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*
Division of Infectious Diseases, Department of Medicine, and
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; and
Department of Microbiology, University of Nevada School of Medicine, Reno, NV 89557
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs specific for C. neoformans capsular GXM can prolong survival, decrease fungal tissue burden, and reduce serum GXM levels in vivo (16, 17, 21, 22, 23, 24, 25, 26, 27, 28); facilitate effector cell function (25, 29, 30, 31, 32); and potentiate the efficacy of antifungal drugs (14, 15, 16, 18). Immunization with the GXM-TT produces high titers of GXM-specific Ab (19, 20, 33). Passive immunization studies using mAbs generated against the GXM-TT have demonstrated that 1) both protective and nonprotective Abs are elicited in response to this glycoconjugate (22, 34); and 2) major determinants of Ab efficacy are isotype (22) and epitope specificity (22, 34, 35). mAbs of the IgG1 isotype are highly protective (14, 15, 16, 17, 33), whereas mAbs of the IgG3 isotype have been shown to be poorly protective or disease enhancing (22, 36). mAbs bind with either a punctate or annular indirect immunofluorescence pattern to the C. neoformans capsule. mAbs that bind with a punctate pattern are consistently nonprotective, whereas those that bind with an annular pattern are either protective or nonprotective depending on their isotype and/or epitope specificity (34, 35). These studies indicate that the GXM-TT vaccine contains epitopes that elicit both protective and nonprotective Abs. The ideal vaccine would contain antigenic determinants that not only stimulate high Ab titers, but selectively elicit only those Abs that have protective characteristics. Identification of epitopes that elicit protective and nonprotective Abs is crucial to the design of such a vaccine.
mAbs possess unique epitope specificities and can thus be used to map
antigenic determinants and identify the type of Abs elicited. Anti-GXM
mAbs have been categorized into five epitope specificity groups,
designated I through V, based on Ig variable gene utilization;
specificity for capsular polysaccharide of C. neoformans
serotypes A, B, C, and D; and recognition of the mAb 2H1 anti-Id
(37). The IgG1 mAbs E1 (17) and 2H1 (22, 38) recognize two epitopes
that can elicit protective Abs. mAb E1 (17) defines group I based on
utilization of VHT15, ability to bind only serotype A GXM,
and inability to recognize the 2H1 anti-Id. Group II, defined by
mAb 2H1, includes mAbs 3C2, 339, and 471, which use VH7183;
bind serotype A, B, C, and D GXM; and recognize the 2H1 anti-Id.
Group III is defined by mAbs 339 and 1255 based on VH10
variable gene element utilization; ability to bind serotype A, B, C,
and D GXM; and inability to recognize the 2H1 anti-Id. mAb 302
defines group IV based on utilization of the VHVGam
variable region, ability to bind only serotypes A and D GXM, and
inability to recognize the 2H1 anti-Id (Table I
) (37).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The derivation and structural characterization of the IgG1 mAbs 2H1, 3C2, 301, 339, 439, 471, and 1255 have been described previously (33, 37, 39, 40). The IgA mAb 18G9 was derived from the same progenitor B cell, uses the same Ig variable gene elements, and has the same fine specificity as mAb 2H1 (38). The IgM mAb 13F1 uses the same Ig variable gene elements but has a different fine specificity relative to mAb 2H1 as a result of somatic mutation (33, 34, 38). mAb ascites was generated by growing hybridoma cells in the peritoneal cavity of pristane-primed BALB/c mice. mAbs 3C2, 302, and 439 were affinity purified on a protein A-Sepharose column; mAbs 339, 471, and 1255 were affinity purified using a GXM-AH-Sepharose column (41); and mAb 2H1 was affinity purified on a protein G column (Pierce, Rockford, IL). mAb concentrations were determined by A280 using an extinction coefficient of 1.42 (42).
Cryptococcal strain and capsular polysaccharide
All studies used the serotype D C. neoformans strain 24067 from the American Type Culture Collection (ATCC; Manassas, VA). C. neoformans ATCC 24067 was selected for these studies because all the mAbs involved bind serotype D GXM (37, 39, 43), we have extensive experience with this strain in mAb protection studies (22, 23, 27, 44), and serotype D strains are pathogenic for humans (45). ATCC 24067 was maintained at 4°C on Sabouraud’s dextrose agar (Difco, Detroit, MI) and grown at 30°C with shaking in Sabouraud’s dextrose broth (Difco). Unless indicated otherwise, logarithmic phase organisms (18–20 h of growth) were used for all experiments. Before use, organisms were washed three times with sterile PBS and counted using a hemacytometer. GXM was purified by cetyltrimethylammonium bromide precipitation (46).
Survival studies
A murine model of i.p. mAb administration and cryptococcal challenge was used (22). One milligram of purified mAb 3C2, 302, 339, 439, 471, or 1255 or 0.25 ml of sterile PBS was administered i.p. to each of 10 adult female A/JCr mice (The Jackson Laboratory, Bar Harbor, ME). Two hours following mAb administration, each mouse was infected i.p. with 1 x 107 C. neoformans. The challenge inoculum was confirmed by plating on Sabouraud’s dextrose agar. Mice were monitored twice daily.
ELISAs
Two ELISAs were used to compare the relative Ag binding characteristics of mAbs 2H1, 3C2, 302, 339, 439, 471, and 1255 in the presence of limiting mAb and GXM concentrations. To examine the effect of limiting GXM concentration, mAb at 10 µg/ml was added to microtiter plates (Corning 25801, Corning, NY) coated with serially diluted GXM. To examine the effect of mAb concentration, mAbs were serially diluted across microtiter plates coated with 10 µg/ml GXM. In both assays, mAb binding was detected with alkaline phosphatase-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL). Assays were developed with 1 mg/ml p-nitrophenyl phosphate (Sigma, St. Louis, MO), and the A405 was determined.
A mAb competition ELISA was used to confirm the epitope specificity grouping of the IgG1 mAbs 2H1, 3C2, 302, 339, 439, 471, and 1255. This assay used IgA mAb 18G9, which has the same epitope specificity as mAb 2H1 (33). A mAb of the IgA isotype was used to facilitate discrimination of the competing mAbs using isotype-specific reagents. The relative binding of each IgG1 mAb to microtiter plates coated with 10 µg/ml GXM in the presence and the absence of 2.5 µg/ml mAb 18G9 was detected using alkaline phosphatase-conjugated goat anti-mouse IgG1. The relative binding of mAb 18G9 to microtiter plates coated with 10 µg/ml GXM in the presence and the absence of 2.5 µg/ml of each IgG1 mAb was detected using alkaline phosphatase-conjugated goat anti-mouse IgA (Southern Biotechnology Associates). The assay was developed with 1 mg/ml p-nitrophenyl phosphate, and the A405 was determined. Competition between mAb 18G9 and IgG1 mAb pairs was considered to occur if the relative binding of either mAb was decreased by the presence of the other mAb. Noncompetition between mAb 18G9-IgG1 mAb pairs was considered to occur if the relative binding of neither mAb was decreased by the presence of the other mAb.
The relative apparent affinity constant of binding to GXM was determined for mAbs 2H1, 3C2, 302, 339, 439, 471, and 1255 by an Ag inhibition ELISA (47). The relative ability of each mAb to bind microtiter plates coated with 10 µg/ml GXM in the presence of various concentrations of competing GXM was detected with alkaline phosphatase-conjugated goat anti-mouse IgG1. The assay was developed using 1 mg/ml p-nitrophenyl phosphate, and the A405 was determined. The apparent affinity constant is defined as the reciprocal Ag concentration at which 50% maximal mAb binding occurs (47). A m.w. of 8 x 105 was used for GXM (2, 48). Although a GXM molecule is probably multivalent, and this method of calculating affinity constants assumes the ligand to be univalent, each of the apparent affinity values was calculated in the same manner and can thus be compared to one another.
Murine macrophage assays
The ability of mAbs 3C2, 302, 339, 439, 471, and 1255 to enhance
effector cell activity (phagocytosis and killing) was studied using the
murine macrophage-like cell line, J774.16 (49). J774.16 cells yield
results comparable to primary murine or human phagocytes in the
evaluation of anti-cryptococcal mAb activity in vitro (25, 29).
J774.16 cells were maintained in medium containing DMEM (Mediatech,
Washington, DC) supplemented with 10% NCTC-109 (Mediatech), 10%
heat-inactivated FCS, and 1% nonessential amino acids (Mediatech). For
both phagocytosis and killing assays, J774.16 cells were plated at a
density of 8 x 104/well on 96-well tissue culture
plates (Falcon 3072, Becton Dickinson, Franklin Lakes, NJ), stimulated
with 500 U of murine rIFN-
(Genzyme, Cambridge, MA)/ml of medium,
and incubated overnight at 37°C. The medium was then replaced with
fresh medium containing 500 U of rIFN- and 3 µg of
Escherichia coli serotype O127:B8 LPS (Sigma)/ml with and
without purified mAb. C. neoformans (1.6 x
104 or 9.0 x 104) was added to each well
to produce an E:T cell ratio of 10:1 or 2:1 for CFU and phagocytosis
assays, respectively. Cocultures of C. neoformans and
J774.16 cells were incubated at 37°C and then processed as described
below for either CFU or phagocytosis determinations.
Killing assays were performed as described previously (16, 50) at mAb concentrations of 0 and 5 µg/ml. Briefly, following incubation of viable C. neoformans with J774.16 cells for 2 h at 37°C, supernatants of each well were collected, and the cells within each well were lysed by incubation at room temperature for 45 min in the presence of 0.1 ml of sterile distilled H2O. The lysate was vigorously aspirated and ejected several times with a micropipette to complete cell disruption. An additional 0.1 ml of sterile PBS was used to rinse each well. The supernatant, lysate, and rinse from each well were pooled, vortexed, diluted, vortexed again, and then spread onto a Sabouraud’s dextrose agar plate to determine the number of CFU per well. This method circumvents artifactual reduction in CFU due to agglutination by a combination of dilution below the agglutination threshold and mechanical disruption (16, 50).
Phagocytosis assays were used to study the effect of mAb concentration, coculture length, and presence of A/JCr mouse serum on attachment and ingestion of C. neoformans by J774.16 cells. The attachment index was defined as the number of extracellular attached organisms divided by the number of J774.16 cells within a field. The ingestion index was defined as the number of intracellular organisms divided by the number of J774.16 cells within a field. Attachment and ingestion indexes were determined in the presence of mAbs 3C2, 302, 339, 439, 471, and 1255 at concentrations of 0, 1.25, 2.5, 5, and 10 µg/ml following 2 h of coincubation at 37°C with viable C. neoformans. The same conditions were used to further study the effect of mAbs 3C2 and 302 at concentrations of 0, 1, 10, and 100 µg/ml. Coculture length was studied using heat-killed C. neoformans incubated in the presence of 2.5 µg/ml mAb 3C2 or 302 for 2, 4, 6, or 8 h at 37°C. Heat-killed organisms were used to eliminate growth during the prolonged coincubation period. Organisms were heat killed via incubation at 55°C for 15 min; plating on Sabouraud’s dextrose agar confirmed <0.04% viability. The effect of A/JCr mouse serum was examined by incubating cocultures of J774.16 and viable C. neoformans in the presence and the absence of 1.25 µg/ml mAb 3C2 or 302 with 25% (v/v) heat-inactivated or normal A/J mouse serum for 2 h at 37°C. Following the length of coculture indicated for each study, the medium within each well was removed and discarded, and the remaining cellular monolayer was fixed with ice-cold methanol. The wells were then washed several times with PBS and stained with a 1/10 dilution of Giemsa (Sigma). The average attachment and ingestion indexes were obtained by counting a minimum of eight fields (two fields per well) within each experimental group using an Olympus light microscope (Olympus, New Hyde Park, NY) at a final magnification of x400. The ability to distinguish attached and ingested organisms by light microscopy was confirmed by fluorescent staining of cocultured organisms with diaethanol (Uvitex 3BSA, Ciba-Geigy, West Caldwell, NJ) (51).
Agglutination
mAb-mediated agglutination of C. neoformans was examined using a 96-well microtiter plate (Corning 25801) containing 1 x 106 organisms/well in the presence of mAb 2H1, 3C2, 302, 339, 439, 471, or 1255 serially diluted over a concentration range of 0.025 to 50 µg/ml in PBS/1% BSA. The agglutination endpoint was defined as the lowest mAb concentration at which agglutination (clumping of organisms) was evident by light microscopic examination (52).
Indirect immunofluorescence
The indirect capsular immunofluorescence pattern of mAbs 3C2, 302, 339, 439, 471, 1255, 2H1, and 13F1 was examined by scanning laser confocal microscopy. Stationary phase C. neoformans were immobilized on poly-L-lysine-coated slides (Sigma) by air-drying. No apparent differences in the immunofluorescence patterns of suspended vs immobilized yeast were observed. Thus, immobilized organisms were used to obtain confocal images due to inherent technical difficulties of imaging migrating cells. Adhered organisms were incubated 3 to 5 h at room temperature in the presence of 100 µg/ml mAb in PBS/1% BSA. Adhered organisms were rinsed with PBS and then incubated with a 1/100 dilution of FITC-conjugated goat anti-mouse IgG1 or IgM (Southern Biotechnology Associates). Adhered organisms were then rinsed with PBS and prepared for viewing by the addition of mounting solution (50% PBS, 50% glycerol, and 0.05 M N-propyl gallate) and a coverslip. Samples were viewed under oil immersion at x600 final magnification with a Bio-Rad MRC 600 scanning laser confocal microscope (Bio-Rad, Hercules, CA). Images were generated by maximum projection of 8 to 14 Z-series sections.
Statistical analyses
Survival data were analyzed by both parametric (log-rank test) and nonparametric (Wilcoxon test) methods using True Epistat (standard version, copyright 1991, BioMedware, Ann Arbor, MI). Both methods yielded comparable p values. In vitro CFU and phagocytosis data were analyzed using the SigmaStat Statistical Analysis System (version 1.01, copyright 1992, Jandel, San Rafael, CA). In vitro CFU and phagocytosis data were initially analyzed by nonparametric ANOVA using the Kruskal-Wallis test. Multiple group comparisons were performed using either Dunn’s method or Student-Newman-Keuls test. Depending on whether the data were normally distributed or not, pairwise comparisons were performed using the Mann-Whitney rank-sum test and/or Student’s t test to obtain p values. Comparisons with p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The binding of mAbs 3C2, 302, 339, 439, 471, and 1255 to GXM was
compared with that of mAb 2H1 by direct Ag binding, Ag inhibition, and
mAb competition ELISAs (Figs. 1 and
2). Figure 1, A and
B, shows the results of two direct Ag binding ELISAs.
The curves presented in Figure 1A show each serially
diluted mAb bound similarly to microtiter plates coated with 10 µg/ml
GXM. Figure 1B illustrates the binding of a constant
amount of each mAb to microtiter plates coated with serially diluted
GXM. In contrast to the results in Figure 1A, those in
B indicate there are two groups of mAbs based
on binding limiting amounts of immobilized GXM. mAbs 3C2, 439, and 471
bound GXM similarly to mAb 2H1, with 50% maximum binding between 0.08
and 0.2 µg/ml GXM. mAbs 302, 339, and 1255 represent a higher
affinity group with 50% maximum binding at 0.02 to 0.04 µg/ml
immobilized GXM. Figure 1C illustrates the results of an
Ag competition ELISA in which mAb binding to microtiter plates coated
with 10 µg/ml GXM is inhibited by soluble GXM. Apparent affinity
constants of each mAb for GXM, calculated using the concentration of
soluble GXM present at 50% maximal mAb binding and a GXM m.w. of
0.8 x 106 g/mol (2, 48), were between 5 and 26
x 107 M-1 (Table I
) with a relative apparent
affinity constant pattern of mAb 1255 > 3C2 > 2H1 >
339 > 439 > 471 > 302.
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and Fig. 2). This
study was facilitated by utilization of the IgA mAb 18G9, which was
derived from the same progenitor B cell as the group II IgG1 mAb 2H1,
has the same epitope specificity as mAb 2H1 for GXM isolated from the
serotype A strain of C. neoformans ATCC 24064 (33), and
competes with mAb 2H1 for binding to GXM (Table I). This assay relies
on detection of binding using isotype-specific reagents; thus, it was
necessary to use a mAb that relative to mAb 2H1, differed in isotype
but had identical specificity. Competition between mAb 18G9 and IgG1
mAb pairs was considered to occur if the relative binding of either mAb
was decreased by the presence of the other mAb. Results are summarized
in Table I; Figure 2 shows examples of competition and noncompetition
between mAb 18G9 and representative IgG1 mAbs. The group II mAbs 3C2,
439, and 471 competed with mAb 18G9 for binding to GXM. Figure 2, A and B, depicts competition between mAbs 3C2 and
18G9. In contrast, the group III mAbs 339 and 1255 and the group IV mAb
302 did not compete with mAb 18G9 for binding to GXM. Figure 2, C and D, depicts noncompetition between mAbs 339
and 18G9.
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The relative abilities of mAbs 3C2, 302, 339, 439, 471, and 1255
to agglutinate C. neoformans ATCC 24067 were studied in
vitro. For these mAbs the agglutination end point ranged from 0.8 to
6.25 µg/ml (Table I). We consider agglutination end points to be
significantly different if there is at least a fourfold difference in
end point concentration. The agglutination end point of mAb 3C2 was
significantly lower than that of mAbs 471, 1255, 302, and 439, and the
agglutination end point of mAb 439 was significantly greater than that
of mAbs 3C2 and 339. Thus, the relative efficacies of these mAbs in
promoting agglutination were mAb 3C2 < 339 < 471, 1255,
302 < 439.
The capsular immunofluorescence patterns of mAbs 3C2, 302, 339, 439,
471, and 1255 were compared with the patterns previously described for
mAbs 2H1 and 13F1 (Fig. 3) (34). Images
generated by maximum projection of serial optical sections revealed
that mAb binding was homogeneous over the entire surface of the
organism (Fig. 3A). Single optical sections revealed that
each mAb had an annular immunofluorescent pattern similar to that of
mAb 2H1 (Fig. 3B). None of the mAbs studied exhibited the
characteristic punctate pattern of mAb 13F1. The immunofluorescent
(Fig. 3B) and phase contrast (Fig. 3C) images of
single optical sections were superimposed to localize mAb binding with
respect to the cell wall and capsule boundary (Fig. 3D). Each mAb appeared to bind predominantly within the
capsule of the organism.
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Eight groups of 10 adult A/J mice each were challenged i.p. with
107 C. neoformans 2 h following i.p.
administration of PBS or 1 mg of affinity-purified mAb (Fig. 4). Passive immunization with mAbs 3C2,
302, 339, 439, 471, and 1255 resulted in significant prolongation of
survival relative to that of the PBS control (p
< 0.05). Mice given mAbs 3C2, 302, 339, 439, 471, or 1255 lived an
average of 36.6, 33, 25.5, 24.9, 17, or 22.6 days, respectively,
compared with 10 days for PBS control mice (Table I). Significant
differences (p < 0.05 for all comparisons)
were observed in the relative abilities of each mAb to prolong
survival, with 3C2 > 439 > 471 and 302 > 471, 1255.
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The murine macrophage-like cell line, J774.16 (49), was used to
determine whether mAbs 3C2, 302, 339, 439, 471, and 1255 potentiate the
antifungal activity of effector cells. Each mAb significantly enhanced
the fungicidal/fungistatic activity of rIFN-- and LPS-stimulated
J774.16 cells cocultured with C. neoformans at a 10:1 E:T
cell ratio for 2 h relative to the control (no mAb)
(p < 0.001). In the presence of mAb 3C2, 302,
339, 439, 471, or 1255 the number of CFU (x103) was
9.45 ± 0.86, 9.77 ± 1.35, 11.20 ± 1.46, 11.17 ±
1.44, 9.82 ± 1.56, or 12.60 ± 2.49, respectively, relative
to 47.44 ± 4.8 x 103 CFU for the no mAb
control.
J774.16 cells were also used to study the effect of mAbs 3C2, 302, 339,
439, 471, and 1255 on attachment and ingestion of C.
neoformans by effector cells. Figure 5, A and B, shows
the results of a study in which rIFN- and LPS stimulated J774.16
cells were cocultured with C. neoformans at a 2:1 E:T cell
ratio for 2 h in the presence of each mAb at 0, 1.25, 2.5, 5, or
10 µg/ml. Figure 5A shows the attachment indexes at each
mAb concentration. mAbs 302, 439, and 471 significantly enhanced
attachment at each mAb concentration. For mAbs 3C2, 339, 439, and 471,
attachment was inversely proportional to the mAb concentration. mAbs
3C2, 339, and 1255 significantly enhanced attachment at the lowest
concentration (1.25 µg/ml) examined. Figure 5B shows the
ingestion indexes (ratio of intracellular organisms to J774 cells) at
each mAb concentration. Relative to the no mAb control, each mAb
significantly enhanced ingestion at each concentration studied
(p < 0.05 for all comparisons). However,
differences were observed in the relative abilities of particular mAbs
to promote ingestion. mAbs 3C2, 339, and 1255 were equally effective at
each mAb concentration tested (p > 0.05 for
all comparisons). However, for mAbs 302, 439, and 471, ingestion was
dependent on mAb concentration. Even at the highest mAb concentration
studied (10 µg/ml), the ingestion indexes of mAbs 302 and 471 were
significantly lower than the ingestion indexes of mAbs 3C2, 339, and
1255 at the lowest mAb concentration studied (1.25 µg/ml;
p < 0.05 for all comparisons). Figure 5, C
and D, shows the results of an experiment designed
to determine whether, given additional coincubation time, rIFN-- and
LPS-stimulated J774.16 cells in the presence of mAb 302 could
efficiently ingest attached organisms. Heat-killed C.
neoformans were incubated at a 2:1 E:T cell ratio for 2, 4, 6, or
8 h in the presence and the absence of 2.5 µg/ml mAb 3C2 or 302.
In the presence of either mAb 3C2 or 302, attachment and ingestion of
C. neoformans by J774.16 cells occurred within 2 h of
coincubation; however, prolongation of coincubation did not
consistently result in increased attachment or ingestion of organisms
(p2; Fig. 5, C and
D). Although addition of either mAb 3C2 or 302 significantly
enhanced both attachment and ingestion of organisms
(p1; Fig. 5, C and
D), mAb 302 was more efficient than mAb 3C2 at enhancing
attachment of organisms (Fig. 5C; p < 0.001
for comparison at each time point), whereas mAb 3C2 was more efficient
than mAb 302 at enhancing the ingestion of organisms (Fig. 5D; p < 0.001 for comparison at each time
point).
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, A and B,
shows the attachment and ingestion indexes, respectively, at mAb
concentrations of 0, 1, 10, and 100 µg/ml. Both mAb 3C2 and 302
significantly enhanced both attachment and ingestion at each
concentration. The attachment indexes for mAb 302 were significantly
greater relative to mAb 3C2 at 1 and 10 µg/ml
(p < 0.001 for both comparisons); however, at
100 µg/ml no difference in attachment was observed
(p = 0.995; Fig. 6A). mAb 302
exhibited a significant increase in ingestion index between 1 and 10
µg/ml (p < 0.001); however, no significant
increase was observed between 10 and 100 µg/ml
(p = 0.292; Fig. 6B). The ingestion
indexes for mAb 302 were significantly lower relative to mAb 3C2 at
each concentration (Fig. 6B). The ingestion index at 100
µg/ml mAb 302 was comparable to that at 1 µg/ml mAb 3C2
(p = 0.758).
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and
8). Figure 7, A and
B, show that in the absence of specific mAb, normal, but not
heat-inactivated, A/J serum alone facilitated attachment, but not
ingestion, of C. neoformans to J774.16 cells. In the
presence of either mAb 3C2 or 302, attachment, but not ingestion, was
significantly enhanced relative to that in the presence of mAb alone by
the addition of heat-inactivated A/J serum
(p1; Fig. 7, A and
B). In contrast, in the presence of either mAb 3C2 or 302,
both attachment and ingestion were enhanced by the addition of normal
A/J serum (p3; Fig. 7, A
and B). Heat-inactivated A/J serum had no effect on
ingestion in the presence of mAb 3C2 (p2
= 0.969; Fig. 7B); however, in the presence of mAb 302 and
heat-inactivated A/J serum, ingestion was significantly reduced
relative to that in the presence of mAb 302 alone
(p2 < 0.001; Fig. 7B). These
results can be visualized in Figure 8. Figure 8, A and
B, shows that in the absence of mAb or normal A/J serum, low
levels of attachment occurred, but ingestion did not. The presence of
normal A/J serum alone facilitated ingestion and increased attachment
(Fig. 8C).Figure 8, D–F, illustrates
that mAb 3C2 alone efficiently promoted ingestion, which was enhanced
by the addition of normal A/J serum. Figure 8G illustrates
that although mAb 302 alone promoted attachment, minimal ingestion was
observed. However, the addition of normal A/J serum enhanced ingestion
in the presence of mAb 302 (Fig. 8I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (37). Using these mAbs we sought to 1) characterize
their Ag binding properties; 2) determine whether IgG1 mAbs
representing groups III and IV could prolong survival against lethal
cryptococcal challenge; and 3) examine the relative efficacies of group
II, III, and IV IgG1 mAbs in mediating effector cell activity in vitro.
The serotype D strain, C. neoformans ATCC 24067, was
selected for our studies because all the IgG1 mAbs involved bind
serotype D GXM (37, 43), and we have used this strain extensively to
study the efficacy of group II mAbs (22, 23, 24, 27, 50). To confirm the validity of the previous epitope specificity grouping, which was based on serotype reactivity and variable region utilization (37), competition assays were performed between each IgG1 mAb and the IgA mAb 18G9. mAb 18G9 is both structurally related to and competes with mAb 2H1. The finding that mAbs 3C2, 439, and 471, but not mAbs 339, 1255, and 302, compete with mAb 18G9 corroborate the previous finding that group II, but not group III and IV, mAbs recognize the mAb 2H1 Id and support the previous grouping of these IgG1 mAbs (37). Direct Ag binding assays confirmed that each of these mAbs bound GXM; however, differential binding characteristics were observed. Each mAb appeared to bind similarly when serially diluted in the presence of a constant amount of immobilized GXM. However, in the presence of limiting amounts of immobilized GXM, the mAbs separated into two groups based on the amount of immobilized GXM present at 50% maximal binding. Relative to the group II mAbs 2H1, 3C2, 439, and 471, the group III and IV mAbs 302, 339, and 1255 had greater reactivity in the presence of low concentrations of immobilized GXM, implying that the group III and IV mAbs may have a higher affinity for serotype D GXM than the group II mAbs. The relative apparent affinity pattern (1255 > 3C2 > 2H1 > 339 > 439 > 471 > 302), however, does not correlate with these differences. Instead, the epitope recognized by the group II mAbs is probably not as available, due to decreased accessibility or density, as the epitopes recognized by the group III and IV mAbs.
The abilities of the IgG1 mAbs 3C2, 302, 339, 439, 471, and 1255 to prolong survival were examined in a murine model of lethal i.p. cryptococcal infection. Relative to the PBS control, each mAb significantly prolonged survival against challenge with C. neoformans ATCC 24067. This observation not only confirms previous observations that group II IgG1 mAbs are protective (22, 27, 38), but also demonstrates that IgG1 mAbs representing groups III (339, 1255) and IV (302) are protective. Significant differences in survival prolongation were observed with regard to the group II mAbs, in which 3C2 > 439 > 471. These differences correlated with the relative apparent affinity pattern for these mAbs (3C2 > 439 > 471), suggesting that affinity may influence the ability of a class II mAb to prolong survival. This observation is consistent with the demonstration that bactericidal activity of serum Abs against Haemophilus influenzae type b capsular polysaccharide is associated with Ab affinity (53, 54). Although a correlation between survival prolongation and affinity was observed for the group II mAbs, such a correlation does not exist between mAbs from different groups, suggesting both variable gene utilization (i.e., epitope recognition) and affinity contribute toward mAb efficacy in vivo. Epitope recognition has been shown to be an important factor for in vivo efficacy of anti-cryptococcal mAbs. The IgM mAbs 12A1 and 13F1 recognize separate GXM epitopes with similar apparent affinities, and yet mAb 12A1 prolongs survival, whereas mAb 13F1 does not (34, 35). The finding that each mAb exhibited a homogeneous annular capsular immunofluorescence pattern supports the previous observation that mAbs that display an annular immunofluorescence pattern with a particular C. neoformans strain prolong survival against the homologous strain (34, 35).
Group II anti-cryptococcal mAbs have been shown to enhance the function of effector cells (29, 44, 55) which likely have a primary role in host defense (56). The relative abilities of group II, III, and IV IgG1 mAbs to enhance phagocytic and fungistatic/fungicidal activities were examined using the murine macrophage-like cell-line, J774.16 (49). Phagocytosis is a process that involves particle attachment followed by ingestion (4). Phagocytic indexes have previously been shown to be relatively insensitive compared with ingestion indexes with regard to discriminating between the quality of mAb-mediated C. neoformans-J774.16 interactions (30). Thus, we separated the components of phagocytosis and reported attachment and ingestion indexes. The abilities of the IgG1 mAbs 3C2, 302, 339, 439, 471, and 1255 to mediate the attachment and ingestion phases of C. neoformans by J774.16 cells were influenced by both mAb concentration and affinity. The high affinity mAbs 3C2, 339, and 1255 facilitated efficient ingestion even at the lowest mAb concentration examined (1.25 µg/ml), whereas ingestion increased in a concentration-dependent manner in the presence of the lower affinity mAbs 302, 439, and 471. In the presence of these lower affinity mAbs, a greater proportion of the organisms was attached to the extracellular surfaces of the phagocytes, whereas in the presence of the higher affinity mAbs, a greater proportion of the organisms was ingested, suggesting that mAb affinity has a role in determining whether organisms undergo ingestion.
The high and low affinity mAbs, 3C2 and 302, respectively, were used to further study the observed differences in attachment and ingestion. Using these two mAbs, the effects of coculture length, complement, and increased mAb concentration were examined. Prolongation of coculture length did not facilitate enhanced ingestion or attachment of heat-killed organisms in the presence of either mAb. Ingestion of C. neoformans occurred within 2 h of introduction to J774 cells; organisms that were attached at 2 h remained attached and did not eventually become ingested. Addition of A/J mouse serum significantly enhanced both attachment and ingestion of organisms in the presence of mAbs 3C2 and 302, but did not abrogate the difference in ingestion observed in the presence of mAb 3C2 vs 302. The increase in ingestion was attributable to the presence of complement, since heat-inactivated serum did not enhance ingestion. Even at high mAb concentrations (100 µg/ml), the phagocytic index of mAb 302 was less than that of mAb 3C2, suggesting that mAb affinity does not solely determine the efficiency with which opsonized particles are ingested.
The "zipper theory" of phagocytosis proposes that attachment involves localized particle-phagocyte contact, whereas ingestion requires extensive interactions that facilitate circumferential enclosure of the particle by the phagocyte pseudopodia (57, 58, 59). Our findings are consistent with this proposal, in that we have demonstrated that although only minimal amounts of mAb are required for attachment to occur, ingestion requires that C. neoformans be opsonized with a greater density of mAb. The ability of J774.16 Fc receptors to interact with the mAb Fc regions may also be determined by where the mAb binds within the C. neoformans capsule. Indeed, ingestion of C. neoformans by J774.16 cells is less efficient in the presence of the IgM mAb 13F1, which exhibits a punctate pattern of capsular immunofluorescence, than in the presence of the IgM mAb 12A1, which exhibits an annual pattern of capsular immunofluorescence (35). Although mAbs 3C2 and 302 both exhibit an annular pattern of immunofluorescence, based on Ig gene utilization and mAb competition assays, each recognizes a different epitope within GXM. Differences in both epitope location and affinity probably contribute to the efficiency with which C. neoformans is phagocytosed when opsonized by these mAbs.
Each of the IgG1 mAbs significantly enhanced the fungistatic/fungicidal activity of J774.16 cells, demonstrating that similar to group II mAbs (30, 60), group III and IV mAbs can enhance the activity of murine macrophages. mAb 3C2, which has the highest relative affinity and is one of the most efficient at enhancing ingestion, was the most effective at enhancing J774.16 fungistatic/fungicidal activity. However, differences observed between the other mAbs did not necessarily correlate with ingestion indexes or affinity. Although the low affinity mAbs 302, 439, and 471 did not facilitate high levels of ingestion, they did facilitate high levels of attachment. The ingestion phase of phagocytosis is a prerequisite for intracellular killing of pathogens; however, extracellular mechanisms of effector cell killing have also been shown to be effective against C. neoformans (55, 61, 62, 63). Even though a low affinity mAb may not be very effective at enhancing ingestion, by facilitating attachment, the target is brought into proximity with the effector cell, which may increase the efficacy of extracellular fungicidal/fungistatic mechanisms. Furthermore, the production of extracellular mediators, such as nitric oxide and superoxide, has been shown to increase in the presence of anti-cryptococcal mAbs and GXM or intact C. neoformans (60, 62).
In summary, our results 1) demonstrate the presence of two additional GXM epitopes (recognized by group III and IV mAbs) associated with prolongation of survival and enhanced effector cell activity, 2) indicate that differences in affinity affect the ability of mAbs to prolong survival in vivo and mediate the attachment and ingestion phases of phagocytosis by effector cells in vitro, 3) suggest that both extracellular and intracellular mechanisms are involved in mAb-mediated effector cell fungicidal/fungistatic activity, and 4) support the association between mAb protective efficacy and binding to C. neoformans with an annular capsular immunofluorescence pattern. Previous studies have shown that IgG1 anti-GXM mAbs can prolong survival (17, 22, 27, 36, 50). This study extends those findings by demonstrating functional differences for IgG1 mAbs that differ in variable gene utilization, epitope specificity, and affinity. Hence, the efficacies of passive and active immunization strategies are likely to depend on both variable and constant region characteristics of GXM-binding mAbs.
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2 Address correspondence and reprint requests to Dr. Jean Mukherjee at the current address, Department of Biomedical Sciences, Division of Infectious Diseases, Tufts University School of Veterinary Medicine, Building 20, 200 Westboro Road, North Grafton, MA 01536.
3 Abbreviations used in this paper: GXM, glucuronoxylomannan; GXM-TT, GXM tetanus toxoid.
Received for publication March 3, 1998. Accepted for publication May 29, 1998.
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