Production and Characterization of Monoclonal Antibodies to Exotoxin ...

Vol. 44, No. 2

INFECTION AND IMMUNITY, May 1984, p. 262-267 0019-9567/84/050262-06$02.00/0 Copyright ©3 1984, American Society for Microbiology

Production and Characterization of Monoclonal Antibodies to Exotoxin A from Pseudomonas aeruginosa D. R. GALLOWAY,* R. C. HEDSTROM, AND 0. R. PAVLOVSKIS Infectious Diseases Program Center, Naval Medical Research Institute, Bethesda, Maryland 20814 Received 21 November 1983/Accepted 28 January 1984

Hybridomas secreting monoclonal antibodies specific for exotoxin A from Pseudomonas aeruginosa strain PA103 were derived from the fusion of spleen cells from mice immunized with: (i) purified exotoxin A, (ii) Formalin-treated exotoxin A, (iii) exotoxin A covalently coupled to Sepharose 4B, or (iv) P. aeruginosa-infected mice. All hybridomas were screened and selected by using an enzyme-linked immunoadsorbent assay. All antibody isotypes were represented (immunoglobulins G, A, and M) as determined by enzyme-linked immunoadsorbent assay. The most productive fusions resulted from immunization with antigens coupled to an insoluble matrix, such as Sepharose 4B, or by infection of mice. Several hybridomas were selected and cloned by limiting dilution. The specificity of the monoclonal antibodies for exotoxin A was demonstrated by indirect immunoprecipitation of 125I-labeled exotoxin A followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and by the immunoblotting technique. The protective ability of certain monoclonal antibodies was demonstrated in vitro by toxin neutralization in tissue culture and in vivo by prolonged survival time in the burned mouse infection model, after passive immunization. One monoclonal antitoxin displayed specificity for PA103-derived exotoxin yet failed to react with exotoxin purified from PAO-PR1 or PAO1, suggesting that structural differences exist between these exotoxins.

Pseudomonas aeruginosa is an opportunistic pathogen and is a leading cause of infection in compromised hosts (1, 5, 26, 27). The virulence associated with this organism has beenl attributed to various factors, one of the most extensively studied being exotoxin A (10, 19). Similar in its mode of action to diphtheria toxin, exotoxin A is synthesized as a proenzyme that catalyzes the transfer of the ADP ribose moiety from NAD into covalent linkage with elongation factor 2. Consequently, exotoxin A is a potent inhibitor of mammalian protein synthesis (9, 10). Further understanding of the role of exotoxin A in the infectious process requires a detailed structural and functional analysis of the molecule. The production of monoclonal antibodies to exotoxin A will provide the necessary reagents required to accomplish such an analysis. Furthermore, the use of monoclonal antitoxin A in passive immunotherapy studies should provide some insight into the contribution of exotoxin A to the infectious process. Previous studies with conventional polyclonal antisera to exotoxin A have shown the importance of antitoxin A in host survival (18, 22, 23), and such antisera have been used for detection of exotoxin A. However, there are many inherent limitations to the production and use of polyclonal antisera, which tend to vary in specificity and quality. The use of monoclonal antibodies overcomes these inherent limitations and provides the means for answering questions regarding functional domains of the exotoxin A molecule. In this paper we describe the isolation and characterization of monoclonal antibodies specific for P. aeruginosa exotoxin A. MATERIALS AND METHODS Purification of exotoxin A. Exotoxin A was purified from a P. aeruginosa PA103 (13) strain variant (PA103/22) provided by S. Leppla. Exotoxin was also purified from toxin-hyperproducing recombinant strains (R. C. Hedstrom, C. R. Funk, 0. R. Pavlovskis, and D. R. Galloway, Abstr. Annu. *

Meet. Am. Soc. Microbiol. 1984, BISI, p. 42) derived from strains PA01 and PAO-PR1 produced by B. H. Iglewski (2). The protein was purified from 50 liters of culture supernatant according to the Leppla procedure (12) with the following modifications (S. Leppla, personal communication). All procedures were performed at 5°C. A 50-liter portion of culture supernatant was diluted with 150 liters of cold deionized water, and 2 liters of DEAE-cellulose (slurried in water) was added. While the suspension was vigorously stirred, 150 ml of 2.0 M HCI, diluted in 4 liters of cold water, was added. After 1 h of stirring, the DE-52 resin was allowed to settle for 2 h and then was collected and washed three times with cold 0.01 M Tris-hydrochloride, pH 8.1 (buffer A). Finally, the washed DE-52 resin was transferred to a funnel and slowly eluted with 2.5 liters of cold 0.15 M Tris-hydrochloride, pH 8.1. The first 500 ml of eluate was discarded. The remaining 2 liters was collected, and 2-mercaptoethanol was added to a final concentration of 5 mM. Exotoxin A was precipitated by the addition of solid ammonium sulfate to 75% saturation. The precipitate was redissolved ahd dialyzed overnight against several liters of buffer A containing 2 mM 2-mercaptoethanol and than applied to a 200-ml DE-52 column equilibrated with the same buffer. After elution from the DE52 column (using a linear gradient of 0.01 to 0.3 M NaCl in buffer A plus 2 mM 2-mercaptoethanol), the exotoxin A fractions were pooled and adjusted to pH 6.8 with 2 M HCI. Exotoxin was then further purified by using a column of hydroxylapatite (Bio-Rad HTP; Bio-Rad Laboratories, Richmond, Calif.) as previously described (12), except that all buffers included 2-mercaptoethanol at a final concentration of 2 mM. Protein peaks were concentrated by precipitation with ammonium sulfate, dialyzed against buffer A, and frozen at -70°C in small fractions. This preparation was used throughout the study. Immunization of mice. NMRI (20) and BALB/c (Charles River Breeding Laboratories, Wilmington, Mass.) mice were selected for hybridoma production. In this study the exotoxin was presented to the mice in several different ways: (i) purified exotoxin A, (ii) a toxoid preparation (17), and (iii)

Corresponding author. 262

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MONOCLONAL ANTIBODIES TO P. AERUGINOSA EXOTOXIN A

exotoxin covalently coupled to Sepharose 4B, as well as exposure to exotoxin through challenge with live organisms (PA220 [18]). Mice were immunized intraperitoneally (i.p.) with 20 ng of exotoxin per mouse on day 1, 30 ng per mouse on day 15, 50 ng per mouse on day 24, 150 ng per mouse on day 39, and a final intravenous boost with 150 ng per mouse on day 47. All i.p. immunizations were done with Freund incomplete adjuvant. Toxoid-immunized mice received an initial intravenous injection of 10 Rxg of toxoid plus 50 pug of N-acetylmuramylL-alanyl-D-isoglutamine (Calbiochem-Behring Corp., La Jolla, Calif.), followed by intramuscular immunizations on days 14, 28, 42, and 56. Exotoxin A was covalently coupled to Sepharose 4B as described by March et al. (14) at a concentration of 5 mg/ml. Mice were immunized i.p. with 0.2 ml of a 10% suspension of toxin-Sepharose beads in phosphate-buffered saline at weekly intervals, receiving four immunizations in total. In addition, all mice received a final booster immunization consisting of 50 ng of exotoxin A in 100 ,ul of phosphatebuffered saline with 60 ,g of N-acetylmuramyl-L-alanyl-Disoglutamine 4 days before fusion. Mice exposed to exotoxin A through P. aeruginosa infection were injected subcutaneously with a 0.1-ml saline suspension containing approximately 107 viable organisms (PA220). Ten days later spleens from surviving mice were utilized for hybridoma production. Production of monoclonal antibodies. Spleens from hyperimmunized mice were aseptically removed and dissociated into a single-cell suspension. Lymphocytes (108 cells) obtained in this manner were fused with the nonsecreting myeloma cell line SP2/0-Agl4 (108 cells) according to the methods of Fazekas de St. Groth and Scheidegger (4). Cells were fused with 50% polyethylene glycol 4000 (J. T. Baker Chemical Co., Phillipsburg, N.J.) and 5% dimethyl sulfoxide in RPMI 1640 without serum at cell ratios of 1:1. The fused cells were resuspended in hybrid selective growth medium (RPMI 1640 medium containing 10% fetal calf serum supplemented with 100 ,uM hypoxanthine, 4 ,uM aminopterin, and 3 ,uM thymidine) and seeded into 96-well microtiter plates (Costar, Cambridge, Mass.) at densities of 2 x 104 myeloma cells per well. Culture supernatants were assayed for antitoxin activity by enzyme-linked immunoadsorbent assay (ELISA) 10 to 12 days later. All initial growth and analysis cultures were carried out in culture plates containing feeder layers of normal mouse peritoneal exudate cells. Antibodyproducing hybridomas were subsequently cloned by limiting dilution on feeder layers. Clones were then grown in largescale culture and as ascitic tumors in BALB/c mice pretreated with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane), irradiated (600 rads), and inoculated i.p. with 106 to 107 hybrid cells. ELISA. Polyvinyl flat-bottom 96-well microtiter plates (Immulon-II; Dynatech Laboratories, Inc., Alexandria, Va.) were coated with 100 pul of purified exotoxin A diluted to a concentration of 500 ng/ml in coating buffer (0.1 M Na2CO3, 0.02% NaN3, pH 9.6) and allowed to incubate overnight at 37°C. The plates were then washed five times with working buffer (WB), 0.036 M borate-buffered saline (pH 7.8) containing 0.5% bovine serum albumin fraction V (CalbiochemBehring) and 0.5% Tween 20 (Sigma Chemical Co., St. Louis, Mo.). Test samples were diluted in WB and added to the coated wells in 100-pI volumes. After a 2-h incubation at 37°C, the wells were washed five times, a 1:2,500 dilution of goat antimouse immunoglobulin (Cappel Laboratories, West Chester, Pa.) in WB was added at a 100-,1u volume to each well, and the plates were incubated once again at 37°C. After

263

2 h the wells were washed five times with WB, and 100 p.1 of 1:2,500 dilution of alkaline phosphatase (Sigma Chemical Co.)-rabbit antigoat immunoglobulin conjugate (17) diluted in WB was added. After a 2-h incubation at 37°C, the plates were rinsed five times with WB and once with 10% diethanolamine. Substrate (100 p.l of 1 mg of p-nitrophenylphosphate per ml in diethanolamine [10%]) was added and the reaction was stopped after 1 h at 37°C, using 200 ,ul of 2 N NaOH. The absorbance was read at 405 nm, using a Titertek multiscan ELISA reader (Flow Laboratories, Inc., Rockville, Md.) Heavy- and light-chain antibody class determinations were carried out by ELISA analysis, using rabbit antimouse immunoglobulin typing sera and alkaline phos-

a

phatase conjugate (Litton Bionetics, Rockville, Md.) to detect antibodies bound to exotoxin A-coated plates. All assays were run in duplicate and utilized normal mouse serum as a negative control. 1251-labeling procedure. Exotoxin A was labeled with 125I (carrier-free Na125I; New England Nuclear, Boston, Mass.) by the IODO-GEN (1,3,4,6-tetrachloro-3,6-diphenylgly-

couril; Pierce Chemical Co., Rockford, Ill.) procedure according to published protocols (6, 15). Reaction products were separated with a 10-ml Sephadex G-25 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) column. In general, a specific activity of 4 x 106 cpm/,ug of protein was obtained for exotoxin A.

Indirect radioimmunoprecipitation procedure. Analysis of monoclonal antitoxin reagents was carried out with an indirect immunoprecipitation procedure utilizing 125I-labeled exotoxin A. The products were analyzed by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Aliquots of monoclonal antitoxin reagent (culture supernatant, purified antitoxin, or ascites fluid) were added to approximately 105 cpm of I251-labeled exotoxin A, brought to a final volume of 0.5 ml, and incubated overnight at 40C with gentle rotation. The 125I-labeled exotoxin-antiexotoxin conjugates were then complexed by the addition of rabbit antimouse immunoglobulin for 1 h with rotation at 4°C. Antigen-antibody conjugates were precipitated with a 10% suspension of staphylococcal protein A-Sepharose (Sigma Chemical Co.) in phosphate-buffered saline containing 1 mg of gelatin (Difco Laboratories, Detroit, Mich.) per ml and 0.02% NaN3, after a 30-min incubation with rotation at 4°C. The beads containing the immunocomplexes were collected by centrifugation and washed three to five times in 50 mM Tris-hydrochloride, pH 7.5, containing 5 mM EDTA, 150 mM NaCl, 1 mg of gelatin per ml, and 0.02% NaN3. Bound 125I-labeled antigens were eluted from the washed beads in 100 pl of SDS sample buffer (described below) by heating for 5 min at 100°C and collected from the supernatant fraction. The resultant material was analyzed by SDS-PAGE followed by autoradiography. PAGE and immunoelectrophoresis. Analysis of SDS-solubilized proteins was carried out by using a discontinuous PAGE procedure which has been previously described (29), using the discontinuous buffer system of Laemmli (11). Gels for immunoblotting analysis were processed by a procedure (7) that is a modification of the method described by Towbin et al. (28). CHO cell cytotoxicity. Chinese hamster ovary (CHO) cells (10) were cultured in RPM1 1640 medium supplemented with 10% fetal calf serum. The cells were plated in microtiter plates at a concentration of 2 x 104 cells per well. Duplicate wells were supplemented with various amounts of antitoxin depending upon experimental protocol. After the addition of antitoxin to the

suspended cells, 0.25 ,ug of exotoxin A was

264

INFECT. IMMUN.

GALLOWAY, HEDSTROM, AND PAVLOVSKIS

added to each well and the plates were incubated at 37°C in 5% CO2 for 72 h. After incubation, the plates were removed and placed at ambient temperature. The plates were then scored for surviving CHO cells by inspection or by reading the absorbance at 560 nm, using a Titertek multichannel spectrophotometer. Living cells produced a change in coloration as pH levels dropped, whereas dead cells produced no coloration change with time (10). Subtracting background absorbance resulted in a semiquantitative profile of net cell survival. In vivo protection studies. The burned mouse infection model (20), initially described by Holder and Jogan (8) and Stieritz and Holder (25), was used to study the protective effect of monoclonal antibodies. The burn was nonlethal and penetrated the musculature only slightly (20). Bacteria in logarithmic-phase growth were suspended shortly before use in saline at a desired concentration, and 0.5-ml volumes were injected subcutaneously in the burn area immnediately after the trauma. At 2 h preinfection and at 10 and 20 h postinfection mice were injected i.p. with 0.3 ml of the desired antitoxin. Control mice received 0.2 ml of undiluted, heatinactivated fetal calf serum (Hem Research, Inc., Rockville, Md.). The infected mice were observed for at least 180 h postinfection. Preparation of rabbit antitoxin. Antitoxin sera were produced in several adult albino rabbits by intravenous injection of 13 ,ug of purified exotoxin in 0.25 ml of sterile saline. Similar immunizations were made on days 4, 8, and 18 after the primary dose. A final subcutaneous injection of 25 pLg of exotoxin in Freund incomplete adjuvant followed 1 week later. The serum from each rabbit was heat inactivated (56°C, 30 min) and stored at -70°C.

a8F

TABLE 1. Fusion-specific efficienciesa .No. of No. of Immunizig antigen antigen

hybridomas tested

specific hybridomas

Specific

efficiency (%)

Exotoxin A 40 1 2.5 Toxoid 53 2 4 Exotoxin379 (69) 36 (25) 10 (40) Sepharose Infection 300 45 15 a Specific efficiencies are calculated as the ratio of specific antitoxin hybridomas to total hybridomas x 100%. Results from two separate exotoxin-Sepharose fusions are reported, as indicated by parentheses. Fusion wells = 960 for each antigen.

RESULTS Production of monoclonal antibodies. Figure 1 illustrates that infected mice recovering from a P. aeruginosa infection develop an increasing antibody titer against exotoxin A. Consequently, spleen cells from the surviving mice were used for hybridoma production. Spleen cells from mice immunized with the various preparations were fused with SP2/0-Ag14 myeloma cells at a 1:1 ratio, according to the procedures outlined by Fazekas de St. Groth and Scheidegger (4). Our experience was that other ratios were not as successful. Table 1 provides a summary of the fusion results and allows a comparison of the efficiencies associated with each of these protocols. Clearly, immunization with exotoxin A coupled to Sepharose 413 or extoxin A associated with infection resulted in a greater fusion efficiency. In these cases the numbers of hybridomas produced was increased, as was the percentage of hybridomas specific for exotoxin A. A similar result has been obtained in the production of monoclonal antibodies to P. aeruginosa elastase (D. R. Galloway, R. C. Hedstrom, and 0. R. Pavlovskis, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, B159, p. 50). When mice were immunized with soluble preparations of exotoxin A considerably fewer hybridomas were generated. To date, 96 antitoxin hybridomas have been identified. Of these, more than 20 have been cloned and stabilized by limiting dilution. TABLE 2. Summary of isolated hybridoma antibodies

I

Antibody

0*

OA

0.2

4

12 16 20 DAYS FIG. 1. ELISA determination of increasing antitoxin serum titer after i.p. injection of 107 viable PA220 cells. Numbers are based on ELISA results of duplicate pooled serum samples from infected mice (0) at a 10-' dilution. O.D., Optical density.

Antibody class

Logl0 titer, ELISAa

Mouse

line

Exotoxin A 6 NMRI IgG1, K TC-2 Toxoid 4 IgG1, K NMRI TC-9.10 Infection 4 IgA, K NMRI TC-15.3 Infection 3 IgM, K NMRI TC-29.4 Infection IgM, K 3 NMRI TC-29.6 Infection IgM, K 3 NMRI TC-31.4 Infection IgM, K 3 NMRI TC-34.1.2 Infection NPb IgM, K NMRI TC-49 Toxin-Sepharose IgM, K 6 NMRI TC-52.6 Toxin-Sepharose IgM, K NP NMRI TC-53.6 Toxin-Sepharose IgM, K NP NMRI TC-53.7 Toxin-Sepharose NP IgM, K NMRI TC-64 Txoin-Sepharose IgM, K NP BALB/c TC-98 Toxin-Sepharose IgM, K NP BALB/c a Titers were determined by measuring the ELISA results, using increasing dilutions of antitoxin from ascitic fluids. The titration endpoint was defined as the highest dilution yielding a value 30% above a normal serum control. b NP, Not performed. TC-1

to

a

Immunogen


VOL. 44, 1984

TABLE 3. Relative exotoxin specificitiesa Avg ELISA OD405 Antibody PA103 PA103 PAO-PR1

1

2

3

4

5

6

7

8

9 10

11

12

w

-200 -116 -92 -68

e.w.

-43

PAO1 Exotoxin A SDS-toxin CRM-toxin exotoxin A

1.1 1.5 1.0 0.9 0.8 1.2 2 2 1.8 1.9 1.8 0.8 2 1.6

TC-1 TC-2 TC-9.10 TC-15.3 TC-24.4 TC-29.6 TC-31.4 TC-34.1.2 TC-49 TC-52.6

TC-53.6 TC-53.7 TC-64 TC-98

0.3 1.1 1.7 0.7 0.5 1.0 2 1.6 1.4 1.8 1.8 0.7 1.8 1.6

1.5 1.3 0.3 0.3 0.2 0.4 1.4 1.4 0.8 0.6 1.0 0.2 1.4 1.2

0.3 1.6 0.9 0.8 0.8 1.1 1.8 1.4 1.5 1.8 1.7 0.7 1.0 1.3

0.4 0.5 0.5 0.1 Normal mouse serum 1.4 1.9 Rabbit antitoxin a Comparative ELISA analysis of monoclonal antitoxin binding to purified exotoxins. Values represent the average ELISA optical density at 405 nm (OD405) of a 10-3 dilution of antitoxin with 50 ng of exotoxin coated onto 96-well microtiter plates. All samples were run in duplicate. CRM, Cross-reactive material.

Properties of antitoxin monoclonal antibodies. The reactivities of the antiexotoxin A monoclonal antibodies were analyzed in several ways (Tables 2 and 3). All hybridomas were initially selected by an ELISA screening procedure with exotoxin A-coated microtiter plates. This assay provides evidence of the direct binding of these antibodies to exotoxin A and was used to estimate the titers of monoclonal

1 2

3

4

5

_

-_

200K-

116K92K68K-

i

43 K31K::

21K-

O

-

FIG. 2. SDS-PAGE analysis and autoradiograph of the indirect immunoprecipitation of "25I-labeled exotoxin A, using (1) rabbit antitoxin, (2) 1251I-labeled exotoxin A standard, (3) normal mouse serum control, and (4 and 5) monoclonal antitoxin TC-15, 20 and 10 ,ul, respectively. Positions corresponding to molecular weight standards are as indicated: 200K, myosin; 116K, P-galactosidase; 92K, phosphorylase B; 68K, bovine serum albumin; 43K, ovalbumin; 31K, carbonic anhydrase; 21K, soybean trypsin inhibitor. (Note: The bands other than the 68K toxin A band in lanes 1, 2, 4, and 5 probably represent precursors, aggregates and fragments of the toxin.

TXN-

-_

1mV

r,f

265

-31 -b

-21 -

df

FIG. 3. Immunoblotting analysis of 10% SDS-PAGE profile of concentrated culture supernatants and purified exotoxin A, using monoclonal antitoxin TC-1 (lanes 1 to 8) and rabbit antitoxin (lanes 9 to 12). The samples are indicated as follows: (1) PAO-PR1; (2) PAO1; (3) PA103; (4) PA103/29; (5) P. putida; (6) 2 p.g of exotoxin A; (7) PA103 cell lysate; (8) PA103/29 cell lysate. Culture supernatant versus rabbit antitoxin; (9) PA103; (10) PA103/29; (11) P. putida; (12) 5 ,ug of exotoxin A.

antitoxins obtained from ascites fluids (Table 2). The ELISA method was also used to determine the isotype of the various monoclonal antibodies under study. It was noted that most of the antibodies are of the immunoglobulin M (IgM) class, although IgG and IgA class monoclonal antibodies were also isolated. An additional property of the monoclonal antibodies investigated was revealed in the indirect immunoprecipitation analysis of 125I-labeled exotoxin A. An example is provided in Fig. 2. All of the monoclonal antibodies tested were able to bind directly to soluble exotoxin A, with the exception of the IgG antibody TC-1 which failed to react with exotoxin by indirect immunoprecipitation (data not shown). However, monoclonal TC-1 was reactive with exotoxin A by immunoblotting analysis of 10% SDS gels (Fig. 3, lane 6) or by ELISA analysis (Table 3). The IgA and IgM monoclonal antibodies, on the other hand, were not as reactive by immunoblotting analysis, possibly because of their large size. ELISA analysis provided evidence that monoclonal TC-1 reacted very well with SDS-denatured exotoxin A whereas some of the other monoclonal antitoxins failed to demonstrate much reactivity against the unfolded toxin (Table 3). In summary, TC-1 demonstrated high specificity and reactivity for exotoxin by immunoblotting and ELISA analysis, yet was unable to bind the conformationally intact toxin molecule in solution. Specificity of monoclonal antibodies. A preliminary analysis of the specificity of monoclonal antitoxins included a survey of various P. aeruginosa strains producing exotoxin A. Culture supernatant fractions were analyzed by the ELISA technique and by immunoblotting, using SDSPAGE. An important observation can be seen in Table 3, which summarizes the ELISA analysis of exotoxin purified from various strains. Obviously, some monoclonal antitoxins demonstrated a selectivity in their reaction to exotoxin A from various strains. Monoclonal TC-1 fails to react with exotoxin A from strain PAO1 and with the immunologically cross-reactive (nontoxic) toxin from strain PAO-PR1, a derivative of PA01 (2). This was further demonstrated by immunoblotting (Fig. 3, lanes 1 and 2). However, TC-1 does react with exotoxin A from strain PA103 and its derivative PA103/29 (16) (Fig. 3, lanes 3 and 4).

266

GALLOWAY, HEDSTROM, AND PAVLOVSKIS

Protection studies with monoclonal antibodies. Experiments were performed to investigate any potential toxinneutralizing activity of the monoclonal antitoxins both in vivo and in vitro. In vitro experiments were conducted with CHO cells. Monoclonal antitoxins were able to protect the CHO cells from purified exotoxin, although monoclonal TC2 apparently does not confer any protection (Fig. 4). In addition, monoclonal antitoxins TC-13, TC-31, and TC-49 also neutralized exotoxin (data not shown). In vivo protection studies were performed with the burned mouse model which has been previously described (18). In these studies BALB/c mice, which are significantly more sensitive to experimental P. aeruginosa infection (16), were used. The results shown in Fig. 5 demonstrate that monoclonal TC-31 significantly increased the survival times of mice receiving the antibody, whereas control mice (using antibovine serum albumin) failed to show any increased survival. On the other hand, TC-1 did not protect the mice against P. aeruginosa infection (data not shown). DISCUSSION We have described the isolation and partial characterization of several monoclonal antibodies to exotoxin A from P. aeruginosa strain PA103. The monoclonal antibodies are of three classes, IgG, IgM, and IgA, although preliminary evidence indicates a predominance of antibodies of the IgM class. The antibodies have been stabilized and produced both in culture and in BALB/c hosts as ascites tumors. More efficient fusions resulted from mice which had been immunized with "immobilized" exotoxin A, i.e., toxin A-Sepharose, and from mice previously infected with viable organisms. Conceivably, these immunizations resulted in "hyperimmunization" as a consequence of the continuous circulation of exotoxin A, leading to an increased proportion of antigen-specific B-lymphocyte blast cells in the spleen at the time of fusion. Evidence in support of this concept has 8

6

E 0 (0 U0

E 26 ~

=

-

-

0

z

4

8

12

16

20

14l Antitoxin

FIG. 4. CHO cell toxicity assay showing the ability of various antitoxins to inhibit the effects of purified exotoxin on cultured CHO cells. The procedure is described in the text. Symbols: Monoclonal antitoxin TC-2 (A); monoclonal antitoxin TC-1 (0); rabbit antitoxin (0). The dashed line indicates the level at which there is unambiguous cell death.

INFECT. IMMUN. 100-

I _

ISt o 0

-

0i) 60I 2-

z

LU

0.4

0

20

40

60

80

100

160

HOURS POST INFECTION

FIG. 5. Survival of burned infected (PA220) mice treated with monoclonal antibody TC-31 (A) or fetal calf serum (0) (15 mice per group). Infective dose: 1.92 x 102 organisms per mouse. Serum was administered ( T ) at 2 h preinfection and 10 and 20 h postinfection.

been provided in the works of Stahli et al. (24), who have shown increased blast cell formation in response to multiple high-dose immunizations with human chorionic gonadotropin. The utility of monoclonal reagents compared with polyclonal antisera was clearly demonstrated when monoclonal antitoxins were compared for their reactivity against culture supernatants from various P. aeruginosa strains. The ability of monoclonal TC-1 to discriminate between the toxin from strain PA01 and its mutant PAO-PR1 and exotoxin A from strain PA103 suggests that these exotoxins possess different antigenic determinants. Data presented in Table 3 show that some of the monoclonal antitoxins are capable of binding to SDS-denatured exotoxin A, indicating that these antibodies probably bind to a linear sequence of amino acids, whereas monoclonal antitoxins which do not bind to SDS-toxin may bind to a conformationally dependent antigenic region. Each of these monoclonal antigenic specificities for exotoxin A will prove useful in studies correlating structural features with functional aspects of the molecule. The in vitro and in vivo protection studies demonstrate the ability of monoclonal antitoxins to prevent the toxic effects of this protein and indicate its importance in Pseudomonas sp. infections. We observed that antibodies TC-1 and TC-31 protected CHO cells from the effects of purified exotoxin, yet TC-1 failed to protect mice in the infection model, whereas antibody TC-31 did provide protection as determined with the in vivo system. The results of protection studies with these two different assay systems must take into consideration the expression or presentation of exotoxin in each system, as well as the class of antibody involved. Furthermore, one must consider the region(s) of exotoxin to which the specificity of the antibody is directed. It is possible, for example, that TC-1 (IgG) binds to a receptor-binding region on the unfolded exotoxin, thus preventing its uptake by CHO cells, whereas TC-31 (IgM) may react with exotoxin associated with P. aeruginosa cells, resulting in complement-mediated clearance of the infecting organism. Studies are in progress to determine the immunogenic regions of exotoxin and to elucidate the mechanism by which monoclonal antibodies may inhibit the effects of this protein. Peptide

VOL. 44, 1984


mapping studies in combination with monoclonal antitoxins should provide clues relating exotoxin structure to function. ACKNOWLEDGMENTS This work was supported by the U.S. Naval Research and Development Command, work unit no. 63750A. 3M463750D808.AB.062. We thank Charles Williams and Valerie Hunter for excellent technical assistance and Gregory Dasch for ELISA reagents and helpful discussion. We acknowledge the excellent editorial services of Donna Boyle in the preparation of this manuscript. We thank B. H. Iglewski for P. aeruginosa strains PAO-PR1 and PA103/29. LITERATURE CITED 1. Cross, A. S., J. C. Sadoff, B. H. Iglewski, and P. A. Sokol. 1980. Evidence for the role of toxin A in the pathogenesis of infection with Pseudomonas aeruginosa in humans. J. Infect. Dis. 142:538-546. 2. Cryz, S. J., R. L. Friedman, and B. H. Iglewski. 1980. Isolation and characterization of a Pseudomonas aeruginosa mutant producing a nontoxic, immunologically crossreactive toxin A protein. Proc. Natl. Acad. Sci. U.S.A. 77:7199-7203. 3. Engvall, E., and P. Perlmann. 1972. Enzyme-linked immunoadsorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109:129-135. 4. Fazekas de St. Groth, S., and D. Scheidegger. 1980. Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 35:1-21. 5. Flick, M. R., and L. E. Cluff. 1976. Pseudomonas bacteremia. Review of 108 cases. Am. J. Med. 60:501-508. 6. Fraker, P. J., and J. C. Speck. 1978. Protein and cell membrane iodinations with a sparingly soluble chloroamide 1,3,4,6-tetrachloro-3,6-diphenylglycouril. Biochem. Biophys. Res. Commun. 80:849-857. 7. Hedstrom, R. C., 0. R. Pavlovskis, and D. R. Galloway. 1983. Antibody response of infected mice to outer membrane proteins of Pseudomonas aeruginosa. Infect. Immun. 43:49-53. 8. Holder, I. A., and M. Jogan. 1971. Enhanced survival in burned mice treated with antiserum prepared against normal and burned skin. J. Trauma 11:1041-1046. 9. Iglewski, B. H., and D. Kabat. 1975. NAD dependent inhibition or protein synthesis by Pseudomonas aeruginosa toxin. Proc. Natl. Acad. Sci. U.S.A. 72:2284-2288. 10. Iglewski, B. H., and J. C. Sadoff. 1979. Toxin inhibitors of protein synthesis: production, purification, and assay of Pseudomonas aeruginosa toxin A. Methods Enzymol. 60:780-793. 11. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 12. Leppla, S. 1976. Large-scale purification and characterization of the exotoxin of Pseudomonas aeruginosa. Infect. Immun. 14:1077-1086. 13. Liu, P. V. 1966. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. II. Effects of lecithinase and protease. J. Infect. Dis. 116:112-116. 14. March, S. C., I. Parikh, and P. Cuatrecasas. 1974. A simplified

15.

16. 17.

18.

19.

20.

21. 22.

23.

24. 25.

26. 27.

28.

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