The monoclonal antibody against the major diagnostic antigen of Paracoccidioides brasiliensis mediates immune protection in infected BALB/c mice challenged intratracheally with the fungus

Abstract

The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.

FIG. 1.

Phagocytosis of yeast cells by J774.16 cells after 12 h in the presence of MAbs against gp43 (3E, 10D, 17D, 19G, 21F, and 32H) compared to that of controls of yeast incubated without MAb (PBS) or with an irrelevant MAb (MAb A4). Each bar represents the average of three measurements, and error bars indicate SD. Experiments were done in triplicate, and different fields were counted. *, significant difference (P < 0.05, determined by analysis of variance and Tukey's honestly significant difference test) compared to the control without MAb.

FIG. 2.

Nitrite measurements in supernatants from phagocytosis assays. P. brasiliensis yeast was cultured with J774.16 cells for 12 h in the presence of MAbs against gp43 (3E, 10D, 17D, 19G, 21F, and 32H), PBS (without MAb), or the irrelevant MAb (A4). Nitrite levels were detected using a Griess assay. *, significant difference (P < 0.05, determined by analysis of variance and Tukey's honestly significant difference test) relative to the PBS control. Error bars denote SD.

FIG. 3.

Lung CFU from mice infected i.t. with 3 × 10⁵ yeast cells and treated with MAbs against gp43 (3E, 10D, 17D, 19G, 21F, and 32H) 24 h prior to infection. Mice were sacrificed after 15 (black bars) or 30 (gray bars) days of infection. Control mice were infected and received PBS (infected only) or an irrelevant MAb (A4). Each bar represents the average count of fungi in the lung, and error bars indicate SD. *, significant difference (P < 0.05, determined by analysis of variance and Tukey's honestly significant difference test) relative to the PBS control.

FIG. 4.

Lung CFU from mice infected i.t. with 3 × 10⁵ yeast cells and treated with MAbs against gp43 (3E or 32H) 30 days after infection. Mice were sacrificed after 45 (black bars) or 60 (gray bars) days of infection. Control mice were infected and received PBS (infected only) or an irrelevant MAb (A4). Each bar represents the average count of fungi in the lung, and error bars indicate SD. *, significant difference (P < 0.05) relative to the PBS control.

FIG. 5.

CFU from lungs (black bars) or spleens (gray bars) of mice infected i.v. with 3 × 10⁵ yeast cells and treated with 1 mg of MAbs against gp43 (3E or 32H) 24 h prior to infection and with 100 μg every week for a month as a maintenance dose. Mice were sacrificed after 30 days of infection. Control mice were infected and received PBS (infected only) or an irrelevant MAb (A4). Each bar represents the average count of fungi in the organ, and error bars indicate SD. *, significant difference (P < 0.05, determined by analysis of variance and Tukey's honestly significant difference test) relative to the PBS control.

FIG. 6.

Histopathology of representative lung sections from mice i.t. infected according to protocol 1 and euthanized at 30 days after infection. (A) Lung section from an untreated infected mouse; (B) lung section from a mouse infected and treated with MAb 32H; (C) lung section from an infected mouse treated with MAb 3E. Hematoxylin-eosin staining, ×10 magnification.

FIG. 7.

Reactivity of peptides NHVRIPIGYWAV(R-CONH₂), -(R-COOH), and P10 (QTLAIAHTLAIRYAN) with MAb 3E by ELISA. Microtiter plates were sensitized with 100 ng of each peptide, and the reaction was developed with MAb 3E. The diamond symbol indicates the background measurement with buffer alone, and the square symbol corresponds to the reactivity of the peptides against the irrelevant antibody.