Article Figures & Data
Figures
- Fig. 1.
Purification of His6-tagged truncated CbpA fragment from recombinant E. coli. (A) SDS-PAGE analysis (12% gel) of protein samples from various stages of the purification procedure. Lanes: 1, lysate of recombinant E. coliexpression construct before induction; 2, 100,000 ×g supernatant of E. coli lysate after a 4-h induction with IPTG before being loaded onto Ni-NTA resin; 3, unbound fraction washed from the resin; 4, purified His6-tagged CbpA fragment (approximate mass, 75 kDa) after elution from Ni-NTA with imidazole. (B) Western immunoblot analysis of corresponding samples in panel A after they were electroblotted onto nitrocellulose. The samples were reacted with anti-His6monoclonal antibody.
- Fig. 2.
Western immunoblot analysis of purified PdB (53 kDa) and His6-tagged CbpA fragment (75 kDa) (A) and of whole-cell lysates of S. pneumoniae D39 (B), isogenic CbpA-negative D39 (4) (C), and isogenic PspA-negative D39 (4) (D) showing specificity of antibody responses to the protein antigens. The proteins were separated by SDS-PAGE and then electroblotted onto nitrocellulose. They were then reacted with sera from the groups of mice immunized with the proteins in different adjuvant combinations. Lane C, Coomassie blue-stained gel showing the relative mobilities of the two purified proteins. Lanes 1 to 8, nitrocellulose membrane strips reacted with sera from mice immunized with AlPO4 adjuvant (lane 1), MPL plus AlPO4(lane 2), PdB in AlPO4 adjuvant (lane 3), PdB in MPL plus AlPO4 (lane 4), CbpA in AlPO4 adjuvant (lane 5), CbpA in MPL plus AlPO4 (lane 6), a combination of PdB and CbpA in AlPO4 adjuvant (lane 7), and a combination of PdB and CbpA in MPL plus AlPO4 (lane 8). The arrows show a band of approximately 60 kDa cross-reacting with the anti-CbpA serum pool.
- Fig. 3.
Survival times for mice after intraperitoneal challenge. Groups of 13 or 14 BALB/c mice were immunized with the indicated antigens and challenged 2 weeks after the third immunization with approximately 1.3 × 105 CFU of the capsular type 2 strain D39. The broken lines denote the median survival time for each group.
- Fig. 4.
Survival times for mice after intraperitoneal challenge. Groups of 14 or 15 BALB/c mice were immunized with the indicated antigens and challenged 2 weeks after the third immunization with approximately 1.3 × 107 CFU of capsular type 2 strain D39. The broken lines denote the median survival time for each group.
Tables
- Table 2.
Statistical comparison of median survival timesa
Immunization group Statistical difference (P value) between indicated groups AlPO4 MPL + AlPO4 PdB + AlPO4 PdB + MPL + AlPO4 CbpA + AlPO4 CbpA + MPL + AlPO4 PdB + CbpA + AlPO4 PdB + CbpA + MPL + AlPO4 AlPO4 <0.025 ≪0.001 ND ≪0.001 ND ≪0.001 ND MPL + AlPO4 <0.001 ND ≪0.001 ND ≪0.001 ND ≪0.001 PdB + AlPO4 NS ND ≪0.01 ≪0.001 ND ≪0.001 ND PdB + MPL + AlPO4 ND ≪0.001 <0.025 ND NS ND NS CbpA + AlPO4 NS ND NS ND NS NS ND CbpA + MPL + AlPO4 ND <0.001 ND <0.05 NS ND NS PdB + CbpA + AlPO4 <0.025 ND <0.01 ND NS ND NS PdB + CbpA + MPL + AlPO4 ND ≪0.001 ND NS ND NS NS ↵a Differences in median survival times between groups of mice immunized with the indicated antigen formulations were analyzed using the Mann-Whitney U test (one tailed). P values are indicated for groups challenged with 1.3 × 105 CFU or 1.3 × 107 CFU (in bold) of capsular type 2 strain D39. ND, not determined; NS, not significant.
