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

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 (V_L-C_L) and heavy chains (V_H-C_H1) 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 × 10⁷ PFU) were incubated overnight at 4°C with 4 × 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 κ light chain as determined with ELISA using anti-human κ 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 (C_H1-hinge-C_H2-C_H3), 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 V_H-C_H1 gene and M1 Fab V_L-C_L 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 × 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 × 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 × 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 × 10⁸ C. albicans 3153A yeast cells. C. albicans 3153A yeast cells at 4 × 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).

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 V_H gene was derived from V_H3-74 with 89% identity, D_H5-24 with 90% identity, and J_H4 with 90% identity. The M1 V_L gene was derived from Vκ2-A3 with 95% identity and Jκ2 with 97% identity.

Conversion of M1 to the full-length IgG1 antibody M1g1 and production of M1g1.

The M1 light-chain gene (V_L-C_L) was inserted into the IgG1 expression vector, and the M1 heavy-chain Fd gene (V_H-C_H1) was ligated to the gene specific for the IgG1 heavy chain (C_H1-hinge-C_H2-C_H3) 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.

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.

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.

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).

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).

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.