Murine Monoclonal Antibodies Specific for Virulent Treponema pallidum

INFECTION AND IMMUNITY, June 1982, p. 1076-1085 0019-9567/82/061076-10$02.00/0

Vol. 36, No. 3

Murine Monoclonal Antibodies Specific for Virulent Treponema pallidum (Nichols) STELLA M. ROBERTSON,1 JOHN R. KETTMAN,' JAMES N. MILLER 2 AND MICHAEL V.

NORGARDl*

Department of Microbiology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235,1 and Department of Microbiology and Immunology, University of California-Los Angeles School of Medicine, Los Angeles, California 900242

Received 19 November 1981/Accepted 3 February 1982

Murine anti-Treponema pallidum (Nichols) lymphocyte hybridoma cell lines secreting monoclonal antibodies against a variety of treponemal antigens have been generated. Hybridomas isolated were of three major types: those that were directed specifically against T. pallidum antigens, those that were directed against treponemal group antigens (as evidenced by their cross-reactivity with T. phagedenis biotype Reiter antigens), and those that cross-reacted with both treponemal as well as rabbit host testicular tissue antigens. The majority (31 of 39 clones) of these anti-T. pallidum hybridomas, which produced monoclonal antibodies of mouse isotypes immunoglobulin G1 (IgGl), IgG2a, IgG2b, IgG3 or IgM, were directed specifically against T. pallidum and not other treponemal or rabbit antigens tested by radioimmunoassay. Four of these T. pallidum-specific hybridomas secreted monoclonal antibodies with greater binding affinity for "aged" rather than freshly isolated intact T. pallidum cells, suggesting a possible specificity for "unmasked" surface antigens of T. pallidum. Six anti-T. pallidum hybridomas produced complement-fixing monoclonal antibodies (IgG2a, IgG2b, or IgM) that were capable of immobilizing virulent treponemes in the T. pallidum immobilization (TPI) test; these may represent biologically active monoclonal antibodies against treponemal surface antigens. Three other hybridomas secreted monoclonal antibodies which bound to both T. pallidum and T. phagedenis biotype Reiter antigens, thus demonstrating a possible specificity for treponemal group antigens. Five hybridoma cell lines were also isolated which produced IgM monoclonal antibodies that cross-reacted with all treponemal and rabbit host testicular tissue antigens employed in the radioimmunoassays. This report describes the construction and characteristics of these hybridoma cell lines. The potential applications of the anti-T. pallidum monoclonal antibodies are discussed.

Syphilis is a chronic, complex, sexually transmitted disease of humans caused by Treponema pallidum. Little is known about the pathogenetic factors or developing immune mechanisms operative during the disease process. Both humoral (5, 6, 30, 49, 50, 54, 57-59, 61, 63) and cellmediated (3, 27-30, 46, 53) mechanisms have been implicated in acquired immunity to syphilis, but their respective roles have not yet been clearly defined. The immunogens of T. pallidum responsible for eliciting protective immunity in both the rabbit animal model (34, 40) and humans (9, 31, 61, 62) have thus far evaded precise identification. Recently, Baseman and Hayes (4) and Alderete and Baseman (2) have characterized the cell surface of T. pallidum by employing radioimmunoprecipitation systems using conventional multivalent antisera (from infected

rabbits or humans) in attempts to elucidate the apparent major immunogenic determinants of virulent treponemes. This work has resulted in the application of sensitive immunological procedures for the possible identification and characterization of relevant immunogenic determinants of T. pallidum. However, it is known from the serological complexity of the humoral immune response during the syphilitic process that sera from infected rabbits or humans consists of a very large and complex array of both specific and cross-reacting antibodies directed against a multitude of treponemal determinants (37, 41, 43, 45, 56). Thus, there are limitations to the use of such antisera for the possible affinity purification of specific T. pallidum immunogens of interest. Because the specific T. pallidum immunogens that remain unidentified and uncharacterized may hold the key to possible vac-

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cines for the control of syphilis, it is therefore desirable to design contemporary analytical systems with the potential to isolate immunogens that can be used for the elucidation of the host immune response mechanisms involved in protective immunity. Lymphocyte hybridoma technology (21, 22) provides a new and innovative way to circumvent major obstacles of the past and the use of multivalent antisera in antigen isolation systems. The production of monoclonal antibodies against T. pallidum is a potentially useful way of providing virtually limitless supplies of monospecific antibodies directed against a variety of treponemal antigens. Furthermore, the use of monospecific antibodies prepared in this manner offers the added potential for accurate identification of T. pallidum in the exudate lesions and tissues of patients with suspected syphilis. Because we are primarily interested in employing monoclonal antibodies against T. pallidum for (i) the identification and isolation of T. pallidum immunogens, (ii) the screening of T. pallidum recombinant DNA clones, (iii) the affinity purification of intact as well as fractionated T. pallidum cells from rabbit tissue, and (iv) the identification of T. pallidum as a diagnostic aid, we have generated a number of lymphocyte hybridomas producing monoclonal antibodies against T. pallidum (Nichols). This report describes the production and characteristics of these anti-T. pallidum hybridoma cell lines. MATERIALS AND METHODS Treponemal strains and antigens. T. pallidum (Nichols) was used for sensitizing mice and as a source of antigen in the radioimmunoassay (RIA). The organisms were cultivated in the testicles of New Zealand White rabbits, previously examined upon receipt for the absence of clinical and serological (VDRL nonreactive) evidence of Treponema paraluis-cuniculi infection; the animals were subsequently housed individually at 18 to 20'C with antibiotic-free food and water given ad libitum. Approximately 12 days after each intratesticular injection of 2 x 107 T. pallidum organisms in 1 ml of serum-saline extraction medium (0.01 M sodium phosphate buffer, pH 7.2, 0.85% [wt/vol] NaCl, 50%o [vol/vol] heat-inactivated [56°C, 30 min] normal rabbit serum) per testicle, treponemes were extracted aerobically from minced testicles by rotary shaking at 180 rpm (23°C) for 30 min using 10 ml of extraction medium per testicle. Gross rabbit testicular tissue debris was removed from the treponemal suspension by centrifugation twice at 500 x g for 10 min at 23°C in a 50-ml conical polypropylene centrifuge tube. Treponemes in suspension were then examined for motility, concentrated by centrifugation at 16,000 x g for 20 min, suspended in phosphate-buffered saline (PBS) (0.01 M sodium phosphate buffer-0.85% NaCl, pH 7.2), and enumerated by dark-field microscopy (39). This isolation procedure decreased the likelihood of removing any relevant antigens that may be "loosely bound" (55). "Fresh" treponemes were

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used immediately upon isolation to prepare RIA plates, which could be stored at -70°C. "Aged" treponemes were produced by storing suspensions of T. pallidum in PBS at 4°C for 7 days before binding to microtiter plate wells for RIA. T. phagedenis biotype Reiter (Reiter treponeme) was used in an RIA to identify antibodies that may be directed against Treponema group antigens. The organisms were maintained at 23°C and transferred monthly in BBL Thioglycollate Medium 135C (without indicator; BBL Microbiology Systems) containing 10%o (vol/vol) heat-inactivated (56°C, 30 min) sterile bovine serum (P. A. Hanff and R. M. Smibert, personal communications). Cells for use in RIA were cultivated for 5 days at 35°C in large screw-capped tubes containing 40 ml of BBL Spirolate Broth (BBL Microbiology Systems) supplemented with 4 ml of sterile bovine serum (total volume of 44 ml per tube). Cells from five tubes were collected by centrifugation, washed twice by centrifugation in 40 ml of sterile PBS, and suspended in PBS at a cell density of approximately 108 cells per ml as determined by dark-field microscopy. A rabbit testicular antigen extract for RIA was prepared from the testicles of normal, uninfected, VDRL-nonreactive rabbits for the detection of monoclonal antibodies that cross-reacted with rabbit antigens or were directed against contaminating rabbit host antigens present in T. pallidum suspensions (used for the sensitization of mice). Testicles were minced and extracted in serum-saline medium as described for T. pallidum, but centrifuged only once for 7 min at 250 x g, yielding a turbid supernatant. Ten microliters of this rabbit antigen preparation was used per microtiter plate well in the RIA. Protein assays (26) indicated that 10 ,ul of this preparation contained about 120 ,ug of total rabbit protein and that 50% of this total protein was due to the normal rabbit serum in the serum-saline extraction medium. Preparations stored for up to 6 months at -20°C appeared to react equally as well as freshly prepared extracts in the RIA. Sonicated suspensions of T. pallidum or T. phagedenis biotype Reiter were also used as RIA antigen to detect antibodies directed against either masked, subsurface, or cytoplasmic treponemal antigens. Treponemal suspensions of approximately 108 cells per ml in PBS were sonicated for a total of 3 min (6 min at 50% pulse) (on ice), using a stepped microtip and a Branson Sonifier model W350 set for an output of 4 (about 40 W) (29; S. A. Lukehart, personal communication). Ten microliters of these sonic extract suspensions was used per microtiter plate well in the RIA. Unused suspensions were stored at -20°C. Mouse plasmacytoma cell line. The mouse plasmacytoma cell line SP2/0-Agl4 (SP2/0), a hybrid cell line derived from SP2/HGLK, which is formed from the hybridization between a BALB/c spleen cell and the myeloma cell line X63/Ag8 (52), was the cell line used in this study. SP2/0 synthesizes no immunoglobulin heavy or light chains (52), is resistant to 20 pFg of 8azaguanine per ml, and is killed in hypoxanthineaminopterin-thymidine (HAT) medium (24). SP2/0 cells were maintained in vitro in Dulbecco modified Eagle medium with high glucose (DMEM) (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 15% (vol/vol) fetal calf serum (Hy-Clone FCS; Sterile Systems, Logan, Utah), 2 mM glutamine, and 50 U of penicillin/streptomycin (GIBCO) per ml.

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Immunization of mice as a source of T. pallidumsensitized lymphocytes and preparation for hybridization. Adult female BALB/c mice (6 to 8 weeks old) were purchased from Jackson Laboratories (Bar Harbor, Maine) and used as a source of splenic lymphocytes producing antibodies against T. pallidum antigens. Mice were initially immunized with freshly prepared T. pallidum cells in PBS (2 x 10' cells per 0.15 ml of PBS) in complete Freund adjuvant (1:1) intraperitoneally. Three additional injections of 2 x 10' freshly isolated organisms (0.3 ml of PBS, intraperitoneally) were given on days 21, 42, and 63, and a final injection of 1.2 x 10' organisms in 0.4 ml of PBS (0.2 ml intraperitoneally and 0.2 ml intravenously) was given on day 84. Three days after the last injection, spleens were removed aseptically from two mice, and the spleen cells were prepared for hybridoma cell fusion by gently teasing them with forceps to prepare a single cell suspension in DMEM. The cells were then washed three times with DMEM by centrifugation at 270 x g for 10 min at 80C. Total cell counts were determined by flow cytometry (Coulter Counter model ZF, Coulter Electronics, Inc., Atlanta, Ga.), and viability was measured by trypan blue exclusion. Cell fusions. SP2/0 plasmacytoma cells and spleen cells from the immunized mice were fused using a modification of the general procedure of Kennett et al. (21) as described in detail by Robertson et al. (51). Plasmacytoma cells were harvested from culture in the logarithmic phase of growth, pelleted by centrifugation at 270 x g for 10 min, and washed three times in DMEM. Approximately 107 SP2/0 cells and the washed spleen cells were mixed together at a ratio of 7 viable spleen cells per viable SP2/0 cell and pelleted by centrifugation. After the supernatant was removed, 0.2 ml of warm (370C) polyethylene glycol (PEG 1000; J. T. Baker Chemical Co., Phillipsburg, N.J.) (35% [wt/vol] in DMEM) was added to the pellet to cause cell fusion. The cells were then pelleted, washed in DMEM, resuspended in 25 to 60 ml of DME-Hy medium (21) at approximately 4 x 106 cells per ml, and plated under the conditions of limiting dilution (23) in 50-SSl volumes in 96-well microtiter plates (Costar Plastics, Vineland, N.J.). This fusion procedure differed from the method of Kennett et al. (21) in that the SP2/0-spleen cell mixture, polyethylene glycol, and DMEM were all kept at 370C for the fusion processes. The cell pellet was loosened with a glass rod to ensure gentle mixing of the cells, and the cells remained in polyethylene glycol for 3 min at 370C and for approximately 5 min at 8°C (during centrifugation). A twofold concentration of HAT selection medium (24) was added to each well (50 ,ul) on day 2, followed by 100 ,ul of HAT on day 4. Hybrids could be observed after 6 to 10 days. Wells which contained growing cells were subdivided into another 96-well plate; cells were grown and transferred into 24-well tissue culture plates (Costar Plastics) for maintenance. Culture supernatants were harvested for use in the screening assays for anti-T. pallidum reactivity and determination of monoclonal isotype. RIA screening assays. To each well of a Cooke 96well microtiter plate (Dynatech Laboratories, Alexandria, Va.) was added 10 ,ul of a suspension or sonic extract of T. pallidum or T. phagedenis biotype Reiter containing the equivalent of 108 cells per ml in PBS or the rabbit testicular extract described earlier. After incubation at 4°C overnight to dryness, the wells were

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washed three times each with 0.2-ml portions of PBS containing 0.02% sodium azide to remove nonadhering antigen. Due to the strong affinity of treponemal and rabbit testicular tissue antigens for polyvinyl chloride and other plastics, it was unnecessary to employ fixatives to promote binding of these antigens to the microtiter plate wells. If desired, however, 20 ,u1 of 10%'ethanol in each well, followed by evaporation to dryness at 37°C, can be utilized for fixation (64). Wells were then precoated with 0.2 ml of PBS + 1% (wt/vol) bovine serum albumin at 4°C overnight and then washed three times with 0.2 ml of PBS + azide. Plates were used on the same day or 1 day after preparation. For the RIA test, 0.05 ml of a 4-day-old hybridoma clone supernatant was added to each well containing a respective antigen preparation. After 3 h at 23°C, the wells were washed three times with 0.2 ml of PBS + azide, followed by the addition of 2.6 x 105 cpm of a mixture of affinity-purified "25I-labeled (19) rabbit antimouse immunoglobulin G (IgG; specific activity of 2.0 x 106 cpm/,ug) and IgM (specific activity of 3.7 x 106 cpm/,ug) in 0.1 ml of PBS + azide + 2% (vol/vol) horse serum per well. Binding of this probe was carried out overnight at 4°C. Wells were then washed extensively with PBS and tap water. After drying of the plates and cutting of the individual wells, counting of the wells was performed in a Nuclear Chicago model 1185 gamma counter for 0.4 min. Antisera used as positive controls in the RIA included (i) mouse anti-T. pallidum serum collected from mice used as a source of splenic lymphocytes in cell fusions and (ii) mouse anti-rabbit testicular extract serum produced by immunizing BALB/c mice with rabbit testicular extract preparations (described earlier). The mouse anti-rabbit testicular extract serum was generated by immunizing mice intraperitoneally with 0.25 ml of testicular extract plus 0.25 ml of complete Freund adjuvant on day 1 and with 0.3 ml of extract on days 30 and 51, followed by collection of the serum on day 72. Monoclonal antibody isotope assays. Mouse antibody isotypes were identified by solid-phase RIA (51). Cooke microtiter plates were coated with goat antimouse immunoglobulin. Culture supernatants were then added and incubated for 3 h at 37°C. lodinated, affinity-purified rabbit anti-mouse heavy chain-specific reagents were added to identify the isotype of antibody bound to the plate. Monoclonal antibodies in the TPI test. Mouse anti-T. pallidum serum, mouse anti-rabbit testicular extract serum, and monoclonal antibodies secreted by anti-T. pallidum hybridomas were tested for their ability to immobilize virulent T. pallidum (Nichols) in the T. pallidum immobilization (TPI) test. The TPI assay was carried out, with minor modifications, as described in the Manual of Tests for Syphilis (8); penicillinase was incorporated in the test procedure due to the presence of penicillin in the hybridoma clone supernatants. Because T. pallidum was not sensitive to streptomycin at the concentrations employed in the medium, removal of streptomycin from the hybridoma clone supernatants was unnecessary.

RESULTS Response of T. paUidum-primed mice as a source of sensitized lymphocytes. Few studies have been performed regarding the inoculation

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TABLE 1. RIA of five representative hybridoma cell lines producing monoclonal antibodies against T. palliduma 1251 counts per 0.4 min with antigens: T pallidum, Reite etr

Clone T. pallidum, T.onpalidumb T.palidum aged

soniCd

isotype Reiter, Rabbt' ~~~~~~~~~~~~~~~~~~~~A Rbbt sonic f

175 9B12 429 173 200 118 2,594 IgG1 280 243 206 219 6F6 233 2,455 IgM 187 274 207 195 200 13D4 2,220 IgG1 270 215 12D10 195 138 153 2,013 IgG2a 226 189 7E3 207 192 195 1,742 IgG3 Control antisera 229 220 298 PBS 252 175 214 NAh 201 194 167 189 225 DMEM 183 NA Mouse anti-T. pallidum' 20,049 20,691 21,274 18,433 12,399 11,424 ND' Mouse anti-rabbitk 18,314 17,973 19,872 15,826 13,646 18,799 ND a The antigen bound to RIA plates appeared to be limiting in most of these assays, since test samples could be diluted 10- to 100-fold and retain undiluted activities. b Intact, freshly isolated T. pallidum. c Intact, aged T. pallidum. d Sonic extract of T. pallidum. ' Intact T. phagedenis biotype Reiter. f Sonic extract of T. phagedenis biotype Reiter. 8 Rabbit testicular tissue extract. I NA, Not applicable. 'A 1:10 dilution in PBS of mouse anti-T. pallidum serum from sensitized mice. ND, Not done. k A 1:10 dilution in PBS of mouse anti-rabbit testicular tissue extract serum.

of mice with virulent T. pallidum. Available information suggests that T. pallidum organisms are rapidly cleared by the mouse immune system (32, 44) and that relatively large doses of T. pallidum are required for the production of significant titers of anti-T. pallidum antibodies. It was therefore necessary to monitor the serological responsiveness of mice to treponemal antigens before using their splenic lymphocytes in cell fusion experiments. The microhemagglutination test for T. pallidum antibodies (MHATP; Ames Division, Miles Laboratories, Elkhart, Ind.) was used to assess the serum reactivity of mice primed with T. pallidum antigens. The MHA-TP rather than the fluorescent treponemal antibody absorption test was employed because of its greater technical simplicity and high degree of sensitivity and specificity observed in the experimental rabbit infection (56). Sera from normal mice and rabbits as comparative controls were MHA-TP nonreactive. Sera from rabbits infected with T. pallidum for 9 months and immune to homologous intradermal challenge exhibited MHA-TP titers of 1:20,480, whereas those from mice primed with T. pallidum and used as a source of lymphocytes for cell fusions yielded titers of 1:1,280. The schedule described for the inoculation of BALB/c mice used as a source of splenic lymphocytes for somatic cell hybridizations yielded the most efficient cell hybridization frequencies. Hybridization frequencies using spleen cells derived from mice with fewer booster injections or

booster injections given over shorter periods of time were poor. Furthermore, hybridomas arising in these experiments produced mainly IgM antibodies and were found to be highly crossreactive or directed primarily against rabbit antigens and not T. pallidum determinants, as determined by RIA (data not shown). Detection of hybridomas secreting monoclonal antibodies against T. pallidum. A total of 39 hybridoma cell lines were identified by RIA as secretors of monoclonal antibodies that reacted with T. pallidum antigens. Reactivity in the RIA was considered positive if 1251 counts per 0.4 min were above 600 (background counts generally were in the order of 200 to 300). On this basis, the monoclonal antibodies arising from anti-T. pallidum hybridomas could be grouped into three major categories. One group of monoclonal antibodies was directed specifically against T. pallidum antigens; among these were monoclonal antibodies also capable of immobilizing virulent T. pallidum organisms in the TPI test. Another group of monoclonal antibodies reacted with both T. pallidum and T. phagedenis biotype Reiter antigens and therefore was apparently directed against treponemal group antigens. A third group of monoclonal antibodies was capable of binding both treponemal and rabbit host testicular tissue antigens and thus was highly cross-reactive with all antigens tested in the RIA. Table 1 presents an example of the RIA protocol used to screen anti-T. pallidum hybrids. The

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TABLE 2. RIA of anti-T. pallidum hybridomas producing monoclonal antibodies against aged, intact treponemesa

Clone

T. pallidum

1251 counts per 0.4 min with antigens:

T. pallidum, aged

11E3 366 1,610 212 8G2 1,031 364 778 5C9 7D7 610 629 a Abbreviations and controls as in Table 1.

data shown are from

T. pallidum, sonic

Reiter

Reiter, sonic

Rabbit

Antibody isotype

1,047 2,223 1,343 1,241

268 154 242 191

381 136 426 215

198 160 275 300

IgG2a IgG1 IgM IgM

of several typical RIA

ed that appeared to produce monoclonal antibodies with greater affinity for possible surface istic of the majority of clones producing mono- determinants of T. pallidum (Table 2). Monocloclonal antibodies specifically against T. palli- nal antibodies from these clones possessed reladum. Monoclonal antibodies from such clones tively high reactivity with both intact, aged as those described in Table 1 did not react well treponemes and sonic extracts of T. pallidum, with intact treponemes. However, they ap- but not with freshly isolated treponemes, with peared to bind preferentially to sonicated anti- one possible exception, clone 7D7. Little or no gens of T. pallidum, suggesting that they may be cross-reactivity with other treponemal or rabbit directed against either intracellular or masked antigens was observed for these monoclonal antigens. Their specificity for T. pallidum deter- antibodies. Despite their apparent increased minants was indicated by their failure to cross- specificity for possible surface components of T. react with T. phagedenis biotype Reiter antigen pallidum, these monoclonal antibodies were also preparations and with rabbit testicular tissue nonreactive in the TPI test. antigens; these monoclonal antibodies were of Table 3 shows RIA data on anti-T. pallidum mouse classes IgG and IgM and were not reachybridoma cell lines producing monoclonal antitive against T. pallidum in the TPI test. The bodies capable of reacting against T. pallidum in polyclonal antisera preparations (mouse anti-T. the TPI test. These clones secreted antibodies of pallidum and mouse anti-rabbit testicular tissue) the mouse isotypes which can fix complement. used as positive controls in the RIA were strong- The antibodies did not react very well, however, ly positive against all antigens tested. This was with either freshly isolated intact or aged intact expected because the mouse anti-T. pallidum treponemes. This observation was surprising in antibodies were derived from mice sensitized view of the reactive TPI test data. with T. pallidum suspensions containing both Because of the multitude of antigenic similartreponemal group antigens (that cross-react with ities known to exist between members of the antigens of T. phagedenis biotype Reiter) as well genus Treponema (41-43, 47, 48) it was not as contaminating rabbit host antigens. Similarly, unexpected that we could generate hybridoma mouse anti-rabbit testicular antibodies also recell lines producing monoclonal antibodies that acted with treponemal antigens due to the pres- cross-reacted with both T. pallidum and T. ence of shared antigens common to both rabbit phagedenis biotype Reiter antigenic determihost tissues and treponemes (41). nants. However, only 3 of 39 anti-T. pallidum Four hybridoma cell lines secreting antibodies hybridomas isolated possessed this characterisspecifically against T. pallidum were also isolat- tic (Table 4). one

tests performed and represent results character-

TABLE 3. RIA of anti-T. pallidum hybridomas secreting monoclonal antibodies reactive in the TPI testa 1251 counts per 0.4 min with antigens: Antibody T. pallidum, sonic Reiter Rabbit T. pallidum, aged T.Cloneistp isotype pallidum Reiter, sonic 215 489 295 401 848 3G5 3,189 IgM 546 230 211 252 397 13C6 2,262 IgG2a 328 213 206 420 469 5A7 1,269 IgM 239 205 448 229 13G10 297 1,199 IgG2a 244 331 249 293 4H7 404 1,164 IgG2b 243 263 344 588 360 13C8 1,114 IgG2b a Abbreviations and controls as in Table 1.

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TABLE 4. RIA of anti-T. pallidum hybridomas secreting monoclonal antibodies that cross-react with T. phagedenis biotype Reitera Clone

T. pallidum

per 0.4 min with antigens: '25I countsT. pallidum, sonic Reiter

450 271 463 264 316 459 a Abbreviations and controls as in Table 1.

13A7

iB11 12G11

603

Reiter, sonic 765

Rabbit 128

522 577

363 819

259 211

T. pallidum, aged

1,942 1,686 1,218

TABLE 5. RIA of hybridomas secreting monoclonal antibodies that bind T. pallidum, T. Reiter, and rabbit testicular antigens 1251 counts per 0.4 min with antigens: Cloneistp T. T. pallidum, aged T. pallidum, sonic pallidum Reiter Reiter, sonic il1 1,773 2,209 4,465 2,201 2,174 12F4 1,220 1,274 3,492 2,352 2,255 689 1870-5A5 1,083 2,521 1,449 1,354 627 6A9 958 706 1,287 1,339 976 772 308 486 581 12E3 a Abbreviations and controls as in Table 1.

A class of hybridoma cell lines was also isolated that produced monoclonal antibodies found to cross-react with all treponemal and rabbit testicular antigens employed in the RIA (Table 5). Because of the limiting dilution procedure employed (23) and the fact that only one antibody isotype was observed for each cell line (except one clone 12H4), it is unlikely that these hybridoma cell lines represent more than one hybridoma clone growing together in culture. It may be of interest that all of these highly crossreactive antibodies were of the IgM class of antibody. It appeared that a greater degree of anti-T. pallidum specificity was exhibited by IgG-seTABLE 6. Summary of monoclonal antibody characteristics from 39 anti-T. pallidum hybridomas No. of clones with antibody specificitiesa: Mouse antibody T. pallidum T. pallidum T. pallidum + Reiter isotype + Reiter + rabbit +Reiter 2 5 7b IgM 2 0 0 IgG3 IgGI 0 0 liC IgG2b 1 3b 0 0 7b,c 0 IgG2a IgGI, IgG2bd 1 0 0 a Abbreviations as in Table 1. b Two of these clones were reactive in the TPI test. c One of these clones was reactive in the MHA-TP test but unreaction in the TPI test. d Clone 1939-12H4 (double producer).

Antibody isotype

IgM IgM IgG2b

phagedenis biotype Antibody Rabbit

isotype

1,676 1,685 1,964

IgM IgM IgM IgM IgM

982 700

creting clones than by those clones secreting IgM. Table 6 summarizes the isotypes of all murine anti-T. pallidum monoclonal antibodies, their specificities, and their frequency of isolation in this study. A majority (24 of 31 clones) of the hybridomas isolated that reacted only against T. pallidum antigens were of IgG subclasses, compared to seven IgM clones with similar properties. Of eight total clones that cross-reacted with either T. phagedenis biotype Reiter or rabbit testicular antigens, seven of these were IgM producers.

DISCUSSION This report describes the isolation and characterization of 39 lymphocyte hybridoma cell lines producing monoclonal antibodies directed against T. pallidum antigens. These hybridomas were of three major types: those that were directed specifically against T. pallidum antigens, those that were directed against treponemal group antigens (as evidenced by crossreactivity with both T. pallidum and T. phagedenis biotype Reiter antigens), and those that cross-reacted with all treponemal and rabbit host tissue antigens employed in the RIA screening assay. The majority (31 clones) of anti-T. pallidum hybridomas generated were directed specifically against T. pallidum antigens. The inoculation schedule for the priming of BALB/c mice with T. pallidum antigens was found to be important. Multiple high-dose inoculations given over a substantial period of time (84 days) were necessary for both the relatively

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high titer of mouse serum MHA-TP antibodies observed and the enlargement of spleens, which yielded adequate numbers of sensitized lymphocytes for cell hybridizations. These observations are consistent with previous studies suggesting that mice respond relatively slowly to stimulation by treponemal antigens, as determined by the TPI (32), the fluorescent treponemal antibody absorption (44), and the automated microhemagglutination (44) tests. Although the MHA-TP titers of T. pallidumsensitized mouse sera were significantly lower than immune rabbit serum titers (about 20-fold), the observed mouse MHA-TP titers were considered high for mouse reactivity against T. pallidum antigens as compared to mouse anti-T. pallidum serological reactivity observed in the past (32, 44). Although little is known about the types of mouse anti-T. pallidum antibodies measured by the MHA-TP test or the relationships of mouse-generated anti-T. pallidum antibodies to those produced in the infected rabbit animal model (or in humans), the MHA-TP test was found to be useful as a general assay for monitoring the overall humoral response of mice sensitized with T. pallidum antigens. A majority of the hybridomas producing monoclonal antibodies directed specifically against T. pallidum and not other antigens (Tables 1 to 3 and 6) responded very well in the RIA to sonicated T. pallidum cells, but poorly against intact treponemes. This finding initially raised the possibility that most of the specific anti-T. pallidum hybridomas may be directed against intracellular rather than surface antigens of the T. pallidum organism. However, it has not been determined whether the sonication process releases intracellular T. pallidum components (against which the monoclonal antibodies are directed), or if sonication actually exposes or "unmasks" surface determinants of T. pallidum that are normally unexposed or coated with host proteins (1) or mucopolysaccharide (11, 12, 14, 15, 20, 25, 60, 65). Indeed, the previously observed binding of these components to the surface of T. pallidum may be an important pathogenetic mechanism that protects the organism from the immune response of the host (16, 17, 30, 33, 35, 38). It is significant that monoclonal antibodies from these clones did not react in the RIA against sonic extracts of T. phagedenis biotype Reiter. Although the treponemal crossreactivity exhibited by three clones (Table 4) supported the common antigenicity between these members of the genus Treponema, the demonstration of monoclonal antibodies from 31 clones directed against determinants unique to T. pallidum provides further support to the concept that these specific antigens may be located on or near the surface rather than in the cytoplasm of the organism. Experiments are in

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progress to assess more adequately the nature of the T. pallidum antigens liberated by sonication. Four hybridoma clones (Table 2) exhibited an increased affinity for antigens of aged, intact T. pallidum cells, suggesting that the aging process may also result in exposing cell surface determinants that subsequently were able to interact with antibody. In support of this observation, it is known that aged treponemes are more reactive in the T. pallidum agglutination, fluorescent treponemal antibody absorption, and TPI tests (17, 36; M. J. Seldeen, Sc.M. Thesis, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Md., 1953), presumably due to the possible shedding of surface coat components that may be of T. pallidum or rabbit host origin, or both (1, 2, 4, 11, 12, 14, 15, 20, 25, 60, 65). It remains possible, however, that the observed increased reactivity for these monoclonal antibodies against aged treponemes may be due to internal antigens released during treponemal lysis as a result of aging for 7 days at 4°C in PBS. Minor lysis, not readily detectable by dark-field microscopy, may provide enough antigen for reactivity in the RIA. It has been our experience, though, that T. pallidum cells are quite resistant to overt lysis under these conditions (M. V. Norgard and J. N. Miller, unpublished data). One of the clones (7D7) (Table 2) that possessed an increased affinity for aged T. pallidum cells also appeared to produce antibody that bound equally well to freshly isolated intact treponemes. This particular hybridoma clone may produce a monoclonal antibody (IgM) capable of interacting with a specific T. pallidum determinant, even in the presence of copious amounts of contaminating host components present on the surface of T. pallidum. The fact that monoclonal antibodies from clone 7D7 reacted more strongly against sonicated rather than whole T. pallidum cells may be due to the release and subsequent exposure of additional surface antigen for available binding of antibody. It was surprising that the anti-T. pallidum hybridomas producing monoclonal antibodies that were reactive in the TPI test were only moderately reactive in the RIA against whole T. pallidum organisms (Table 3). This is evident from a comparison of the data in Tables 1 and 3, and suggests that T. pallidum antigens responsible for eliciting TPI antibodies in vivo may be present only at a relatively low surface density or also may have limited exposure on the treponemal cell surface. In either case, the TPI test combines the use of guinea pig complement and specific anti-T. pallidum antibody for the immobilization of motile, virulent treponemes; it is conceivable that appropriate specificity and the ability to fix complement may be the only re-

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quirements necessary for complement-mediated immobilization, even if the specific T. pallidum antigens involved in this interaction occur infrequently on the surface of the T. pallidum cell. The demonstrated ability to isolate hybridomas producing monoclonal antibodies capable of immobilizing virulent treponemes in vitro has led to exciting possibilities, including a more accurate elucidation of the mechanism of the TPI

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the cross-reactive monoclonal antibodies described in this report arose as the result of specific rabbit antigen stimulation because T. phagedenis biotype Reiter was cultivated in medium containing bovine rather than rabbit serum. The anti-T. pallidum monoclonal antibodies from hybridomas generated in this study have several potentially important applications. Among the most significant of these applications is the use of monoclonal antibodies in radioimmunoprecipitation systems (2, 4) designed to identify and isolate the major immunogenic determinants of T. pallidum. This laboratory is also employing monoclonal antibodies in solidphase RIAs for the identification and characterization of Escherichia coli recombinant DNA clones expressing T. pallidum genes encoding for treponemal immunogens (M. V. Norgard, H.-F. Kung, and J. N. Miller, manuscript in preparation). A more practical application of these monoclonal antibodies may also be found in their ability to be used in affinity column chromatography (10) to separate and purify treponemal organisms (or their constituent components) from contaminating rabbit testicular host tissues. The efficiency of the affinity column purification method should allow for the isolation of T. pallidum organisms that retain their surface antigens even if loosely bound (1, 40). Such treponemes would be more economical to prepare while preserving their antigenic characteristics, and therefore may be useful as protective immunogens, as antigens in specific serological tests for syphilis, or as both. Additionally, the use of monoclonal antibodies for the specific identification of T. pallidum in the exudative lesions and tissues of patients with syphilis would offer the physician a diagnostic tool with the potential capability of replacing the equivocal dark-field microscope examination procedure.

test. The very fact that anti-T. pallidum monoclonal antibodies generated in this study were capable of immobilizing T. pallidum in the TPI test is of added significance. Information is accumulating to suggest it is feasible to construct lymphocyte hybridomas and isolate monoclonal antibodies that retain biological activities other than just that of antigen binding. For example, it has recently been shown by Briles et al. that IgM monoclonal antibodies raised against either type 3 pneumococcal polysaccharide or phosphocholine are protective when injected intraperitoneally before intravenous injection of virulent type 3 Streptococcus pneumoniae into mice (7). All of the anti-T. pallidum monoclonal antibodies isolated that were reactive in the TPI test in this study were of the mouse antibody isotypes capable of fixing complement. It is therefore tempting to speculate as to whether these antibodies are capable of (i) inactivating T. pallidum, as measured by the in vitro-in vivo neutralization assay (6); (ii) passive protection (5, 50, 54, 57, 59, 63); and (iii) blocking the attachment of virulent treponemes to host tissues (13, 18). The generation of anti-T. pallidum hybridomas producing monoclonal antibodies that cross-reacted with either T. phagedenis biotype Reiter or rabbit host testicular antigens, or both, was expected from the degree of shared antigens between T. pallidum and T. phagedenis biotype Reiter (42, 43, 47, 48), as well as the presence of contaminating rabbit host testicular tissue found in T. pallidum suspensions used to sensitize ACKNOWLEDGMENTS mice. There is also evidence that normal rabbit We thank J. Uhr for encouraging this work, E. Hansen and testicular tissue possesses some antigens in his staff for helpful discussions, Carolyn Frisch for performing common with T. pallidum and T. phagedenis Lowry assays, and Nancy H. Bishop and Philip A. Hanff for (41). Thus it was not unexpected to isolated their assistance in conducting the TPI assays. The skillful of R. E. Shanks, Elizabeth Coffee, and Nancv monoclonal antibodies that were reactive with assistance Wischkaemper has been appreciated. We thank D. Marcouboth T. pallidum and T. phagedenis biotype lides for careful typing of the manuscript. Reiter antigens (Table 4), or with all of the This work was partially supported by World Health Organitreponemal and rabbit testicular antigens tested zation agreements V3/181/64 and V3/181/26 to M.V.N. and respectively, and by Public Health Service grants AI(Table 5). Except in one case, all of these J.N.M., 11851, AI-12601, AI-16692, and CA-23115 from the National hybridomas secreted IgM antibodies; the impli- Institutes of Health. S.M.R. was a Fellow of the Arthritis cations of this result are unclear. There is also Foundation. no present method to determine whether the LITERATURE CITED monoclonal antibodies originally arose as a result of rabbit rather than treponemal antigen 1. Alderete, J. F., and J. B. Baseman. 1979. Surface-associated host proteins on virulent Treponema pallidum. Instimulation during the immunization of mice. Immun. 26:1048-1056. Due to the contamination of treponemes with 2. fect. Alderete, J. F., and J. B. Baseman. 1980. Surface characrabbit testicular antigens, stimulation by either terization of virulent Treponema pallidum. Infect. Imwas possible. It is unlikely, however, that any of mun. 30:814-823.

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