Analysis of human monoclonal antibodies elicited by vaccination with a Cryptococcus neoformans glucuronoxylomannan capsular polysaccharide vaccine.

The Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) has been conjugated to tetanus toxoid (GXM-TT) as an investigational vaccine. GXM-TT elicits antibodies that are protective in C. neoformans-infected mice. In an effort to characterize the fine specificity and molecular structure of human GXM-TT-elicited antibodies, we generated two GXM monoclonal antibodies (MAbs) from peripheral blood lymphocytes of a volunteer GXM-TT recipient and studied serum GXM antibody idiotype expression in 10 additional vaccinees. The MAbs, 2E9 and 3B6, are the immunoglobulin M(lambda) isotype and bind capsular polysaccharides of C. neoformans serotypes other than the serotype A that was used for immunization. Neither antibody competes with murine GXM MAbs for antigen binding, suggesting that the human MAbs recognize a different epitope. The B-cell superantigen staphylococcal protein A binds both MAbs, and human immunodeficiency virus gp120 binds 2E9. MAb nucleic acid sequence analysis revealed that both antibodies use an identical V lambda 1a-J lambda genetic element with different, somatically mutated, members of the VH3 gene family and different DH and JH gene elements. The gene elements used by both MAbs occur in fetal B-lymphocyte repertoires, autoantibodies, and other polysaccharide antibodies. Post-GXM-TT vaccination GXM antibodies from 10 additional vaccinees expressed a shared idiotype defined by rabbit antiserum raised against MAb 2E9. Our data suggest that the human GXM antibody response is restricted and raise questions regarding the importance of specific variable-region elements and superantigens in the generation of human antibody responses to encapsulated pathogens.     

Protective and nonprotective human immunoglobulin M monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan manifest different specificities and gene use profiles

The features of protective murine antibodies to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been rigorously investigated; however, the characteristics of protective human antibodies to GXM have not been defined. We produced monoclonal antibodies (MAbs) from XenoMouse mice (transgenic mice that express human immunoglobulin M [IgM], IgG2, and kappa) which were immunized with a C. neoformans serotype D strain 24067 GXM-diphtheria toxoid conjugate. This study reports the specificity and efficacy of three human IgM MAbs, G14, G15, and G19, generated from these mice. Each MAb was specific for GXM, but G14 and G19 had different specificity based on their binding to serotype A strain H99 and SB4 GXMs, to which G15 did not bind. Nucleic acid sequence analysis revealed that G15 uses V(H)3-64 in the germ line configuration. G14 and G19 use V(H)6-1, which has somatic mutations. All of the MAbs use V kappa DPK22/A27. Studies of MAb efficacy in BALB/c mice showed that administration of 0.1 mg, but not 1 or 0.01 mg, of G15 prolonged survival against lethal C. neoformans strain 24067 challenge, whereas G14 and G19 were not protective at any dose. This panel of MAbs illustrates that serotype D GXM has epitopes that elicit human antibodies that can be either protective or nonprotective. Our findings suggest that V(H) gene use may influence GXM specificity and efficacy, and they provide insights into the possible contribution that V(H) gene use may have in resistance and susceptibility to cryptococcosis.     

人抗—HBsFab段基因的序列分析及表达

对已建的噬菌体抗体库中分离的人抗－ＨＢｓ克隆进行了序列分析和表达研究，发现１９个克隆都具有相同的重链可变区基因，轻链可变区基因有４个相同，其余１５个未进行序列测定。ＤＮＡ序列分析表明ＶＨ、ＪＨ分属ＶＨＶＨⅢ和ＪＨ６，Ｄ段为二个Ｄ基因的融合，ＶＫ，ＪＫ属于ＶＫＩ和ＪＫ４。     

Monoclonal antibodies to Cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice.

Immunoglobulin G1 (IgG1) monoclonal antibodies (MAbs) to the capsular glucuronoxylomannan (GXM) were studied for their ability to modify the course of intravenous Cryptococcus neoformans infection in mice. A/J mice were given intraperitoneal injection of 1.0 mg of either a GXM-binding IgG1 MAb (2H1 or 2D10 gamma 1) or the irrelevant isotype-matched control MAb 36-65 prior to intravenous infection. Parameters used to study antibody efficacy were lung and brain tissue fungal burden, lung and brain weights, serum GXM levels, and histopathological examination of lung, brain, heart, kidney, and spleen tissues. Mice given GXM-binding MAb had significantly reduced lung tissue fungal burden as measured by CFU. In contrast to the reduction in lung tissue burden, the reduction in brain tissue burden was small and did not achieve statistical significance. Serum GXM levels were reduced in mice receiving GXM-binding MAb. Histopathological examination revealed reduced numbers of granulomas and C. neoformans organisms in the lungs, brains, and kidneys of MAb 2H1-treated mice relative to control mice. The lungs and brains of mice receiving GXM-binding MAb weighed significantly less than those of control animals, consistent with the reduced inflammation noted histologically. Subendocardial inflammation and kidney cortical infarctions were present in control infected mice but not in MAb 2H1-treated mice. Immunocytochemical staining for polysaccharide antigen revealed a marked reduction in the amount of tissue polysaccharide in mice treated with MAb 2H1 relative to control mice. The results support an useful role for passive antibody administration in C. neoformans infections.     

Monoclonal antibodies specific for immunorecessive epitopes of glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans, reduce serotype bias in an immunoassay for cryptococcal antigen

Immunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to remove O-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population.     

Structural basis of stimulatory anti-idiotypic antibodies

In order to design and produce effective vaccines based upon the idiotype network hypothesis of Jerne, a thorough understanding of the biological and structural aspects underlying the stimulating activities of anti-idiotypic antibodies is needed. Here we determined the nucleotide sequence of the variable heavy and light chain regions of two monoclonal anti-idiotypic antibodies which induce different anti-phosphorylcholine responses. The nucleotide sequences of the variable domains of two monoclonal anti-TEPC 15 (T15) antibodies (F6-3 and 4C11) were determined by the primer extension and Maxam-Gilbert techniques. The nucleotide sequence data show that 4C11 and F6-3 have homologous VH segments and JH segments, but different D regions. The VH segments of both clones belongs to the J558 VH family. Most of the differences among the VH segments are located in CDR2. The VK segments of 4C11 and F6-3 are homologous to the VK gene group 4 and group 8, respectively. Comparison of the sequences of 4C11 and F6-3 with other published anti-idiotype antibodies shows that there is no preferential utilization of immunoglobulin genes. An analysis of the distribution of charged residues and hydropathic comparison studies were used to interpret the sequence of 4C11 in terms of the biological mimicry of antigenic stimulation.     

Molecular and Idiotypic Analyses of the Antibody Response to Cryptococcus neoformans Glucuronoxylomannan-Protein Conjugate Vaccine in Autoimmune and Nonautoimmune Mice

The antibody response to Cryptococcus neoformans capsular glucuronoxylomannan (GXM) in BALB/c mice frequently expresses the 2H1 idiotype (Id) and is restricted in variable gene usage. This study examined the immunogenicity of GXM-protein conjugates, V (variable)-region usage, and 2H1 Id expression in seven mouse strains: BALB/c, C57BL/6, A/J, C3H, NZB, NZW, and (NZB x NZW)F(1) (NZB/W). All mouse strains responded to vaccination with GXM conjugated to tetanus toxoid (TT), the relative magnitude of the antibody response being BALB/c approximately C3H > C57BL/6 approximately NZB approximately NZW approximately NZB/W > A/J. Analysis of serum antibody responses to GXM with polyclonal and monoclonal antibodies to the 2H1 Id revealed significant inter- and intrastrain differences in idiotype expression. Thirteen monoclonal antibodies (MAbs) (two immunoglobulin M [IgM], three IgG3, one IgG1, three IgG2a, two IgG2b, and two IgA) to GXM were generated from one NZB/W mouse and one C3H/He mouse. The MAbs from the NZB/W mouse were all 2H1 Id positive (Id(+)) and structurally similar to those previously generated in BALB/c mice, including the usage of a V(H) from the 7183 family and Vkappa5.1. Administration of both 2H1 Id(+) and Id(-) MAbs from NZB/W and C3H/H3 mice prolonged survival in a mouse model of cryptococcosis. Our results demonstrate (i) that V-region restriction as indicated by the 2H1 Id is a feature of both primary and secondary responses of several mouse strains; and (ii) that there is conservation of V-region usage and length of the third complementarity-determining region in antibodies from three mouse strains. The results suggest that V-region restriction is a result of antibody structural requirements necessary for binding an immunodominant antigen in GXM.     

Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide.

Cryptococcus neoformans is surrounded by a capsular polysaccharide. There are at least four known serotypes of the polysaccharide. The objective of this study was to produce monoclonal antibodies (MAbs) that could be used to study the distribution of epitopes among the serotypes of C. neoformans. BALB/c mice were immunized with cryptococcal polysaccharides of serotype A or D that were coupled to sheep erythrocytes. Splenocytes were isolated, and hybridomas secreting MAbs specific for cryptococcal polysaccharides were isolated. Two hybridomas, designated MAbs 439 and 1255, were produced from mice immunized with serotype A polysaccharide. One hybridoma, designated MAb 302, was produced from mice immunized with serotype D polysaccharide. All three antibodies were of the immunoglobulin G1 isotype. MAb 302 showed a specificity for serotypes A and D in Ouchterlony diffusion, agglutination, and opsonophagocytosis assays. MAb 1255 was reactive with polysaccharides and cells of serotypes A, B, and D. MAb 439 was reactive with polysaccharides and cells of serotypes A, B, C, and D. The reactivity of these MAbs closely matched the distribution of epitopes among cryptococcal polysaccharides predicted in previous studies of polyclonal antibodies reactive with cryptococcal polysaccharides. The ability to produce a MAb against an epitope shared by all four serotypes may have value for the detection of cryptococcal antigens in body fluids.     

Molecular basis for immunoglobulin M specificity to epitopes in Cryptococcus neoformans polysaccharide that elicit protective and nonprotective antibodies

The protective efficacy of antibodies (Abs) to Cryptococcus neoformans glucuronoxylomannan (GXM) is dependent on Ab fine specificity. Two clonally related immunoglobulin M monoclonal Abs (MAbs) (12A1 and 13F1) differ in fine specificity and protective efficacy, presumably due to variable (V)-region sequence differences resulting from somatic mutations. MAb 12A1 is protective and produces annular immunofluorescence (IF) on serotype D C. neoformans, while MAb 13F1 is not protective and produces punctate IF. To determine the Ab molecular determinants responsible for the IF pattern, site-directed mutagenesis of the MAb 12A1 heavy-chain V region (V(H)) was followed by serological and functional studies of the various mutants. Changing two selected amino acids in the 12A1 V(H) binding cavity to the corresponding residues in the 13F1 V(H) altered the IF pattern from annular to punctate, reduced opsonic efficacy, and abolished recognition by an anti-idiotypic Ab. Analysis of the binding of the various mutants to peptide mimetics revealed that different amino acids were responsible for GXM binding and peptide specificity. The results suggest that V-region motifs associated with annular binding and opsonic activity may be predictive of Ab efficacy against C. neoformans. This has important implications for immunotherapy and vaccine design that are reinforced by the finding that GXM and peptide reactivities are determined by different amino acid residues.     

Variable efficacy of passive antibody administration against diverse Cryptococcus neoformans strains.

The efficacy of monoclonal antibody (MAb 2H1) against diverse strains of Cryptococcus neoformans was studied in a murine model of intravenous infection. For six of eight strains, administration of MAb prior to infection prolonged survival of mice. For two strains, 371 and SB4A, administration of MAb prior to infection did not prolong survival in multiple experiments with inocula ranging from 10(2) to 10(6) yeast cells per mouse. Mice infected with strains 371 and SB4A had fewer CFU than non-MAb-treated controls, but the CFU reduction was not sufficient to affect survival. Serum glucuronoxylomannan (GXM) levels varied for the different C. neoformans strains. For mice that did not receive MAb 2H1, there was a positive correlation between lung fungal burden and serum GXM levels. MAb 2H1-treated mice had significantly reduced serum GXM levels. The results indicate that the efficacy of MAb 2H1 administration in prolonging survival and/or reducing organ CFU varies with the C. neoformans strain.     

Sequence changes at the V-D junction of the VH1 heavy chain of anti- phosphocholine antibodies alter binding to and protection against Streptococcus pneumoniae

X-linked immune deficient (Xid) mice fail to produce anti- phosphocholine (PC) antibodies even after immunization with Streptococcus pneumoniae. Consequently, Xid mice are extremely susceptible to infection with S. pneumoniae, PC-specific B cells appear to undergo clonal deletion in Xid mice; however, a new thymus-dependent form of PC, 6-(O-phosphocholine)hydroxyhexanoate (EPC), can rescue PC- specific B cells from the bone marrow presumably by providing T cell help before clonal deletion. Analysis of PC-specific IgG hybridomas from Xid mice revealed utilization of several V-D junctional variants of the VH1 gene segment rearranged to different D and JH gene segments. The majority of Xid anti-PC antibodies exhibit an Asp-->Gly95H replacement at the V-D junction. These Gly95H VH1 variants associate with kappa 1C L chains to produce anti-PC antibodies that: (1) have low relative affinity for PC, (ii) are heteroclitic for nitrophenylphosphocholine and (iii) fall to bind to or provide protection against S. pneumoniae. Single prototypic V-D variants of the T15 idiotype (Asp95H), M603 idiotype (Asn95H) and M167 idiotype (Asp95H- Ala96H) were also induced in Xid mice. The M603-like and M167-like antibodies bound to and protected against S. pneumoniae even though they exhibited Kas for PC which were lower than T15 idiotype+ antibodies. These data demonstrate that small changes in the V-D junctional sequence of the T15 (VH1) heavy chain alter L chain usage and the structure of the PC binding site so that the PC expressed on S. pneumoniae is no longer recognized.      

Immunogenicity and efficacy of Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan peptide mimotope-protein conjugates in human immunoglobulin transgenic mice

Peptide mimotopes of capsular polysaccharides have been proposed as antigens for vaccines against encapsulated pathogens. In this study, we determined the antibody response to and efficacy of P13, a peptide mimetic of the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM), in mice that produce human antibodies. P13 was conjugated to tetanus toxoid (TT) or diphtheria toxoid (DT) and administered subcutaneously in Alhydrogel with or without CpG to mice transgenic for human immunoglobulin loci (XenoMouse mice) and expressing either immunoglobulin G2 (IgG2) (G2 mice) or IgG4 (G4 mice). Mice were vaccinated and revaccinated two or three times. The serum antibody responses of the mice to GXM and P13 and antibody idiotype expression were analyzed by an enzyme-linked immunosorbent assay. The results showed that both P13-TT and P13-DT were antigenic, inducing a mimetic response to P13 in both G2 and G4 mice, and immunogenic, inducing a mimotope response including VH3 (idiotype)-positive antibodies to GXM in G2 but not G4 mice. CpG led to higher titers of IgG to P13 and GXM in P13-TT-vaccinated G2 mice. C. neoformans challenge of P13-protein conjugate-vaccinated and control G2 mice induced anamnestic IgG- and VH3-positive responses to GXM and was associated with a significantly decreased risk of death and a prolongation of survival in P13-DT-vaccinated mice compared to phosphate-buffered saline-treated or protein carrier-vaccinated mice. These findings reveal that P13 elicited a human antibody response with VH3 expression in human immunoglobulin transgenic mice that has been observed for human antibodies to GXM and support the concept that peptide mimotope-based vaccines may hold promise for the treatment of C. neoformans infections.     

In Vitro and In Vivo Stability a Cryptococcus neoformans Glucuronoxylomannan Epitope That Elicits Protective Antibodies

The monoclonal antibody (MAb) 2H1 defines an epitope in Cryptococcus neoformans capsular glucuronoxylomannan (GXM) that can elicit protective antibodies. In murine models of cryptococcosis, MAb 2H1 administration prolongs survival and reduces fungal burden but seldom clears the infection. The mechanism by which C. neoformans persists and escape antibody-mediated clearance is not understood. One possibility is that variants that do not bind MAb 2H1 emerge in the course of infection. Using an agglutination-sedimentation protocol, we recovered a variant of strain 24067 that did not agglutinate, could not be serotyped, and had marked reduction in GXM O-acetyl groups. Binding of MAb 2H1 to 24067 variant cells produced a different immunofluorescence pattern and lower fluorescence intensity relative to the parent 24067 cells. Addition of MAb 2H1 to 24067 variant cells had no effect on cell charge. Phagocytic assays demonstrated that MAb 2H1 was not an effective opsonin for the 24067 variant. The 24067 variant was less virulent than the 24067 parent strain in mice, and MAb 2H1 administration did not prolong survival in animals infected with the variant strain. To investigate whether variants which do not bind MAb 2H1 are selected in experimental infection, three C. neoformans strains were serially passaged in mice given either MAb 2H1 or no antibody. Analysis of passaged isolates by agglutination assay, flow cytometry, and indirect immunofluorescence revealed changes in MAb 2H1 epitope expression but no clear trend with regards to gain or loss of MAb 2H1 epitope. C. neoformans variants with reduced MAb 2H1 epitope content can be isolated in vitro, but persistence of infection in mice given MAb 2H1 does not appear to be a result of selection of escape variants that lack the MAb 2H1 epitope.     

Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C

Abstract

Clostridium tetani causes a life-threatening infectious disease by production of tetanus neurotoxin (TeNT), a 150?kDa molecule composed of light (LC) and heavy chain (HC) polypeptides. The TeNT HC contains an N-terminal domain critical for LC translocation and a C-terminal toxin receptor-binding domain known as fragment C. Despite extensive investigations on epitope specificity of anti-TeNT antibodies, the immunodominant neutralizing epitopes of the toxin are poorly defined. This study describes the generation and characterization of four monoclonal antibodies (MAb) specific for TeNT. The characteristics of each MAb were explored in terms of isotype, specificity, affinity, and immuno-globulin heavy chain variable region (IGHV) gene usage using ELISA, Western blotting, and sequencing techniques. The toxin neutralizing activity of the MAbs was also investigated using the in vitro GT1b neutralizing assay. The data demonstrated that all MAbs bind to tetanus toxin and toxoid. Sub-fragments binding analysis showed that two MAbs react with fragment C, one with both fragment C and LC, and one with LC. Only the two fragment C-specific MAbs were able to neutralize the toxin. Sequencing of the expressed VH and VL genes revealed rearrangements of various VH and VL gene segments in all hybridoma clones. Clonality of the hybridomas was also confirmed by a competition assay that showed recognition of distinct epitopes by these MAbs. The results suggest the importance of TeNT fragment C in terms of immunogenicity and toxin neutralization activity.     

Idiotypic Replica of an Anti-Human Tumour-Associated Antigen Monoclonal Antibody DNA Sequence Comparison between Ab1 and Ab3

2G-3 is an anti-anti-idiotypic MoAb (Ab3) obtained upon immunization with a monoclonal Ab2 (A3B10), which behaves as the 'internal image' of CaMBr1, a saccharide epitope defined by MoAb MBr1 (Ab1). CaMBr1 is expressed on glycoconjugates of the human mammary carcinoma cell line MCF-7 and on normal and neoplastic mammary gland epithelial cells. Ab1 and Ab3, although exhibiting, in many respects, superimposable paratopic and idiotopic specificities, show a non-identical fine immunoreactivity, since 2G-3 has a preferential reactivity with the saccharidic epitope mounted on glycoproteins, while MBr1 reacts with both glycoproteins and glycolipids. V-region sequence analysis has shown that: (i) the VK genes employed belong to different families (VK1 and VK10); (ii) the JK2 segment is shared by the two L chains (thus a high degree of homology is observed between VK CDR3s); (iii) the VH genes employed derive from the same family VHIIB/J558 (but show CDR homology only in CDR2); (iv) different JH region genes are employed. These data, together with the comparison of deduced secondary structure parameters, give further evidence for the possible production of similar combining sites using different VH and VL germ-line genes.     

Variable region sequences and idiotypic expression of a protective human immunoglobulin M antibody to capsular polysaccharides of Neisseria meningitidis group B and Escherichia coli K1.

We determined the heavy (H)- and light (L)-chain variable (V) region nucleotide and translated amino acid sequences of the human immunoglobulin M(kappa) monoclonal antibody (MAb) 5E1, which is specific for the polysaccharide capsule of Escherichia coli K1 and Neisseria meningitidis group B (poly[alpha(2-->8)-N-acetylneuraminic acid]) and which is protective in animal models of infection. The 5E1 VH gene is a member of the VHIIIb family and is 97% homologous to the 9.1 germ line gene. The 5E1 VL gene is a member of the kappa I subgroup and is 98% homologous to the germ line gene, 15A, also known as KLO12. The VL and/or VH genes used by 5E1 are highly homologous to the V genes encoding antibodies to the Haemophilus influenzae type b polysaccharide and to antibodies reactive with self-antigens such as erythrocyte "i," DNA, and thyroid peroxidase. We also produced three murine anti-idiotype (Id) MAbs against 5E1. All three anti-Ids recognize a minor subset of antimeningococcal B polysaccharide antibodies present in serum from normal adults. Two of the anti-Ids define distinct Ids associated with antibodies having kappa I-15A V regions. These 15A-associated Ids are expressed by some heterologous human antimeningococcal B polysaccharide MAbs, and they also are independently expressed by two human MAbs that are specific for either the H. influenzae b polysaccharide or the i erythrocyte antigen and that utilize the kappa I-15A V region. Taken together, these data indicate that the 5E1 antibody uses V regions that recur in the human antibody repertoires to this polysaccharide and to structurally dissimilar polysaccharides and autoantigens. Thus, the poor immunogenicity of poly[alpha(2-->8)-N-acetylneuraminic acid] cannot be explained by the unavailability of certain critical VH and VL genes required for generation of antibody response.     

Restricted usage of VH and V kappa genes by murine monoclonal antibodies against 3-fucosyllactosamine

We are studying the structure and regulation of murine antibodies against the 3-fucosyllactosamine antigenic determinant. Analysis of the sequences of seven BALB/c IgM, kappa monoclonal antibodies (mAb), obtained from four fusions, indicates that these antibodies exhibit restriction in their usage of VH and VL genes. Based on a combination of mRNA sequences and Southern filter hybridization data, all seven light chains are encoded by V kappa 24B and J kappa 1 gene segments. Complete mRNA sequences of the heavy chains revealed that all seven mAb are encoded by VH441, six antibodies are encoded by JH4 and one uses a JH3 gene segment. The VH441 gene segment and all seven mAb contain a potential glycosylation site at Asn 58 in complementarity-determining region (CDR)2. In contrast to the similarity of the VH regions, the heavy chain CDR3 segments exhibit considerable heterogeneity. They are encoded by three D segments, they vary in length from 7-9 amino acids and display differences in their deduced amino acid sequences. The VH441 gene segment also encodes antibodies against four other carbohydrate antigens, levan, galactan, dextran and galactosyl globoside. The use of a single gene segment to encode antibodies against five different antigens suggests that the domain encoded by VH441 might be particularly well adapted for forming sites that bind carbohydrate determinants. Glycosylation of CDR2 might contribute to the unique properties of this VH domain.     

Antigenic and biological characteristics of mutant strains of Cryptococcus neoformans lacking capsular O acetylation or xylosyl side chains

Cryptococcus neoformans is surrounded by an antiphagocytic polysaccharide capsule whose primary constituent is glucuronoxylomannan (GXM). Three prominent structural features of GXM are single xylosyl and glucuronosyl side chains and O acetylation of the mannose backbone. Isogenic pairs of O-acetyl-positive and O-acetyl-negative strains (cas1 Delta) as well as xylose-positive and xylose-negative strains (uxs1 Delta) of serotype D have been reported. The cas1 Delta strains were hypervirulent, and the uxs1 Delta strains were avirulent. The goal of this study was to examine the effects of the cas1 Delta and uxs1 Delta mutations on the following: (i) binding of anti-GXM monoclonal antibodies (MAbs) in capsular quellung reactions, (ii) activation of the complement system and binding of C3, (iii) phagocytosis by neutrophils, and (iv) clearance of GXM in vivo. The results showed that loss of O acetylation produced dramatic changes in the reactivities of five of seven anti-GXM MAbs. In contrast, loss of xylosylation produced a substantive alteration in the binding behavior of only one MAb. O-acetyl-negative strains showed no alteration in activation and binding of C3 from normal serum. Xylose-negative strains exhibited accelerated kinetics for C3 deposition. Loss of O acetylation or xylosylation had no effect on phagocytosis of serum-opsonized yeast cells by human neutrophils. Finally, loss of O acetylation or xylosylation altered the kinetics for clearance of GXM from serum and accumulation of GXM in the liver and spleen. These results show that O acetylation and/or xylosylation are important for binding of anti-GXM MAbs, for complement activation, and for tissue accumulation of GXM but do not impact phagocytosis by neutrophils.     

Monoclonal antibodies reactive with immunorecessive epitopes of glucuronoxylomannan,the major capsular polysaccharide of Cryptococcus neoformans

Cryptococcus neoformans is surrounded by an antiphagocytic capsule whose primary constituent is glucuronoxylomannan (GXM). An epitope shared by GXM serotypes A, B, C, and D is immunodominant when mice are immunized with serotype A GXM. In contrast, an epitope shared only by serotypes A and D is immunodominant when mice are immunized with serotype D. Hybridomas secreting antibodies reactive with subdominant epitopes were identified through a positive-negative screening procedure in which antibody-secreting colonies were characterized by reactivity with both the immunizing polysaccharide and GXMs from each of the four major serotypes. In this manner, a monoclonal antibody (MAb) that was reactive with an epitope shared only by serotypes A and B was identified and designated F10F5. Such an epitope has not been described previously. Immunization of mice with de-O-acetylated serotype A GXM generated a hybridoma that secreted an antibody, designated F12D2, that was reactive with all four serotypes. Unlike previously described monoclonal and polyclonal panspecific antibodies, the reactivity of MAb F12D2 was not altered by de-O-acetylation of GXM. These results indicate that there are at least two panspecific GXM epitopes; one epitope is dependent on O acetylation for antibody reactivity, and the other is independent of O acetylation. This study identifies strategies for production of MAbs that are reactive with subdominant or cryptic GXM epitopes and provides new information regarding the antigenic makeup and the humoral immune response to GXM, an essential virulence factor that is a target for active and passive immunization.