A 70-kilodalton recombinant heat shock protein of Candida albicans is highly immunogenic and enhances systemic murine candidiasis

Abstract

The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-gamma), IL-12, and IL-10 but not IL-4 production by CD4+ lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-gamma was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-gamma upon in vitro stimulation of CD4+ cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement of Candida infection seemed to occur in the mouse model used.

FIG. 1

(a) Molecular mass, definition, and location of the gene fragments encoding the CaHsp70 products used as immunogens throughout this study. A, CaHsp70; B, CaHsp70-Cter; C, CaHsp70-Nter; D, 39.4-kDa N-terminal fragment. The arrow indicates the 5′→3′ direction of transcription. N-6xhis, six-histidine tag. (b) Immunoblot reaction with the CaHsp70 recombinant products indicated in panel a of serum from mice immunized with CaHsp70, CaHsp70-Cter, and CaHsp70-Nter (blots a, b, and c, respectively). One microgram of each recombinant product was electrophoresed on an SDS-polyacrylamide gel and electrotransferred to a nitrocellulose membrane. The reaction was performed with a 1:1,000 dilution of each antiserum and visualized with a phosphatase-conjugated second antibody as detailed in Materials and Methods. Arrowheads indicate molecular mass markers (in kilodaltons).

FIG. 2

Proliferation of splenocytes from CD2F1 mice not immunized or immunized with CaHsp70, CaHsp70-Cter, and CaHsp70-Nter and stimulated in vitro with the respective antigen (10 μg/ml) or ConA (0.1 μg/ml). (a and b) Two independent experiments. Nonstimulated splenocyte cultures never incorporated more than 800 cpm, and these values were subtracted. Error bars indicate standard deviations.

FIG. 3

Proliferation of human PBMC from two independent donors following in vitro stimulation with the indicated antigen or IL-2. The concentrations of CaHsp70 stimulants were as in the legend to Fig. 2. The mannoprotein fraction (MP-F2) was used at 10 μg/ml, and IL-2 was used at 100 U/ml. Nonstimulated PBMC cultures never incorporated more than 350 cpm, and these values were subtracted. Error bars indicate standard deviations.

FIG. 4

Kidney histopathology of mice immunized with CaHsp70 (a) or not immunized (adjuvant) and challenged with C. albicans. In both cases, hyphal cells were seen clustering with inflammatory cells both in cortical tissue and outside parenchymal tissue.

FIG. 5

Cytokine production by purified CD4⁺ splenocytes cultured in vitro with splenic adherent macrophages and incubated in the presence of ConA (0.1 μg/ml), HCA (5 × 10⁵ cells), CaHsp70 (10 μg/ml), or CaHsp70-Cter (10 μg/ml). Cytokine levels were determined by cytokine-specific ELISAs. ∗, below the detection limit of the assay, indicated by a less-than symbol on the y axis. Error bars indicate standard deviations.

FIG. 6

Cytokine production by purified CD4⁺ splenocytes of immunized mice, cultured in vitro with splenic adherent macrophages and incubated in the presence of HCA (5 × 10⁵ cells), mannoprotein fraction MP-F2 (25) (10 μg/ml), CaHsp70 (10 μg/ml), or CaHsp70-Cter (10 μg/ml). The animals were immunized against the CaHsp70 products as described in Materials and Methods, and their spleens were removed 3 days after the last immunizing antigen dose. The animals whose CD4⁺ cells were stimulated in vitro with HCA or MP-F2 had been immunized with a low-virulence nongerminating variant of C. albicans (PCA-2) or MP-F2, respectively, following previously published methods (25). Cytokine levels were determined by cytokine-specific ELISAs. ∗, below the detection limit of the assay, indicated by a less-than symbol on the y axis. Error bars indicate standard deviations.