The IgM mAbs 12A1 and 13F1 are protective and nonprotective, respectively, against lethal Cryptococcus neoformans infection in mice. To better understand the variables that contribute to IgM efficacy against C. neoformans, we studied the effects of inoculum size, route of infection, and Ab dose for each of these mAbs. mAb 13F1 did not prolong survival under any condition studied. mAb 12A1 prolonged survival after the administration of certain Ab doses after i.p. infection with defined inocula and promoted phagocytosis, agglutination, and the formation of inflammatory cell rings around yeast cells in vivo. Large Ab doses of mAb 12A1 resulted in either no protection or enhanced infection, consistent with a prozone-like effect. Investigation of this phenomenon revealed that the fungal cell was protected against microbicidal nitrogen-derived oxidants when large amounts of Ab were bound to the C. neoformans capsule. mAb 12A1 was opsonic in vitro for peritoneal, but not splenic or alveolar macrophages. In summary, our results indicate that IgM efficacy against C. neoformans is a function of the route of infection, inoculum, and Ab dose and is associated with its ability to promote opsonization, agglutination, and phagocytic ring formation in vivo. The occurrence of the prozone-like phenomenon implies that high Ab titers are not necessarily beneficial in assuring protection against certain pathogens and that caution should be exercised in using high Ab titer as a measure for vaccine efficacy.
Cryptococcus neoformans is the only pathogenic fungus with a polysaccharide capsule that is essential for virulence (1, 2). The main component of the capsule is glucuronoxylomannan (GXM),3 which is antiphagocytic in vitro and has been demonstrated to cause a variety of deleterious effects to immune function (1). Despite its antiphagocytic properties in vitro, C. neoformans is a facultative intracellular pathogen in vivo (3). Although it is a relatively rare cause disease in normal individuals, C. neoformans is a relatively frequent cause of chronic meningitis in patients with impaired immune function (reviewed in Ref. 4). The hallmark of an effective tissue response to C. neoformans is granuloma formation, and there is general consensus in the field that cellular immunity is critically important for host defense (4). However, there is now a large body of evidence that humoral immunity can make an important contribution to host defense, as evidenced by data from four independent research groups showing that administration of mAbs to the C. neoformans capsule can modify the course of infection in mice (5, 6, 7, 8).
The availability of a large and well-characterized collection of murine mAbs to C. neoformans has facilitated the dissection of how certain Ab characteristics contribute to Ab efficacy. The efficacy of Ab against C. neoformans is a function of isotype and epitope specificity (reviewed in Refs. 9, 10, 11). Protective and nonprotective IgG Abs have been described, but the mechanism of protection is not well understood. The best evidence that specificity plays a critical role in Ab-mediated protection against C. neoformans comes from studies with a pair of IgM mAbs known as 12A1 and 13F1 (12). These two IgMs originated from the same B cell, use the same V region, but differ in certain complementarity-determining region amino acid sequences as a result of somatic mutations, which in turn result in differences in specificity (12). mAbs 12A1 and 13F1 both protect against infection with serotype A isolates, but only mAb 12A1 protects against serotype D infection (13). mAbs 12A1 and 13F1 produce annular and punctate fluorescence patterns with serotype D isolates, respectively (14). These patterns reflect the formation of different types of Ag-Ab complexes in the C. neoformans capsule (15). The interaction of each mAb with the capsule also produces qualitatively different types of capsular reactions (16). Both mAbs activate the classical complement pathway, but mAb 12A1 suppresses capsular activation of the alternative complement pathway (16). The differences in immunofluorescence-binding pattern, capsular reaction, competition studies, and peptide mimotope reactivity indicate that these two mAbs bind to different epitopes in GXM (12, 13, 17).
In this study, we evaluated the influence of dose, inoculum, and route of infection on the protective efficacy of IgM mAbs and correlate the results with in vitro measurement of Ab function. A better understanding of IgM efficacy against C. neoformans is important because IgM to GXM is common in human sera (18, 19, 20, 21), can protect against this pathogen (7, 8), and does not cause acute lethal toxicity when administered to infected mice (22). The results indicate that IgM Ab-mediated protection occurs only for certain Ab doses, inocula, and routes of infection. At high Ab doses, protection is abolished possibly as a result of the type of interactions made by Ab with the C. neoformans capsule in conditions of Ab excess.
Materials and Methods
C. neoformans strain 24067 (serotype D) was obtained from the American Type Culture Collection (Manassas, VA). This strain was selected for study because it was the strain used to define differences in protective efficacy between mAbs 12A1 and 13F1 (12). Cultures were grown in Sabouraud dextrose broth (Difco, Detroit, MI) at 30°C overnight with moderate shaking (200 rpm). Yeast cells were washed three times, suspended in PBS, and counted in a hemocytometer before infection.
The IgM mAbs 12A1 and 13F1 have been described previously (12, 13, 23). IgM ascites was generated in BALB/c mice given i.p. injections of mAb 12A1 or 13F1 hybridomas. Endotoxin concentration in ascites was <1 ng/ml as measured by the Limulus amebocyte test (BioWhittaker, Walkersville, MD). mAb concentrations were determined by ELISA relative to isotype-matched standards. IgM was purified by ammonium sulfate precipitation followed by size exclusion chromatography using a HiPrep Sephacryl S-300 high resolution (HR26/60) gel (Pharmacia Biotech, Uppsala, Sweden). The column was equilibrated with endotoxin-free PBS and the separation was monitored by UV absorbance. Fractions containing IgM were pooled and concentrated. The IgG1 mAb 18B7 was used as a positive control for phagocytosis experiments. For description of mAb 18B7, see Ref. 24 .
A/JCr, C57BL/6, and BALB/c (6- to 8-wk-old male) were obtained from the National Cancer Institute (Frederick, MD) and The Jackson Laboratory (Bar Harbor, ME). CD4-deficient mice (C57BL/6-parental) were donated by Matthew D. Scharff (Bronx, NY) from our institution. The mAbs were administrated i.p. as either purified IgM or ascites 5 or 30 min before infection. In one experiment, yeast cells were incubated with mAb and then injected i.p. Control groups were given NSO (nonproducing plasmacytoma line) or ascites containing irrelevant IgM with specificity for mycobacterial lipoarabinomannan (25). Mice were infected by either i.p., i.v., or intratracheal (i.t.) routes. Intraperitoneal infection was done as described previously (7, 26, 27). Intravenous infection was done by injection of 106 or 107 yeast cells in PBS via the lateral tail vein. For i.t. infection, mice were anesthetized with 70 mg/kg sodium pentobarbital (Anpro Pharmaceutical, Arcadia, CA), their necks were hyperextended, and the trachea was exposed at the level of the thyroid and infected with 106 or 107 yeast cells in PBS using a 26-gauge needle. The incisions were sutured with 5-0 silk. Intraperitoneal infections were done by injections of 105, 106, 107, and 108 yeast cells in PBS.
In vitro phagocytosis
Peritoneal, splenic, and alveolar macrophages from BALB/c and A/JCr mice were cultured in DMEM with 10% heat-inactivated FCS, 10% NCTC-109 medium (Life Technologies, Grand Island, NY), and 1% nonessential amino acids (Cellgro, Washington, DC). Other phagocytosis experiments used the J774.16 line, which is a macrophage-like cell line derived from a reticulum cell sarcoma (28). The protocol for in vitro phagocytosis was as described in earlier studies (29, 30) with minor modifications. Cells were plated in 96-well tissue culture plates (Falcon; Becton Dickinson, Franklin Lake, NJ) at a density of 105 cells/well in 96-well culture plates and stimulated with 50 U/ml recombinant murine IFN-γ (Genzyme, Cambridge, MA) and LPS (Sigma, St. Louis, MO). Briefly, macrophages were stimulated with 50 U/ml IFN-γ/ml and incubated at 37°C overnight. The medium in each well was then replaced with fresh medium containing 50 U/ml IFN-γ and 1 μg/ml LPS, and C. neoformans cells were added at a ratio of 5:1 macrophages to fungi. Phagocytosis was measured in the presence or absence of purified mAbs (0–1000 μg/ml). After the addition of C. neoformans, the cells were incubated at 37°C for 2 h, washed several times with sterile PBS, fixed with cold absolute methanol, and stained with a 1/20 solution of Giemsa. PBS was used as a negative control and mAb 18B7 (IgG1) was used as a positive control. Phagocytic indices were determined with a microscope at a magnification of ×600 (Nikon diaphot; Nikon, Garden City, NJ). The phagocytic index is defined by: PI = P × F, where P is the percentage of phagocytic macrophage and F is the average of yeast cells per macrophage. Experiments were done in triplicate and five or eight different fields were counted.
In vivo phagocytosis
Mice were given either mAbs 12A1 or 13F1 or PBS i.p. 5 min before infection with 107 yeast cells. Two hours after infection, mice were killed and peritoneal macrophages were obtained by peritoneal lavage with PBS. Cells were washed three times with PBS and their number was determined by counting with a hemocytometer. Approximately 3 × 105 cells/ml were transferred to DME10 (10% heat-inactivated FCS, 10% NCTC-109 medium, 1% nonessential amino acids. and DMEM; Life Technologies), placed in a 96-well tissue culture plate, and incubated for 1 h in a 10% CO2 incubator at 37°C to allow adherence of macrophages to the plastic support. Inspection of the cell exudate by light microscopy revealed that phagocytosis had already occurred before adherence to the plastic. The monolayer was then washed three times with sterile PBS, fixed with cold absolute methanol for 30 min at ambient temperature, washed again three times with sterile PBS, and stained with a 1/20 Giemsa solution (Sigma). Phagocytic indices were determined with a microscope at a magnification of ×600 (Nikon diaphot; Nikon) as described above. Experiments were done in triplicate and each involved counting five or eight different fields.
Determination of serum GXM concentration
Serum was analyzed for GXM content at 3, 18, 24, and 48 h after infection. The serum GXM level was determined by capture ELISA as described elsewhere (31). Briefly, serum was digested with proteinase K (1 mg/ml) overnight at 37°C and the GXM concentration was measured relative to a GXM standard of known concentration by ELISA using mAbs 2D10 (IgM) and 2H1 (IgG1) for capture and detection, respectively, as described previously (31).
C3 in the serum of mice infected with C. neoformans was measured by both immunodiffusion and capture ELISA. For immunodiffusion, goat anti-mouse C3 (Cappel, Aurora, OH) and serum were placed in 1% agarose gels and the gel was stained with Coomassie to visualize precipitin lines. For capture ELISA, polystyrene plates (Falcon) were coated with goat anti-mouse C3 diluted 1/5000 (Cappel) in PBS and the plate was blocked with 1% BSA in PBS. Serum was then serially diluted in 1% BSA followed by the addition of peroxidase-conjugated goat anti-mouse C3 (Cappel) diluted 1/25,000. The reaction was developed by the addition of o-phenylenediamine (Sigma; 5 mg in 25 ml of 0.07 M citrate buffer (pH 5.0) and 0.01% H2O2). After the color developed, the reaction was stopped by addition of 2 M H2SO4, and the absorbance at 492 nm was measured in a Labsystem Multiscan reader (Labsystem, Helsinki, Finland). Each step was followed by incubation for 1.5 h at 37°C. The plates were washed in a Skanwasher400 (Skatron, Lier, Norway) between each step with 0.05% Tween 20 (polyoxyethyllenesorbitan monolaurate) in PBS. Each plate was washed three times between each step with the exception of the last step before the addition of the substrate solution in which five washes were used. Complement deposition on the C. neoformans capsule was used as a functional measure for the complement system. Yeast cells were incubated with serum or diluted serum for 2 h at 37°C, placed on a glass slide, and fixed overnight at ambient temperature. The slides were then incubated with FITC-labeled goat anti-mouse C3 (Cappel) and examined using an Olympus IX 70 microscope (Olympus America, Melville, NY). In other experiments, yeast cells were recovered from the peritoneal washes of infected mice given 1 mg of mAb 12A1, washed, and stained simultaneously for both C3 with FITC-labeled goat anti-mouse C3 (Cappel) and IgM with rhodamine-labeled goat anti-mouse IgM (Cappel).
Organ fungal burden
The fungal burden in infected mice was measured by determining organ CFUs (1 colony = 1 CFU). The entire lung, liver, spleen, and brain were removed, weighed, and homogenized in 10 ml of sterile PBS. Dilutions of the organ homogenate were spread on Sabouraud dextrose agar and colonies were counted after incubating the plates for at least 2 days at 30°C. CFUs were determined for mice that died or at day 3 infection with 107 yeast cells.
Binding capacity of C. neoformans capsule for the mAb 12A1
The binding capacity of the C. neoformans capsule for mAb 12A1 was determined by incubating yeast cells with various concentrations of Ab and measuring the free Ab by ELISA. C. neoformans strain 24067 was grown in Sabouraud dextrose broth (Difco) at 30°C overnight with moderate shaking (200 rpm). Yeast cells were washed three times, suspended in PBS, and counted in a hemocytometer before use. The diameter of the capsule was examined at ×1000 under oil immersion in an india ink suspension. The distance from the cell to the outer margin capsule and the cell diameter (not including the capsule) was measured by using an eyepiece grid with a resolution of 0.5 μm. A suspension of 107 yeast cells was placed in a 50-ml polypropylene conical tube (Becton Dickinson, Franklin Lake, NJ) previously blocked with 1% BSA and incubated with different amounts of purified mAb 12A1 at 37°C for 1 h with constant shaking. After incubation, the supernatant was collected and free mAb 12A1 was determined by ELISA relative to isotype-matched standards.
Fungicidal activity of nitrogen-related oxidants in the presence and absence of mAb 12A1
The ability of nitrogen-related oxidants to kill C. neoformans in the presence and absence of mAb 12A1 was determined as described previously (32, 33). The experimental design involved incubating equal numbers of cells (106 yeast cells) with or without mAb 12A1 in 25 mM succinic acid (pH 4.17) for 15 min at 37°C with shaking, followed by the addition of 0.5 mM NaNO2. Controls included C. neoformans suspended in either 25 mM succinic acid or 0.5 mM NaN02. Cells were plated in Sabouraud dextrose agar at various times to determine their viability and the colonies were counted after incubating plates for at least 2 days at 30°C. The concentration of nitrite in the aliquots collected at the different times was determined by absorbance at 540 nm using the Greiss reagent. All experiments were performed in triplicate.
Pairwise comparison between groups was done by the t test using Primer of Statistics-The Program (McGraw-Hill, New York, NY) after significance was established by ANOVA. Survival analysis was performed using log rank analysis (SPSS, Chicago, IL).
Previous studies have established that administration of mAb 12A1, but not 13F1, prolongs survival in murine cryptococcosis when given at a dose of 1 mg/mouse before infection. However, the influence of different Ab doses on survival with a certain inoculum or with different inocula has not been examined previously. The efficacy of mAbs 12A1 in prolonging the survival of mice given i.p. infection was a function of both dose and inoculum (Fig. 1,A). Average survival for mice given 1 mg of mAb 12A1 and 108 yeast cells was longer than that of control mice receiving no Ab, consistent with previous observations. However, administration of 1 mg of mAb 12A1/mouse reduced the average survival of Ab-treated mice relative to control mice for those infected with 107, 106, and 105 yeast cells. At the lower mAb 12A1 doses, significant prolongation in survival was observed in mice receiving 107 cells but not in groups infected with 106 or 105 yeast/mouse. Similar results were observed when the Ab was administered 5 min before infection or when the yeast cells were incubated with Ab in vitro and then injected i.p. (data not shown). For mice treated with mAb 13F1, survival was prolonged in the group receiving a 1-mg dose before infection with 107 organisms, but the effect did not reach statistical significance (Fig. 1 B). When evaluated in i.v. and i.t. models of infection, mAb 12A1 did not prolong the survival of infected mice for any combination of dose and inoculum studied (data not shown). Administration of either mAb 12A1 or 13F1 at doses of 0.1 or 1 mg/mouse to CD4−/− mice did not prolong survival even though Ab prolonged survival in the parental C57BL/6 strain (data not shown).
Effect of mAb 12A1 on serum GXM
Serum GXM levels were measured in mice given 1.0, 0.5, 0.1, and 0.0 mg of mAb 12A1 followed by infection with 107 yeast cells. In mice given no mAb, the level of serum GXM rose rapidly after infection (Table I). In contrast, in mice given mAb, no serum GXM was measurable until 18 h after infection. At 24 h, the level of serum GXM in mice given mAb 12A1 was inversely proportional to the amount of mAb administered (Table I). However, by 48 h, the level of GXM in the mAb-treated groups was higher in the mice that received 1.0 and 0.5 mg than in mice that received 0.1 mg, consistent with the survival data showing longer survival for the mice that received 0.1 mg of the mAb.
|Time (h) .||Serum GXM Level (μg/ml) .||.||.||.|
|.||1.0 mg .||0.5 mg .||0.1 mg .||Control .|
|3||0||0||0||40 ± 1.3|
|18||0||0||0||126 ± 4.5|
|24||2.6 ± 0.02||32.7 ± 3.3||155 ± 10.9||326 ± 11.0|
|48||213 ± 34.3||283 ± 27.8||113 ± 8.1||1013 ± 225|
|Time (h) .||Serum GXM Level (μg/ml) .||.||.||.|
|.||1.0 mg .||0.5 mg .||0.1 mg .||Control .|
|3||0||0||0||40 ± 1.3|
|18||0||0||0||126 ± 4.5|
|24||2.6 ± 0.02||32.7 ± 3.3||155 ± 10.9||326 ± 11.0|
|48||213 ± 34.3||283 ± 27.8||113 ± 8.1||1013 ± 225|
Organ CFU early in infection and at death
To explore the mechanisms for the Ab-mediated effects on survival, organ CFUs were determined in mice given mAb 12A1 at day 3 of infection or shortly after death. Analysis of organ CFUs at day 3 of infection revealed that dissemination of infection from the peritoneal cavity had occurred in all mouse groups regardless of the administration of Ab or Ab dose used (Fig. 2,a). The pattern of organ CFU per gram of tissue was very similar in all groups irrespective of mAb 12A1 administration or dose (Fig. 2,a). Mice receiving 1 mg of mAb 12A1 had significantly higher brain CFUs than those receiving no Ab or lower doses of mAb 12A1 (Fig. 2,a). By the time of death, the relative pattern of organ fungal burden had changed such that brain CFUs had continued to increase consistent with death due to meningoencephalitis (Fig. 2 b).
We investigated whether the prozone-like phenomenon observed in passive protection experiments involving large doses of mAb 12A1 reflected complement depletion as a consequence of Ab administration. Serum C3 was measured before infection and at 3, 18, 24, and 48 h after infection for mice given 0.0, 0.1, 0.5, and 1.0 mg of mAb 12A1 by ELISA and immunodiffusion. No difference in serum C3 concentration was observed among Ab-treated and control groups (data not shown). Analysis of C3 deposition on the capsule of C. neoformans after incubation in serum from the same mice also revealed no difference between Ab-treated and control groups (data not shown). To establish whether large doses of mAb 12A1 interfered with complement deposition in vivo yeast cells from mice given 1 mg of mAb 12A1 were recovered from the peritoneum and stained for C3 and IgM (Fig. 3). C. neoformans cells from mice given mAb 12A1 stained brightly throughout the capsule whereas cells from control mice given no IgM showed only weak C3 staining localized primarily to the cell wall (data not shown).
In vivo phagocytosis
To determine whether mAbs 12A1 and 13F1 were opsonic in vivo, we studied the peritoneal exudates for evidence of internalized cryptococci. The phagocytic index for peritoneal macrophages from mice given mAb 12A1 was significantly higher than macrophages from mice receiving the control mAb (Fig. 4). In contrast to mAb 12A1, administration of mAb 13F1, did not promote phagocytosis in vivo (Fig. 4). Larger numbers of phagocytic rings consisting of collections of macrophages and neutrophils surrounding yeast cells were observed in the peritoneum of mice receiving mAb 12A1 relative to those receiving mAb 13F1 or saline (Fig. 5). Similarly, the number of extracellular agglutinated yeast cells in mice receiving mAb 12A1 was significantly higher than in mice given mAb 13F1 (Fig. 5). No agglutination was observed in peritoneal exudates from mice given saline.
In vitro phagocytosis
We investigated the effect of the mAb dose on the phagocytic index in vitro using both primary macrophages and J774.16 macrophage-like cells. mAb 12A1 was opsonic for peritoneal macrophages but not alveolar or spleen macrophages (Fig. 6). In contrast, mAb 13F1 was either not opsonic or minimally opsonic and mAb 18B7 (IgG1) was opsonic for macrophages from all three anatomical sites. To determine dose-response effects of mAb 12A1 on C. neoformans opsonization, we measured the phagocytic index at various Ab concentrations using peritoneal macrophages (Fig. 7). Phagocytosis was observed at all mAb 12A1 concentrations but the phagocytic index was maximal at 100 μg/ml and declined for Ab levels >100 μg/ml (Fig. 7). A similar trend was observed with mAb 18B7 (Fig. 7). Similar results were observed when the experiment was done with J774.16 macrophage-like cells (data not shown).
Monoclonal Abs 12A1 binding by C. neoformans cells
Absorption studies were done to determine the Ab binding capacity of C. neoformans cells for mAb 12A1. Microscopic analysis of strain 24067 cells, in the conditions described above, revealed an cell average diameter of the 5.05 ± 0.75 μm and capsule average diameter of the 2.52 ± 0.67 μm, consistent with previous measurements (34). A suspension of 107 cells was saturated by 70–100 μg of mAb 12A1. Assuming a molecular mass of 950,000 Da for IgM, we calculate that each cell of strain 24067 cells grown in vitro in the conditions used in this study and estimate that saturation occurs after the binding of ∼4.4 × 107 molecules of IgM.
Effect of mAb 12A1 on fungicidal activity of nitrogen-related oxidants
We evaluated the hypothesis that Ab binding to the capsule could influence the susceptibility of C. neoformans to nitrogen-related oxidants. NO− and related products produced by murine immune effectors cell have been shown to be fungistatic and fungicidal for C. neoformans (33, 35, 36). The experimental design involved measuring CFUs after incubation of C. neoformans cells with and without mAb 12A1 in the presence and absence of serum followed by exposure to nitrogen-related oxidants as described previously (32). Killing of C. neoformans by nitrogen-related oxidants was highly efficient in the presence of 10 μg/ml mAb 12A1 but higher concentrations impeded killing (Fig. 8). Control experiments revealed that the differences in killing were not due to differences in nitrite concentration between the various groups (data not shown).
Previous studies of Ab efficacy against C. neoformans have shown that Ab-mediated protection is dependent on many factors, including Ab isotype, specificity, the state of host T cell function, and the cryptococcal strain (for reviews, see Refs. 9, 10, 11, 37). In this study, we explored the relationship between the IgM dose and protective efficacy. Although it has been proposed that a minimum amount of specific serum IgG is necessary for protection against certain pathogens (38), there is also evidence that Ab-protective efficacy can decline in conditions of Ab excess. A prozone-like “zone effect” was described for Streptococcus pneumoniae whereby administration of amounts larger than the optimally protective dose results in loss of protection (39, 40). As early as 1913, Cole (41) noted that Ab protection studies did not obey the law of multiple proportions with respect to infecting inocula. These observations from the early 20th century appear to have been largely forgotten.
The efficacy of mAb 12A1 in prolonging survival was dramatically dependent on the mAb dose and optimal protection was observed for mice given 100 μg of mAb12A1/mouse and infected i.p. with 107 or 108 C. neoformans cells. However, administration of larger quantities of mAb 12A1 significantly shortened survival. This observation was reproducible and suggested a prozone-like effect similar to that described earlier for S. pneumoniae (40). Interestingly, at the lower inocula of 106 or 105 yeast cells/mouse, no protection was observed with the mAb 12A1 doses given, possibly because these mAb dose-inocula interactions still resulted in Ab excess in light of the fact that the total Ab-binding capacity of 107 cells was <100 μg. Our observation of a prozone-like phenomenon after the administration of a mAb rather than polyclonal immune serum, as noted historically, suggests that it may represent a general principle of the relationship between Ab dose and protective efficacy for a given pathogen.
To our knowledge, the phenomenon of prozone-like effects in passive Ab studies have never been explained and we considered several potential mechanisms. The absence of phagocytosis at high Ab concentrations is not the explanation because efficient phagocytosis was observed both in vivo and in vitro at high Ab concentrations. The possibility that infusion of large amounts of IgM would trigger complement depletion was explored but deemed to be unlikely since we found no differences in the serum complement component C3 in mice treated with mAb 12A1 and controls. Furthermore, we detected no difference in the ability of serum from Ab-treated mice and control mice to deposit C3 in the C. neoformans capsule. Yeast cells recovered from mice given large amounts with mAb 12A1 stained brightly for C3 relative to cells from control mice, indicating that this IgM promoted complement deposition in the capsule in vivo. These observations indicated that the complement system was active in mAb 12A1-treated mice and we sought other explanations for the prozone-like effect. Next, we evaluated whether Ab binding could interfere with killing by nitrogen-derived oxidants of the type made by mouse phagocytic cells. At high concentrations, mAb 12A1 binding to the C. neoformans reduced susceptibility to oxidant killing. mAb 12A1 binding to the capsule is known to cause changes in the refractive index (16) and capsule structure (42). We interpret the reduced susceptibility to nitrogen-derived oxidant killing in the presence of high concentrations of mAb to reflect the existence of a proteinaceous coating to the capsule, which can have the paradoxical effect of protecting the yeast cell against oxidative killing. In this regard, we note that prozone-like effects were observed at Ab doses greater than 100 μg, which are near the saturation dose for the inoculum used. Hence, the prozone-like effect may reflect saturation binding of mAb 12A1 in the capsule of mice given large doses of Ab.
The mAb 12A1 was protective in mice lethally infected with C. neoformans only when both Ab and infection were administered i.p. In contrast, mAb 13F1 was not protective in any model studied. Serum GXM was measurable in all mice given mAb 12A1 at 24 h of infection and the serum GXM level was inversely proportional to the Ab dose administered. The low Ag levels in Ab-treated mice are consistent with previous data showing that specific IgM can clear serum Ag by promoting deposition in the liver (43). Since the serum half-life of IgM in normal mice in the absence of Ag is only 2 days (44), we attribute the appearance of serum GXM after 24 h as indicative of declining levels of IgM through normal Ig metabolism and possibly consumption of Ab by the infective process. The in vivo phagocytosis studies showed that the mAb 12A1 was opsonic, agglutinating, and promoted phagocytic cell ring formation around yeast cells in the peritoneal cavity. In contrast, mAb 13F1 was weakly agglutinating and there was no significant phagocytosis or phagocytic ring formation relative to mice given PBS. Since IgM-mediated macrophage phagocytosis has been associated with increased killing of C. neoformans, the protective effect of mAb 12A1 may be derived in part from its ability to promote phagocytosis by peritoneal macrophages. Furthermore, phagocytic ring formation in peritoneal exudates of C. neoformans infections has been associated with enhanced fungal killing and possibly earlier granuloma formation (45, 46, 47). The occurrence of in vivo phagocytosis, agglutination, and phagocytic ring formation with mAb 12A1 but not 13F1 suggests mechanisms for Ab-mediated protection. Agglutination alone is unlikely to be the protective mechanism since IgG3-agglutinating Abs have not been protective in this model (7, 48). Furthermore, studies of IgM-mediated protection against Candida albicans indicate that agglutination of yeast forms is not sufficient for protective efficacy (49).
C. neoformans yeast agglutination was prominent in the peritoneum of mice given mAb 12A1, but not those receiving either mAb 13F1 or PBS. Surprisingly, there were no striking differences in liver, lung, and spleen CFUs for mice receiving various doses of mAb and control mice 3 days after infection. This implied that prolongation of survival by the lower and middle range doses of mAb 12A1 was not a result of simple containment of infection in the peritoneal cavity or reduction in organ tissue burden. For mice receiving 1 mg of mAb 12A1, there was a statistically significant 5- to 8-fold increase in brain CFUs relative to control mice, suggesting an explanation for the accelerated mortality observed with large doses of Ab administration. Consistent with the observation of Ab-mediated enhancement of infection at this dose, mice given 1 mg of mAb 12A1 had higher serum GXM levels than mice given 0.1 mg at 48 h after infection. To investigate the mechanism of death, mice were necropsied shortly after death and their lung, liver, and brain CFU burden was determined. Control mice had more lung CFU than brain CFU at the time of death. In contrast, mAb 12A1-treated mice had brain CFUs which were at least 2 log10 higher than control mice. For the mAb 12A1-treated mice that received large doses and were not protected, the higher brain CFUs probably reflect continued replication of the higher brain burden observed at day 3. For the mAb 12A1-treated mice that received smaller doses and lived longer than control mice, we attribute the higher brain CFU to progressive meningoencephalitis resulting from early dissemination to the CNS.
Administration of mAb 12A1 before i.v. or i.t. infection did not prolong survival. This result contrasts with the observation that IgG1 mAbs can mediate protection in i.p., i.v., intracerebral, and i.t. models of infection (7, 50, 51). To investigate this observation, we compared the opsonic efficacy of mAb 12A1 with a protective IgG1 mAb for peritoneal, splenic, and alveolar macrophages. Tissue macrophages are intimately associated with C. neoformans in murine infection and macrophages are the major phagocytic cell for this fungus (3, 52). mAb 12A1 was opsonic for peritoneal but not splenic or alveolar macrophages, whereas the IgG1 mAb was opsonic for all these types of tissue-derived macrophages. Given that opsonization is probably an important mechanism for the IgM-mediated protection, the inability of mAb to opsonize C. neoformans for splenic and alveolar macrophages suggests a potential explanation for the inability of this Ab to protect against i.v. and i.t. infection. Functional differences in antifungal efficacy have been described for macrophages from different body sites (53). The mechanism of IgM-mediated phagocytosis in the absence of complement is not understood and may involve cellular receptors or changes in the capsule properties that reduce its antiphagocytic properties. mAbs 12A1 and 13F1 have been shown to bind at different sites in the capsule of C. neoformans (15), suggesting that the lack of opsonic efficacy of mAb 13F1 to opsonize yeast cells may reflect an inability to interact with host cell receptors or to promote changes in the capsule structure will make it less antiphagocytic. In this regard, a receptor for IgM-mediated phagocytosis has been described in peritoneal macrophages (54). We are currently studying the mechanism responsible for this effect.
The efficacy of IgG1 and IgG3 mAbs against C. neoformans is dependent on T cell function (55). To evaluate whether the same applies to IgM, we studied the effect of passive immunization with mAbs 12A1 and 13F1 in CD4-deficient mice. Neither Ab prolonged survival in CD4-deficient mice. Numerous studies have established that cell-mediated immunity is essential for host defense against C. neoformans and that granuloma formation is associated with control of infection (27, 56). Although the mechanism by which IgM-mediated protection is dependent on T cells was not studied, we hypothesize that the effects of IgM are not sufficient to prolong survival in mice that are unable to mount an effective cell-mediated response.
We are cognizant that our results were obtained in a model of i.p. infection which does not represent the physiological route of C. neoformans infection. However, the mouse i.p. model is a time-honored system that has consistently identified protective Abs against other respiratory pathogens such as S. pneumoniae and Neisseria meningitides (48). Nonphysiological models of infection have proven useful historically for the identification of protective Abs (48). For our purposes, this model is the only option available since IgM was not protective against C. neoformans in the i.v. and i.t. infection models. An important measure of the usefulness of this model is the fact that it can distinguish between protective and nonprotective Abs against C. neoformans (7).
In summary, the results observed with Ab protection experiments against C. neoformans reveal a complex relationship among Ab isotype, epitope specificity, dose, and functional status of CD4 T cells. The most straightforward explanation of our results is that mAb 12A1 is protective because it mediates phagocytosis, agglutination, and phagocytic ring formation in vivo, which slows the course of infection and translates into a prolongation in survival despite little or no effect on organ CFUs. Consistent with this view, mAb 13F1 is not protective because it does not mediate phagocytosis and/or phagocytic ring formation in vivo. The Ab dose can have a dramatic prozone-like effect whereby larger doses are less protective than smaller doses. In vivo studies suggest that the reduced survival observed in mice receiving large doses of mAb 12A1 was a result of enhanced brain infection. In vitro studies suggest an explanation for the prozone based on the observation that large amounts of Ab in the capsule reduce opsonic efficacy and can interfere with the microbicidal activity of nitrogen-derived oxidants. Thus, a better understanding of the prozone effect may be essential for evaluating vaccine efficacy and designing better vaccines and for developing Ab-based therapies. In this regard, we note that a highly immunogenic vaccine which elicited high titers of Abs to GXM in mice was not protective and in some experiments vaccinated mice had accelerated infection (57, 58). Our results indicate the need for additional studies to investigate the relationship between Ab dose and protective efficacy and suggest that vaccines which elicit very high titers of certain Abs may not be protective through prozone-like effects.
We are very grateful to Dr. Liise-anne Pirofski for critically reading this manuscript.
A.C. is supported by National Institutes of Health awards AI33774, AI3342, and HL-59842-01 and a Burroughs Wellcome Development Therapeutics Award. C.P.T. was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo Grant 99/00185-1.
Abbreviations used in this paper: GXM, glucuronoxylomannan; i.t., intratracheal.