Interplay between protective and inhibitory antibodies dictates the outcome of experimentally disseminated Candidiasis in recipients of a Candida albicans vaccine

Abstract

Mice immunized with heat-inactivated, whole yeast-form cells (Y cells) of Candida albicans developed intense, specific humoral and cell-mediated immune responses. However, they were modestly protected against a lethal challenge by the fungus, and their sera did not confer passive protection upon nonimmunized animals. Surprisingly, this immune serum conferred an elevated degree of passive protection to normal and SCID mice when preadsorbed on whole C. albicans cells. After adsorption, no antibodies specific to mannoprotein (MP)-rich extracts or secretions were detected by indirect enzyme-linked immunosorbent assay and no serum reaction with the fungal cell surface was seen in immunofluorescence assays. However, this serum had totally preserved the level of other antibodies, in particular those reacting with beta-1,3 and beta-1,6 glucan (GG). The hypothesis that anti-GG antibodies contributed to the passive protection was suggested by the following circumstantial evidence: (i) mice immunized with C. albicans cells treated with dithiothreitol and protease (YDP cells), which exposed GG on their surfaces and generated anti-GG but not anti-MP antibodies, were substantially protected against a lethal fungus challenge; (ii) the sera, and their immunoglobulin fractions, of mice immunized with YDP cells transferred protection to nonimmune animals; and (iii) this passive protection was substantially abolished by preadsorption on GG but not on intact cells. Overall, our findings demonstrate that some anti-Candida antibodies can block the protective potential of immune serum, a potential to which anti-GG antibodies appear to contribute. Our observations may also help explain why subjects with elevated anti-Candida antibody titers, inclusive of anti-MP and anti-GG antibodies, remain nonetheless susceptible to invasive candidiasis.

FIG. 1.

IFA reactivity of anti-Y-cell or anti-YDP-cell serum with C. albicans and effect of treatment with DTT and proteinase K on the antigenic array of the fungal cell surface. Untreated Y cells (a, c, and e) or DTT-proteinase-treated YDP cells (b, d, and f) were immunostained as described in Materials and Methods with the anti-Y-cell (a and b) or anti-YDP-cell (c and d) serum or with a monoclonal antibody (AF1) (e and f). Anti-Y-cell and anti-YDP-cell sera were used at a dilution of 1:200; the monoclonal antibody AF1 was used at a dilution of 1:5,000. Magnification, ×1,000. The figure represents the results of five repeated experiments.

FIG. 2.

In vitro proliferative response of splenocytes of mice immunized with Y cells or YDP cells. Individual splenocyte cultures from four mice for each immunization group were stimulated in vitro with Y or YDP cells or with ConA, as described in Materials and Methods. Proliferative responses were evaluated after 3 days by measuring [³H]thymidine incorporation. Values are mean stimulation indexes ± standard deviations measured for each experimental group compared to unstimulated control cultures. The asterisks indicate a significant difference (∗, P < 0.05; ∗∗, P < 0.001; ANOVA and Bonferroni's multiple t test) with respect to values measured in adjuvant (Adj)-treated animals. All other differences in the proliferative response were not significant. The data are from a representative experiment of three with similar results.

FIG. 3.

Different protective effect of Y- or YDP-cell immunization against murine disseminated candidiasis. (A and B) Survival rates of Y or YDP cell-vaccinated mice compared to those of control, nonimmunized mice. Mice (6 for panel A and 15 for panel B) were immunized with Y or YDP cells or with adjuvant (Adj) only and challenged i.v. with 10⁶ (A) or 2 × 10⁶ (B) Candida cells. Data represent percent survival, recorded daily for 60 days postchallenge. Differences in survival rates (on day 60) between YDP cell- and adjuvant- or Y cell-immunized animals were found to be statistically significant (P < 0.05) as assessed by Fisher's exact test. For the statistical significance of the MST (in days), see the text. The data in panel A refer to a single experiment, while the data in panel B represent the results of two separate experiments. (C) Kidney invasion in Y or YDP cell-vaccinated mice following an i.v. challenge with C. albicans. Groups of mice immunized with Y or YDP cells were challenged i.v. with 10⁶ Candida cells. On day 7 postchallenge, three mice per group were sacrificed and fungal invasion in the left kidney was evaluated by individual CFU counts. Values are weighted means of CFU count measured in each group of animals ± standard deviations. Probability, as indicated in the graph, was evaluated by Kruskal-Wallis ANOVA and Bonferroni-type nonparametric multiple comparison. The data are from a representative experiment of two with similar results (independent of the data in panels A and B).

FIG. 4.

Effect of preadsorption with particulate glucan on the protective action of anti-YDP-cell serum against a systemic fungal challenge. Control unadsorbed or preadsorbed serum was given i.p. (0.5 ml/mouse) to three mice per group 2 h before an i.v., sublethal challenge with C. albicans (5 × 10⁵ cells/mouse). Kidney invasion was assessed 48 h postchallenge by individual CFU counts of individual kidneys. Data are weighted means of CFU counts measured in mice from each experimental group. Statistical assessment was performed by Kruskal-Wallis ANOVA and Bonferroni-type nonparametric multiple comparison. The data are the results of one representative experiment out of three with similar results. Adj, adjuvant.

FIG. 5.

Effect of preadsorption with intact Y cells on the protective anti-Candida activity of anti-Y-cell or anti-YDP-cell serum. Mice (three per group) were injected i.p. with 0.5 ml of the indicated serum. Two hours later, the animals were challenged i.v. with 5 × 10⁵ cells of C. albicans. Kidney invasion was evaluated 48 h postchallenge by individual CFU counts of individual kidneys. Data represent weighted means of CFU counts for each experimental group. Statistical comparisons were made by Kruskal-Wallis ANOVA and Bonferrroni-type nonparametric multiple comparison. The data are the results of one representative experiment out of two with similar results. Adj, adjuvant.