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.     

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.     

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.     

Monoclonal antibodies against Haemophilus lipopolysaccharides: clone DP8 specific for Haemophilus ducreyi and clone DH24 binding to lacto-N-neotetraose.

Mouse monoclonal antibodies (MAbs) DP8 [immunoglobulin G1(kappa)] and DH24 [immunoglobulin M(kappa)], which are specific for Haemophilus ducreyi lipopolysaccharide (LPS), were generated by fusing mouse myeloma NS0 cells with spleen cells of BALB/c mice immunized with a total membrane preparation of H. ducreyi. MAb DP8 reacted in whole-cell enzyme immunoassay (EIA) and colony dot immunoblotting with all 50 strains of H. ducreyi but not with any other bacteria tested, which suggests an exposed and species-specific epitope on the H. ducreyi cell surface. This conclusion was supported by the finding that DP8 bound to all six H. ducreyi LPSs tested but not to any of the Haemophilus influenzae or enterobacterial LPSs or synthetic glycoconjugates. The MAb DH24 bound to 43 of 50 strains of H. ducreyi and to few strains of H. influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis, as evaluated by whole-cell EIA and colony dot immunoblotting. The MAb DH24 reacted with five of the six H. ducreyi LPSs tested and with the lacto-N-neotetraose (Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc) series of synthetic glycoconjugates, as determined by EIA. By using polysaccharides obtained after both mild acidic hydrolysis and strong alkali treatment and dephosphorylated samples as inhibitors of the MAbs binding to H. ducreyi LPS antigens, it could be shown that phosphate groups were essential for the binding of DP8 to LPS but that they did not affect antigenic recognition by DH24. None of the MAbs bound to isolated lipid A, but aggregation caused by the fatty acids of lipid A was essential for epitope recognition.     

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.     

Detection of Neisseria gonorrhoeae by dot-enzyme immunoassay using monoclonal antibodies

A highly sensitive and specific dot-enzyme immunoassay for the detection of Neisseria gonorrhoeae was developed using a pool of monoclonal antibodies (MAbs). The MAbs were obtained following immunization of mice with lithium acetate extracted outer membrane (OM) preparations. Western immunoblotting experiments demonstrated that MAbs NG26 and NG38, both IgG2a, reacted with lipopolysaccharides (LPS) and with the major OM protein, P1, respectively, MAb NG28, an IgG3, did not react in Western immunoblotting, MAbs NG28 and NG38 failed to react with OM treated with proteolytic enzymes or with semi-purified preparation of LPS. MAb NG26 reacted with the same LPS preparation. Binding radioimmunoassay with live bacteria showed that all the MAbs adsorbed to cell surface-exposed antigenic determinants. The limit of detection of the dot-enzyme immunoassay was between 1 and 4 x 10(4) cfu per dot. Using a panel of 177 strains of N. gonorrhoeae, MAbs NG28 and NG38 recognized only P1A and P1B strains respectively. MAb NG26 reacted with P1A, P1B and non-typable strains. These MAbs did not react with other Neisseria species or other bacterial species. Using this pool, the dot-enzyme immunoassay had a sensitivity of 93.2% and a specificity of 100%.     

Rat monoclonal antibodies to mouse IgG1, IgG2a, IgG2b, and IgG3 subclasses, and kappa chain isotypic determinants

Rats of the LOU/Ws1 strain were immunized with mixtures of mouse monoclonal antibodies (MAbs) of various isotypes, and their spleen cells were fused with the rat myeloma Y3.Ag1.2.3 or the mouse myeloma X63.Ag8.653. From four fusion experiments, we have selected 14 rat MAbs that exhibited selective binding to either IgG2a, IgG2b, IgG1, and IgG3 subclasses or to kappa chain isotypic determinants. Cross-blocking studies revealed that three rat MAbs identified distinct determinants on the Fc fragment of the MAb H10-81.10 (A.TH, Igh-1e). By contrast, the IgG1, IgG2b, IgG3, and kappa isotypes defined by the mAbs analyzed in this study were found to be in close spatial relationship. These rat MAbs bound IgG2a, IgG2b, or IgG1 mouse MAbs, expressing the Igh-1e,j,a, or c, Igh-3a or b, or Igh-4a or b allelic specificities, respectively.     

Two chicken monoclonal antibodies specific for heterophil Hanganutziu-Deicher antigens.

Two chicken monoclonal antibodies (MAbs), HU/Ch2-7 and HU/Ch6-1, against heterophil Hanganutziu-Deicher (HD) antigens with N-glycolylneuraminic acid (NeuGc) at a terminal carbohydrate were established by cell fusions using chicken B cell lines lacking thymidine kinase and spleen cells from chickens immunized with II3NeuGc alpha-LacCer (HD3). The reactivities of these MAbs against several gangliosides including NeuGc-containing glycosphingolipids were examined by a thin-layer chromatography/immunostaining method. MAb HU/Ch2-7 (IgG) reacted strongly with HD3 and IV3NeuGc alpha-nLc4Cer (HD5) and weakly with VI3NeuGc alpha-nLc6Cer (HD7) and 4-O-acetyl-HD3. HU/Ch6-1 (IgG) reacted with HD3 and HD5, but did not react with the other HD antigens. The reactivities of these MAbs against HD antigen were greatly reduced by pre-treatment of the antigen with neuraminidase. These MAbs did not react with N-acetylneuraminic acid-containing gangliosides (GM1 and GM3). These results indicate that these two chicken MAbs are directed toward the antigenic epitope containing the NeuGc.     

Monoclonal antibodies to hog thyroglobulin recognizing disulfide-dependent conformational structures

Monoclonal antibodies (MAbs) to hog thyroglobulin (Tg) were obtained by fusion of myeloma cells with B-lymphocytes from mice immunized with the protein. The five MAbs were classified into three groups which recognized different determinants; (1) MAb 4, (2) MAbs 6 and 10, and (3) MAbs 16 and 18. MAb 16 was IgG2b and the others IgG1. The immunoreactivity of Tg to the five MAbs was lost after reduction of the protein under denaturing conditions but remained after limited digestion by proteolytic enzymes. Tryptic fragments were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrophoretically blotted and stained by a peroxidase-linked second antibody. Various sized tryptic fragments reacted similarly with the three MAbs which had different determinant specificities. All the MAb-reactable fragments in the tryptic digest completely lost their immunoreactivity after reduction of their disulfide linkages.     

Molecular and idiotypic analysis of antibodies to Cryptococcus neoformans glucuronoxylomannan.

Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) form the basis of two potential therapeutic intervention strategies, i.e., conjugate vaccines and passive antibody therapy. To better understand the molecular basis of the antibody response, the heavy- and light-chain immunoglobulin variable region (VH and VL, respectively) sequences of seven monoclonal antibodies (MAbs) to GXM were determined. Rabbit anti-idiotypic serum was made to the previously characterized murine MAb 2H1 and used to study MAb 2H1 idiotype expression in other GXM-binding MAbs and immune sera. MAb E1 originated from a C3H/HeJ mouse immunized with C. neoformans serotype A polysaccharide. MAbs 471, 1255, 339, 3C2, 386, and 302 originated from BALB/c mice immunized with polysaccharide of serotypes A, A, B, C, D, and D, respectively, conjugated to sheep erythrocytes. In the E1, VH uses V11 from the T15 gene family and JH3 and has a D segment of three amino acids, and the VL uses a VKSer-like gene family element and JK5. In MAbs 471 and 3C2, the VH uses VH7183-like gene family elements and JH2 and has D segments of seven amino acids, and the VL uses VK5.1 and JK1. In MAbs 1255 and 339, the VH uses VH10-like gene elements and JH4 and has six codon D segments, and the VL uses a VK21-like gene element and JK5. In MAbs 302 and 386, respectively, the VH uses VHGAM-like gene elements and JH2 and JH3 and has six and four codon D segments, and VL uses VK4/5-like gene elements and JK1.VH usage, MAb 2H1 idiotype expression, and fine specificity mapping define a minimum of three GXM epitopes which elicit protective antibodies. The results confirm that the antibody response is highly restricted, suggest a close relationship between molecular structure and serological properties, and provide insight into protein structural motifs important for GXM binding.     

Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis.

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.     

Production and characterization of murine monoclonal antibodies to Histoplasma capsulatum yeast cell antigens

Four monoclonal antibodies (MAbs) were produced by immunizing mice with a disrupted yeast cell homogenate of Histoplasma capsulatum. MAbs 1 and 2 reacted only with the yeast cell antigens of H. capsulatum and Blastomyces dermatitidis, whereas MAbs 3 and 4 showed broader cross-reactivity. MAb 3 cross-reacted with B. dermatitidis, Paracoccidioides brasiliensis, Sporothrix schenckii, and Candida albicans, and MAb 4 cross-reacted with B. dermatitidis, C. albicans, Coccidioides immitis, Aspergillus fumigatus, and Mycobacterium tuberculosis. All four MAbs exhibited unique specificity when reacted with three different strains of H. capsulatum (G217B, A811, and P-IN). MAb 1 belonged to the IgG2b subclass, MAb 3 belonged to the IgG1 subclass, and MAbs 2 and 4 belonged to the IgG3 subclass. MAbs 1, 2, and 3 formed bands in the Western immunoblot assay; the two dominant distinct bands had apparent molecular masses of 72 and 62 kilodaltons.     

Monoclonal antibodies against Candida tropicalis mannan: antigen detection by enzyme immunoassay and immunofluorescence.

Three strains of mice were immunized with Candida tropicalis cell walls, and antibodies against mannan were detected by indirect enzyme immunoassay (EIA) in 3 of 9 BALB/c mice, 4 of 11 C57BL/6 mice, and 4 of 8 CFW mice. Responding mice produced immunoglobulin M (IgM), but IgG was not detected in their sera. Fusion of the high-responder BALB/c mouse with a plasmacytoma cell line resulted in 41 clones secreting antimannan monoclonal antibodies (MAbs). Four clones selected for propagation included one IgM and one IgG MAb that reacted with mannans of Candida albicans serotypes A and B and of C. tropicalis and two IgM MAbs specific for an epitope only in the mannans of C. albicans serotype A and C. tropicalis. One of the IgM MAbs, CB6, was an effective substitute for rabbit antibodies in the double-antibody sandwich EIA to detect antigenemia produced in rabbits infected with C. albicans A or C. tropicalis. It could function either as the peroxidase-conjugated indicator antibody or as the capture antibody. Two MAbs, CB6 (C. tropicalis and C. albicans A specific) and AC3 (C. tropicalis and C. albicans A and B specific), functioned in place of polyclonal antisera in the serotyping of C. albicans by immunofluorescence. There was 95.8% agreement in the results of serotyping using MAbs as reagents compared with rabbit antisera. Competitive inhibition in EIA between CB6 and monospecific antisera against C. albicans factors 1, 4, and 6 indicated that CB6 binds to an epitope which is probably factor 6. Serologic similarity between factor 4 and the binding site of MAb AC3 was also determined.     

Production and characterization of monoclonal antibodies against human thyroglobulin

Spleen cells of Biozzi-HR mice immunized with human thyroglobulin (hTg) were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty monoclonal antibodies (MAbs) selected by an enzyme immunoassay (indirect ELISA) were produced, purified and characterized. The equilibrium association constant (Ka) of one of the MAbs, determined by Scatchard analysis of the ELISA data, was found to be 2 X 10(9) M-1; the Ka of the other MAb, estimated from titration curves by comparison with the aforementioned MAb, ranged from 8 X 10(9) M-1 to 6 X 10(7) M-1. The reaction between the MAb and hTg was not inhibited by thyroxin (T4), triiodothyronine (T3) and triiodothyropropionic acid (DT3). Species specificity of the MAb was studied using bovine and porcine Tgb. The topology of the MAb was investigated by competitive inhibition immunoassays. Seven distinct antigenic regions were identified.     

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.     

Characterisation and epitope analysis of monoclonal antibodies to virions of clover yellow vein and Johnsongrass mosaic potyviruses

Summary Mouse monoclonal antibodies (MAbs) against the Australian B strain of clover yellow vein (C1YVV-B) and the JG strain of Johnsongrass mosaic (JGMV) potyviruses were produced, characterised and the epitopes with which they reacted were deduced. Using intact particles of C1YVV a total of ten MAbs were obtained which reacted strongly with C1YVV-B in an enzyme-linked immunosorbent assay and Western blots. Four of these MAbs (1, 2, 4, and 13) were found to be ClYVV-specific, as they reacted with all five C1YVV strains from Australia and the U.S.A. but not with 11 strains of bean yellow mosaic (BYMV), pea mosaic (PMV), and white lupin mosaic (WLMV) viruses which, together with C1YVV, form the BYMV subgroup of potyviruses. These MAbs failed to react with eight other potyvirus species, including six which infect legumes like the viruses in the BYMV subgroup. The C1YVV MAb 10 was found to be BYMV subgroup-specific. It reacted strongly with 15 of the 16 strains of viruses in the subgroup and gave no reaction with eight other potyviruses. The other five C1YVV MAbs reacted with varying degrees of specificity with the BYMV subgroup viruses and also with other potyviruses. Eight of the C1YVV MAbs (1, 2, 4, 5, 13, 17, 21, and 22) reacted with the intact coat proteins only and not with the truncated (minus amino terminus) coat protein of C1YVV suggesting that the epitopes for these MAbs are located in the surface-exposed, amino-terminal region of the C1YVV coat protein. Comparison of published coat protein sequences of BYMV and C1YVV isolates indicated that the epitopes for the four ClYVV-specific MAbs may be in the amino-terminal region spanning amino acid residues 18 to 30, whereas those for the other four MAbs may be located in the first 17 amino-terminal amino acid residue region. The epitopes that reacted with BYMV subgroup-specific MAb 10 and MAb 30 which reacted with 20 of the 24 potyvirus isolates, are probably located in the core region of C1YVV coat protein as these MAbs reacted with the intact as well as truncated coat protein of C1YVV. Analysis, in Western blot immunoassay, of 17 MAbs raised against virions of JGMV revealed that only two MAbs (1–25 and 4–30) were JGMV-specific, whereas others displayed varying degrees of specificity to different potyviruses. When these MAbs were screened against the intact and truncated (minus 67 amino-terminal amino acid residues) coat proteins of JGMV, the two JGMV-specific MAbs reacted only with the intact coat protein, whereas the other MAbs reacted with the intact as well as with truncated coat proteins, in Western blots. These results suggest that the epitopes for the two JGMV-specific MAbs are located in the surface-exposed amino-terminal 67 amino acid residue region and those for the cross-reactive MAbs are contained in the conserved core region of the JGMV coat protein. Screening of potyvirus MAbs against intact and truncated coat proteins thus appears to be a simple procedure to select virus-specific MAbs to potyviruses.     

Monoclonal antibodies delineate multiple epitopes on the O antigens of Salmonella typhi lipopolysaccharide.

Fifteen monoclonal antibodies (MAbs) directed against Salmonella typhi were produced and characterized. The specificities of the antibodies were determined by their binding patterns in an enzyme immunoassay, with a panel of lipopolysaccharides isolated from different bacteria. Seven MAbs reacted with S. typhi, Salmonella enteritidis, and Salmonella dublin (all belonging to serogroup D). One MAb also reacted with Salmonella paratyphi A and S. paratyphi B. Five MAbs reacted with S. typhi, S. enteritidis, S. dublin, and S. paratyphi B. Two MAbs did not bind to any lipopolysaccharide but showed reactivity with bacterial sonic extracts isolated from S. typhi, S. paratyphi A, S. paratyphi B, Escherichia coli, and Shigella sonnei. These antibodies would be helpful in studying the complexity of antigenic determinants expressed by S. typhi and the nature of the antibody response during typhoid and paratyphoid fevers and also in the diagnosis of the disease.     

Production and characterization of two monoclonal antibodies to human pancreatic trypsin 1

Two monoclonal antibodies (MAb) G6 and A8 directed against human trypsin 1 have been produced by hybridization of myeloma cells with spleen cells of OF1 immunized mice. Antibodies were screened by radioimmunoassay. Monoclonal antibodies were purified by affinity chromatography on Protein A-Sepharose, and we found that MAb G6 was of the IgG2b class and MAb A8 of the IgG2a class. Both MAbs had a high affinity for trypsin 1 with the respective affinity constants equal to 1.3 x 10(8) l/mol for G6 and 3.2 x 10(7) l/mol for A8. Epitope specificity was studied by western blotting, using human trypsinogens 1 and 2. Both MAbs gave a positive reaction with native trypsinogen 1 and no reaction with the same protein after reduction. Only MAb G6 reacted with trypsinogen 2 in the native form. Its affinity for trypsin 2 was found similar to that for trypsin 1 with a constant equal to 2.7 x 10(7) l/mol. Both antibodies appeared directed against conformational and not sequential epitopes.     

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.     

Cryptococcus neoformans serotype A glucuronoxylomannan-protein conjugate vaccines: synthesis, characterization, and immunogenicity.

We synthesized Cryptococcus neoformans serotype A glucuronoxylomannan (GXM) conjugate vaccines under conditions suitable for human use to prevent disseminated cryptococcosis. The purified, sonicated GXM was derivatized with adipic acid dihydrazide through either hydroxyl or carboxyl groups and then covalently bound to tetanus toxoid (TT) or Pseudomonas aeruginosa exoprotein A (rEPA). The immunogenicity of these conjugates was evaluated in BALB/c and general purpose mice by subcutaneous injection in saline. The conjugates elicited higher GXM antibody responses than GXM alone. Booster immunoglobulin G (IgG) and IgM responses were elicited by all conjugates in BALB/c mice. The conjugates prepared through hydroxyl activation (GXM-TT2 and GXM-rEPA) were more immunogenic than the one prepared through carboxyl activation (GXM-TT1). GXM antibody response was enhanced by the administration of monophosphoryl lipid A 2 days following the injection of GXM-TT2 (P less than 0.03). The conjugates also elicited IgG antibodies to the carrier proteins. Gel diffusion tests using conjugate-induced hyperimmune sera and chemically modified GXMs suggested that the specificity of GXM-TT1-induced antibodies was conferred by the O-acetyl groups. Hyperimmune sera generated by GXM-TT2 precipitated with the chemically unmodified and the de-O-acetylated GXMs but not with the carboxyl-reduced and de-O-acetylated GXM. GXM-TT2-induced hyperimmune serum also precipitated with the capsular polysaccharides of C. neoformans serotypes D, B, and C. The conjugate vaccines prepared through hydroxyl activation of the GXM are sufficiently immunogenic and appear to be suitable for clinical evaluation.