An immunological study of the diphtheria toxin molecule

Immunochemi.~try, 1972, Vol. 9, pp. 891-.°,06. Pergamon Press. Printed in Great Britain

AN I M M U N O L O G I C A L STUDY OF T H E D I P H T H E R I A T O X I N MOLECULE* A. M. PAPPENHEIMER, Jr., TSUYOSHI UCHIDA and ANNABEL AVERY HARPER Biological Laboratories, Harvard University, Cambridge, Mass. 02138, U.S.A. (Received 17January 1972)

Abstract-Quantitative immunochemical studies of the reaction between horse and rabbit antitoxins with purified fragment A and fragment B polypeptides isolated from nicked diphtheria toxin, and with two non-toxic serologically related, mutant proteins, crm45 and crm~aT, have made it possible to map the locations of certain antigenic determinants along the toxin molecule. Antibodies directed against the fragment A portion of the molecule inhibit the enzymic activity of isolated fragment A and of reduced and alkylated nicked toxin in vitro, but fail to neutralize toxin in vivo. Antibodies against determinants on fragment B, especially.those located on its 17,000 dalton C-terminus, have a high affinity for toxin and neutralize toxicity in vivo by preventing its attachment to the sensitive cell membrane, but do not interact with fragment A. Evidence is presented that the avidity of an antitoxin is inversely related to its anti-fragment A content. Since anti-fragment A fails to neutralize the action of toxin in vivo, maximal avidity is possible only in sera that contain no anti-fragment A. It is further suggested that most of the antigenic determinants of fragment A are masked in intact toxin, but become uncovered as a result of degradation of the more labile fragment B portion of the molecule either as a result of proteolytic action during storage in vitro, or in the animal that is being immunized. Diphtheria toxin is synthesized and released extracellularly as a single polypeptide chain of 62,000 daltons by C. diphtheriae lysogenic for/3tox+-phage (Gill and Dinius, 1971). Intact newly-synthesized diphtheria toxin is enzymically inactive in vitro (Gill and Pappenheimer, 1971; Drazin et al., 1971). W h e n subjected to brief t r e a t m e n t with trypsin in the presence of a thiol, the molecule is 'nicked' by hydrolysis of a single peptide bond located in the loop f o r m e d by the N-terminal of its two disulfide bridges. T h e resulting enzymically active, nicked and reduced toxin, consists of two fragments, A and B of 24,000 and 38,000 daltons respectively, that remain held together by weak interactions (Gill and Dinius, 1971; Collier and Kandel, 1971). Both fragments play a different but essential role in toxicity. T h e NAD-transferase II-ADP ribose transferase activity of toxin (Gill et al., 1969; Honjo et al., 1968, 1971)is located on the thermostable N-terminal f r a g m e n t A portion of the molecule, whereas the labile f r a g m e n t B, containing three half-cystines and the C-terminus, appears to be required for attachment to the sensitive cell and for penetration of f r a g m e n t A to the cytoplasm (Baseman et al., 1970; Uchida et al., 1971, 1972). T h e two fragments can be separated f r o m one a n o t h e r in 6M urea solution by gel filtration. *Aided by Grant No. 09006 from National Institutes of Health and Grant No. GB 18919 from the National Science Foundation. 891

892

A. M. PAPPENHEIMER, Jr. et al.

It has recently become possible to isolate a number of purified non-toxic proteins that are serologically related to toxin from culture filtrates of C. diphtheriae lysogenized with tox-crm+-/3 phages that carry a mutation in the toxin structural gene. O f particular interest have been two cross reacting mutant proteins: (a) crm45, which lacks the C-terminal 17,000 amino acid sequence of fragment B, but which upon treatment with trypsin in the presence of thiol yields enzymically active fragment A (Uchida et al., 1971) and (b) crmlaT, which contains a missense mutation in its fragment A leading to complete loss of enzymic activity (Uchida et al., 1972). The 62,000 dalton crm197 protein, in which fragment B appears to be unaltered, is immunologically indistinguishable from toxin itself. In this paper we report the results of quantitative studies comparing the reactions of various antibodies in horse and rabbit antitoxins with purified diphtheria toxin, with purified isolated fragments A and B, and with the above two purified non-toxic mutant proteins. We have also examined, quantitatively, the behavior of rabbit antisera prepared against purified fragment A, crm45 and crm~a7 when tested against the various toxin-related proteins. From these immunochemical studies, it has been possible to map the approximate location of antigenic determinants on various regions of the toxin molecule. Moreover, by comparing inhibition of enzymic activity with neutralization of toxicity, antitoxins have been shown to contain antibodies that differ functionally from one another. Finally, the studies we are reporting suggest a new mechanism to explain differences between antitoxins of high versus low avidity. MATERIALS AND METHODS

C-Y medium The medium used for production of toxin and crm-proteins from C7 strains was prepared as follows: 10g Casamino acids (Difco certified), 20g yeast extract (Difco) and 5g KH2PO4 were dissolved in 1 1. distilled water. After addition of 2ml 50% CaCI2.2H20, the pH was adjusted to 7.4 and the solution was brought to boiling and filtered. Two ml solution II and 1 ml solution III (Mueller and Miller, 1941) were added to the deferrated medium which was then dispensed in 100ml amounts in liter Erlenmeyer flasks and autoclaved at 115° for 20 min. Culture The stock lysogenic culture, (C7(/345) or C7(/3197), wild type C7(/3)), or PW8 strain, in late exponential growth phase was inoculated into C-Y medium containing 2% deferrated maltose to an initial OD590 of about 0"05 as measured in a Bausch and Lomb Spectronic 20 (ca, 5 × l0 T organisms/ml). Flasks were incubated at 35°C on a rotary shaker at 200 rev/min for 16-17 hr. At this time the OD had reached 12-15 and the cultures were harvested. Purification of crm-proteins Cultures were centrifuged at 10,000g for 15 rain. The bacteria-free supernates were brought to 0.75 or to 0-65 saturation with ammonium sulfate for precipitation of crm45 or crm197 respectively and allowed to stand 24-48 hr in the cold. The precipitates were collected, dissolved in 0.01M sodium phosphate

Immunochemistry of Diphtheria Toxin

893

pH 7"2 and then equilibrated by dialysis against the same buffer. After removal of a small amount of insoluble material by centrifugation, the solutions were passed through a DE52 column and eluted by a NaCI gradient in 0.01M phosphate. Crm45 emerged as a distinct peak when the NaC1 concentration reached 0.125-0" 13M. Both intact monomeric toxin and crmlaT, on the other hand, were eluted between 0.075 and 0.08M NaC1. The eluates were brought to 0.63 saturation with (NH4)2SO4. At this concentration, any fragment A formed by proteolytic degradation remains in the supernate. The precipitates were dissolved, reequilibrated with 0.01M phosphate buffer, rechromatographed on DE52 and reprecipitated with ammonium sulfate. The final solutions were made up in 0.01M sodium phosphate buffer to contain about 1-2% protein. Crm19~ showed an absorbency at 280 nm of about 270Lf per OD unit. Figure 1 shows that both crm45 and crmla7 purified in this way gave major bands at positions corresponding to molecular weights of 45,000 and 62,000 respectively when analysed by SDS-polyacrylamide gel electrophoresis even in the presence of a thiol. Only traces of fragment A (24,000 daltons) could be detected and more than 90 per cent of the protein in both preparations shown in Fig. 1 moved as a single unnicked polypeptide chain.* F ormalinization of crmla7 (B lass et al., 1967; Linggood et al., 1963)

One ml purified crm~9~ (1300 Lf/ml) was dialysed overnight against a large volume of 0.067M phosphate buffer, pH 7.8, containing 0.025M lysine. The solution was removed from the dialysis bag and brought to 0"2% formalin and stored in a volume of 2.5ml for 2 weeks at room temperature. Toxins Several preparations of toxin from different sources were used. Intact 'unnicked' toxin was prepared from culture filtrates of PW8 grown on C-Y medium and harvested within 10 hr during declining growth phase. The toxin (40 Lf/ml) was purified by ammonium sulfate precipitation, and ion exchange chromatography as described above and then stored in the frozen state. ? Toxin No. 223 was purified in this laboratory from a concentrated crude preparation generously sent us by Dr. R. Y. Gottschall of the Michigan State Department of Health. Other preparations were a crystalline nicked toxin sent us some years ago by Dr. M. Yoneda of Osaka University and a purified toxin isolated from a preparation made in Utrecht. Lot No. 18 (Massachusetts Department of Health) was a fraction that eluted from a DEAE column with 0.1M phosphate at pH 7 and the toxin was present almost entirely in the dimeric form (Goor, 1968; Relyveld, 1970) with a sedimentation constant of 6.6S. Carboxy-methylated (C-M) toxin 0.97mi crystalline toxin (97% nicked, OD276nm = 1.65, ca. 600 Lf/ml) in *A component present in trace amounts and moving slightly behind crm197 can be seen in both gels. Its molecular weight is about 67,000. It is synthesized by all C7 strains whether toxigenic or not and is unrelated to toxin (Uchida et al., 1971). t Repeated freezing and thawing of concentrated purified toxin should be avoided since we have found that this treatment invariably results in the formation of nicked toxin and eventually in reduced toxicity.

894

A. M. PAPPENHEIMER, Jr. et al.

0.05M Tris at pH 8 containing 0.4raM EDTA were placed in a 10ml rubber stoppered flask and flushed with nitrogen gas for 15 min. Then 10ftl dithiothreitol (DTT) and 10/~1 1M sodium iodoacetate were added and the flask was again flushed with nitrogen. After 90 min at room temperature in the dark, 10/~1 1M mercaptoethanol was added and the solution was dialysed overnight in the cold against 0. IM Tris at pH 8. The final solution (0.8ml) had an OD276 = 1.48 and 560LF/ml. Its toxicity was 20-30MLD/Lf as compared with 40-50MLD/ Lf before carboxymethylation. The toxicity of reduced toxin before carboxymethylation was also about 40-50 per cent that of toxin before reduction. The ADP-ribose transferase activity per mole of C-M toxin determined with Nethylmaleimide inactivated transferase II (Gill and Pappenheimer, 1971) as substrate was the same as that of free fragment A. 125I- labeled proteins Purified toxin, fragment A and crm45 preparations were trace labeled with approximately one atom 125I per mole by a slight modification (Pappenheimer and Brown, 1968) of the method of Greenwood et al. (1963).

Preparation offragments A and B To 5ml of a purified preparation of diphtheria toxin containing 1500Lf/ml was added 100ftl 0.1M EDTA and 100/.tl dithiothreitol (DTT). The solution was brought to pH 8 with 1M Tris and 50/~1 of 0.1% crystalline trypsin in 0.001N HC1 was added. After 10 min in the water bath at 37°C, the reaction was stopped by addition of 10ftl of 1% soy bean trypsin inhibitor and 200~1 of a 0.7% alcoholic solution of phenylmethylsulfonyl fluoride. 1.8g urea ('ultrapure' SchwarzMann, Orangeburg, N.Y.) was added and the mixture was then chromatographed through a Sephadex GI50 column (void volume = 48ml) equilibrated with 6M urea containing 0.012M methylamine and lmM D T T at pH 6.8. Three peaks absorbing at 276nm were identified. The first which emerged with the void volume was discarded. T h e second, mainly fragment B, was collected in a volume of 17ml. The third peak, fragment A, measured 23ml and contained virtually all of the ADP-ribose transferase activity associated with the original toxin. Both fractions were precipitated by dialysis against 0.8 saturated ammonium sulfate. The precipitates in a volume of about lml were dialysed against the 6M u r e a - D T T - C H 3 N H 2 buffer and rechromatographed through G150. Fragment A emerged as a single peak which was precipitated with ammonium sulfate, dissolved in a small volume of 0.02N acetate buffer at pH 4.5 and then dialysed against the same buffer. A precipitate which formed redissolved when the solution was brought to p H 7-3 with 0.5M Na2HPO4 and heated to 85°C for 5 min. The clear solution had an OD (276nm) = 3.7 and ran as a single sharp, enzymically active, band on SDS-polyacrylamide gels as expected for fragment A, 24,000 daltons. Because of its instability, fragment B was stored dissolved in 6M urea. It had no toxicity when tested for inhibition of leucine uptake by HeLa cells and only a trace of ADP-ribose transferase activity.

crm19 ,

ili!i

Fig. 1. SDS-polyacrylamide 10% gel electrophoresis of purified crm4~ and crm197 in 0"lM sodium phosphate, pH 7"2, stained with Coomassie blue. Five ~1 samples containing 8/~g and 10/zg protein, respectively, in buffer containing 50raM DTT were applied.

Fig. 2A, B. Immunodiffusion against horse antitoxin SA No. 10 (500 units/ml) in agar containing 0.5M urea in buffered saline. The antigen solutions in each case contained 0"2-0"5rag protein per ml.

(Facingpage 894)

Immunochemistry of Diphtheria Toxin

895

Toxicity MLD's were determined by subcutaneous injection into guinea pigs weighing 250±25g. Excess toxin in supernates from the precipitin reaction was assayed using 2ml volumes of suspension-cultures of HeLa cells in glass roller tubes. The supernate to be tested (usually 25-50/LI) was added, and after 3 hr rotation at 5 rev/min at 37°C, 14C-L-Ieucine was added (0'2/LCi per tube). After a further 3hr, 20/LI 0·5M EDTA was added, the cells were collected on Millipore filters, washed three times with balanced salt solution and then three times with 5% TCA. They were glued to planchets, dried and counted in a gas flow counter. When excess toxin was present the counts dropped to 10-20 per cent of the control. Enzymic activity NAD-transferase II ADP-ribose transferase actlVlty was assayed using transferase II prepared from rabbit reticulocytes and I4C-adenine NAD as previously described (Gill and Pappenheimer, 1971). In the case of reduced and carboxymethylated toxin, its enzymic activity was determined in the absence of thiol using an inactive transferase II preparation, the free-SH groups of which had been blocked by treatment with 40mM N-ethylmaleimide (NEM) as described elsewhere (Gill and Pappenheimer, 1971). Antisera Horse diphtheria antitoxic globulin, SAlO, containing 500 flocculating units per ml was kindly supplied by the Antitoxin and Vaccine Laboratories of the Massachusetts Department of Public Health through the courtesy of Mr. Leo Levine. The rabbit antitoxin was a globulin fraction isolated from the serum of a single rabbit that had been immunized with formol toxoid prepared from recrystallized diphtheria toxin. It contained 250 units per ml as determined by rabbit skin test. Antisera against purified fragment A and crm-proteins were prepared in rabbits. The primary injection of antigen (O'1-0'5mg) was given subcutaneously in complete Freund adjuvant (CFA). One month later, a booster injection in incomplete adjuvant was given. Booster i~ections were continued at intervals until a suitable titer had been reached. Bleedings were taken 7-10 days after injection. Globulin fractions were prepared by precipitation with 0·5 saturated ammonium sulfate followed by dialysis against phosphate buffered saline (PBS). The toxin neutralizing potency of the various antisera was assayed by the rabbit intracutaneous method of Fraser (1931) using the standard National Institutes of Health antitoxin diluted to 0·01 unit per ml for comparison. Quantitative precipitation andflocculation reactions A constant amount of serum, equivalent to 10-25 units of rabbit or horse antitoxic globulin, was added to a series of tubes. Increasing amounts of antigen were added and the total volume was made up to 0'2ml with PBS. After 2-3 hr at room temperature and overnight in the cold, the precipitates were centrifuged, washed three times with chilled PBS, drained and dissolved in exactly

896

A. M. PAPPENHEIMER,

Jr.

et al.

0'3ml 0'05N KOH. The OD at 280nm was then determined. The supernates were assayed for toxicity, ADP-ribose transferase activity and, when 125I-Iabeled antigens were used, were counted in a Picker Liquimat gamma counter. RESULTS

Immunodiffusion Figure 2A shows that fragments A and B are immunologically distinct when tested against antitoxin by immunodiffusion. Both fragments form lines of partial identity with toxin and with purified crm 197 , and the latter protein could not be distinguished serologically from toxin. Figure 2B shows that fragment B forms a line of partial identity with crm45 when tested against horse antitoxin. As previously reported (Uchida et al., 1971), fragment A forms a similar line of partial identity with crm45 which in turn shows spur formation when tested against toxin and the same antitoxin. Quantitative precipitation ofantitoxin by toxin and related proteins The differences in reactivity between purified nicked toxin, fragment A, crm45 and crm 197 proteins revealed by immunodiffusion were studied in detail by quantitative precipitation using rabbit and horse antitoxins. Figure 3 shows that toxin itself gave a typical precipitin curve with rabbit antitoxic globulin. There was a sharp maximum when 25Lf (50JLg) toxin was added to O'lml of the antitoxic globulin, which thus contains 250 units/ml by in vitro test. Analysis of the supernates with transferase II for ADP-ribose transferase activity showed that up to and including the point of maximum precipitation, more than 99 per cent of the enzymic activity was precipitated. As indicated by the arrows, excess enzyme activity and excess toxicity (for HeLa cells) was detected in the supernates once the maximum was exceeded. Estimation of the neutralizing potency of this antitoxin by the rabbit intracutaneous method also gave a value of 250 ± lO per cent units per ml. Thus its in vivo: in vitro ratio is close to 1·0 and the serum is therefore of high avidity (Jerne, 1951; Raynaud, 1966). Figure 3 also shows that crm45 gave a curve of similar shape with the rabbit antitoxin but only precipitated 75 per cent of the total antibodies. Therefore, approximately 25 per cent of the precipitable antibodies in this serum are directed against antigenic determinants located on the C-terminal 17,000 dalton amino acid sequence of the toxin molecule that is lacking in crm 45 . Once again, less than I per cent of the enzymic activity of crm45 remained in the supernate until the point of maximum precipitation had been exceeded, as indicated by the arrows. Finally, Fig. 3 shows that antibodies directed against determinants on isolated fragment A account for only about 30 per cent of the total precipitable antibody. In Fig. 4 is plotted the quantitative precipitation of antibodies in horse antitoxic globulin SAlO by the various proteins. As expected, toxin itself gave the flocculation type of curve that is characteristic of most horse antiprotein sera of high titer. Optimal flocculation occurred with 20Lf (40JLg) toxin, but significant excess enzymic activity and toxicity were not detected until between 25 and 30Lf had been added to 20 flocculating units of the antitoxin. The toxin-neutralizing potency of SAlO, as determined by the rabbit intradermal method, was equivalent to 25 ± 10% units per m!. Horse antitoxin SAJO has a relatively high

897

Immunochemistry of Diphtheria Toxin 1·2

ILOS Toxin or crm 45 or fragment A equivalent

Fig. 3. Quantitative precipitation of rabbit antitoxic globulin TV.• crystalline toxin MY. x crm 45 • 0 fragment A. 0·4

D

~Q. .~ 0·2

/

,A;\

j

c. \)

;;:

I

'u

~

(f)

0·1

'\

L~I 20

40

60

80

fLgs Toxin or crm or fragment A equivalent

Fig. 4. Quantitative precipitation of horse antitoxic globulin SA No. 10. • crystalline toxin MY. 0 crm l97 . X crm 45 . 0 fragment A. avidity, therefore, with an in vivo: in vitro (Lr/Lf) ratio of about 1·2. The open squares that superpose upon the toxin flocculation curve were obtained using a purified preparation of the non-toxic crm 197-protein. Thus crm 197 behaves serologically like toxoid and is quantitatively indistinguishable immunochemicalIy from toxin at least with this particular antitoxin. The crm45-protein precipitated about 70 per cent of the total antitoxin from SAlO and fragment A precipitated about 30 per cent. It should be stressed at this point that we have only examined the quantitative behavior of one rabbit and one horse antitoxin as shown in Figs. 3 and 4 and different antitoxins may vary in the proportion of their total antibody that can be precipitated by fragment A or crm45' Thus a much-studied horse antitoxic

898

A. M. PAPPENHEIMER,

Jr.

et

at.

globulin preparation (No. 5353) (Pappenheimer and Yoneda, 1957) of particularly high avidity contains only traces of antibody directed against determinants on fragment A.

Properties ofcrm4s-absorbed antitoxins The antibodies that remain after absorption of an antitoxin with crm4S are directed against determinants located on the C-terminal 17,000 amino acid sequence of the toxin molecule. It is this portion of the molecule that appears to be required for attachment to the membrane of sensitive cells. After absorption with an amount of crm 4s-protein just sufficient to bring the rabbit antitoxin to the point of maximum precipitation or horse antitoxin SAlO into the equivalence zone, only 25 and 30 per cent respectively of the precipitable antibody remained in the supernate (Figs. 3 and 4). However, when the antitoxic activity of the absorbed supernate was assayed by rabbit intracutaneous test, a significantly higher amount of toxin-neutralizing antibody was found than would have been expected from the precipitation data. In short, the in vivo: in vitro ratio of the antitoxin that remained had increased by about 20-30 per cent in both serums. Such an increase in neutralizing activity would be expected, if (as will be shown below) antibodies directed against determinants located on the fragment A N-terminal sequence of the toxin fail to neutralize its toxicity. The existence of non-neutralizing antibodies in diphtheria antitoxic sera was first recognized some years ago by Raynaud (1959, 1966). If, as generally supposed (Pappenheimer and Brown, 1968; Strauss and Hendee, 1959) antitoxin acts by preventing toxin from reaching the sensitive cell membrane, it is not surprising that antibodies directed against determinants located on the C-terminal portion of the molecule, presumably needed for attachment, should be highly effective in neutralizing its toxicity. We would not expect, however, that the same antibodies would be equally effective in inhibiting the enzymic activity located on fragment A at the other end of the polypeptide chain. As expected, neither crm4s-absorbed rabbit nor crm4Sabsorbed horse antitoxin had any effect on the enzymic activity of isolated, purified fragment A. Unfortunately, it is not possible to test directly the effect of antitoxic antibodies on the enzymic activity of fragment A when it is attached to fragment B as in toxin. Activation ordinarily requires the addition of excess thiol to nicked toxin in order to reduce its disulfide linkages (Collier and Kandel, 1971; Gill and Pappenheimer, 1971) and we have found that complexes of antitoxin with fragment A or with nicked toxin are dissociated in the presence of thiols with liberation of active enzyme. Therefore, in order to test the effect of crm4s-absorbed antitoxin on the activity of fragment A while still part of the toxin molecule, it was necessary to use nicked toxin that had been reduced and carboxymethylated and to use transferase II in which the thiol groups had been blocked by treatment with NEM. Under these conditions, as shown in Table 1, the absorbed rabbit and the absorbed horse antitoxins behave very differently from one another. Before absorption, both antitoxins inhibit to titer the ADPribose transferase activity of C-M-toxin. After absorption with crm45, horse antitoxin SAlO no longer inhibits. The crm 4s-absorbed rabbit antitoxin, on the other hand, still inhibits the enzymic activity of C-M-toxin to titer, even though

899

Immunochemistry of Diphtheria Toxin

Table 1. Inactivation of ADP-ribosylating activity of reduced and carboxymethylated nicked toxin by horse and rabbit antitoxins, before and after absorption with crm 45 protein a Units/ml added to an equal vol. 9Lf/ml CM-toxin

Activityb cpm

0

1330

Rabbit antitoxin (unabsorbed)

13 6·5 3·3 1-6

768 1020 1130

Rabbit antitoxin (crm45-absorbed)

40 20 10 5 2·5 1·3

270 336 lost 440 1070 1108

80 75

14 7 3·5 1·8

235 643 1170 1190

82 52 12

60 30 IS 7·5

1250 1315 1320 1205

6

Antitoxin Control

Horse antitoxin SAIO (unabsorbed)

Horse antitoxin SAIO (crm 45 -absorbed)

;~56

Per cent Inhibition

73 42 24 IS

67 20 17

]()

I I 9

a25,.d CM-toxin (9Lf/ml) + 25ILI of antitoxin dilution incubated for 1 hr at 25°C. Then SILl of each mixture was tested for residual NAD-transferase II ADP-ribose transferase activity using NEM-treated transferase 11 as substrate. The control was normal rabbit y-globulin. bTCA precipitable 14C-ADP-ribosyl-NEM-transferase II formed after 10 min at 37°C under standard conditions for toxin enzyme assay (see Methods). the same serum has no inhibitory effect whatsoever on the activity of free fragment A. The reason for this difference between the two antitoxins has not been determined. It is possible that the antibodies remaining in absorbed horse antitoxin are of high affinity for determinants on fragment B. Their interaction could cause a conformational change, whereby the bonds holding the two fragments together in C-M-toxin are weakened. As a result free fragment A would be released and fragment B-antibody aggregates formed. In the case of the rabbit antibody, the two fragments remain together in the aggregated antigen-antibody complex, thus preventing fragment A from reacting with its substrate, transferase II.

Antijragment A serum Two rabbits, Rl and R2 were immunized with purified fragment A. They both received repeated booster doses over a period of 4 months and both produced precipitating antibodies. The quantitative reactions of the y-globulin fraction prepared from the final bleeding from R2 are shown in Fig. 5 using

900

A. M. PAPPENHEIMER,

Jr.

et

at.

0·5 ~

re

00·4

o

I

SO'3 ~

Q.

'0 ~0'2 u

iO. 1 VI

Antigen added (normalized arbitrary oolls)

Fig. 5. Quantitative precipitation of anti-fragment A globulin. • toxin. o crm45 . X fragment A.

purified fragment A, crm45 and a toxin preparation as preopItants. The quantities of the three antigens added are expressed in arbitrary units, chosen to show that the points for all three lie on the same curve. This was necessary in order to correct for impurities still present in the crm45 preparation and because, as discussed below, only a variable fraction of most toxin preparations is precipitated by anti-fragment A, even in the region of excess antibody. The method of plotting is justified, since absorption of the serum with anyone of the three antigens, removed antibodies against the other two. As increasing amounts of fragment A or of crm45 were added, more than 99 per cent of the enzymic activity was precipitated by the antiserum until the point of maximum precipitation had been exceeded. The crm 45 preparation used as antigen in Fig. 5 was only partially purified and when trace-labeled with 1251, only 30 per cent of the label was precipitable. Later, with more highly purified crm45 such as shown in Fig. I, more than 90 per cent of the label was precipitated by antifragment A. The purified 1251-labeled-fragment A preparation was 87 per cent precipitable. In contrast to fragment A and crm45' the behavior of toxin preparations when tested against anti-fragment A was inconsistent and puzzling. Despite the fact that all the toxin preparations tested were at least 90 per cent precipitable by diphtheria antitoxins, anti-fragment A precipitated only a fraction of label from 125I-trace labeled toxin. The exact per cent of label precipitated varied widely (anywhere from 25 to 80 per cent) depending upon the particular preparation. For example, 4Lf of 125I-Iabeled crystalline toxin MY added to lO0J.LI of the y-globulin from R2 precipitated only about 70-80 per cent of the total anti-fragment A antibody. Yet despite the presence of excess antibody, tests on the supernate revealed that 50 per cent of the 125I-Iabeled toxin still remained free in the supernate. That it was free and not complexed with antibody is shown by the fact that it emerged as a single retarded peak from a Sephadex GlOO column as expected for a molecule of 62,000 daltons. This was further confirmed by tests for enzymic activity which showed that only about 40 per cent

Immunochemistry of Diphtheria Toxin

901

had been precipitated. Similarly, about 60 per cent of the toxicity as determined by guinea pig MLD, failed to precipitate. Despite the fact that antifragment A did precipitate an appreciable fraction of toxin from this and other preparations, the serum showed no toxin-neutralizing potency at all « 0·01 unit/ml) when tested by the rabbit intracutaneous test. Finally, the fact that excess toxin was required to precipitate all the fragment A antibody could not be attributed to the presence of free fragment A liberated by degradation, since SDS polyacrylamide gel electrophoresis failed to reveal more than traces of a 24,000 dalton component or other degradation products, even when the gels were overloaded. The puzzling quantitative behavior of toxin preparations when tested against anti-fragment A can be explained by the tendency of purified toxins to form dimers (Goor, 1968; Relyveld, 1969) and higher aggregates (D. M. Gill, personal communication). In Fig. 6, are compared the precipitin curves for four different toxin preparations with anti-fragment A y-globulin R2. Of the two preparations that precipitate the least antibody, toxin MY was a completely nicked crystalline, monomeric preparation with sedimentation constant 4·2S as determined in the Spinco Model E ultracentrifuge. The other toxin had been freshly prepared from a filtrate of a growing culture of the PW8 strain. It was unnicked and was assumed to be in the monomeric form since it was eluted at low salt concentration from a DE52 column. The fraction from Lot No. 18, which required a high ionic strength for elution was almost entirely in the dimeric form with a sedimentation constant of 6·6S. Finally, the preparation designated 'Dutch toxin' which had been repeatedly frozen and thawed contained a good deal of aggregated as well as some degraded material and had lost 0·6

/LrJS Toxin added

Fig. 6. Precipitation of anti-fragment A glot tions. 0 'Dutch' (aggregated). • Lot No. 18B MY (monomer, nicked)... TV (monc>

IMM Vol. 9 No. 9-D

902

A. M. PAPPENHEIMER,

Jr. et at.

a significant proportion of its original toxicity. Figure 6 shows clearly that the preparations containing dimeric or aggregated toxin are far more effective than monomeric toxin in precipitating anti-fragment A. In fact, it is possible that the precipitation that occurred with excess MY or fresh unnicked toxins can be attributed to small amounts of toxin that were present in aggregated form and that the monomeric form is not precipitated at all.

Rabbit antisera against crm45- and crm197-proteins Like isolated fragment A, purified crm45- and crm 197-proteins proved to be relatively poor antigens in the rabbit and repeated booster doses were necessary to obtain good precipitating antisera. A y-globulin fraction from the rabbit which responded best to prolonged immunization with crm 45 , contained appreciable amounts of antibody directed against diphtherial proteins unrelated to toxin when tested by immunodiffusion. Nevertheless, the globulin contained approximately 2mg/ml antibody specifically precipitable by either purified crm 45 or toxin. In an avid serum this amount of precipitable antibody would be equivalent to about 125 units/ml of neutralizing antitoxin. Since only 30 units/ml were found by rabbit intradermal assay, the serum was of low avidity. At least 70 per cent of the total precipitable antitoxin was precipitable by purified fragment A, leaving all of the toxin-neutralizing antibodies in the supernate. Antisera prepared against crm197 were likewise of low avidity and after repeated injections also contained precipitating antibodies directed against diphtherial proteins unrelated to toxin. Despite the fact that the purified crm19r protein is quantitatively indistinguishable from toxin (Fig. 4) only 1 and 4 units/ ml, respectively, were present in globulin fractions prepared from antisera produced by intensive immunization of two rabbits. From the amount of antibody specifically precipitated by toxin, at least 10 times this amount of neutralizing antitoxin would have been expected. The in vivo: in vitro ratios of both these antitoxins were thus less than 0·1. Of the total antibody precipitated by purified toxin, at least 90 per cent was precipitable by isolated fragment A, leaving all of the neutralizing antitoxin in the supernate. In other words, the antisera against crm45- and crm197-proteins behaved as if most of the antibody were specifically directed against determinants located on free fragment A. Antigenicity offormalinized crm197 It seemed possible that the superiority of toxoid over crm197 with respect to production of toxin-neutralizing antibodies might be due to the stabilizing effect of formalinization in the former case protecting it from proteolytic degradation. Two rabbits were given a sensitizing dose of 130Lf of formalinized crm 197 in CFA followed 1 month later by a booster dose of 260Lf in incomplete adjuvant. A control rabbit received a similar course with untreated crm197 except that a 2'5-fold greater amount of antigen was injected with each dose. One week after the booster injection, both rabbits that had received formalinized crm197 had produced precipitating antitoxin of high avidity with neutralizing titers of about '0 and 120 units/ml. In the former serum no precipitating antibodies against "lgment A .could be detected. Maximum precipitation in the latter case required Lf toxin per ml of serum and less than 15 per cent of the total antibody was

Immunochemistry of Diphtheria Toxin

903

precipitable by fragment A. The in vivo: in vitro ratios were thus close to 1·0 for both antitoxins. The rabbit immunized with untreated crml97 produced 16 units/ml of neutralizing antitoxin, but in this case the serum was of low avidity. At least 50 per cent of the total antibody was non-neutralizing and was precipitable by isolated fragment A. DISCUSSION It was first shown by Pope and Stevens (1958a, b, c) that when crystalline diphtheria toxin is subjected to a variety of degradative procedures, such as boiling, treatment with alkali at pH 12, with certain proteolytic enzymes or with alkaline sodium sulfite, and then examined by immunodiffusion against antitoxin, several lines of precipitation (up to four) may appear, all of which form lines of partial identity with pure toxin. These observations were confirmed by Raynaud and his coworkers (1959, 1966) and Relyveld and Raynaud (1959), who correctly interpreted them as indicating breakdown of the toxin molecule into fragments upon which different antigenic determinants were located. Raynaud obtained evidence for at least 4 different precipitating antibodies in antitoxic horse sera. One of them, At, was directed against a fragment tt which could be separated by gel filtration from the degradation products of toxin that had been treated with alkaline sodium sulfite in 6M urea containing 0·2 mg/ml phenylmercuric borate. The isolated tt fragment precipitated a relatively minor, non-neutralizing fraction of the total antibody from antitoxic sera. The toxinneutralizing antibodies that remained in the supernate after absorption with t b showed a corresponding small but definite increase in specific avidity or in vivo: in vitro ratio. Because of its stability and because it interacts only with nonneutralizing antibodies present in antitoxic sera, it is probable that the t r fragment isolated by the French workers is similar to if not identical with fragment A. It is also likely that the 'alkali stable', 'pepsin stable' and 'trypsin stable' toxin-derived antigens of Pope and Stevens, all consisted mainly of fragmentA. It has long been known that antitoxins vary greatly in the firmness with which they bind toxin and thus in the degree of protection conferred. This property, termed avidity, can be measured by comparing the toxin-neutralizing potency of antitoxins at different dilutions. Thus, increasing relative amounts of a nonavid antitoxin will be required to neutralize a given toxin as the system becomes more and more dilute. In his classic thesis on the quantitative aspects of standardization of diphtheria antitoxins, Jerne (1951) concluded that avidity was determined by the equilibrium constant of the primary reversible interaction between toxin and antitoxin, i.e. T + A ~ T A. From the molar ratio of antitoxin needed to neutralize toxin at the high dilutions used in the rabbit intracutaneous assay, he calculated that the association constant, K A , for this reaction must reach values of 101t l/mole or more for antitoxins of high avidity. Jerne noted that antibodies formed early in the course of immunization tended to be of low avidity with values for Kl estimated to be of the order of 10 8 1/mole. At the time Jerne's study was carried out, the extreme heterogeneity of immunoglobulins had not been recognized and it was not realized that even immunization against a single molecular species of protein leads to the production of many antibodies

A. M. PAPPENHEIMER, Jr. et al.

904

with distinct specificities. The present study suggests that the avidity of an antitoxin is determined by the proportion of the total antitoxin that is directed against determinants located on the fragment A portion of the toxin molecule. Table 2 shows that antitoxins of high avidity contain little or no anti-fragment A whereas those of low avidity contain a high proportion of anti-fragment A. The table shows that without exception there is a close reciprocal correlation between avidity and anti-fragment A content. It is not known whether the formation of anti-fragment A antibodies found in most antitoxins is stimulated by intact toxin (or toxoid) molecules or whether they arose because free fragment A was already present in the toxin preparation before its conversion to toxoid with formalin. We strongly suspect the latter to be the case. Fragment A, whether free or combined with B as in intact toxin, is highly resistant to denaturation and to the action of proteolytic enzymes. In contrast, fragment B is extremely sensitive to denaturation and to proteolytic breakdown. For this reason, stored preparations of toxin, even after purification, gradually lose toxicity and accumulate free fragment A and other breakdown products of intermediate molecular weight which contain fragment A. This can readily be demonstrated by polyacrylamide gel electrophoresis. It seems likely that in undegraded toxin (or toxoid), most of the antigenic determinants located on fragment A are masked or buried just as is the enzymic active site. Indeed, the fact that monomeric toxin precipitates anti-fragment A poorly or not at all, Table 2. Relation of anti-fragment A content to avidity in antitoxic sera

Antitoxin No. 5353 (Horse) SA No. 10 (Horse) SA No. 10 (crmwabsorbed)

Avidity in vivo: in vitro ratio a

anti-fragment A as per cent of total toxin- precipitable antibodies

> 1·2

< 20

1·2 > 1·2

30

1·0 1·2

30

0·25 0·1 0·1 0'5

70

TUyG (rabbit) TUyG (crm 45 -absorbed) anti-crm 45 anti-crm 197 No.1 anti-crm 197 No.2 anti-crm197 No.3 anti-crm197 formalinized No.1 anti-crm197 formalinized No.2 anti-fragment A

~

~

1·0 1·0

o

o o

> 90 > 90 50

o 15 100

a In vivo titer was determined by rabbit skin test. The in vitro titer is given as the amount of toxin in Lf units required for optimal flocculation in the case of horse antitoxin or the Lf units required for maximal precipitation of rabbit antitoxins except in those antitoxins of high anti-fragment A content where precipitation of toxin was incomplete. In the latter case, the in vitro titer was calculated from the maximum amount of antibody precipitable by toxin assuming 15 IJ-g antibody protein per antitoxin unit (Cohn and Pappenheimer, 1949).

Immunochemistry of Diphtheria Toxin

905

suggests that in the whole toxin molecule no more than one determinant may be available for interaction with antibody and for this reason precipitation can occur only when toxin is in the dimeric or aggregated form (Fig. 6). Immunization of rabbits with freshly prepared purified crm45 or crml97proteins produced antitoxins of low avidity and relatively high anti-fragment A content (Table 2). Since neither preparation contained significant amounts of free fragment A, it seems likely that rapid breakdown of the fragment B portion of the mutant proteins occurred in vivo. * When crm 197 , treated with formaldehyde as in conversion of toxin to toxoid, was used as antigen, it produced antitoxins of high avidity with little anti-fragment A after primary stimulation followed by a single booster dose. It is probable that formalinization of toxin or of crm 197 stabilizes the molecules so that they become more resistant to denaturation and proteolytic breakdown. In diphtheria toxoid at least 30 per cent of the e-amino groups are blocked (Blass, 1964) and it has been shown that methylene bridges linking lysine to tyrosine are formed (Blass et al., 1967). It is possible that such covalent bridges may form between fragments A and B, thereby increasing still further, stability and resistance to proteolysis. It will be recalled that following intravenous injection into rabbits or guinea pigs, the fate of toxin trace-labeled with 1251 or 131 1 is quite different from that oflabeled toxoid (Masouredis, 1960; Baseman et al., 1970). The latter is taken up preferentially by tissues of the reticuloendothelial system and is eliminated more slowly than is toxin. Toxin, like BSA, is rapidly eliminated from the tissues in parallel with its elimination from the blood stream (Baseman et al., 1970). One is tempted to speculate that a comparison of protein antigens before and after formalinization might reveal striking differences in specificity of the antibodies produced. We have observed that when toxin is complexes with antitoxin, it is protected from the action of trypsin at neutral pH. It may be for this reason, that antitoxin produced in man by immunization with toxin-antitoxin complexes, such as were used before the introduction of formal toxoid, is of high avidity. For similar reasons, antitoxin developed as a result of 'natural' immunization is of high avidity. The present studies have shown that neutralization of toxin by antitoxin depends upon interaction with antibodies directed against determinants located on fragment B, especially those located on the 17,000 dalton C-terminus that appear to be necessary for attachment to the sensitive cell membrane. Antitoxin thus acts by preventing toxin from attaching to the cell membrane, rather than by inhibiting its enzymic activity. In fact, horse antitoxin (SAlO), after absorption by crm 45 of all precipitating antibodies except those directed against determinants on the 17,000 dalton C-terminus, neutralizes with maximal avidity even though having no effect on the ribosylating activity of nicked, reduced and alkylated toxin. In the absence of antitoxin, toxin becomes fixed to sensitive cells even at very high dilution; concentrations between lO-l3M and lO- 12M have been shown to inhibit growth of sensitive mammalian cells in culture (Lennox *In crm45, even the unnicked molecule has enzymic activity in the presence of a thiol, so that it is possible that antigenic determinants located on fragment A are exposed in the intact molecule. Nevertheless, it is readily nicked and its B fragment is highly sensitive to proteolytic breakdown.

906

A. M. PAPPENHEIMER, Jr. et ai.

and Kaplan, 1957; Solotorovsky and Gabliks, 1963) and a mere 108 molecules suffices to cause a visible skin reaction in rabbits. It would thus appear that the affinity of specific sites on sensitive cells for toxin is very high. It follows, therefore, that in order to compete with these sites for toxin, the affinity of neutralizing antitoxin for toxin must also be very high. Since antifragment A fails to neutralize toxin in the rabbit intradermal assay, the value of K A for formation of the toxin-anti-fragment A complex is probably at least 3 or 4 orders of magnitude less than that of the toxin-neutralizing antitoxin complex. Acknowiedgement- We are grateful to Dr. D. Michael Gill for helpful suggestions and

criticism. REFERENCES Baseman J. B., Pappenheimer A. M. Jr., Gill D. M. and Harper A. A. (1970)]. expo Med. 132,1138. Blass J. (1964) Bioiogie medicaie 53, 202. Blass J., Bizzini B. and Raynaud M. (1967) Bull. soc. Chim. (France) 10, 3957. Cohn M. and Pappenheimer A. M.Jr. (1948)]. Immun. 63, 291. Collier R.J. and KandelJ. (1971)J. bioi. Chem. 246,1496. Drazin R., KandelJ. and Collier R.J. (1971)]. bioi. Chem. 246,1504. Fraser D. T (1931) Trans. R. Soc. Canada Sec. V25, 175. Gill D. M., Pappenheimer A. M. Jr., Brown R. and KurnickJ. (1969)]. expo Med. 129, 1. Gill D. M. and Dinius L. L. (1971)]. bioi. Chem. 246,1485. Gill D. M. and Pappenheimer A. M..lr. (1971)]. bioi. Chem. 246,1492. Goor, R. S. (1968) Nature, Lond. 217,1051. Greenwood F. C., HunterW. M. andGloverJ. S. (1963) Biochem.]. 89,114. Honjo T, Nishizuka Y., Hayaishi O. and Kato 1. (1968)]. bioi. Chem. 243, 3553. Honjo T, Nishizuka Y., Kato I. and Hayaishi O. (1971)]. bioi. Chem. 246, 4251. .Ierne, N. K. (1951) Acta Path. Microbioi. Scand. suppi. LXXXVII. Lennox E. S. and Kaplan A. S. (1957) Proc. Soc. expo Bio!' Med. 95, 700. Lingood F. V., Stevens M. F., Fulthorpe A. J., Woiwod A. J. and Pope C. G. (1963) Brit.]. expo Path. 44, 177. Masouredis S. P. (1960)]. Immun. 85,623. Mueller J. H. and Miller P. A. (1941)]. Immun. 40, 21. Pappenheimer A. M..lr. and Yoneda M. (1957) Brit.]. expo Path. 38,194. Pappenheimer A. M.,Jr. and Brown R. (1968)]. expo Med. 127, 1073. Pope C. G. and Stevens M. F. (1958a) Brit.]. expo Path. 39,150. Pope C. G. and Stevens M. F. (1958b) Brit.]. expo Path. 39, 386. Pope C. G. and Stevens M. F. (l958c) Brit.]. expo Path. 39,490. Raynaud M., BlassJ. and Turpin A. (1957) C. r. Acad. Sci. 245, 862. Raynaud M. (1959) in Mechanisms of Hypersensitivity (Edited by Shaffer J. H., Lo Grippo G. A. and Chase M. W.), p. 26. Little Brown, Boston, Mass. Raynaud M. (1966) Proc. Fed. European Biochem. Soc. Vol. I, p. 197. Pergamon Press, Oxford. Relyveld E. H. and Raynaud M. (1959) Annis. Inst. Pasteur 96, 537. Relyveld E. H. (1970) C. r. Acad. Sci. Paris D270, 410. Solotorovsky M. and Gabliks J. (1963) in Cell Culture in the Study ofBacteriai Disease (Edited by Solotorovsky M.), p. 5. Rutgers University Press, New Brunswick, New Jersey. Strauss N. and Hendee E. D. (1959)]. expo Med.l09, 145. Uchida T, Gill D. M. and Pappenheimer A. M.Jr. (1971) Nature, Lond. 233,8. Uchida T., Pappenheimer A. M. Jr. and Harper A. A. (1972) Sciencel75, 901.