Monoclonal antibodies against peptidorhamnomannans of Scedosporium apiospermum enhance the pathogenicity of the fungus.

Scedosporium apiospermum is part of the Pseudallescheria-Scedosporium complex. Peptidorhamnomannans (PRMs) are cell wall glycopeptides present in some fungi, and their structures have been characterized in S. apiospermum, S. prolificans and Sporothrix schenckii. Prior work shows that PRMs can interact with host cells and that the glycopeptides are antigenic. In the present study, three monoclonal antibodies (mAbs, IgG1) to S. apiospermum derived PRM were generated and their effects on S. apiospermum were examined in vitro and in vivo. The mAbs recognized a carbohydrate epitope on PRM. In culture, addition of the PRM mAbs increased S. apiospermum conidia germination and reduced conidial phagocytosis by J774.16 macrophages. In a murine infection model, mice treated with antibodies to PRM died prior to control animals. Thus, PRM is involved in morphogenesis and the binding of this glycopeptide by mAbs enhanced the virulence of the fungus. Further insights into the effects of these glycopeptides on the pathobiology of S. apiospermum may lead to new avenues for preventing and treating scedosporiosis.

Monoclonal antibody to fungal glucosylceramide protects mice against lethal Cryptococcus neoformans infection.

Glucosylceramides (GlcCer) are involved in the regulation of Cryptococcus neoformans virulence. In the present study, we demonstrate that passive immunization with a monoclonal antibody to GlcCer significantly reduces host inflammation and prolongs the survival of mice lethally infected with C. neoformans, revealing a potential therapeutic strategy to control cryptococcosis.

Vaccination of bovines with recombinant Boophilus Yolk pro-Cathepsin.

Boophilus Yolk pro-Cathepsin (BYC) is an aspartic proteinase found in Boophilus microplus eggs that is involved in the embryogenesis and has been tested as antigen to compose an anti-tick vaccine. The vaccine potential of a recombinant BYC expressed in Escherichia coli (rBYC) was investigated. rBYC was purified and used to immunize Hereford cattle. The sera of bovines immunized with rBYC recognized the native BYC with a titer ranging from 125 to 4000. Furthermore, immunized bovines challenged with 20,000 larvae presented an overall protection of 25.24%. The partial protection obtained against B. microplus infestation with the recombinant protein immunization was similar to the already described for native BYC immunization.

Purification and antigenicity of two recombinant forms of Boophilus microplus yolk pro-cathepsin expressed in inclusion bodies.

The tick Boophilus microplus is a bovine ectoparasite present in tropical and subtropical areas of the world and the use of vaccines is a promising method for tick control. BYC is an aspartic proteinase found in eggs that is involved in the embryogenesis of B. microplus and was proposed as an important antigen in the development of an anti-tick vaccine. The cDNA of BYC was amplified by PCR and cloned for expression in two forms with and without thioredoxin fusion protein (Trx), coding recombinant proteins named rBYC-Trx and rBYC, respectively. Expression, solubility, and yields of the two forms were analyzed. The recombinant proteins were expressed in inclusion bodies (IBs) and three denaturant agents (N-lauroyl sarcosine, guanidine hydrochloride, and urea) were tested for IBs solubilization. The N-lauroyl sarcosine solubilized 90.4 and 92.4% of rBYC-Trx and rBYC IBs, respectively, and was the most efficient denaturant. Two recombinant forms were affinity-purified by Ni2+-Sepharose under denaturing conditions. After dialysis, the yield of soluble protein was 84.1% for r-BYC-Trx and 5.9% for rBYC. These proteins were immune-reactive against sera from rabbit, mouse, and bovine previously immunized with native BYC, which confirms the antigenicity of the recombinant BYCs expressed in the Escherichia coli system.

A monoclonal antibody to glucosylceramide inhibits the growth of Fonsecaea pedrosoi and enhances the antifungal action of mouse macrophages.

Fungal glucosylceramides (GlcCer) are conserved lipid components in a large variety of pathogenic and non-pathogenic fungal species, but their biological functions are still unclear. Recent studies demonstrate that GlcCer are immunologically active components inducing the production of antifungal antibodies. In this work, a major GlcCer was purified and characterized from mycelial forms of Fonsecaea pedrosoi, the most frequent causative agent of chromoblastomycosis. As determined by fast atom bombardment mass spectrometry (FAB-MS) analysis, the purified molecule was identified as the conserved structure N-2'-hydroxyhexadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. A monoclonal antibody (Mab) against this structure was used in indirect immunofluorescence with the different morphological stages of F. pedrosoi. Only the surface of young dividing cells was recognized by the antibody. Treatment of F. pedrosoi conidia with the Mab against GlcCer resulted in a clear reduction in fungal growth. In addition, the Mab also enhanced phagocytosis and killing of F. pedrosoi by murine cells. These results suggest that, in F. pedrosoi, GlcCer seem to be cell wall components targeted by antifungal antibodies that directly inhibit fungal development and also enhance macrophage function, supporting the use of monoclonal antibodies to GlcCer as potential tools in antifungal immunotherapy.

Glucosylceramides in Colletotrichum gloeosporioides are involved in the differentiation of conidia into mycelial cells.

Glucosylceramides (GlcCer) were extracted from the plant pathogen Colletotrichum gloeosporioides and purified by several chromatographic steps. By using electrospray ionization mass spectrometry and nuclear magnetic resonance, GlcCer from C. gloeosporioides were identified as N-2'-hydroxyoctadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine and N-2'-hydroxyoctadecenoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against these structures were produced and used as tools for the evaluation of the role of GlcCer in the morphological transition of C. gloeosporioides. In the presence of antibodies to GlcCer, the differentiation of conidia into mycelia was blocked. Since GlcCer is present in several plant pathogens, the inhibitory activity of external ligands recognizing these structures may be applicable in other models of fungal infections.