Multivalent Pneumococcal Protein Vaccines Comprising Pneumolysoid with Epitopes/Fragments of CbpA and/or PspA Elicit Strong and Broad Protection

Abstract

Immunization with the pneumococcal proteins pneumolysin (Ply), choline binding protein A (CbpA), or pneumococcal surface protein A (PspA) elicits protective responses against invasive pneumococcal disease in animal models. In this study, we used different mouse models to test the efficacy of a variety of multivalent protein-based vaccines that comprised various combinations of full-length or peptide regions of the immunogens Ply, CbpA, or PspA: Ply toxoid with the L460D substitution (referred to herein as L460D); L460D fused with protective peptide epitopes from CbpA (YPT-L460D-NEEK [YLN]); L460D fused with the CD2 peptide containing the proline-rich region (PRR) of PspA (CD2-L460D); a combination of L460D and H70 (L460D+H70), a slightly larger PspA-derived peptide containing the PRR and the SM1 region; H70+YLN; and other combinations. Each mouse was immunized either intraperitoneally (i.p.) or subcutaneously (s.c.) with three doses (at 2-week intervals) of the various antigen combinations in alum adjuvant and then challenged in mouse models featuring different infection routes with multiple Streptococcus pneumoniae strains. In the i.p. infection sepsis model, H70+YLN consistently provided significant protection against three different challenge strains (serotypes 1, 2, and 6A); the CD2+YLN and H70+L460D combinations also elicited significant protection. Protection against intravenous (i.v.) sepsis (type 3 and 6A challenge strains) was largely dependent on PspA-derived antigen components, and the most protection was elicited by H70 with or without L460D or YLN. In a type 4 intratracheal (i.t.) challenge model that results in progression to meningitis, antigen combinations that contained YLN elicited the strongest protection. Thus, the trivalent antigen combination of H70+YLN elicited the strongest and broadest protection in diverse pneumococcal challenge models.

FIG 1

Schematic representation of CbpA and PspA derivatives used in vaccine constructs. (A) Full-length CbpA (top) and regions used to make each construct, YPT and NEEK (bottom). The dotted lines indicate disulfide bridges formed by dual substitutions made in YPT (V333C and K386C) and NEEK (K364C and V439C) to engender a constrained structure of native CbpA. Fusion constructs of peptides with L460D are shown. (B) Full-length PspA (top) and regions used to make each vaccine construct: pUAB055, CD2, H70, and CD2-L460D (bottom).

FIG 2

Properties of vaccine constructs. (A) SDS-PAGE analysis of purified recombinant proteins stained with Coomassie blue. A molecular size marker ladder is also shown. (B) In vitro hemolytic activity of L460D, L460D fusions, and wild-type Ply. The indicated dilutions of Ply and L460D constructs were incubated with 1% washed human erythrocytes for 30 min at 37°C. After centrifugation, hemoglobin release was quantitated by absorbance at 540 nm. Triton X-100- and PBS-treated erythrocytes were used as controls for 100% and 0% lysis, respectively.

FIG 3

Immune responses in mice. (A, C, and D) Serum IgG responses to the various antigens were measured by ELISA using L460D (A), rCbpA (C), or H70 (D) as a coating antigen. BALB-C mice, gray bars; CD1 mice, black bars. The data are shown as the geometric mean (GM) titer, with the error bars denoting the 95% confidence intervals (CI). ND, not determined. (B) Functional activity of L460D antibodies elicited by various vaccine constructs was measured by the ability to neutralize 50% of the hemolytic activity of a standard dose of wild-type Ply. The data are shown as the GM antihemolytic titers. In this assay, the average hemolytic titer of the alum control group was 16.

FIG 4

Survival time of vaccinated mice in three sepsis models. (A to C) Intraperitoneal sepsis model. Mice were challenged with strain D39 (A), 1861 (B), or P9 (C). The solid lines denote the median survival time for each group, and mice that survived to 21 days postinfection were plotted as alive. Significant protection compared to that with the alum control group is indicated in each panel by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (D and E) Intravenous sepsis model. Mice were challenged with strain DBL6A (D) or A66.1 (E). The solid lines denote the median survival time for each group, and mice that survived to 21 days postinfection were plotted as alive. (F) Intratracheal sepsis/meningitis model. Mice were challenged with strain TIGR4X. The solid lines denote the median survival time for each group. The number of mice in each group and the number of mice alive at day 14 are depicted at the top of the panel.

FIG 5

Organ-specific analysis of vaccine protection. (A) Percentage of mice infected with strain TIGR4X that developed meningitis in each vaccine group, as determined by positive cerebrospinal fluid (CSF) culture (mean ± standard deviation). (B) Bacterial density in the nasopharynx of mice surviving to day 14 postinfection with TIGR4X (mean ± standard deviation). (C) Focal pneumonia: Mice were challenged with EF3030, and lungs were harvested at day 5 for CFU recovery. The solid lines denote the group medians. For all graphs, significant differences relative to the alum control group are indicated by asterisks (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).