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Cryptococcus neoformans is an encapsulated yeastlike fungus that has emerged as a leading pathogen in immunocompromised individuals, including 6 to 8% of patients with AIDS (reviewed in reference 3). There is strong evidence that C. neoformans synthesizes cell wall-associated melanin during infections (6, 7, 9). Antibodies to melanin are generated during cryptococcal infection (6). Melanins are thought to contribute to virulence by protecting C. neoformans against host effector mechanisms, such as macrophage-mediated phagocytosis, oxidants, and microbicidal peptides (reviewed in reference 1).

We have recently generated murine monoclonal antibodies (MAbs) to melanin and used them to study melanization of C. neoformans in vivo (7, 9). In this study we investigated whether the MAbs to melanin have protective efficacy against C. neoformans infections in mice. Our results indicate that the MAbs to melanin can prolong the survival of and reduce the fungal burden in lethally infected mice.

The data in this paper are from a thesis to be submitted by Ángel L. Rosas in partial fulfillment of the requirements for the degree of doctor of philosophy in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y.

C. neoformans strain 24067 (serotype D) was obtained from the American Type Culture Collection (Rockville, Md.), and strain H99 (serotype A) was obtained from John Perfect (Durham, N.C.). C. neoformans cultures were grown in Sabouraud dextrose broth (Difco, Detroit, Mich.) for 2 days at 30°C in a rotary shaker at 150 rpm. Ten 8- to 10-week-old female C57BL/6 mice (National Cancer Institute, Rockville, Md.) per group were passively immunized with ascites containing 1 mg of MAb 6D2 or 11B11 and infected with strain 24067. Ascites from NSO, the nonproducing mouse myeloma fusion partner of the hybridomas, was used as a negative control. At 30 min after immunization, the mice were infected intravenously with 2 × 10⁷ cells. Comparisons of survival of infected mice immunized with the MAbs to that of melanin- or NSO ascites-treated mice were analyzed using log rank analysis (SPSS Inc., Chicago, Ill.). P values of less than 0.05 were considered significant. The mice were housed in the animal facility of Albert Einstein College of Medicine, and all experimental procedures adhered to protocols approved by the Animal Care and Use Committee. Mice given MAb to melanin lived significantly longer than control mice (P < 0.05; Fig. 1A). The experiment was performed three times with similar results. A fourth experiment was done using mice infected intravenously with 2 × 10⁵ cells of strain H99 and employing the immunoglobulin M (IgM) MAb 5C11 with specificity for mycobacterial lipoarabinomannan (3) as a negative control (Fig. 1B). Again, mice given MAb to melanin lived significantly longer than control mice given the irrelevant IgM.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   (A) Passive immunization with MAb 6D2 or 11B11 to melanin prolongs the survival of mice infected with C. neoformans strain 24067 in comparison to mice given NSO ascites (10 mice per group). (B) Passive immunization with MAb 6D2 or 11B11 prolongs the survival of mice infected with C. neoformans strain H99 in comparison to mice given MAb 5B11 with specificity for mycobacterial lipoarabinomannan (10 mice per group).

C. neoformans infections were also performed to determine whether immunization with the MAbs to melanin reduced the fungal burden in infected mice. Ten female C57BL/6 mice per group were immunized and infected as described above. At day 7 after infection, the surviving mice were killed, and the lungs and brains were collected and homogenized by mechanical grinding in 5 ml of phosphate-buffered saline (PBS, pH 7.4) to recover the fungal cells. Cells were then plated on Sabouraud dextrose agar (Difco) for 3 days at 30°C. P values were calculated with Student's t test using Primer of Statistics: The Program, version 3.0 (McGraw-Hill Inc., New York, N.Y.) for comparison of fungal burden in mice immunized with MAb 6D2 or 11B11 to that in mice treated with NSO ascites. P values of less than 0.05 were considered significant. Mice given MAbs to melanin had significantly lower fungal burdens than control mice (P < 0.001; Table 1).

To investigate whether the MAbs to melanin had an effect on melanized C. neoformans cells, C. neoformans 24067 cells were grown in minimal medium (15 mM dextrose, 10 mM MgSO₄, 29.4 mM KH₂PO₄, 13 mM glycine, and 3 µM vitamin B₁ [pH 5.5]) with or without 1 mM L-3,4-dihydroxyphenylalanine (L-dopa) (Sigma Chemical Co., St. Louis, Mo.) for 7 days at 30°C. L-Dopa served as the substrate for melanization. Cells were then collected, washed, and suspended in minimal medium with L-dopa. A suspension containing 5,000 melanized or nonmelanized fungal cells (100 µl) was plated in 96-well tissue culture plates in duplicate (Becton Dickinson, Cockeysville, Md.) in the presence of ascites containing MAb 6D2 or 11B11 (100 to 0.78 µg/ml). Ascites with MAb 5C11 (100 µg/ml) to mycobacterial lipoarabinomannan served as a negative control. After 2 days of incubation at 30°C, the cells were collected, diluted 1:10 in PBS, and counted using a hemacytometer. The counting was facilitated by the fact that MAbs to melanin do not cause agglutination of C. neoformans (9). Incubation of the MAbs to melanin with in vitro-melanized C. neoformans 24067 significantly reduced the growth rate of the fungal cells in comparison to growth with MAb 5C11 (Fig. 2). No differences in growth rate were observed with nonmelanized C. neoformans 24067 cells incubated with the MAbs to melanin or MAb 5C11 (data not shown). This result is of particular interest because previous studies have shown that C. neoformans can grow on purified capsule-binding MAbs (2). In a similar experiment, in vitro-melanized C. neoformans 24067 was incubated with the melanin-binding peptide 4B4 (YERKFWHGRH) or the control peptide P601G (DGASYSWMYGA) (6). A significant reduction in the growth rate of melanized C. neoformans was observed when the cells were incubated with the melanin-binding peptide 4B4 in comparison to cells grown with the control peptide (Fig. 2). The growth experiments with the MAbs and the melanin-binding peptide were performed three times with similar results. Hence, it appears that binding of melanin-binding protein to melanized cells, whether in the form of antibody or peptide, can inhibit melanized cell growth.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Incubation of MAbs to melanin or melanin-binding peptide reduces the growth rate of in vitro-melanized C. neoformans cells in comparison to growth with MAb 5C11 or a control peptide. Data points represent the mean of measurements for two wells for each concentration from a single experiment.

In summary, the results from these experiments demonstrate that the MAbs to melanin have protective efficacy against C. neoformans infections in mice. The observation that MAbs to melanin mediate protection against C. neoformans infection is consistent with and supportive of the premise that the fungus melanizes in tissue (1). C. neoformans is surrounded by a polysaccharide capsule (reviewed in reference 4), and binding of the MAbs to the melanin occurs in the cell wall area (7, 9). The MAbs to melanin do not induce phagocytosis of in vitro-melanized C. neoformans cells by the murine macrophage-like cell line J774.16 (unpublished observations), and therefore it is unlikely that the mechanism of protection involves induction of phagocytosis of the fungus by host effector cells. The MAbs to melanin are not agglutinating, and this excludes yeast cell clumping as a mechanism for the effects observed in in vitro and in vivo. The MAbs to melanin and the melanin-binding peptides can diffuse through the capsule to bind at the cell surface (6, 9), and they both were able to reduce the growth rate of melanized C. neoformans cells in vitro. This suggests a mechanism of MAb-mediated protection by which binding of the MAbs to melanized C. neoformans in vivo could alter the properties of the melanin in the cell wall and thereby interfere with cell growth and replication in the presence of this polymer. This mechanism may be similar to that recently described for antibodies to glucosylceramide, which also inhibited C. neoformans growth after binding to the cell wall (8). Furthermore, our results suggest that antibody responses to melanin elicited by C. neoformans infection may be useful in host defense.