Immunization with a ZmpB-based protein vaccine could protect against pneumococcal diseases in mice

Abstract

Zinc metalloprotease B (ZmpB) is present in all isolated pneumococcal strains and contributes to the pathogenesis of pneumococcal infection. In this study, recombinant ZmpB was cloned and expressed in Escherichia coli. The expression of ZmpB by different pneumococcal strains was detectable by Western blotting with antisera raised to recombinant ZmpB. Flow cytometry analysis demonstrated that anti-ZmpB polyclonal antibodies could bind to the cell surface of the pneumococcal strains analyzed. Both recombinant ZmpB protein and anti-ZmpB polyclonal antibodies significantly inhibited the adhesion of Streptococcus pneumoniae D39 to A549 cells. In mouse models, mucosal immunization with recombinant ZmpB could significantly reduce pneumococcal lung colonization caused by S. pneumoniae serotypes 19F and 14 and significantly increase mice survival times following invasive pneumococcal challenge with different pneumococcal strains, including serotypes 2, 3, 6B, and 14. Furthermore, intraperitoneal immunization with recombinant ZmpB in combination with the recombinant pneumolysin mutant (DeltaA146 Ply) and heat shock protein 40 (DnaJ) could enhance the protection against pneumococcal infection compared to protection provided by single-protein antigens. Passive immunization with hyperimmune antisera against these three antigens also demonstrated that the combination of three hyperimmune antisera could provide better protection than single antisera. Taken together, our results suggest that ZmpB is a good candidate pneumococcal vaccine antigen.

FIG. 1.

Purification of rZmpB protein from E. coli. (A) SDS-PAGE analysis (10% gel) of protein samples. Lanes: 1, lysate of untransformed E. coli expression strain; 2, lysate of recombinant E. coli expression construct before induction; 3,supernatant of E. coli lysate centrifuged at 100,000 × g after a 4-h induction with IPTG before being loaded onto Ni-NTA resin; 4, protein markers; 5, purified His6-tagged ZmpB protein (approximate mass, 90 kDa) after elution from Ni-NTA. (B) Western blot analysis of purified rZmpB. SDS-PAGE (10%) was loaded with recombinant SUMO protein (lane 1) and rZmpB (lane 2) purified from E. coli. (C) Western blot analysis of pneumococcal lysates. SDS-PAGE (10%) was loaded with whole-cell lysates obtained from ZmpB mutant D39 (lane 1), wild-type D39 (lane 2), CMCC 31436 (lane 3), CMCC 31207 (lane 4), CMCC 31614 (lane 5), and CMCC 31693 (lane 6). (D) Western blot analysis of pneumococcal supernatants. SDS-PAGE (10%) was loaded with concentrated supernatants of late-exponential-phase broth cultures of ZmpB mutant (lane 1) and wild-type D39 (lane 2). PVDF membrane strips then were reacted with anti-ZmpB sera. Apparent molecular mass in kilodaltons is indicated by arrows.

FIG. 2.

Flow-cytometric analysis of different S. pneumoniae isolates. Flow cytometry analysis was performed using anti-ZmpB sera obtained from immunized mice. Shown are overlays of flow-cytometric histograms demonstrating the staining of D39, CMCC 31436, CMCC 31207, CMCC 31614, CMCC 31693, and ZmpB mutant D39 with anti-ZmpB or preimmune control sera.

FIG. 3.

Analysis of S. pneumoniae adhesion to A549 cells. (A) Adhesion of ZmpB mutant and wild-type D39 cells to A549 cells. The ZmpB mutant and wild-type D39 were added to A549 cells and incubated for 1 h. (B) The effect of rZmpB on S. pneumoniae adhesion. rZmpB at serial concentrations ranging from 0 to 50 μg/ml was added to the cells and incubated for 1 h. S. pneumoniae D39 (1 × 10⁶ CFU) and then were added to A549 cells. Recombinant SUMO protein was used as the control protein. (C) The effect of antibodies against ZmpB on S. pneumoniae adhesion. S. pneumoniae D39 (1 × 10⁶ CFU) was added to A549 cells before or after 30 min of incubation with antisera obtained from mice immunized with recombinant ZmpB. The sera from preimmune mice were used as the control. All adhesion experiments were performed in triplicate, and the total numbers of adherent pneumococci (CFU/well) were recorded. Statistical analysis was performed using 1-way ANOVA. ** and ***, P < 0.01 and < 0.001, respectively, compared between groups denoted by horizontal lines.

FIG. 4.

Immune responses elicited by mucosal immunization with rZmpB. The mice were intranasally immunized with rZmpB, with CT as the mucosal adjuvant. (A) The levels of specific total IgG, IgG1, IgG2a, IgG2b, IgG3, or IgA against rZmpB in sera or saliva samples were tested by ELISA. The antibody titers were defined as the reciprocal of the dilution of sera giving a 2.1-fold value of the highest absorbance value versus the background level at 450 nm. The antibody titers were measured 7 days after the third immunization. (B) Effect of mucosal immunization with rZmpB on cytokine production in splenocytes. Seven days after the last immunization, the splenocytes (1 × 10⁵ cells/well) were cultured in the absence (uninduced) or in the presence (induced) of 5 μg of rZmpB for 72 h at 37°C. After 72 h, the culture supernatants were assayed for the levels of IFN-γ, IL-10, and IL-17A by ELISA. *, P < 0.05 compared between groups denoted by horizontal lines.

FIG. 5.

Protection against pneumococcal pneumonia by mucosal immunization with rZmpB. Mice immunized with rZmpB by the nasal route were intranasally challenged with pneumococcal strain CMCC 31693 (1.0 × 10⁷ CFU) (A) or CMCC 31614 (1.5 × 10⁶ CFU) (B). The lung colonization of individual mice was determined at day 3 after challenge. Each dot represents one mouse. The horizontal lines indicate the median CFU per lung. ***, P < 0.001 compared between groups denoted by horizontal lines.

FIG. 6.

Survival times for mice after intranasal challenge. Groups of 12 BALB/c mice were immunized intranasally with the indicated antigens and challenged 14 days after the third immunization with D39 (7.0 × 10⁷ CFU) (A), CMCC 31436 (6.0 × 10⁸ CFU) (B), CMCC 31207 (3.0 × 10⁸ CFU) (C), or CMCC 31614 (2.0 × 10⁸ CFU) (D). Each dot represents one mouse. The horizontal lines denote the median survival time for each group. ***, P < 0.001 compared between groups denoted by horizontal lines.

FIG. 7.

Survival times for mice after intranasal or intraperitoneal challenge. Groups of 12 BALB/c mice were immunized intraperitoneally with the indicated antigens. Vaccinated mice then were challenged with 2.0 × 10⁸ CFU of D39 intranasally (A) or 1 ×10⁷ CFU of D39 intraperitoneally (B) after the third immunization. Each dot represents one mouse. The horizontal lines denote the median survival times for each group. *, P < 0.05 compared between groups denoted by horizontal lines.

FIG. 8.

Survival times for mice after intraperitoneal challenge. Groups of 12 BALB/c mice were immunized with the indicated antisera containing specific polyclonal antibodies against the indicated antigens and challenged with 1 ×10⁷ CFU of D39 intraperitoneally. Each dot represents one mouse. The horizontal lines denote the median survival time for each group. *, P < 0.05 compared between groups denoted by horizontal lines.