Ssa1-targeted antibody prevents host invasion by Candida albicans

Abstract

Introduction: Candida albicans is a commensal fungus that colonizes most healthy individuals' skin and mucosal surfaces but can also cause life-threatening invasive infections, particularly in immunocompromised patients. Despite antifungal treatment availability, drug resistance is increasing, and mortality rates remain unacceptably high. Heat shock protein Ssa1, a conserved member of the Hsp70 family in yeast, is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and enables C. albicans to cause maximal damage to host cells and induces disseminated and oropharyngeal disease.

Result: Here we discovered a mouse monoclonal antibody (mAb 13F4) that targeting C. albicans Ssa1 with high affinity (EC₅₀ = 39.78 ng/mL). mAb 13F4 prevented C. albicans from adhering to and invading human epithelial cells, displayed antifungal activity, and synergized with fluconazole in proof of concept in vivo studies. mAb 13F4 significantly prolonged the survival rate of the hematogenous disseminated candidiasis mice to 75%. We constructed a mAb 13F4 three-dimensional structure using homology modeling methods and found that the antigen-binding fragment (Fab) interacts with the Ssa1 N-terminus.

Discussion: These results suggest that blocking Ssa1 cell surface function may effectively control invasive C. albicans infections and provide a potential new treatment strategy for invasive fungal infections.

Keywords: Candida albicans; Ssa1; antibody; antifungal; systemic infection.

Figure 1

The screening of hybridoma cell line. The affinity of cell supernatant with Ssa1 was tested by ELISA (part of the experimental data). The positive clones in the first (A) and the second (B) screening were selected and amplified. After twice screenings (C,D) of the cell supernatant in the 24-well plate, the positive clones are selected for subcloning.

Figure 2

The binding assay of mAb 13F4 targeting C. albicans Ssa1. (A) The binding affinity of cell supernatant with Ssa1 was tested by ELISA. The mAb 13F4 is the top clone. (B) The cell supernatant of mAb 13F4 was purified and tested by ELISA. The concentration of antibodies is 10 μg/mL in the first well. Coating 0.1 μg/well Ssa1 protein in 96-well plate. EC₅₀ = 39.78 ng/mL. (C) The binding affinity of mAb 13F4 with C. albicans ssa1 Δ/Δ and ssa1Δ/Δ::SSA1 strains. The concentration of antibodies is 50 μg/mL in the first well. Coating ~10⁷ CFU C. albicans in 96-well plate. mAb 13F4 could specifically recognize the Ssa1 on the cell wall surface of C. albicans. EC₅₀ = 14.40 ug/mL. (D) C. albicans were incubated with mAb 13F4 of 128 μg/mL, 64 μg/mL, 32 μg/mL or 0 μg/mL, respectively. The samples were performed using a BD FACSVerse and analysis was performed using FlowJo analysis software.

Figure 3

Blocking Ssa1 in cell wall can reduce the adhesion and endocytosis of epithelial cells by C. albicans. (A) Adhesion and endocytosis of FaDu cells by C. albicans. Live C. albicans was co-cultured with the FaDu cells grown on confocal dish for 90 min. After staining with CFW (1 μg/mL) and PSA-FITC (20 μg/mL) for 10 min, the samples were viewed by confocal laser scanning microscope directly. Scale bar represents 20 μm. Fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC), calcofluor white (CFW) and overlay are shown individually. (B,C) C. albicans SC5314 interacted with FaDu cells and Caco-2 cells, respectively, to observe the adhesion between the fungus and different epithelial cells. The experiment was repeated twice, and the data was expressed by mean ± SD. One-way ANONA test was used to analyze the statistical significance of the difference in adhesion rate between the groups. ^*p < 0.05.

Figure 4

Blocking Ssa1 in cell wall can reduce the damage of host epithelial cells by C. albicans. C. albicans SC5314 interacted with KB cells (A) and FaDu cells (B) respectively, and the amount of LDH released in the supernatant was measured to assess the degree of damage to epithelial cells by C. albicans. mAb 13F4 could significantly inhibited the damage of KB cells by C. albicans. The experiment was repeated three times, and the data was expressed by mean ± SD. One-way ANONA test was used to analyze the statistical significance of the difference in LDH release rate between the groups. LDH, lactate dehydrogenase. ^**p < 0.01, ^*p < 0.05.

Figure 5

Blocking of Ssa1 is required for the reduction of C. albicans systemic infection. C57BL/6 mice were infected with 1 × 10⁶ CFU SC5314 in 200 μL sterile PBS via lateral tail vein. (A) Survival of C57BL/6 mice infected with the indicated strains was monitored for 12 days (n = 8 per group). A log-rank test was used to evaluate survival curve equality. Data are representative of three independent experiments. (B) Quantification of the fungal burden in kidney tissues of C57BL/6 mice (n = 8 per group) infected with indicated C. albicans at day 2. Data are representative of three independent experiments. Horizontal lines represent mean values, and whiskers representing standard deviations. ^**p < 0.01 versus the result for the control (one way analysis of variance). (C,D) Representative PAS (for C. albicans) and H&E (for the inflammatory cells influx and the extent of tissue necrosis) staining of kidneys from C57BL/6 mice infected with indicated strains at day 2. Arrows indicate C. albicans filaments in the tissues. ^***p < 0.001, ^**p < 0.01.

Figure 6

The structure of Ssa1-mAb 13F4 Fab complex. (A) Three-dimensional structure of the complex between monomeric Ssa1 (purple) and mAb 13F4 Fab [HC (green)/LC (orange)]. Interface between mAb 13F4 HC (green) and Ssa1 (purple) (B) and mAb 13F4 LC (orange) and Ssa1 (purple) (C). Both chains of the Fab interact with Ssa1 and create hydrogen bonds (dotted lines). Residue Y132, L133 interact with heavy chain (HC) and D30 interacts with light chain (LC) bу π–π stacking interaction.