Humans Surviving Cholera Develop Antibodies against Vibrio cholerae O-Specific Polysaccharide That Inhibit Pathogen Motility

We analyzed polyclonal antibody responses in plasma from 10 adults recovering from cholera caused by V. cholerae O1 Ogawa in Bangladesh (see Table S1 in the supplemental material). These patients all developed IgG, IgA, and IgM responses against V. cholerae O1 OSP (P < 0.01 for all antibody isotypes) (Fig. 1A). The anti-V. cholerae O1 Ogawa agglutination activity was present in plasma across a range of dilutions in individual patients (1:64 to >1:8) (Table 1). Using high-speed video dark-field microscopy, we then assessed motility after incubating V. cholerae O1 with acute and convalescent-phase heat-inactivated plasma at subagglutinating concentrations (1:256) under the same conditions used for the agglutination assays. There was a significant decrease in motility when V. cholerae organisms were mixed with convalescent plasma compared to that with acute plasma (P = 0.001) (Fig. 1B). Adsorbing plasma with purified V. cholerae OSP eliminated its capacity to inhibit V. cholerae motility (Fig. 1B).

We next isolated IgG, IgA, and IgM antibody fractions from these plasma samples and focused our analyses on the IgG fraction, the most abundant isotype recovered. Using conditions that replicated those used in the video microscopy, we did not detect agglutination in 2-fold dilutions of purified IgG starting at 0.5, 1, or 2.5 μM depending on sample availability (Table 1). Starting at a concentration of 2.5 μM, we found the impact of pooled IgG on V. cholerae motility was concentration dependent and detectable at a subagglutinating concentration of 0.25 μM (Fig. 2A) (P ≤ 0.05) and independent of effects on V. cholerae viability (see Fig. S1). Purified IgG fractions from individual patients had similar anti-OSP reactivity (Fig. 2B) (P ≤ 0.01), and inhibition of V. cholerae motility at 0.25 μM was abrogated by adsorbing the IgG with purified V. cholerae OSP (Fig. 2C) (P ≤ 0.05). The absence of bacterial clumping/agglutination at 0.25 μM IgG was confirmed by microscopy. Thus, polyclonal IgG anti-OSP antibodies in convalescent patient sera inhibit the cholera pathogen’s motility at concentrations that do not cause the organism to agglutinate.

To assess whether antibody-based cross-linking was required for inhibition of V. cholerae motility, we generated fragment antigen-binding (Fab) fragments from the IgG fractions of individual patients. In contrast to intact IgG antibodies, which both bound V. cholerae OSP (Fig. 2B) (P < 0.01) and blocked V. cholerae motility (Fig. 2C) (P ≤ 0.05), Fab fragments bound OSP (Fig. 2D) (P ≤ 0.01) but did not inhibit motility (Fig. 2D), suggesting that multivalent binding of OSP by antibody molecules is important for impeding motility. Supporting this idea, we found that dimeric IgA and polymeric IgM fractions also inhibited V. cholerae motility in an OSP-dependent fashion (see Fig. S2) (P ≤ 0.001).

We next turned our attention to analyses of V. cholerae-specific monoclonal antibodies isolated from intestinal mucosa-homing plasmablasts from the peripheral blood of cholera patients sampled 7 days after they presented for clinical care in Bangladesh (22). We previously cloned (as human IgG1) and characterized >100 monoclonal antibodies from this collection (22). For the present analysis, we selected 4 previously characterized antibodies, including one monoclonal antibody with high affinity for V. cholerae OSP (G1), two antibodies with low affinity for V. cholerae OSP (A4 and B4), and one specific for V. cholerae flagellin A (AT11) (22). The OSP-specific monoclonal IgG antibodies were previously shown to recognize both Ogawa and Inaba antigens (22). Monoclonal IgG antibodies were assessed for agglutinating activity via 2-fold dilutions beginning at 2.048 μM (0.320 mg/ml). If no agglutination was evident at this dilution, the plasma result was marked as >2.048 μM. One low-affinity OSP-specific IgG monoclonal antibody (B4) had agglutinating activity at 1.024 μM, whereas the other three monoclonal antibodies (G1, A4, and AT11) did not have detectable agglutinating activity at the highest concentration assessed (2.048 μM) (Table 1). Similar to our findings using polyclonal antibodies described above, we found that the anti-OSP monoclonal antibodies also blocked V. cholerae motility in a concentration-dependent manner, including at subagglutinating concentrations, whereas the anti-flagellin antibody did not block motility (Fig. 3) (P ≤ 0.001 to 0.05) (see Movies S1, S2, and S3). Inhibition of “shooting star” motility at the single bacterial cell level (without agglutination and clumping) was demonstrated by analysis of freeze-frame images of V. cholerae in the presence of human monoclonal OSP-specific antibodies but not in the presence of the anti-flagellin antibody (Fig. 3 and 4; Movies S1, S2, and S3). There were no apparent differences in the capacities of the high- versus low-affinity OSP-specific antibodies to inhibit V. cholerae motility (Fig. 3 and 4). Scanning electron microscope imaging of V. cholerae revealed that the OSP-specific monoclonal antibody promoted flagellar tethering, but tethering was occasionally observed in the presence of flagellum-specific monoclonal antibody (see Fig. S3). Occasional bacterial blebbing was also observed in scanning electron microscopic analyses with both antibodies. Our attempts to confirm the potential requirement for antibody mediated cross-linking using the monoclonal antibodies were confounded by aggregation of monoclonal Fab fragments under all assessed nonreducing conditions.

We used a neonatal mouse challenge model to determine whether OSP-specific monoclonal antibody G1 alters the survival of suckling mice after lethal challenge with wild-type V. cholerae O1 strain C6706. Mixture of G1 with C6706 prior to orogastric inoculation led to a marked reduction in death of the mice (Fig. 5A) (P < 0.001). To assess how the effect of OSP-specific antibody on motility impacts V. cholerae survival and localization in the intestine, we carried out competition studies where the colonization of V. cholerae C6706lacZ⁻ (a strain that colonizes as well as the wild type) was compared to either (i) a C6706lacZ⁺ transposon mutant that was flagellated but nonmotile (motB::Kan^r) or (ii) a C6706lacZ⁺ transposon mutant that was motile but rough and lacking OSP due to deficient perosamine synthesis (VC0244::Kan^r). The motilities of the comparator and rough strains were equivalent, whereas the motB::Kan^r strain was confirmed as nonmotile. Relative to that for the C6706lacZ⁻ strain, the nonmotile motB::Kan^r mutant had a marked thousand-fold colonization defect in the proximal small intestine and a less pronounced 100-fold defect in the distal small intestine (Fig. 5B). When an OSP-specific monoclonal antibody was added to the inocula mixture at a subagglutinating concentration, the competitive disadvantage of the nonmotile mutant was significantly ameliorated in the proximal but not distal small intestine (Fig. 5B) (P < 0.001). This reduction in the competitive defect related to a decrease in the recovery of motile V. cholerae from the proximal small intestine in the presence of OSP-specific antibody G1 (see Table S3). In contrast, addition of the anti-flagellin monoclonal antibody to the inocula did not alter the competitive advantage of motile V. cholerae compared to that of the nonmotile mutant in either the proximal or distal intestine (Fig. 5B). These observations suggest that the inhibition of motility by the anti-OSP antibody impedes V. cholerae’s capacity to colonize the proximal small intestine.

In similar experiments where the comparator smooth C6706lacZ⁻ strain was competed with the rough (OSP⁻) mutant, we found that the rough strain had an ∼100-fold colonization defect in both the proximal and distal small intestine (Fig. 5C). Notably, recovery of the smooth comparator strain decreased in the presence of OSP-specific monoclonal antibody, in part abolishing the disadvantage of the rough strain in both the proximal and distal small intestine (Fig. 5C) (P < 0.001) (Table S3). In marked contrast, the anti-flagellin monoclonal antibody did not alter the competitive indices found in intestinal samples.

Venous blood was collected at the acute phase (day 2) and convalescent phase of infection (day 7) from 10 adult patients (age, 18 to 55 years) presenting to the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) hospital with culture-confirmed V. cholerae O1 El Tor Ogawa infection. Patients were treated with intravenous fluids and/or oral rehydration solution and antimicrobials at the discretion of the attending physician. This study was approved by the Research and Ethical Review Committees of the icddr,b and the human studies committee of Massachusetts General Hospital.

The V. cholerae O1 strains used in this study are listed in Table S2 in the supplemental material (42). Bacterial strains were grown at 37°C in Luria-Bertani (LB) medium with or without antibiotics as appropriate. Antibiotics and their concentrations were streptomycin (Sm) at a concentration of 100 μg/ml and kanamycin (Km) at 45 μg/ml. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) (200 μg/ml) plus isopropyl β-d-1-thiogalactopyranoside (IPTG; 0.1 mM) were used for blue/white colony screening. For experiments with the rough strain, bacteria were resuspended in 0.5% NaCl to prevent autoagglutination after growth in LB medium.

IgG was purified from heat-inactivated human plasma using protein G HP spin trap columns (GE Healthcare) and then incubated with CaptureSelect IgA affinity matrix (Thermo Scientific) to remove contaminating IgA per the manufacturer’s instructions. IgA and IgM antibodies were then subsequently purified by sequentially running the flowthrough from the protein G column onto columns loaded with CaptureSelect IgA and IgM affinity matrix (Thermo Scientific). The quantity and purity of IgG, IgA, and IgM isotype fractions were determined by enzyme-linked immunosorbent assay (ELISA) using standard curves of ChromPure human IgG, IgA, and IgM (Jackson ImmunoResearch).

Fab fragments of IgG were generated using the Fab preparation kit (Pierce) per the manufacturer’s instructions. Briefly, immobilized papain was used to digest the desalted and purified IgG, and the resultant Fab fragments were purified using a Protein A Plus spin kit (Pierce). The purity of the Fab fragments was assessed by Western blotting with 100 ng of sample per well and direct probing with anti-IgG F(ab)₂ and anti-IgG Fc conjugated to horseradish peroxidase (Jackson ImmunoResearch).

Recombinant human monoclonal antibodies were generated from the cholera-induced day-7 plasmablast population of V. cholerae-infected Bangladeshi patients by single-cell expression as previously described (22). For this analysis, we used the following previously characterized human IgG monoclonal antibodies: high-affinity anti-OSP G1 (CF21.2.G01), low-affinity anti-OSP A4 (CF21.1.A04), low-affinity anti-OSP B4 (CF21.1.B04), and anti-FlaA-flagellin AT11 (AT11.1.B12) (22).

OSP from V. cholerae O1 Ogawa strain PIC158 was purified and conjugated to bovine serum albumin (BSA) as previously described (43).

Vibriocidal assays were performed on heat-inactivated plasma samples as previously described (44), and the target strain V. cholerae O1 Ogawa O395 was used. Heat-inactivated plasma and exogenous guinea pig complement (EMD Millipore) were incubated with the target strain. Vibriocidal titers were defined as the reciprocal of the highest serum dilution resulting in a 50% reduction in optical density at 600 nm (OD₆₀₀) compared to that of no serum controls.

OSP responses were measured using an ELISA as previously described (44). Microplates were coated with 100 ng/well of O1 Ogawa OSP-BSA. Samples were added to plates (plasma, 1:50; purified IgG, 10 μM; purified Fab, 1 μM), and IgG responses were detected with goat anti-human IgG conjugated to horseradish peroxidase [anti-IgG F(ab)₂ for Fab] (Jackson ImmunoResearch). IgM and IgA anti-OSP reactivity of plasma was measured using goat anti-human IgM and IgA conjugated to horseradish peroxidase (Jackson ImmunoResearch). Peroxidase activity was measured with the substrate 2,2-azinobis (ethylbenzthiazolinesulfonic acid) for plasma, o-phenylenediamine for purified IgG/M/A, and SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific) for purified Fab fragments.

Agglutination assays were performed as previously described with modification to match conditions used in assessing motility via high-speed microscopy (22). Briefly, V. cholerae O1 classical Ogawa strain O395 was grown to mid-log phase in bovine heart infusion medium. Bacteria were pelleted, washed twice with phosphate-buffered saline (PBS), and diluted to an OD₆₀₀ of 0.1 with PBS. In a U-bottom microtiter plate blocked with BSA, 25 μl of bacterial samples was mixed with equal volumes of day-7 patient samples to a final starting dilution/concentration of plasma 1:8; purified IgG at 2.5, 1, or 0.5 μM depending on sample availability, purified IgG Fab at 1 μM, or monoclonal antibody starting at a concentration of 2.048 μM (0.320 mg/ml). The minimum agglutinating titer was determined by serial 2-fold dilutions of antibody sample. Plates were incubated at room temperature for 5 min and imaged using a UV imaging system (ChemiDoc; Bio-Rad) to assess agglutination. If no agglutination was evident, the value was marked as greater than the most concentrated value.

V. cholerae O1 classical Ogawa strain O395 was grown to mid-log phase in LB medium. Bacterial samples, adjusted to an OD₆₀₀ of 0.1, were added to equal volumes of PBS or patient’s sample (heat-inactivated convalescent plasma or purified IgG for final antibody concentration of 1:256 for plasma and 0.25 μM and 2.5 μM for purified IgG). The mixture was incubated for 5 min at 37°C. The effect of the antibody on bacterial viability was determined by serially diluting samples 10-fold and enumerating bacteria on LB plates. Experiments were performed in quadruplicates.

V. cholerae motility was assessed using a modification of a previously reported approach (13). Briefly, V. cholerae O1 O395 were grown to mid-log-phase in LB broth, and the OD₆₀₀ was adjusted to 0.1. This OD was found to approximate 3 × 10⁶ CFU/ml. Heat-inactivated convalescence antibodies were mixed with bacterial sample at a 1:1 dilution of acute or convalescence antibodies (final antibody concentrations of plasma and purified IgG were at subagglutinating levels: 1:256 (plasma), 0.25 μM [polyclonal IgG], and 0.5 μM [Fab generated from purified polyclonal IgG]), and 10 μl of the mixture was placed on a standard glass slide. Concentration dependence was also assessed for purified polyclonal IgG and monoclonal antibodies using 10-fold serial dilutions (2.5 to 0.025 μM for purified IgG; 2.5 to 0.00025 μM for monoclonal antibodies) using slides blocked with BSA. Motility inhibition of pooled samples of IgM and IgA was assessed at 0.25 μM; sample volumes were too low to assess motility individually for each patient using these antibody isotypes. PBS mixed 1:1 with bacteria was used as a negative control. Slides were incubated for 5 min at 37°C before visualization of motility by dark-field microscopy (Nikon Eclipse Ti-E inverted microscope stand, Nikon Plan-Fluor 40×/0.75 lens objective, Nikon TI-DF dark-field dry condenser [numerical aperture, 0.95 to 0.80], Hamamatsu ORCA-ER camera, and MetaMorph imaging software). Images were taken at 400-ms exposure time over 100 frames with readout time frame of 111.72 ms (movie play time of 1/30th s per frame). Frames 1, 50, and 98 were frozen as still shots, and bacteria were counted as lines (motile bacteria) or dots (nonmotile bacteria). Results were expressed as percent motile (motile/total bacteria) and averaged across the three screen shots. To confirm that the antimotility effect was secondary to OSP-specific antibodies, we performed experiments with antibody samples that were first adsorbed with OSP-BSA. Antibody samples were first incubated overnight with OSP-BSA before mixing with the bacterial sample for microscopy. Adsorption was performed using a 1:100 molar ratio (antibody/OSP) for purified antibody, and 250 μg of OSP-BSA for diluted plasma samples. PBS mixed with OSP-BSA was used as a negative control. All assays were performed in at least triplicates.

Scanning electron microscopy (SEM) was performed at the Harvard University Center for Nanoscale Systems (CNS) using a FESEM Supra55VP microscope with an SE2 laser. V. cholerae O1 O395 was imaged in the presence or absence of anti-flagellin monoclonal antibody (AT11, 0.025 μM) at a magnification range of 45,300 to 46,700 or anti-OSP monoclonal antibody (G1, 0.005 μM) at a magnification range of 63,800 to 66,500.

The mouse neonatal V. cholerae challenge assay was used to assess protection afforded by OSP-specific antibodies as previously described (6). Briefly, 3- to 5-day-old CD-1 mice were separated from their dams for 3 h and then orally inoculated with 50 μl of a mixture containing 10⁹ V. cholerae O1 Inaba C6706 and PBS or anti-OSP monoclonal antibody (G1) at a final concentration of 0.25 μM. This strain was used due to the availability of mutant strains used in the competition assays (see below). Mice were then housed away from the dams at 30°C and monitored for survival for 30 h. All mouse studies were approved by the MGH Institutional Animal Care and Use Committee.

A single colony of each V. cholerae strain was inoculated into LB medium with Sm for C6706lacZ⁻ (referred to below as wild type [WT]) and LB medium with Sm and Kan for C6706lacZ⁺ transposon mutants (rough [VC0244::Kan^r] and nonmotile [motB::Kan^r]) and incubated at 37°C overnight on a roller drum. Cultures were then regrown to mid-log phase, and the rough and comparator wild-type (WT) strains were mixed together 1:1 (10⁵ organisms) and resuspended in 50 μl of LB medium without antibiotic mixed with PBS or 0.025 μM monoclonal antibody G1. A higher inoculum of the nonmotile strain was required to enable sufficient recovery after infection for enumeration and calculation of a competitive index (input, 10⁶ mutant and 10⁴ comparator). Three- to 5-day-old CD-1 infant mice were inoculated by oral gavage. After 20 h, the pups were sacrificed, and the proximal and distal thirds of the small intestines were isolated and mechanically homogenized in 5 ml of LB medium. Serial dilutions were plated on LB with Sm, X-Gal, and IPTG to enumerate bacteria and determine the input and output ratios of the comparator WT and competing rough or nonmotile strains. The competitive index was calculated as the output ratio of competing strain/WT strain divided by the input ratio of competing strain/WT. The data represent infections of multiple litters on different days that were pooled for analysis. All mouse studies were approved by the MGH Institutional Animal Care and Use Committee.

Results were expressed as medians and compared by a Wilcoxon matched-pairs signed-rank sums test or Mann-Whitney U test for within group and between group comparisons as appropriate. Results of the survival curve analysis were compared by log rank testing. The threshold for statistical significance was a two-tailed P value of <0.05. Prism 6 was used for all statistical analyses.