Monoclonal antibodies targeting Candida disrupt biofilms and inhibit growth across global clinical isolates

Abstract

Candida species are a major cause of severe fungal infections, particularly in immunocompromised individuals and hospitalized patients. Among these, Candida auris stands out as an emerging "superbug", responsible for life-threatening bloodstream infections with mortality rates up to 60%. This multidrug-resistant, hospital-acquired pathogen has been declared a serious global health threat, complicating effective treatment and increasing mortality risks. Our research has identified universal monoclonal antibodies (mAbs) targeting Candida albicans cell wall epitopes, which are highly homologous to those in other pathogenic Candida species, including C. auris. Passive transfer of mAbs-C3.1 (anti-β-1,2-mannotriose) and 9F2 (anti-phosphoglycerate kinase 1)-significantly extended survival and improved fungal clearance in a complement C5-deficient A/J murine model. Additionally, C3.1 and 9F2 mAbs show direct fungicidal effects against C. auris isolates, presenting a promising therapeutic option against this critical threat.

Keywords: Immunology; Medical microbiology; Mycology.

Graphical abstract

Figure 1

C3.1 mAb-mediated attenuation of proinflammatory response (A) Fungal burden in kidney, brain, and heart of A/J mice on days 1, 3, and 7 post-infection. Mice were treated prophylactically with DPBS (200 μL), IgG3 isotype control (12 mg/kg in 200 μL DPBS), or C3.1 mAb (12 mg/kg in 200 μL DPBS) before being challenged with a sublethal dose of C. auris isolate AR-0389. (B) Serum levels of proinflammatory cytokines (IFN-γ, IL-6, and TNF-α) were measured by multiplex cytokine assay on days 1, 3, and 7 post-challenge. C3.1 treatment significantly reduced IFN-γ (p < 0.01), IL-6 (p < 0.05), and TNF-α (p < 0.05) levels on both days 3 and 7 compared to controls. IL-1β was undetectable in all groups. Data are shown as mean (A) or mean ± SD (B), with individual data points included for cytokine levels. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. Sample sizes: n = 10 (A), n = 5 (B). ∗p < 0.05; ∗p < 0.01; ∗∗∗p < 0.001; ns = not significant.

Figure 2

Binding properties of mAbs C3.1 and 9F2 against Candida auris epitopes (A) ELISA binding curves of C3.1 against β-1,2-mannotriose (β-Man₃) in purified form and from mannan cell wall extract. (B) Surface plasmon resonance (SPR) sensorgram of C3.1 binding to immobilized β-Man₃. (C) SPR-derived equilibrium dissociation constant (KD) of C3.1 for β-Man₃. Data was measured using a one-to-one two-state fit model. (D) Serum levels of C3.1 in C. auris-infected vs. uninfected A/J mice over time. (E) Serum levels of 9F2 in C. auris-infected vs. uninfected A/J mice. (F) ELISA inhibition curves of C3.1 binding to β-Man₃ and 9F2 binding to a 15-aa peptide. IC₅₀ values were determined by nonlinear regression analysis, showing 9F2 exhibits a significantly lower IC₅₀ (higher affinity) compared to C3.1. Data are representative of two independent experiments.

Figure 3

Enhanced phagocytosis of Candida auris by RAW 264.7 macrophages mediated by C3.1 and 9F2 monoclonal antibodies (mAbs) (A) Schematic of phagocytosis assay. RAW macrophages were plated in a 96-well plate at a density of 4 x 10⁴ cells/well. FITC-stained C. auris yeast cells were incubated with (B) Phagocytosis assays were conducted using C. auris strains AR-0389 (top panel) and AR-0386 (bottom panel) in the presence of C3.1 and 9F2 mAbs. Colony-forming units (CFU) of live C. auris cells were quantified to assess phagocytic activity. Candida control represents C. auris alone, and macrophage control represents macrophages without antibody treatment. Both C3.1 and 9F2 mAbs significantly increased phagocytosis of C. auris by RAW 264.7 cells compared to isotype controls (IgG3 and IgG2a) and untreated controls. The data are shown as mean CFU ±SD. Statistical significance was determined by one-way ANOVA: ∗∗∗p < 0.001. These results indicate the ability of C3.1 and 9F2 to enhance opsonophagocytosis of C. auris, contributing to their fungicidal efficacy.

Figure 4

MAb-mediated growth inhibition of Candida auris isolates from clades I–IV Candida auris yeast cells were incubated with (A) 100 μg/mL of C3.1 mAb, (B) 100 μg/mL 9F2 mAb, (A and B) nonspecific isotype control mAb, or DPBS vehicle for 18 h. In a separate experiment, serial dilutions of (C) C3.1 or (D) 9F2 mAb for 18 h. In both experiments, cell suspensions were then plated onto GYEP agar plates and colony forming units (CFUs) were quantified after 48 h. (A–D) 18-h growth as a percentage of CFUs of yeast incubated with DPBS vehicle control. β-Man₃ = β-1,2-mannotriose, Pgk1 = phosphoglycerate kinase 1. (A–D) Data are mean ± SD. (A, B) n = 6–9. (C and D) n = 3. (A–D) One-way ANOVA was used to identify significant differences. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗p < 0.0001, ns = not significant. ND = not detectable.

Figure 5

Metabolic inhibition and fungicidal activity of C3.1 and 9F2 monoclonal antibodies (mAbs) against Candida auris AR-0389 biofilms (A) Representative images showing the effect of C3.1 and 9F2 mAbs on C. auris biofilm viability compared to IgG3 and IgG2a isotype controls. Treatment with C3.1 or 9F2 resulted in substantial biofilm disruption and cellular debris, indicating fungicidal activity. Confocal microcopy images of C. auris yeast cells after 18 h incubation with 100 μg/mL of C3.1, 9F2, or isotype control Ab. (B) Dose-dependent reduction in metabolic activity of C. auris biofilms by C3.1 and 9F2 mAbs, as measured by XTT assay. Mean absorbance values (A_475−650) are shown for each concentration, indicating biofilm viability. Both mAbs significantly inhibited metabolic activity in a dose-dependent manner, with 9F2 achieving complete inhibition at 1.6 mg/mL and C3.1 at 1.0 mg/mL. Error bars represent the standard deviation from triplicate wells. These results demonstrate the potent fungicidal effects of C3.1 and 9F2 on pre-formed C. auris biofilms, highlighting their potential as therapeutic agents against biofilm-associated C. auris infections.