Production and Characterization of Monoclonal Antibodies against Enterocytozoon bieneusi Purified from Rhesus Macaques
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感染与免疫杂志 2005年第8期
Division of Infectious Diseases, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536
New England Regional Primate Research Center, Southborough, Massachusetts 01772
ABSTRACT
Enterocytozoon bieneusi spores derived from rhesus macaque feces were purified by serial salt-Percoll-sucrose-iodixanol centrifugation, resulting in two bands with different specific densities of 95.6% and 99.5% purity and with a recovery efficiency of 10.8%. An ultrastructural examination revealed typical E. bieneusi spores. Twenty-six stable hybridomas were derived from BALB/c mice immunized with spores and were cloned twice by limiting dilution or growth on semisolid medium. Four monoclonal antibodies (MAbs), reacting exclusively with spores, were further characterized. These MAbs specifically reacted with spores present in stools of humans and macaques, as visualized by immunofluorescence, and with spore walls, as visualized by immunoelectron microscopy. A blocking enzyme-linked immunosorbent assay and Western blotting revealed that the epitope recognized by 8E2 was different from those recognized by 7G2, 7H2, and 12G8, which identified the same 40-kDa protein. These MAbs will be valuable tools for diagnostics, for epidemiological investigations, for host-pathogen interaction studies, and for comparative genomics and proteomics.
INTRODUCTION
Enterocytozoon bieneusi is clinically the most significant microsporidian parasite associated with persistent diarrhea and wasting in individuals with AIDS (4, 10, 39). E. bieneusi has also been identified in immunologically healthy patients with diarrhea (3, 12, 19, 21, 31, 35) and in individuals receiving immunosuppressive therapy (15, 18, 26, 30, 34). E. bieneusi has also been described as infecting other mammalian species, including both immunologically normal and simian immunodeficiency virus (SIV)-infected macaques (Macaca mulatta, Macaca cyclopies, and Macaca nemestrina) (17, 23, 24, 32). E. bieneusi is found within the cytoplasm of epithelial cells of the gallbladder, bile ducts, and the small intestine, causing a proliferative cholecystitis, serositis, cholangiohepatitis, and enteropathy, respectively, in humans with human immunodeficiency virus (HIV)/AIDS (11, 25, 28, 29) and macaques with SIV/AIDS (6, 7). We have previously shown that E. bieneusi strains isolated from macaques and humans are morphologically, genetically, and antigenically indistinguishable (7, 24). Human- and rhesus-derived E. bieneusi sequences share 99.5% nucleic acid sequence identity over a 2.0-kb fragment of the ribosomal gene complex (5). However, recent data from our laboratory demonstrated that spores from these two mammal-infecting species have different specific densities and different karyotypes (unpublished data).
In the absence of the ability to propagate E. bieneusi in vitro or in vivo (38), feces from infected humans or rhesus macaques are the only available source of spores. Purification has not been easy because of the size of the spores. Several methods to purify spores from feces have been described by other laboratories (1, 8, 20) as well as by our group (33). Two monoclonal antibodies (MAbs) against human E. bieneusi have been reported (2), but they are unavailable commercially. To our knowledge, the production of MAbs against E. bieneusi isolates of rhesus macaque origin has not been reported.
In this communication, we describe the concentration and purification of spores from feces of macaques in sufficient quantities to generate several well-characterized specific MAbs.
MATERIALS AND METHODS
Fecal samples. All rhesus macaques (Macaca mulatta) were housed at the New England Regional Primate Research Center in accordance with the standards of the American Association of Laboratory Animal Care and Harvard Medical School's Animal Care and Use Committee. Fecal samples were collected daily and were analyzed for E. bieneusi shedding by nested PCR according to a previously described procedure (5, 24, 40, 41). For purification and MAb production, feces from SIV-infected rhesus macaques were collected in phosphate-buffered saline (PBS) and stored at 4°C for further processing.
Purification of E. bieneusi spores. (i) Salt-Percoll-sucrose centrifugation. Fecal specimens were processed as described previously (33), with the following modifications. Briefly, feces were homogenized in 0.01 M PBS, pH 7.2 to 7.4 (1:5 to 1:10), and serially filtered through American standard sieves (pore sizes, 425, 180, 100, and 63 μm; Newark Wire Cloth Company, Newark, NJ). The spores were pelleted at 3,200 x g for 40 min and washed four times with distilled water (3,200 x g, 20 min). The pellet was mixed with PBS and saturated sodium chloride (the final concentration of sodium chloride was 75%) and centrifuged at 1,000 x g for 15 min. In order to increase the recovery of spores, the pellet was processed again with a final sodium chloride concentration of 85%. The middle layer was collected, its sodium chloride concentration was adjusted to 50%, and the spores were pelleted at 3,200 x g for 30 min. The pellet was washed one more time (3,200 x g, 20 min) and then resuspended in PBS. For Percoll centrifugation, the samples were mixed with 72% isotonic Percoll (3 ml spores plus 30 ml 72% Percoll) and centrifuged for 60 min at 14,000 x g. The layer above the sediments (B, 2 to 2.5 ml) and the middle layer (M, 26 ml) were collected separately and washed one more time (3,200 x g, 20 min). Both layers were loaded on a 30 to 60% (wt/wt) sucrose gradient and centrifuged for 24 h at 77,000 x g. Spores were collected with a 20-gauge needle and pelleted at 77,000 x g for 60 min. Spores were washed twice with PBS (18,000 x g, 3 min).
(ii) Preformed iodixanol gradient centrifugation. OptiPrep density gradient medium (60% [wt/vol] solution of iodixanol in water) was purchased from Sigma (St. Louis, MO). The above spores were loaded on a 10 to 50% (wt/vol) preformed iodixanol gradient with 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4, and centrifuged for 60 min at 30,000 x g (16, 37). The spores were collected and resuspended in PBS. The recovery of spores at each step was monitored by an immunofluorescence assay (IFA) with rabbit polyclonal antibodies against human E. bieneusi as described previously (33).
TEM. Purified spores were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 18 to 24 h. The samples were rinsed in buffer and postfixed in 1% osmium tetroxide containing 0.8% potassium ferricyanide for 1 h. Samples were completely dehydrated in a graded series of ethanol. The spores were infiltrated with epoxy plastics according to the Mollenhauer formulation (27) and then cured at 60°C for 48 h. The blocks were sectioned on a Leica Ultracut R microtome, and the grids were stained with saturated uranyl acetate and lead citrate. Grids were viewed and photographed on a Phillips CM-10 electron microscope. The purity of the spores was examined under a low magnification by transmission electron microscopy (TEM). The purities of different bands were calculated by counting spores, bacteria, and other debris on each section.
Production of monoclonal antibodies. Three adult (6-week-old) female BALB/c mice were bled and immunized intraperitoneally four times at 2-week intervals with 4 x 107 spores per 100 μl emulsified at a 1:1 ratio with Freund's complete adjuvant (Calbiochem, La Jolla, CA) for the first inoculation and with Freund's incomplete adjuvant (Sigma, St. Louis, MO) for subsequent immunizations. Mouse humoral immune responses were monitored with an indirect enzyme-linked immunosorbent assay (ELISA) and IFA. Before fusion, immunized mice were boosted intravenously with 4 x 107 spores in 50 to 100 μl of PBS, and spleen cells were harvested and fused with SP2/0 cells as described previously (14). Two fusions were performed. For the first fusion, fused cells were seeded in six 96-well plates, and their supernatants were screened by ELISA 8 or 9 days after fusion. Hybridomas in positive wells were cloned two or three times by limiting dilution as described previously (13). For the second fusion, fused cells were incubated for 16 h at 37°C in a 5% CO2 humidified incubator and then were selected and cloned using a commercially prepared semisolid hypoxanthine-aminopterin-thymidine-containing medium (StemCell, Vancouver, Canada) according to the manufacturer's manual (9), with modifications. Briefly, fused cells were mixed with semisolid hypoxanthine-aminopterin-thymidine-containing medium and distributed into eight 100-mm tissue culture dishes. On days 12 to 14, 830 single clones were picked and plated in 96-well plates. Indirect ELISA and IFA, as detailed below, were used to screen supernatants after 3 or 4 days of culture. Positive clones were checked for isotypes by using a ClonalCell InstantCHEK one-minute isotyping kit (StemCell, Vancouver, Canada) as described by the manufacturer's protocol. Both ELISA- and IFA-positive clones were cloned a second time by using the same semisolid medium.
ELISA. An indirect ELISA was used to monitor mouse immune responses, to screen supernatants for clones, and to isotype MAbs. For antigen preparation, 4.2 x 108 purified spores in 1 ml PBS (purity = 99.5%) were mixed with 200 μl of 0.1-mm glass beads (BioSpec Products, Inc., Bartlesville, OK) and beat at 5,000 rpm for 2 min. The disrupted spores were frozen and thawed twice. After centrifugation at 300 x g for 1 min, the supernatant was collected. The protein concentration in the supernatant was determined using a Lowry protein assay kit (22), and the supernatant was stored at –20°C. Antigen concentrations were optimized by crisscross serial dilution analysis. Indirect ELISA was performed by standard procedures, as follows. Briefly, an EIA plate (Corning, NY) was coated overnight with 100 μl of antigens (0.5 μg/ml) in PBS with 0.05% sodium azide and then blocked with PBS containing 0.05% Tween 20 and 0.1% bovine serum albumin (PBST). The primary antibody was added, incubated for 60 min, and then washed three times with PBST. For antibody screening, horseradish peroxidase (HRP)-labeled anti-mouse immunoglobulin G (IgG) (heavy plus light chains) (Southern Biotech Inc., Birmingham, AL) was added and incubated for 60 min. For isotyping, HRP-labeled anti-mouse IgG1, IgG2a, IgG2b, IgG3, or or light chain was used. For color development, the TMB peroxidase substrate system (KPL, Gaithersburg, MD) was added, developed for 20 to 30 min at room temperature, and read at a wavelength of 650 nm before being stopped or 450 nm after being stopped.
Immunofluorescence assay. One microliter of purified spores (about 4,000 to 6,000 spores) or homogenized feces was mounted on a slide, air dried, and fixed by heat or in ice-cold acetone. Undiluted supernatants were transferred to slides and incubated for 30 min. The slides were then washed in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG+IgM (Molecular Probes, Eugene, OR) at a dilution of 1:500, with Evans blue as the counterstain. The slides were incubated at room temperature for 30 min and then washed. After being mounted, the slides were examined with epifluorescence illumination.
Characterization of monoclonal antibodies. MAb isotypes were determined with a ClonaCell InstantCHEK one-minute isotyping kit (StemCell, Vancouver, Canada) and then further determined by ELISA as described above. The epitopes recognized by MAbs 8E2, 7G2, 7H2, and 12G8 were determined by blocking ELISA. Briefly, plates were coated and blocked as described above. The MAbs 8E2, 7G2, 7H2, and 12G8 and complete Dulbecco's modified Eagle's medium were added to testing wells and control wells, respectively. After 30 min of incubation, MAb 8E2, 7G2, 7H2, or 12G8 was added to the corresponding wells and incubated for another 30 min. After the wells were washed, HRP-labeled anti-mouse IgG1, IgG2a, and IgG2b were added to the corresponding wells and incubated for 60 min. After another wash, the color was developed as described above. The inhibition rate was calculated as follows: inhibition rate (%) = (ODcontrol – ODtest)/ODcontrol x 100, where OD is the optical density.
Western blot analysis. Purified spores were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in Laemmli sample buffer, and 2 x 108 spores were subjected to SDS-PAGE on a 5 to 15% Tris-glycine polyacrylamide gel. Electrophoresis, the transfer to nitrocellulose, and blocking were performed under standard conditions. Culture supernatants with MAb 8E2, 7G2, 7H2, or 12G8 from T150 flasks were used as the primary antibodies. Mouse preimmune and immune sera (1:100 dilution) were used as negative and positive controls, respectively. Medium was used as a blank control. The secondary antibody, goat anti-mouse IgG linked to HRP (Southern Biotech Inc., Birmingham, AL), was detected using the TMB membrane peroxidase substrate system (KPL, Gaithersburg, MD).
Immunogold labeling. For each monoclonal antibody and control (PBS or a corresponding isotype), 200 μl of partially purified spores (1 x 108) was incubated with 1 ml of supernatant or PBS for 60 min and pelleted at 13,000 x g for 3 min. The pellet was washed once with PBS and incubated with 1 ml (1:30) goat anti-mouse IgG labeled with 10-nm gold particles (Sigma, St. Louis, MO) for 60 min. After being washed, the pellet was processed according to the above procedures for TEM.
Cross-reactivity studies. The reactivities of the mouse MAbs with purified E. bieneusi from human stool, cultured Encephalitozoon intestinalis, Encephalitozoon hellum, and Encephalitozoon cuniculi, and common bacteria and yeasts present in feces of human and macaque origins were assessed by IFA.
RESULTS
Purification of E. bieneusi spores. The purification and recovery of spores at each step were monitored by IFA. From sieving to washing, the average recovery rate of spores was approximately 70%. The recovery of spores was about 60% after salt centrifugation, 70% after Percoll centrifugation, 40% after sucrose gradient centrifugation, and 99% after iodixanol gradient centrifugation. The mean recovery rate throughout the procedure for four experiments was 10.8%. After centrifugation, the samples were resolved into two groups, i.e., band 1 (including B1 and M; specific density, 1.146 to 1.156) and band 2 (B2; specific density, 1.165 to 1.175) (Fig. 1). Purified spores were kept for at least 6 months at 4°C, with no loss in numbers or morphology.
TEM. Under a low magnification (x5,850) (Fig. 2), the purities of B1 (or M) and B2 were 95.6% and 99.5%, respectively. Although spores had different specific densities in bands M (B1) and B2, there were no apparent structural differences observed by TEM, which showed the typical five to seven polar tubes (Fig. 2, inset).
Monoclonal antibody production. Preimmunization sera did not react with any purified spores in IFA and showed very low ODs (<0.02) when assayed by ELISA. All mice began to produce antibodies after the first intraperitoneal injection. The largest antibody response was raised after the third injection. All three mice demonstrated no difference in their immune responses to purified spores. After the third injection, the serum IgG titer was maintained between 104 and 105 (OD450 = 1.0). The major isotype was IgG. IgA antibodies were detected at low levels, and IgM antibodies increased temporarily in the first 4 weeks and then decreased rapidly to a lower level.
For the first fusion experiment, 132 wells were positive by ELISA. Eight hybridomas were cloned two or three times by limiting dilution. For the second fusion experiment, 17 hybridomas were cloned two times by growth in semisolid medium and one time by limiting dilution. A total of 26 clones were obtained through two fusions. Their basic characteristics are detailed in Table 1. Four clones secreting IgG were passaged over 3 months and expanded to CELLine CL-1000 (IBS INTEGRA Biosciences Inc., Chur, Switzerland) for large-scale production.
Characterization of selected monoclonal antibodies (8E2, 7G2, 7H2, and 12G8). (i) IFA. MAbs 8E2, 7G2, 7H2, and 12G8 were selected for further testing against purified spores and fecal samples of monkey and human origins and were confirmed by PCR and a polyclonal rabbit E. bieneusi-specific serum (5, 33, 40, 41) (Fig. 3).
(ii) Ultrastructural immunolocalization. The four MAbs were shown by immune EM to label the spore walls, but not colocalized bacteria, of partially purified spores (Fig. 4). No gold particles were observed when the spores were incubated with the gold-labeled secondary antibody alone or with a corresponding isotype control.
(iii) Blocking ELISA for epitope mapping and Western blotting. HRP-labeled antibodies against mouse IgG1, IgG2a, and IgG2b were used to determine optical densities (of inhibited wells or control wells) because 8E2 (IgG1), 7G2 (IgG2a)/7H2 (IgG2a), and 12G8 (IgG2b) had different isotypes. 7G2, 7H2, and 12G8 slightly blocked the reaction of 8E2 (their inhibition rates were 17.82%, 14.59%, and 24.39%, respectively). In contrast, 8E2 did not block 7G2, 7H2, or 12G8. 7G2 and 7H2 inhibited 12G8 by 33.9% and 29.06%, respectively. Similarly, 12G8 inhibited 7G2 and 7H2 by 49.5% and 33.49%, respectively (Fig. 5). Western blotting revealed that 8E2 failed to recognize the targeted protein, while 7G2, 7H2, and 12G8 recognized the same protein (molecular mass, 40 kDa) (Fig. 6). Taken together, it appears that the epitope recognized by 8E2 is different from the epitope(s) recognized by 7G2/7H2 and 12G8.
Cross-reactivity. The IFA results revealed that 8E2, 7G2, 7H2, and 12G8 have no cross-reactivity to E. intestinalis, E. hellum, E. cuniculi, or common bacteria and yeasts derived from human or monkey feces.
DISCUSSION
The lack of ability to propagate E. bieneusi in the laboratory, a key step for the generation of large quantities of purified antigens for MAb production, has been a major obstacle. An alternative was to develop methods for the concentration and purification of spores directly from stools of heavily infected mammalian hosts. Heavily infected hosts are those with immunodeficiencies such as HIV/AIDS for humans and SIV/AIDS for macaques, who at the terminal phase of their illness excrete large quantities of E. bieneusi spores in the stool. As a consequence, we have developed methods for the concentration and purification of spores from human patients with chronic voluminous watery diarrhea using salt-sucrose gradient centrifugation or Percoll-sucrose gradient centrifugation (33). These methods were further modified, as reported in this communication, to further facilitate the purification of spores from semisolid macaque stools. Other investigators had purified spores from human stool by using discontinuous Percoll gradients and sterilized them for immunization with antibiotics (2). They purified spores by immunoaffinity expanded-bed adsorption (1), which is not available commercially. A lower yield of purified spores was obtained by using gradient and cell-sorting techniques (20).
The method we used for macaque feces included salt-Percoll-sucrose centrifugation combined with preformed iodixanol gradient centrifugation. This helped to generate large quantities of highly purified spores from macaque feces. During purification, the salt centrifugation step was performed twice with different sodium chloride concentrations, and only the middle layer rather than the supernatant was collected after centrifugation. In earlier experiments, we found that spores derived from rhesus macaques can be centrifuged into saturated sodium chloride (specific density, 1.20) at 3,200 x g for 30 min (unpublished data). Therefore, we maintained a 50% sodium chloride concentration when the spores were pelleted from the middle layer, which considerably reduced the pellet size and increased the efficiency of the original procedure. In the original Percoll centrifugation protocol, only the near-bottom layer above the sediments was collected, which averaged a recovery rate of 30%. The modified Percoll centrifugation step increased the recovery rate from 30% to 70% by collecting both the near-bottom layer (30%) and the middle layer (40%). Highly purified spores were obtained by preformed 10 to 50% iodixanol gradient centrifugation, an approach not previously used for the purification of E. bieneusi spores. OptiPrep (Sigma, St. Louis, MO) is a sterile and endotoxin-free solution of 60% (wt/vol) iodixanol in water. Customized isosmotic working solutions can be easily prepared for the purification of macromolecules, viruses, and cellular organelles (16, 37). By combining the above procedures, we were able to purify large quantities of E. bieneusi spores derived from rhesus macaques, which excrete considerably fewer spores than infected humans. In four separate experiments, we purified a total of 7.0 x 109 spores, with 95.6% and 99.5% purity for the two bands, from approximately 800 g of monkey feces collected from one animal. The average recovery was about 10.8%. Purified spores can be stored at 4°C for at least 6 months without a loss in the total number and intensity, as judged by IFA and immune EM or double staining with 5,6-carboxyfluorescein diacetate succinimidyl ester and IFA with MAbs (unpublished data). By using iodixanol gradients, the spores were resolved into two specific-density groups, demonstrating for the first time the existence of two different specific densities of E. bieneusi spores derived from the same animal. However, no ultrastructural differences between these two groups were observed by EM. Whether spores with different densities differ biologically is unclear.
In 1997, Tzipori et al. (36) established persistent infections of E. bieneusi derived from a human with HIV/AIDS in SIV-infected rhesus macaques. The natural occurrence of E. bieneusi infections in healthy and SIV-infected macaques (24) led to the subsequent use of the rhesus macaque as a model of this infection (36). These studies were conducted using the cumbersome PCR method for the detection and monitoring of E. bieneusi infection. The development of the MAbs described in this communication is therefore a major breakthrough for investigators working on this infection with humans and macaques.
We also report here the development of a high-throughput ELISA to screen MAbs by using antigens derived from purified spores, which was readily modified for epitope mapping and for identifying carbohydrate determinants (unpublished data). Of the 26 clones derived from two fusions, four lines were shown by IFA and immune EM to react exclusively with the spore wall by recognizing different epitopes, as shown by ELISA and Western blotting. It is clear that 7G2, 7H2, and 12G8 recognized the same protein by Western blotting, but whether 7G2, 7H2, and 12G8 target the same or different epitopes needs to be further investigated. It is noteworthy that 8E2 did not recognize any protein, presumably owing to the denatured SDS-PAGE. The IFA assay using these four characterized MAbs was highly specific for E. bieneusi spores in stool specimens from humans and monkeys, with no background interfering fluorescence. These results were consistent with nested PCR results.
This is the first report of the development of MAbs against E. bieneusi derived from rhesus macaques which cross-react with E. bieneusi strains that infect humans. These MAbs will help determine the tissue distribution of E. bieneusi in SIV-infected and healthy macaques and will facilitate further investigations on this clinically most significant microsporidium in mammals.
ACKNOWLEDGMENTS
This project was supported by NIH grants PO1DK55510 and R21AI52792 and by EPASTAR grant R828043.
The support and dedication of the technicians at the New England Regional Primate Research Center, who cared for the rhesus macaques and collected the fecal samples, are appreciated.
REFERENCES
1. Accoceberry, I., M. Thellier, A. Datry, I. Desportes-Livage, S. Biligui, M. Danis, and X. Santarelli. 2001. One-step purification of Enterocytozoon bieneusi spores from human stools by immunoaffinity expanded-bed adsorption. J. Clin. Microbiol. 39:1947-1951.
2. Accoceberry, I., M. Thellier, I. Desportes-Livage, A. Achbarou, S. Biligui, M. Danis, and A. Datry. 1999. Production of monoclonal antibodies directed against the microsporidium Enterocytozoon bieneusi. J. Clin. Microbiol. 37:4107-4112.
3. Albrecht, H., and I. Sobottka. 1997. Enterocytozoon bieneusi infection in patients who are not infected with human immunodeficiency virus. Clin. Infect. Dis. 25:344.
4. Canning, E. U., and W. S. Hollister. 1990. Enterocytozoon bieneusi (Microspora): prevalence and pathogenicity in AIDS patients. Trans. R. Soc. Trop. Med. Hyg. 84:181-186.
5. Carville, A., K. Mansfield, G. Widmer, A. Lackner, D. Kotler, P. Wiest, T. Gumbo, S. Sarbah, and S. Tzipori. 1997. Development and application of genetic probes for detection of Enterocytozoon bieneusi in formalin-fixed stools and in intestinal biopsy specimens from infected patients. Clin. Diagn. Lab. Immunol. 4:405-408.
6. Chalifoux, L. V., A. Carville, D. Pauley, B. Thompson, A. A. Lackner, and K. G. Mansfield. 2000. Enterocytozoon bieneusi as a cause of proliferative serositis in simian immunodeficiency virus-infected immunodeficient macaques (Macaca mulatta). Arch. Pathol. Lab. Med. 124:1480-1484.
7. Chalifoux, L. V., J. MacKey, A. Carville, D. Shvetz, K. C. Lin, A. Lackner, and K. G. Mansfield. 1998. Ultrastructural morphology of Enterocytozoon bieneusi in biliary epithelium of rhesus macaques (Macaca mulatta). Vet. Pathol. 35:292-296.
8. Challier, S., S. Brown, C. Ombrouck, I. Desportes-Livage, D. De Nay, and M. Gentilini. 1994. Flow cytometry as a possible method of isolation of spores of the microsporidian Enterocytozoon bieneusi. J. Eukaryot. Microbiol. 41:27S.
9. Davis, J. M., J. E. Pennington, A. M. Kubler, and J. F. Conscience. 1982. A simple, single-step technique for selecting and cloning hybridomas for the production of monoclonal antibodies. J. Immunol. Methods 50:161-171.
10. Desportes, I., Y. Le Charpentier, A. Galian, F. Bernard, B. Cochand-Priollet, A. Lavergne, P. Ravisse, and R. Modigliani. 1985. Occurrence of a new microsporidan: Enterocytozoon bieneusi n.g., n. sp., in the enterocytes of a human patient with AIDS. J. Protozool. 32:250-254.
11. French, A. L., L. M. Beaudet, D. A. Benator, C. S. Levy, M. Kass, and J. M. Orenstein. 1995. Cholecystectomy in patients with AIDS: clinicopathologic correlations in 107 cases. Clin. Infect. Dis. 21:852-858.
12. Gainzarain, J. C., A. Canut, M. Lozano, A. Labora, F. Carreras, S. Fenoy, R. Navajas, N. J. Pieniazek, A. J. da Silva, and C. del Aguila. 1998. Detection of Enterocytozoon bieneusi in two human immunodeficiency virus-negative patients with chronic diarrhea by polymerase chain reaction in duodenal biopsy specimens and review. Clin. Infect. Dis. 27:394-398.
13. Galfre, G., and C. Milstein. 1981. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 73:3-46.
14. Gefter, M. L., D. H. Margulies, and M. D. Scharff. 1977. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet. 3:231-236.
15. Goetz, M., S. Eichenlaub, G. R. Pape, and R. M. Hoffmann. 2001. Chronic diarrhea as a result of intestinal microsporidiosis in a liver transplant recipient. Transplantation 71:334-337.
16. Graham, J. M. 2002. OptiPrep density gradient solutions for mammalian organelles. ScientificWorldJournal 2:1440-1443.
17. Green, L. C., P. J. Didier, L. C. Bowers, and E. S. Didier. 2004. Natural and experimental infection of immunocompromised rhesus macaques (Macaca mulatta) with the microsporidian Enterocytozoon bieneusi genotype D. Microbes Infect. 6:996-1002.
18. Guerard, A., M. Rabodonirina, L. Cotte, O. Liguory, M. A. Piens, S. Daoud, S. Picot, and J. L. Touraine. 1999. Intestinal microsporidiosis occurring in two renal transplant recipients treated with mycophenolate mofetil. Transplantation 68:699-707.
19. Gumbo, T., I. T. Gangaidzo, S. Sarbah, A. Carville, S. Tzipori, and P. M. Wiest. 2000. Enterocytozoon bieneusi infection in patients without evidence of immunosuppression: two cases from Zimbabwe found to have positive stools by PCR. Ann. Trop. Med. Parasitol. 94:699-702.
20. Kucerova, Z., H. Moura, G. J. Leitch, R. Sriram, C. Bern, V. Kawai, D. Vargas, R. H. Gilman, E. Ticona, A. Vivar, and G. S. Visvesvara. 2004. Purification of Enterocytozoon bieneusi spores from stool specimens by gradient and cell sorting techniques. J. Clin. Microbiol. 42:3256-3261.
21. Lopez-Velez, R., M. C. Turrientes, C. Garron, P. Montilla, R. Navajas, S. Fenoy, and C. del Aguila. 1999. Microsporidiosis in travelers with diarrhea from the tropics. J. Travel Med. 6:223-227.
22. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.
23. Mansfield, K. G., A. Carville, D. Hebert, L. Chalifoux, D. Shvetz, K. C. Lin, S. Tzipori, and A. A. Lackner. 1998. Localization of persistent Enterocytozoon bieneusi infection in normal rhesus macaques (Macaca mulatta) to the hepatobiliary tree. J. Clin. Microbiol. 36:2336-2338.
24. Mansfield, K. G., A. Carville, D. Shvetz, J. MacKey, S. Tzipori, and A. A. Lackner. 1997. Identification of an Enterocytozoon bieneusi-like microsporidian parasite in simian-immunodeficiency-virus-inoculated macaques with hepatobiliary disease. Am. J. Pathol. 150:1395-1405.
25. McWhinney, P. H., D. Nathwani, S. T. Green, J. F. Boyd, and J. A. Forrest. 1991. Microsporidiosis detected in association with AIDS-related sclerosing cholangitis. AIDS 5:1394-1395.
26. Metge, S., J. T. Van Nhieu, D. Dahmane, P. Grimbert, F. Foulet, C. Sarfati, and S. Bretagne. 2000. A case of Enterocytozoon bieneusi infection in an HIV-negative renal transplant recipient. Eur. J. Clin. Microbiol. Infect. Dis. 19:221-223.
27. Mollenhauer, H. H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39:111-114.
28. Pol, S., C. Romana, S. Richard, F. Carnot, J. L. Dumont, H. Bouche, G. Pialoux, M. Stern, J. F. Pays, and P. Berthelot. 1992. Enterocytozoon bieneusi infection in acquired immunodeficiency syndrome-related sclerosing cholangitis. Gastroenterology 102:1778-1781.
29. Pol, S., C. A. Romana, S. Richard, P. Amouyal, I. Desportes-Livage, F. Carnot, J. F. Pays, and P. Berthelot. 1993. Microsporidia infection in patients with the human immunodeficiency virus and unexplained cholangitis. N. Engl. J. Med. 328:95-99.
30. Rabodonirina, M., M. Bertocchi, I. Desportes-Livage, L. Cotte, H. Levrey, M. A. Piens, G. Monneret, M. Celard, J. F. Mornex, and M. Mojon. 1996. Enterocytozoon bieneusi as a cause of chronic diarrhea in a heart-lung transplant recipient who was seronegative for human immunodeficiency virus. Clin. Infect. Dis. 23:114-117.
31. Sandfort, J., A. Hannemann, H. Gelderblom, K. Stark, R. L. Owen, and B. Ruf. 1994. Enterocytozoon bieneusi infection in an immunocompetent patient who had acute diarrhea and who was not infected with the human immunodeficiency virus. Clin. Infect. Dis. 19:514-516.
32. Schwartz, D. A., D. C. Anderson, S. A. Klumpp, and H. M. McClure. 1998. Ultrastructure of atypical (teratoid) sporogonial stages of Enterocytozoon bieneusi (Microsporidia) in naturally infected rhesus monkeys (Macacca mulatta). Arch. Pathol. Lab. Med. 122:423-429.
33. Sheoran, A. S., X. Feng, S. Kitaka, L. Green, C. Pearson, E. S. Didier, S. Chapman, J. K. Tumwine, and S. Tzipori. 2005. Purification of Enterocytozoon bieneusi from stools and production of specific antibodies. J. Clin. Microbiol. 43:387-392.
34. Sing, A., K. Tybus, J. Heesemann, and A. Mathis. 2001. Molecular diagnosis of an Enterocytozoon bieneusi human genotype C infection in a moderately immunosuppressed human immunodeficiency virus-seronegative liver-transplant recipient with severe chronic diarrhea. J. Clin. Microbiol. 39:2371-2372.
35. Sobottka, I., H. Albrecht, J. Schottelius, C. Schmetz, M. Bentfeld, R. Laufs, and D. A. Schwartz. 1995. Self-limited traveller's diarrhea due to a dual infection with Enterocytozoon bieneusi and Cryptosporidium parvum in an immunocompetent HIV-negative child. Eur. J. Clin. Microbiol. Infect. Dis. 14:919-920.
36. Tzipori, S., A. Carville, G. Widmer, D. Kotler, K. Mansfield, and A. Lackner. 1997. Transmission and establishment of a persistent infection of Enterocytozoon bieneusi, derived from a human with AIDS, in simian immunodeficiency virus-infected rhesus monkeys. J. Infect. Dis. 175:1016-1020.
37. Van Veldhoven, P. P., E. Baumgart, and G. P. Mannaerts. 1996. Iodixanol (Optiprep), an improved density gradient medium for the iso-osmotic isolation of rat liver peroxisomes. Anal. Biochem. 237:17-23.
38. Visvesvara, G., G. J. Leitch, N. J. Pieniazek, A. J. Da Silva, S. Wallace, S. B. Slemenda, R. Weber, D. A. Schwartz, L. Gorelkin, C. M. Wilcox, et al. 1995. Short-term in vitro culture and molecular analysis of the microsporidian Enterocytozoon bieneusi. J. Eukaryot. Microbiol. 42:506-510.
39. Weber, R., H. Kuster, R. Keller, T. Bachi, M. A. Spycher, J. Briner, E. Russi, and R. Luthy. 1992. Pulmonary and intestinal microsporidiosis in a patient with the acquired immunodeficiency syndrome. Am. Rev. Respir. Dis. 146:1603-1605.
40. Zhu, X., M. Wittner, H. B. Tanowitz, A. Cali, and L. M. Weiss. 1994. Ribosomal RNA sequences of Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis: phylogenetic construction and structural correspondence. J. Eukaryot. Microbiol. 41:204-209.
41. Zhu, X., M. Wittner, H. B. Tanowitz, D. Kotler, A. Cali, and L. M. Weiss. 1993. Small subunit rRNA sequence of Enterocytozoon bieneusi and its potential diagnostic role with use of the polymerase chain reaction. J. Infect. Dis. 168:1570-1575.(Quanshun Zhang, Inderpal )
New England Regional Primate Research Center, Southborough, Massachusetts 01772
ABSTRACT
Enterocytozoon bieneusi spores derived from rhesus macaque feces were purified by serial salt-Percoll-sucrose-iodixanol centrifugation, resulting in two bands with different specific densities of 95.6% and 99.5% purity and with a recovery efficiency of 10.8%. An ultrastructural examination revealed typical E. bieneusi spores. Twenty-six stable hybridomas were derived from BALB/c mice immunized with spores and were cloned twice by limiting dilution or growth on semisolid medium. Four monoclonal antibodies (MAbs), reacting exclusively with spores, were further characterized. These MAbs specifically reacted with spores present in stools of humans and macaques, as visualized by immunofluorescence, and with spore walls, as visualized by immunoelectron microscopy. A blocking enzyme-linked immunosorbent assay and Western blotting revealed that the epitope recognized by 8E2 was different from those recognized by 7G2, 7H2, and 12G8, which identified the same 40-kDa protein. These MAbs will be valuable tools for diagnostics, for epidemiological investigations, for host-pathogen interaction studies, and for comparative genomics and proteomics.
INTRODUCTION
Enterocytozoon bieneusi is clinically the most significant microsporidian parasite associated with persistent diarrhea and wasting in individuals with AIDS (4, 10, 39). E. bieneusi has also been identified in immunologically healthy patients with diarrhea (3, 12, 19, 21, 31, 35) and in individuals receiving immunosuppressive therapy (15, 18, 26, 30, 34). E. bieneusi has also been described as infecting other mammalian species, including both immunologically normal and simian immunodeficiency virus (SIV)-infected macaques (Macaca mulatta, Macaca cyclopies, and Macaca nemestrina) (17, 23, 24, 32). E. bieneusi is found within the cytoplasm of epithelial cells of the gallbladder, bile ducts, and the small intestine, causing a proliferative cholecystitis, serositis, cholangiohepatitis, and enteropathy, respectively, in humans with human immunodeficiency virus (HIV)/AIDS (11, 25, 28, 29) and macaques with SIV/AIDS (6, 7). We have previously shown that E. bieneusi strains isolated from macaques and humans are morphologically, genetically, and antigenically indistinguishable (7, 24). Human- and rhesus-derived E. bieneusi sequences share 99.5% nucleic acid sequence identity over a 2.0-kb fragment of the ribosomal gene complex (5). However, recent data from our laboratory demonstrated that spores from these two mammal-infecting species have different specific densities and different karyotypes (unpublished data).
In the absence of the ability to propagate E. bieneusi in vitro or in vivo (38), feces from infected humans or rhesus macaques are the only available source of spores. Purification has not been easy because of the size of the spores. Several methods to purify spores from feces have been described by other laboratories (1, 8, 20) as well as by our group (33). Two monoclonal antibodies (MAbs) against human E. bieneusi have been reported (2), but they are unavailable commercially. To our knowledge, the production of MAbs against E. bieneusi isolates of rhesus macaque origin has not been reported.
In this communication, we describe the concentration and purification of spores from feces of macaques in sufficient quantities to generate several well-characterized specific MAbs.
MATERIALS AND METHODS
Fecal samples. All rhesus macaques (Macaca mulatta) were housed at the New England Regional Primate Research Center in accordance with the standards of the American Association of Laboratory Animal Care and Harvard Medical School's Animal Care and Use Committee. Fecal samples were collected daily and were analyzed for E. bieneusi shedding by nested PCR according to a previously described procedure (5, 24, 40, 41). For purification and MAb production, feces from SIV-infected rhesus macaques were collected in phosphate-buffered saline (PBS) and stored at 4°C for further processing.
Purification of E. bieneusi spores. (i) Salt-Percoll-sucrose centrifugation. Fecal specimens were processed as described previously (33), with the following modifications. Briefly, feces were homogenized in 0.01 M PBS, pH 7.2 to 7.4 (1:5 to 1:10), and serially filtered through American standard sieves (pore sizes, 425, 180, 100, and 63 μm; Newark Wire Cloth Company, Newark, NJ). The spores were pelleted at 3,200 x g for 40 min and washed four times with distilled water (3,200 x g, 20 min). The pellet was mixed with PBS and saturated sodium chloride (the final concentration of sodium chloride was 75%) and centrifuged at 1,000 x g for 15 min. In order to increase the recovery of spores, the pellet was processed again with a final sodium chloride concentration of 85%. The middle layer was collected, its sodium chloride concentration was adjusted to 50%, and the spores were pelleted at 3,200 x g for 30 min. The pellet was washed one more time (3,200 x g, 20 min) and then resuspended in PBS. For Percoll centrifugation, the samples were mixed with 72% isotonic Percoll (3 ml spores plus 30 ml 72% Percoll) and centrifuged for 60 min at 14,000 x g. The layer above the sediments (B, 2 to 2.5 ml) and the middle layer (M, 26 ml) were collected separately and washed one more time (3,200 x g, 20 min). Both layers were loaded on a 30 to 60% (wt/wt) sucrose gradient and centrifuged for 24 h at 77,000 x g. Spores were collected with a 20-gauge needle and pelleted at 77,000 x g for 60 min. Spores were washed twice with PBS (18,000 x g, 3 min).
(ii) Preformed iodixanol gradient centrifugation. OptiPrep density gradient medium (60% [wt/vol] solution of iodixanol in water) was purchased from Sigma (St. Louis, MO). The above spores were loaded on a 10 to 50% (wt/vol) preformed iodixanol gradient with 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4, and centrifuged for 60 min at 30,000 x g (16, 37). The spores were collected and resuspended in PBS. The recovery of spores at each step was monitored by an immunofluorescence assay (IFA) with rabbit polyclonal antibodies against human E. bieneusi as described previously (33).
TEM. Purified spores were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 18 to 24 h. The samples were rinsed in buffer and postfixed in 1% osmium tetroxide containing 0.8% potassium ferricyanide for 1 h. Samples were completely dehydrated in a graded series of ethanol. The spores were infiltrated with epoxy plastics according to the Mollenhauer formulation (27) and then cured at 60°C for 48 h. The blocks were sectioned on a Leica Ultracut R microtome, and the grids were stained with saturated uranyl acetate and lead citrate. Grids were viewed and photographed on a Phillips CM-10 electron microscope. The purity of the spores was examined under a low magnification by transmission electron microscopy (TEM). The purities of different bands were calculated by counting spores, bacteria, and other debris on each section.
Production of monoclonal antibodies. Three adult (6-week-old) female BALB/c mice were bled and immunized intraperitoneally four times at 2-week intervals with 4 x 107 spores per 100 μl emulsified at a 1:1 ratio with Freund's complete adjuvant (Calbiochem, La Jolla, CA) for the first inoculation and with Freund's incomplete adjuvant (Sigma, St. Louis, MO) for subsequent immunizations. Mouse humoral immune responses were monitored with an indirect enzyme-linked immunosorbent assay (ELISA) and IFA. Before fusion, immunized mice were boosted intravenously with 4 x 107 spores in 50 to 100 μl of PBS, and spleen cells were harvested and fused with SP2/0 cells as described previously (14). Two fusions were performed. For the first fusion, fused cells were seeded in six 96-well plates, and their supernatants were screened by ELISA 8 or 9 days after fusion. Hybridomas in positive wells were cloned two or three times by limiting dilution as described previously (13). For the second fusion, fused cells were incubated for 16 h at 37°C in a 5% CO2 humidified incubator and then were selected and cloned using a commercially prepared semisolid hypoxanthine-aminopterin-thymidine-containing medium (StemCell, Vancouver, Canada) according to the manufacturer's manual (9), with modifications. Briefly, fused cells were mixed with semisolid hypoxanthine-aminopterin-thymidine-containing medium and distributed into eight 100-mm tissue culture dishes. On days 12 to 14, 830 single clones were picked and plated in 96-well plates. Indirect ELISA and IFA, as detailed below, were used to screen supernatants after 3 or 4 days of culture. Positive clones were checked for isotypes by using a ClonalCell InstantCHEK one-minute isotyping kit (StemCell, Vancouver, Canada) as described by the manufacturer's protocol. Both ELISA- and IFA-positive clones were cloned a second time by using the same semisolid medium.
ELISA. An indirect ELISA was used to monitor mouse immune responses, to screen supernatants for clones, and to isotype MAbs. For antigen preparation, 4.2 x 108 purified spores in 1 ml PBS (purity = 99.5%) were mixed with 200 μl of 0.1-mm glass beads (BioSpec Products, Inc., Bartlesville, OK) and beat at 5,000 rpm for 2 min. The disrupted spores were frozen and thawed twice. After centrifugation at 300 x g for 1 min, the supernatant was collected. The protein concentration in the supernatant was determined using a Lowry protein assay kit (22), and the supernatant was stored at –20°C. Antigen concentrations were optimized by crisscross serial dilution analysis. Indirect ELISA was performed by standard procedures, as follows. Briefly, an EIA plate (Corning, NY) was coated overnight with 100 μl of antigens (0.5 μg/ml) in PBS with 0.05% sodium azide and then blocked with PBS containing 0.05% Tween 20 and 0.1% bovine serum albumin (PBST). The primary antibody was added, incubated for 60 min, and then washed three times with PBST. For antibody screening, horseradish peroxidase (HRP)-labeled anti-mouse immunoglobulin G (IgG) (heavy plus light chains) (Southern Biotech Inc., Birmingham, AL) was added and incubated for 60 min. For isotyping, HRP-labeled anti-mouse IgG1, IgG2a, IgG2b, IgG3, or or light chain was used. For color development, the TMB peroxidase substrate system (KPL, Gaithersburg, MD) was added, developed for 20 to 30 min at room temperature, and read at a wavelength of 650 nm before being stopped or 450 nm after being stopped.
Immunofluorescence assay. One microliter of purified spores (about 4,000 to 6,000 spores) or homogenized feces was mounted on a slide, air dried, and fixed by heat or in ice-cold acetone. Undiluted supernatants were transferred to slides and incubated for 30 min. The slides were then washed in PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG+IgM (Molecular Probes, Eugene, OR) at a dilution of 1:500, with Evans blue as the counterstain. The slides were incubated at room temperature for 30 min and then washed. After being mounted, the slides were examined with epifluorescence illumination.
Characterization of monoclonal antibodies. MAb isotypes were determined with a ClonaCell InstantCHEK one-minute isotyping kit (StemCell, Vancouver, Canada) and then further determined by ELISA as described above. The epitopes recognized by MAbs 8E2, 7G2, 7H2, and 12G8 were determined by blocking ELISA. Briefly, plates were coated and blocked as described above. The MAbs 8E2, 7G2, 7H2, and 12G8 and complete Dulbecco's modified Eagle's medium were added to testing wells and control wells, respectively. After 30 min of incubation, MAb 8E2, 7G2, 7H2, or 12G8 was added to the corresponding wells and incubated for another 30 min. After the wells were washed, HRP-labeled anti-mouse IgG1, IgG2a, and IgG2b were added to the corresponding wells and incubated for 60 min. After another wash, the color was developed as described above. The inhibition rate was calculated as follows: inhibition rate (%) = (ODcontrol – ODtest)/ODcontrol x 100, where OD is the optical density.
Western blot analysis. Purified spores were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in Laemmli sample buffer, and 2 x 108 spores were subjected to SDS-PAGE on a 5 to 15% Tris-glycine polyacrylamide gel. Electrophoresis, the transfer to nitrocellulose, and blocking were performed under standard conditions. Culture supernatants with MAb 8E2, 7G2, 7H2, or 12G8 from T150 flasks were used as the primary antibodies. Mouse preimmune and immune sera (1:100 dilution) were used as negative and positive controls, respectively. Medium was used as a blank control. The secondary antibody, goat anti-mouse IgG linked to HRP (Southern Biotech Inc., Birmingham, AL), was detected using the TMB membrane peroxidase substrate system (KPL, Gaithersburg, MD).
Immunogold labeling. For each monoclonal antibody and control (PBS or a corresponding isotype), 200 μl of partially purified spores (1 x 108) was incubated with 1 ml of supernatant or PBS for 60 min and pelleted at 13,000 x g for 3 min. The pellet was washed once with PBS and incubated with 1 ml (1:30) goat anti-mouse IgG labeled with 10-nm gold particles (Sigma, St. Louis, MO) for 60 min. After being washed, the pellet was processed according to the above procedures for TEM.
Cross-reactivity studies. The reactivities of the mouse MAbs with purified E. bieneusi from human stool, cultured Encephalitozoon intestinalis, Encephalitozoon hellum, and Encephalitozoon cuniculi, and common bacteria and yeasts present in feces of human and macaque origins were assessed by IFA.
RESULTS
Purification of E. bieneusi spores. The purification and recovery of spores at each step were monitored by IFA. From sieving to washing, the average recovery rate of spores was approximately 70%. The recovery of spores was about 60% after salt centrifugation, 70% after Percoll centrifugation, 40% after sucrose gradient centrifugation, and 99% after iodixanol gradient centrifugation. The mean recovery rate throughout the procedure for four experiments was 10.8%. After centrifugation, the samples were resolved into two groups, i.e., band 1 (including B1 and M; specific density, 1.146 to 1.156) and band 2 (B2; specific density, 1.165 to 1.175) (Fig. 1). Purified spores were kept for at least 6 months at 4°C, with no loss in numbers or morphology.
TEM. Under a low magnification (x5,850) (Fig. 2), the purities of B1 (or M) and B2 were 95.6% and 99.5%, respectively. Although spores had different specific densities in bands M (B1) and B2, there were no apparent structural differences observed by TEM, which showed the typical five to seven polar tubes (Fig. 2, inset).
Monoclonal antibody production. Preimmunization sera did not react with any purified spores in IFA and showed very low ODs (<0.02) when assayed by ELISA. All mice began to produce antibodies after the first intraperitoneal injection. The largest antibody response was raised after the third injection. All three mice demonstrated no difference in their immune responses to purified spores. After the third injection, the serum IgG titer was maintained between 104 and 105 (OD450 = 1.0). The major isotype was IgG. IgA antibodies were detected at low levels, and IgM antibodies increased temporarily in the first 4 weeks and then decreased rapidly to a lower level.
For the first fusion experiment, 132 wells were positive by ELISA. Eight hybridomas were cloned two or three times by limiting dilution. For the second fusion experiment, 17 hybridomas were cloned two times by growth in semisolid medium and one time by limiting dilution. A total of 26 clones were obtained through two fusions. Their basic characteristics are detailed in Table 1. Four clones secreting IgG were passaged over 3 months and expanded to CELLine CL-1000 (IBS INTEGRA Biosciences Inc., Chur, Switzerland) for large-scale production.
Characterization of selected monoclonal antibodies (8E2, 7G2, 7H2, and 12G8). (i) IFA. MAbs 8E2, 7G2, 7H2, and 12G8 were selected for further testing against purified spores and fecal samples of monkey and human origins and were confirmed by PCR and a polyclonal rabbit E. bieneusi-specific serum (5, 33, 40, 41) (Fig. 3).
(ii) Ultrastructural immunolocalization. The four MAbs were shown by immune EM to label the spore walls, but not colocalized bacteria, of partially purified spores (Fig. 4). No gold particles were observed when the spores were incubated with the gold-labeled secondary antibody alone or with a corresponding isotype control.
(iii) Blocking ELISA for epitope mapping and Western blotting. HRP-labeled antibodies against mouse IgG1, IgG2a, and IgG2b were used to determine optical densities (of inhibited wells or control wells) because 8E2 (IgG1), 7G2 (IgG2a)/7H2 (IgG2a), and 12G8 (IgG2b) had different isotypes. 7G2, 7H2, and 12G8 slightly blocked the reaction of 8E2 (their inhibition rates were 17.82%, 14.59%, and 24.39%, respectively). In contrast, 8E2 did not block 7G2, 7H2, or 12G8. 7G2 and 7H2 inhibited 12G8 by 33.9% and 29.06%, respectively. Similarly, 12G8 inhibited 7G2 and 7H2 by 49.5% and 33.49%, respectively (Fig. 5). Western blotting revealed that 8E2 failed to recognize the targeted protein, while 7G2, 7H2, and 12G8 recognized the same protein (molecular mass, 40 kDa) (Fig. 6). Taken together, it appears that the epitope recognized by 8E2 is different from the epitope(s) recognized by 7G2/7H2 and 12G8.
Cross-reactivity. The IFA results revealed that 8E2, 7G2, 7H2, and 12G8 have no cross-reactivity to E. intestinalis, E. hellum, E. cuniculi, or common bacteria and yeasts derived from human or monkey feces.
DISCUSSION
The lack of ability to propagate E. bieneusi in the laboratory, a key step for the generation of large quantities of purified antigens for MAb production, has been a major obstacle. An alternative was to develop methods for the concentration and purification of spores directly from stools of heavily infected mammalian hosts. Heavily infected hosts are those with immunodeficiencies such as HIV/AIDS for humans and SIV/AIDS for macaques, who at the terminal phase of their illness excrete large quantities of E. bieneusi spores in the stool. As a consequence, we have developed methods for the concentration and purification of spores from human patients with chronic voluminous watery diarrhea using salt-sucrose gradient centrifugation or Percoll-sucrose gradient centrifugation (33). These methods were further modified, as reported in this communication, to further facilitate the purification of spores from semisolid macaque stools. Other investigators had purified spores from human stool by using discontinuous Percoll gradients and sterilized them for immunization with antibiotics (2). They purified spores by immunoaffinity expanded-bed adsorption (1), which is not available commercially. A lower yield of purified spores was obtained by using gradient and cell-sorting techniques (20).
The method we used for macaque feces included salt-Percoll-sucrose centrifugation combined with preformed iodixanol gradient centrifugation. This helped to generate large quantities of highly purified spores from macaque feces. During purification, the salt centrifugation step was performed twice with different sodium chloride concentrations, and only the middle layer rather than the supernatant was collected after centrifugation. In earlier experiments, we found that spores derived from rhesus macaques can be centrifuged into saturated sodium chloride (specific density, 1.20) at 3,200 x g for 30 min (unpublished data). Therefore, we maintained a 50% sodium chloride concentration when the spores were pelleted from the middle layer, which considerably reduced the pellet size and increased the efficiency of the original procedure. In the original Percoll centrifugation protocol, only the near-bottom layer above the sediments was collected, which averaged a recovery rate of 30%. The modified Percoll centrifugation step increased the recovery rate from 30% to 70% by collecting both the near-bottom layer (30%) and the middle layer (40%). Highly purified spores were obtained by preformed 10 to 50% iodixanol gradient centrifugation, an approach not previously used for the purification of E. bieneusi spores. OptiPrep (Sigma, St. Louis, MO) is a sterile and endotoxin-free solution of 60% (wt/vol) iodixanol in water. Customized isosmotic working solutions can be easily prepared for the purification of macromolecules, viruses, and cellular organelles (16, 37). By combining the above procedures, we were able to purify large quantities of E. bieneusi spores derived from rhesus macaques, which excrete considerably fewer spores than infected humans. In four separate experiments, we purified a total of 7.0 x 109 spores, with 95.6% and 99.5% purity for the two bands, from approximately 800 g of monkey feces collected from one animal. The average recovery was about 10.8%. Purified spores can be stored at 4°C for at least 6 months without a loss in the total number and intensity, as judged by IFA and immune EM or double staining with 5,6-carboxyfluorescein diacetate succinimidyl ester and IFA with MAbs (unpublished data). By using iodixanol gradients, the spores were resolved into two specific-density groups, demonstrating for the first time the existence of two different specific densities of E. bieneusi spores derived from the same animal. However, no ultrastructural differences between these two groups were observed by EM. Whether spores with different densities differ biologically is unclear.
In 1997, Tzipori et al. (36) established persistent infections of E. bieneusi derived from a human with HIV/AIDS in SIV-infected rhesus macaques. The natural occurrence of E. bieneusi infections in healthy and SIV-infected macaques (24) led to the subsequent use of the rhesus macaque as a model of this infection (36). These studies were conducted using the cumbersome PCR method for the detection and monitoring of E. bieneusi infection. The development of the MAbs described in this communication is therefore a major breakthrough for investigators working on this infection with humans and macaques.
We also report here the development of a high-throughput ELISA to screen MAbs by using antigens derived from purified spores, which was readily modified for epitope mapping and for identifying carbohydrate determinants (unpublished data). Of the 26 clones derived from two fusions, four lines were shown by IFA and immune EM to react exclusively with the spore wall by recognizing different epitopes, as shown by ELISA and Western blotting. It is clear that 7G2, 7H2, and 12G8 recognized the same protein by Western blotting, but whether 7G2, 7H2, and 12G8 target the same or different epitopes needs to be further investigated. It is noteworthy that 8E2 did not recognize any protein, presumably owing to the denatured SDS-PAGE. The IFA assay using these four characterized MAbs was highly specific for E. bieneusi spores in stool specimens from humans and monkeys, with no background interfering fluorescence. These results were consistent with nested PCR results.
This is the first report of the development of MAbs against E. bieneusi derived from rhesus macaques which cross-react with E. bieneusi strains that infect humans. These MAbs will help determine the tissue distribution of E. bieneusi in SIV-infected and healthy macaques and will facilitate further investigations on this clinically most significant microsporidium in mammals.
ACKNOWLEDGMENTS
This project was supported by NIH grants PO1DK55510 and R21AI52792 and by EPASTAR grant R828043.
The support and dedication of the technicians at the New England Regional Primate Research Center, who cared for the rhesus macaques and collected the fecal samples, are appreciated.
REFERENCES
1. Accoceberry, I., M. Thellier, A. Datry, I. Desportes-Livage, S. Biligui, M. Danis, and X. Santarelli. 2001. One-step purification of Enterocytozoon bieneusi spores from human stools by immunoaffinity expanded-bed adsorption. J. Clin. Microbiol. 39:1947-1951.
2. Accoceberry, I., M. Thellier, I. Desportes-Livage, A. Achbarou, S. Biligui, M. Danis, and A. Datry. 1999. Production of monoclonal antibodies directed against the microsporidium Enterocytozoon bieneusi. J. Clin. Microbiol. 37:4107-4112.
3. Albrecht, H., and I. Sobottka. 1997. Enterocytozoon bieneusi infection in patients who are not infected with human immunodeficiency virus. Clin. Infect. Dis. 25:344.
4. Canning, E. U., and W. S. Hollister. 1990. Enterocytozoon bieneusi (Microspora): prevalence and pathogenicity in AIDS patients. Trans. R. Soc. Trop. Med. Hyg. 84:181-186.
5. Carville, A., K. Mansfield, G. Widmer, A. Lackner, D. Kotler, P. Wiest, T. Gumbo, S. Sarbah, and S. Tzipori. 1997. Development and application of genetic probes for detection of Enterocytozoon bieneusi in formalin-fixed stools and in intestinal biopsy specimens from infected patients. Clin. Diagn. Lab. Immunol. 4:405-408.
6. Chalifoux, L. V., A. Carville, D. Pauley, B. Thompson, A. A. Lackner, and K. G. Mansfield. 2000. Enterocytozoon bieneusi as a cause of proliferative serositis in simian immunodeficiency virus-infected immunodeficient macaques (Macaca mulatta). Arch. Pathol. Lab. Med. 124:1480-1484.
7. Chalifoux, L. V., J. MacKey, A. Carville, D. Shvetz, K. C. Lin, A. Lackner, and K. G. Mansfield. 1998. Ultrastructural morphology of Enterocytozoon bieneusi in biliary epithelium of rhesus macaques (Macaca mulatta). Vet. Pathol. 35:292-296.
8. Challier, S., S. Brown, C. Ombrouck, I. Desportes-Livage, D. De Nay, and M. Gentilini. 1994. Flow cytometry as a possible method of isolation of spores of the microsporidian Enterocytozoon bieneusi. J. Eukaryot. Microbiol. 41:27S.
9. Davis, J. M., J. E. Pennington, A. M. Kubler, and J. F. Conscience. 1982. A simple, single-step technique for selecting and cloning hybridomas for the production of monoclonal antibodies. J. Immunol. Methods 50:161-171.
10. Desportes, I., Y. Le Charpentier, A. Galian, F. Bernard, B. Cochand-Priollet, A. Lavergne, P. Ravisse, and R. Modigliani. 1985. Occurrence of a new microsporidan: Enterocytozoon bieneusi n.g., n. sp., in the enterocytes of a human patient with AIDS. J. Protozool. 32:250-254.
11. French, A. L., L. M. Beaudet, D. A. Benator, C. S. Levy, M. Kass, and J. M. Orenstein. 1995. Cholecystectomy in patients with AIDS: clinicopathologic correlations in 107 cases. Clin. Infect. Dis. 21:852-858.
12. Gainzarain, J. C., A. Canut, M. Lozano, A. Labora, F. Carreras, S. Fenoy, R. Navajas, N. J. Pieniazek, A. J. da Silva, and C. del Aguila. 1998. Detection of Enterocytozoon bieneusi in two human immunodeficiency virus-negative patients with chronic diarrhea by polymerase chain reaction in duodenal biopsy specimens and review. Clin. Infect. Dis. 27:394-398.
13. Galfre, G., and C. Milstein. 1981. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 73:3-46.
14. Gefter, M. L., D. H. Margulies, and M. D. Scharff. 1977. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet. 3:231-236.
15. Goetz, M., S. Eichenlaub, G. R. Pape, and R. M. Hoffmann. 2001. Chronic diarrhea as a result of intestinal microsporidiosis in a liver transplant recipient. Transplantation 71:334-337.
16. Graham, J. M. 2002. OptiPrep density gradient solutions for mammalian organelles. ScientificWorldJournal 2:1440-1443.
17. Green, L. C., P. J. Didier, L. C. Bowers, and E. S. Didier. 2004. Natural and experimental infection of immunocompromised rhesus macaques (Macaca mulatta) with the microsporidian Enterocytozoon bieneusi genotype D. Microbes Infect. 6:996-1002.
18. Guerard, A., M. Rabodonirina, L. Cotte, O. Liguory, M. A. Piens, S. Daoud, S. Picot, and J. L. Touraine. 1999. Intestinal microsporidiosis occurring in two renal transplant recipients treated with mycophenolate mofetil. Transplantation 68:699-707.
19. Gumbo, T., I. T. Gangaidzo, S. Sarbah, A. Carville, S. Tzipori, and P. M. Wiest. 2000. Enterocytozoon bieneusi infection in patients without evidence of immunosuppression: two cases from Zimbabwe found to have positive stools by PCR. Ann. Trop. Med. Parasitol. 94:699-702.
20. Kucerova, Z., H. Moura, G. J. Leitch, R. Sriram, C. Bern, V. Kawai, D. Vargas, R. H. Gilman, E. Ticona, A. Vivar, and G. S. Visvesvara. 2004. Purification of Enterocytozoon bieneusi spores from stool specimens by gradient and cell sorting techniques. J. Clin. Microbiol. 42:3256-3261.
21. Lopez-Velez, R., M. C. Turrientes, C. Garron, P. Montilla, R. Navajas, S. Fenoy, and C. del Aguila. 1999. Microsporidiosis in travelers with diarrhea from the tropics. J. Travel Med. 6:223-227.
22. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.
23. Mansfield, K. G., A. Carville, D. Hebert, L. Chalifoux, D. Shvetz, K. C. Lin, S. Tzipori, and A. A. Lackner. 1998. Localization of persistent Enterocytozoon bieneusi infection in normal rhesus macaques (Macaca mulatta) to the hepatobiliary tree. J. Clin. Microbiol. 36:2336-2338.
24. Mansfield, K. G., A. Carville, D. Shvetz, J. MacKey, S. Tzipori, and A. A. Lackner. 1997. Identification of an Enterocytozoon bieneusi-like microsporidian parasite in simian-immunodeficiency-virus-inoculated macaques with hepatobiliary disease. Am. J. Pathol. 150:1395-1405.
25. McWhinney, P. H., D. Nathwani, S. T. Green, J. F. Boyd, and J. A. Forrest. 1991. Microsporidiosis detected in association with AIDS-related sclerosing cholangitis. AIDS 5:1394-1395.
26. Metge, S., J. T. Van Nhieu, D. Dahmane, P. Grimbert, F. Foulet, C. Sarfati, and S. Bretagne. 2000. A case of Enterocytozoon bieneusi infection in an HIV-negative renal transplant recipient. Eur. J. Clin. Microbiol. Infect. Dis. 19:221-223.
27. Mollenhauer, H. H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39:111-114.
28. Pol, S., C. Romana, S. Richard, F. Carnot, J. L. Dumont, H. Bouche, G. Pialoux, M. Stern, J. F. Pays, and P. Berthelot. 1992. Enterocytozoon bieneusi infection in acquired immunodeficiency syndrome-related sclerosing cholangitis. Gastroenterology 102:1778-1781.
29. Pol, S., C. A. Romana, S. Richard, P. Amouyal, I. Desportes-Livage, F. Carnot, J. F. Pays, and P. Berthelot. 1993. Microsporidia infection in patients with the human immunodeficiency virus and unexplained cholangitis. N. Engl. J. Med. 328:95-99.
30. Rabodonirina, M., M. Bertocchi, I. Desportes-Livage, L. Cotte, H. Levrey, M. A. Piens, G. Monneret, M. Celard, J. F. Mornex, and M. Mojon. 1996. Enterocytozoon bieneusi as a cause of chronic diarrhea in a heart-lung transplant recipient who was seronegative for human immunodeficiency virus. Clin. Infect. Dis. 23:114-117.
31. Sandfort, J., A. Hannemann, H. Gelderblom, K. Stark, R. L. Owen, and B. Ruf. 1994. Enterocytozoon bieneusi infection in an immunocompetent patient who had acute diarrhea and who was not infected with the human immunodeficiency virus. Clin. Infect. Dis. 19:514-516.
32. Schwartz, D. A., D. C. Anderson, S. A. Klumpp, and H. M. McClure. 1998. Ultrastructure of atypical (teratoid) sporogonial stages of Enterocytozoon bieneusi (Microsporidia) in naturally infected rhesus monkeys (Macacca mulatta). Arch. Pathol. Lab. Med. 122:423-429.
33. Sheoran, A. S., X. Feng, S. Kitaka, L. Green, C. Pearson, E. S. Didier, S. Chapman, J. K. Tumwine, and S. Tzipori. 2005. Purification of Enterocytozoon bieneusi from stools and production of specific antibodies. J. Clin. Microbiol. 43:387-392.
34. Sing, A., K. Tybus, J. Heesemann, and A. Mathis. 2001. Molecular diagnosis of an Enterocytozoon bieneusi human genotype C infection in a moderately immunosuppressed human immunodeficiency virus-seronegative liver-transplant recipient with severe chronic diarrhea. J. Clin. Microbiol. 39:2371-2372.
35. Sobottka, I., H. Albrecht, J. Schottelius, C. Schmetz, M. Bentfeld, R. Laufs, and D. A. Schwartz. 1995. Self-limited traveller's diarrhea due to a dual infection with Enterocytozoon bieneusi and Cryptosporidium parvum in an immunocompetent HIV-negative child. Eur. J. Clin. Microbiol. Infect. Dis. 14:919-920.
36. Tzipori, S., A. Carville, G. Widmer, D. Kotler, K. Mansfield, and A. Lackner. 1997. Transmission and establishment of a persistent infection of Enterocytozoon bieneusi, derived from a human with AIDS, in simian immunodeficiency virus-infected rhesus monkeys. J. Infect. Dis. 175:1016-1020.
37. Van Veldhoven, P. P., E. Baumgart, and G. P. Mannaerts. 1996. Iodixanol (Optiprep), an improved density gradient medium for the iso-osmotic isolation of rat liver peroxisomes. Anal. Biochem. 237:17-23.
38. Visvesvara, G., G. J. Leitch, N. J. Pieniazek, A. J. Da Silva, S. Wallace, S. B. Slemenda, R. Weber, D. A. Schwartz, L. Gorelkin, C. M. Wilcox, et al. 1995. Short-term in vitro culture and molecular analysis of the microsporidian Enterocytozoon bieneusi. J. Eukaryot. Microbiol. 42:506-510.
39. Weber, R., H. Kuster, R. Keller, T. Bachi, M. A. Spycher, J. Briner, E. Russi, and R. Luthy. 1992. Pulmonary and intestinal microsporidiosis in a patient with the acquired immunodeficiency syndrome. Am. Rev. Respir. Dis. 146:1603-1605.
40. Zhu, X., M. Wittner, H. B. Tanowitz, A. Cali, and L. M. Weiss. 1994. Ribosomal RNA sequences of Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis: phylogenetic construction and structural correspondence. J. Eukaryot. Microbiol. 41:204-209.
41. Zhu, X., M. Wittner, H. B. Tanowitz, D. Kotler, A. Cali, and L. M. Weiss. 1993. Small subunit rRNA sequence of Enterocytozoon bieneusi and its potential diagnostic role with use of the polymerase chain reaction. J. Infect. Dis. 168:1570-1575.(Quanshun Zhang, Inderpal )