Rapid Effects of a Protective O-Polysaccharide-Specific Monoclonal IgA on Vibrio cholerae Agglutination, Motility, and Surface Morphology

Abstract

b2D6 is a dimeric monoclonal immunoglobulin A (IgA) specific for the nonreducing terminal residue of Ogawa O-polysaccharide (OPS) of Vibrio cholerae. It was previously demonstrated that 2D6 IgA is sufficient to passively protect suckling mice from oral challenge with virulent V. cholerae O395. In this study, we sought to define the mechanism by which 2D6 IgA antibody protects the intestinal epithelium from V. cholerae infection. In a mouse ligated-ileal-loop assay, 2D6 IgA promoted V. cholerae agglutination in the intestinal lumen and limited the ability of the bacteria to associate with the epithelium, particularly within the crypt regions. In vitro fluorescence digital video microscopy analysis of antibody-treated V. cholerae in liquid medium revealed that 2D6 IgA not only induced the rapid (5-to 10-min) onset of agglutination but was an equally potent inhibitor of bacterial motility. Scanning electron microscopy showed that 2D6 IgA promoted flagellum-flagellum cross-linking, as well as flagellar entanglement with bacterial bodies, suggesting that motility arrest may be a consequence of flagellar tethering. However, monovalent 2D6 Fab fragments also inhibited V. cholerae motility, demonstrating that antibody-mediated agglutination and motility arrest are separate phenomena. While 2D6 IgA is neither bactericidal nor bacteriostatic, exposure of V. cholerae to 2D6 IgA (or Fab fragments) resulted in a 5-fold increase in surface-associated blebs, as well an onset of a wrinkled surface morphotype. We propose that the protective immunity conferred by 2D6 IgA is the result of multifactorial effects on V. cholerae, including agglutination, motility arrest, and possibly outer membrane stress. C holera is a life-threatening disease that remains endemic in many parts of the world (1-3). The etiological agent of cholera, Vibrio cholerae, is a noninvasive, Gram-negative bacterium that is acquired by humans via the fecal-oral route. Following ingestion, V. cholerae colonizes the mucosal surfaces of the small intestines, a process that is facilitated by the bacterium's single polar flagellum (4-7). Adherence to the epithelial surface requires expression of the toxin-coregulated pilus (TCP), in addition to other virulence factors (8), most notably, a potent ADP-ribosylating toxin known as cholera toxin (CT). CT disrupts chloride secretion within intestinal epithelial cells, inducing profuse water and electrolyte secretion and ultimately resulting in the hallmark "rice water diarrhea" associated with cholera. Cholera outbreaks frequently occur when water sanitation is disrupted, either following natural disasters or seasonally in areas where V. cholerae is endemic (9). The recent cholera outbreak in Haiti following the 2010 earthquake highlighted the ongoing potential of V. cholerae to cause mass causalities, as it resulted in more than half a million infected individuals and more than 7,000 deaths (10). Due to the rapid onset of symptoms and limited treatment options, control of cholera in many parts of the globe, particularly...

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

“…Infection and Immunity support for the latter mechanism was reported using a monoclonal anti-LPS antibody (14). However, in that study, Fab fragments generated from IgA maintained their ability to inhibit motility, raising questions as to what the mechanism of motility inhibition is.…”

“…Furthermore, because antibodies can have multiple effects on bacteria, it is unclear if protection is mediated solely through blocking of motility or is instead multifactorial. Because motility is inhibited at antibody concentrations too low to cause agglutination, we hypothesized that IgG binds to the O-antigen of the outer membrane sheath surrounding the V. cholerae polar flagellum and imposes a crimping force, or alternatively it cross-links the sheath and flagellum to the cell body, as recently demonstrated for IgA (14), so that the flagellum no longer functions properly. This suggests that the bivalent structure of IgG is required for motility inhibition, much like its property for agglutination.…”

A monoclonal antibody that targets the conserved core/lipid A region of lipopolysaccharide affects motility and reduces intestinal colonization of both classical and El Tor Vibrio cholerae biotypes

“…Mid-log phase cultures of strain O395 expressing GFP were mixed with ZAC-3 IgG then seeded on glass microscope slides and monitored by fluorescent digital video microscopy for 30 min, as described [14]. We found that ZAC-3 IgG treatment resulted in almost instantaneous motility arrest: >95% of the bacteria had stopped swimming within 2 min of antibody exposure (Figure 1Bii; Movies S1–2).…”

“…Although the precise mechanisms by which antibodies limit the ability of V. cholerae to colonize the intestinal epithelium remains unknown, two groups have reported a correlation between the ability of OPS-specific IgA antibodies to arrest V. cholerae flagellum-based motility in vitro and protection in vivo [11, 12]. Indeed, we recently demonstrated that the Ogawa-specific IgA mAb, 2D6, which was shown to protect mice against classical O1 V. cholerae strain O395, is also an effective inhibitor of bacterial motility in semi-solid and liquid media [14]. …”

Highlights of the 8th International Conference on Vaccines for Enteric Diseases: the Scottish Encounter To Defeat Diarrheal Diseases

“…In the area of cholera immunity, Nicholas Mantis, from the Wadsworth Center, showed how IgA monoclonal antibodies specific for the O antigen or the lipid A portion of the V. cholerae LPS inhibited flagellumbased motility and altered the bacterial outer membrane (Fig. 2) and proposed that secretory IgA deploys multiple effects that will ultimately impair the ability of the bacterium to colonize the intestinal epithelium (23).…”