Date Published: March 7, 2019
Publisher: Public Library of Science
Author(s): Jae Seung Yang, So Jung An, Mi Seon Jang, Manki Song, Seung Hyun Han, Nicholas J Mantis.
Serum vibriocidal antibody assays have long been used to evaluate the immunogenicity of cholera vaccines formulated with killed whole-cell Vibrio cholerae. However, the antibody isotypes responsible for the serum vibriocidal activity are not fully characterized. In this study, we examined 20 clinical serum samples obtained from human subjects who had been vaccinated with a killed, whole-cell cholera vaccine and a positive control, human convalescent sera with high vibriocidal activity, to determine which isotype antibody is associated with the vibriocidal activity. Antibody isotypes from pooled convalescent sera were fractionated by size-exclusion column chromatography, and the major vibriocidal activity was detected in the IgM fraction. Depletion of IgM antibodies in the convalescent sera produced a significant (P<0.05) decrease in vibriocidal activity (16-fold decrease), whereas only a small change was observed with depletion of IgG or IgA. In addition, anti-LPS IgM antibody showed the highest correlation with vibriocidal activity (Spearman correlation coefficient r = 0.846) among antibody isotypes against heat-killed V. cholerae, lipopolysaccharide (LPS), or major outer membrane protein (Omp U), while total IgG, IgA, or IgM antibody level was not correlated with vibriocidal activity in the 20 human clinical serum samples. Furthermore, human convalescent sera significantly (P<0.001) inhibited the attachment of V. cholerae to HT-29, a human intestinal epithelial cell in vitro. Interestingly, IgM-depleted convalescent sera could not effectively inhibit bacterial adherence compared with non-depleted sera (P<0.05). Finally, bacterial adhesion was significantly inhibited by sera with high vibriocidal titer compared with low-titer sera (P = 0.014). Collectively, we demonstrated that anti-V. cholerae LPS IgM is highly correlated with serum vibriocidal activity and it could be a surrogate antibody isotype representing protective antibodies against V. cholerae.
Vibrio cholerae causes acute diarrhea by cholera toxin-mediated intestinal fluid secretion in humans. Humoral immunity rather than cellular immunity has been considered to play a role in protection against cholera because the bacteria do not invade the intestinal epithelial barrier and are unable to survive intracellularly . Humoral immunity against V. cholerae has long been focused on V. cholerae LPS for protective immunity due to its association with protection in humans . In addition, mucosal and systemic humoral responses against outer membrane protein , cholera toxin B subunit , and toxin-coregulated pilus (TCP)  have also been induced in cholera patients.
Serum vibriocidal antibody titer has been widely used to evaluate protective immunity to cholera in populations suffering from infection or in those administered cholera vaccines [28, 29]. Previous reports have indicated that serum IgG is an important isotype for protection against cholera [6, 30]. However, the serum antibody isotypes responsible for vibriocidal antibody activity are not fully understood. In the present study, we found that total IgG, IgA, or IgM antibody level was not associated with vibriocidal titer, but V. cholerae LPS-specific IgM was highly correlated with its bactericidal activity in the clinical specimen. The correlation was further demonstrated by showing that the fractions responsible for vibriocidal activity were IgM rather than IgG or IgA, and that depletion of IgM showed significant decrease in vibriocidal titer, which was not shown with either IgG or IgA depletion. In addition, sera with high vibriocidal titer and anti-V. cholerae LPS IgM level substantially inhibited the attachment of V. cholerae to intestinal epithelial cells, while sera with low vibriocidal titer or IgM-depleted sera did not. All these data indicate that serum anti-V. cholerae LPS IgM is responsible for vibriocidal activity and inhibition of bacterial adhesion to intestinal mucosa.