Monoclonal immunoglobulin G1 directed against Aspergillus fumigatus cell wall glycoprotein protects against experimental murine aspergillosis

Abstract

Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall, which, being the outermost part of the cell, mediates the host-fungus interplay. For these reasons much effort has focused on the discovery of useful inhibitors of cell wall glucan, chitin, and mannoprotein biosynthesis. In the absence of a wide-spectrum, safe, and potent antifungal agent, a new strategy for antifungal therapy is directed towards the development of monoclonal antibodies (MAbs). In the present study the MAb A9 (immunoglobulin G1 [IgG1]) was identified from hybridomas raised in BALB/c mice immunized with cell wall antigen of Aspergillus fumigatus. The immunoreactive epitopes for this IgG1 MAb appeared to be associated with a peptide moiety, and indirect immunofluorescence microscopy revealed its binding to the cell wall surface of hyphae as well as with swollen conidia. MAb A9 inhibited hyphal development as observed by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (25.76%), reduced the duration of spore germination, and exerted an in vitro cidal effect against Aspergillus fumigatus. The in vivo protective efficacy of MAb A9 was also evaluated in a murine model of invasive aspergillosis, where a reduction in CFU (>4 log(10) units) was observed in kidney tissue of BALB/c mice challenged with A. fumigatus (2 x 10(5) CFU/ml) and where enhanced mean survival times (19.5 days) compared to the control (7.1 days) and an irrelevant MAb (6.1 days) were also observed.

FIG. 1.

Immunofluorescence photograph of A. fumigatus swollen and germinated conidia stained with MAb A9. Fluorescence was uniformly distributed over the entire surface of the fungal cells.

FIG. 2.

Characteristic protein profile of the cell wall of A. fumigatus on 12% SDS-PAGE. Strips (left to right): 1, standard molecular mass marker; 2, complete protein profile on gel stained with Coomassie blue; 3, immunogenic proteins of A. fumigatus as detected by immunoblotting using pooled sera from patients with aspergillosis; 4, detection of immunogenic protein of the A. fumigatus cell wall probed with MAb A9; and 5, PAS-stained immunogenic protein (as in strip 4), demonstrating it to be a glycoprotein.

FIG. 3.

Detection of the same antigenic protein in A. fumigatus (lane 1), A. flavus (lane 2), A. niger (lane 3), and A. terreus (lane 4) by immunoblotting using MAb A9.

FIG. 4.

In vitro fungicidal activity of MAb A9 against A. fumigatus, A. flavus, A. terreus, and A. niger measured by the reduction in number of CFU compared to the control with an irrelevant MAb. Values are means of triplicate determinations ± standard errors of the means. Differences in fungicidal activity between A. fumigatus spores treated with MAb A9 and irrelevant MAb were found to be statistically significant (P < 0.0001) as determined by the Student t test.

FIG. 5.

Dose-response bar graph generated from FACS analysis, showing the percentage of dead cells at different concentrations of MAb A9 and concanavalin A, which causes agglutination. The figure shows that antibody-mediated agglutination is insufficient for killing of A. fumigatus conidia. Data presented are the averages from three experiments.

FIG. 6.

In vitro effect of MAb A9 on germination of A. fumigatus conidia. Swollen A. fumigatus conidia (1.0 × 10³) were incubated at 37°C with 50 mg of MAb A9 in 100 μl RPMI 1640. At selected time intervals, percent germination was calculated. A total of 100 conidia per field were counted at a magnification of ×400, and the mean value from three independent experiments was calculated. ⧫, control; ▪, irrelevant IgG; ▴, MAb A9.

FIG. 7.

Alveolar macrophages (J774 cell line) showing phagocytosis of A. fumigatus conidia. (Left) Conidia without any treatment (with MAb A9) were cocultured with macrophages and served as a control. (Middle) Conidia treated with MAb A9 for 18 h and then cocultured with macrophages, showing more phagocytosed conidia than the control. (Right) Conidia treated with MAb A9 which was heated to 56°C for inactivation of complement and then cocultured with macrophages exhibited phagocytosis similar to that in the middle panel, indicating complement-independent phagocytosis.

FIG. 8.

Effect of MAb A9 on the reduction in CFU from kidney tissue of experimental mice. A reduction in CFU of >4 log₁₀ units was observed in kidney tissue of BALB/c mice challenged with A. fumigatus (2.0 × 10⁵ cells per mouse) and MAb A9 administered prophylactically 2 h before infection via the tail vein. The difference between MAb A9-treated groups and the control group (without treatment) was significant (P < 0.001 according to Student t test).

FIG. 9.

Protective effect of MAb A9 against systemic aspergillosis in experimental mice. Survival rates of MAb A9-vaccinated mice compared to those of control mice (▪), mice treated with an irrelevant IgG (⧫), and MAb A9-immunized mice (▴). Data represent % survival recorded daily for 26 days postchallenge. Differences in survival rates (on day 26) between MAb A9- and irrelevant IgG-immunized mice were found to be statistically significant (P < 0.001 as assessed by the log rank test).