Detection of Antibodies against Paracoccidioides brasiliensis Melanin inIn VitroandIn VivoStudies during Infection

Abstract

Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) -chain and four IgM melanin-binding MAbs. The five IgG1 -chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensisinfected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and crossreactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.

Occurrence of Ochroconis and Verruconis Species in Clinical Specimens from the United States

“…This is an unusual phenomenon that occurs in a few other fungi, such as Neoscytalidium hyalinum, Aspergillus flavus, Ophiostoma floccosum, Ophiostoma piceae, and Ophiostoma pluriannulatum (53)(54)(55), which in general are involved almost exclusively in skin and nail disease (56,57). The melanin plays an important role in fungal pathogenesis, and its absence often generates less virulent isolates than with pigmented ones (47,(58)(59)(60). Although we could not demonstrate that this isolate was the etiological agent of the infection, the presence of albino isolates recovered from deep tissues might suggest that this fungus has an additional mechanism of pathogenicity, apart from that of melanin.…”

Fungal Melanin: What do We Know About Structure?

“…Antibodies ( Rosas et al, 2000a ; Youngchim et al, 2004 ; Urán et al, 2011 ) and peptide ligands ( Nosanchuk et al, 1999 ) have been generated to fungal melanin. Immunofluorescence studies utilizing some of these reagents reveal that there is diffuse, homogeneous binding along the surface of melanins isolated from different fungi, which suggests that there are conserved repeating units serving as epitopes to react with these reagents.…”

Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy

“… 24 and 8C3 IgG was produced in our laboratories as described in Urán et al . 27 . Anti-murine CTLA4 mAb 9D9 for the immunotherapy treatment arm was purchased from BioXCell (West Lebanon, NH, USA) and IgG control mAb – from Creative Diagnostics (NYC, NY, USA).…”

“…IgGs are generally more amenable to pre-clinical development including chimerization or humanization and large scale manufacturing for clinical trials compared to mAbs with IgM isotype. More recently, our laboratories generated an IgG type mAb (8C3) to melanin from another pathogenic fungus, Paracoccidioides brasiliensis 27 . Here we report the results of the comparative structural investigation of the novel 8C3 IgG and the previous generation 6D2 IgM and 8C3 evaluation as a potential RIT agent in aggressive murine melanoma.…”