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. 1998 Apr;66(4):1547–1553. doi: 10.1128/iai.66.4.1547-1553.1998

Bivalency Is Required for Anticapsular Monoclonal Antibodies To Optimally Suppress Activation of the Alternative Complement Pathway by the Cryptococcus neoformans Capsule

Thomas R Kozel 1,*, Randall S MacGill 1, Kevin K Wall 1
PMCID: PMC108087  PMID: 9529080

Abstract

Encapsulated cells of Cryptococcus neoformans are potent activators of the alternative complement pathway. Previous studies found that monoclonal antibodies (MAbs) specific for the major capsular polysaccharide, termed glucuronoxylomannan (GXM), can markedly suppress the ability of the capsule to accumulate C3 from normal human serum via the alternative pathway. The present study examined the abilities of F(ab)2 and Fab fragments of three MAbs (MAbs 439, 3C2, and 471) to mediate the suppressive effect. The results showed that F(ab)2 fragments of all three MAbs suppressed activation and binding of C3 via the alternative pathway in a manner similar to that of intact antibodies. In contrast, Fab fragments of MAb 439 and MAb 3C2 showed no suppressive activity, and Fab fragments of MAb 471 were markedly reduced in suppressive activity. Indeed, there was an earlier accumulation of C3 on encapsulated cryptococci in the presence of the Fab fragments. Study of subclass switch families of MAb 439 and MAb 471 found that MAbs of an immunoglobulin G (IgG) subclass with increased flexibility in the hinge region (IgG2b) had less suppressive activity than MAbs of IgG subclasses with less flexibility (IgG1 or IgG2a). Taken together, these results indicate that cross-linking of the capsular matrix is an essential component in suppression of the alternative complement pathway by anti-GXM MAbs.

The capsule of the pathogenic yeast Cryptococcus neoformans is a powerful activator of the alternative complement pathway (8, 16). Incubation of encapsulated cryptococci in normal human serum (NHS) leads to deposition of 107 to 108 molecules of C3 onto the typical yeast cell (18, 38); the capsule itself is the site for C3 binding (19, 21). Such activation and binding of C3 is due solely to the action of the alternative pathway (20, 21, 37). Binding of C3 via the alternative pathway in NHS is characterized by a delay of approximately 4 to 6 min before bound C3 is readily detectable (20).

The major component of the cryptococcal capsule is the high-molecular-weight polysaccharide glucuronoxylomannan (GXM). Several anti-GXM monoclonal antibodies (MAbs) have been shown to provide a measure of protection in a murine model of cryptococcosis (9, 24, 30). In the accompanying report, we have examined the ability of anti-GXM MAbs to initiate the classical pathway, leading to accelerated deposition of C3 onto the yeast (17). These studies showed that most anti-GXM MAbs promote early deposition of C3 fragments into the capsule. However, depending on the epitope specificity of the MAb, some anti-GXM MAbs markedly reduced the apparent rate of amplification of bound C3, with the net result that fewer C3 molecules bound to the cell over a 20- to 30-min incubation period than would have bound in the absence of the antibody. When classical pathway initiation was blocked by the use of EGTA to chelate Ca2+ (12, 28), antibodies with the suppressive epitope specificity almost completely blocked the normal alternative pathway activation and binding of C3 that would have occurred in the absence of the MAbs.

The ability of an antibody to block antibody-independent activation of the alternative pathway is without an obvious parallel in the literature. There are several potential mechanisms for antibody-mediated suppression. First, the antibody could bind to and occlude specific sites on the capsule that might be preferred acceptors for metastable C3b. Second, the capsule could contain specific domains that regulate the ability of the capsule to activate the alternative pathway. Antibodies specific for such regulatory domains could influence the ability of the cell to activate the alternative pathway. Finally, multivalent antibody could cross-link the capsule in a manner that prevents effective amplification. For example, binding of a multivalent antibody could reduce the ability of metastable C3b to diffuse from sites of C3 convertase activity. We have reasoned that the first two mechanisms for antibody-induced suppression of C3 binding would be mediated by intact antibody, F(ab)2 fragments of the antibody, and Fab fragments. In contrast, inhibition that is dependent on cross-linking of the capsular matrix would be mediated by intact antibodies and F(ab)2 fragments but not by Fab fragments.

The objective of our study was to examine three anticapsular MAbs that suppress alternative pathway-dependent C3 binding. The suppressive activities of intact antibodies, F(ab)2 fragments, and Fab fragments were compared. The results showed that intact antibodies and F(ab)2 fragments of the antibodies suppressed accumulation of C3 fragments on the capsule. In contrast, Fab fragments of the suppressive antibodies showed markedly reduced or no ability to block alternative pathway activation by the capsule; indeed, Fab fragments derived from suppressive antibodies accelerated activation and binding of C3 via the alternative pathway.

MATERIALS AND METHODS

Yeast cells.

Unless otherwise indicated, formalin-killed cells of C. neoformans 388, an encapsulated strain of serotype A, were used throughout the study. The conditions for growth, formalin inactivation and storage of the cells have been described elsewhere (38).

Serum and serum proteins.

Peripheral blood samples were collected from at least 10 adult volunteer donors. The sera were isolated, pooled, and stored at −85°C until use. C3 was isolated from frozen human plasma as described elsewhere (19, 36). C3 was labeled with 125I by the Iodogen (Pierce, Rockford, Ill.) procedure (13) according to the manufacturer’s directions.

Antibody production and fragmentation.

Three anti-GXM MAbs were used in this study, i.e., MAbs 439, 3C2, and 471. The characteristics of these antibodies have been previously described (1, 2, 10, 35). The MAbs were isolated from mouse ascites fluid and purified as described elsewhere (31). All three antibodies have similar properties, including (i) reactivity with GXM serotypes A, B, C and D, (ii) belonging to the same molecular group as described by Casadevall et al. (2), (iii) immunoglobulin G1 (IgG1) isotype, (iv) modest ability to facilitate early activation and binding of C3 to the cryptococcal capsule via the classical pathway (17), and (v) strong ability to suppress activation and binding of C3 to the capsule via the alternative pathway (17). Subclass switch families (IgG1→IgG2b→IgG2a) of the cell lines secreting MAbs 439 and 471 were produced as described elsewhere (33), and the antibodies were isolated from ascites fluids (31).

F(ab)2 and Fab fragments were prepared by using the ImmunoPure IgG1 Fab and F(ab′)2 preparation kit (Pierce) according to the manufacturer’s directions. Briefly, the intact antibody was digested with ficin under conditions optimized for production of Fab or F(ab)2 fragments from murine IgG1, and the fragments were separated from whole IgG and Fc fragments by affinity chromatography with immobilized protein A (Pierce). The MAb 471 Fab fragments were further purified by molecular sieve chromatography on Superdex 200 (Pharmacia Biotech Inc., Piscataway, N.J.) and affinity chromatography on protein A-Sepharose CL-4B (Pharmacia Biotech). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the F(ab)2 and Fab fragments followed by staining with Coomassie Brilliant Blue showed protein bands of the expected molecular weights and an absence of contaminating proteins.

Analysis of MAb binding to yeast cells.

The numbers of whole antibodies, F(ab)2 fragments, and Fab fragments binding to cells of strain 388 were determined by use of 125I-labeled proteins as described elsewhere (17). The numbers of MAb molecules bound per cell were calculated according to the method of Scatchard (32).

Kinetic analysis of C3 binding.

The kinetics for activation and binding of C3 fragments to cryptococcal cells were assessed in 1.5-ml reaction mixtures consisting of (i) 40% NHS, (ii) GVB-Mg-EGTA (5 mM sodium Veronal-buffered saline [pH 7.3] containing 0.1% gelatin, 10 mM EGTA, and 10 mM MgCl2), (iii) 125I-labeled C3 sufficient to provide a specific activity of 50,000 cpm/μg of C3 for the mixture of labeled and unlabeled C3 in the serum, (iv) anti-GXM MAbs or their fragments at 50 or 200 μg/ml, and (v) 6.0 × 105 cryptococcal cells. The tubes containing all reagents except the cryptococcal cells were prewarmed for 5 min at 37°C, and the reaction was initiated by addition of the yeast cells. Samples were withdrawn in duplicate at various time intervals, and the amount of specifically bound C3 was determined as described elsewhere (17). Binding data are reported as the number of C3 molecules per yeast cell versus incubation time. The time to 50% of maximum binding was determined by use of a four-parameter logistic equation which was calculated with the assistance of SigmaPlot 3.0 for Windows (Jandel Scientific, San Rafael, Calif.).

Immunofluorescence analysis of C3 binding patterns.

Reaction mixtures were prepared as described above for the C3 kinetic binding assay, with the exception that radiolabeled C3 was not included. Samples were taken at various time intervals and stained for the presence of bound C3 by use of fluorescein isothiocyanate (FITC)-labeled antiserum to human C3 (Kent Laboratories Inc., Redmond, Wash.) by previously described procedures (17, 20).

The pattern of C3 deposition was determined by epifluorescence microscopy with a Nikon Eclipse E800 microscope with an oil immersion objective of ×100. Images were collected at 0.4-μm intervals with a Photonic Science integrating charge-coupled device camera (Millham, United Kingdom) and Image Pro Plus version 2.0 image analysis software (Media Cybernetics, Silver Spring, Md.). Unless otherwise indicated, images shown within a series of experiments were collected with an identical number of integrations and identical gain settings. Deconvolution of the images was done with MicroTome IP for Windows 95 version 5.1 (VayTek, Inc., Fairfield, Iowa) operating within an Image Pro Plus version 3.0 environment. Final assembly of the images was done with CorelDRAW 7. The images are shown as a projection of a 1.2-μm section through the center of the cell.

RESULTS

Effect of intact MAbs, F(ab)2 fragments, and Fab fragments on the kinetics of alternative-pathway-dependent C3 deposition.

MAbs 439, 3C2, and 471 all suppress the ability of encapsulated cryptococci to activate and bind C3 via the alternative pathway (17). This suppression occurs regardless of whether the cells are live or formalin killed (data not shown). An initial experiment evaluated the abilities of F(ab)2 and Fab fragments of these antibodies to influence the kinetics of C3 accumulation on the cells. Cryptococcal cells were incubated in the presence of 10 mM Mg-EGTA with NHS and 50 or 200 μg of intact antibody, F(ab)2 fragments, or Fab fragments per ml. Samples were taken after various incubation times, and the amounts of bound C3 were determined.

The results (Fig. 1) showed that incubation of encapsulated cryptococci with NHS alone showed little or no binding for the first 4 min. This was followed by a burst of binding in which the amount of bound C3 rapidly accumulated until approximately 3 × 107 molecules were bound per yeast cell when the elapsed incubation time reached approximately 10 min. Addition of either 50 or 200 μg of intact MAb per ml produced a marked suppression of both the apparent rate of accumulation of bound C3 as well as the amount of C3 that bound over a 25-min incubation period. All three MAbs had a suppressive effect. These results confirm our report that MAbs 439, 3C2, and 471 suppress alternative-pathway-mediated activation and binding of C3 (17). Similar results occurred when F(ab)2 fragments of the MAbs were incorporated into the incubation mixture. These results indicate that the Fc portion of the antibody is not necessary to mediate suppression of C3 binding.

FIG. 1.

FIG. 1

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Effect of intact antibodies, F(ab)2 fragments, and Fab fragments of MAbs 439, 3C3, and 471 on the kinetics of activation and binding of C3 fragments to encapsulated cryptococci via the alternative pathway. Cryptococci were incubated with 40% NHS containing 125I-C3, 10 mM Mg-EGTA, and 50 or 200 μg of intact antibody, F(ab)2 fragments, or Fab fragments per ml. Samples were taken at various times, and the amounts of bound C3 were determined.

No suppression was observed in the presence of Fab fragments of either MAb 439 or 3C2. Instead, there was a reduction of the initial lag from 4 to approximately 2 min. Once past the lag, there was rapid accumulation of C3 on the yeast cells at a rate that resembled the rate observed for NHS alone. The time to 50% of maximum binding was 5.8 min when cryptococci were incubated with NHS alone and was reduced to 3.9 min when they were incubated with NHS and 50 or 200 μg of Fab fragments of MAbs 439 or 3C2 per ml.

Fab fragments of MAb 471 produced an effect that differed from the effect produced by Fab fragments of MAbs 439 and 3C2. At the highest concentration (200 μg/ml), there was a suppression of the rate of accumulation of C3 on the yeast cells. However, the level of suppression was markedly less than the level of suppression produced by comparable amounts of either intact or F(ab)2 fragments of MAb 471. At 50 μg/ml, Fab fragments of MAb 471 produced a slight reduction in the lag time before measurable amounts of C3 bound to the cells, but a reduction in the overall rate and total amount of C3 accumulation was also observed. We considered the possibility that the Fab fragments of MAb 471 contained undigested or only partially digested antibodies. If this were the case, the undigested antibodies could produce an overall effect that was suppressive. As a consequence, the Fab fragments of MAb 471 were passed over a Superdex 200 molecular sieve column and protein A-Sepharose in an effort to eliminate contaminants. Neither passage over the molecular sieve alone nor use of the molecular sieve and protein A had any effect on the ability of Fab fragments of MAb 471 to mediate modest suppression of C3 binding.

One explanation for the failure of Fab fragments to block alternative-pathway-mediated C3 binding is the possibility that the Fab fragments bind poorly to the encapsulated cells. As a consequence, we radiolabeled the intact antibodies and their F(ab)2 and Fab fragments and determined the level of binding of each protein according to the method of Scatchard (32). This analysis allowed a calculation of the maximum number of protein molecules bound under saturating conditions as well as the number bound under the conditions used in the C3 binding experiments (50 and 200 μg/4 × 105 yeast cells). The results (Table 1) showed that each of the antibodies, as well as their cleavage fragments, readily bound to the yeast cells. Indeed, the Fab fragments exhibited a two- to eightfold increase in numbers of bound molecules compared to the intact antibodies.

TABLE 1.

Binding of MAbs and MAb fragments to encapsulated cryptococci as determined by Scatchard analysis

IgG fragment and MAb No. of molecules/cell under saturating conditions No. of molecules/cell at 50 μg/4 × 105 yeast cells No. of molecules/cell at 200 μg/4 × 105 yeast cells
Intact
 439 6.4 × 107 5.8 × 107 6.3 × 107
 3C2 4.4 × 107 4.1 × 107 4.4 × 107
 471 3.5 × 107 3.4 × 107 3.5 × 107
F(ab)2
 439 8.2 × 107 7.7 × 107 8.1 × 107
 3C2 8.8 × 107 6.5 × 107 8.2 × 107
 471 1.1 × 108 7.8 × 107 1.0 × 108
Fab
 439 5.6 × 108 4.9 × 108 5.4 × 108
 3C2 3.3 × 108 2.7 × 108 3.1 × 108
 471 2.4 × 108 1.7 × 108 2.2 × 108
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The above results showed strikingly different effects of intact MAbs and their Fab fragments. Intact antibodies suppressed alternative-pathway-mediated activation and binding of C3, whereas Fab fragments of MAbs 439 and 3C2 accelerated C3 binding at the early time points. This raised a question as to which is the dominant effect, suppression or facilitation. An experiment was done in which cryptococci were incubated with NHS in the presence of Mg-EGTA, to which was added either (i) no antibody, (ii) intact MAb 3C2 (50 μg/ml), (iii) Fab fragments of MAb 3C2 (50 μg/ml), or (iv) both intact MAb 3C2 and its Fab fragments (50 μg each per ml). The results (Fig. 2) showed the expected findings that intact antibody suppressed and the Fab fragment facilitated C3 binding. The combination of intact antibody and Fab fragments produced an overall effect of suppression, although this result was slightly diminished from the suppression observed with intact antibody alone. A replicate experiment produced an identical result (not shown).

FIG. 2.

FIG. 2

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Effect of mixing intact antibodies and Fab fragments on the kinetics of activation and binding of C3 fragments to encapsulated cryptococci via the alternative pathway. Cryptococci were incubated with 40% NHS containing 125I-labeled C3, 10 mM Mg-EGTA, and 50-μg/ml intact MAb 3C2 or Fab fragments of MAb 3C2 either alone or in combination. Samples were taken at various times, and the amounts of bound C3 were determined.

Effect of intact MAbs, F(ab)2 fragments, and Fab fragments on the sites for alternative-pathway-mediated C3 deposition.

Previous studies found that initiation of the alternative pathway by encapsulated cryptococci is characterized by a focal deposition of C3 molecules in which the initial foci appear to expand with incubation time to eventually fill the capsule (20). As a consequence, an experiment was done to assess the effects of intact antibodies, F(ab)2 fragments and Fab fragments on the focal initiation patterns that would normally occur in the absence of the antibodies. Encapsulated cryptococci were incubated for various times with NHS or NHS combined with intact IgG, F(ab)2 fragments, or Fab fragments of MAb 3C2 (50 μg/ml). Cryptococci that were incubated with NHS for 2 min showed very limited amounts of bound C3 that occurred as minute foci at apparently random sites in the capsule (Fig. 3). At 4 min, the foci had expanded and appeared as larger patches that were beginning to coalesce. At 8 min, the C3 bound uniformly throughout the capsule.

FIG. 3.

FIG. 3

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Effect of intact antibodies, F(ab)2 fragments, and Fab fragments of MAb 3C2 on the sites of binding of C3 fragments to encapsulated cryptococci via the alternative pathway. Cryptococci were incubated with 40% NHS, 10 mM Mg-EGTA, and 50-μg/ml intact antibody, F(ab)2 fragments, or Fab fragments. Samples were taken at various times, and the sites of C3 binding were determined by use of FITC-labeled anti-human C3. Conditions used for image acquisition were varied to optimally visualize the binding of C3 to each cell: ∗, 5, image integrations and gain of 0; ∗∗, 25 image integrations and gain of 0; ∗∗∗, 25 image integrations and gain of 9.

Intact antibodies and F(ab)2 fragments of the antibodies produced similar effects. C3 binding was focal in an effect that closely mimicked the results observed in the absence of antibody. However, the apparent rate of expansion of the foci was much lower than that observed in the absence of antibody. At 4 min of incubation, foci remained quite distinct, and there was little tendency toward coalescence of the sites. At 8 min, there was a somewhat uniform distribution of the C3 in the capsule; however, sites of more intense focal binding were present on some cells, e.g., the cell shown for incubation with NHS and intact IgG for 8 min (Fig. 3). Although not readily evident from the photographs, the intensity of fluorescence was much less with cells incubated in the presence of either intact antibody or the F(ab)2 fragments; a greater number of integrations were required to produce a printable image.

Incorporation of Fab fragments of MAb 3C2 into the reaction mixture produced an effect that differed from the results found with NHS alone or with NHS and either intact antibody or F(ab)2 fragments. C3 was readily detectable on cells incubated for 2 min with NHS and the Fab fragments. Although the C3 was rather uniformly distributed throughout the capsule, there were patches of intense fluorescence that suggested appreciable amplification of the initial focal deposits of C3. By 4 min, the cells showed dense deposition of C3 throughout the capsule.

Effect of IgG subclass on the ability to regulate C3 deposition via the alternative pathway.

MAbs 439, 3C2, and 471 are all of the IgG1 isotype. Previous studies found that these antibodies also produced suppression of the rate of C3 accumulation when the classical pathway was operative, i.e., when Mg-EGTA was not present. In contrast, IgG2a and IgG2b subclass switch variants of MAbs 439 and 471 markedly enhanced activation and binding of C3 via the classical pathway (17). This raised a question as to the abilities of the subclass switch antibodies to mediate suppression of alternative pathway dependent activation and binding of C3, i.e., binding in the presence of Mg-EGTA. This is a particularly relevant question in view of the need for bivalency for suppression (Fig. 1) and the observation that antibodies of different IgG subclasses display differences in the flexibilities of their hinge regions (5, 26).

Encapsulated cryptococci were incubated with NHS in the presence of Mg-EGTA, to which was added (i) no antibody or (ii) 50 or 200 μg of MAb 471 or MAb 439 per ml having the IgG1, IgG2a, or IgG2b isotype, and the kinetics for activation and binding of C3 were determined. The results (Fig. 4) showed that antibodies of the IgG1 and IgG2a subclasses were similarly suppressive. The IgG2b subclass switch antibodies of both MAb 471 and MAb 439 were less suppressive than either the IgG1 or IgG2a isotype.

FIG. 4.

FIG. 4

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Effect of IgG subclass on the kinetics of activation and binding of C3 fragments to encapsulated cryptococci via the alternative pathway. Cryptococci were incubated with 40% NHS containing 125I-labeled C3, 10 mM Mg-EGTA, and 50 or 200-μg/ml subclass switch antibodies (IgG1→IgG2b→IgG2a) of MAbs 439 and 471. Samples were taken at various times, and the amounts of bound C3 were determined.

DISCUSSION

The present study considered two potential mechanisms for suppression of the alternative pathway by anticapsular MAbs. In the first mechanism, the capsule is viewed as a mosaic of specific domains that are essential for activation of the alternative pathway. The specific domains could represent preferred binding sites for metastable C3b (29) and could therefore be essential to efficient initiation and/or amplification of the alternative pathway. Alternatively, the specific domains could promote activation of the alternative pathway by favoring the binding or activity of factor B or by suppressing the binding or activity of factor H. If specific regulatory domains exist, an antibody reactive with such domains could suppress activation of the alternative pathway. If this mechanism is operative, we have reasoned that inhibition should occur regardless of whether the suppressive antibody is used as the intact antibody or as F(ab)2 or Fab fragments of the antibody.

The second general mechanism for suppression of the alternative pathway is inhibition of the amplification process through a mechanical or physical alteration of the capsular environment. According to this mechanism, the suppressive antibody could cross-link the capsular matrix and thereby alter its physical properties. This could occur in an epitope-specific manner if some epitopes are exposed in positions that permit such cross-linking while other epitopes might be exposed in positions in which the necessary cross-linking could not occur. Cross-linking could suppress activation of the alternative pathway in several ways. First, the antibody could create a shell at the surface of the capsule and block penetration of complement components to sites beneath the shell. Second, the cross-linking could create mini-cages within the capsule that would allow activation within each cage but would not permit amplification beyond the cage. Finally, cross-linking could reduce the rate of diffusion of macromolecules through the capsular matrix such that diffusion of metastable C3b from activation sites would be retarded. Given the short half-life of metastable C3b, estimated at 60 μS (34), decreased diffusion rates could greatly reduce expansion of focal initiation sites. Regardless of the specific mechanism by which a physical alteration in the capsular matrix occurs, cross-linking of the capsular matrix would require a multivalent antibody; suppression would occur with intact antibodies and their F(ab)2 fragments but would not occur with their Fab fragments.

Our results showed that F(ab)2 fragments of all three MAbs readily suppressed alternative-pathway-mediated activation and binding of C3 to the cryptococcal cells. In contrast, Fab fragments of two of the three MAbs (MAbs 439 and 3C2) were not suppressive. Fab fragments of the third antibody (MAb 471) showed markedly reduced suppressive activity, but some suppressive activity remained. These results support the argument that cross-linking of the capsular matrix is a critical component of antibody-mediated suppression of the alternative pathway. The mildly anomalous behavior of Fab fragments of MAb 471 may be related to the fact that the serotype specificity of MAb 471 is slightly different from the specificities of MAbs 3C2 and 439 (1). Although it is a less likely explanation, our data do not exclude the alternative possibility that regulation of C3 deposition is a function of a large domain on the polysaccharide located near, but not at, the binding site of the suppressive MAb. In such a case, the adjacent regulatory domain might be blocked by the larger IgG or F(ab)2 molecules but not the Fab fragment. However, this latter explanation is not consistent with the contribution of IgG isotype to the suppressive effect (Fig. 4).

Reduced binding to the capsule is an alternative explanation for the lack of suppression by Fab fragments. An analysis of the binding of intact antibodies, F(ab)2 fragments, and Fab fragments to cryptococcal cells showed that all three forms of the MAbs readily bound to the cells under the conditions used to assess suppressive activity. Indeed, approximately two to eight times more Fab molecules bound to the cells than did intact molecules. The reduced molecular size of the Fab fragments may allow increased access by Fab fragments to binding sites within the capsular matrix. Alternatively, cross-linking of the capsule by intact antibodies may block binding of subsequent antibody molecules.

Because cross-linking of the capsule is a critical component of antibody-mediated suppression, we examined the suppressive activity of isotype switch (IgG1→IgG2b→IgG2a) families of MAbs 471 and 439. These immunoglobulin isotypes differ in the lengths of the upper hinge sequences (IgG2b > IgG2a > IgG1) and in the flexibility of the hinge region (also IgG2b > IgG2a > IgG1) (5, 26). Results of the comparison showed that MAbs of the IgG1 and IgG2a isotypes strongly suppressed alternative-pathway-mediated binding of C3. The IgG2b variants of MAbs 471 and 439 were also suppressive, but the levels of suppression were reduced and required more antibody to produce the suppressive effect than did the IgG1 and IgG2a antibodies. Notably, the IgG2b switch antibody of MAb 471 also has a reduced ability to precipitate GXM compared to the IgG1 and IgG2a antibodies (33). It is not clear why an antibody with increased hinge flexibility should exhibit decreased suppression of the alternative pathway. Perhaps antibodies with rigid hinge regions produce tighter cross-linking than antibodies with more flexibility in the hinge.

An unexpected activity of the Fab fragments was facilitation of early C3 binding via the alternative pathway. Fifty percent of maximum binding occurred 2 min earlier in the presence of Fab fragments of MAbs 439 and 3C2 than in the absence of the MAbs. This accelerated binding of C3 fragments in the presence of Fab fragments was readily evident when immunofluorescence was used to evaluate the sites for deposition of C3. Cryptococci incubated for 2 min in NHS containing Fab fragments of MAb 3C2 showed easily discernible C3 that was distributed over the surface of the capsule. In contrast, cryptococci incubated for 2 min in NHS alone showed a few very faint foci of C3.

To date, we have not examined potential mechanisms by which Fab fragments might facilitate the alternative pathway. In a converse of possible mechanisms for inhibition by antibody, the Fab fragments could provide favorable binding sites for metastable C3b. Alternatively, the Fab fragments could facilitate the binding or activity of factor B or could block the binding or activity of factor H. Reduced binding of factor H to particle-bound C3b has been shown to distinguish particulate activators of the alternative pathway from nonactivators (11, 14, 15, 27).

These studies further illustrate the complexity of the interaction between anti-GXM antibodies and the cryptococcal capsule. Previous studies found that MAbs reactive with cryptococcal serotypes A, B, C, and D suppress alternative-pathway-mediated C3 binding, whereas MAbs reactive with cryptococcal serotypes A and D are not suppressive (17). Epitope-specific effects of anti-GXM antibodies have also been demonstrated with other systems. Some anti-GXM IgM MAbs are protective in a murine model of cryptococcosis, whereas IgM MAbs with another specificity are not protective (22, 25). MAbs with the protective specificity produce an annular pattern of binding in the capsule; MAbs with the nonprotective specificity produce a punctate pattern (22, 25). We speculate that these diverse biological activities have a common molecular mechanism. Identification of a unifying mechanism may prove important in the selection of MAbs for use in passive immunization (4, 9, 23, 24, 30) and in vaccine design for prevention of cryptococcosis (3, 6, 7). Such studies also have the potential to contribute to our understanding of the mechanism of action of antibody-dependent protection against encapsulated bacteria.

ACKNOWLEDGMENT

This work was supported by Public Health Service grant AI 14209 from the National Institute of Allergy and Infectious Diseases.

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