Antigens of selected Acanthamoeba species detected with monoclonal antibodies

Antigens of selected Acanthamoeba species detected with monoclonal antibodies

International Journal for Parasitology 35 (2005) 981–990 www.elsevier.com/locate/ijpara Antigens of selected Acanthamoeba species detected with monoc...

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International Journal for Parasitology 35 (2005) 981–990 www.elsevier.com/locate/ijpara

Antigens of selected Acanthamoeba species detected with monoclonal antibodies* Marian L. Turner, Emma J. Cockerell, Helen M. Brereton, Paul R. Badenoch, Melinda Tea, Douglas J. Coster, Keryn A. Williams* Department of Ophthalmology, Flinders University of South Australia, Adelaide, SA 5042, Australia Received 28 January 2005; received in revised form 14 March 2005; accepted 31 March 2005

Abstract Acanthamoeba species are ubiquitous soil and freshwater protozoa that have been associated with infections of the human brain, skin, lungs and eyes. Our aim was to develop specific antibodies to aid in rapid and specific diagnosis of clinically important isolates. Mice were variously immunised with live mixtures of Acanthamoeba castellanii strain 112 (AC112) trophozoites and cysts, or with sonicated, formalinfixed or heat-treated trophozoites, or with a trophozoite membrane preparation. Eight hybridoma cell lines secreting monoclonal antibodies reactive with A. castellanii epitopes were generated. Seven of the new antibodies (designated AMEC1-3 and MTAC1-4) were isotyped as IgMk and one (MTAC5) as IgG1k. All of the novel antibodies bound to AC112 cysts, and MTAC4 and MTAC5 also bound to trophozoites as measured by flow cytometry on unfixed cells. Single chain antibody fragments that retained parental antibody binding characteristics were engineered from three of the hybridomas (AMEC1, MTAC3 and MTAC4). Four monoclonal antibodies (AMEC1, AMEC3, MTAC1, MTAC3) bound reliably to unfixed cysts of clinical isolates of A. castellanii (two strains) and Acanthamoeba polyphaga (two strains), belonging to Pussard-Pons morphological group II, and to Acanthamoeba lenticulata and Acanthamoeba culbertsoni, belonging to PussardPons morphological group III. None of the antibodies bound to cysts or trophozoites of the environmental group I species, Acanthamoeba tubiashi. Antibodies AMEC1, MTAC3, MTAC4 and MTAC5 reacted with buffered formalin-fixed AC112 by immunohistochemistry, and also stained Acanthamoeba in sections of infected rat cornea and buffered formalin-fixed, paraffin-embedded infected human cornea. These antibodies may be useful in diagnosing pathogenic Acanthamoeba species in clinical specimens, provided that cysts are present. q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Acanthamoeba; Trophozoite; Cyst; Monoclonal antibody; Cornea

1. Introduction Acanthamoeba species are ubiquitous free-living protozoa capable of opportunistic infection in humans (MarcianoCabral and Cabral, 2003). The life cycle of the organism alternates between the active, proliferating trophozoite and the dormant cyst. The genus has been divided by the Pussard-Pons system into three groups according to cyst morphology. * Nucleotide sequence data reported in this paper are available in the GenBanke, EMBL and DDBJ databases under the accession numbers AY661777, AY661778, AY661779, AY661780, AY661781, AY661782, AY661783, AY661784, AY661785, AY661786, AY661787, AY661788, AY661789. * Corresponding author. Tel.: C618 204 5047; fax: C618 8277 0899. E-mail address: [email protected] (K.A. Williams).

Many species share antigenic epitopes and immunological separation of species has proved difficult (Marciano-Cabral and Cabral, 2003). Fourteen distinct Acanthamoeba nuclear 18S ribosomal DNA sequences (Rns) have been identified and the genus is now additionally divisible by genotype (Gast et al., 1996; Stothard et al., 1998). Pussard-Pons group II species are the most prevalent in clinical and environmental isolates (Walochnik, 2000), but group III species have also been implicated in human infections. Clinical isolates of Acanthamoeba have thus far been restricted to Pussard-Pons classification groups II and III; most have been genotyped as Rns T3, T4 or T11 (Daggett et al., 1985). Group I Acanthamoeba species are not known to be pathogenic. Acanthamoeba have been associated with diseases of the brain, eye, skin and lung. Acanthamoeba keratitis, a painful

0020-7519/$30.00 q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2005.03.015

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and potentially blinding condition, is the most common such infection, with contact lens wear being the primary risk factor (Seal et al., 1992; Radford et al., 2002). Early-stage Acanthamoeba keratitis closely resembles other corneal infections, particularly herpes simplex virus keratitis, leading to delays in correct diagnosis and appropriate management. Initial treatment with anti-viral therapy and corticosteroids may in fact exacerbate Acanthamoeba infections (McClellan et al., 2001). Current diagnostic techniques for Acanthamoeba keratitis rely primarily on microbiological culture and microscopic identification, the sensitivity of which has been estimated at 65% (Claerhout and Kestelyn, 1999). Histochemical (Epstein et al., 1986; Stothard et al., 1999) and PCR-based (Schroeder et al., 2001) diagnostics are also available. These techniques require biopsies, lavages or corneal scrapes to be taken, and do not provide immediate results. Confocal microscopy shows some promise as an adjunctive technique for the diagnosis of Acanthamoeba keratitis (Kaufman et al., 2004). We sought to develop a panel of antibodies that might aid in the diagnosis of Acanthamoeba infections. Eight hybridomas secreting distinct monoclonal antibodies to Acanthamoeba were developed by immunising mice with a clinical isolate of the group II species Acanthamoeba castellanii, and immunoglobulin single chain variable domain (scFv) fragments were engineered from three of these hybridomas. Characterisation of the antibodies revealed differences in binding profiles to cells of various species and strains of Acanthamoeba, including clinical isolates from two of three morphological groups. Furthermore, several of the antibodies reacted with both unfixed cells and with cells in formalin-fixed tissues, and may thus be of diagnostic value.

2. Materials and methods 2.1. Acanthamoeba cultures Amoebae strains were obtained from the Australian reference laboratory for free-living amoebae at the Australian Water Quality Centre, Bolivar, South Australia,

Australia. The species name, laboratory reference number, American Type Culture Collection (ATCC) number (where available), Pussard-Pons group assignation, Rns genotype (Stothard et al., 1998), and original source are listed in Table 1. Amoebae were grown in undisturbed axenic culture at 23 8C in proteose peptone-yeast extract-nucleic acid-folic acid-hemin (PYNFH) broth. The medium was made to the ATCC modifications (American Type Culture Collection, 1984) of the original formula of Laverde and Brent (1980) with the addition of 82 nM d-biotin, 100 IU/ml penicillin and 100 mg/ml streptomycin sulphate. Trophozoite cultures were maintained by passaging a proportion of cells into fresh medium every 4–6 days. Cysts were obtained by centrifugation of mixed cultures at 650!g for 5 min. The cell pellet was washed once in 0.9% weight/volume (w/v) sodium chloride, resuspended in Page’s amoeba saline (PAS) (Lauderdale et al., 1999) and left at 23 8C for a minimum of 7 days to promote encystment. To assess the purity of trophozoite or cyst populations, cells were mixed 1:1 with 0.1% w/v trypan blue and examined under the phase contrast microscope. Trophozoites were identified by morphology and failure to stain with trypan blue. Cysts were distinguished by characteristic morphology including the presence of a double cell wall (Fig. 1) and by staining with trypan blue. Cell populations of R98% cysts or trophozoites were used for all experiments. 2.2. Acanthamoeba preparations for immunisation of mice A. castellanii strain AC112 trophozoite suspensions were used unfixed, or heat treated at 65 8C for 15 min, or fixed in 10% buffered formalin for 15 min, or sonicated for 30 s. An extract of trophozoite plasma membrane was prepared according to a modification of a published method (Clarke et al., 1988). In brief, 1!108 trophozoites were disrupted in a Dounce homogeniser in 10 mM Tris pH 7.5 containing 0.35 M sucrose. Plasma membranes were re-extracted twice in 0.35 M sucrose in 10 mM Tris pH 6.9. Pooled supernatants were readjusted to 0.3 M sucrose in 10 mM Tris pH 6.9 and centrifuged at 750!g at 4 8C for 20 min. The pellet was resuspended in 40 ml 0.25 M sucrose in 10 mM Tris pH 6.9 and the suspension centrifuged at 590!g at 4 8C for 20 min.

Table 1 Acanthamoeba species and strains used in this study Species

AWQC no.

ATCC no.

Pussard-Pons group

Acanthamoeba tubiashi Acanthamoeba castellanii Acanthamoeba castellanii Acanthamoeba polyphaga

AC064 AC112 AC021 AC019

30867 – 30868 30873

I II II II

Acanthamoeba polyphaga Acanthamoeba lenticulata Acanthamoeba culbertsoni

AC010 AC230 AC001

30871 – 301171

II III III

AWQC, Australian Water Quality Centre; ATCC: American Type Culture Collection.

Rns genotype

Origin

Reference

T8 T4 T4 T4

Freshwater, USA Keratitis, Australia Keratitis, England Keratitis, England

T4 T5 T10

Freshwater, USA Keratitis, Australia Cell culture, USA

Daggett et al. (1985) Roussel et al. (1985) Jones et al. (1975) Nagington and Richards (1976) Page (1967) Flint et al. (2003) Culbertson et al. (1959)

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supernatants were screened by flow cytometry for reactivity against unfixed AC112 cysts and trophozoites. Selected hybridomas were subjected to two rounds of cloning at limiting dilution to achieve monoclonality. The isotype of the monoclonal antibodies was determined using an IsoStrip Mouse Monoclonal Antibody Isotyping kit (Roche Diagnostics Corporation, Alameda, CA, USA). 2.5. Collection of mouse serum Blood was collected from all immunised mice immediately after euthanasia, and from six female age-matched BALB/c mice that had been housed under identical conditions but that had not been immunised with Acanthamoeba preparations. Serum was separated and stored in aliquots at K20 8C. 2.6. Antibody fragment engineering Fig. 1. Cysts of Acanthamoeba stained with trypan blue and showing the typical double cell wall: (A) Acanthamoeba tubiashi, Pussard-Pons group I; (B) Acanthamoeba castellanii, Pussard-Pons group II; (C) Acanthamoeba lenticulata, Pussard-Pons group III. Original magnification!1000. Scale barZ5 mm.

The final pellet was resuspended in 1 ml 10 mM Tris pH 6.9 in 0.9% w/v NaCl and the protein concentration adjusted to 1 mg/ml. 2.3. Mammalian cells The human lens epithelial cell line SRA 01/04 was the kind gift of Professor V. Reddy, Kellogg Eye Center, University of Michigan, Ann Arbor, MI. Erythrocytes were separated from heparinised blood taken from an adult rat. The blood was centrifuged at 500!g for 10 min and the buffy coat removed. Erythrocytes were washed twice by centrifugation in 0.9% w/v NaCl. 2.4. Monoclonal antibody production All animal experimentation was carried out with the approval of the Flinders University Animal Welfare Committee. Eight female BALB/c mice aged 10 weeks, bred within the institution, were injected i.p. with 200 ml of antigen preparation containing 3!106 live or heat-treated or formalin-fixed trophozoites, or the sonicated fraction of 3!106 trophozoites, or 100 mg trophozoite membrane preparation, or a mixture of live trophozoites and cysts. Each preparation was emulsified with ImmunEasy Mouse Adjuvant (Qiagen Pty Ltd, Doncaster, Vic, Australia) according to the manufacturer’s instructions. Mice received identical booster injections at 2-week intervals for 6 weeks. Three days after the final injection, mice were killed and splenocytes were fused with the murine myeloma cell line P3X63Ag8.75 by addition of polyethylene glycol (Donohoe et al., 1995). Hybridomas were selected in hypoxanthine, aminopterin and thymidine medium, and culture

Whole cellular cDNA was prepared from the hybridoma cell lines using Amersham Quick-Prep mRNA isolation and First Strand cDNA synthesis kits (Amersham Biosciences, Castle Hill, NSW, Australia) according to the manufacturer’s instructions. Single chain antibody fragments (scFvs) in the orientation variable light chain (VL)-linker-variable heavy chain (VH) with a 20 amino acid linker (Gly4Ser)4 were constructed using degenerate primer mixes for the amplification of VH and VL domains from hybridoma cDNA (Plu¨ckthun et al., 1996). A PCR clamp using a small peptide nucleic acid was used to inhibit amplification of aberrant light chain from the myeloma fusion partner (Carroll et al., 1988; Cochet et al., 1999). Fragments assembled by splice-overlap-extension PCR were cloned into the bacterial expression vector pHB400, which contains a high-level expression promoter, appends a 6-histidine tag (his-tag) to the fragments, and co-expresses the periplasmic chaperonin Skp (Mavrangelos et al., 2001). Escherichia coli HB2151 (Pharmacia Biotech, Piscataway, USA) was transformed with pHB400 and colonies were cultured in medium containing 1 mM isopropyl-b-thiogalactopyranoside to induce protein expression. Colonies expressing scFv inserts were identified by colony blot detection of the histag. The soluble fraction of bacterial sonicate was analysed for expression of his-tagged protein by slot blot and for binding activity to Acanthamoeba by flow cytometry. Cultures of pHB400 clones containing inserts encoding irrelevant scFv specificities were used as negative controls for Acanthamoeba binding activity. 2.7. Antibody sequencing The coding sequences of the variable light and variable heavy domains of the monoclonal antibodies, amplified by PCR using degenerate primer mixes (Plu¨ckthun et al., 1996), were sequenced bidirectionally using an Applied Biosystems 3100 Genetic Analyser. For scFvs, plasmid

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DNA of pHB400 clones with a scFv insert was prepared. The scFv inserts were sequenced bidirectionally and compared with parental antibody sequences. 2.8. Monoclonal antibodies Monoclonal antibodies used as positive and negative controls for flow cytometry and immunohistochemistry were produced as undiluted supernatants from stationaryphase hybridomas and were supplemented with 0.02 M sodium azide. Cell line P3X63Ag8 (X63: unknown specificity; IgG1 isotype; negative control) was obtained from the American Type Culture Collection (Manassas, VA, USA); hybridoma OX-18 (anti-major histocompatibility complex class I monomorphic determinant; IgG1 isotype; positive control) and OX-38 (anti-rat CD4; IgG2a isotype; negative control) was obtained from the European Collection of Animal Cell Cultures (Porton Down, Wiltshire, UK). Hybridoma LION1 (anti-rabbit myeloid antigen; IgM isotype; negative control) was generated within the laboratory (Williams et al., 1992). 2.9. Flow cytometry Amoebae for flow cytometry were harvested by centrifugation at 650!g for 5 min, washed once in PAS supplemented with 0.02 M sodium azide (PAS-azide), counted and resuspended in PAS-azide at a density of 1! 107 cells/ml. All incubations were carried out for 30 min at 4 8C. For hybridoma screening and for testing the reactivity of monoclonal antibodies and mouse sera, aliquots of 5! 105 cells were incubated with hybridoma supernatant or with dilutions of sera in PAS-azide. Cells were washed with PAS-azide at 4 8C and incubated with an affinity purified, fluorescein isothiocyanate (FITC)-conjugated F(ab)2 fragment of sheep anti-mouse immunoglobulin (Ig) secondary antibody (Silenus, VIC, Australia) detecting all mouse Ig, or with FITC-conjugated goat anti-mouse IgG (Fc) fragment (Beckman Coulter, Fullerton, CA) detecting mouse IgG. Cells were then washed with PAS-azide at 4 8C and fixed in 4% w/v paraformaldehyde in Dulbecco’s A phosphatebuffered saline (PBS) containing 5 mM sodium azide (PBS-azide). Fluorescence intensity was measured on a Becton-Dickinson FACScan flow cytometer using a 488 nm argon ion laser. PAS-azide and isotype-matched monoclonal antibodies of irrelevant specificity (LION1, OX-38) were used as negative controls. A similar protocol was used for rat erythrocytes or SRA cells as target cells, except that PBS-azide was used in place of PAS-azide. For detection of cell-bound scFv, cells were incubated with mouse monoclonal anti-poly histidine antibody (clone His-1; Sigma), washed, incubated with biotinylated goat anti-mouse Ig (Dako-Cytomation, Glostrup, Denmark), washed, and incubated with streptavidin R-phycoerythrin conjugate (Molecular Probes, Eugene, OR). A scFv of irrelevant specificity was used as the negative control.

2.10. Infection of rat corneas with Acanthamoeba Corneas of outbred Sprague-Dawley rats bred within the institution were inoculated with 104 trophozoites of A. castellanii AC112 and 106 viable Corynebacterium xerosis as previously described (Badenoch et al., 1990). Rats were inspected daily at the operating microscope and euthanased by overdose of inhalation anaesthetic halothane once keratitis was clinically evident. Eyes were fixed in paraformaldehyde-lysine-periodate (PLP), frozen and sections cut at 8 mm on a cryostat as described elsewhere (Smith et al., 1999). 2.11. Immunohistochemistry Sections of rat cornea were incubated for 10 min at room temperature with 10% v/v normal swine serum (Commonwealth Serum Laboratories, Melbourne, Australia) in PBS, followed by the primary antibody for 18 h. Sections were washed with 0.2% w/v gelatin in PBS, incubated for 30 min with biotinylated goat anti-mouse Ig (DAKO-Cytomation) diluted 1 in 500 in PBS containing 1% v/v normal rat serum, washed, and then incubated for 30 min with horseradish peroxidase-conjugated streptavidin (DAKO-Cytomation) diluted 1 in 1000 in PBS. Sections were washed, developed for 5 min in 9 mM Tris–HCl buffer (pH 7.6) containing 40 mM sodium azide, 20 mM 3,3 0 -diaminobenzidine tetrahydrochloride, 9 mM imidazole and 0.07% v/v hydrogen peroxide (Sigma Chemical Company), and counter-stained with haematoxylin. Where scFvs were used as the primary antibody, another step was included. Sections were incubated for 30 min with mouse monoclonal anti-poly histidine antibody (clone His-1; Sigma) and washed, prior to incubation with the biotinylated secondary antibody. Acanthamoeba cells (2!104) were air dried on slides and fixed with PLP, 100% acetone, 100% methanol, 95% ethanol, buffered formalin, or 4% paraformaldehyde for 10 min at room temperature. The slides were then stained with monoclonal antibodies or scFvs as described above. Four sections of human cornea from a patient who had suffered Acanthamoeba keratitis, diagnosed by culture and histology, were available from an archival tissue block. The cornea had been removed at the time of therapeutic penetrating keratoplasty and had been fixed in buffered formalin and paraffin-embedded. Immunohistochemistry was performed essentially as above, but with normal human serum replacing the rat serum in the diluent. An archival section stained with Giemsa was also available. 2.12. GenBank accession numbers GenBank accession numbers for sequences of the variable heavy and light chain domains of five new monoclonal antibodies (AMEC2 and 3, MTAC1, 2 and 5) and three new scFvs are as follows: AMEC2 VH: AY661780, VL: AY661785; AMEC3 VH: AY661781,

M.L. Turner et al. / International Journal for Parasitology 35 (2005) 981–990

VL: AY661786; MTAC1 VH: AY661782, VL: AY661787; MTAC2 VH: AY661783, VL: AY661788; MTAC5 VH: AY661784, VL: AY661789; scFvAMEC1: AY661777; scFvMTAC3: AY661778; scFvMTAC4: AY661779.

3. Results 3.1. Production of monoclonal antibodies and scFvs to A. castellanii Eight hybridomas producing monoclonal antibodies (AMEC1-3, MTAC1-5) to cell surface epitopes of A. castellanii were generated from immunised mice. The use of a general-purpose anti-mouse Ig antibody together with an IgG-specific selective secondary antibody was found to be an efficient method of screening hybridoma supernatants for antibodies of specific isotype by flow cytometry. Seven of the new antibodies were isotyped as IgMk and one (MTAC5) as IgG1k. The patterns of reactivity of the eight antibodies with intact, unfixed A. castellanii AC112 cysts and trophozoites as determined by flow cytometry are shown in Table 2. All antibodies bound to cysts and one, MTAC4, also bound to trophozoites (Table 2, Fig. 2A, 2B). Additionally, MTAC5 bound very weakly to trophozoites. No antibodies were generated that bound to trophozoites only. None of the antibodies were reactive with rat erythrocytes or with a human lens epithelial cell line (representative histograms shown in Fig. 2C, 2D). Sequence analysis of the immunoglobulin VL and VH chain domains showed that all antibodies differed from each other. Sera from naı¨ve mice and from Acanthamoeba-immunised mice used to produce hybridomas were analysed by flow cytometry for binding to A. castellanii AC112 cysts Table 2 Reactivity of monoclonal antibodies with Acanthamoeba castellanii AC112 as determined by flow cytoometry Antibody AMEC1 AMEC2 AMEC3 MTAC1 MTAC2 MTAC3 MTAC4 MTAC5 a

Immunogena Live trophozoites, cysts Live trophozoites, cysts Live trophozoites, cysts Sonicated trophozoites Formalin-fixed trophozoites Sonicated trophozoites Heat-treated trophozoites Trophozoite membrane prepn

Isotype

Reactivity with cysts

trophozoites

IgMk

C

K

IgMk

C

K

IgMk

C

K

IgMk

C

K

IgMk

C

K

IgMk

C

K

IgMk

C

C

IgG1k

C

G

All immunising antigens were derived from A. castellanii AC112 trophozoites.

985

and trophozoites (Fig. 3). Naı¨ve mice possessed significant levels of Acanthamoeba-reactive serum antibody, with binding above background levels observed to a serum dilution of at least 1/200 for cysts and to a dilution of at least 1/50 for trophozoites when an anti-mouse Ig reagent was used for detection of all immunoglobulin subclasses, and to a dilution of at least 1/200 for cysts and 1/10 for trophozoites when an anti-mouse IgG-specific reagent was used. Immunisation with each of the Acanthamoeba preparations increased serum antibodies with binding specificity for cysts and trophozoites (Fig. 3). ScFvs (scFvAMEC1, scFvMTAC3 and scFvMTAC4) were generated from three of the new monoclonal antibodies. Sequence analysis demonstrated that the constructs had the intended scFv orientation and linker structure, and that the VH and VL domains showed complete amino acid identity with the corresponding parental sequences. Using the his-tag engineered into the constructs for detection purposes, the scFvs were found to retain antigen binding to Acanthamoeba by flow cytometry. Representative flow cytometry histograms showing reactivity of scFvMTAC4 and scFvMTAC3 with AC112 trophozoites and cysts, respectively, are presented in Fig. 2(E) and (F). Sequences of the VL and VH domains for five of the antibodies (AMEC2, AMEC3, MTAC1, MTAC2 and MTAC5) and the three scFvs (scFvAMEC1, scFvMTAC3 and scFvMTAC4) have been deposited in GenBank. 3.2. Reactivity of monoclonal antibodies to different Acanthamoeba strains and species To examine species and strain specificity, the eight new monoclonal antibodies were next tested by flow cytometry against trophozoites of a second isolate of A. castellanii and against trophozoites of other Acanthamoeba species, including two strains of another representative of PussardPons group II (Acanthamoeba polyphaga), and a representative of each of groups I (A. tubiashi) and III (A. lenticulata). MTAC4 reacted identically with trophozoites of both strains of A. castellanii but with no other species examined; none of the other antibodies reacted strongly with unfixed trophozoites of any species by flow cytometry. Antibody reactivity against cysts of different species and strains was then examined (Table 3). In all cases, reactivity to the second strain of A. castellanii (AC021) was identical with that to AC112. Six of the antibodies (AMEC1, 2 and 3, and MTAC1, 3 and 5) were also consistently reactive with cysts of both strains of A. polyphaga. The remaining two antibodies, MTAC2 and MTAC4, showed variable reactivity to cysts of this species. Five of the antibodies were consistently reactive with cysts of A. lenticulata, one antibody (MTAC2) was consistently unreactive with this species and two antibodies (AMEC2 and MTAC4) showed variable reactivity. Six of the eight antibodies were also tested

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Fig. 2. Flow cytometry histograms demonstrating reactivity of eight monoclonal antibodies with (A) cysts and (B) trophozoites of unfixed Acanthamoeba castellanii AC112, and lack of binding of MTAC1 to (C) rat erythrocytes and (D) human lens epithelium cell line SRA (red lines). Monoclonal antibodies AMEC1-3 and MTAC2-5 were similarly unreactive with rat erythrocytes and SRA cells. Flow cytometry histograms showing binding of (E) scFvMTAC4 to trophozoites of A. castellanii AC112, and (F) scFvMTAC3 to cysts of A. castellanii AC112 (red lines). An isotype-matched murine monoclonal antibody of irrelevant specificity (A, B, C, D) or scFv of irrelevant specificity (E, F) is shown in each overlay (control, blue lines).

M.L. Turner et al. / International Journal for Parasitology 35 (2005) 981–990

LION1

LION1

Fig. 3. The binding of sera from naı¨ve and immunised mice to Acanthamoeba cysts and trophozoites, measured by flow cytometry with an anti-mouse Ig secondary reagent (immune serum, naı¨ve serum) or with an anti-mouse IgG secondary reagent (immune serum IgG, naı¨ve serum IgG) on (A) Acanthamoeba castellanii AC112 cysts and (B) A. castellanii AC112 trophozoites. Data points represent the meanGS.E. of mean fluorescence intensities (MFIs) of sera from six naı¨ve mice or from eight immunised mice. The MFI of the PAS negative control was subtracted from the test MFIs (cyst PAS MFIZ3, trophozoite PAS MFIZ6 for anti-mouse Ig secondary antibody; cyst PAS MFIZ3, trophozoite PAS MFIZ3 for anti-mouse IgG secondary antibody).

against cysts of the group III species A. culbertsoni: all bound strongly. None of the eight antibodies bound to A. tubiashi cysts. 3.3. Immunohistochemistry on Acanthamoeba trophozoites and cysts, and on sections of normal and Acanthamoebainfected rat eyes Using an indirect immunoperoxidase technique, MTAC4 (the only antibody to react strongly with trophozoites by flow cytometry) exhibited strong staining of A. castellanii AC112 trophozoites (Fig. 4). Some reactivity above background was also observed with AMEC1, MTAC3 and MTAC5 on trophozoites (data not shown). MTAC4 showed strong positive staining (Fig. 4) and AMEC3 and MTAC5 showed intermediate positive staining (data not shown) of A. castellanii AC112 cysts irrespective of the method of fixation, although methanol

987

A

MTAC4

B

C

MTAC4

D

Fig. 4. Indirect immunoperoxidase staining on methanol-fixed Acanthamoeba castellanii AC112 (A, B) cysts and (C, D) trophozoites, with MTAC4 (B, D) and LION1, a negative control monoclonal antibody of IgM isotype (A, C). No counterstain was applied to the preparations. Original magnification!320. Scale barZ10 mm.

was considered the fixative of choice. The negative control antibodies were either negative (X63) or very weakly positive (LION1) on trophozoites and on cysts. Immunohistochemistry using bacterial extracts of the scFv constructs was plagued with high backgrounds resulting from binding of the secondary anti-histidine tag reagent, but each of the three scFv constructs was considered to exhibit some positive staining on cysts and scFvMTAC4 on trophozoites (data not shown). Corneas taken from rats with fulminant Acanthamoeba keratitis were PLP-fixed and compared with similarly fixed but uninfected, normal rat corneas. The positive control (OX-18) showed specific positive staining on all corneas. Seven of the eight anti-Acanthamoeba monoclonal antibodies showed little or no staining on normal rat cornea when compared with the isotype-matched negative controls (X63, LION1); considerable background staining was observed with AMEC3. All of the monoclonal antibodies showed some background staining on infected corneas, but an acceptable signal to background ratio was obtained with MTAC4 and MTAC5, and stained trophozoites were visible in sections of infected corneas (data not shown).

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Table 3 Reactivity of monoclonal antibodies to different strains and species of Acanthamoeba cysts as determined by flow cytometry Species (strain)

No. exptsa

AMEC1

AMEC2

AMEC3

MTAC1

MTAC2

MTAC3

MTAC4

MTAC5

Acanthamoeba tubiashi (AC064) Acanthamoeba castellanii (AC021) Acanthamoeba polyphaga (AC019) Acanthamoeba polyphaga (AC010) Acanthamoeba lenticulata (AC230) Acanthamoeba culbertsoni (AC001)

3

K

K

K

K

K

K

K

K

2

C

C

C

C

C

C

C

C

9

C

C

C

C

Gb

C

Gc

C

2

C

C

C

C

Gd

C

C

C

5

C

Ge

C

C

K

C

Gf

C

3

C

C

C

C

C

C

NT

NT

NT, not tested. a Number of occasions that cysts were tested. b MTAC2 reacted with A. polyphaga (AC019) on five occasions and failed to react on four occasions. c MTAC4 reacted with A. polyphaga (AC019) on four occasions and failed to react on five occasions. d MTAC2 reacted with A. polyphaga (AC010) on one occasion and failed to react on one occasion. e AMEC2 reacted with A. lenticulata on three occasions and failed to react on two occasions. f MTAC4 reacted weakly with A. lenticulata on three occasions and failed to react on three occasions.

3.4. Immunohistochemistry on sections of an Acanthamoeba-infected human eye Sections of formalin-fixed, paraffin-embedded human cornea known to contain many trophozoites and a few cysts were stained with AMEC1, MTAC3, MTAC4 and MTAC5. Strong staining of Acanthamoeba cells was observed with AMEC1, MTAC3, MTAC4 (Fig. 5) and moderate staining with MTAC5. Cysts were clearly stained by all of these antibodies, as well as some intermediate forms of Acanthamoeba. MTAC4 stained cells with the morphology of trophozoites.

4. Discussion Of eight novel monoclonal antibodies produced to A. castellanii, six (AMEC1-3, MTAC1-3) were IgM antibodies reactive with cysts only, one (MTAC4) was an IgM antibody reactive with both trophozoites and cysts, and one (MTAC5) was an IgG antibody reactive with cysts and weakly reactive with trophozoites by flow cytometric analysis. MTAC3 and AMEC1 also reacted with some intermediate forms that were not able to be identified unambiguously as ‘cysts’ or ‘trophozoites’ by immunohistochemistry on buffered formalin-fixed tissue. All antibodies reacted with native, surface-expressed epitopes, as well as with fixed cells. Each antibody showed a different amino acid sequence of immunoglobulin variable domains, and differences in patterns of reactivity with different species and strains of Acanthamoeba were also evident.

Of the seven strains of Acanthamoeba examined here, four were from clinical sources (Table 1). A fifth strain, the A. culbertsoni type strain, was isolated originally as a contaminant in tissue culture, but this species is known to be pathogenic for humans (Martinez, 1991). Based on their reactivity with cysts of different species and strains, the novel antibodies fell into two groupings. Five antibodies (AMEC1, AMEC3, MTAC1, MTAC3 and MTAC5) reacted reliably by flow cytometry with unfixed cysts of four potentially pathogenic species (five to six strains) of amoebae classified to Pussard-Pons groups II and III, but not to a group I isolate. A second grouping contained an antibody, AMEC2, which bound reliably to the group II species but variably to one of the group III species and not to a group I species, and two antibodies, MTAC2 and MTAC4, which bound reliably to A. castellanii but variably to another group II species, A. polyphaga, weakly or not at all to one species in group III, and not to a species in group I. The variable reactivity of AMEC2, MTAC2 and MTAC4 with cysts of specific strains is of interest, suggesting that the epitopes identified by these antibodies may be related to stage of encystment. Group I Acanthamoeba species are genetically very divergent from those of the other two Pussard-Pons groups, and reclassification of members of this group into a separate genus has been suggested (Stothard et al., 1998). It was thus of interest that none of the eight antibodies we generated reacted with trophozoites or cysts of a group I isolate, although a number were broadly reactive with cysts of group II and III isolates.

M.L. Turner et al. / International Journal for Parasitology 35 (2005) 981–990

Fig. 5. Indirect immunoperoxidase staining with MTAC3, MTAC4 and AMEC1 on sections of an Acanthamoeba-infected human cornea. No counterstain was applied to the preparations. The anterior surface of the cornea is on the left. (A) MTAC3; (B) MTAC4; (C) MTAC4; (D) AMEC1; (E) MTAC3; (F) Giemsa-stained section of the same cornea shown for comparative purposes. Original magnification: !120 (panels A, B, F), !320 (panels C, D, E). Scale barZ10 mm.

The finding that normal unimmunised mice showed serum reactivity to both cysts and trophozoites is suggestive of prior exposure and was not unexpected, given the ubiquity of Acanthamoeba within the environment. The preponderance of anti-cyst reactive monoclonal antibodies produced from the hybridoma fusions was, however, somewhat unexpected, given that most of the immunising preparations were derived from trophozoites. However, Acanthamoeba cysts and trophozoites do share common antigens, as previously reported (McClellan et al., 2002) and as evidenced by the generation of MTAC4 in this study. Kennett et al. (1999) immunised mice with live trophozoites and produced 43 monoclonal antibodies that recognised trophozoite surface epitopes. The reactivity of these antibodies to cysts was not discussed. However, 656 hybridoma supernatants were screened to isolate the 43 antibodies (Kennett et al., 1999). Classification and species identification of Acanthamoeba is best carried out by genetic and molecular methods (Stothard et al., 1998; Khan et al., 2002; Flint et al., 2003), but these techniques do not necessarily lend themselves well to rapid diagnosis of clinical isolates. In the context of

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clinical Acanthamoeba infections, antibodies reactive against both unfixed and fixed epitopes of trophozoites and cysts from potentially pathogenic species but unreactive against non-pathogenic species might be useful diagnostic tools. Cross-reactive monoclonal antibodies to A. castellanii and A. polyphaga have previously been described (Flores et al., 1990), and Acanthamoeba-specific antibody fragments have also been generated (Khan et al., 2000; Paget et al., 2000). One such antibody fragment showed binding to three reportedly pathogenic species (A. castellanii and two unidentified species) but not to reportedly non-pathogenic species (Acanthamoeba astronyxcis, Acanthamoeba royreba, Acanthamoeba palestinensis, Acanthamoeba griffini) (Khan et al., 2000). Of the new antibodies we describe, AMEC1, AMEC3, MTAC3 and MTAC5, which showed strong and consistent binding to cysts of all potentially virulent strains examined, may thus be of diagnostic usefulness provided cysts are present in the specimen. The use of a cocktail of antibodies might increase their diagnostic potential. However, AMEC3 produced background staining on normal cornea and is thus unsuitable for use on corneal sections. MTAC4, which is reactive with trophozoites of A. castellanii, and with cysts of A. castellanii and some other clinical isolates, exhibited particularly strong, clear reactivity on unfixed and fixed cells by immunohistochemistry and may have diagnostic potential for buffered formalin-fixed sections.

Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, the Ophthalmic Research Institute of Australia, and the Flinders Medical Centre Foundation. The authors thank Bret Robinson from the Australian Water Quality Centre, Bolivar, Australia for providing reference strains of Acanthamoeba, Graham Nye and Alex Szabo from Gribbles Pathology, Wayville, Australia, for providing sections of human cornea, and Kirsty Marshall for expert technical assistance.

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