Evaluation of antibody responses to outer membrane vesicles (OMVs) and killed whole cell of Vibrio cholerae O1 El Tor in immunized mice

Abstract

Background and objectives: Cholera disease remains an important global health problem affecting 3-5 million subjects worldwide. Outer membrane vesicles (OMVs) have been found in a variety of Gram-negative bacteria and act as protective transport vesicles. The aim of this study was to evaluate Immune responses against Vibrio cholerae O1 El Tor clinical strain OMV and compare it with killed whole cell (KWC), complex of (KWC-OMV) as well as the internationally licensed oral cholera vaccine, Dukoral, in serum and intestinal secretions of mice.

Materials and methods: OMVs were prepared by using modified detergent-centrifugation procedure from V. cholerae O1 El Tor clinical strain from 2005 outbreak. The ultrastructure and content of OMVs were investigated via the Scanning Electron Microscopy (SEM) and SDS-PAGE analysis. Three doses of oral immunization were adjusted and total IgG and IgA in serum and intestinal secretion were measured by enzyme-linked immunosorbent assay (ELISA).

Results: Extracted OMVs from the V. cholerae were spherical vesicles with a size ranging from 10 to 300 nm. OMV-immunized mice showed an increased level of total IgG and IgA both in serum and intestinal secretion when compared to the negative controls. Also, there existed a higher level of secretory IgA than the total IgG, suggesting the most of protection against V. cholerae colonization provided by sIgA.

Conclusion: Our findings revealed that oral immunization with V. cholerae OMVs might induce a long-term immunity, especially when administered in combination with KWC. This study tested the adjuvant activity of OMVs and may be useful in future nano vaccine research.

Keywords: Dukoral vaccine; Killed whole cell; Outer membrane vesicle; Vibrio cholerae O1 El Tor.

Fig. 1.

(A, B) Scanning electron micrograph of VC492 outer membrane vesicles (Bar = 50–300 nm), OMV size is shown in panel (A)

Fig. 2.

VC492 analyzed OMVs by gel electrophoresis, SDS-PAGE followed by a R250 Blue Coomassie stain. OMVs of VC492 (left line, shown by arrows) and the molecular weight marker in kDa (right line).

Fig. 3.

Total IgG and IgA titers in immunized and non-immunized mice sera within 56 days. Four vaccine regimens increased antibody titers (total IgG and IgA) in sera, indicating significant antibody responses in treatment groups in comparison with negative controls after each immunization,) P value < 0.05). IgA levels against VC492 (KWC-OMV) regimen showed significant differences compared to current vaccine, (*P value < 0.05).

Fig. 4.

Titers of total IgG (A) and sIgA (B)immunoglobulin in immunized and non-Immunized mice after 35 days. In Panel A: all immunized groups, except those received OMV, showed significant antibody levels compared to control groups, *P value < 0.05, and in panel B: Significantly higher antibody levels were found in immunized groups than the control groups, *P value < 0.05.