Tritrichomonas foetus: Preparation of monoclonal antibodies with effector function

ExPERIMENTALPARASITOLOGY62,266-274(1986)

Tritrichomonas

foetus:

Preparation of Monoclonal Effector Function

Antibodies

with

DONALD E. BURGESS Laboratory of Immunology, Department of Veterinary Science, Montana State UniversiTy, Bozeman, Montana 59717, U.S.A. (Accepted for publication 6 March 1986) BURGESS, D. E. 1986. Tritrichomonas foetus: Preparation of monoclonal antibodies with effector function. Experimental Parasitology 62, 266-274. Monoclonal antibodies were prepared against Tritrichomonas foetus and characterized with regard to binding and immune effector activities. Nine of 27 monoclonal antibodies which reacted with T. foetus appeared to bind to the surface of live parasites. Immunoelectron microscopy confirmed the surface binding of two of these. At least six of these surface reactive monoclonal antibodies facilitated complement mediated lysis of T. foetus and one acted as an opsonin for phagocytosis by peripheral blood bovine monocytes. Five surface reactive monoclonal antibodies identified a molecule of approximately 150,000 relative molecular weight on Western blots of whole parasite preparations. These results collectively suggest the 150,000 relative molecular weight molecule may be an important target antigen for the immune response to T. foe&s. 0 1986 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Tritrichomonasfoetus; Protozoa, parasitic; Antibodies, monoclonal; Antigens, surface; Molecular weight, relative (M,); Immunofluorescence assay, indirect (IFA); Dulbecco’s modified phosphate buffered saline (DPBS); Millonig’s phosphate buffered saline (M-PBS); Complement (C); Plastic adherent bovine mononuclear cells (BMC); Peripheral blood leukocytes (PBL); Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).

efficacy (Skirrow et al. 1985). Similar infection rates have been observed in herds Tritrichomonas foetus, an obligate proto- in Montana (Burgess, unpublished data) zoan parasite of the bovine, causes a vene- and, despite the availability of effective real disease in the female host character- drugs, T. foe&s remains prevalent in beef ized by mild endometritis and occasionally herds in the United States. Such infection pyometra and abortion (Parsonson et al. rates can have considerable impact on beef 1976). In the male bovine, T. foetus ap- herd productivity and undoubtedly causes pears to be confined almost exclusively to considerable economic loss to the U.S. the preputial cavity (Hammond and Bart- beef industry each year. lett 1943) and is usually present as an Although extensive morphological (reasymptomatic infection. Bulls may be con- viewed by Honingberg 1978) and physiosistent carriers by the age of 3 years and logical (Shorb 1964; Wang et al. 1983a,b) thus represent the main source of infection. studies on T. foetus have been published, The incidence of T. foetus appears to be little is known of the immune response to about 8% in bulls (Abbitt and Meyerholz this organism or the nature of its antigenic 1979) although extensive surveys have not components. Earlier reports, however, inbeen performed recently. In a recent re- dicate that some degree of acquired resisport, 38% of 19.5beef bulls in California tance to T. foetus develops in female bowere found to have T. foetus and two treat- vines after active infection or parenteral ments with ipronidazole resulted in 100% immunization (Andrews 1938; Morgan 266 0014-4894/86$3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in my form reserved.

Tritrichomonasfoetus:

EFFECTORMONOCLONALANTIBODIES

1947; Honigberg 1978). More recently, Clark et al. (1983b, 1984) demonstrated successful immunization of bulls with Formalin fixed crude antigen preparations and membrane glycoprotein preparations of T. foetus administered in a mineral oil adjuvant. No data, however, were presented on the mechanism of resistance or the target antigens of the immune response in the vaccinated animals. To begin our studies of the immune response to T. foetus in the bovine, we have prepared monoclonal antibodies to a recent field isolate of T. foetus in order to define the antigens relevant to protective immunity. In this report, we describe the preparation of these monoclonal antibodies, some of which bind to the surface of this protozoan and have effector activity against this parasite. We also present data which suggests a major target of these surface reactive monoclonal antibodies is an antigen of approximately 150,000 M, MATERIALSANDMETHODS Two isolates of Tritrichomonas foetus were used throughout this study. The BP-4 strain, isolated by Diamond in 1956 at the University of Maryland, was obtained from the American Type Culture Collection (ATCC 30003; Rockville, MD, USA). A Montana strain of T. foetus, isolated in 1984 from a bovine uterus submitted to the Veterinary Diagnostic Laboratory, Montana State University and provisionally designated MT84-685, was also used in several experiments and to prepare monoclonal antibodies. Isolates were grown at 37 C in T-25 plastic tissue culture flasks (Corning Glass, Corning, NY, USA) and passaged at 3 to 4 day intervals in Diamond’s medium (Diamond 1957), containing 10% newborn bovine serum (v/v) and 20 p,g/ml of gentamycin sulfate. Monoclonal antibodies were generated as follows. Female BALB/ByJ mice (Jackson Laboratories, Bar Harbor, ME, USA), 6-12 weeks old, were immunized with approximately 10’ T. foetus (MT84-685) mixed 1:l with complete Freund’s adjuvant by intraperitoneal injection. Antibody titers were determined for test bleedings from each mouse and compared to preimmunization serum samples by an IFA as described previously (Burgess et al. 1984) using acetone fixed slides of T. foetus and fluorescein-conjugated, goat IgG anti-mouse IgG (H&L chain), (F-anti-mouse Ig, Cooper Biomedical, Malvern, PA, USA). When

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antibody titers fell to the prebleed serum titers, mice were boosted by intravenous inoculation of 2 x IO7T. foetus equivalents of frozen thawed (6 cycles, liquid nitrogen: 37 C) antigen in DPBS pH 7.2 (GIBCO, Grand Island, NY, USA) 3 days prior to fusion by methods published previously (Kohler and Milstein 1975; Galfre et al. 1977; Trucco et ai. 1978). Primary culture supernates were screened by the IFA, positive cultures were selected, and hybridoma clones were prepared by limiting dilution. Positive clones were selected by testing the supemates by the IFA and expanded in 75 cm2 flasks (Corning). Supemates were harvested by centrifugation (SOOg,10 min, 4 C) and precipitated by 50% saturated (NH&SO, overnight at 4 C. The precipitates were dissolved in distilled water (10% of the original supernate) and dialyzed against several changes of 50 vol of DPBS at 4 C. Concentrated monoclonal antibodies were stored at - 70 C or at 4 C with 0.01% NaNN,added as preservative. To assess surface binding of monoclonal antibodies to T. foetus, live T. foetus were exposed to the desired dilution of each monoclonal antibodies in Diamond’s medium for 30 min at 37 C. Treated T. foetus were washed in DPBS 3 x by centrifugation (4OOg,10 min, 20 C) and resuspended in DPBS to the original volume. An equal volume of 4% Formalin in DPBS was added and the mixture incubated 2 hr at room temperature. Each suspension was then washed 2 x in DPBS, resuspended in DPBS, and F-anti-mouse Ig (preadsorbed with 10’ washed, Formalin fixed T. foetusiml) was added to a final dilution of 1:20. This mixture was incubated at room temperature for 15 min, washed twice in DPBS, and the samples were mounted as described by Osborn and Weber (1982) and examined by fluorescence microscopy. For immunoelectron microscopy, samples were treated similarly except that, after monoclonal antibody treatment, T. foe&s samples were washed in 0.85% NaCl, lightly fixed for 40 min at room temperature in 0.2% glutaraldehyde in M-PBS, then exposed to undiluted ferritin conjugated goat anit-mouse yglobulin (Fe-anti-mouse Ig;IA-2306, E-Y Labs, San Mateo, CA, USA) for 30 min at room temperature. Samples were washed in M-PBS, pelleted, fixed in 2.5% glutaraldehyde overnight, and processed for transmission electron microsocopy as described previously (Speer et al. 1983). To asses the effector capability of monoclonal antibodies, three methods were used: (1) C mediated lysis, (2) agglutination, and (3) adherence/phagocytosis by bovine monocytes. To test the ability of monoclonal antibodies to facilitate C mediated lysis and agglutination, T. foetus were suspended in serial dilutions of each monoclonal antibody in a flat bottomed 96-well plate (Costar) in Diamond’s medium and incubated at 37 C for 30 min. After incubation, each well was evaluated for the presence of agglutina-

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DONALD E. BURGESS

tion of live organisms according to the number of T. foetus per clump: O-3, negative; 4-10, I; >lO, +. These plates were then washed 2~ in DPBS, and a l/10 dilution of guinea pig C absorbed with T. foetus or unabsorbed rabbit C (Cappel Labs) was added. Plates were reincubated and examined at 30 min, 1 hr, and 3 hr. Controls included wells without additives or with only C added. The plates were examined by inverted phase contrast microscopy, and the presence of C mediated lysis was indicated by large numbers (>.50% of the organisms present) being rounded up and immobile. Neither organisms without added C, nor those with added C without monoclonal antibodies, showed any evidence of damage as they were morphologically normal and quite motile throughout the assay. To evaluate the opsonizing ability of the monoclonal antibodies, T. foetus were treated as described above for agglutination except at nonagglutinating dilutions then washed in DPBS once, then RPM1 1640 containing 10% fetal bovine serum (supplemented RPMI), and exposed to cultures of BMC established 24 hr earlier from a virgin bull. The BMC were prepared by isolation of PBL on ficoll-sodium ditrizoate (Histopaque, Sigma Chemical Company, St. Louis, MO, USA) cushions as described previously (Baker and Knoblock 1982), cultured in S-well culture chamber slides (Lab Tek, Miles Scientific, Naperville, IL, USA) for 24 hr followed by washing 3 x in supplemented RPM1 immediately prior to exposure to either T. foetus treated with monoclonal antibodies or untreated T. foe&s. At 30 min, 1 hr, or 3 hr of incubation at 37 C after addition of T. foe&s, slides were removed, observed by inverted phase contrast microscopy, washed 5 x with warm DPBS, fixed and stained with Giemsa’s stain. Slides were further evaluated by bright field microscopy. Adherence was scored by the presence of numerous live T. foetus tightly bound to BMC and as illustrated by Fig. 3. Little or no adherence was observed in BMC cultures containing untreated T. foetus. The molecules to which selected monoclonal antibodies were bound were identified by separation of antigens by SDS-PAGE as described by Laemmli (1970) followed by electrophoretic transfer onto the nitrocellulose (Towbin et al. 1979) and probing of the nitrocellulose by a modified, solid phase ELISA (Johnson et al. 1984) as described (Burgess and Jerrells 1985). By comparison to the molecular weight standards (B-galactosidase, bovine serum albumin, and ovalbumin; Sigma), M, were estimated.

(data not shown). A total of nine bound to the surface T. foetus as assessed by the live IFA (Fig. 1, Table I). No more than 50% of the T. foetus organisms in the MT84-685 isolate, however, displayed fluorescence when treated with any one of the 27 monoclonal antibodies or surface reactive monoclonal antibodies (Fig. 1A). These results suggest considerable phenotypic heterogeneity of the expression of epitopes among individual organisms in this isolate. In similar experiments the binding of two monoclonal antibodies, 1.5C2.3 and l.lA3.1, was examined by immunoelectron microscopy. After exposure of live T. foe&s to 1X2.3, binding was evident from the heavy ferritin deposits on the cell surface (Fig. 2A) as compared to the control with no antibody present (Fig. 2B). Similar results also occurred with l.lA3.1 (data not shown) which is also surface reactive by live IFA (Table I). To assess the effector function of surface reactive monoclonal antibodies, their ability to cause agglutination, C mediated lysis, and to facilitate cytoadherence/ phagocytosis by BMC was examined. Three monoclonal antibodies agglutinated live T. foe&s and six monoclonal antibodies caused C mediated lysis of T. foetus (Table I) as evidence by clumping or rounding up of the cells and loss of motility, respectively (assessed by phase contrast microscopy). Both guinea pig C absorbed with T. foetus and unabsorbed rabbit C could lyse treated T. foetus but did not affect cell morphology or mobility in the absence of antibody. Phase contrast observation of cultures of 24 hr adherent BMC exposed to T. foe&s treated with monoclonal antibody for 3 hr followed by washing revealed tightly adherent, live T. foetus (data not shown). RESULTS Subsequent fixation and staining of these The supernatants of a total of 36 cloned cultures with Giemsa’s stain confirmed hybridomas were tested for monoclonal an- these observations as shown in Fig. 3 for tibodies, and 27 were positive by the IFA 1.lA3.1 which illustrates a T. foe&s organism tightly bound to BMC (arrow). on acetone fixed Tritrichomonas foe&s

Tritrichomonasfoetus:

EFFECTORMONOCLONALANTIBODIES

I3~. 1. Micrographs of Tvitrichomonas foetus after treatment (live) with monoclonal antiba Idy 1.1A3.1 followed by F-anti-mouse Ig: Phase contrast (A) and fluorescence (B) of the same microsccw tie1d. Agglutinated organisms are clearly fluorescent while free organisms (arrow) are not. Bar in dicat es 40 urn.

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DONALD E. BURGESS TABLE I Summary of Monoclonal Antibody Reactions with Tritrichomonas foetus

Hybridoma

Live IFA”

l.lA3.1 l.lA3.3 1.1Bl.l l.lB1.3 1.3A3.1 1.3A3.4 1x2.2 1X2.3 1.8C6.1

+ + + + + + + + +

Live agglutination

Cb Mediated lysis

Apparent M,” of antigen (X 103)

+ +

+ + +

1.50 150

+

+ + !I

1.50 150 150

n IFA = indirect immunofluorescence. b C = complement. c M, = relative molecular weight.

reacted with the surface of live T. foetus as detected by IFA (Fig. 1) and immunoelectron microscopy (Fig. 2). In addition to binding data, studies on the functional activity of selected monoclonal antibodies suggested that bovine peripheral monocytes can interact with monoclonal antibody treated T. foetus and adhere tightly or phagocytose this protozoa (Fig. 3) and certain monoclonal antibodies can facilitate C mediated lysis of T. foe&s (Table I). The interaction of bovine leukocytes, particularly neutrophils, with T. foetus in the urogenital tract has been suggested by the reports of apparent phagocytosis of T. foe&s (Hammond and Bartlett 1945) and by the observations of Parsonson et al. (1976) of large numbers of neutrophils and macrophages present in the stratum compactum of the uterus of experimentally infected, pregnant bovines. In experimental infections of T. foetus in murine hosts, Ito et al. (1975) examined adherence/phagocytosis of T. foetus by mouse peritoneal macrophages taken from immunized mice chalDISCUSSION lenged by intraperitoneal injection of live We have prepared monoclonal antibodies T. foe&s 3 hr prior to removal of exudates against a Tritrichomonas foetus isolate des- and light and electron microscopic obserignated MT84-685 and assessed the binding vation. Phagocyte uptake of T. foetus was and effector characteristics of these anti- most likely facilitated by specific immunoglobulin, present in the immunized mice, bodies. Several monoclonal antibodies

To assess the nature of the molecule bound by surface reactive monoclonal antibodies, whole cell lysates of T. foe&s were separated by SDS-PAGE, electroblotted onto nitrocellulose, and probed by monoclonal antibodies. Figure 4 illustrates the binding of two monoclonal antibodies, 1.X2.3 and 1.3A3.4, to a molecule of 150,000 M,, whereas a third monoclonal (l.lB1.3) did not identify an antigen on the Western blot although it was surface reactive by IFA (Table I). Five monoclonal antibodies, shown to bind to the surface of live T. foetus by IFA (Table I), identified the 150,000 M, molecule on Western blots in lysates of the MT84-685 strain (Table I) and in lysates of the BP-4 strain (data not shown). Certain surface reactive monoclonal antibodies, however, did not appear to react with any antigens in Western blots of whole cells of either strain (e.g., l.lB1.3, Fig. 4, Table I for MT-685; data not shown for BP-4).

Tritrichomonasfoetus:

EFFECTORMONOCLONALANTIBODIES

FIG. 2. Electron micrographs of Tvkrichomon& foetus after treatment (live) with monoclonal antibody. 1X2.3 (A) or no antibody (B) and subseqtlent treatment with Fe-anti-mouse Ig. Note heavy deposits of ferritin on the antibody treated T. foetd11s(arrow). Bar indicates 0.5 pm.

FIG. 3. Binding of T~itrichomonas foe&s treated with monoclonal antibody (l.lA3.1) to bovine peripheral blood mononuclear cells in vitro. Note the T. foerus adjacent to the monocyte nucleus (arrow). Bar indicates 30 pm.

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FIG. 4. Binding of monoclonal antibodies to 150,000 M, antigen (arrow) on a Western blot prepared after SDS-PAGE separation (10% gel) of whole Tvitrichomonas foetus antigen preparations. Lane 1, 1X2.3; lane 2, 1.3A3.4; lane 3, 1.1Bl.l. Numbers at the left indicate that relative position of molecular weight markers (116,000, B-galactosidase; 66,000, bovine serum albumin; 45,000, ovalbumin).

which bind to the parasite and acted as an opsonin for phagocytosis. Although early studies have shown that serum antibodies can be induced by intrauterine inoculation of a saline extract of T. foetus (Kerr and Robertson 1943), and protective immunity against challenge with live T. foetus can be induced by repeated parenteral inoculation of live T. foetus (Morgan 1947), little is known of the precise mechanism of protective immunity to T. foetus in the bovine. Experimental immunization of rabbits with T. foetus or T. vaginalis resulted in specific phagocytosis in vitro of the species used for immunization suggesting the role of specific opsonins in the immune mechanism against this parasite (Stepkowski 1961, cited in Honigberg 1978). Our results (Fig. 3) clearly indicate specific antibody can mediate adherence of T. foetus to cells of the immune system such as monocytes. Since exposure of T. foe&s to bovine Ig could occur at times such as during estrus, an opsonin effect could be one effector mechanism of the immune response to T. foetus in the bovine. The role of antibody and immunocytes in

protective immunity to T. foetus, however, remains unclear and deserves further study. Whatever the mechanisms of immunity to T. foetus, the target antigen(s) are most likely surface molecules. For this reason, we have concentrated on the analysis of monoclonal antibodies which bind to the surface of T. foetus. Some of these monoclonal antibodies identified a molecule from whole cell extracts of T. foetus of 150,000 M,. (Fig. 4). These results suggest this molecule or a component of this molecule may be located on the surface of T. foetus. Further work is in progress to define additional antigens of T. foetus and to confirm the cellular location of the 150,000 M, antigen. Recently, monoclonal antibodies prepared against Trichomonas vaginalis have been described which reacted with the surface of live T. vaginalis (Torian et al. 1984). One monoclonal also reacted with internal determinants present in T. gallinae, Giardia lamblia, and T. foetus but not with the surface of any of these protozoa. This antibody identified a 62,000 M, molecule on Western blots of T. vaginalis. In a subsequent report, these authors (Connelly et al. 198.5)described a surface antigen to T. vaginalis with components of 115,000, 58,000, and 64,000 M,, purified by immunoadsorption with monoclonal antibodies and subsequently detected by polyvalent rabbit antisera on Western blots. Interestingly, these workers also noted that the surface antigen was present on some, but not all, T. vaginalis isolates. We have not detected subunits or other molecules of lower M, with any of our monoclonals (Burgess, unpublished observations) although clearly we do not have markers for all the possible surface molecules of T. foetus since no more than 50% of the T. foetus organisms in the MT84-685 strain were labeled by any one monoclonal antibody (see Results, text). These results suggest antigenic epitope heterogeneity in this

Tvitvichomonasfoetus:

EFFECTORMONOCLONALANTIBODIES

population and agree with published results with T. vaginalis (Torian et al. 1984). Thus, either the absence of this epitope(s) from the surface or intramolecular variation could account for these findings. In the former, either developmental changes in culture or the presence of at least two antigenie types could explain our results. Distinct serotypes of T. foetus have been described previously (Pierce 1949), and work is currently under way in our laboratory to determine if these monoclonal antibodies display similar binding patterns on clones of the MT84-685 strain and other strains of T. foe&s. In the latter case, a slightly different molecule, lacking this particular epitope, could be present, and we are conducting blocking assays to test this possibility by determining which monoclonal antibodies are specillc for unique epitopes. Recently, Clark and co-workers (Clark et al. 1983b, 1984) have demonstrated that bulls up to the age of 5 years can be protected from T. foetus infection by subcutaneous immunization with whole parasites or glycoproteins prepared from membrane fractions. The glycoprotein fraction was prepared by concanavalin A-Sepharose 4-B affinity chromatography and given after infection in three doses of 160 p,g each at monthly intervals to four animals. Three animals cleared their infection by 2 weeks after the second injection and remained free of infection after a subsequent challenge. These experiments clearly demonstrate that vaccination against T. foetus can cure infection and protect against reinfection. Development of a suitable immunization procedure would eliminate the need for costly drug treatment as well as reduce the economic losses caused by infertility and lengthened calving intervals (Clark et al. 1983a) due to infections with Tuitrichomonas foetus. ACKNOWLEDGMENTS The author thanks Dr. C. A. Speer, Electron Microsopy Laboratory, Department of Veterinary Science,

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Montana State University for the preparation of electron micrographs of Tritrichomonas foetus. The expert technical assistance of Mr. Kenneth Knoblock in preparing the hybridomas used in this study is greatly appreciated. I also thank Ms. Christy Bind1 and Ms. Merrie Mendenhall for excellent preparation of the manuscript. This research was supported by the Montana State University Agricultural Experiment Station, Project No. 417 and published as Journal Series No. J-1753.

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