Anti-CotH3 antibodies protect mice from mucormycosis by prevention of invasion and augmenting opsonophagocytosis

Abstract

Mucorales are fungal pathogens that cause mucormycosis, a lethal angioinvasive disease. Previously, we demonstrated that Rhizopus, the most common cause of mucormycosis, invades endothelial cells by binding of its CotH proteins to the host receptor GRP78. Loss of CotH3 renders the fungus noninvasive and attenuates Rhizopus virulence in mice. Here, we demonstrate that polyclonal antibodies raised against peptides of CotH3 protected diabetic ketoacidotic (DKA) and neutropenic mice from mucormycosis compared to mice treated with control preimmune serum. Passive immunization with anti-CotH3 antibodies enhanced neutrophil inlfux and triggered Fc receptor-mediated enhanced opsonophagocytosis killing of Rhizopus delemar. Monoclonal antibodies raised against the CotH3 peptide also protected immunosuppressed mice from mucormycosis caused by R. delemar and other Mucorales and acted synergistically with antifungal drugs in protecting DKA mice from R. delemar infection. These data identify anti-CotH3 antibodies as a promising adjunctive immunotherapeutic option against a deadly disease that often poses a therapeutic challenge.

Fig. 1. CotH antibody titers in mucormycosis patients are not different from those in healthy subjects, while rabbit anti-CotH3 polyclonal antibodies specifically bind to Mucorales.

(A) ELISA plates coated with rCotH3 from Escherichia coli or the 16-mer peptide showing low and similar anti-CotH3 antibody titers in sera collected from mucormycosis patients (11 total) or healthy volunteers (12 total). Colored symbols indicate sera collected from the same patient at different days (numbers) following diagnosis (day 0). (B) Flow cytometry analysis revealed that different concentrations of anti-CotH3 polyclonal antibody (and not the preimmune IgG antibody) bound both R. delemar spores and germlings with high consistency but not with A. fumigatus. The different doses of antibodies ranged from the highest (100 μg/ml) to the lowest (3 μg/ml). (C) The polyclonal antibodies also bound to a number of other fungi belonging to the order Mucorales.

Fig. 2. Anti-CotH3 polyclonal antibodies protect mice from R. delemar intratracheal infection.

Intraperitoneal treatment with a single dose of the polyclonal antibody (Ab), 24 hours after intratracheal infection of neutropenic mice (A to C) or DKA mice (D and E), protected them from R. delemar infection. Survival of neutropenic (A) or DKA mice (D) was conducted using 10 mice per group with confirmed inhaled inoculum of 1.2 × 10⁴ for (A) and 6.3 × 10³ for (D). *P < 0.05 versus mice treated with preimmune IgG. A single dose of 30 μg of anti-CotH3 IgG resulted in a significant reduction of lung and brain fungal burden in neutropenic (B) (inhaled inoculum of 3.2 × 10³) and DKA mice (E) (inhaled inoculum of 3.6 × 10³) when compared to isotype-matched control IgG at the same dose. Mice were euthanized 72 hours after infection. Hematoxylin and eosin staining of lung and brain sections harvested from neutropenic mice (C) revealed extensive tissue edema and hemorrhage in the lungs of mice treated with isotype-matched IgG, whereas mice treated with anti-CotH3 IgG had normal architecture (lower magnification). Abundant fungal hyphae invading lungs of mice treated with isotype-matched IgG, but not anti-CotH3 IgG, could be detected (higher magnification). Brains from isotype-matched IgG-treated, but not anti-CotH3 IgG-treated, mice showed mucormycosis angioinvasion. Scale bars, 300 μm (C).

Fig. 3. Anti-CotH3 antibodies protect neutropenic mice from infection by five other Mucorales.

Neutropenic mice were infected intratracheally with Rhizomucor (A), Apophysomyces (B), Lichtheimia (C), Cunninghamella (D), or M. circinelloides (E) and then, 24 hours later, treated with 30 μg of anti-CotH3 IgG or isotype-matched control IgG. Survival of mice served as an end point. Inhaled inocula were 1.6 × 10³, 3.7 × 10³, 4.6 × 10³, 2.2 × 10³ (average inoculum of two experiments), and 2.2 × 10³ (average inoculum of two experiments) for Rhizomucor (A), Apophysomyces (B), Lichtheimia (C), Cunninghamella (D), or M. circinelloides (E), respectively. Ten mice per group were used, except for (D) and (E), in which 20 mice per group were used. *P < 0.05.

Fig. 4. Anti-CotH3 antibodies stimulate immune recognition of R. delemar in vivo and in vitro through opsonophagocytosis.

(A) Higher MPO levels detected in lungs and spleens harvested from DKA mice (n = 5 per group) infected with R. delemar (inhaled inoculum of 1.6 × 10³ spores) and treated with 30 μg of anti-CotH3 IgG versus preimmune IgG. Treatment with the antibodies started 24 hours after infection, and the mice were euthanized 72 hours after infection. Data are presented as raw MPO values in nanograms per gram of tissue or normalized to burden of infection in each individually marked organ in nanograms per gram per log₁₀ colony-forming unit (CFU). (B) Anti-CotH3 IgG enhanced the ability of PMNs harvested from normal or DKA mice in phagocytizing R. delemar ex vivo. (C) Incubation of PMNs harvested from DKA with anti-CotH3 IgG, but not preimmune IgG, enhanced their ability to damage R. delemar ex vivo as determined using the XTT assay. (D) Confocal imaging using LysoTracker Red staining confirms the activation of PMN by anti-CotH3 IgG showing acidification and maturation of the phagolysosome after phagocytosis of the fungal spores. (E) Anti-CotH3 IgG-activated PMNs harvested from DKA mice generated significantly higher amounts of ROS compared to control conditions including PMNs alone, PMNs + R. delemar, or PMNs + R. delemar + preimmune IgG. Blocking of Fc receptors (Fc-R) of PMNs harvested from DKA mice with Fc receptor antibodies abrogates the ability of PMNs to damage R. delemar even in the presence of anti-CotH3 IgG. (F) Conversely, F(ab)′2 anti-CotH3 IgG fragments fail to enhance the DKA mouse PMN–mediated damage activity of R. delemar. (G) Neutrophil depletion by Ly6G antibody completely reverses the enhanced survival of DKA mice (n = 9 per group except for mice treated with anti-CotH3 IgG + Ly6G antibody, which had 8) infected with R. delemar and treated with anti-CotH3 IgG (given at 30 μg 24 hours after infection). *P < 0.05 versus all groups, **P < 0.001 versus preimmune IgG, and P = 0.1 versus Ly6G antibody–treated mice. Data in (A) to (C), (E), and (F) are expressed as median ± interquartile changes.

Fig. 5. Monoclonal antibodies against CotH3 protect DKA and neutropenic mice from R. delemar and other Mucorales.

Treatment with 30 μg of three different clones of anti-CotH3 monoclonal antibodies (mAb) (C1 to C3), 24 hours after intratracheal R. delemar infection, protected DKA mice (n = 9 to 10 mice per group) (A) and neutropenic mice (n = 10 to 15 per group) (B) from lethal mucormycosis. *P < 0.05 versus isotype-matched control IgG; **P < 0.05 versus isotype-matched control IgG, C1 clone, and polyclonal IgG. C2 clone also protected neutropenic mice from mucormycosis due to M. circinelloides (C) and L. corymbifera (D). n = 10 per group for mice in (C) and (D). Confirmed inhaled inocula were 5.6 × 10³, 2.2 × 10³, 1.6 × 10³, and 2.1 × 10³ for (A) to (D), respectively.

Fig. 6. C2 monoclonal IgG1 synergistically protects DKA mice from R. delemar when combined with antifungal drugs.

DKA mice were infected with R. delemar (confirmed inhaled inoculum of 3.9 × 10³) and, 48 hours later, treated with either a single dose of 30 μg of C2 monoclonal IgG1, posaconazole (POSA) (30 mg/kg, twice daily) for 7 days, or a combination of both. End points were survival (A) (n = 9 to 14 per group) or tissue fungal burden in lungs (B) or brain (C) as determined by quantitative polymerase chain reaction (PCR). (D) The same model was used with similar treatment except that posaconazole treatment was substituted with LAmB (10 mg/kg per day for 4 days) (n = 9 to 10 per group). *P < 0.05 versus isotype-matched IgG, **P < 0.008 versus all other arms, ^‡P < 0.006 versus isotype-matched IgG, ^¥P < 0.03 versus isotype-matched IgG or C2, and P = 0.05 versus LAmB.