A multifunctional anti-O-Antigen human monoclonal antibody protects against Shigella sonnei infection in vivo

Abstract

Shigellosis is a global public health challenge that mostly affects low- and middle-income countries and causes considerable morbidity and mortality among children under 5 y of age. Multi- and extensively drug-resistant Shigella sonnei strains associated with recent outbreaks in high-income countries exacerbate the problem and have prompted the World Health Organization to include Shigella spp. among the high-risk pathogens for which novel prophylactic and therapeutic tools are urgently needed. Among the most promising and cutting-edge solutions, monoclonal antibodies are gaining considerable attention in the infectious diseases field. Here, we report the discovery of human monoclonal antibodies against S. sonnei, a species whose prevalence is constantly increasing worldwide and is associated with frequent drug-resistant infections. We isolated antibodies generated in response to an experimental S. sonnei vaccine followed by a controlled human infection and screened them by using a panel of high-throughput assays. We identified a molecule which exhibited potent bactericidal activity in vitro, inhibition of invasion of epithelial cells and conferred full protection from S. sonnei infection in vivo. Overall, our study provides a candidate antibody that can rapidly progress to industrial development for application as a prophylactic, therapeutic, and diagnostic tool against shigellosis.

Keywords: O-Antigen; Shigella sonnei; antimicrobial resistance; human monoclonal antibodies; shigellosis.

Fig. 1.

Binding profile and in vitro functional activity of C-0302B17. (A) Binding of C-0302B17 to S. sonnei 53G detected by confocal fluorescence microscopy. DAPI (blue) stained the bacterial nucleoid, whereas the green signal (A488) indicates the mAb. The scale bar corresponds to 2 µm. Quantification of the A488 signal intensity of the Hexa1 and Hexa1-LS binding to the bacterial strains is reported in the graphs. Statistical analysis performed using the unpaired t test resulted in a significant difference (P < 0.0001) between the strains for each mAb. (B) Serum Bactericidal Assay performed with OAg-positive S. sonnei 53G in the presence of BRC and increasing concentrations (X-axis) of the Hexa1 or Hexa1-LS versions of C-0302B17. Luminescence units are on the Y-axis. N = 3 biological and technical replicates. (C) Time-lapse imaging of OAg-positive S. sonnei 53G upon incubation with BRC and different concentrations of C-0302B17. Images were taken by the Opera Phenix confocal microscope at the different time points (in minutes) indicated in the figure. The scale bar corresponds to 10 µm.

Fig. 2.

Impact of C-0302B17 Hexa1-LS on bacterial phagocytosis and invasion of epithelial cells. (A) Opsonophagocytosis assay carried out with differentiated THP-1 cells and OAg-positive S. sonnei 53G in the presence of increasing concentrations of the mAb indicated on the X-axis. The Y-axis reports the fold uptake, compared to the no mAb condition, of the number of bacteria adherent and internalized by cells counted in each experimental condition. The two-way ANOVA statistical test was performed to compare each dose to the no mAb condition: *P < 0.05; **P < 0.01. Images show the DNA of THP-1 cells (blue), the cell membranes (red), and the bacteria used for the infection (yellow). (Scale bar, 10 µm.) N = 2 biological and technical replicates. (B) Invasion assay performed with HeLa cells and OAg-positive S. sonnei 53G. The graph reports the average and SD of relative fluorescence units calculated from three independent replicates as a function of the different concentrations of the tested mAb. (C) GoC model. The images are representative of three replicates for each experimental condition. Monoclonal antibody concentration is indicated on each image. The scale bar corresponds to 750 µm. (D) GoC model. The graph illustrates the average and SD of the area of the foci of infection visualized and imaged through confocal microscopy when the GoC was infected with S. sonnei 53G expressing GFP. Values were calculated from three independent replicates. One-way ANOVA with comparison to the no mAb control sample was performed: ***P = 0.0007 (10 µg/mL); ***P = 0.0003 (50 µg/mL); ****P < 0.0001 (100 µg/mL); ****P < 0.0001 (500 µg/mL).

Fig. 3.

In vivo efficacy of C-0302B17 Hexa1-LS in murine models of infection and pharmacokinetic study. (A) Experimental design of the C-0302B17 in vivo efficacy study. Animals were administered intraperitoneally with 1, 5, or 20 mg/Kg of the mAb 1 d before infection. They were challenged with 2.5 × 10⁶ CFU of S. sonnei Moseley intranasally and monitored for 15 d. (B) Results of the C-0302B17 in vivo efficacy study in a murine model of pneumonia. The graph reports the percentage of survival of the different groups of animals (Y-axis) upon bacterial challenge as a function of time (X-axis). “mAb” indicates C-0302B17, the negative controls were represented by the isotype control mAb and by PBS only. PBS: phosphate buffer saline. The log-rank (Mantel–Cox) test demonstrated that survival curves of the mAb-treated groups were significantly different from the isotype control-treated group with the following P values: P = 0.0288 for the 1 mg/Kg dose, P = 0.0003 for the 5 mg/Kg dose, P < 0.0001 for the 20 mg/Kg dose. The difference between the isotype control group and the PBS-treated animals was not statistically significant. (C) Pharmacokinetic study of C-0302B17 in mice. The graphs display the results of the ELISA carried out on serum samples from mice administered with C-0302B17, isotype control mAb, or PBS. mAb doses are indicated on top of each graph. Time points are indicated on the X-axes, mAb concentration is on the Y-axes. Relevant statistics by one-way ANOVA with Tukey’s multiple comparisons test are indicated on the graphs: *P = 0.0248, **P < 0.009. (D) Experimental design of the septicemia infection model. Mice received the candidate antibody via the intravenous route 24 h before intraperitoneal challenge with S. sonnei Moseley. Animal survival was monitored over 7 d, spleens were collected at the end of the experiment for CFU enumeration. (E) Results of the C-0302B17 efficacy study in a murine model of septicemia. The log-rank (Mantel–Cox) test demonstrated that the survival curve of the mAb-treated group was significantly different from the negative control with P = 0.0027. (F) CFU counted in the spleens of animals infected with S. sonnei Moseley and either mock-treated or administered with the mAb C-0302B17 before bacterial challenge. The number of CFU in the spleens of treated animals was below the detection limit and therefore is not displayed in the figure. The Mann–Whitney test demonstrated significant difference with P = 0.0079.

Fig. 4.

STD NMR binding studies between S. sonnei OAg and mAb C-0302B17. (A) STD NMR (black) of S. sonnei O-Antigen and mAb C-0302B17 and proton NMR reference spectrum (red). W, X, Y, and Z are the four sugar residues involved in the binding (see panel B); in the STD NMR spectrum, the respective proton signals of the binding epitopes are indicated. (B) Chemical structure of the S. sonnei core (boxed) and OAg with the highest STD response highlighted to delineate the binding epitope. The heatmap indicates the percentage of STD measured in the study which has been calculated by (I0−Isat)/I0, where (I0−Isat) was the signal intensity and I0 was the peak intensity of the reference spectrum. The diagram and the corresponding legend illustrate the residues which constitute the S. sonnei core and OAg repeating unit.