[Frontiers in Bioscience, 3, d136-151, February 1, 1998]

	[Frontiers in Bioscience, 3, d136-151, February 1, 1998] Reprints PubMed CAVEAT LECTOR
	*MECHANISM OF ACTION OF ANTIBODY TO CAPSULAR POLYSACCHARIDE IN CRYPTOCOCCUS NEOFORMANS* INFECTION Marta Feldmesser¹and Arturo Casadevall^1,2** 1Departments of Medicine (Division of Infectious Diseases) and Microbiology and Immunology², Albert Einstein College of Medicine, Golding Building Room 701, 1300 Morris Park Ave., Bronx, NY 10461 Received 1/5/98 Accepted 1/9/98 7. MABS TO THE CAPSULE OF C. NEOFORMANS 7.1 Description Since the mid 1980s, mAbs to the cryptococcal capsule have been produced by at least four independent laboratories. Murine mAbs have been produced from infected mice (79), and from mice immunized with GXM-TT (79), purified CNPS (80), and CNPS conjugated to sheep erythrocytes (81). In addition, human mAbs have been generated from volunteers vaccinated with GXM-TT (82) and mouse-human chimeric Abs have been constructed (83). Protective, nonprotective and disease-enhancing mAbs have been described, and mAb epitope and isotype are both important determinants of mAb efficacy (84). Study of these Abs has provided new knowledge about the antibody response to this organism. Comparison of molecular characteristics of mAbs produced in response to infection with a serotype A strain and the GXM-TT conjugate vaccine showed that Ig variable gene usage is highly restricted, as most of the antibodies use the same V_H7183 gene element, have a seven codon CDR3 and utilize J_H2, V_K5.1 and J_K1 gene elements (85). Of two IgM mAbs originating from the same B cell that differ only by somatic mutations in the variable regions, one is protective while the other is not (86). Differences in a few amino acid residues, particularly in the heavy chain complementary determining region 2 (CDR2), are associated with variation in fine specificity and protective efficacy of the mAbs (87). Based upon their ability to confer protection, their fine specificity and molecular structure, mAbs to GXM have been classified into six groups (88). Further study of the fine specificity of mAbs developed by hybridoma technology has used phage display peptide libraries to determine peptide binding motifs in antigen binding sites. This technique can potentially identify mimotopes that elicit mAbs to the protective epitopes on the capsular polysaccharide (89). "Protective" mAbs to GXM prolong survival following i.v., intraperitoneal (i.p.), intracranial (i.c.), and i.t. infection with a variety of C. neoformans strains (84,90-93). They also provide additional antifungal benefit when administered in conjunction with several antifungal agents (94-98). Nonprotective and disease-enhancing mAbs are generally defined by their effect on survival relative to saline or to isotype matched control mAbs. While the mAbs are cross-reactive with all serotypes, the ability of mAb to prolong survival and to reduce CFU varies with the cryptococcal strain (99) and, in murine infection models, with the mouse strain (100). Immunofluorescent binding patterns of mAb to the cryptococcal capsule vary, and an annular fluorescence pattern has been associated with a protective response (87). While mAbs have also been produced to other cryptococcal targets, including galactoxylomannan contained in the capsule and cell wall (101) and to cytoplasmic Ags (102,103), further study of the effects of these mAbs has been limited. The remainder of the discussion is limited to mAbs to GXM. 7.2 The role of isotype The mAbs to GXM that have been produced are isotype restricted, though mAbs of every isotype have been produced by isotype switching (104). Hybridomas produced from infected mice are predominantly of the IgM class, consistent with the classification of GXM as a T independent Ag, while those produced following immunization are most commonly IgG (85). mAb isotype affects the ability of the mAb to be protective (figure 2). In one family of mAbs derived from the same B cell, IgG1 mAbs prolong survival, reduce tissue CFU and serum GXM in murine infection, while IgG3 mAbs do not. Isotype switching of a disease-enhancing IgG3 resulted in production of a protective IgG1 (105). Studies using knockout mice show that CD4+ cells are required for the protective effect of the IgG1 mAb, but not for the disease enhancing effect of IgG3. IgG3-mediated disease enhancement requires CD8+ cells. In interferon (IFN)-gamma knockout mice, the IgG1 is no longer protective and the IgG3 does not reduce survival (106). Figure 2. Overview of the roles of Fc regions of Ab in C. neoformans infection. Diagram highlights the role of Ab molecules as bridges between effector cells and C. neoformans cells. Noted are the roles of Fc and Fv regions in Ab function and cell types associated with Ab-mediated protection are listed. The impact of isotype on the ability of mAb to enhance cryptococcal phagocytosis and killing by macrophages has been studied by several groups with some variance in results. For example, with one family of mAbs derived from the same B cell that have the same idiotype and use identical variable region genes, the relative opsonic ability of the mAbs is IgG1=IgG3>IgG2a>IgG2b>IgM>IgA (107). However, the ability of these mAbs to increase cryptococcal killing by the murine macrophage-like cell line J774.16 is IgG1>IgG2a>IgM>IgG3>IgG2b>IgA (107). Their relative ability to agglutinate cryptococci does not correlate with protective efficacy (84). Another group that produced a family of isotype-switch variants found that the relative ability of their mAbs to opsonize C. neoformans for phagocytosis was IgG2a>IgG1>IgG2b (108). However, while with this family, the IgG2a and IgG2b subclasses reproducibly reduced CFU in the lung and spleen in murine i.v. infection, survival was not prolonged by any of the isotypes (109). Thus, Ab isotype clearly plays a role in the ability of mAbs with the same fine specificities to protect mice in murine infection, as well as to promote opsonization and fungal killing by murine macrophages and macrophage-like cell lines. The reason for differences in protective ability of mAb subclasses is not understood. The explanation for the varying effects of isotype between mAb families is also unknown, but may relate to the fine specificities of the mAbs involved, differences in the cell lines used in these studies or to the cryptococcal strains (107). The importance of isotype is not surprising since isotype mediates many biological effects of Ab, including the ability to fix complement, facilitation of Ab-dependent cellular cytotoxicity (ADCC) and relative avidity. Different isotypes bind to different Fc receptors (FcRs), which may result in initiation of different signal transduction pathways (110). Differences in the isotypes that are best at mediating each of these effects suggest that macrophage cell killing and phagocytosis are not the only mechanism of mAb action. Further, the findings presented above illustrate that isotype-specific effects are dependent not only on FcR binding, but on the presence of other components of the immune response, such as T cell recruitment and cytokine production.

[Frontiers in Bioscience, 3, d136-151, February 1, 1998]
Reprints
PubMed
CAVEAT LECTOR

MECHANISM OF ACTION OF ANTIBODY TO CAPSULAR POLYSACCHARIDE IN CRYPTOCOCCUS NEOFORMANS INFECTION

Marta Feldmesser¹and Arturo Casadevall^1,2

1Departments of Medicine (Division of Infectious Diseases) and Microbiology and Immunology², Albert Einstein College of Medicine, Golding Building Room 701, 1300 Morris Park Ave., Bronx, NY 10461

Received 1/5/98 Accepted 1/9/98

7. MABS TO THE CAPSULE OF C. NEOFORMANS

7.1 Description

Since the mid 1980s, mAbs to the cryptococcal capsule have been produced by at least four independent laboratories. Murine mAbs have been produced from infected mice (79), and from mice immunized with GXM-TT (79), purified CNPS (80), and CNPS conjugated to sheep erythrocytes (81). In addition, human mAbs have been generated from volunteers vaccinated with GXM-TT (82) and mouse-human chimeric Abs have been constructed (83). Protective, nonprotective and disease-enhancing mAbs have been described, and mAb epitope and isotype are both important determinants of mAb efficacy (84). Study of these Abs has provided new knowledge about the antibody response to this organism. Comparison of molecular characteristics of mAbs produced in response to infection with a serotype A strain and the GXM-TT conjugate vaccine showed that Ig variable gene usage is highly restricted, as most of the antibodies use the same V_H7183 gene element, have a seven codon CDR3 and utilize J_H2, V_K5.1 and J_K1 gene elements (85). Of two IgM mAbs originating from the same B cell that differ only by somatic mutations in the variable regions, one is protective while the other is not (86). Differences in a few amino acid residues, particularly in the heavy chain complementary determining region 2 (CDR2), are associated with variation in fine specificity and protective efficacy of the mAbs (87). Based upon their ability to confer protection, their fine specificity and molecular structure, mAbs to GXM have been classified into six groups (88). Further study of the fine specificity of mAbs developed by hybridoma technology has used phage display peptide libraries to determine peptide binding motifs in antigen binding sites. This technique can potentially identify mimotopes that elicit mAbs to the protective epitopes on the capsular polysaccharide (89).

"Protective" mAbs to GXM prolong survival following i.v., intraperitoneal (i.p.), intracranial (i.c.), and i.t. infection with a variety of C. neoformans strains (84,90-93). They also provide additional antifungal benefit when administered in conjunction with several antifungal agents (94-98). Nonprotective and disease-enhancing mAbs are generally defined by their effect on survival relative to saline or to isotype matched control mAbs. While the mAbs are cross-reactive with all serotypes, the ability of mAb to prolong survival and to reduce CFU varies with the cryptococcal strain (99) and, in murine infection models, with the mouse strain (100). Immunofluorescent binding patterns of mAb to the cryptococcal capsule vary, and an annular fluorescence pattern has been associated with a protective response (87).

While mAbs have also been produced to other cryptococcal targets, including galactoxylomannan contained in the capsule and cell wall (101) and to cytoplasmic Ags (102,103), further study of the effects of these mAbs has been limited. The remainder of the discussion is limited to mAbs to GXM.

7.2 The role of isotype

The mAbs to GXM that have been produced are isotype restricted, though mAbs of every isotype have been produced by isotype switching (104). Hybridomas produced from infected mice are predominantly of the IgM class, consistent with the classification of GXM as a T independent Ag, while those produced following immunization are most commonly IgG (85). mAb isotype affects the ability of the mAb to be protective (figure 2). In one family of mAbs derived from the same B cell, IgG1 mAbs prolong survival, reduce tissue CFU and serum GXM in murine infection, while IgG3 mAbs do not. Isotype switching of a disease-enhancing IgG3 resulted in production of a protective IgG1 (105). Studies using knockout mice show that CD4+ cells are required for the protective effect of the IgG1 mAb, but not for the disease enhancing effect of IgG3. IgG3-mediated disease enhancement requires CD8+ cells. In interferon (IFN)-gamma knockout mice, the IgG1 is no longer protective and the IgG3 does not reduce survival (106).

Figure 2. Overview of the roles of Fc regions of Ab in C. neoformans infection. Diagram highlights the role of Ab molecules as bridges between effector cells and C. neoformans cells. Noted are the roles of Fc and Fv regions in Ab function and cell types associated with Ab-mediated protection are listed.

The impact of isotype on the ability of mAb to enhance cryptococcal phagocytosis and killing by macrophages has been studied by several groups with some variance in results. For example, with one family of mAbs derived from the same B cell that have the same idiotype and use identical variable region genes, the relative opsonic ability of the mAbs is IgG1=IgG3>IgG2a>IgG2b>IgM>IgA (107). However, the ability of these mAbs to increase cryptococcal killing by the murine macrophage-like cell line J774.16 is IgG1>IgG2a>IgM>IgG3>IgG2b>IgA (107). Their relative ability to agglutinate cryptococci does not correlate with protective efficacy (84). Another group that produced a family of isotype-switch variants found that the relative ability of their mAbs to opsonize C. neoformans for phagocytosis was IgG2a>IgG1>IgG2b (108). However, while with this family, the IgG2a and IgG2b subclasses reproducibly reduced CFU in the lung and spleen in murine i.v. infection, survival was not prolonged by any of the isotypes (109).

Thus, Ab isotype clearly plays a role in the ability of mAbs with the same fine specificities to protect mice in murine infection, as well as to promote opsonization and fungal killing by murine macrophages and macrophage-like cell lines. The reason for differences in protective ability of mAb subclasses is not understood. The explanation for the varying effects of isotype between mAb families is also unknown, but may relate to the fine specificities of the mAbs involved, differences in the cell lines used in these studies or to the cryptococcal strains (107). The importance of isotype is not surprising since isotype mediates many biological effects of Ab, including the ability to fix complement, facilitation of Ab-dependent cellular cytotoxicity (ADCC) and relative avidity. Different isotypes bind to different Fc receptors (FcRs), which may result in initiation of different signal transduction pathways (110). Differences in the isotypes that are best at mediating each of these effects suggest that macrophage cell killing and phagocytosis are not the only mechanism of mAb action. Further, the findings presented above illustrate that isotype-specific effects are dependent not only on FcR binding, but on the presence of other components of the immune response, such as T cell recruitment and cytokine production.