The murine monoclonal antibody (MAb) 18B7 [immunoglobulin G1(κ)] is in preclinical development for treatment ofCryptococcus neoformans infections. In anticipation of its use in humans, we defined the serological and biological properties of MAb 18B7 in detail. Structural comparison to the related protective MAb 2H1 revealed conservation of the antigen binding site despite several amino acid differences. MAb 18B7 was shown by immunofluorescence and agglutination studies to bind to all four serotypes of C. neoformans, opsonize C. neoformans serotypes A and D, enhance human and mouse effector cell antifungal activity, and activate the complement pathway leading to deposition of complement component 3 (C3) on the cryptococcal capsule. Administration of MAb 18B7 to mice led to rapid clearance of serum cryptococcal antigen and deposition in the liver and spleen. Immunohistochemical studies revealed that MAb 18B7 bound to capsular glucuronoxylomannan in infected mouse tissues. No reactivity of MAb 18B7 with normal human, rat, or mouse tissues was detected. The results show that both the variable and constant regions of MAb 18B7 are biologically functional and support the use of this MAb in human therapeutic trials.

Monoclonal antibodies (mAbs) to the polysaccharide capsule of Cryptococcus neoformans can prolong survival in mice. However, the properties of antibodies that mediate protection are not fully understood. The IgM mAbs 12A1 and 13F1 originated from the same B cell and differ only by somatic mutations in their variable regions; yet mAb 12A1 protects against serotype D infection, while mAb 13F1 does not. Phage peptide display libraries were used to analyze the fine specificity of these two mAbs. The selection of distinct peptide motifs from identical libraries confirmed that mAbs 12A1 and 13F1 bound to two distinct epitopes. Immunofluorescence and immunoelectron microscopy studies revealed differences in antibody localization within the capsule of serotype D strain; mAb 12A1 bound to the outer rim of the capsule resulting in an annular pattern, whereas mAb 13F1 bound throughout the capsule and had a punctate appearance. The difference in the binding pattern of mAb 12A1 and 13F1 was not observed on serotype A organisms, where both mAbs bound to the capsule with an annular fluorescence pattern. The fluorescence pattern of binding correlated with protective efficacy; mAb 13F1 prolonged survival of mice infected with the J11 serotype A strain (annular fluorescence), but not serotype D strains (punctate pattern). Annular binding, but not punctate binding, was associated with increased opsonic efficacy for phagocytosis of C. neoformans by J774.16 macrophage-like cells. The correlation between capsular binding pattern, opsonic activity, and ability to prolong survival suggests that the efficacy of anticryptococcal antibodies is dependent upon where they bind in the polysaccharide capsule.

Voriconazole is a broad-spectrum triazole that offers extended activity against molds and yeasts that are not susceptible to earlier azole-type drugs. Recent studies indicate that melanization can severely reduce the susceptibility of certain antifungal drugs, but there is no information as to whether voriconazole is vulnerable to this effect. The activity of voriconazole on C. neoformans was assessed by MIC analysis and time-kill assays for melanized and nonmelanized cells. Cell morphology, capsule release, and phagocytosis of C. neoformans were studied in the presence or absence of subinhibitory concentrations of voriconazole. Voriconazole was fungicidal at concentrations of >8 g/ml in vitro against the strains of C. neoformans examined, and its efficacy was not diminished by melanization. Cells grown in subinhibitory concentrations of voriconazole had smaller cellular and capsular volumes than cells grown in the absence of drug. The induction of the capsule by serum was not affected by voriconazole. Cells grown in subinhibitory concentrations of voriconazole released their capsule and were phagocytosed at rates comparable with yeast grown without the antifungal. The high activity of voriconazole against both melanized and nonmelanized cells results suggest that voriconazole may be a particularly valuable drug for cryptococcosis.Cryptococcus neoformans is a relatively frequent cause of serious fungal infections in immunocompromised patients. The prevalence in the United States of cryptococcal meningoencephalitis in patients with AIDS receiving retroviral therapy is currently estimated to be Ͻ2% (23) but is Ͼ30% in areas of South East Asia and Sub-Saharan Africa (29). Patients with AIDS complicated by cryptococcosis often respond poorly to treatment and, in the setting of continued immunosuppression, require lifelong maintenance therapy since currently available antifungal agents seldom eradicate these fungal pathogens in the setting of severe immune suppression (16,41).Voriconazole, a synthetic derivative of fluconazole, is a broad-spectrum triazole antifungal that inhibits cytochrome P450-dependent 14␣-lanosterol demethylation, which is a critical step in fungal cell membrane ergosterol synthesis. Voriconazole demonstrates excellent in vitro activity against C. neoformans (17, 31) and achieves good levels in cerebrospinal fluid (35). Voriconazole is not currently licensed for use in cryptococcosis and no clinical trials have evaluated its efficacy for cryptococcal disease. There is limited published information regarding the clinical use of voriconazole for cryptococcosis (12, 30). In a study by Perfect et al., voriconazole therapy resulted in a 39% response rate in 18 patients with refractory cryptococcal meningoencephalitis (30).Given recent evidence that melanization can significantly reduce the efficacy of certain antifungal drugs against C. neoformans (36), we evaluated the activity of voriconazole against both melanized and nonmelanized yeast cells. Although we previously did not see a protective effect...

Cryptococcus neoformans has a polysaccharide capsule composed primarily of glucuronoxylomannan (GXM). This study focuses on the morphology of both encapsulated and non-encapsulated organisms in the presence and absence of monoclonal antibodies (mAbs) and serum proteins, and the effect of glucose on capsular polysaccharide release. Examination of the encapsulated C. neoformans strains 24067 and 34873 by scanning electron microscopy (SEM) revealed globular cells covered with a loose fibrillar network which was most prominent during the early stationary phase. In the presence of GXM-binding mAbs or serum the capsule border became distinct and bud scars were evident in the fibrillar network. In contrast, SEM of strain 52817, a non-encapsulated mutant of 34873 revealed ovoid cells devoid of a fibrillar network with bud scars and small surface protrusions. mAb 2H1 bound to cells from strains 24067 and 34873 but not 52817. No GXM was detected in supernatants of 52817 culture. For several strains, there was significantly more GXM in culture supernatants using high glucose media. In summary, our results indicate: i) SEM methods for studying capsular structure in C. neoformans; ii) no reactivity by GXM-binding mAb with a non-encapsulated strain; iii) the presence of distinctive bud scars in both encapsulated and non-encapsulated cells; and iv) dependence of GXM concentration on glucose concentration in culture media. The implications of these results are discussed.

Amphotericin B (AmB) and fluconazole (FLU) are the major antifungal drugs used in the treatment of cryptococcosis. Both drugs are believed to exert their antifungal effects through actions on cell membrane sterols. In this study we investigated whether AmB and FLU had other, more subtle effects on C. neoformans that could contribute to their therapeutic efficacy. C. neoformans cells were grown in media with subinhibitory concentrations of either AmB or FLU and analyzed for cellular charge, phagocytosis by macrophages with antibody and complement opsonins, appearance by scanning electron and light microscopies, and release of the capsular polysaccharide glucuronoxylomannan into the culture medium. Growth in the presence of either AmB or FLU resulted in major reductions in cellular charge, as measured by determination of the zeta potential. Phagocytosis studies demonstrated that exposure of C. neoformans to subinhibitory concentrations of AmB or FLU enhanced phagocytosis by macrophages. Scanning electron microscopy revealed that a large proportion of cells had an altered capsular appearance. Cells grown in medium with either AmB or FLU were smaller and released more glucuronoxylomannan into the culture medium than cells grown without antibiotics. The results suggest additional mechanisms of action for AmB and FLU that may be operative in body compartments where drug levels do not achieve the MICs. Furthermore, the results suggest mechanisms by which AmB and FLU can cooperate with humoral and cellular immune defense systems in controlling C. neoformansinfections.

Cryptococcus neoformans var. neoformansstrains have historically been divided into serotypes A and D on the basis of reactivity with rabbit sera. Previously, we noted that two murine immunoglobulin M monoclonal antibodies (MAbs) to the capsular glucuronoxylomannan produced different indirect immunofluorescence (IF) patterns, described as annular and punctate, when bound to C. neoformans cells from different strains. In this study, we examined the reactivity of these two MAbs, known as 12A1 and 13F1, with 20 C. neoformans var.neoformans strains, of which 13 were serotype A and 7 were serotype D. For all strains, MAb binding was studied by IF and agglutination assays. In addition, we blindly tested the IF patterns of 22 C. neoformans var. neoformans strains. For selected strains, MAb binding was studied by flow cytometry (FACScan) and phagocytosis assays. The epitopes recognized by MAbs 12A1 and 13F1 were found in all of the strains. MAb 12A1 binding produced an annular IF pattern with all of the strains, irrespective of the serotype classification. MAb 13F1 binding produced annular binding with all of the serotype A strains and punctate binding with 19 of 20 serotype D strains. In general, the punctate IF pattern was associated with lower fluorescence intensity, a requirement for higher antibody concentrations to produce yeast cell agglutination, and lower opsonic efficacy. Our results provide strong support for the existing classification of two serological types for strains assigned to varietyneoformans and indicate qualitative and quantitative antigenic differences among serotype A and D strains.

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Cryptococcus neoformans serotypes A and D are responsible for the overwhelming majority of infections in patients with AIDS. The genetic relationship between the serotypes is poorly understood, but there are significant differences in the epidemiology and clinical presentation of serotype A and D infections. We evaluated the genetic relationship between reference C. neoformansstrains belonging to serotypes A and D by analyzing theirURA5 sequences and restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element 1 (CNRE-1) probe. The results were compared to those previously obtained for isolates from Brazil and New York City by the same typing methods, and dendrograms were generated. Serotype A and D strains produced distinct RFLP patterns consistent with their separation into two major clusters in the dendrogram generated on the basis of RFLP data. Similarly, serotype A and D strains clustered independently on the basis of the nucleotide sequences of their URA5 genes. Pairwise comparisons revealed average numbers of nucleotide differences within serotypes A and D of 3.0 ± 1.7 and 7.2 ± 3.4, respectively (P < 0.0001), and between serotypes A and D of 41.9 ± 2.7. In summary, our results indicate phylogenetic differences between the two serotypes of C. neoformans var. neoformans and suggest that these serotypes could probably be considered different varieties of C. neoformans.