Pertussis Vaccine Candidate Based on Outer Membrane Vesicles Derived From Biofilm Culture

Abstract

Outer membrane vesicles (OMV) derived from Bordetella pertussis-the etiologic agent of the resurgent disease called pertussis-are safe and effective in preventing bacterial colonization in the lungs of immunized mice. Vaccine formulations containing those OMV are capable of inducing a mixed Th1/Th2/Th17 profile, but even more interestingly, they may induce a tissue-resident memory immune response. This immune response is recommended for the new generation of pertussis-vaccines that must be developed to overcome the weaknesses of current commercial acellular vaccines (second-generation of pertussis vaccine). The third-generation of pertussis vaccine should also deal with infections caused by bacteria that currently circulate in the population and are phenotypically and genotypically different [in particular those deficient in the expression of pertactin antigen, PRN(-)] from those that circulated in the past. Here we evaluated the protective capacity of OMV derived from bacteria grown in biofilm, since it was observed that, by difference with older culture collection vaccine strains, circulating clinical B. pertussis isolates possess higher capacity for this lifestyle. Therefore, we performed studies with a clinical isolate with good biofilm-forming capacity. Biofilm lifestyle was confirmed by both scanning electron microscopy and proteomics. While scanning electron microscopy revealed typical biofilm structures in these cultures, BipA, fimbria, and other adhesins described as typical of the biofilm lifestyle were overexpressed in the biofilm culture in comparison with planktonic culture. OMV derived from biofilm (OMVbiof) or planktonic lifestyle (OMVplank) were used to formulate vaccines to compare their immunogenicity and protective capacities against infection with PRN(+) or PRN(-) B. pertussis clinical isolates. Using the mouse protection model, we detected that OMVbiof-vaccine was more immunogenic than OMVplank-vaccine in terms of both specific antibody titers and quality, since OMVbiof-vaccine induced antibodies with higher avidity. Moreover, when OMV were administered at suboptimal quantity for protection, OMVbiof-vaccine exhibited a significantly adequate and higher protective capacity against PRN(+) or PRN(-) than OMVplank-vaccine. Our findings indicate that the vaccine based on B. pertussis biofilm-derived OMV induces high protection also against pertactin-deficient strains, with a robust immune response.

Keywords: Bordetella pertussis; biofilm; outer membrane vesicles; planktonic; protection; vaccine.

Figure 1

Biofilm formation by B. pertussis Tohama I strain (gray column) and the clinical isolate BpAR106 (black column) on an abiotic surface. Biofilm biomass was quantified at 96 h of growth by staining with crystal violet and absorbance was measurement at OD 595 nm. Each value represents the mean from three independent experiments and the bars indicate standard deviation. Statistically significant differences in Student’s t-test between the clinical isolate and B. pertussis Tohama I biomass absorbance are indicated by * (p < 0.05).

Figure 2

(A) Scanning electron microscopy of B. pertussis BpAR106 biofilm culture on abiotic surface at 24h and 96h of growth. For observation through the FEI Quanta 200 electron microscope (24,000 x magnification), the samples were dried by the critical point technique and coated in gold. (B) To visualize the OMV, an electronic zoom of panel (A) is shown. White arrows were used to mark the presence of OMV.

Figure 3

(A) Volcano plot of protein samples from the clinical isolate B. pertussis BpAR106 grown in biofilm (Biof) or planktonic (Plank) condition. -Log₁₀ of the p-value associated with the magnitude of the fold-change of a given protein between the biofilm and planktonic growth conditions is plotted on the ordinate as a function of the log₂ of that fold change on the abscissa. M = log₂(fold-change). The overexpressed proteins are indicated in red (M ≥ 1), and the underexpressed proteins in blue (M ≤ −1) based on p<0.05. The proteins located on the 0 line correspond to the ON/OFF proteins since those proteins did not have an associated p-value. (B) Representation of the different clusters of orthologous groups (COGs) containing the proteins differentially expressed under biofilm culture condition of B. pertussis BpAR106. In the figure, the bar length represents the number of overexpressed (black) or underexpressed (gray) proteins within each COG among the total differentially expressed proteins.

Figure 4

(A) SDS-PAGE (12.5%) of OMV derived from B. pertussis BpAR106 grown under planktonic or biofilm culture conditions. Molecular weights are indicated at the left. (B) Lipo-oligosaccharides content in OMVplank and OMVbiof suspensions normalized by total protein content. (C) Scanning electron micrographs of negative stained OMV obtained from B. pertussis BpAR106 grown under planktonic or biofilm culture conditions (scale bar: 200 nm).

Figure 5

Anti-B. pertussis antibodies induced by 2-dose vaccination schedules. (A) Anti-B. pertussis IgG titers as well as the IgG isotypes were measured 14 days after the second vaccination dose. The titers are expressed as the geometric mean of the data from each group. (B) The avidity of IgG antibodies was also measured 14 days after the second dose and is represented as percentages of eluted B. pertussis-specific antibodies after treatment with increasing concentrations of ammonium thiocyanate (NH₄SCN). The asterisk indicates statistically significant difference with p < 0.05. (C) Immunoblotting of total proteins of B. pertussis BpAR106 separated by 12.5% (w/v) SDS-PAGE and probed with the polyclonal antiserum obtained from immunized mice. The sera are designated according to the OMV-based vaccine used to immunize the mice. (D) Immunoblotting of BpAR106 LOS from both culture condition here tested separated by 12.5% (w/v) SDS-PAGE and probed with the polyclonal antiserum obtained from immunized mice. The sera are designated according to the OMV-based vaccine used to immunize the mice.

Figure 6

Protection against B. pertussis PRN(-) isolates induced with OMVbiof- or OMVplank-based vaccines in a mouse model. BALB/c mice were immunized (i.m) twice, 2 weeks apart. Mice were challenged with sublethal doses (5 x 10⁷ CFU/40 μl) of B. pertussis BpAR106 PRN(+) (A), USA PRN(-) B. pertussis clinical isolate (B) or Argentinian PRN(-) B. pertussis clinical isolate (C), 2 weeks after the second immunization with OMV-based vaccine. Non-immunized animals were included as negative control of protection. Three independent experiments were performed for each strain/isolate. Results from one representative experiment are shown. Results depicted are means of 5 mice per group sacrificed at 7 days post-challenge. The dashed line indicates the lower limit of detection. The number of bacteria recovered from mouse lungs is expressed as the average log₁₀ CFU ± SEM (error bars) per lung. Data obtained were analyzed statistically by using one-way analysis of variance (ANOVA) followed by Bonferroni´s multiple comparison test (GraphPadPrism^®). For panel (A) significant differences among the treatments with p < 0.001 were detected. For panels (B, C) significant differences among the treatments with p < 0.05 were detected.