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Immunity Provided by an Outer Membrane Vesicle Cholera Vaccine Is Due to O-Antigen-Specific Antibodies Inhibiting Bacterial Motility - PubMed

  • ️Fri Jan 01 2016

Immunity Provided by an Outer Membrane Vesicle Cholera Vaccine Is Due to O-Antigen-Specific Antibodies Inhibiting Bacterial Motility

Zhu Wang et al. Infect Immun. 2016.

Abstract

An outer membrane vesicle (OMV)-based cholera vaccine is highly efficacious in preventing intestinal colonization in the suckling mouse model. Immunity from OMVs comes from immunoglobulin (Ig), particularly IgG, in the milk of mucosally immunized dams. Anti-OMV IgG renders Vibrio cholerae organisms immotile, thus they pass through the small intestine without colonizing. However, the importance of motility inhibition for protection and the mechanism by which motility is inhibited remain unclear. By using both in vitro and in vivo experiments, we found that IgG inhibits motility by specifically binding to the O-antigen of V. cholerae We demonstrate that the bivalent structure of IgG, although not required for binding to the O-antigen, is required for motility inhibition. Finally, we show using competition assays in suckling mice that inhibition of motility appears to be responsible for most, if not all, of the protection engendered by OMV vaccination, thus providing insight into the mechanism of immune protection.

Keywords: Vibrio cholerae; cholera; flagellar motility; immunity; lipopolysaccharide; mucosal vaccines; outer membrane vesicles.

Copyright © 2016 American Society for Microbiology.

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Figures

FIG 1
FIG 1

Inhibition of V. cholerae motility is the primary mechanism of immune protection. Two types of nonmotile mutant, flagellated but nonmotile (ΔmotY) (A) and aflagellate (ΔflaACEDB) (B), were competed against the motile wild-type strain (WT) in either naive or immune pups. Five-day-old suckling mice born to mice mock immunized or immunized with OMVs produced by strains indicated on the x axes were challenged with equal mixtures of the competing V. cholerae strains. The competitive index was calculated as the ratio of nonmotile to wild type after correcting for the input ratio. Each symbol represents sample from one mouse. Bars are medians. Statistical analysis was done using Mann-Whitney U test with Kruskal-Wallis test for multiple-variable groups (*, P < 0.0001).

FIG 2
FIG 2

Both OMV and LPS from mutant strains of V. cholerae lack O antigen. (A) Protein and LPS content of outer membrane vesicles produced by mutant strains of V. cholerae. Five micrograms of OMVs with or without SDS and proteinase K (Prot. K) were incubated at 55°C overnight. An aliquot of 2.5 μg was loaded on a 4 to 12% gradient SDS-PAGE gel. After electrophoretic separation, the gel was stained with silver. (B) Ten microliters of serial dilutions of purified LPS from wild-type O1 El Tor E7946, a rough mutant derivative (ΔwbeL), and O139 serogroup strain MO10 were spotted in triplicate onto a nitrocellulose membrane and air dried. The membrane was probed using primary anti-O1-antigen antibody and Cy5-conjugated, anti-IgG secondary antibody.

FIG 3
FIG 3

Transmission electron microscopy examination of outer membrane vesicle morphology. OMVs purified from the strains indicated were diluted to a concentration of 1 μg/μl in PBS. Grids were floated in OMV solution for 1 min, washed with 2% acidic uranyl acetate, and blotted dry before visualization under TEM. Scale bars are 100 nm.

FIG 4
FIG 4

Inhibition of V. cholerae motility requires intact IgG antibody binding. Wild-type V. cholerae was incubated with commercial IgG that is specific against O-antigen at subagglutinating concentrations. Percentages of motile bacteria after antibody addition were recorded (A), and the relative amounts of IgG or Fab bound to bacteria were calculated using fluorescently labeled antibody (B). Each symbol represents samples from one experiment. Bars are means with standard deviations.

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