Antibody response that protects against disseminated candidiasis.

We previously showed that surface mannans of Candida albicans function as adhesins during yeast cell attachment to mouse splenic marginal zone macrophages. The mannan adhesin fraction was encapsulated into liposomes and used to vaccinate mice over a 5- to 6-week period. Circulating agglutinins specific for the fraction correlated with increased resistance to disseminated candidiasis. Antiserum from vaccinated animals protected naive BALB/cByJ mice against C. albicans serotype A and B strains and Candida tropicalis. Antiserum also protected SCID mice against disseminated disease. The serum protective ability was stable at 56 degrees C, but this ability was adsorbed by C. albicans cells. The antiserum was divided into three fractions after separation by high-performance liquid chromatography. One fraction contained all of the agglutinin activity and transferred resistance to naive mice. A second fraction also transferred resistance. Two monoclonal antibodies (MAbs) specific for candidal surface determinants were obtained. MAb B6.1 is specific for a mannan epitope in the adhesin fraction, and MAb B6 is specific for a different epitope in the fraction. Both MAbs are immunoglobulin M, and both strongly agglutinate candidal cells, but only MAb B6.1 protected both normal and SCID mice against disseminated candidiasis. In one experiment, 10 normal mice were given MAb B6.1 and challenged with yeast cells. Six mice survived the 67-day observation period; 4 of the survivors were cured as evidenced by the lack of CFU in the kidney and spleen. Our studies show that antibodies against certain cell surface antigens of C. albicans help the host resist disseminated candidiasis.     

A Vaccine and Monoclonal Antibodies That Enhance Mouse Resistance to Candida albicans Vaginal Infection

We previously reported that a vaccine composed of liposome-mannan complexes of Candida albicans (L-mann) stimulates mice to produce protective antibodies against disseminated candidiasis. An immunoglobulin M (IgM) monoclonal antibody (MAb), B6.1, specific for a β-1,2-mannotriose in the complexes protects against the disease, whereas MAb B6 does not. In the present study, the vaccine and MAbs B6.1 and B6 were tested for the ability to protect against Candida vaginal infection, established by intravaginal (i.vg.) inoculation of yeast cells in mice maintained in pseudoestrus. Fungal CFU in each vagina was determined to assess the severity of infection. Mice vaccinated before infection developed about 62% fewer vaginal CFU than nonimmunized controls. Naive mice that received polyclonal antiserum (from vaccinated mice) i.vg. before infection had 60% fewer CFU than controls. The serum protective factor was stable at 56°C, but C. albicans cells absorbed this factor. Mice given MAb B6.1 i.vg. after infection was established had fewer Candida CFU in vaginal tissue than control mice given buffer instead of antibody. MAbs B6.1 and B6 given intraperitoneally before infection protected mice, but MAbs preabsorbed with yeast cells did not. MAb B6.1 also protected against C. tropicalis vaginal infection, but MAb B6 did not. The protective activities of MAbs B6.1 and B6 appeared to be specific because an irrelevant IgM carbohydrate-specific MAb and an irrelevant IgG protein-specific MAb were not protective; also, MAb B6.1 did not affect development of vaginal chlamydial infection. These studies show that an appropriate antibody response, or administration of protective antibodies, can help the host to resist Candida vaginal infection.     

Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies.

We previously reported that the immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1 protects mice against disseminated candidiasis, whereas the IgM MAb B6 does not. Both MAbs are specific for an adhesin fraction isolated from the cell surface of Candida albicans, but their epitope specificities differ. In the present study, we examined the surface locations of both epitopes and obtained structural information regarding the B6.1 epitope. Immunofluorescence confocal microscopic analysis of C. albicans yeast forms showed that epitope B6.1 is displayed rather homogeneously over the entire cell surface, whereas epitope B6 appears to have a patchy distribution. Both antibodies were essentially nonreactive with the surfaces of mycelial forms of the fungus, indicating that neither epitope is expressed on the surfaces of these forms. For isolation of the B6.1 epitope, the adhesin fraction consisting of cell surface phosphomannan was subjected to mildly acidic (10 mM HCl) hydrolysis and was fractionated into acid-labile and acid-stable portions by size exclusion chromatography. Antibody blocking experiments showed that the B6.1 epitope is an acid-labile moiety of the phosphomannan and that the B6 epitope is located in the acid-stable fraction. The B6 epitope appeared to be mannan because it was stable to heat (boiling) and protease treatments but was destroyed by alpha-mannosidase digestion. The B6.1 epitope eluted from the size exclusion column in two fractions. Mass spectroscopic analyses showed that one fraction contained material with the size of a mannotriose and that the other was a mixture of mannotriose- and mannotetraose-size substances. Dose response inhibition tests of the fractions indicated that the B6.1 epitope is associated with the mannotriose. Nuclear magnetic resonance (NMR) spectroscopic analysis of the epitope yielded data consistent with a beta-(1-->2)-linked mannotriose. The fine structure of the B6 epitope is under investigation. Information derived from these investigations will be useful both in understanding protective versus nonprotective antibody responses to C. albicans and in improving anti-Candida vaccine formulations.     

A monoclonal antibody to Candida albicans enhances mouse neutrophil candidacidal activity.

A monoclonal antibody (MAb) to Candida albicans (MAb B6.1) that protects against candidiasis and the nonprotective MAb B6 were compared for ability to support neutrophil (polymorphonuclear leukocyte [PMN]) candidacidal activity. Both MAbs are immunoglobulin M, and each recognizes distinct C. albicans mannan cell wall determinants. PMN candidacidal activity was assessed by transmission electron microscopy and by an in vitro killing assay. The results indicated that MAb B6.1, but not MAb B6, enhances ingestion and killing of yeast cells by PMN in the presence of serum complement.     

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.     

Hybridoma passage in vitro may result in reduced ability of antimannan antibody to protect against disseminated candidiasis

We previously reported the enhanced resistance of monoclonal antibodies B6.1 (an immunoglobulin M [IgM]) and C3.1 (an IgG3) against experimental candidiasis. Both MAbs recognize the same fungal epitope. We have since found that a highly passaged B6.1 hybridoma (hp-B6.1) resulted in antibody that has little protective potential. The potential clinical applicability of the antibody and our interest in understanding antibody protection against candidiasis led us to investigate an explanation for this phenomenon. Antibody genetic structure of hp-B6.1, the original hybridoma clone (ori-B6.1) stored frozen since 1995, a subclone of hp-B6.1 that produces protective antibody, the IgG3-producing hybridoma, and a nonprotective IgG1-producing hybridoma were compared. Variable region gene sequences of heavy (V(H)) and light chains showed genetic instability of V(H) chains with only the hp-B6.1; the V(H) sequences from ori-B6.1 and the subclone were, however, identical. Activation-induced cytidine deaminase levels were greatest in the B6.1 hybridomas, which may explain the instability. The constant region CH3 domain remained unchanged, implying normal N-glycation and complement-fixing potential, and antibody binding affinities appeared unchanged. Complement fixation assays surprisingly showed that ori-B6.1 antibody fixes C3 more rapidly than does hp-B6.1 antibody. The V(H) region primary structure may affect complement activation, which could explain our result. Indeed, antibody from the hp-B6.1 subclone fixed complement like antibody from ori-B6.1. These results show that the greatest protection occurs when antimannan antibodies possess the dual abilities of recognizing the appropriate carbohydrate epitope and rapidly fixing complement; loss of the latter property results in the loss of protective potential by the antibody.     

Human recombinant antimannan immunoglobulin G1 antibody confers resistance to hematogenously disseminated candidiasis in mice

Mannan is a major cell wall component found in Candida species. Natural antimannan antibody is present in sera from most normal adults, but its role in host resistance to hematogenously disseminated candidiasis is unknown. The purpose of this study was to develop recombinant human antimannan antibody and to study its protective function. A phage Fab display combinatorial library containing Fab genes from bone marrow lymphocytes was screened with Candida albicans yeast cells and chemically purified mannan. One antimannan Fab, termed M1, was converted to a full-length immunoglobulin G1 antibody, M1g1, and M1g1 was produced in CHO cells. The M1g1 epitope was found in C. albicans serotypes A and B, Candida tropicalis, Candida guilliermondii, Candida glabrata, and Candida parapsilosis. Its expression was active at both 23 degrees C and 37 degrees C and uniform over the cell surface. BALB/c mice passively immunized with M1g1 were more resistant than control mice to a lethal hematogenous infection by C. albicans, as evidenced by extension of survival in an M1g1 dose-dependent manner (P, 0.08 to <0.001) and by reduction in number of infection foci and their size in the kidney. In vitro studies found that M1g1 promoted phagocytosis and phagocytic killing of C. albicans yeast cells by mouse peritoneal macrophages and was required for activation of the mouse complement cascade. Thus, human antimannan antibody may have a protective role in host resistance to systemic candidiasis.     

Mouse antibody to phosphocholine can protect mice from infection with mouse-virulent human isolates of Streptococcus pneumoniae.

Previous studies have demonstrated that mouse antibodies to phosphocholine (PC) can protect mice against fatal infection caused by several, but not all, mouse-virulent laboratory strains of Streptococcus pneumoniae. Because the pneumococcal strains used in previous studies had been mouse passed and were propagated for many years outside of humans, it was not known whether antibody to PC would be able to protect mice against S. pneumoniae freshly isolated from humans. In the present study, we examined the ability of an immunoglobulin G (IgG) monoclonal antibody (MAb) to PC to protect against infections in mice caused by 14 pneumococcal strains of capsular types 3, 4, 6A, and 6B. Nine of these strains were selected as the most virulent strains for mice from a group of 69 fresh clinical isolates. Five were mouse-passed laboratory strains. Mouse IgG3 MAb to PC was able to exhibit protective effects (survival or increased time to death) against infection with virtually all of the strains injected intravenously and against infection with 70% of the strains injected intraperitoneally. The protective effects of antibody to PC appeared to be partially dependent on capsular type. MAb to PC was most effective against capsular type 3 strains and least effective against type 4 strains. With type 3 and type 4 strains, MAb to PC could frequently protect against larger numbers of CFU injected intravenously than intraperitoneally. For capsular type 6A and 6B strains the reverse was true.     

Candida-specific antibodies during experimental vaginal candidiasis in mice

Protective host defense mechanisms against vaginal Candida albicans infections are poorly understood. Although cell-mediated immunity (CMI) is the predominant host defense mechanism against most mucosal Candida infections, the role of CMI against vaginal candidiasis is uncertain, both in humans and in an experimental mouse model. The role of humoral immunity is equally unclear. While clinical observations suggest a minimal role for antibodies against vaginal candidiasis, an experimental rat model has provided evidence for a protective role for Candida-specific immunoglobulin A (IgA) antibodies. Additionally, Candida vaccination-induced IgM and IgG3 antibodies are protective in a mouse model of vaginitis. In the present study, the role of infection-induced humoral immunity in protection against experimental vaginal candidiasis was evaluated through the quantification of Candida-specific IgA, IgG, and IgM antibodies in serum and vaginal lavage fluids of mice with primary and secondary (partially protected) infection. In na?ve mice, total, but not Candida-specific, antibodies were detected in serum and lavage fluids, consistent with lack of yeast colonization in mice. In infected mice, Candida-specific IgA and IgG antibodies were induced in serum with anamnestic responses to secondary infection. In lavage fluid, while Candida-specific antibodies were detectable, concentrations were extremely low with no anamnestic responses in mice with secondary infection. The incorporation of alternative protocols-including infections in a different strain of mice, prolongation of primary infection prior to secondary challenge, use of different enzyme-linked immunosorbent assay capture antigens, and concentration of lavage fluid-did not enhance local Candida-specific antibody production or detection. Additionally, antibodies were not removed from lavage fluids by being bound to Candida during infection. Together, these data suggest that antibodies are not readily present in vaginal secretions of infected mice and thus have a limited natural protective role against infection.     

Electric and chemical fusions for the production of monoclonal antibodies reacting with the in-vivo growth phase of Candida albicans

To obtain monoclonal antibodies (MAbs) directed preferentially against the pathogenic phase of Candida albicans, mice were immunised with germ tubes of C. albicans serotype A, strain VW.32, killed by exposure to ultraviolet (UV) irradiation. Fusions were performed either by the standard chemical procedure with polyethylene glycol, or by electric discharge following linkage of the myeloma and lymphocyte cells with a Concanavalin A-mannoprotein bridge. The preliminary characteristics of one MAb obtained from each of these fusions are described. An IgM antibody (3B7) obtained from the chemical fusion reacted with a polysaccharide antigen that was heterogeneously distributed on both in-vitro and in-vivo forms of C. albicans. This MAb agglutinated different strains of C. albicans irrespective of their serotype. An IgG1 antibody (3G6) that had been obtained from the electric fusion was found to react in vitro with a proteinaceous antigen located only on the germ tubes of strain VW.32. However, MAb 3G6 displayed strong reactivity against all growth forms of C. albicans in vivo and reactivity extended to other strains.     

Vaccination with recombinant N-terminal domain of Als1p improves survival during murine disseminated candidiasis by enhancing cell-mediated, not humoral, immunity

Candida spp. are opportunistic fungal pathogens that are among the most common causes of nosocomial bloodstream infections. The mortality attributable to disseminated candidiasis is 40 to 50% despite antifungal therapy. Clearly, new strategies are needed to prevent this life-threatening infection. Because risk factors for disseminated candidiasis are well defined and frequently of limited duration, vaccination is an appealing prophylactic strategy. We have identified a cell surface protein, Als1p, that mediates adherence of Candida albicans to a variety of human substrates and plastic. Here we report that immunizing BALB/c mice with the recombinant N-terminal domain of Als1p (rAls1p-N) improved survival during a subsequent challenge with a lethal inoculum of C. albicans. The protective 20-mug dose of rAls1p-N significantly increased Candida stimulation of Th1 splenocytes and increased in vivo delayed-type hypersensitivity. In contrast, antibody titers did not correlate with protection. Finally, the vaccine was not protective in T-cell-deficient mice but was protective in B-cell-deficient mice. These data indicate that the mechanism of action of the rAls1p-N vaccine is stimulation of cell-mediated, rather than humoral, immunity against C. albicans. The majority of efforts to date have focused on the development of passive immunization strategies to prevent or treat disseminated candidiasis. In contrast, our results provide proof of principle for vaccination with an adhesin of C. albicans and emphasize the potential for cell-mediated immune modulation as a prophylactic or therapeutic strategy against disseminated candidiasis.     

Capacity of passively administered antibody to prevent establishment of Brucella abortus infection in mice.

In contrast to immunity against some other facultative intracellular parasites, protective immunity against Brucella abortus is mediated in mice by antibodies as well as by cell-mediated immune responses. It was the purpose of this study to determine whether antibody alone would prevent infection with B. abortus. The majority (82%) of CD-1 outbred mice infected with 100 CFU of virulent B. abortus 2308 preincubated with graded quantities of an O polysaccharide-specific IgG2a monoclonal antibody (MAb) were free of infection 1. 2, 4, and 6 weeks later, based on detection limits of 13 brucellae per spleen and 39 per liver. Infection was present in 95% of control animals. Similar results were obtained with a challenge dose of 500 CFU, but with a challenge dose of 5,000 CFU, infection became established even with the highest concentration of MAb used (50 micrograms of MAb per 5,000 brucellae). Pretreatment with an O polysaccharide-specific IgG1 MAb or with convalescent-phase serum diminished but did not prevent establishment of infection by 100 CFU of B. abortus. A majority of culture-negative mice tested 6 weeks after infection were serologically negative, which could have signified either the absence of previous infection or the early elimination of infection. In an in vitro test system, all of the antibody preparations were efficient in opsonizing B. abortus. Effective killing of the organism by unelicited mouse peritoneal macrophages occurred in conventional but not in endotoxin-free medium, suggesting that activated macrophages were required for killing of opsonized B. abortus. These results emphasize the potential importance of antibodies in the immunoprophylaxis of brucellosis and suggest that the design of a successful vaccine will require the induction of antibodies not only of appropriate specificity but also of the optimal isotype for mediating protective functions.     

Modulation of cell surface-associated mannoprotein antigen expression in experimental candidal vaginitis.

The monoclonal antibody (MAb) AF1 recognizes an oligosaccharide epitope present on highly immunogenic and immunomodulatory mannoproteins (MP) of Candida albicans. The expression of this epitope (AF1-MP) during experimental candidal vaginitis was studied in two strains of C. albicans (3153 and CA-2) which were equally vaginopathic but differed in the mode of hypha formation in the vagina. In both strains, immunofluorescence of vaginal samples, taken 1 h after challenge, revealed an intense, MAb AF1-specific labelling of the yeast cells. This labelling was very scarce in fungal cells taken at 24 h and on subsequent days during the development of filamentous forms. Electron-microscopic gold immunolabelling observations showed that molecules carrying AF1-MP spanned the entire cell wall in the initial yeast cells but were absent on the cell surface and in the outermost, capsular layer of the cell wall of the germ tubes and filamentous forms. In both strains, at any time and for any form of intravaginal growth, AF1-MP was clearly expressed in the cytoplasm and cytoplasmic vesicles, and was fully incorporated into the inner layers of the cell wall. As seen by immunofluorescence, the vaginal fluid from C. albicans-infected rats did not hinder the expression of AF1-MP on the yeast cells surface in vitro. In electron-microscopic gold immunolabelling, a hypha-specific MAb (3D9) labelled the surface of the hyphal but not of the yeast cells of C. albicans harvested from rat vagina. Overall, these data strongly suggest that cell surface expression of MP antigen is modulated during intravaginal growth and morphogenesis of C. albicans.     

Production, characterization, and epitope mapping of a monoclonal antibody against aspartic proteinase of Candida albicans

A monoclonal antibody (MAb; MAb CAP1) that was reactive with extracellular aspartic proteinase of Candida albicans (CAP) was produced. The MAb showed strong sensitivity and reactivity to CAP but not to the aspartic proteinases of Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus or to human cathepsin D or porcine pepsin. The epitope of the CAP recognized by the MAb was the proteinaseous part of CAP and the putative epitope of the MAb was located in the Asp77 to Gly103 sequence. This antibody could be useful for the characterization of CAP and would be a valuable probe for the detection of CAP antigen in the sera of patients with invasive candidiasis.     

The effects of monoclonal antibodies against iC3b receptors in mice with experimentally induced disseminated candidiasis.

CR3 (iC3b receptor), composed of CD11b/CD18, is a beta 2 integrin. A protein that shares antigenic and structural homology with the alpha-chain of CD11b/CD18 has been isolated from the surface of Candida albicans. This molecule is thought to be essential in the pathogenesis of disseminated candidiasis. To evaluate the effects of anti-iC3b receptor antibodies on adhesion between human dermal microvascular endothelial cells (HDMEC) and C. albicans, and in treatment of candidal infection, a binding assay of C. albicans to cultured HDMEC was performed in vitro. An anti-iC3b receptor-specific monoclonal antibody was administered to mice infected with C. albicans. The mice were monitored for mortality and renal involvement by culture and histopathological findings. Flow cytometric analysis demonstrated surface expression of iC3b receptor on C. albicans. The adherence of C. albicans to HDMEC was significantly decreased by treatment with anti-iC3b receptor antibodies. Anti-iC3b receptor antibodies significantly increased the survival time and rate while lowering the renal fungal burden. The iC3b receptors are involved in the adherence of C. albicans to vascular endothelial cells and are likely to be involved in the pathogenesis of disseminated candidiasis. The increased survival in mice infected with C. albicans after treatment with anti-iC3b receptor antibodies indicates that this modality may be beneficial for future development of a new therapy for candidiasis.     

A monoclonal antibody directed against a Candida albicans cell wall mannoprotein exerts three anti-C. albicans activities

Antibodies are believed to play a role in the protection against Candida albicans infections by a number of mechanisms, including the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity. Although some of these biological activities have been demonstrated individually in monoclonal antibodies (MAbs), it is not clear if all these anti-C. albicans activities can be displayed by a single antibody. In this report, we characterized a monoclonal antibody raised against the main target of salivary secretory immunoglobulin A in the cell wall of C. albicans, which exerts three anti-C. albicans activities: (i) inhibition of adherence to HEp-2 cells, (ii) inhibition of germination, and (iii) direct candidacidal activity. MAb C7 reacted with a proteinic epitope from a mannoprotein with a molecular mass of >200 kDa predominantly expressed on the C. albicans germ tube cell wall surface as well as with a number of antigens from Candida lusitaniae, Cryptococcus neoformans, Aspergillus fumigatus, and Scedosporium prolificans. MAb C7 caused a 31.1% inhibition in the adhesion of C. albicans to HEp-2 monolayers and a 55.3% inhibition in the adhesion of C. albicans to buccal epithelial cells, produced a 38.5% decrease in the filamentation of C. albicans, and exhibited a potent fungicidal effect against C. albicans, C. lusitaniae, Cryptococcus neoformans, A. fumigatus, and S. prolificans, showing reductions in fungal growth ranging from 34.2 to 88.7%. The fungicidal activity showed by MAb C7 seems to be related to that reported by antibodies mimicking the activity of a killer toxin produced by the yeast Pichia anomala, since one of these MAbs also reacted with the C. albicans mannoprotein with a molecular mass of >200 kDa. Results presented in this study support the concept of a family of microbicidal antibodies that could be useful in the treatment of a wide range of microbial infections when used alone or in combination with current antimicrobial agents.     

Vaccine and monoclonal antibody that enhance mouse resistance to candidiasis

Previously we showed that antibodies specific for the glycan β-1,2-mannotriose [β-(Man)₃] on the cell surface of Candida albicans protect mice against disseminated candidiasis (H. Xin, S. Dziadek, D. R. Bundle, and J. E. Cutler, Proc. Natl. Acad. Sci. U. S. A. 105:13526–13531, 2008). Furthermore, six 14-mer peptides that are within the N-terminal portion of C. albicans wall proteins were conjugated to the glycan in an attempt to create immunogenic glycopeptide conjugates. By a dendritic cell (DC)-based immunization approach, all were immunogenic and three of the six conjugates induced a high degree of protection in mice. Interestingly, whereas all six peptides induced antibody responses when used alone to pulse DCs for subsequent immunizations, three peptides induced protection, and one in particular, peptide Fba (derived from fructose-bisphosphate aldolase), induced robust protective responses and is the focus of the current work. Fba peptide is not restricted by the major histocompatibility complex class II (MHC-II), as it induced anti-Fba antibodies in mice of different H-2 haplotypes and in rabbits. Furthermore, the peptide induced protection against disease caused by different C. albicans strains. Partial protection was achieved when alum was used in place of DCs for Fba immunizations. The passive transfer of immune sera from Fba-vaccinated mice, but not immune serum preabsorbed with fungal cells, conferred protection in naïve mice. This result, along with our finding that a monoclonal antibody specific for the peptide, E2-9 (IgM), protected mice against candidiasis, provide strong evidence that antibodies contribute to protection. Our work demonstrates the utility of cell wall peptides alone or as glycopeptides in vaccines designed for the induction of immunity against candidiasis and monoclonal antibodies as a rapid immunoprotective approach against the disease.     

A monoclonal antibody against hamster tracheal mucin, which recognizes N-acetyl-galactosamine containing carbohydrate chains as an epitope

Airway mucin that is present in airway secretion, plays an important role in host-defense by trapping airborne particles and removing them by mucociliary transport system. For the study of mucin, it is crucially important to have antibodies specific against mucin because other commonly used methods such as histologic stain for the detection of mucin usually suffer from varying levels of nonspecificity. In this study, we produced a monoclonal antibody (MAb) against hamster airway mucin, which is one of the most commonly used animal species for the study of mucin in vitro, and characterized its immunological properties along with the determination of the epitope it recognizes. The MAb, which was named MAb HTA, was IgM isotype and specific against mucin from both in vitro cell culture and in vivo airway secretion. In Western blot, MAb HTA specifically recognized high molecular weight airway mucin, which was also confirmed by the appearance of peak profile of immunological signal only on void volume fraction in Sepharose CL-4B gel filtration chromatography. It also immunoprecipitated high molecular weight hamster airway mucin with the aid of antimouse IgM agarose. In immunohistochemical stain of hamster trachea, it showed strong signal on airway epithelium and also on the mucin secreting goblet cell granules. The immunological signal was greatly increased by the treatment of endotoxin, which has been reported to cause airway secretory cell metaplasia. The MAb HTA recognized carbohydrate chains containing N-acetyl-galactosamine, one of the linking sugars of airway mucin, as an epitope. Treatment of mucin with N-acetyl-galactosaminidase caused great reduction of immunological signal. To the best of our knowledge, this is the first to report a MAb that recognizes N-acetylgalactosamine, a linking sugar of airway mucin. The specificity of MAb HTA against airway mucin and the clear demonstration of the epitope it recognizes should greatly aid the pharmacological and biochemical study of mucin in various physiological and pathological situations.     

Production and characterization of monoclonal antibodies to cell wall antigens of Aspergillus fumigatus

Two murine monoclonal antibodies (MAbs) against Aspergillus fumigatus were produced and characterized. Splenocytes from cell wall-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with mannans of Candida albicans serotypes A or B or Candida tropicalis, indirect immunofluorescence with C. albicans- or A. fumigatus-infected tissues, indirect immunofluorescence with smears of other pathogenic fungi, Western blotting (immunoblotting) with the lectin concanavalin A or BS-1 from the seeds of Bandeirea simplicifolia, and immunoelectron microscopy. MAb 1 did not cross-react with Candida mannan and recognized a periodate-sensitive, pronase- and heat-resistant epitope in CA antigen and three mannose- and galactose-containing components (80, 62, and 49 kilodaltons) of a mycelial homogenate. Immunoelectron microscopy demonstrated binding of MAb 1 to the inner cell wall and intracellular membranes of hyphae and conidia of A. fumigatus. Circulating antigen was detected in experimental invasive aspergillosis by inhibition enzyme immunoassay with MAb 1 and CA antigen. MAb 40 was a nonprecipitating antibody cross-reactive with Candida species, and competition for an epitope located diffusely in the cell wall of A. fumigatus hyphae was demonstrated by incubating MAb 40 with mannan of C. albicans serotype A. These results suggest that MAb 1 recognizes immunodominant oligogalactoside side chains of A. fumigatus galactomannan, while MAb 40 binds to mannopyranosyl side chains common to A. fumigatus galactomannan and C. albicans mannan.     

An anti-idiotype antibody which mimics the inner-core region of lipopolysaccharide protects mice against a lethal challenge with endotoxin.

Recently, we described the generation and characterization of an Armenian hamster Ab2 beta anti-idiotype monoclonal antibody (MAb4G2) specific for the binding site of a mouse monoclonal antibody, MAbY1-4A6, directed against the conserved 2-keto-3-deoxyoctulosonate (Kdo)-containing inner-core region of lipopolysaccharide (LPS) (S. K. Field, M. Pollack, and D. C. Morrison, Microb. Pathog. 15:103-120, 1993). In that study, mice and hamster immunized with MAb4G2 generated serum immunoglobulin G and M (IgG and IgM) antibodies which cross-react with Salmonella minnesota R595-chemotype rough mutant LPS (Re-LPS). In this report, we demonstrate that in C3Heb/FeJ mice, MAb4G2 elicits an immune response which is characterized by specific binding of antibody to Re-LPS, as assessed by enzyme-linked immunosorbent assay. The practical use of MAb4G2 as a potentially effective therapeutic agent against gram-negative bacterial sepsis is suggested by the demonstration that immunization of these mice with MAb4G2 results in significant protection of D-galactosamine-sensitized animals against an otherwise lethal dose of Re-LPS. Assessment of the temporal changes in Re-LPS-specific serum antibody titers from mice immunized with MAb4G2 or Re-LPS over a 40-day period indicates that immunization with Re-LPS elicits significantly higher titers of serum IgM antibodies compared with those in animals immunized with MAb4G2. Conversely, two immunizations with MAb4G2 result in an up to 10-fold increase in anti-Re-LPS-specific IgG serum antibody titers relative to those obtained in mice immunized with Re-LPS. Nineteen days after the secondary boost with MAb4G2, anti-Re-LPS-specific IgG serum antibody titers were significantly higher (three- to fourfold) compared with those in Re-LPS-treated animals. Initial immunization with the anti-idiotype antibody primes animals for enhanced secondary responses to Re-LPS, as assessed by the titers of anti-Re-LPS-specific IgG profiles. These data suggest the potential utility of MAb4G2 as a candidate vaccine against the lethal properties of gram-negative bacterial LPS.