (NM) is an encapsulated gram-negative diplococcus, which can be classified into 13 serogroups based on the antigenicity of their capsular polysaccharide (CPS). Most pathogenic strains belong to serogroup A, B, C, Y, W135 and X. In addition to the variation on the CPS, many outer membrane proteins of these organisms are also subjected to antigenic and/or phase variation consequently limiting the effectiveness of single antigen vaccines. One way to obtain a broadly protective vaccine against NM infection is to use multi-component vaccines. The meningococcal lipoprotein Ag473 as a vaccine candidate has previously been demonstrated. In this study, the effectiveness of an Ag473-based multi-component vaccine (MP) including four minor outer membrane proteins (mOMPs), Ag5407, Ag-lyi, P64k(F) and Ag-IIC3(F) to protect mice against group B meningococcal disease was evaluated. Groups of mice were immunized with Ag473, Ag473 plus mOMPs (MP), or PBS. After confirming the presence of anti-NM specific antibodies in Ag473 and MP-immunized mice by ELISA, mice were challenged with the serogroup B strain Nm22209-6R3. With 300 CFU challenge dose, the survival rates for PBS-, Ag473-, and MP-immunized mice were 20%, 40%, and 80%, respectively. When increase the challenge dose to 1500 CFU, the survival rate of the Ag473-immunized group reduced to 20% while 80% of the MP-immunized mice remained to be protected. The results show that adding of the minor proteins, although each alone does not exhibit protection, to the Ag473 can significantly improve the protection effectiveness. In addition, the epitope on Ag5407, the target antigen of the monoclonal antibody (mAb) 54-07 which binds to the meningococcal surface, was defined by gene fragmentation. The results indicate that the functional epitope lies within the C-terminal 67 amino acid residues. Deletion of five residues from either N- or C-terminus of this region destroyed the antigenicity completely suggesting that the mAb 54-07 recognizes a conformational epitope. Because Ag5407 is a ribosomal subunit and detected only on a small portion of meningococcal surfaces suggesting that this protein may have other functions. To address this question, plasmid pEN11-LS5407 encoding a lipidated Ag5407 was introduced into group B MC58 and W135 NM1996-020 strains. The surface expression of the lipidated Ag5407 in the transformants grown in the presence of IPTG was confirmed by flow cytometry. Functional analysis suggested that the surface expression of Ag5407 enhances the adhesion of meningococci to human epithelial cells but does not affect the serum sensitivity of meningococcal strains. Finally, this study accidently discovered that chloramphenycol acetyltransferase, the selectable marker on the plasmid pEN11, may use the capsular polysaccharide of W135 as the acetyl group acceptor in the absence of chloramphenical. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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