INFECTION AND IMMUNITY, Nov. 1992, p. 4534-4541 0019-9567/92/114534-08$02.00/0 Copyright C 1992, American Society for Microbiology

Vol. 60, No. 11

Protective Murine Monoclonal Antibodies to Cryptococcus neoformans JEAN MUKHERJEE,1 MATTHEW D. SCHARFF,1 AND ARTURO CASADEVALL2* Department of Cell Biology' and Departments of Medicine (Division of Infectious Diseases) and Microbiology and Immunology,2 Albert Einstein College of Medicine, 1300 Mormis Park Avenue, Bronx, New York 10461 Received 11 June 1992/Accepted 25 August 1992 Several murine monoclonal antibodies (MAbs) specific for the capsular glucuronoxylomannan of Cryptococcus neoformans were studied for their capacity to confer protection when passively administered to lethally infected mice. The MAb group studied recognized at least three distinct epitopes and included immunoglobulin M (IgM), IgG3, IgGl, and IgA isotypes. The protection model used A/J and BALB/c mice infected intraperitoneally with 108 cryptococci. The MAbs were administered either immediately preceding or, in one experiment, 24 to 48 h prior to infection. Protective efficacy was assessed by the ability of passively administered MAbs to prolong the survival of lethally infected mice. Three IgM MAbs, each of which recognized a distinct epitope, were able to prolong survival of lethally infected mice to different extents. A set of IgM, IgG3, IgGl and IgA MAbs which utilize the same immunoglobulin gene elements and were derived from the same B-cell clone exhibited significant class differences in protective efficacy with IgA, IgGl > IgM > IgG3. The results confirm that protective MAbs against C. neoformans capsular polysaccharide exist and strongly suggest that both epitope specificity and isotype are important determinants of protective efficacy.

Cryptococcus neoformans is a ubiquitous fungus that can cause life-threatening human infections. Immunocompromised individuals, including those with AIDS, are at particular risk (17), and approximately 10% of patients with AIDS develop meningoencephalitis (10, 17, 68). C. neoformans has a polysaccharide capsule which is a determinant of virulence (39) that is shed and accumulates in blood and tissues during human infections (1, 2). Cell-mediated mechanisms are believed to play the major role in host resistance to C. neoformans (5, 11, 32, 37, 44, 45, 48, 60). In contrast, the role of antibody in resistance is less clear. The fact that patients with serum anticapsular antibodies have a better prognosis than those without (19) suggests a role for antibody in protection. However, with the exception of rare cases (17, 35), cryptococcal infections are not associated with human antibody deficiencies. B-celldeficient mice are also not especially susceptible to cryptococcal infection (49). Studies by Graybill et al. (33) and Gadebusch (28), however, demonstrated that passive administration of polyclonal rabbit immune sera to mice conferred some protection from subsequent challenge with cryptococci. Louria and Kaminiski (46) reported that passive antibody administration did not affect either survival time or the number of cryptococci in brain tissue. Passive therapy with monoclonal antibodies (MAbs) in murine models of lethal C. neoformans infection has also yielded contradictory results. Dromer et al. (24) reported that passive administration of the anticryptococcal immunoglobulin Gl (IgGl) MAb El produced a sixfold increase in survival time of lethally infected complement-deficient DBA/2 mice. In addition, this MAb was also effective in potentiating the activity of the antifungal drug amphotericin B (22, 23). In contrast, Sanford et al. (59) used IgG2a and IgG2b isotype switch variants of another IgGl MAb in passive therapy of lethally infected mice, but none of the antibodies prolonged survival. *

Immunization studies have also yielded contradictory results, since immunization with highly immunogenic cryptococcal polysaccharide conjugates has not been protective in mice (30) but immunization with fractionated capsular components has provided some protection (31). In contrast to the conflicting in vivo data, the role of antibody in enhancing cellular clearance of cryptococci in vitro has been amply demonstrated. Specific anticapsular antibodies enhance natural killer cell inhibition of C. neoformans growth (47, 51). Anticryptococcal antibody enhances phagocytosis and killing by murine peritoneal cells (40, 43) and stimulated human peripheral blood monocytes (44) and also facilitates killing by nonadherent peripheral blood mononuclear cells, including natural killer cells (16, 18, 47). The finding that many AIDS patients lack specific anticryptococcal IgG antibodies (21) raises the possibility that a defect in the humoral immunity of these patients contributes to their marked susceptibility for C. neoformans infections. The overall aim of our work was to identify protective anticryptococcal antibodies which could potentially be used in the prevention and therapy of human cryptococcosis. We have examined the protective efficacy of several murine MAbs that differ in epitope specificity and isotype to determine the properties of anticryptococcal MAbs that are important for protection. The results indicate that both protective and nonprotective anticryptococcal antibodies exist and strongly suggest that antibody isotype and epitope specificity are important determinants of protective efficacy. (The data in this paper are from a thesis to be submitted by J.M. in partial fulfillment of the requirements of the degree of doctor of philosophy in the Sue Golding Graduate Division of Medical Science, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y.)

MATERIALS AND METHODS Abbreviations. Abbreviations used are as follows: GXM, glucuronoxylomannan; CDR, complementarity-determining

Corresponding author. 4534

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TABLE 1. Summary of MAb isotype, serotype recognition, and variable-gene usage Serotype binding MAb

7B13 4H3 21D2r 2D10 3E5 2H1 18G9 17E12

Isotype

IgM(k) IgG3(A) IgM(K) IgM(K) IgG3(K) IgGl(K) IgA(K) IgGl(K)

A

B

C

D

_ -

_ -

_ -

+ + + + + +

+ + + + + +

+

+ + + + + + + +

+ + +

+ +

VH

JH

VL

JL

441 441 7183:50.1

3 3 2 2 2 2 2 2

VX2 Vx1 VK5.1 VKS.1

JX2 JA1

7183b 7183 7183 7183 7183

a MAb 21D2 was originally reported to recognize only serotypes A and D (8) but has subsequently been found to recognize VH7183 is a member of the 7183 VH family different from the VH7183:50.1 member used by MAb 21D2.

VK5.1 VKS.1 VKS.1 VKS.1

JKI JK1 JK1 JK1 JK1 JK1

all four serotypes (6).

b

region; CNPS, C. neoformans capsular polysaccharide; CNPS-D, serotype D capsular polysaccharide; VH, heavy chain variable region; PBS, 0.02 M phosphate-buffered saline, pH 7.2; i.p., intraperitoneal; and ELISA, enzymelinked immunosorbent assay. C. neoformans strains. Two strains were used in protective studies. Serotype D (ATCC 24067) was obtained from the American Type Culture Collection, Rockville, Md. Strain GH was isolated from the cerebrospinal fluid of an AIDS patient with cryptococcal meningitis at Bronx Municipal Hospital Medical Center. Strain GH expresses both serotype A and D epitopes (8). The strains were maintained in Sabouraud dextrose agar slants at 4°C. Cultures for protection experiments were grown in Sabouraud broth at 37°C with moderate shaking for 4 to 5 days. The yeast cells were collected by centrifugation and washed three times in sterile PBS before inoculation of mice. Inoculum size was based on hemacytometer counts which were confirmed by plating on Sabouraud agar. Plating efficiencies varied from 30 to 60%. We attribute the relatively low plating efficiencies to the fact that using the hemacytometer we counted budding organisms twice and a single colony will result from a budding organism when plated on agar. MAbs. The MAbs used in this study have been described previously (6, 8). Ascitic fluid was prepared in the Hybridoma Facility of the Cancer Center at our institution by injection of 107 hybridoma cells into the peritoneum of pristane-primed BALB/c mice. The concentration of antibody of the relevant isotype in ascitic fluids was determined by an ELISA relative to standards of the same isotype and of known concentration. Ten MAbs, including IgM, IgG3, IgGl, and IgA isotypes, were used in protection experiments. Table 1 lists the MAbs used and their isotypes, serotype specificity, and variable-gene usage. All bind the GXM fraction of cryptococcal polysaccharide. MAbs 7B13 and 4H3, generated in response to infection with strain GH, have A light chains and bind only to CNPS-D. On the basis of serotype specificity and molecular structure, MAbs 7B13 and 4H3 bind an epitope different from that bound by the others (8). MAbs 2D10, 3E5, 2H1, 18G9, 17E12, and 21D2 bind to polysaccharides of the four main serotypes (6, 8). The 2D10, 3E5, 2H1, and 18G9 MAbs were generated in response to immunization with a conjugate of serotype A (ATCC 24064) GXM and tetanus toxoid, and all recognize the same epitope since (i) they have similar profiles of reactivity with the various serotypes, (ii) they share the same gene elements (including CDR3) in their construction (50), (iii) their respective hybridomas trace their ancestry to one B cell (50), and (iv) the representative MAbs 2H1 and 18G9 do

not bind de-O-acetylated GXM and competitively inhibit each other's binding to CNPS (6). It is noteworthy that 2D10, 3E5, 2H1, and 18G9 have somatic mutations, some of which result in replacement substitutions and, hence, their variable regions have some primary amino acid sequence differences. MAb 17E12 binds the same antigenic determinant and is structurally very similar to the 2D10 family members but differs in that it was derived from a different parental B cell and has amino acid sequence differences within its CDR3 (50). MAb 21D2, generated in response to infection with strain GH, recognizes an epitope different from that recognized by the 2D10 group, since it binds de-O-acetylated GXM and uses a different VH7183 variable gene element (VHSO.1) which confers a different antigen binding site sequence (8). In summary, our MAbs bind to at least three distinct epitopes which are defined by 7B13 (and 4H3), 2D10 (and 3E5, 2H1, 18G9, and 17E12), and 21D2. To determine the relative amount of MAb needed for agglutination of cryptococci, MAbs 2D10, 3E5, 2H1, and 18G9 were serially diluted across a microtiter plate blocked with 1% bovine serum albumin and 0.5% horse serum. A constant number of serotype D (ATCC 24067) C. neoformans cells were added to each well, and the plate was gently agitated for 1 h at room temperature. Relative agglutination was visually assessed independently by the three authors by determining the lowest MAb concentration needed for visible clumping of cryptococci to occur. Animal protection experiments. Female A/J and BALB/c mice were obtained from the National Cancer Institute. Mice were given MAb as ascitic fluid via i.p. injection. Each experiment utilized 10 mice per MAb or control group. Control groups received i.p. injections of PBS or NSO ascitic fluid. NSO is the nonproducing mouse myeloma fusion partner of our hybridomas. Hybridoma ascitic fluid concentrations of antibody of the relevant isotype were as follows: 2D10, 1.6 mg of IgM per ml; 2H1, 5.1 mg of IgGl per ml; 3E5, 2.8 mg of IgG3 per ml; 18G9, 4.7 mg of IgA per ml; and 17E12, 4.8 mg of IgGl per ml. Ascitic fluid from the NSO myeloma contained polyclonal antibody concentrations of IgM, IgG3, IgGl, and IgA of 35, 10, 1, and 2 p,g/ml, respectively. On the basis of these values, it is assumed that most of the relevant isotype in the ascitic fluid is the MAb. The MAbs were administered either following a time lapse of about 30 min or, in one experiment, in two doses 24 and 48 h preceding i.p. infection with 108 cryptococci. Mice were observed daily. Results of each experiment were analyzed by a log-rank analysis program written by Chee Jen Chang using the SAS statistical package. Serum polysaccharide. The relative concentration of circu-

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INFECT. IMMUN.

100 O

80

cc

60

z

TABLE 2. Summary of average survival of experimental groups

X 7B13- IgMk 2D10- IgMk * 21 D2 - IgMk * PBS

Figure

Control of MAb

Avg survival Isotype s(days)

1

PBS 7B13 21D2 2D10

IgM(X) IgM(K)

O

Z 40

p 3E5 [IgG3] = 18G9 [IgA] > 2H1 [IgGl]) was proportional to the relative antibody avidity but did not correlate with protection. Although the IgA antibody was the most effective isotype on a weight basis when administered just prior to infection, it has a shorter (100 h) intravascular half-life than IgGl (200 h) (56), such that more IgA than IgGl would have to be administered passively to achieve comparable long-term concentrations. To more closely mimic what occurs in vivo following immunization or passive administration, we injected into mice equal volumes of ascitic fluid containing half as much IgA as IgGl (Fig. 2B), and in this situation, the IgGl appears to be more effective (Table 2). The effect of passive MAb administration on circulating polysaccharide levels was studied. On day 5 of infection in the experiment shown in Fig. 2A, sera from five mice of each group were pooled and the concentration of directly detectable CNPS-D was measured. Although this method may not detect antibody-complexed CNPS-D, at least antibody-detectable circulating CNPS-D in the pooled serum of each group was inversely proportional to the protection conferred, with control (PBS) > IgG3 (3E5) > IgM (2D10) > IgA (18G9) > IgGl (2H1) (Fig. 2A). This probably reflects the amount of replication of the organism that had occurred in these animals and the degree to which the different antibodies were able to persist and clear the polysaccharides. We studied three animals from the isotype comparison experi-

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A

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CD 100 z > 80

C: 60

C>:

0.

cn

z

0i

z40 w

o 20 w

C

Passively Administered mAb

O DAYS AFTER INFECTION

B cD z

100 ~80

mAbs:

o 2D10- IgMk a 3E5 - IgG3k X 2H1 IgGlk

C) Co

40

A

W

20

18G9 - IgAk

-* NS PBS

W-

0

10

20 30 DAYS AFTER INFECTION

40 50150 350

FIG. 2. Survival of A/J mice after i.p. treatment with the clonally related MAbs 2D10 [IgM(K)], 3E5 [IgG3(K)], 2H1 [IgGl(K)], and 18G9 [IgA(K)] or NSO myeloma ascitic fluid and infection with 108 serotype D (ATCC 24067) C. neoformans cells. (A) Survival curves after i.p. treatment with 1 mg of MAb 2D10, 3E5, 2H1, or 18G9 or 250 p.l of PBS. The inset of panel A shows the concentrations of circulating CNPS-D of a pooled subset of each treatment group from panel A 5 days following infection and MAb treatment. The MAb-treated mice show a reduction in the levels of circulating CNPS-D relative to levels in PBS control mice. (B) Survival curves after i.p. treatment with 0.6 mg of 2H1 IgGl, 0.3 mg of 18G9 IgA MAb, or 250 Il1 of NSO ascitic fluid. See Table 2 for average lengths of survival.

ment shown in Fig. 2A that were still alive at 200 days and

found that C. neoformans could be cultured from the brain and lungs of only one of these three mice. This suggests that at least in some animals, a single dose of antibody facilitated complete clearance of the cryptococcal infection even when it resulted from a massive inoculum of 108 yeast cells per

tent mice and also to determine whether an IgGl MAb generated from a different B cell was protective, BALB/c mice were given 1 mg of MAb 17E12 i.p. immediately prior to infection with 108 serotype D cryptococci. Administration of 17E12 significantly prolonged survival of lethally infected BALB/c mice relative to that of controls (Fig. 4 and Table 2).

mouse.

To determine whether the IgG3 isotype and/or the epitope specificity of MAb 3E5 contributed towards the minimal protective efficacy of this MAb, we examined this issue further by comparing MAb 3E5 with another IgG3 MAb, 4H3. MAb 4H3 differs from 3E5 in structure and epitope specificity and has a structure very similar to and the same serotype specificity as the 7B13 (IgM(X)] MAb (Table 1), which is protective (Fig. 1). When administered immediately prior to infection, the 4H3 MAb was not protective and MAb 3E5 conferred a marginal prolongation in survival (Fig. 3A and Table 2). When administered in two doses 24 and 48 h prior to infection with the same strain of cryptococcus, neither 3E5 nor 4H3 was protective and 4H3 administration decreased survival relative to that of the NSO control (Fig. 3B and Table 2). This experiment provides additional evidence that the IgG3 subclass is either not protective or only marginally protective. Ability to protect normal mice. The IgGl MAb El of Dromer et al. was protective against cryptococcosis in the complement-deficient DBA/2 mouse strain (24) but not protective in BALB/c mice (25), and the IgGl MAb of Sanford et al. did not prolong survival of Swiss Webster mice (59). To determine whether our MAbs could protect immunocompe-

DISCUSSION The model for evaluating the protective efficacy of anticryptococcal MAbs used A/J and BALB/c mice given i.p. injections of antibody and C. neoformans. Both A/J and BALB/c mice were used because these strains differ in susceptibility to C. neoformans. A/J mice are more. susceptible to cryptococcal infection, presumably because of a deficiency of complement component C5 (58). We chose to administer the MAbs and C. neoformans at approximately the same time into the peritoneum because this approach has been used successfully to evaluate the protective efficacy of passive antibody administration against encapsulated bacteria, including Streptococcus pneumoniae (4, 36, 63, 67) and Escherichia coli Kl (66). In addition, this same model was successfully used by Graybill et al. (33) to demonstrate protection against C. neoformans by passive administration of polyclonal rabbit sera. A serotype D (ATCC 24067) strain of C. neoformans was used because all our MAbs recognize CNPS-D (Table 1), allowing us to compare their relative protective efficacies. We used prolongation of survival as the measure of passive MAb protective efficacy. Prolongation of survival

4538

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MUKHERJEE ET AL.

A

(D3

100 * 3E5-IgG3k * 4H3- IgG3% O PBS A NSO

z 80

C

60

z

40

cn w

0

c

w a-

20

B z

cc Cl) z LL

20 a.

0 14 16 12 18 20 4060 80 100 10 DAYS AFTER INFECTION FIG. 3. Survival of A/J mice after i.p. treatment with the IgG3 MAbs 3E5 and 4H3. (A) Survival curves after i.p. administration of 1 mg of 3E5 or 4H3 or 250 ,ul of PBS immediately prior to infection with 108 serotype D (ATCC 24067) C. neoformans cells; (B) survival curves after i.p. administration of 1 mg of 3E5 or 4H3 or 250 ,ul of NSO myeloma ascitic fluid in two doses 24 and 48 h prior to infection with 108 serotype D (ATCC 24067) C. neoformans cells. See Table 2 for average lengths of survival.

0

2

4

6

8

may reflect the ability of the MAbs to prevent dissemination of the challenge inoculum from the peritoneum. Passive administration of MAb often resulted in long prolongation of survival, but many of the animals ultimately died. This probably reflects the conversion of a rapidly lethal acute infection into a chronic infection. It is likely that passive administration of MAb prolongs survival by facilitating fungal clearance by cellular defense mechanisms, given the in vitro data that antibody enhances phagocytosis (43) and mediates fungistasis by natural killer cells (18, 47). In addition, binding of antibody to the capsule of C. neofonnans may prevent adhesion of the fungus to glycosphingolipid receptors on endothelial cells (38), thereby decreasing infectivity. In this regard, passive MAb administration is known to be very effective in decreasing cryptococcal colony counts in the organs of infected mice (24, 59). However,

100 z

regardless of the mechanism or site of protection, the finding that some of our antibodies are highly protective whereas others are not indicates that we can distinguish between protective and nonprotective MAbs with this model. We observed considerable variation in the survival of treated and control mouse groups between experiments (Table 2). The cause for this variability is not understood. The experiments described here were performed over the course of 1 year, and the variations in interexperiment survival could reflect subtle differences in the pathogenicity of the yeast and/or differences between animal lots. Nevertheless, controls were done with each experiment, and this model has allowed us to distinguish highly protective from less protective MAbs. The finding that three IgM MAbs each recognizing different epitopes within the capsular polysaccharide vary in the * 17E12-IgGlk o NSO

80

60 cn

Z 40

{L 20_

DAYS AFTER INFECTION

FIG. 4. Survival of BALB/c mice after i.p. treatment with 1 mg of the IgGl anti-CNPS MAb 17E12 or 250 pl of NSO myeloma ascitic fluid immediately preceding infection with 108 serotype D (ATCC 24067) C. neoformans cells. See Table 2 for average lengths of survival.

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ability to prolong survival (Fig. 1) raises the possibility that epitope specificity is an important determinant of protective efficacy. The fact that 2H1 [a protective IgGl(K) MAb] does not bind de-O-acetylated CNPS-A GXM (6) suggests that acetyl groups are either part of the epitope recognized or necessary for the structural integrity of the epitope. It is remarkable that the IgM MAbs, 21D2 and 2D10, are structurally very similar in that each uses a VH7183 family member, has a 7-amino-acid D, and uses JH2, VK5.1, and JK1" (50), yet 2D10 is more protective than 21D2. Thus, the differences in protective efficacy could be the result of different amino acids encoded by the VH used by 2D10 or the replacement mutations in both the VH and light chain variable region of 2D10. The sequence differences between 2D10 and 21D2 may result in different fine specificities. If epitope specificity is important for protective efficacy, it could explain the divergent observations made by Dromer et al. (24) and Sanford et al. (59) using passive murine MAbs in protection studies. The El MAb used by Dromer et al. (24) is primarily specific for serotype A polysaccharide (26), whereas the MAb used by Sanford et al. (59) binds all four serotypes. These differences in serotype binding suggest different epitope specificities for these MAbs. Differences in the epitope specificities and/or isotype compositions (see below) of polyclonal sera used in passive protection experiments may also account for the contradictory observations that passive therapy is protective (28, 33) and nonprotective (46). The role of isotype in protective efficacy was studied by comparing the survival of lethally infected mice to which MAbs differing in isotype were passively administered. Members of the MAb set 2D10 (IgM), 2H1 (IgGl), 3E5 (IgG3), and 18G9 (IgA) were derived from one B-cell clone, and all bind polysaccharide from the four serotypes. These MAbs use the same variable gene elements (Table 1) but differ in constant-region class. Since each MAb has several replacement somatic mutations in both CDRs and frameworks, we cannot rule out subtle differences in fine specificity, but we have no evidence for this. Among this set, the IgA MAb was more protective on a weight basis than the IgGl and IgM MAbs and the IgG3 MAb was much less protective than the others. The differences in protective efficacy of this closely related family of antibodies strongly suggest that isotype is a major contributor to the protective efficacy of anticryptococcal capsule-specific antibodies. Similar observations have been made with isotype switch variant MAbs in E. coli and S. pneumoniae infections (3, 53). In addition, Sanford et al. (59) reported differences in the capacity of IgGl, IgG2a, and IgG2b switch variants of an anticryptococcal MAb to decrease organ colony counts in lethally infected mice, even though the antibodies did not prolong survival. The finding that two IgG3 MAbs which recognize the same epitopes as protective antibodies were either minimally or not protective was surprising, since this isotype is highly protective against the encapsulated bacterium S. pneumoniae (3) and polymerizes upon binding to bacterial antigens, thereby increasing its avidity (34). The relative ineffectiveness of the IgG3 subclass contrasts with the efficacy of the IgGl subclass. The differences in protective efficacy shown by these isotype-variant MAbs strongly suggest that Fcmediated effector functions contribute to the protective efficacy. The Fc region confers specific effector functions, such as complement fixation activity, the ability to facilitate antibody-dependent cell-mediated cytotoxicity, relative avidity (61), and utilization of different Fc receptors. Differ-

4539

ent Fc receptors on murine macrophages have been described for IgG3 (Fcly3R), IgG2a (Fc-y2.R), and IgG1-IgG2b (Fc-yl/,y2bR) (13-15, 65). The difference in protective efficacy between the IgGl and IgG3 subclasses may be due to recognition and signal transduction by different receptors (13, 14, 57). Thus, binding of cryptococci opsonized with the IgGl MAb 2H1 to macrophage Fc-yjR could conceivably lead to phagocytosis and effective fungal killing, whereas the binding of cryptococci opsonized with the IgG3 MAb 3E5 to FcY3R may be less effective in the elimination of the organism. Although there is no IgM Fc receptor, this isotype may be effective because of its relatively high avidity and ability to activate complement, which has been shown to enhance phagocytosis of cryptococci by macrophages (5, 48, 60). In fact, IgM is superior to the IgG subclasses in activating complement-mediated killing of E. coli (52). The protective efficacy of IgA against C. neoformans was surprising, for IgA does not activate the classical complement pathway and its Fc receptor is present only on alveolar macrophages (29) and leukocytes (9). However, IgA does activate the alternate pathway of complement (55) which is essential for opsonization of C. neofornans (12, 20, 42) and binds a receptor present on liver cells (64) which may effectively facilitate clearance of the organism from the intraperitoneal space. At a practical level, since the intravascular metabolism of IgA and IgM molecules is much faster than that of IgG (56), a much larger dose of IgA or IgM relative to IgG MAb would have to be administered to attain persistent levels of IgA and IgM in vivo. Therefore, it may be easier to achieve protection with IgG, and this is illustrated for the IgGl MAb 2H1 in Fig. 2B. Since antibody-mediated agglutin'ation and clumping of the challenge inoculum could conceivably prevent dissemination of the cryptococci from the peritoneum, we studied the relative ability of our MAbs to induce clumping of cryptococci. We found that the relative ability of the 2D10 family of MAbs to agglutinate cryptococci (2D10 [IgM] > 3E5 [IgG3] = 18G9 [IgA] > 2H1 [IgGl]) was associated with antibody avidity but did not correlate with protective efficacy, confirming a similar observation by Diamond (16). Thus, agglutination of the challenge inoculum within the peritoneal cavity is unlikely to be the mechanism by which our MAbs protect. Circulating cryptococcal GXM persists for 1 week in serum, presumably because of poor immune clearance and a lack of enzymes capable of degrading the polysaccharide (41). Passive MAb therapy may be useful in clearance of CNPS, especially in AIDS patients who appear to have difficulty in the ability to eliminate serum polysaccharides (27). In addition, it has recently been shown that CNPS augments human immunodeficiency virus type 1 infectivity in vitro (54) and that the inhibitory effect of CNPS on phagocytosis blocks macrophage-mediated antigen processing of encapsulated strains and thus inhibits T-cell function (11). The ability of MAbs to decrease circulating CNPS levels could be beneficial to the host by directly facilitating

efficient phagocytosis and indirectly enhancing T-cell-mediated immune clearance. Animal model selection may be the critical parameter in demonstrating protective efficacy of passively administered antibody against C. neoformans infections. The dose, route, and timing of both antibody and challenge inocula as well as the cryptococcal and murine strains used are potential variables in these experiments. The exact contribution of each of these parameters to experimental outcome remains to be determined. Previous passive immunization studies against cryptococcal infection have demonstrated that the

4540

MUKHERJEE ET AL.

results are dependent on the administration sites of the antibody and challenge inocula as well as the complement status of the mouse strain. Protection is more likely to occur if both the organism and the antibody are administered at about the same time and by the same route (28, 33), as we have done in most of the experiments reported here. i.p. antibody administration and intravenous C. neoformans challenge have been shown to result in increased survival of C5-deficient (24, 25) but not CS-competent (25, 59) mice. However, protection has been demonstrated with CS-competent mice when both antibody and C. neoformans are given i.p. (28, 33). Our results, showing that our IgGl MAbs are protective in both complement-deficient A/J mice and non-complement-deficient BALB/c mice confirms this latter observation. Moreover, our MAbs are presumably protective against both the fatal pneumonia and the meningoencephalitis which develop in C5-deficient and C5-competent mice, respectively, given large fungal loads (25). All the animal models for antibody protection of cryptococcosis are artificial, and it is unclear which, if any, best approximates the physiological role of anticapsular antibody in protection. Nonetheless, passive antibody administration can significantly prolong survival in some cases (24, 28, 33), and even in models in which no prolongation of survival occurred, some measure of protection was conferred, as reflected by decreased organ cryptococcus colony counts (59). We have identified both protective and nonprotective MAbs by using a murine model of C. neoformans infection. Protective MAbs included those generated from both infected and GXM-protein conjugate-immunized mice (6). However, the most effective MAbs were generated from mice immunized with the GXM-tetanus toxoid conjugate and were the product of a T-cell-dependent immune response (6). Our results indicate that antibody class is important in protection and suggest that further studies should be done to dissect the roles of isotype and epitope by using families of switch variants generated in vitro in which the binding sites are identical. The possibility that epitope specificity is important for protective efficacy suggests that antigenic differences (62) between strains may be markers of virulence.

ACKNOWLEDGMENTS We thank Terri Kelly, Susan Buhl, Gerardo Gomez, and Yoon Young Jhang for expert technical assistance in various aspects of this work and Nancy Drenzyk for help in preparing the manuscript. We thank L. Pirofsky for critical reading of the manuscript. We also acknowledge Chee Jen Chang for invaluable assistance in the statistical analysis of the data presented here. This work was supported by NIH grants CA39838-07, CA1333020, and AI10702-20. J.M. is supported by NIH training grant 2T32C809173-16. M.D.S. is supported in part by the Harry Eagle Chair provided by the Women's Division of Albert Einstein College of Medicine. A.C. was supported by a Pfizer Postdoctoral Fellowship. REFERENCES 1. Bennett, J. E., H. F. Hasenclever, and B. S. Tynes. 1964. Detection of cryptococcal polysaccharide in serum and spinal fluid. Value in diagnosis and prognosis. Trans. Assoc. Am. Physicians 77:14. 2. Bloomfield, W., M. A. Gordon, and D. F. Elmendorff, Jr. 1963. Detection of Cryptococcus neoformans antigen in body fluids by latex agglutination. Proc. Soc. Exp. Biol. Med. 114:64. 3. Briles, D. E., J. L. Claflin, K. Schroer, and C. Forman. 1981. Mouse IgG3 antibodies are highly protective against infection with Streptococcus pneumoniae. Nature (London) 294:88-89. 4. Briles, D. E., C. Forman, S. Hudak, and J. L. Claflin. 1982. Anti-phosporylcholine antibodies to the T15 idiotype are opti-

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