Thomas R. Kozel

The polysaccharide capsule ofCryptococcus neoformansis a powerful activator of the complement system. The goal of the present study was to assess serum and cellular variables that influence the sites for C3 binding within the capsular matrix. Confocal microscopy using fluorophore-labeled polyclonal anti-C3 and anticapsular monoclonal antibodies and rosetting of fluorescent microspheres coated with anti-C3 were used to identify sites of C3 binding relative to the capsular edge. The results showed that the source of serum was a major variable influencing localization of C3. C3 bound at or very near the capsular edge in the case of human serum. C3 deposition was further from the capsule edge with guinea pig and rat sera; in the case of mouse serum, there was no binding of C3 in the outer region of the capsule. Addition of human C3 to mouse serum led to deposition of the C3 at the capsular edge, indicating that distinct properties of mouse and human C3 account for the differential localization of C3. Finally, the density of the capsular matrix was an important variable in determining sites for C3 deposition. Yeast cells with a high concentration of polysaccharide near the capsule edge supported deposition of mouse C3 at or near the capsular edge, whereas cells with a low matrix density showed deposition well beneath the edge. Taken together, these results indicate that the spatial deposition of C3 within the capsular matrix is a complex process that is influenced by the serum source and the density of the capsular matrix.

Mannan-binding lectin (MBL) is a component of the innate immune system. The goal of the present study was to evaluate binding of MBL to Candida albic

Glucuronoxylomannan (GXM), the major component of the capsular polysaccharide ofCryptococcus neoformans, is essential to virulence of the yeast. Previous studies found that the interaction between GXM and phagocytic cells has biological consequences that may contribute to the pathogenesis of cryptococcosis. We found that GXM binds to and is taken up by murine peritoneal macrophages. Uptake is dose and time dependent. Examination of the sites of GXM accumulation by immunofluorescence microscopy showed that the pattern was discontinuous and punctate both on the surfaces of macrophages and at intracellular depots. Although resident macrophages showed appreciable accumulation of GXM, uptake was greatest with thioglycolate-elicited macrophages. A modest stimulation of GXM binding followed treatment of resident macrophages with phorbol 12-myristate 13-acetate, but treatment with lipopolysaccharide or gamma interferon alone or in combination had no effect. Accumulation of GXM was critically dependent on cytoskeleton function; a near complete blockade of accumulation followed treatment with inhibitors of actin. GXM accumulation by elicited macrophages was blocked by treatment with inhibitors of tyrosine kinase, protein kinase C, and phospholipase C, but not by inhibitors of phosphatidylinositol 3-kinase, suggesting a critical role for one or more signaling pathways in the macrophage response to GXM. Taken together, the results demonstrate that it is possible to experimentally enhance or suppress binding of GXM to macrophages, raising the possibility for regulation of the interaction between this essential virulence factor and binding sites on cells that are central to host resistance.

Bacillus anthracisis surrounded by an antiphagocytic polypeptide capsule composed of poly γ-d-glutamic acid (γDPGA). γDPGA has been identified recently as a potential target for vaccine development. Studies of the role of γDPGA in disease have been hampered by the poor Ab response to this antigen and the lack of immunochemical reagents. As a consequence, neither the extent of γDPGA production during anthrax nor the protective activity of γDPGA Abs in inhalation anthrax are known. Here we report production of IgG Abs to γDPGA in mice following an immunization regimen using γDPGA in combination with agonist mAbs to CD40. mAbs were produced that are specific for γDPGA. Passive immunization with γDPGA mAbs protected >90% of mice in a pulmonary model of anthrax that was lethal in control mice (P< 0.0001). Use of γDPGA mAb in an antigen detection immunoassay found that the appearance of γDPGA in serum coincided with the emergence of bacteremia. These studies identify CD40 stimulation as a means for production of Ab and generation of mAbs against a weakly immunogenic antigen and demonstrate that the capsule is an effective target for immunoprotection and for antigen detection in the diagnosis of anthrax.

Summary: Many microbes are surrounded by phagocytosis‐inhibiting capsules. We took advantage of the large size of the polysaccharide capsule of the pathogenic yeast Cryptococcus neoformans to examine capsular architecture and the relationship between molecular architecture and the interaction of the capsule with potentially opsonic serum proteins. Our experimental design used complementary approaches in which (i) assessment of permeability to macromolecules of different Stokes radii; (ii) determination of the binding of Fab fragments of anticapsular antibodies as a measure of matrix density; (iii) capsular deconstruction by treatment with dimethyl sulphoxide; and (iv) evaluation of capsule plasticity, were used to probe the molecular structure of the capsule. The results showed that the capsule is a matrix with a variable porosity that increases with distance from the cell wall. A high density of the matrix at the capsule interior prevents penetration of large macromolecules to sites near the cell wall. In contrast, the capsular edge that is the interface with phagocytes presents capsular polysaccharide in a very low density that exhibits considerable plasticity and permeability to macromolecules. Notably, the capsule of yeast cells harvested from infected tissue showed a greater matrix density than yeast cells grown in vitro under capsule induction conditions.

Sera from normal adult humans may contain high levels of antibody reactive withCandida albicansmannan. This study examined selected biological activities of such antibodies, focusing on sera that were collected from 34 donors and analyzed individually. The results showed that antimannan titers were normally distributed. Reactivity as determined by enzyme-linked immunosorbent assay with serotype A mannan generally paralleled reactivity with serotype B. Analysis of the kinetics for activation of the complement system and deposition of complement component 3 (C3) onto serotype A and serotype B cells showed a decrease in the lag time that occurred before the onset of rapid accumulation of C3 that correlated with increasing antimannan titers. In contrast, there was a decrease in the overall rate of accumulation of C3 on serotype A cells that was strongly correlated with increasing antibody titers; serotype B cells showed no such decrease. An evaluation of the contribution of mannan antibody to opsonophagocytic killing showed that mannan antibody in individual sera and antimannan immunoglobulin G (IgG) affinity purified from human plasma contributed to killing by neutrophils in a dose-dependent fashion in the absence of a functional complement system. However, affinity-purified antibody in very high concentrations was inhibitory to both complement-dependent and complement-independent opsonophagocytosis, and this finding suggests a prozone-like effect. In contrast, if the complement system was functional, antimannan IgG was not needed for opsonophagocytic killing. These results suggest that naturally occurring mannan antibodies and the complement system are functionally redundant for opsonophagocytic killing by neutrophils.

Cryptococcus neoformansis surrounded by an antiphagocytic capsule whose primary constituent is glucuronoxylomannan (GXM). An epitope shared by GXM serotypes A, B, C, and D is immunodominant when mice are immunized with serotype A GXM. In contrast, an epitope shared only by serotypes A and D is immunodominant when mice are immunized with serotype D. Hybridomas secreting antibodies reactive with subdominant epitopes were identified through a positive-negative screening procedure in which antibody-secreting colonies were characterized by reactivity with both the immunizing polysaccharide and GXMs from each of the four major serotypes. In this manner, a monoclonal antibody (MAb) that was reactive with an epitope shared only by serotypes A and B was identified and designated F10F5. Such an epitope has not been described previously. Immunization of mice with de-O-acetylated serotype A GXM generated a hybridoma that secreted an antibody, designated F12D2, that was reactive with all four serotypes. Unlike previously described monoclonal and polyclonal panspecific antibodies, the reactivity of MAb F12D2 was not altered by de-O-acetylation of GXM. These results indicate that there are at least two panspecific GXM epitopes; one epitope is dependent on O acetylation for antibody reactivity, and the other is independent of O acetylation. This study identifies strategies for production of MAbs that are reactive with subdominant or cryptic GXM epitopes and provides new information regarding the antigenic makeup and the humoral immune response to GXM, an essential virulence factor that is a target for active and passive immunization.

Cryptococcus neoformansis surrounded by an antiphagocytic polysaccharide capsule whose primary constituent is glucuronoxylomannan (GXM). Three prominent structural features of GXM are single xylosyl and glucuronosyl side chains and O acetylation of the mannose backbone. Isogenic pairs ofO-acetyl-positive andO-acetyl-negative strains (cas1Δ) as well as xylose-positive and xylose-negative strains (uxs1Δ) of serotype D have been reported. Thecas1Δstrains were hypervirulent, and theuxs1Δstrains were avirulent. The goal of this study was to examine the effects of thecas1Δanduxs1Δmutations on the following: (i) binding of anti-GXM monoclonal antibodies (MAbs) in capsular quellung reactions, (ii) activation of the complement system and binding of C3, (iii) phagocytosis by neutrophils, and (iv) clearance of GXM in vivo. The results showed that loss of O acetylation produced dramatic changes in the reactivities of five of seven anti-GXM MAbs. In contrast, loss of xylosylation produced a substantive alteration in the binding behavior of only one MAb.O-acetyl-negative strains showed no alteration in activation and binding of C3 from normal serum. Xylose-negative strains exhibited accelerated kinetics for C3 deposition. Loss of O acetylation or xylosylation had no effect on phagocytosis of serum-opsonized yeast cells by human neutrophils. Finally, loss of O acetylation or xylosylation altered the kinetics for clearance of GXM from serum and accumulation of GXM in the liver and spleen. These results show that O acetylation and/or xylosylation are important for binding of anti-GXM MAbs, for complement activation, and for tissue accumulation of GXM but do not impact phagocytosis by neutrophils.

Incubation of encapsulated cryptococci with monoclonal antibodies (MAbs) specific for glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans , produces two distinct capsular quellung-type reactions termed rim and puffy. The type of capsular reaction that occurs is determined by the epitope specificity of the MAb and the serotype of the yeast cell. Several biological activities, including opsonic activity, complement activation, and protective efficacy, are associated with the type of capsular reaction produced by a MAb. The goal of this study was to examine the reactivities of two families of anti-GXM MAbs with serotype A and D capsular polysaccharides in several immunochemical assays, including agglutination, immunofluorescence, quantitative precipitation, and enzyme-linked immunosorbent assay, in an effort to identify serological assays that are predictive of the capsular quellung reaction. The results showed that the type of capsular reaction (rim versus puffy) is a qualitative assessment of antibody-capsule interaction that cannot be predicted on the basis of a serological assay. The results further showed that antibody reactivity demonstrated in one serological assay is not necessarily predictive of results in another assay, particularly in cases where one assay examines antibody-capsule interactions, e.g., agglutination, and another assay examines interaction of antibody with soluble GXM. Taken together, the results suggest caution in interpretation of immunochemical assays for anti-GXM antibodies and recommend the use of multiple assays formats when studying anticryptococcal antibodies.

Monoclonal antibodies (MAbs) reactive with glucuronoxylomannan (GXM), the major capsular polysaccharide of the yeast Cryptococcus neoformans , produce distinct capsular reactions when viewed by differential interference contrast microscopy. These reactions depend on the epitope specificity of the antibody. Opsonic activities of immunoglobulin G1 (IgG1) MAbs that produce patterns termed rim and puffy were examined. Rim-pattern MAbs are reactive with an epitope shared by GXM serotypes A, B, C, and D. Puffy-pattern MAbs are reactive only with serotypes A and D. In phagocytosis assays, using serotype A cells and resident murine peritoneal macrophages, rim-pattern MAbs were markedly more opsonic than puffy-pattern MAbs. F(ab′) ₂ fragments of rim-pattern MAbs were synergistic with heat-labile factors in normal human serum for opsonization of the yeast. F(ab′) ₂ fragments of puffy-pattern MAbs were also synergistic with normal serum in opsonization but at a much lower level than fragments of rim-pattern MAbs. Normal serum alone was not opsonic. F(ab′) ₂ fragments of rim-pattern MAbs, but not puffy-pattern MAbs, stimulated phagocytosis of encapsulated cryptococci in the absence of serum. This serum-independent opsonic action of F(ab′) ₂ fragments was abrogated by pretreatment of macrophages with purified GXM, suggesting the involvement of a phagocyte GXM receptor. The results indicate that (i) there are multiple mechanisms by which anticapsular IgG MAbs facilitate phagocytosis of encapsulated cryptococci, (ii) some anti-GXM antibodies are opsonic in an Fc-independent manner, and (iii) opsonic activity correlates with the capsular reaction and occurs in an epitope-specific manner.