Background: Developing immunotherapies for fungal keratitis is a high priority. A conserved surface polysaccharide, poly-N-acetyl glucosamine (PNAG), is expressed by a variety of bacterial pathogens. Antibodies to a specific glycoform of PNAG protect animals against infections caused by E. coli S. aureus, A. baumannii and Burkholderia spp. We analyzed fungal pathogens for expression of PNAG and used a mouse model of ocular keratitis caused by Aspergillus, and Fusarium to determine if PNAG was a target for protective antibody in the eye.Methods: Expression of PNAG on Candida, Fusarium, Aspergillus, Cryptococcus and Saccharomyces cells was assessed by immunofluorescence (IF) using a human IgG1 MAb specific to PNAG. Specificity of binding to PNAG was ascertained by showing loss of reactivity following degradation by the PNAG-specific hydrolase enzyme Dispersin B and periodate oxidation. Cross reactivity with fungal glycans was tested by ELISA inhibition. Keratitis was induced by scratching the corneas of anesthetized female C57Bl/6 mice followed by inoculation with ~105-107cfu/eye of A. flavus, and F. solani. Normal and immune polyclonal goat antibody to PNAG, or a PNAG-specific or control human IgG1 MAb were injected IP 4 h after infection and additional antibody applied topically 24 h and 32 h after infection. Twenty four to 48 h post-infection corneas were scored for pathology on a scale of 0 to 4, then excised, and fungal cfu determined. Results: The human IgG1 MAb to PNAG, but not a control IgG1 MAb, was readily observed by immunofluorescence to bind to cells of multiple fungal pathogens. Binding was lost following Dispersin B and periodate treatment but not inhibited by beta-glucans, indicating no cross reactivity between PNAG and fungal glucans. A. flavus, and F. solani infected mice treated therapeutically by IP injection plus topical application of either polyclonal antibody to PNAG or the human IgG1 MAb had markedly reduced fungal levels in the eye and reduced corneal pathology. Conclusions: PNAG is produced by a multitude of fungal pathogens and antibody to PNAG demonstrated therapeutic efficacy in mice with A. flavus, or F. solani keratitis, indicating in vivo expression of PNAG and the potential to prevent or treat fungal eye infections by vaccines and immunotherapeutics to PNAG.
Full conference title:
- ASM 115th (2015)