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Cryptococcus neoformans is an encapsulated yeast mainly responsible for meningitis, particularly in AIDS patients (2). Many clinical, histopathological, and experimental data prove that cellular immunity makes a critical contribution to host defense against C. neoformans (1). During the past decade, several laboratories have studied the role of humoral immunity, especially that elicited by capsular polysaccharide, and found some monoclonal antipolysaccharide antibodies to be protective against murine cryptococcosis (9, 11, 19). In contrast, very few studies have investigated the antibody response to protein antigens (4, 12-14), and no study has looked at the role of antibody response or its kinetics during the course of cryptococcosis.

We used a murine model of disseminated cryptococcosis to analyze the kinetics of the humoral response and to look for indicators predictive of the outcome (15). In this model, outbred mice exhibit individual patterns of susceptibility to C. neoformans infection, independently of the inoculum size. Some of the mice develop acute, disseminated and rapidly lethal infections, whereas others survive for several weeks with limited chronic infection, thus allowing comparison of the antibody responses as a function of outcome.

Outbred male OF1 mice (Iffa-Credo Laboratories, l'Arbresle, France; mean body weight, 18 to 20 g) were infected with C. neoformans isolate NIH52D (10⁴ to 10⁶ yeasts/animal in groups of 12 mice that could be identified individually, in three independent experiments). Survival was recorded daily until sacrifice by CO₂ inhalation (up to day 84 after inoculation). Blood was drawn weekly from the lateral tail vein (34 µl) and immediately used for blood culture (10 µl), as previously reported (15), and for immunoblotting (24 µl). For mice that were sacrificed, blood was drawn by cardiac puncture, buffy coats were cultured, and plasma samples were stored at 20°C until assayed. For each experiment, noninfected control mice housed under the same conditions were used.

The cytosol and membrane extracts were prepared as previously described (4) from strain NIH52D and after heat stress (13). The resulting cytosol (C52D) extract and membrane (M52D) extract were aliquoted and stored at 20°C. The same procedure was used to obtain cytosol and membrane fractions from an equal (vol/vol) mixture of 11 epidemiologically unrelated recent clinical isolates of C. neoformans.

The electrophoreses were run on precast polyacrylamide preparative gels in the Mini-Protean II system, and proteins were transferred onto nitrocellulose membranes, as recommended by the manufacturer (Bio-Rad Laboratories, Ivry-sur-Seine, France). Immunoblotting was performed with a multiscreen apparatus (Bio-Rad), using either plasma or whole blood from infected and control mice, peroxidase-conjugated antimouse immunoglobulin G (heavy plus light chains) antibody (Bio-Sys, Compiègne, France), and a chemiluminescent substrate. The band patterns were analyzed after digitalization using Taxotron software (P. D. Grimont, Institut Pasteur, Paris, France). For each mouse, only bands that were observed on two separate days or bands that were as intense as those obtained with the positive control serum were considered positive. In addition, for the analysis of bands associated with survival or death, the decision regarding the existence of a given band was based on its detection in samples from at least 10 mice. Statistical analyses were performed using Statview II software (Abacus Concepts, Inc., Berkeley, Calif.) and nonparametric tests. The level of significance was 0.05.

Course of C. neoformans infection in OF1 mice. The course of the infection was the same as that observed previously (15). All 25 mice that died of the infection had at least one positive blood culture during the study and died during the acute phase of the infection before day 32 after inoculation. In contrast, all survivors during the chronic phase of the infection had negative blood cultures at the time of the sacrifice.

Kinetics of the antibody response in C. neoformans-infected mice. In preliminary experiments, it was determined that similar data were obtained when plasma samples diluted 1:100 or supernatants of the blood drawn on the same day from the same mice immediately diluted 1:50 in blocking buffer and centrifuged were used. Therefore, subsequent experiments were conducted with whole blood from living animals diluted 1:50 or with plasma from sacrificed animals diluted 1:100.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Correlation between fungemia and the magnitude of the antibody response to C. neoformans protein antigens (i.e., the number of bands) detected in the blood sample drawn before death of infected OF1 mice (M52D [rs = 0.848] and C52D [rs = 0.605]).

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Evolution of the antibody response during the course of C. neoformans infection in three arbitrarily selected OF1 mice. The antibodies, detected sequentially by immunoblotting using the C. neoformans M52D extract, are represented as band patterns generated by Taxotron software. The day of the sampling is shown at the top of each lane.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Comparison of percentages of antibody-positive sera from NIH52D-infected OF1 mice recognizing selected C. neoformans protein antigens according to the outcome of the infection. Sera obtained from survivors () and future nonsurvivors () during the acute phase of the infection were compared by immunoblotting on C52D and M52D protein extracts from C. neoformans. *, P < 0.05 versus the other group (Fisher's exact test).