Monoclonal antibodies to heat shock protein 60 alter the pathogenesis of Histoplasma capsulatum

Abstract

Heat shock proteins with molecular masses of approximately 60 kDa (Hsp60) are widely distributed in nature and are highly conserved immunogenic molecules that can function as molecular chaperones and enhance cellular survival under physiological stress conditions. The fungus Histoplasma capsulatum displays an Hsp60 on its cell surface that is a key target of the cellular immune response during histoplasmosis, and immunization with this protein is protective. However, the role of humoral responses to Hsp60 has not been fully elucidated. We generated immunoglobulin G (IgG) isotype monoclonal antibodies (MAbs) to H. capsulatum Hsp60. IgG1 and IgG2a MAbs significantly prolonged the survival of mice infected with H. capsulatum. An IgG2b MAb was not protective. The protective MAbs reduced intracellular fungal survival and increased phagolysosomal fusion of macrophages in vitro. Histological examination of infected mice showed that protective MAbs reduced the fungal burden and organ damage. Organs of infected animals treated with protective MAbs had significantly increased levels of interleukin-2 (IL-2), IL-12, and tumor necrosis factor alpha and decreased levels of IL-4 and IL-10. Hence, IgG1 and IgG2a MAbs to Hsp60 can modify H. capsulatum pathogenesis in part by altering the intracellular fate of the fungus and inducing the production of Th1-associated cytokines.

FIG. 1.

Serological responses to immunizations of mice with rHsp60 selected to generate hybridomas. The results were similar for second immunized mice. A comparison of the titers of preimmune serum and sera obtained 2 weeks after each immunization is shown. The bars are the averages of three independent ELISA experiments, and the error bars indicate standard deviations. *, P < 0.01 for comparisons of preimmune serum and serum obtained after the first immunization; **, P < 0.01 for a comparison of sera obtained after the first and second immunizations, calculated by analysis of variance and adjusted by using the Bonferroni correction.

FIG. 2.

MAb-labeled Hsp60 on the cell surface of H. capsulatum var. capsulatum: immunofluorescence and bright-field microscopy showing labeling of H. capsulatum var. capsulatum by MAbs to Hsp60. (A and C) Representative images of diffuse binding with MAb 11D1. (B and D) Punctate binding with MAb 12D3. (E) Immunoblot showing binding of MAbs to rHsp60 protein at 60 kDa. rHsp60 hyperimmune mouse serum was used as a positive control. (F) Representative curves for MAb binding to rHsp60 of yeast cells as determined by indirect ELISA. (G and H) Representative immunogold-labeled transmission electron micrographs for control MAb (G) and MAb 12D3 (H), showing the distribution of Hsp60 on the cell wall and surface of the yeast. Clusters of gold balls labeling Hsp60 consistent with vesicular transport are indicated by arrows.

FIG. 3.

Representation of H. capsulatum var. capsulatum Hsp60 with associated binding sites for the IgG isotype MAbs. (A) Sequence showing the mapped peptide residues. The different colors correspond to the different binding regions. (B) Localization of the epitopes in the three-dimensional structure of H. capsulatum var. capsulatum Hsp60 generated by homology modeling using GroEL as a template. Binding sites of MAbs 11D1 (orange), 6B7 (yellow), and 7B6 (yellow) are localized in the protein cleft, whereas the recognition sites of the other MAbs, MAbs 4E12 (blue), 13B7 (blue), and 12D3 (green), are on the external surface of the protein. (C) Epitope distribution in the linear model.

FIG. 4.

MAbs can modify the intracellular fate of H. capsulatum var. capsulatum. (A) Effect of MAbs to Hsp60 on H. capsulatum var. capsulatum phagocytosis by J774.16 cells. (B) Blockage of Fc receptors reduced phagocytosis. (C) Blockage of the CD11 receptor decreased phagocytosis in control groups and in the IgG2a groups, although phagocytosis was significantly greater in all the MAb groups than in the controls. (D) Effect of MAbs to Hsp60 on H. capsulatum var. capsulatum killing by J774.16 cells. In panels A to D, the values are the averages of three independent experiments, and the error bars indicate standard deviations. Each experiment was done in quadruplicate. *, P < 0.001 for comparisons between MAbs and controls; **, P < 0.001 for a comparison between MAb 7B6 and the other MAbs to Hsp60. (E to H) Colocalization of H. capsulatum var. capsulatum yeast cells in phagosomes as determined using the FITC-dextran contents of lysosomes. MAbs 11D1 (E), 12D3 (F), and 13B7 (G) induced phagolysosomal fusion within J774.16 cells. In contrast, the rate of fusion with MAb 7B6 (H) was much lower.

FIG. 5.

MAbs to Hsp60 affect the pathogenesis of histoplasmosis. (A) Intraperitoneal injection of 500 μg of IgG1 (11D1 and 6B7) or IgG2a (4E12, 12D3, and 13B7) MAbs 2 h prior to infection significantly prolonged survival (P < 0.05 for a comparison to controls), but injection of IgG2b MAb 7B6 did not prolong survival. (B) IgG1 and IgG2a MAbs were protective when they were preincubated with H. capsulatum var. capsulatum yeast cells prior to infection (P < 0.05), and the IgG2b MAb was not protective. Each panel shows survival results representative of three similar experiments. (C to E) Numbers of CFU in (C) lungs, (D) spleens, and (E) livers at 7 and 14 days after sublethal intranasal challenge with 5 × 10⁶ H. capsulatum var. capsulatum yeast cells for mice treated intraperitoneally with selected MAbs to Hsp60 or an irrelevant MAb. *, P < 0.001 at 7 days postinfection; **, P < 0.001 at 14 days postinfection. (F to M) IgG1 and IgG2a MAbs to Hsp60 decrease inflammation and the fungal burden in tissues. (F to I) Lung sections obtained after 7 days of infection; (J to M) lung sections obtained after 14 days of infection. The lungs of mice treated with protective MAbs 11D1 and 12D3 exhibited peribronchiolar inflammation at day 7 (G and H, respectively) and resolving inflammation at day 14 (K and L, respectively). In contrast, sections of lungs from the control group and MAb 7B6-treated animals exhibited diffuse, dense inflammation with increased tissue infiltration and early granuloma formation at 7 days (I). Although in the lungs of control mice there was evidence of reduced inflammation at day 14 (J), mice treated with MAb 7B6 had progressive necrotizing pneumonia (M).