A Pseudomonas aeruginosa-Derived Particulate Vaccine Protects against P. aeruginosa Infection

Abstract

Despite numerous efforts to develop an effective vaccine against Pseudomonas aeruginosa, no vaccine has yet been approved for human use. This study investigates the utility of the P. aeruginosa inherently produced polyhydroxyalkanaote (PHA) inclusions and associated host-cell proteins (HCP) as a particulate vaccine platform. We further engineered PHA inclusions to display epitopes derived from the outer membrane proteins OprF/OprI/AlgE (Ag) or the type III secretion system translocator PopB. PHA and engineered PHA beads induced antigen-specific humoral, cell-mediated immune responses, anti-HCP and anti-polysaccharide Psl responses in mice. Antibodies mediated opsonophagocytic killing and serotype-independent protective immunity as shown by 100% survival upon challenge with P. aeruginosa in an acute pneumonia murine model. Vaccines were stable at 4 °C for at least one year. Overall, our data suggest that vaccination with subcellular empty PHA beads was sufficient to elicit multiple immune effectors that can prevent P. aeruginosa infection.

Keywords: Pseudomonas aeruginosa; polyhydroxyalkanoate; protective immunity; vaccine.

Figure 1

Schematic overview of the production and immunological evaluation of PHA bead vaccines. PHA beads coated with the selected P. aeruginosa candidate antigens were produced from P. aeruginosa PAO1ΔCΔ8ΔF mutant strain. Plasmid harboring strains were grown under PHA accumulating conditions to mediate overproduction of the fusion protein and subsequent PHA bead self-assembly. Formation of PHA, Ag-PHA and PHA-PopB beads resulted in the display of the selected antigens covalently linked to the PHA synthase along with the HCPs. PHA beads were isolated by mechanical disruption and subsequent purification. Mice were vaccinated with sterile PHA beads via subcutaneous injection with Alhydrogel as an adjuvant. Immunological responses and survival after intranasal challenge with the P. aeruginosa strain N13 were measured.

Figure 2

Production and biophysical characterization of PHA beads. (A) Schematic representation of hybrid genes encoding fusion proteins for the production of PHA, Ag-PHA and PHA-PopB beads in P. aeruginosa PAO1ΔCΔ8ΔF strain. MW (Molecular weight). phaC1 is the PHA synthase gene. (B) Table showing PHA beads composition and yield data. (C) TEM images for accumulation and size analysis of PHA beads in whole-cell and isolated PHA beads fractions. (D) Size of PHA beads before and after formulation with Alhydrogel adjuvant. Polydispersity indices are shown above the beads. (E) Zeta potential of PHA beads before and after formulation with Alhydrogel adjuvant. The particle size and zeta-potential of each PHA beads were measured three times by Zetasizer Nano ZS. Each data point of measurement represents the mean ± SEM. The values are shown in Table S3.

Figure 3

Composition of PHA bead vaccines isolated from engineered P. aeruginosa. (A) GC-MS for quantification and compositional analysis of PHA beads shown as percentage of CDW or BDW (CDW, cellular dry weight; BDW, bead dry weight). 3-HB (C4), 3-hydroxybutanoate; 3-HH (C6), 3-hydroxyhexanoate; 3-HO (C8), 3-hydroxyoctanoate; 3-HD (C10), 3-hydroxydecanoate; 3-HDD isomer (C12), 3-hydroxydodecanoate isomer; 3-HDD (C12), 3-hydroxydodecanoate; 3-HTD (C14), 3-hydroxytetradecanoate; and 3-HHD (C16), 3-hydroxyhexadecanote. P. aeruginosa PAO1ΔCΔ8ΔF is the negative control empty cells. (B) Protein profile analysis of whole-cell lysate and the PHA beads separated by SDS-PAGE and gel stained with Coomassie Blue. PHA (63.2 kDa), Ag-PHA (77.7 kDa), and PHA-PopB (86.4 kDa) fusion proteins (in red arrows) along with the HCPs were isolated from P. aeruginosa PAO1 ΔCΔ8ΔF strain containing the respective plasmids. Selected co-purifying HCPs were numbered I-IX. The PHA and PHA fusion proteins were confirmed by mass spectrometry (Table S4). (C) Table summarizing the identification of the nine PHA bead associated HCPs by MS. Complete list is shown in Table S5.

Figure 4

Antigen-specific antibody responses in mice vaccinated with different PHA beads. (A) Antigen-specific total IgG, IgG1 or IgG2c antibody responses measured by ELISA. Each data point represents results from 12 mice ± SEM. Statistical analysis was done by one-way ANOVA with statistical significance (p < 0.05) indicated by letter-based representation of pairwise comparisons between groups using Tukey’s post hoc test (i.e., data with different letters are statistically significant). (B) Specific antigen recognition by immunoblot using pooled antisera from PHA bead-vaccinated mice (n = 12 per group). PhaC1, Ag-PhaC1 and PhaC1-PopB are in red arrows.

Figure 5

PHA bead vaccines induced functional antibodies and protective immunity. a-c OPK assay against P. aeruginosa (A) strain PAO1, (B) strain PAO1ΔpslA, and (C) serotype O11 strain 9882-80. Percent killing was calculated relative to results obtained in assays run in the absence of serum/antibody. Results are one representative experiment of three independent experiments performed. (D) Survival of the vaccinated FVB/N mice after challenge with the serotype O6 P. aeruginosa strain N13. Mice (n = 12 per group) were immunized SC with Alhydrogel, PHA bead + Alhydrogel, Ag-PHA bead + Alhydrogel, or PHA-PopB bead + Alhydrogel, once every 2 weeks for 6 weeks (3 doses), and then intranasally challenged 3 weeks later.