Article Figures & Data
Figures
- FIG. 1.
Regions of whole PspA (top) and portions of the full protein used in the constructs (bottom; see labels on the left) used in this paper. Amino acid sequences to the right of the construct diagrams are distinguished by region as follows: unbolded amino acids represent PspA sequence outside the proline-rich region, bolded amino acids represent proline-rich regions, underlined amino acids represent the NPB within a PR region, and boxed amino acids represent a hypothesized epitope for MAb PR-5C4.7. Diagrams in this figure are not drawn to scale.
- FIG. 2.
Reactivity of MAb to PR regions. Western blot of PR recombinant proteins reacted with MAb to PR regions (upper panels). Flow cytometry analysis of binding of MAb to pneumococcal strain D39 and its ΔpspA, ΔpspC, and ΔpspA ΔpspC mutants (lower panels). The ΔpspA ΔpspC mutant bound significantly less antibody than the wild type for all three MAb that bound the wild type (P < 0.0001 by ANOVA). Results for the Dunnett's multiple-comparison test against the wild-type control are as follows: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Comparisons to the ΔpspA ΔpspC double mutant are as follows: +, P < 0.05; ++, P < 0.01; and +++, P < 0.001.
- FIG. 3.
Active immunization with PR recombinant proteins protects against i.v. infection with pneumococcal type 3 strain 3JYP2670. CBA/N mice immunized with Imject alum control, vector tag protein, PR+NPB, PR-NPB, or NPB were infected i.v. with type 3 strain 3JYP2670 and monitored for the number of days to moribundity. Mice plotted as “Alive” were still not moribund at 21 days postinfection. None of the “Alive” mice showed ruffled fur, a hunched back, or any other signs of illness. All three proteins were found to protect significantly against infection compared to a pool of the alum and two vector controls (for Kruskal Wallis, P < 0.0001; P values were <0.001 [***] and <0.05 [*] compared to the pooled control using Dunn's multiple-comparison post hoc test). None of the control groups protected significantly more than any of the others in a separate post hoc analysis. The sequence of the PR region of infection strain 3JYP2670 PspA is distinguished by regions as in Fig. 1.
- FIG. 4.
Surface binding of MAb (bars) and passive protection against infection (symbols) for pneumococcal challenge strain WU2. Monoclonal antibodies for each panel are listed on the x axis, and the sequence of the single PR region in the PspA of strain WU2 is given below. For each antibody, the log binding to the infection strain determined by flow cytometry is indicated as a bar graph along the left y axis, and the number of days to reach moribundity is indicated as a dot plot along the right y axis. During challenge, mice were injected with antibody and infected i.v. 1 h later. Two MAb, PR-1A4.7 and PR-6A5.12, protected significantly compared to the control group that received no antibody (Kruskal Wallis P value, 0.0002; **, P < 0.01 using Dunn's multiple-comparison test to the control). MAb 2A4, which interacts with the alpha-helical region of family 1 PspA, was used as a positive control. The protective MAb (PR-1A4.7, PR-6A5.12, and 2A4) bound significantly more strongly to the infection strain (WU2) than did the nonprotective MAb (PR-5C4.7) or the saline (no-antibody) control (ANOVA P value, <0.001. ***, P < 0.01 compared to nonprotective strains using Tukey's post hoc test).
- FIG. 5.
Surface binding of MAb (bars) and passive protection against infection (symbols) for pneumococcal challenge strain BG12730. Monoclonal antibodies for each panel are listed on the x axis, and the sequence of the PR regions in the PspA and the PspC of strain BG12730 is given below the x axis. Mice indicated with solid symbols were scored as moribund and euthanized due to neurologic symptoms. Mice indicated with open symbols were scored as moribund and euthanized due to a surface temperature of <24°C. For each antibody, the binding to the infection strain determined by flow cytometry is indicated as a bar graph along the left y axis, and the number of days to reach moribundity is indicated as a dot plot along the right y axis. During challenge, mice were injected with antibody and infected i.v. 1 h later. In this challenge, MAb PR-5C4.7 protected significantly well compared to the control group that received no antibody (Kruskal Wallis P value, 0.01; **, P < 0.01 using Dunn's multiple-comparison test to the control). Monoclonal antibody 2A4, which does not bind the PspA of this strain, was included as a negative control.
Tables
- TABLE 1.
Strains used in this studya
Strain Sequence reference Wild type Capsule NPB PspA PspA PspC Family Clade D39 8, 21 NA 2 + + 1 2 JY53 46 D39 2 Tru + NA NA TRE118 19 D39 2 + Ins/dup 1 2 TRE121.3 19 D39 2 Ins/dup Ins/dup NA NA 3JYP2670 This paper NA 3 + − 2 4 WU2 21 NA 3 + − 1 2 BG12730 This paper NA 6A − + 2 3 AC094 21 NA + Unknown 1 1 TIGR4 41 NA 4 − + 2 3 BG9739 21 NA 1 1 ↵ a NA, not applicable; Tru, a truncated version of PspA is expressed that does not contain choline-binding repeats and is secreted from the bacterial surface; Ins/dup, insertion duplication to interrupt protein production; +, presence of the NPB in the molecule; −, absence of the NPB in the molecule.
- TABLE 2.
Binding of polyclonal sera from immunized mice to PR+NPB-6× measured by ELISA
Antigen Avg binding (±SE)b P valuea Alum PR+NPB PR+NPB 2.72 (0.050) <0.0001 PR-NPB 1.97 (0.065) <0.0001 <0.0001 NPB 1.72 (0.080) <0.0001 <0.0001 pET41 0.851 (0.084) >0.05 <0.0001 Alum 0.620 (0.025) <0.0001 ↵ a Binding of polyclonal sera to PR+NPB-6× was significantly different by ANOVA (P < 0.0001). P values in the table are Tukey's post hoc comparisons of immunization antigen groups to either an alum-immunized group or a PR+NPB-immunized group. PR-NPB and NPB groups did not have significantly different binding.
↵ b Measured by antibody index.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Table S1. Plasmids expressing recombinant PR proteins.
MS Word document, 39K.
- Supplemental file 1
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Table S1. Plasmids expressing recombinant PR proteins.
