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Infection and Immunity, January 2006, p. 362-369, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.362-369.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Human Recombinant Antimannan Immunoglobulin G1 Antibody Confers Resistance to Hematogenously Disseminated Candidiasis in Mice

Mason X. Zhang,1* M. Charlotte Bohlman,2 Carol Itatani,1 Dennis R. Burton,3 Paul W. H. I. Parren,3,{dagger} Stephen C. St. Jeor,2 and Thomas R. Kozel2

Department of Biological Sciences, California State University, Long Beach, California 90840,1 Department of Microbiology and Immunology, University of Nevada School of Medicine, Reno, Nevada 89557,2 Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, California 920373

Received 11 August 2005/ Returned for modification 21 September 2005/ Accepted 8 October 2005


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ABSTRACT
 
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°C and 37°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.


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INTRODUCTION
 
Candida albicans and several other Candida species, including C. glabrata, C. tropicalis, and C. parapsilosis, are the etiological agents for life-threatening hematogenously disseminated candidiasis (33, 34). Common to all Candida species is the presence of mannan, a major cell wall component that consists of O-linked oligomannosides and N-linked mannose polysaccharides (36). Mannan is immunogenic. In fact, naturally occurring antibodies reactive with specific Candida mannan epitopes are present in the serum of most normal individuals without regard to gender, race, or age (22, 24, 44), but they vary with regard to quantity, immunoglobulin class, or binding specificity (22, 44; T. Kozel, M. Zhang, J. Guesford, and M. Gates, Abstr. 100th Gen. Meet. Am. So. Microbiol. 2000, abstr. F-48, p. 329, 2000). Previously, we showed that affinity-purified, pooled human antimannan immunoglobulin G (IgG) is required for complement opsonization of Candida yeast cells through either the classical or alternative complement pathways (22, 23, 43, 44). However, little is known about the role of human antimannan antibody in host defense against hematogenously disseminated candidiasis.

The hypothesis that human antimannan antibody may have a protective role has been supported by studies with murine antimannan antibody by Cutler (7). Han and Cutler found that immunization of mice with Candida mannan induced protective immunity against hematogenously disseminated candidiasis (12). Furthermore, transfer of serum from the immunized mice conferred protection against systemic candidiasis (12). This antibody-mediated protection was confirmed with passive immunization using a monoclonal IgM antibody (B6.1) against Candida mannan (12) and its murine IgG3 variant (15). Further studies revealed that antimannan antibody B6.1 enhances mouse neutrophil candidacidal activity (5) and that protection by B6.1 or its murine IgG3 isotype variant requires host complement (14).

Additional support for the importance of human antimannan antibody is provided by a clinical observation that passive immunization of liver transplant patients with purified total human IgG antibody significantly reduced the incidence of fungal infections, including candidiasis (35). Given that appreciable amounts of naturally occurring antimannan IgG are present in the general population (22, 24, 44), this observation provides an indirect support for the use of human antimannan IgG as a passive immunization agent.

Despite these studies, direct evidence for a protective role of human antimannan antibody has been lacking. We approached this question by developing full-length recombinant monoclonal human antimannan antibodies and by studying their protective functions. In this paper, we report the following: (i) construction of a full-length recombinant human IgG1 antimannan antibody (M1g1) from a Fab fragment developed with the phage Fab display technique, (ii) a broad binding specificity of M1g1 for C. albicans and several other Candida species and the expression pattern for the M1g1 epitope, (iii) the protective efficacy of M1g1 in BALB/c mice against hematogenously disseminated candidiasis, and (iv) the ability of M1g1 to mediate phagocytosis and killing of Candida yeast cells by mouse peritoneal macrophages and to activate the mouse complement system. Our findings suggest that naturally occurring antimannan antibody may influence host resistance to disseminated candidiasis.


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MATERIALS AND METHODS
 
Strains and purification of Candida mannans. Yeast cells of the Candida strains C. albicans serotype A (ATCC 36801); C. albicans 3153A, derived from a clinical isolate (provided by L. Chaffin, Texas Tech University Health Sciences Center, Lubbock); C. albicans serotype B (ATCC 36803); C. tropicalis (ATCC 750); C. guilliermondii (ATCC 34134); C. glabrata (ATCC 48435); C. parapsilosis (ATCC 10232); C. krusei (ATCC 14243); C. kefyr (ATCC 34137); and Saccharomyces cerevisiae (ATCC 26786) were maintained and grown as described previously(18, 23). Briefly, yeast cells of each strain were grown in 3 ml of GYEP (2% glucose, 1% peptone, 0.3% yeast extract) at 37°C, unless indicated otherwise, passaged daily three times, and then used to initiate a large broth culture. Each large culture was shaken overnight at 37°C, inactivated by 1 h of treatment with 1% formaldehyde at room temperature, harvested by centrifugation, washed, resuspended in phosphate-buffered saline (PBS; pH 7.2) (1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 154 mM NaCl) containing 0.02% azide, and stored at 4°C.

Purification of Candida mannan utilized standard procedures (21, 32) with a few minor changes, as described previously (44). Briefly, yeast cells prepared as described above were treated with acetone for removal of lipids and then heated in water for 5 h at 121°C. Water-soluble mannan was precipitated with Fehling solution, and mannan in the precipitate was released with Amberlite IR-120 (H+) resin (Acros Organics N.V., Fair Lawn, NJ). The mannan solution was neutralized, dialyzed against water at 4°C, and lyophilized. C. albicans yeast mannan obtained through this method typically contains approximately 95% carbohydrate as mannose and about 2% protein (20).

Identification of genes for antimannan Fab fragment (M1). The phage Fab display technique was used to isolate genes for human immunoglobulin Fab fragments against Candida (1, 2, 26). Briefly, total RNA was isolated from bone marrow lymphocytes from a healthy adult and was reverse transcribed into cDNA. From the cDNA, genes for light chains (VL-CL) and heavy chains (VH-CH1) were amplified with PCR and inserted into separate cloning sites of phagemid pComb3H (2). The cloning site for the heavy-chain fragment gene is upstream of a truncated gene for the carboxyl-terminal domain of coat protein III that displays one monovalent Fab on the phage particle (1, 2). The phagemids were transformed into Escherichia coli XL-1 Blue and rescued with helper phage VCSM13 (Stratagene, La Jolla, CA). Phage particles (5 x 107 PFU) were incubated overnight at 4°C with 4 x 108 yeast cells of C. albicans (ATCC 36801 and ATCC 36803), and yeast-bound phage particles were eluted. The eluted phages were used to reinfect E. coli cells. The selection cycle was repeated at least five times to enrich the phage display library for particles displaying anti-Candida Fab fragments. The enrichment was monitored for an increase in titers of infectious phage particles and for binding activity of the phage particles for immobilized Candida mannan with an enzyme-linked immunosorbent assay (ELISA) (44). One of the bacterial transformants produced a Fab fragment that exhibited a high binding affinity for purified mannan from C. albicans ATCC 36801, and the antimannan Fab was named M1. Soluble M1 Fab fragment was immunoaffinity isolated with a mannan A-Sepharose 4B column (44). Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed an expected molecular size for M1, and Western blot analysis confirmed its identity with anti-human Fab antibody (SouthernBiotech, Birmingham, AL). M1 was found to contain {kappa} light chain as determined with ELISA using anti-human {kappa} antibody (SouthernBiotech, Birmingham, AL) and by comparison of the sequence of the M1 light-chain gene to the database of human immunoglobulins (IgBLAST, National Center for Biotechnology Information). The GenBank accession numbers for the M1 light-chain and heavy-chain variable regions are DQ286419 and DQ286420, respectively.

Construction and production of the full-length antimannan IgG1 antibody M1g1. M1 Fab was converted to a full-length IgG1 antibody by use of the expression vector pIgG1. The vector contains the gene for IgG1 heavy-chain domains (CH1-hinge-CH2-CH3), two separate cytomegalovirus promoters/enhancers for stoichiometric expression of light and heavy chains, and a gene for glutamine synthetase that allows selection of multiple insertions of pIgG1 in transfected mammalian cells in the presence of a glutamine synthetase inhibitor. M1 Fab VH-CH1 gene and M1 Fab VL-CL gene were sequentially inserted into the expression vector, yielding the gene construct pM1g1 for the antimannan IgG1 antibody. The DNA sequence of the entire heavy-chain gene or the light-chain gene was determined and analyzed with IgBLAST. To produce soluble antibody, pM1g1 was introduced into a Chinese hamster ovary cell line (ATCC CHO-K1) with Lipofectin according to the manufacturer's procedure (Invitrogen, Carlsbad, CA). Transfected cells were selected in glutamine-free Glasgow minimum medium (Sigma, St. Louis, MO) with the glutamine synthetase inhibitor methionine sulfoximine (4). Production of soluble M1g1 was detected in the growth medium by ELISA with Candida mannan in the solid phase and with anti-human IgG antibody (SouthernBiotech, Birmingham, AL). Stable transfectants were purified by limiting dilution, and robust antibody-producing clones were established. The Integra CL 1000 cultivation system was employed for research-scale production of M1g1 antibody in accordance with the manufacturer's instruction (Integra Biosciences, Wallisellen, Switzerland). M1g1 was purified by affinity chromatography using protein A per the manufacturer's procedure (Amersham Biosciences, Piscataway, NJ), dialyzed against PBS, concentrated, filtration sterilized, and stored at –80°C.

Characteristics of recombinant antimannan M1g1 antibody. The molecular size and identity of affinity-purified M1g1 were determined by using goat antibody specific for human IgG light or heavy chain (SouthernBiotech, Birmingham, AL) with standard SDS-PAGE and Western blot procedures using myeloma IgG1 as a standard (The Binding Site, Birmingham, England). IgG1 subclass identity was established for M1g1 with ELISA as described previously (44), where microtiter wells were first coated with purified mannan of C. albicans 3153A, and mannan-bound M1g1 was detected with monoclonal antibodies (MAbs) specific for each of the four IgG subclasses (SouthernBiotech, Birmingham, AL). To determine the pattern of expression of the M1g1 epitope on the cell surface, yeast cells of Candida species and S. cerevisiae were grown at either 37°C or 23°C as described above and incubated for 30 min at 37°C with 2 µg M1g1/ml, and yeast-bound M1g1 was visualized with goat anti-human IgG-fluorescein isothiocyanate (FITC) (SouthernBiotech, Birmingham, AL) and immunofluorescence microscopy (44).

Effect of M1g1 passive immunization on prevention of hematogenously disseminated candidiasis. Six-week-old female BALB/c mice (Harlan, San Diego, CA) were used. Initial assays were conducted to determine the time-dependent appearance and concentration of serum human antibody M1g1 following intraperitoneal injection. Mice were injected intraperitoneally with M1g1, and blood samples from the tail vein in 20 µl were taken at different times and diluted immediately in 100 µl PBS. Titers of M1g1 were estimated with ELISA using mannan as the antigen. It was found that M1g1 titers in the blood reached a peak 3 to 4 hours after injection. The protective efficacy of M1g1 was determined by the method of Han and Cutler for murine antimannan antibody (12). Mice were first given intraperitoneally either different doses of M1g1 in 0.5 ml Dulbecco's PBS (DPBS; Cambrex, East Rutherford, NJ) or the buffer alone and then 4 hours later were challenged intravenously with a lethal dose of 106 C. albicans 3153A yeast cells. The mice were monitored twice daily for moribundity or death following the procedures established by the Animal Welfare Board at California State University—Long Beach. Kaplan-Meier survival curves were constructed and compared statistically with the log rank test by use of SigmaStat (Systat Software, Point Richmond, CA).

Effect of M1g1 treatment on the histological pattern of C. albicans infection in the kidney. Six-week-old female BALB/c mice were injected intraperitoneally either with DPBS alone or the buffer containing 1 mg M1g1/mouse and challenged intravenously 4 hours later with a lethal dose of 106 yeast cells of C. albicans 3153A. Mouse kidneys were harvested 24 or 48 h postchallenge, fixed, embedded in paraffin, sectioned, mounted on SuperFrost slides (Fisher Scientific, Pittsburgh, PA), and stained with Grocott's methenamine silver (GMS) to reveal C. albicans infection foci (clusters of Candida cells). The GMS-stained sections were examined systematically to measure both the number of Candida infection foci and the size of each focus with a calibrated ocular micrometer. The number of infection foci and their sizes for M1g1-treated mice and control mice were compared statistically with the Mann-Whitney rank sum test by use of SigmaStat.

Effect of M1g1 on phagocytosis and phagocytic killing of C. albicans yeast cells. Peritoneal macrophages were isolated from 6-week-old female BALB/c mice and incubated overnight at 1.5 x 105 cells per well in RPMI 1640 containing 10% fetal calf serum at 37°C with 5% CO2. Nonadherent cells were removed by washing with RPMI 1640. For phagocytic killing assays, 3 x 105 C. albicans 3153A yeast cells were added to each well in 96-well culture plates containing the mouse peritoneal macrophages and incubated for 60 min at 37°C with 5% CO2 in 100 µl RPMI 1640 containing various amounts of M1g1. The plates were washed with DPBS, frozen at –80°C in water for lysis of the macrophages (39), thawed at 37°C, and vigorously vortexed to disrupt possible yeast clumps, and Candida CFU were determined on GYEP-agar plates. For phagocytosis assays, 3 x 105 C. albicans 3153A yeast cells were added to each well in 16-well culture slides (Nalge Nunc International, Rochester, NY) containing the mouse peritoneal macrophages in 100 µl RPMI 1640 containing various amounts of M1g1 and incubated for 30 min at 37°C with 5% CO2. Following washing with DPBS, macrophage-ingested or attached yeast cells were visualized microscopically with Wright's-Giemsa stain, and the average number of yeast cells per macrophage was determined from 10 or more microscopic fields that contained a total of 300 or more macrophages. The effects of M1g1 on phagocytosis and phagocytic killing were compared statistically between the doses with the Tukey test by use of SigmaStat.

M1g1-mediated activation of the mouse complement system. A procedure previously developed for analysis of human C3 deposition onto Candida yeast cells was used for M1g1-mediated activation of the mouse complement system (44). Briefly, serum pooled from 10-week-old female BALB/c mice was purchased from Harlan (Madison, WI) and absorbed two times, each with 5 x 108 C. albicans 3153A yeast cells. C. albicans 3153A yeast cells at 4 x 106 were incubated for 30 min at 37°C with 5 µg M1g1 in 1 ml VBS (5 mM sodium Veronal-142 mM NaCl, pH 7.3) and removed by centrifugation. The M1g1-opsonized yeast cells were resuspended at 37°C in 1 ml of a complement activation buffer containing VBS, 0.1% gelatin, 1.5 mM CaCl2, and 1 mM MgCl2; yeast-absorbed normal mouse serum was added to the buffer at a final concentration of 40% to initiate activation of the complement cascade. Deposition of mouse C3 on the yeast cell surface was detected with anti-mouse C3 MAb-FITC (Cedarlane Laboratories, Ontario, Canada) and visualized with immunofluorescence microscopy (44).


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RESULTS
 
Isolation of human genes for the antimannan Fab M1. We used the phage Fab display technique to identify human antimannan Fab genes. The phage Fab display combinatorial library was constructed from genes isolated from bone marrow lymphocytes donated by a healthy male adult. The donor's serum endpoint titer of antimannan A antibody was 1/43,000, compared to 1/44,000 for a serum pool from 40 healthy adults. Screening of the library with C. albicans yeast cells and chemically purified Candida mannan identified an antimannan Fab fragment, named M1. Analysis of the DNA sequence of M1 genes with IgBLAST showed that the M1 VH gene was derived from VH3-74 with 89% identity, DH5-24 with 90% identity, and JH4 with 90% identity. The M1 VL gene was derived from V{kappa}2-A3 with 95% identity and J{kappa}2 with 97% identity.

Conversion of M1 to the full-length IgG1 antibody M1g1 and production of M1g1. The M1 light-chain gene (VL-CL) was inserted into the IgG1 expression vector, and the M1 heavy-chain Fd gene (VH-CH1) was ligated to the gene specific for the IgG1 heavy chain (CH1-hinge-CH2-CH3) in the expression vector. DNA sequence analysis of the M1g1 expression vector confirmed the presence of the entire light- and heavy-chain genes in correct locations and revealed a complete agreement of the cloned gene sequences for the conserved constant domains with the sequences at IgBLAST. The expression vector was then transfected into CHO cells for in vitro production of the full-length antimannan antibody M1g1.

Characteristics of M1g1. The Fab M1 was reactive with chemically purified mannan. To determine whether M1g1 retained its binding specificity for mannan, M1g1 was serially diluted from 0.2 µg/ml in microtiter wells precoated with mannan from C. albicans 3153A yeast cells, and mannan-bound M1g1 was detected with MAb specific for human IgG1. M1g1 was found to bind to the purified mannan in a dose-dependent manner (Fig. 1, top), and this binding of M1g1 to the immobilized mannan could be inhibited with soluble mannan (data not shown). Thus, M1g1 retains its binding specificity for mannan.



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  		FIG. 1. Subclass specificity and binding characteristics of M1g1. (Top) Mannan binding and IgG subclass specificity. MAb M1g1 was serially diluted in microtiter wells containing immobilized mannan of C. albicans 3153A. The reactivity of mannan-bound M1g1 was determined with a horseradish peroxidase-conjugated MAb specific for human IgG1, IgG2, IgG3, or IgG4 and quantified by determining the absorbance at 450 nm. (Bottom) Patterns of M1g1 binding to yeast cells of C. albicans 3153A. Yeast cells were incubated with 0 or 2 µg/ml M1g1 and washed. Yeast-bound M1g1 was detected with affinity-purified anti-human IgG-FITC and visualized with fluorescence microscopy and differential interference contrast microscopy (DIC).

The IgG1 subclass identity for M1g1 was established by ELISA with purified mannan in the solid phase. Reactivity of M1g1 with MAbs specific for human IgG2, IgG3, or IgG4 was at the background level and was independent of the M1g1 doses (Fig. 1, top). Affinity-purified M1g1 also exhibited molecular sizes expected for the whole IgG1 antibody and for the heavy or light chain of IgG1 (3), as shown by SDS-PAGE under both reducing and nonreducing conditions and by Western blot analysis using myeloma IgG1 as a standard (data not shown).

The binding of M1g1 to yeast cells of C. albicans 3153A was determined by immunofluorescence microscopy and was found to be uniform over the cell surface (Fig. 1, bottom). This apparent uniform binding pattern for M1g1 was similar to that observed with the protective murine monoclonal antimannan antibody B6.1, but it was in contrast to a patchy binding pattern observed with the nonprotective murine monoclonal antimannan antibody B6 (12, 13).

Broad distribution of M1g1 mannan epitope among Candida species. Candida mannan is immunogenic. However, mannan epitopes recognized by the human antibody response have not been characterized. In the rabbit system, the antibody response to Candida mannan has been shown to be diverse, recognizing at least 10 epitopes known as factors (36). Immunofluorescence microscopy was used to compare the distribution of the M1g1 epitope to that of the mannan factors found in yeast cells of different Candida species. M1g1 was found to bind to yeast cells of serotype A and B strains of C. albicans, as well as strains of C. tropicalis, C. guilliermondii, C. glabrata, and C. parapsilosis, but not to yeast cells of C. krusei, C. kefyr, or Saccharomyces cerevisiae (Table 1). In addition to the broad distribution of the M1g1 epitope, the pattern of M1g1 binding to the yeast cells of M1g1-reactive strains was uniform and similar to the pattern observed with C. albicans 3153A (Fig. 1, bottom). Finally, no discernible difference was observed in the M1g1 binding patterns on yeast cells that had been grown at 23°C or at 37°C (Table 1). Thus, the M1g1 epitope appears to be a rather common, stable component of Candida mannan.


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  		TABLE 1. Comparison of the distributions of mannan epitopes among Candida species for human M1g1 antibody and for rabbit antibody response

Effect of M1g1 passive immunization on prevention of hematogenously disseminated candidiasis. We utilized a murine model of hematogenously disseminated candidiasis to assess protection by M1g1 (12). Ten 6-week-old female BALB/c mice per group were passively immunized intraperitoneally with various amounts of affinity-purified recombinant M1g1 in DPBS or with DPBS alone as a control and then lethally challenged intravenously 4 h later with 106 yeast cells of C. albicans 3153A. Survival was closely monitored for a period of 70 days. All control mice given DPBS alone died within 22 days postinfection, with a mean survival time of 14 days (Fig. 2). In contrast, there was a significant (P < 0.001) prolongation of survival when mice were treated with 0.25, 1, or 4 mg of MAb. Mice treated with 0.063 mg M1g1 were also more resistant to the lethal infection than control mice, but the difference between these two groups was not statistically significant (P = 0.08). Thus, M1g1 has a dose-dependent protective role in host defense against hematogenously disseminated candidiasis.



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  		FIG. 2. Effect of passive immunization with M1g1 on the survival of mice lethally challenged intravenously with C. albicans yeast cells. Ten mice per group were treated intraperitoneally with DPBS alone or DPBS containing human antimannan M1g1 at 0.063, 0.25, 1, or 4 mg per mouse. Four hours later, the mice were challenged intravenously with 106 C. albicans 3153A yeast cells. Kaplan-Meier survival curves were compared for statistical significance with the log rank test between the control group (DPBS) and each of the M1g1-treated groups. Data from one of three representative experiments are shown.

Effect of M1g1 treatment on the histological pattern of C. albicans infection in the kidney. The severity of Candida infection in the kidneys of mice that had been treated with either 1 mg M1g1 in DPBS or the buffer alone was measured in terms of the number of infection foci (clusters of C. albicans cells) and the size of each infection focus (Fig. 3). It was found that 24 h postinfection, clusters of C. albicans cells were readily ascertainable in both the treated and the control mice. However, the number of infection foci was much lower in the treated mice than in the control mice (P < 0.001) 24 h or 48 h postinfection (Fig. 3, upper left).



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  		FIG. 3. Effect of M1g1 passive immunization on the number and size of infection foci in kidneys of mice with hematogenously disseminated candidiasis. (Upper left) Number of infection foci (clusters of C. albicans cells). (Lower left) Averaged relative sizes of infection foci. Kidneys were retrieved either 24 h postinfection from control mice (M1and M2) and M1g1-treated mice (M3 and M4) or 48 h postinfection from control mice (M5 and M6) or M1g1-treated mice (M7 and M8). Kidney sections were prepared and stained with GMS. At least 30 microscopic fields were examined for each mouse, and the number of infection foci and their sizes were determined. The size of an infection focus was scored as follows: 1, <10 ocular micrometer units (<77 µm); 2, 11 to 29 units (85 to 223 µm); 3, 30 to 49 units (231 to 377 µm); and 4, >50 units (>385 µm). The Mann-Whitney rank sum test was used to compare the M1g1-treated mice to the control mice. Data are reported as means ± standard errors of the means. (Upper right) A GMS-stained section from control mouse M5. (Lower right) A GMS-stained section from M1g1-treated mouse M7. Arrows indicate infection foci. Data from one of two representative experiments are shown.

The effect of antimannan M1g1 on the size of individual infection foci was also noticeable. We used a calibrated ocular micrometer to categorize infection foci into four size groups, ranging from 1 being the smallest to 4 being the largest (Fig. 3, lower left). The relative sizes of infection focus were similar in the kidney sections from both treated and control mice 24 h postinfection (Fig. 3, lower left, 24-h sections). However, the infection foci in the control mice appeared to expand faster as the infection progressed to 48 h than those in the treated mice (P < 0.001) (Fig. 3, lower left, 48-h sections). This apparent more-invasive infection by C. albicans in the control mice was also visible as a denser mycelial growth in the kidney (Fig. 3, upper right) than that in the treated mice (Fig. 3, lower right). These observations together suggested a reduction in the severity of Candida infection in the kidney by M1g1 treatment.

Effect of M1g1 on phagocytosis and phagocytic killing of C. albicans yeast. Phagocytosis and phagocytic killing of yeast cells by peritoneal macrophages from BALB/c mice were evaluated in the presence of 0, 1, 10, or 100 µg M1g1. The addition of M1g1 at higher amounts significantly enhanced both phagocytosis as measured with either the phagocytosis index (P < 0.001) (Fig. 4, left) or the number of yeast cells per macrophage (P < 0.01) (Fig. 4, center) and phagocytic killing as measured with the number of CFU (P < 0.05) (Fig. 4, right).



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  		FIG. 4. Effect of M1g1 on phagocytosis and phagocytic killing of C. albicans yeast by mouse macrophages. Yeast cells were incubated with mouse peritoneal macrophages in the presence of various amounts of M1g1 (100, 10, 1, or 0 µg/ml). The numbers of macrophage-ingested or attached yeast cells were determined microscopically with Wright's-Giemsa staining, and phagocytic killing was determined by counting the numbers of CFU. Data are reported as the means ± standard errors of the means, and pairwise comparisons using the Tukey test were performed. (Left) Phagocytosis index (percentage of macrophages in association with yeast cells); (center) numbers of yeast cells per macrophage; (right) numbers of CFU recovered from yeast cells incubated with mouse macrophages. Data from one of three representative experiments are shown.

M1g1-mediated activation of the mouse complement system. The critical role for the complement system in host defense against disseminated candidiasis has been well established in experimental animal models (9, 14, 16, 25), and the protective efficacy of the murine antimannan IgM antibody B6.1 and its murine IgG3 variant against hematogenously disseminated candidiasis has been shown to require an intact complement system (14). To determine whether the human recombinant antimannan M1g1 mediates initiation of the mouse complement system, we examined deposition patterns for mouse C3 on the surface of C. albicans 3153A yeast cells. Pooled normal mouse serum from 10-week-old female BALB/c mice was used as a source of the mouse complement and absorbed with C. albicans yeast cells to remove natural complement activators. Yeast cells incubated for 6 or 10 min in the absorbed serum containing no exogenous antimannan antibody showed no detectable bound C3 (Fig. 5). In contrast, yeast cells opsonized with 5 µg M1g1/ml readily activated the complement system, leading to a simultaneous deposition of mouse C3 onto the cell surface at multiple sites after a 6-min incubation (Fig. 5). These initial foci expanded to cover the entire cell surface following a 10-min incubation. Thus, human antimannan antibody M1g1 activates the mouse complement system, leading to C3 opsonization of C. albicans yeast cells.



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  		FIG. 5. Requirement of M1g1 for opsonization of yeast cells by mouse C3. Yeast cells of C. albicans 3153A were incubated with 0 or 5 µg M1g1/ml and transferred to a C3 binding reaction mixture containing 40% yeast-absorbed normal mouse serum. Patterns of C3 deposition onto the yeast cells following a 6- or 10-min incubation were visualized with FITC-conjugated MAb specific for mouse C3 and with fluorescence microscopy and differential interference contrast microscopy (DIC). Data are shown from one of three representative experiments.


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DISCUSSION
 
Anti-Candida mannan antibody is naturally present in sera of most individuals, but little is known about its role in host resistance to systemic candidiasis. Our approach to this question was to develop recombinant human monoclonal antimannan antibodies and to study their functions. In this study, we describe the generation of a recombinant human antimannan IgG1 antibody (M1g1). To our knowledge, M1g1 is the first full-length human monoclonal antibody against C. albicans. We also demonstrate its protective role in a murine model of hematogenously disseminated candidiasis (Fig. 2 and 3) and its ability to promote phagocytosis and phagocytic killing of C. albicans yeast cells and to mediate complement opsonization of the yeast (Fig. 4 and 5). These observations suggest a protective role for human antimannan antibody and extend previous observations of murine antimannan antibody-mediated immunity against systemic candidiasis (12, 15).

Analysis of expression of the M1g1 epitope reveals three characteristics (Table 1 and Fig. 1). First, the M1g1 epitope has an apparent uniform distribution on the cell surface (Fig. 1, bottom). This uniform pattern of distribution is identical to the distribution described for the epitope recognized by the protective murine antimannan antibody B6.1 (13). The uniform binding pattern suggests a dense distribution of epitopes displayed on the yeast surface, which is in contrast to the patchy distribution of a mannan epitope recognized by the nonprotective murine antibody B6 (13). Second, expression of the M1g1 epitope is active in yeast cells under different growth temperatures (Table 1). Among the 10 mannan epitopes or factors that have been described for the rabbit system (36), factor 1, containing {alpha}-1,2-linked oligomannosyl residues, is present in all Candida species, and its expression is temperature independent (31). Factors 5 and 6 contain ß-1,2-linked oligomannosyl residues, and their expression is temperature dependent (31, 38); factor 5 is found in two Candida species, and factor 6 is found in three Candida species (36). Thus, the observation that growth temperature at either 23°C or 37°C had no discernible effect on the expression of the M1g1 epitope on yeast cells from various Candida species suggests that this epitope may be a stable component of Candida mannan. Therefore, the M1g1 epitope may be present on the surface of yeast cells during initial Candida infection and may play a role in early events in the pathogenesis of hematogenously disseminated candidiasis, providing a rationale for an antibody-mediated clearance of extracellular pathogens upon their entry into the bloodstream. Third, the M1g1 epitope has a broad distribution among Candida species (Table 1). Among the 10 mannan epitopes recognized by the rabbit antibody response, factor 1 is common to all Candida species as well as S. cerevisiae. Factor 4 is the most common Candida-specific mannan epitope and is present in four Candida species, including C. albicans. However, the M1g1 epitope was found in yeast cells of both serotype A and B strains of C. albicans as well as strains of C. tropicalis, C. guilliermondii, C. glabrata, and C. parapsilosis but not in yeast cells of S. cerevisiae. Thus, M1g1 appears to recognize one of the most common Candida mannan epitopes that have been reported. Given that C. albicans and many other Candida species can cause systemic candidiasis (33, 34), a broad binding specificity for M1g1 is an attractive feature when one considers potential prophylactic or therapeutic applications of passive immunization against candidiasis.

We utilized a murine model of hematogenously disseminated candidiasis to assess protection by M1g1. Mice that had been passively immunized with M1g1 were more resistant to the lethal infection by C. albicans 3153A yeast cells and survived significantly longer (Fig. 2). Furthermore, histopathological analysis of the kidneys of M1g1-treated mice showed a significant reduction in both the number of initial infection foci and the expansion of the foci as the infection progressed (Fig. 3). These observations are consistent with previous studies of murine antimannan antibodies. Experimental mice passively immunized with the protective murine monoclonal antibody B6.1 had fewer CFU of C. albicans recovered from the kidney 48 h postinfection and survived longer than the sham-treated control mice (12, 14).

How M1g1 confers resistance to systemic candidiasis is currently unknown. It is possible that M1g1 blocks the binding sites in mannan that are required for initial adherence of the yeast cells to the endothelium for dissemination into deep tissues. This is an attractive hypothesis given that Candida mannan has a demonstrated role in adhesion (6, 8, 17, 30) and that the stable and active expression of the M1g1 epitope in yeast cells suggests its role in early events in the pathogenesis of systemic candidiasis. Indeed, immunization of mice with Candida mannan induced protective immunity against disseminated candidiasis, and transfer of the serum from the immunized mice conferred protection against candidiasis (12). It is also possible that M1g1-opsonized yeast cells facilitate generation of a protective inflammatory response that includes enhanced phagocyte activity and activation of the complement system. Our in vitro assays showed that M1g1 promotes phagocytosis and phagocytic killing of C. albicans yeast cells by murine peritoneal macrophages (Fig. 4) and that M1g1 is required for C3 deposition onto the yeast cells (Fig. 5). The latter observation supports and extends our previous observation that activation of the human complement system by C. albicans yeast cells requires antimannan antibody (43, 44). Studies by Cutler and collaborators demonstrated that the protective murine antimannan IgM antibody B6.1 enhances mouse neutrophil candidacidal activity in the presence of normal mouse serum (5) and that protection by B6.1 or its murine IgG3 variant requires an intact complement system (14). However, antibody-mediated protection against systemic candidiasis has also been observed with a recombinant human antibody fragment specific for heat shock protein 90 (HSP90) that is free of the Fc region (27, 29). The protective mechanism for the Fc-free anti-HSP90 fragment has not been completely understood (29). Additional C. albicans epitopes that have been studied for antibody-mediated protection against systemic candidiasis include ß-glucan (37), a 58-kDa mannoprotein (41), and secreted aspartic proteinases (40).

Clearly, more well-characterized human monoclonal antimannan antibodies are needed to fully understand the function of the apparently ubiquitous presence of antimannan antibody in the general population (22, 24, 44). We utilized the phage display library approach to identification of human Fabs against mannan. This method has been widely used to generate Fabs against protein antigens. Anticarbohydrate antibodies selected through this method have been limited largely because of their relatively low affinity.

Is it possible to isolate more unique antimannan Fabs from antibody phage libraries? We believe that it is. First, the human antimannan antibody response targets a diverse array of mannan epitopes. We previously utilized a cross-absorption method and showed the presence of naturally occurring human antimannan antibodies that were reactive with epitopes similar to mannan antigenic factors 5 and 6 defined in the rabbit system (Kozel et al., Abstr. 100th Am. Soc. Microbiol., 2000). Second, diverse germ line variable-region genes are available for antimannan antibodies. In addition to the antimannan M1 Fab that is described here, a human antimannan single-chain variable fragment {lambda}2-18 (scFv{lambda}2-18) has been previously described (11, 42). Expression of these two antimannan fragments differs in usage of the germ line genes. A {kappa} gene is utilized for the M1 light-chain variable region, whereas a {lambda} gene is utilized for the scFv{lambda}2-18 light-chain variable region; the gene for the M1 heavy-chain variable region is derived from VH3-74, whereas the gene for the scFv{lambda}2-18 heavy-chain variable region is derived from VH3-64. Although epitope specificities for these two antimannan fragments remain to be defined, they are likely distinct in binding specificity given that they are encoded by different germ line genes. Third, antimannan Fab fragments of low affinity that cannot be selected when monovalently displayed on phage particles may be recovered when displayed multivalently where the overall antigen-binding avidity of each Fab-displaying phage increases (1, 2, 19). Affinity of Fab fragments selected by the multivalent display method may then be improved through mutagenesis of the heavy- and light-chain variable domains, and Fabs of improved affinity can be selected by the monovalent display method (10). Finally, antimannan antibodies with higher affinity may be present in some individuals. The affinity of the M1 Fab has not been determined. However, the M1 Fab was identified from a naïve human antibody library that was constructed from bone marrow lymphocytes donated by an adult whose serum level of natural antimannan antibody was that found in the general population. It is conceivable that some individuals with higher titers of antimannan antibody and in particular those who have recovered from systemic candidiasis will likely possess a repertoire of antimannan antibodies that is more diverse in both binding specificity and affinity than that possessed by a naïve, nonimmunized host.

The observations presented here with the recombinant human antimannan antibody M1g1 indicate that naturally occurring antimannan antibody may influence host resistance to systemic candidiasis. They also add to a growing body of evidence that supports an important role for human antibody-mediated immunity in host resistance to systemic candidiasis (28, 29, 35).


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ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health grants AI052139 and GM063119-02S2 (M.X.Z.) and AI14209, AI37194, and AI44784 (T.R.K.).

The following students at California State University—Long Beach contributed to this research: Adeola Adeseun, Jose Baeza, Charisse Bales, Gayle Boxx, Sindy Chavez, Augusto Cigliano, David Nguyen, Casey Nishiya, Chimenum Nyeche, Hosanna Or, Patrik Schöffler, Joshua Steichen, and Jennifer Weier. Support for student research included the Howard Hughes Medical Institute Undergraduate Honors Thesis Program in Biological Sciences (52002663) and the National Institutes of Health Bridges Program.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, California State University—Long Beach, 1250 Bellflower Blvd., Long Beach, CA 90840. Phone: (562) 985-4819. Fax: (562) 985-8878. E-mail: mzhang2{at}csulb.edu. Back

Editor: A. Casadevall

{dagger} Present address: Genmab B.V., Utrecht, The Netherlands. Back


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Infection and Immunity, January 2006, p. 362-369, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.362-369.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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