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A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles - PubMed

  • ️Thu Jan 01 2015

A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles

Deborah R Leitner et al. Front Microbiol. 2015.

Abstract

Enteric infections induced by pathogens like Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) remain a massive burden in developing countries with increasing morbidity and mortality rates. Previously, we showed that the immunization with genetically detoxified outer membrane vesicles (OMVs) derived from V. cholerae elicits a protective immune response based on the generation of O antigen antibodies, which effectively block the motility by binding to the sheathed flagellum. In this study, we investigated the potential of lipopolysaccharide (LPS)-modified and toxin negative OMVs isolated from V. cholerae and ETEC as a combined OMV vaccine candidate. Our results indicate that the immunization with V. cholerae or ETEC OMVs induced a species-specific immune response, whereas the combination of both OMV species resulted in a high-titer, protective immune response against both pathogens. Interestingly, the immunization with V. cholerae OMVs alone resulted in a so far uncharacterized and cholera toxin B-subunit (CTB) independent protection mechanism against an ETEC colonization. Furthermore, we investigated the potential use of V. cholerae OMVs as delivery vehicles for the heterologously expression of the ETEC surface antigens, CFA/I, and FliC. Although we induced a detectable immune response against both heterologously expressed antigens, none of these approaches resulted in an improved protection compared to a simple combination of V. cholerae and ETEC OMVs. Finally, we expanded the current protection model from V. cholerae to ETEC by demonstrating that the inhibition of motility via anti-FliC antibodies represents a relevant protection mechanism of an OMV-based ETEC vaccine candidate in vivo.

Keywords: Vibrio cholerae; combined vaccine; enteric pathogens; enterotoxigenic Escherichia coli; outer membrane vesicles; vaccines.

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Figures

Figure 1
Figure 1

EΔmsbB OMVs are less endotoxic. Shown is the induction of TNF-α (A), IL-1β (B), and IL-6 (C) in RAW macrophages after treatment with OMVs (1 μg/ml) derived from EWT or EΔmsbB compared to the non-stimulated control group and normalized to the housekeeping gene 36B4. Each data set represents the median from at least six independent experiments. The error bars indicate the interquartile range of each data set. Significant differences between the data sets are marked by asterisks (P < 0.05; Mann–Whitney U test).

Figure 2
Figure 2

Detection of CT and LT using immunoblot analysis. Immunoblots were loaded with TCA precipitated supernatant of VWT (A, lane 1), VΔmsbBΔctxAB (A, lane 2), EWT (B, lane 1) or EΔmsbBΔeltA (B, lane 2) and incubated with a LT cross-reactive anti-CT polyclonal antiserum (Sigma). The arrows to the right indicate the sizes of the A-subunit (a) as well as the B-subunit (b) of CT and LT. Lines to the left indicate the molecular masses of the protein standards in kDa. A corresponding Kang-stained gel (Kang et al., 2002) loaded with equal amounts as the immunoblot served as loading control and is presented in Figure S1.

Figure 3
Figure 3

OMV protein profiles of the OMV donor strains. Depicted are the protein profiles of purified OMVs derived from VWT (lane 1), VΔmsbBΔctxAB (lane 2), EWT (lane 3) and EΔmsbBΔeltA (lane 4). Samples (approximately 7 μg protein each) were separated by SDS-PAGE (15% gels) and protein bands were visualized according to Kang et al. (2002). Lines to the left indicate the molecular masses of the protein standard in kDa.

Figure 4
Figure 4

Temporal immune responses to V. cholerae and ETEC OMVs. Shown are the median titers over time of IgM (A,D), IgG1 (B,E) and IgA (C,F) antibodies to OMVs derived from V. cholerae (A–C) and ETEC (D–F) in sera from mice intranasal immunized with VΔmsbBΔctxAB OMVs (dashed and dotted line), EWT OMVs (dashed line), EΔmsbBΔeltA OMVs (dotted line), an OMV mix (black solid line) and from the non-vaccinated control group (gray solid line) (n = 8 for each group). The error bars indicate the interquartile range of each data set for each time point.

Figure 5
Figure 5

Quantification of the immune response against V. cholerae and ETEC induced in OMV-immunized mice. (A,B) Half-maximum total Ig titers to OMVs (A) derived from V. cholerae and ETEC as well as to purified V. cholerae or ETEC LPS (B) in sera collected at day 86 from mice intranasal immunized with VΔmsbBΔctxAB OMVs, EWT OMVs, EΔmsbBΔeltA OMVs, and an OMV mix (n = 8 for each group). (C) Median IgA titers to OMVs derived from V. cholerae and ETEC extracted from fecal pellets collected at day 86 from the immunization groups (VΔmsbBΔctxAB OMVs, EWT OMVs, EΔmsbBΔeltA OMVs, and the OMV mix) as well as from the non-vaccinated control group (n = 8 for each group). (D) IgG1 titers to OMVs derived from V. cholerae and ETEC in stomach contents collected from litters born from intranasal immunized female mice of the immunization groups (VΔmsbBΔctxAB OMVs, EWT OMVs, EΔmsbBΔeltA OMVs, and the OMV mix) as well as the non-vaccinated control group during the challenge period (day 67–78). The error bars indicate the interquartile range of each data set and the hash key that the result of the data set was below the limit of detection. Significant differences between the data sets are marked by asterisks (P < 0.05; Kruskal–Wallis test and post-hoc Dunn's multiple comparisons).

Figure 6
Figure 6

Characterization of the antibody response against surface proteins of V. cholerae and ETEC in sera from OMV-immunized mice. Representative immunoblots were loaded with V. cholerae (VWT) as well as ETEC (EWT) whole cell lysates (WCL) and OMVs (approximately 7 μg protein each) as indicated above each blot and incubated with sera collected at day 86 from mice immunized with VΔmsbBΔctxAB OMVs (A), EWT OMVs (B), EΔmsbBΔeltA OMVs (C), and an OMV mix (D). Lines to the left indicate the molecular masses of the protein standards in kDa. A corresponding Kang-stained gel (Kang et al., 2002) loaded with equal amounts as the immunoblot served as loading control and is presented in Figure S2.

Figure 7
Figure 7

Challenge of neonates born to OMV immunized mice with V. cholerae and ETEC. Shown are the numbers of recovered CFU/small intestine for neonates born to mice immunized with VΔmsbBΔctxAB OMVs, EWT OMVs, EΔmsbBΔeltA OMVs, and an OMV mix as well as the non-vaccinated control group, which were challenged with either V. cholerae (A) or ETEC (B) (n = 11 for each group). When no bacteria were recovered, the number of CFU was set to the limit of detection of 10 CFU/small intestine (dotted line). The error bars indicate the interquartile range of each data set. Significant differences between the data sets are indicated by asterisks (P < 0.05; Kruskal–Wallis test and post-hoc Dunn's multiple comparisons).

Figure 8
Figure 8

Quantification of the immune response of V. cholerae OMVs expressing ETEC surface antigens. (A) Depicted are the IgG1 titers in sera collected at day 86 from mice intranasal immunized with VΔmsbBΔctxAB CFA/I OMVs, VΔmsbBΔctxABΔflaA hybrid FlaA-FliC OMVs and from the non-vaccinated control group to V. cholerae OMVs (n≥8 for each group). The error bars indicate the interquartile range of each data set and the hash key that the result of the data set was below the limit of detection. Significant differences between the data sets are indicated by asterisks (P < 0.05; Kruskal–Wallis test and post-hoc Dunn's multiple comparisons). (B,C) Shown are the IgG1 titers to purified His-CfaB (B) and His-FliC (C) in sera collected at day 86 from mice intranasal immunized with VΔmsbBΔctxAB CFA/I OMVs, VΔmsbBΔctxABΔflaA hybrid FlaA-FliC OMVs, EΔmsbBΔeltA OMVs, an OMV mix, or the non-vaccinated control group (n≥8 for each group). The error bars indicate the interquartile range of each data set and the hash key that the result of the data set was below the limit of detection. Significant differences between the data sets are indicated by asterisks (P < 0.05; Kruskal–Wallis test and post-hoc Dunn's multiple comparisons).

Figure 9
Figure 9

Challenge of neonates born to mice immunized with V. cholerae OMVs expressing ETEC antigens. Shown are the numbers of recovered CFU/small intestine for neonates born to mice immunized with VΔmsbBΔctxAB CFA/I OMVs, VΔmsbBΔctxABΔflaA hybrid FlaA-FliC OMVs as well as the non-vaccinated control group mice, which were challenged with either V. cholerae (A) or ETEC (B) (n≥11 for each group). When no bacteria were recovered, the number of CFU was set to the limit of detection of 10 CFU/small intestine (dotted line). The error bars indicate the interquartile range for each data set. Significant differences between the data sets are indicated by asterisks (P < 0.05; Kruskal–Wallis test and post-hoc Dunn's multiple comparisons).

Figure 10
Figure 10

Inhibition of motility in vitro correlates with the presence of anti-FliC antibodies and contributes to protection in vivo. (A) Representative images using ETEC mixed with non-vaccinated control sera (a), sera of VΔmsbBΔctxAB OMV immunized mice (b), sera of EΔmsbBΔeltA OMV immunized mice (c), sera of the OMV mix group (d), sera of VΔmsbBΔctxAB CFA/I OMV immunized mice (e), sera of VΔmsbBΔctxABΔflaA hybrid FlaA-FliC OMV immunized mice (f), and anti-FliC antibodies (g). The bacterial motility was visualized by fluorescence microscopy. Motile bacteria appeared as swirls and lines and non-motile bacteria as dots. Bars, 50 μm. (B) Quantification of the bacterial motility of ETEC cells in the presence of non-vaccinated control sera, sera of VΔmsbBΔctxAB OMV immunized mice, sera of EΔmsbBΔeltA OMV immunized mice, sera of the OMV mix group, sera of VΔmsbBΔctxAB CFA/I OMV immunized mice, sera of VΔmsbBΔctxABΔflaA hybrid FlaA-FliC OMV immunized mice and anti-FliC antibodies. Each symbol represents an independent experiment, and the horizontal bar indicates the median of each data set. When no bacteria were visible, the number was set to the limit of detection of 1 bacterium/field (dotted line). Significant differences between the data sets are indicated by asterisks (P < 0.05; Kruskal–Wallis test and post-hoc Dunn's multiple comparisons). (C) Passive immunization of neonates born to naïve dams using ETEC WT mixed with sera of the non-vaccinated control group, sera of VΔmsbBΔctxAB OMV immunized mice, sera of EΔmsbBΔeltA OMV immunized mice, sera of the OMV mix group or anti-FliC antibodies (n≥6 for each group). The error bars indicate the interquartile range of each data set. Significant differences between the data sets are indicated by asterisks (P < 0.05; Kruskal–Wallis test and post-hoc Dunn's multiple comparisons).

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