Mapping of the T-cell epitope in the major 43-kilodalton glycoprotein of Paracoccidioides brasiliensis which induces a Th-1 response protective against fungal infection in BALB/c mice

Abstract

The 43-kDa glycoprotein of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis, the prevalent systemic mycosis of Latin America. Apart from eliciting high antibody titers, gp43 is also immunodominant in delayed-type hypersensitivity reactions in infected animals and humans. The cellular immune response in mice to gp43 administered in complete Freund's adjuvant involves CD4+ Th-1 lymphocytes, secreting gamma interferon (IFN-gamma) and interleukin 2 (IL-2) but not IL-4 and IL-10. The T-cell epitope of this antigen was mapped to a 15-amino-acid peptide (P10) based on lymphoproliferations with primed cells from three different haplotypes and on a computer-assisted protein analysis. The structural requirements of the T-cell epitope were determined by assaying a series of P10 analogous and truncated peptides. Only 12-mer or longer sequences were active, confirming presentation by major histocompatibility complex II. The HTLAIR inner core of P10 is the essential domain of the epitope, with various flanking regions possible. Immunization of mice with both gp43 and P10 led to vigorous protection against intratracheal challenge by virulent P. brasiliensis, with a >200-fold decrease in lung CFU and halting of dissemination to the spleen and liver. The protective effect of P10 is mainly attributed to an IFN-gamma-mediated cellular immune response. Unlike gp43, which induces an antibody response compatible with both Th-1 and Th-2 activation in infected BALB/c mice, P10 does not induce a humoral response. Protection by gp43 and P10 was characterized by a few well-demarcated lung granulomas with numerous nonviable yeast forms or resolved lesions with no detectable fungal cells.

FIG. 1

gp43-stimulated dose-dependent proliferation (± standard deviation) of LN cells from BALB/c mice sensitized with a single injection of gp43 in CFA (open circles). Lymphoblasts from mice sensitized with CFA only (diamond) and cells from mice sensitized with gp43 and stimulated with the culture medium (supplemented RPMI) only (circle with cross) were the negative controls.

FIG. 2

Proliferation (plus standard deviation) of gp43-sensitized BALB/c LN cells induced by 25 peptides spanning the whole sequence of the native gp43 molecule. P10, the only responding peptide, has 15 amino acids. The peptides were tested at 10 μg/ml. C, control with no antigen added; PH, phytohemagglutinin at 5 μg/ml; CA, concanavalin A at 2 μg/ml (positive controls); gp43 was at 50 μg/ml.

FIG. 3

Proliferation (plus standard deviation) with gp43 (12.5 μg/ml) and P10 (2 μg/ml) of popliteal and inguinal LN cells recruited by injection of CFA into the footpads of BALB/c mice infected intraperitoneally with 10⁹ yeast forms/ml/mouse of virulent P. brasiliensis 18 for 45 days. The controls were LN cells from infected animals with no antigen added.

FIG. 4

Comparative proliferative response to gp43 and P10 of LN cells from A/Sn (black bars), BALB/c (cross-hatched bars), and C57-BL (open bars) mice sensitized subcutaneously with both gp43 and P10 in CFA.

FIG. 5

Graphic analysis of a 60-amino-acid fragment (represented by the scale at the bottom) from the gp43 sequence containing P10. (A) Jameson-Wolff antigenic index; (B) Kyte-Doolittle hydrophilicity plot (averaged to 11 amino acid sequences; negative values indicate hydrophobicity); (C) Eisenberg alpha, amphipathic regions; (D) I-A^d regions, Sette MHC II motifs; (E) Emini surface probability plot.

FIG. 6

Proliferation response (plus standard deviation) of LN cells from BALB/c mice primed with P10 to analogous and truncated peptides based on the P10 amino acid sequence. Only the peptides with 12 or more amino acids were active. CO, control with no antigen.

FIG. 7

Requirement (plus standard deviations) in the nine-amino-acid core sequence for proliferation of P10-primed lymphocytes. Maximal responses were obtained with structures containing the HTLAIR hexapeptide. CO, control with no antigen.

FIG. 8

Protective effect of immunization with gp43 and P10 against intratracheal infection by virulent P. brasiliensis Pb18. 1 and 3, 1 and 3.5 months of infection, respectively; L, lung; S, spleen; V, liver; C, control with Freund’s adjuvant alone; G, gp43 immunization; P, P10 immunization. In each system five to six BALB/c mice were used. The arrow indicates that with five animals, one had 195,000 CFU/g of lung tissue and the others had more than 200,000 CFU/g of lung tissue. Error bars indicate the standard deviations.

FIG. 9

Protective effect of gp43 and P10 immunization in intratracheally infected mice (3 × 10⁵ virulent P. brasiliensis yeast forms). (A) Lung section from a control nonimmunized BALB/c mouse showing confluent epithelioid granulomata forming a paracoccidioma-like lesion with a great number of viable yeast cells. (B) Dissemination to the liver of P. brasiliensis in a control nonimmunized mouse; confluent epithelioid granulomas with fungal cells are shown. (C) A single, well-demarcated epithelioid granuloma, with a prominent mononuclear cellular halo, encircling viable and nonviable yeast forms in a lung section from a gp43-immunized animal. (D) Apparently resolved granuloma, containing few macrophages and no fungal cells, in a lung section of a P10-immunized mouse.