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coli family of enterotoxins
DLAGNMICROBIOLINFECTDIS 1983;1:129-138
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Immunological Differences Among the Cholera/ Coli Family of Enterotoxins* Ben A. Marchlewicz** and Richard A. Finkelstein
Pure enterotoxins from two strains of Vibrio cholerae and choleragen-reloted heat-labile enterotoxins (LTs) from strains of Escherichia coli of human and porcine origin were examined by Ouchterlony-type immunodiffusion assays and by neutralization tests in Y-I adrenal cells using specific and immunopurified antisera. In accordance with previous findings, the results indicated that each of the toxins shared antigens with each of the others and that each, in addition, possessed unique antigenic determinants. The present study, however, indicates further that enterotoxin-type specific ant/bodies, in some instances, account for a major portion of the neutralizing activity. The full extent and importance of the antigenic drift in the cholera/ coli family of ADP-ribosylating, adenylate cyclase-activating, heat-labile enterotoxins remains to be determined. The results will be pertinent to efforts to develop broad-spectrum antitoxic immunity as well as in the development of methods for the rapid detection and identification of enterotoxigenic strains.
INTRODUCTION The heat-labile enterotoxin(s) (LTs) of Escherichia coli and the enterotoxin (choleragen) of Vibrio cholerae are similar to each other immunologically, structurally, and functionally (Clements and Finkelstein, 1978a, b, 1979; Clements et al., 1960; Dallas and Falkow, 1980; Gyles and Barnum, 1969; Holmes et al., 1960; and Kunkel and Robertson, 1979). These enterotoxins bind, through their B-regions, with host cell membrane receptors containing ganglioside GM1 (Holmgren, 1973; Moss and Vaughan, 1979) and activate adenylate cyclase, leading to increased intracellular levels of cyclic 3', 5'-adenosine monophosphate (cAMP) (Evans et al., 1972; Moss and Vaughan, 1979). However, despite their overall similarity, structural and immunological differences have been observed among choleragen (CT), LT produced by a human strain of enterotoxigenic E. coli (H-LT), and LT produced by porcine strains of enterotoxigenic E. cell (P-LT) (Geary et al., 1982; Gill et al., 1901; Honda et al., 1981a, 1981b). The neutralization of a porcine E. coil LT by antibody to cholera enterotoxin was first reported by Gyles and Barnum (1969). Since then, there have been several reports
*This work was presented in part at the International Symposium on Bacterial Diarrheal Diseases, Osaka, Japan, 23-25 March 1982. **Present address: Abbott Laboratories, Chicago, IL From the Department of Microbiology, University of Missouri School of Medicine, Columbia, Missouri. Address reprint requests to: Richard A. Finkelstein, Ph.D., Professor and Chairman, University of Missouri-Columbia, M-264 Medical Science Building, Columbia, Me 65212. Received November 3, 1982; accepted February 15, 1983.
© 1983Elsevier SciencePublishing Co., Inc., 52 Vanderbilt Avenue,New York, NY 10017
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of the neutralization of LT activity by cholera antitoxins in rabbit ileal loops (Smith and Sack, 1973}, in permeability factor assays (Evans et al., 1973}, and on adrenal cells (Donta, 1974}. In those studies, crude preparations of LT were used and the degree of neutralization was not quantitated. Nalin et al. (1974} examined the antibody activity against crude LT and CT (in permeability factor assays and by hemagglutination, respectively} in serum from individuals immunized with cholera toxoid and reported that the titers rose in parallel. However, whereas Stoliar et al. (1976} reported that secretory IgA from colostrum of Guatemalan women neutralized LT and CT to similar extents, Holmgren et al. {1976} found that Pakistani milk was more active against LT than against CT. Pierce {1977} immunized rats intraperitoneally with 100 p.g of partially purified formalin-treated cholera toxoid in Freund's complete adjuvant followed by intraduodenal boosting with two doses of 40 mg each of a crude cholera toxoid. Whereas the immunized rats exhibited reduced intestinal secretory responses to crude cholera toxin at doses of 1.5 and 6 mg, resistance to a crude E. coli LT preparation, while evident at low dose challenge (0.75 mg), was insignificant at 4-fold higher challenge. More recently, Svennerholm and Ahren (1982} likewise found that anticholera toxin antibodies were much more protective against live V. cholerae challenges, in rabbit ileal loops, than they were against challenge with live LT-producing E. coll. Thus, earlier observations suggested that although the enterotoxins are immunologically related, there are differences among them which could be an important factor in efforts to develop effective broad-spectrum antitoxic immunogens. In this report, we extend earlier findings by comparing the relative effectiveness of homologous and heterologous sera, as well as immunopurified sera, in the neutralization of biological activity of pure CT, H-LT, and P-LT.
MATERIALS AND METHODS Toxins
CT, H-LT, and P-LT were purified to homogeneity as described previously (Finkelstein et al., 1971; Geary et al., 1982; Clements and Finkelstein, 1979}. Purified cholera toxin from E1 Tor biotype, Ogawa serotype Vibrio cholerae strain 3083 (Honda and Finkelstein, 1979}, was also tested. Unless otherwise indicated, CT results were those obtained with toxin from classical biotype, Inaba serotype strain 569B. In immunodiffusion assays, modified from Ouchterlony (1949}, the purified toxins were used at 20 ~g per 50 p.l well. Sera
The sara used in this study included the U.S. Standard Lot 1 goat anticholera toxin obtained from Carolyn Hardegree (1979}; the Swiss Serum and Vaccine Institute (SSVI} equine anticholera immunoglobulins obtained from Carl Miller at NIH; equine anticholeragenoid (Finkelstein, 1970}; goat anticholeragenoid (Clements and Finkelstein, 1978a}; goat anticholeragen Lot G5156-11 Chemo-Sero-Therapeutic Research Institute (CSTRI}, Kumamoto, Japan, and CSTRI equine anti-choleragen Lot E1006-10-A21, both obtained from N. Ohtomo; specific rabbit antisera directed against isolated choleragen A and B subunits (Finkelstein et al., 1974a}, respectively, prepared by M. Boesman-Finkelstein in this laboratory; goat anti-H-LT {Geary et al., 1982) directed against LT isolated from strain H74-114; and goat anti-P-LT (Clements et al., 1980} directed against LT isolated from strain 711/FILT. A rabbit antiserum against choleragenoid from strain 3083 (Vasil et al., 1974} was also used.
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Neutralization Tests Neutralization tests were performed using the Y-1 adrenal cell assay (Donta et al., 1974; Sack a n d Sack, 1975). Briefly, serial dilutions of the sera were made i n Ham F-10 m e d i u m (Sack a n d Sack, 1975). A 100 ~1 sample containing 10 EDso doses of toxin i n Ham F-10 m e d i u m was added to the diluted sera, for CT, 1 EDso = 6pg; for both LTs, 1 EDso -- 12pg after trypsin activation as described in Clements and Finkelstein (1979). The toxin-sera mixtures were incubated at room temperature for 15 min. The culture m e d i u m on the Y-1 cells was removed by aspiration and toxin-sera mixtures were added to each well (final volume 200 p.1). The Y-1 cell plates were i n c u b a t e d at 37°C for 6 hrs i n a 5% CO2 atmosphere. After incubation, each plate was scored for cell rounding, observed using an inverted phase contrast microscope. The e n d p o i n t of the titration was the highest dilution of serum which allowed a 50% r o u n d i n g of the Y-1 cells. The neutralization data, i n Tables 1 and 2, are summarized as the relative potencies of the heterologous reactions, as compared with the homologous system (set at 1.0), and can thus be read as percent cross-reactivity. The actual titers of the homologous reactions are also provided.
TABLE 1. Homologous a n d Heterologous Neutralizing Activity of Various Antisera Against CT, H-LT, a n d P-LT Enterotoxina Antiserum U.S. standard cholera goat antitoxin (Lot 1) S.S.V.I. cholera antitoxin Goat anticholeragenoid Horse anticholeragenoid Goat anticholeragen (G5156-11) Horse anticholeragen (E 1006-10-A-21) Rabbit anticholera A subunit Rabbit anticholera B subunit Rabbit anticholeragenoid (3083) Goat anti-H-LT (H74-114) Goat anti-P-LT (711/FILT)
569B-CT
3083-CT
H-LT
P-LT
I b (3.4)c (1.3-10.2) e 1 (8.1) (3.2-10.2) 1 (12) (12) 1 (5.1) (2.5-10.2) 1 (8.1) (5.1-10.2) 1 (5.1) (5.1) 1 (0.64) (0.3-1.3) 1 (4.1) (2.5-10.2) 1 (10.9) (5.1-20.2)/ 0.43 (10.2-20.5) 0.12 (0.6-1.3)
0.60a (2.0)/ 0.06 (0.3-1.3)/ n.d.
0.75d (1.3-10.2) 0.10 (0.6-1.3) 0.70 (7.8-9.0) 0.12 (0.3-1.2) 0.89 (5.0-10.2) 2.0 (2.8-20.4) 0.50 (0.32) 1.80 (2.6-20.2) 0.33 (2.5-10.2)/ I b (30.6) (20-40) 0.21 (0.3-1.3)
0.28 (0.3-2.5) 0.06 (0.02-1.2) 0.05 (0.2-1,0) 0.09 (0.04-0.6) 0.19 (1.3-2.5) 0.42 (1.3-10.2) 0.50 (0.32) 0.09 (0.3-0.8) 0.125 (0.6-2.5)/ 0.34 (2.5-20.1) I b (6.67) (2.6-10.2)
0.50 (1.3-5.1) / n.d. n.d. n.d. n.d. 1 (10.2)/ 0.50 (10.2-40.9) f 0.11 (0.8)/
°10 EDsosof each toxin were added per 200 ~1 in each well (1 ED~oof CT, 6.25 pg/well;H-LT, 12.5 pg/ well; P-LT 12.5 pg/well) bArbitrarilyset at unity CActualtiter x 104 (geometric mean of 6 titrations) dRalative activity in comparisonwith homologoussystem {set at unity) "Range of values x 104 (6 titrations) tRange of yalues x 104 (3 titrations)
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2. Effect of I m m u n o a f f i n i t y A d s o r p t i o n o n N e u t r a l i z i n g A c t i v i t y of A n t i s e r a A g a i n s t C h o l e r a g e n o i d , H-LT, a n d P-LT
TABLE
Treatment Antiserum Goat anticholeragenoid
Adsorbed with
Eluted from
--
--CT -H-LT -P-LT --H-LT -CT -P-LT --P-LT -CT -H-LT
CT --
H-LT -P-LT -Goat anti-H-LT (H74-114)
--
H-LT -CT --
P-LT -Goat anti-P-LT (711/FILT)
--
P-LT -CT --
H-LT --
Enterotoxins ° CT
H-LT
P-LT
I b (1.2) c 0.003 1.0 0.35 0.35 0.50 0.20 0.36 0.04 0.22 0.03 0.25 0.13 0.13 0.04 0.006 0.07 0.003 0.03 0.07 0.06
0.70 d 0.003 0.65 0.006 0.50 0.50 0.50 1 b (3.0) 0.009 0.67 0.34 0.67 0.10 0.50 0.08 0.01 0.05 0.03 0.05 0.001 0.01
0.05 0.01 0.007 0.02 0.08 0.005 0.10 0.28 0.01 0.17 0.09 0.05 0.008 0.22 I b (1.3) 0.006 0.70 0.58 0.32 0.24 0.35
~Concentration of toxin as described in Table 1 bArbitrarily set at unity CActual titer x 105 (geometric mean of 3 titrations) dRelative activity in comparison with homologous system (data from two experiments)
Immunoaffinity
Column
Chromatography
I m m u n o a f f i n i t y c o l u m n c h r o m a t o g r a p h y w a s p e r f o r m e d as p r e v i o u s l y d e s c r i b e d ( C u a t r e c a s a s a n d A n f i n s e n , 1971; H o n d a et al., 1981a). 10 m g s a m p l e s of t h e p u r e t o x i n s CT, H-LT, o r P-LT, w e r e e a c h c o u p l e d to a p p r o x i m a t e l y I g m of c y a n o g e n b r o m i d e - a c t i v a t e d S e p h a r o s e 4B ( P h a r m a c i a F i n e C h e m i c a l s , Inc.). E a c h of t h r e e g o a t a n t i s e r a , a n t i c h o l e r a g e n o i d , a n t i - H - L T , a n d anti-P-LT, w a s a p p l i e d to a n i m m u n o a f f i n i t y c o l u m n o n w h i c h t h e h o m o l o g o u s t o x i n w a s c o u p l e d . E l u t i o n w i t h 0.5 M NaCI p l u s 0.2 M g l y c i n e - H C l , p H 2.7 y i e l d e d s p e c i f i c h o m o l o g o u s a n t i b o d y p r e p a r a t i o n s , w h i c h w e r e c a p t u r e d i n 4 v o l u m e s of T E A N buffer, p H 7.9 (Geary et al., 1982), a n d t h e n c o n c e n t r a t e d b y m e m b r a n e u l t r a f i l t r a t i o n a n d l y o p h i l i z e d . S a m p l e s of e a c h s e r u m w e r e a l s o a p p l i e d to i m m u n o a f f i n i t y c o l u m n s o n w h i c h t h e h e t e r o l o g o u s t o x i n s w e r e b o u n d . T h e f l o w t h r o u g h ( a d s o r b e d sera) c o n t a i n e d i m m u n o g l o b u l i n s d i r e c t e d against unique antigenic determinants not found on the bound toxin. The eluted i m m u n o g l o b u l i n r e c o g n i z e d c o m m o n d e t e r m i n a n t s o n all t h e t o x i n s .
RESULTS A l t h o u g h t h e r e is s o m e v a r i a t i o n a m o n g r e p e a t e d a s s a y s , t h e d a t a i n T a b l e 1 s h o w that sera directed against CT antigens neutralized H-LT considerably more effectively t h a n t h e y n e u t r a l i z e d P-LT. F o r e x a m p l e , t h e U.S. S t a n d a r d C h o l e r a A n t i t o x i n a n d
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the goat anticholeragenoid or anticholeragen sera (G5156-11) neutralized H-LT 70-90% as effectively as they neutralized CT. The same sera neutralized P-LT only 5-28% as effectively as they neutralized CT. Some anticholera sera, e.g., horse anticholeragen (E1006-A-21) and rabbit anticholeragen B subunit, appeared to neutralize H-LT better than the homologous toxin. These differences, which were at most twofold, may be attributable to experimental variability, or they could reflect fundamental differences in the availability or number of common antigenic sites recognized by the particular antisera. With the exceptions of the horse anticholeragen and the rabbit anticholera A subunit, each of the sera against cholera toxin antigens neutralized the porcine LT much less effectively than they neutralized CT or H-LT, with the relative potencies vs. P-LT ranging from 3-19%. The rabbit anticholera A subunit serum, although of lower titer than the other sera tested, neutralized both H-LT and P-LT to a similar degree, suggesting that exposed determinants of the A subunits of these three toxins may be more closely related than the B subunits. The higher relative potency of the horse anticholeragen for P-LT may thus reflect its anti-A activity. Although the goat anti-H-LT was more effective against the homologous toxin, it was relatively equally potent against both CT and P-LT (43% and 34%, respectively). Its content of anti-subunit A activity is not yet known. Antiserum against the porcine-LT was relatively ineffective against both CT and H-LT (12% and 21%, respectively), even though it had been shown to contain precipitating antibody against P-LT subunit A (Clements et al., 1980). Interestingly, some variation in cross-neutralization of cholera toxin from two different strains of V. cholerae was also revealed by this study. For example, the U.S. Standard cholera goat antitoxin and the horse anticholeragenoid neutralized CT isolated from strain 3083 only 60% and 50% as effectively as they neutralized CT from strain 569B. The S.S.V.I. cholera antitoxin was even less effective in neutralizing 3083 CT (6% when compared to 569B CT). In contrast, rabbit anti-3083-choleragenoid was equally effective for neutralization of 569B CT and 3083 CT. The heterologous antisera (anti-H-LT, or anti-P-LT) neutralized both CTs to approximately the same titer. The immunological specificities of selected goat serum preparations were examined in Ouchterlony double diffusion tests (Figure 1). The unchromatographed sara (Figure 1, A1, B1, C1) recognized all three toxins with typical spur formation, indicating the presence of unique antigenic determinants in the homologous systems. Similar spurring was also seen when immunoaffinity purified sera were reacted with heterologous toxins (Figure 1, A2, B2, C2}. In each case, the immunoadsorbed serum did not recognize the toxin bound to the column. Anti-H-LT serum, eluted from a choleragen column (A4), gave a line of identity with all three toxins. However, the anti-H-LT serum, which had been adsorbed with CT (A3}, recognized H-LT and, to a lesser degree, P-LT. Thus, H-LT and P-LT have common antigenic determinants which are not present on CT. The CT-adsorbed anti-P-LT serum (A5} recognized PLT more strongly than it recognized H-LT. Thus, even though the LTs have shared determinants not present on CT, they each also have unique specificities. The choleragen-eluted anti-P-LT (A6) precipitated CT and H-LT but gave a barely visible line of precipitation with P-LT. Anti-CT adsorbed with H-LT (B3) recognized CT but only gave a minimal reaction with P-LT while the eluted antibody (B4} recognized all the antigens similarly. Anti-P-LT adsorbed with H-LT (B5) recognized CT very weakly but still reacted with P-LT. The eluted serum (B6) was weakly reactive with all the toxins. These observations further indicate that CT and H-LT are more closely related antigenically than are CT and P-LT. Further, anticholeragenoid, adsorbed with P-LT
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FIGURE 1. CT, H, and P denote choleragen, human and porcine LT respectively. A1 = whole anticholeragenoid; A2 = immunopurified anticholeragenoid; A3 = anti-H-LT adsorbed with choleragen; A4 -- anti-H-LT eluted from choleragen; A5 = anti-P-LT adsorbed with choleragen; A6 = anti-P-LT eluted from choleragen. B1 = whole anti-H-LT; B2 = immunopurified antiH-LT; B3 = anticholeragenoid adsorbed with H-LT; B4 = anticholeragenoid eluted from antiH-LT; B5 = anti-P-LT adsorbed with H-LT; B6 = anti-P-LT eluted from H-LT. C1 = whole anti-P-LT; C2 = immunopurified anti-P-LT; C3 = anticholeragenoid adsorbed with P-LT; C4 -- anticholeragenoid eluted from P-LT; C5 = anti-H-LT adsorbed with P-LT; C6 = anti-H-LT eluted from P-LT. (Faint bands, such as those between A6 and P, B3 and CT and P, B5 and CT, and around B6, did not reproduce well in the photographs.)
(C3), still r e c o g n i z e d t h e o t h e r t o x i n s strongly. T h u s , m u c h of the a n t i b o d y is d i r e c t e d against c o m p o n e n t s w h i c h are n o t a v a i l a b l e o n P-LT a l t h o u g h , o n e l u t i o n (C4), t h e a d s o r b e d a n t i b o d i e s are s t r o n g l y r e a c t i v e w i t h all t h r e e toxins. A d s o r p t i o n of a n t i - H - L T w i t h P-LT (C5) left a c t i v i t y against b o t h CT a n d H-LT, a n d t h e e l u t e d a n t i b o d y w a s again r e a c t i v e w i t h all t h r e e t o x i n s (C6).
Immunological Differences Among Cholera/Coli
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Neutralization studies with these immunospecific sera (Table 2) yielded conclusions similar to those derived from the Ouchterlony assays. Adsorption of goat anticholeragenoid serum with either H-LT or P-LT removed most of the homologous antibody activity, but left nearly original titers of neutralizing activity against the other toxins. The eluted antibody was effective in neutralization of all three toxins. Similarly, with anti-H-LT, a single adsorption with the heterologous toxins removed most of the neutralizing activity for the individual adsorbing toxin but left residual homologous and heterologous activity. In the case of adsorption with P-LT, 10% of the activity against H-LT remained, but the titer against CT was only slightly affected. Similar trends were observed with the anti-P-LT although, because of the relatively low cross-neutralization activity, the results were less striking. DISCUSSION
The present data confirm and extend previous reports on both the immunologic relatedness as well as the immunologic differences among members of the cholera/ coli family of ADP-ribosylating, adenylate cyclase-activating, heat-labile enterotoxins. Ouchterlony-type precipitin analyses and neutralization tests with specifically immunoaffinity purified and adsorbed antisera, indicate that the pure enterotoxins (CT, H-LT, and P-LT) examined all share common antigens and, at the same time, have their own unique antigenic determinants. The results also reinforce previous observations (Finkelstein et al., 1974b; Vasil et al., 1974) which suggested that there may be some antigenic differences even in cholera toxins isolated from different strains of V. cholerae. The data also indicate that enterotoxin-type specific antibodies may be more important in neutralization, and possibly also in protection, than has previously been recognized. For example, as shown in Table 2, adsorption of anticholeragenoid with H-LT removes most of its neutralizing activity against H-LT (as expected), but leaves a third of its activity against CT. Similarly, adsorption of anti-H-LT with CT leaves about a third of the homologous activity. The observations are compatible in general with the operational model depicted in Figure 2. CT, H-LT, and P-LT are each depicted as having common determinants which are shared with the other two (Figure 2, C). Each has unique determinants (Figure 2 (clear areas)). Each may also share determinants with one, but not the other toxin (Figure 2, A, B, and D), and choleragan and H-LT appear to be more closely related to each other than P-LT is to each of the others. The observed immunologic differences probably reflect differences in the amino acid composition of the three toxins (Geary et al., 1982), as well as differences in the conformation of the toxin molecules and availability of antigenic sites. However, it should also be recognized that the model is somewhat simplistic in that it does not take into account the fact that the toxins may have multiple unique and specific antigenic determinants which may be recognized, by antibody, following some immunizing regimens in some animal species, but not in others. An example of this point is the Swiss Serum and Vaccine Institute cholera antitoxin (Table 1), which has a high titer of neutralizing activity against the homologous toxin, but relatively low activity against cholera enterotoxin from strain 3083 (and the other LTs), while other sara, e.g., U.S. standard cholera goat antitoxin and horse anticholeragenoid, exhibit different patterns. It will therefore be of interest to apply the absolute specificity of hybridomaderived monoclonal antibodies to the identification of the common and the unique antigenic determinants of each of the subunits of the various toxin species. Such
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B.A. Marchlewicz a n d R.A. Finkelstein
'.'.'.'.'.'.~ i::i:!:i:i:i:i::.:. .. . • e°° e°o°e e°°° o•
CT
iiiiiiiiiiiiiiiiiiiii P--LT
A-@ B-© C - i
D-@ FIGURE Z. Artistic representation of the distribution of unique (clear areas) and common (shaded areas) antigenic determinants of CT, H-LT, and P-LT. A = antigen(s) shared by CT and H-LT, exclusively; B = antigen(s) shared by CT and P-LT, exclusively; C = antigen(s) common to all three toxins; D = antigen(s} shared by P-LT and H-LT exclusively.
sera w i l l be useful in r a p i d detection and identification of these enter•toxins. It will also be i m p o r t a n t to d e t e r m i n e the full extent of the antigenic and structural drift in the cholera/coli family of heat-labile enter•toxins. These observations also have implications for the d e v e l o p m e n t of b r o a d - s p e c t r u m antitoxic i m m u n i t y . For example, specific protective i m m u n i t y against a cholera e n t e r • t o x i n m a y not be as effective against E. coil LT (or, for that matter, cholera e n t e r • t o x i n s from other strains of V. chalerae) as was p r e v i o u s l y generally presumed. The results m a y also lead to the d e v e l o p m e n t of synthetic antigens w h i c h will be capable of affording equal protection against all the m e m b e r s of the family.
We appreciate the technical assistance of Mr Dennis Palmer in the preparation of Y-1 adrenal cell cultures. This work was supported in part by U.S. Public Health Service grants AI-16776 and AI-17312 (to R.A.F.) under the U.S.-Japan Cooperative Medical Science Program from the National Institute of Allergy and Infectious Diseases.
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ADDENDUM Following s u b m i s s i o n of this paper, Robb et al. (Infect I m m u n 38:267, 1982) reported the isolation of a total of seven hybridoma cell lines w h i c h produced m o n o c l o n a l antibodies against cholera toxin. Of these, five were stated to b i n d to the A s u b u n i t s of CT, P-LT, and H-LT a n d had slight neutralizing activity against CT. The other two b o u n d to the B s u b u n i t s of CT, but not P-LT or H-LT, a n d had higher neutralizing activity against CT than did the anti-A m o n o c l o n a l sera. Neutralizing activity against the E. coli LTs was not tested. As far as they go, their results are i n accord with the present observations.
REFERENCES Clements JD, Finkelstein RA (1978a) Immunological cross-reactivity between a heat-labile enterotoxin(s) of Escherichia coli and subunits of Vibrio cholerae enterotoxin. Infect Immun 21:1036. Clements JD, Finkelstein RA (1978b) Demonstration of shared and unique immunological determinants in enterotoxins from V. eholerae and Escherichia coli. Infect Immun 22:709. Clements JD, Finkelstein RA (1979) Isolation and characterization of homogeneous heat-labile enterotoxin with high specific activity from Eseherichia coli cultures. Infect Immun 24:760. Clements JD, Yancey RJ, Finkelstein RA (1980) Properties of homogeneous heat-labile enterotoxins from Escherichia coli. Infect Immun 29:91. Cuatrecasas P, Anfinsen CB (1971) Affinity chromatography. Methods. Enzymol 22:345. Dallas WS, Falkow S (1980) Amino acid sequence homology between cholera toxin and Escherichia coli heat-labile toxin. Nature 288:499. Donta S (1974) Neutralization of cholera enterotoxin-induced steroidogenesis by specific antibody. J Infect Dis 129:284. Donta ST, Moon HW, Whipp SC (1974) Detection of heat-labile Eseherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science 183:334. Evans DJ, Chen LC, Curlin GT, Evans DG (1972) Stimulation of adenylate cyclase by Escherichia coli enterotoxin. Nature (London) New Biology 236:137. Evans DG, Evans DJ Jr, Gorbach SJ (1973) Identification of enterotoxigenic Escherichia coli and serum antitoxin activity by the vascular permeability factor assay. Infect Immun 8:731. Finkelstein RA (1970) Monospecific equine antiserum against cholera exo-enterotoxin. Infect Immun 2:691-697. Finkelstein RA, Boesman M, Neoh SH, LaRue MK, Delaney R (1974a) Dissociation and recombination of the subunits of the cholera enterotoxin (choleragen). J Immunol 113:145. Finkelstein RA, Fujita K, LoSpalluto JJ (1971) Procholeragenoid: an aggregated intermediate in the formation of choleragenoid. J Immunol 107:1043. Finkelstein RA, Vasil ML, Holmes RK (1974b) Studies on toxinogenesis in Vibrio cholerae. I. Isolation of mutants with altered toxigenicity. J Infect Dis 129:117. Geary SJ, Marchlewicz BA, Finkelstein RA (1982) Comparisons of heat-labile enterotoxins from porcine and human strains of Escherichia col/. Infect Immun 36:215. Gill DM, Clements JD, Robertson DC, Finkelstein RA (1981) Subunit number and arrangement in Escherichia coli heat-labile enterotoxin. Infect Immun 33:677. Gyles CL, Barnum DA (1969) A heat-labile enterotoxin from strains of Escherichia coli enteropathogenic for pigs. J Infect Dis 120:419. Hardegree MC (1979) Joint report of working groups for standardization of antitoxins. In Proceedings of the 14th Joint Conference of the U.S.-Japan Cooperative Medical Science Program Symposium on Cholera, Eds., K. Takeya and Y. Zinnaka. Tokyo: Fuji Printing Co., pp. 165-168. Holmes RK, Bramucci MC, Twiddy EM (1980) Genetic and biochemical studies of heat-labile enterotoxin of Escherichia coli. In Proceedings of the 15th Joint Conference on Cholera,
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U.S.-Japan Cooperative Medical Science Program Symposium NIH Publication No. 80-2003, pp. 187-201. Holmgren J (1973) Comparison of the tissue receptors for Vibrio cholerae and Escherichia call enterotoxins by means of gangliosides and natural cholera toxoid. Infect Immun 8:851. Holmgren J, Hanson LA, Carlson B, Lindblad BS, Rahimtoola J (1976) Neutralizing antibodies against gscherichia coli and Vibrio cholerae enterotoxins in human milk from a developing country. Scand J Immunol 5:867. Honda T, Finkelstein RA (1979): Selection and characteristics of a Vibrio cholerae mutant lacking the A (ADP-ribosylating) portion of the cholera enterotoxin. Proc Natl Acad Sci USA 76:2052. Honda T, Takeda Y, Miwatani T (1981a) Isolation of specific antibodies which react only with homologous enterotoxins from Vibrio cholerae and Escherichia coll. Infect Immun 34:333. Honda T, Tsuji T, Takeda Y, Miwatani T (1981b) Immunological nonidentity of heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli. Infect Immun 34:337. Kunkel SL, Robertson DC (1979) Purification and chemical characterization of the heat-labile enterotoxin produced by enterotoxigenic Escherichia coll. Infect Immun 25:586. Moss JJ, Garrison PH, Richardson SH (1979) Gangliasides sensitize fibroblasts to Escherichia coli heat-labile enterotoxin. J Clin Invest 64:381. Moss J, Vaughan M (1979) Activation of adenylate cyclase by choleragen. Ann Rev Biochem 48:581. Nalin DR, A1-Mahmud A, Curlin G, Ahmed A, Peterson JW (1974) Cholera toxoid boosts serum Escherichia coli antitoxin in humans. Infect Immun 10:747. Ouchterlony O (1949) Antigen-antibody reactions in gels. Acta Pathol Microbiol Scand 26:507. Pierce NF (1977) Protection against challenge with Escherichia coli heat-labile enterotoxin by immunization of rats with cholera toxin/toxoid. Infect lmmun 18:338. Sack DA, Sack RB (1975) Test for enterotoxigenic Escherichia coli in miniculture. Infect Immun 11:334. Smith NW, Sack RB (1973) Immunologic cross-reactions of enterotoxins from Escherichia coil and Vibrio cholerae. J Infect Dis 127:164. Stoliar OA, Pelley RP, Kaniecki-Green E, Klaus MH, Carpenter CCJ (1976) Secretory IgA against enterotoxins in breast-milk. Lancet, 1:1258. Svennerholm AM, Ahren C (1982) Immune protection against enterotoxigenic E. col/: search for synergy between antibodies to enterotoxin and somatic antigens. Acta Pathol Microbial Scand Sect C 90:1. Vasil ML, Holmes RK, Finkelstein RA (1974) Studies on toxinogenesis in Vibrio cholerae. II. An in vitro test for enterotoxin production. Infect Immun 9:195.
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Immunological Differences Among the Cholera/ Coli Family of Enterotoxins* Ben A. Marchlewicz** and Richard A. Finkelstein
Pure enterotoxins from two strains of Vibrio cholerae and choleragen-reloted heat-labile enterotoxins (LTs) from strains of Escherichia coli of human and porcine origin were examined by Ouchterlony-type immunodiffusion assays and by neutralization tests in Y-I adrenal cells using specific and immunopurified antisera. In accordance with previous findings, the results indicated that each of the toxins shared antigens with each of the others and that each, in addition, possessed unique antigenic determinants. The present study, however, indicates further that enterotoxin-type specific ant/bodies, in some instances, account for a major portion of the neutralizing activity. The full extent and importance of the antigenic drift in the cholera/ coli family of ADP-ribosylating, adenylate cyclase-activating, heat-labile enterotoxins remains to be determined. The results will be pertinent to efforts to develop broad-spectrum antitoxic immunity as well as in the development of methods for the rapid detection and identification of enterotoxigenic strains.
INTRODUCTION The heat-labile enterotoxin(s) (LTs) of Escherichia coli and the enterotoxin (choleragen) of Vibrio cholerae are similar to each other immunologically, structurally, and functionally (Clements and Finkelstein, 1978a, b, 1979; Clements et al., 1960; Dallas and Falkow, 1980; Gyles and Barnum, 1969; Holmes et al., 1960; and Kunkel and Robertson, 1979). These enterotoxins bind, through their B-regions, with host cell membrane receptors containing ganglioside GM1 (Holmgren, 1973; Moss and Vaughan, 1979) and activate adenylate cyclase, leading to increased intracellular levels of cyclic 3', 5'-adenosine monophosphate (cAMP) (Evans et al., 1972; Moss and Vaughan, 1979). However, despite their overall similarity, structural and immunological differences have been observed among choleragen (CT), LT produced by a human strain of enterotoxigenic E. coli (H-LT), and LT produced by porcine strains of enterotoxigenic E. cell (P-LT) (Geary et al., 1982; Gill et al., 1901; Honda et al., 1981a, 1981b). The neutralization of a porcine E. coil LT by antibody to cholera enterotoxin was first reported by Gyles and Barnum (1969). Since then, there have been several reports
*This work was presented in part at the International Symposium on Bacterial Diarrheal Diseases, Osaka, Japan, 23-25 March 1982. **Present address: Abbott Laboratories, Chicago, IL From the Department of Microbiology, University of Missouri School of Medicine, Columbia, Missouri. Address reprint requests to: Richard A. Finkelstein, Ph.D., Professor and Chairman, University of Missouri-Columbia, M-264 Medical Science Building, Columbia, Me 65212. Received November 3, 1982; accepted February 15, 1983.
© 1983Elsevier SciencePublishing Co., Inc., 52 Vanderbilt Avenue,New York, NY 10017
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of the neutralization of LT activity by cholera antitoxins in rabbit ileal loops (Smith and Sack, 1973}, in permeability factor assays (Evans et al., 1973}, and on adrenal cells (Donta, 1974}. In those studies, crude preparations of LT were used and the degree of neutralization was not quantitated. Nalin et al. (1974} examined the antibody activity against crude LT and CT (in permeability factor assays and by hemagglutination, respectively} in serum from individuals immunized with cholera toxoid and reported that the titers rose in parallel. However, whereas Stoliar et al. (1976} reported that secretory IgA from colostrum of Guatemalan women neutralized LT and CT to similar extents, Holmgren et al. {1976} found that Pakistani milk was more active against LT than against CT. Pierce {1977} immunized rats intraperitoneally with 100 p.g of partially purified formalin-treated cholera toxoid in Freund's complete adjuvant followed by intraduodenal boosting with two doses of 40 mg each of a crude cholera toxoid. Whereas the immunized rats exhibited reduced intestinal secretory responses to crude cholera toxin at doses of 1.5 and 6 mg, resistance to a crude E. coli LT preparation, while evident at low dose challenge (0.75 mg), was insignificant at 4-fold higher challenge. More recently, Svennerholm and Ahren (1982} likewise found that anticholera toxin antibodies were much more protective against live V. cholerae challenges, in rabbit ileal loops, than they were against challenge with live LT-producing E. coll. Thus, earlier observations suggested that although the enterotoxins are immunologically related, there are differences among them which could be an important factor in efforts to develop effective broad-spectrum antitoxic immunogens. In this report, we extend earlier findings by comparing the relative effectiveness of homologous and heterologous sera, as well as immunopurified sera, in the neutralization of biological activity of pure CT, H-LT, and P-LT.
MATERIALS AND METHODS Toxins
CT, H-LT, and P-LT were purified to homogeneity as described previously (Finkelstein et al., 1971; Geary et al., 1982; Clements and Finkelstein, 1979}. Purified cholera toxin from E1 Tor biotype, Ogawa serotype Vibrio cholerae strain 3083 (Honda and Finkelstein, 1979}, was also tested. Unless otherwise indicated, CT results were those obtained with toxin from classical biotype, Inaba serotype strain 569B. In immunodiffusion assays, modified from Ouchterlony (1949}, the purified toxins were used at 20 ~g per 50 p.l well. Sera
The sara used in this study included the U.S. Standard Lot 1 goat anticholera toxin obtained from Carolyn Hardegree (1979}; the Swiss Serum and Vaccine Institute (SSVI} equine anticholera immunoglobulins obtained from Carl Miller at NIH; equine anticholeragenoid (Finkelstein, 1970}; goat anticholeragenoid (Clements and Finkelstein, 1978a}; goat anticholeragen Lot G5156-11 Chemo-Sero-Therapeutic Research Institute (CSTRI}, Kumamoto, Japan, and CSTRI equine anti-choleragen Lot E1006-10-A21, both obtained from N. Ohtomo; specific rabbit antisera directed against isolated choleragen A and B subunits (Finkelstein et al., 1974a}, respectively, prepared by M. Boesman-Finkelstein in this laboratory; goat anti-H-LT {Geary et al., 1982) directed against LT isolated from strain H74-114; and goat anti-P-LT (Clements et al., 1980} directed against LT isolated from strain 711/FILT. A rabbit antiserum against choleragenoid from strain 3083 (Vasil et al., 1974} was also used.
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Neutralization Tests Neutralization tests were performed using the Y-1 adrenal cell assay (Donta et al., 1974; Sack a n d Sack, 1975). Briefly, serial dilutions of the sera were made i n Ham F-10 m e d i u m (Sack a n d Sack, 1975). A 100 ~1 sample containing 10 EDso doses of toxin i n Ham F-10 m e d i u m was added to the diluted sera, for CT, 1 EDso = 6pg; for both LTs, 1 EDso -- 12pg after trypsin activation as described in Clements and Finkelstein (1979). The toxin-sera mixtures were incubated at room temperature for 15 min. The culture m e d i u m on the Y-1 cells was removed by aspiration and toxin-sera mixtures were added to each well (final volume 200 p.1). The Y-1 cell plates were i n c u b a t e d at 37°C for 6 hrs i n a 5% CO2 atmosphere. After incubation, each plate was scored for cell rounding, observed using an inverted phase contrast microscope. The e n d p o i n t of the titration was the highest dilution of serum which allowed a 50% r o u n d i n g of the Y-1 cells. The neutralization data, i n Tables 1 and 2, are summarized as the relative potencies of the heterologous reactions, as compared with the homologous system (set at 1.0), and can thus be read as percent cross-reactivity. The actual titers of the homologous reactions are also provided.
TABLE 1. Homologous a n d Heterologous Neutralizing Activity of Various Antisera Against CT, H-LT, a n d P-LT Enterotoxina Antiserum U.S. standard cholera goat antitoxin (Lot 1) S.S.V.I. cholera antitoxin Goat anticholeragenoid Horse anticholeragenoid Goat anticholeragen (G5156-11) Horse anticholeragen (E 1006-10-A-21) Rabbit anticholera A subunit Rabbit anticholera B subunit Rabbit anticholeragenoid (3083) Goat anti-H-LT (H74-114) Goat anti-P-LT (711/FILT)
569B-CT
3083-CT
H-LT
P-LT
I b (3.4)c (1.3-10.2) e 1 (8.1) (3.2-10.2) 1 (12) (12) 1 (5.1) (2.5-10.2) 1 (8.1) (5.1-10.2) 1 (5.1) (5.1) 1 (0.64) (0.3-1.3) 1 (4.1) (2.5-10.2) 1 (10.9) (5.1-20.2)/ 0.43 (10.2-20.5) 0.12 (0.6-1.3)
0.60a (2.0)/ 0.06 (0.3-1.3)/ n.d.
0.75d (1.3-10.2) 0.10 (0.6-1.3) 0.70 (7.8-9.0) 0.12 (0.3-1.2) 0.89 (5.0-10.2) 2.0 (2.8-20.4) 0.50 (0.32) 1.80 (2.6-20.2) 0.33 (2.5-10.2)/ I b (30.6) (20-40) 0.21 (0.3-1.3)
0.28 (0.3-2.5) 0.06 (0.02-1.2) 0.05 (0.2-1,0) 0.09 (0.04-0.6) 0.19 (1.3-2.5) 0.42 (1.3-10.2) 0.50 (0.32) 0.09 (0.3-0.8) 0.125 (0.6-2.5)/ 0.34 (2.5-20.1) I b (6.67) (2.6-10.2)
0.50 (1.3-5.1) / n.d. n.d. n.d. n.d. 1 (10.2)/ 0.50 (10.2-40.9) f 0.11 (0.8)/
°10 EDsosof each toxin were added per 200 ~1 in each well (1 ED~oof CT, 6.25 pg/well;H-LT, 12.5 pg/ well; P-LT 12.5 pg/well) bArbitrarilyset at unity CActualtiter x 104 (geometric mean of 6 titrations) dRalative activity in comparisonwith homologoussystem {set at unity) "Range of values x 104 (6 titrations) tRange of yalues x 104 (3 titrations)
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2. Effect of I m m u n o a f f i n i t y A d s o r p t i o n o n N e u t r a l i z i n g A c t i v i t y of A n t i s e r a A g a i n s t C h o l e r a g e n o i d , H-LT, a n d P-LT
TABLE
Treatment Antiserum Goat anticholeragenoid
Adsorbed with
Eluted from
--
--CT -H-LT -P-LT --H-LT -CT -P-LT --P-LT -CT -H-LT
CT --
H-LT -P-LT -Goat anti-H-LT (H74-114)
--
H-LT -CT --
P-LT -Goat anti-P-LT (711/FILT)
--
P-LT -CT --
H-LT --
Enterotoxins ° CT
H-LT
P-LT
I b (1.2) c 0.003 1.0 0.35 0.35 0.50 0.20 0.36 0.04 0.22 0.03 0.25 0.13 0.13 0.04 0.006 0.07 0.003 0.03 0.07 0.06
0.70 d 0.003 0.65 0.006 0.50 0.50 0.50 1 b (3.0) 0.009 0.67 0.34 0.67 0.10 0.50 0.08 0.01 0.05 0.03 0.05 0.001 0.01
0.05 0.01 0.007 0.02 0.08 0.005 0.10 0.28 0.01 0.17 0.09 0.05 0.008 0.22 I b (1.3) 0.006 0.70 0.58 0.32 0.24 0.35
~Concentration of toxin as described in Table 1 bArbitrarily set at unity CActual titer x 105 (geometric mean of 3 titrations) dRelative activity in comparison with homologous system (data from two experiments)
Immunoaffinity
Column
Chromatography
I m m u n o a f f i n i t y c o l u m n c h r o m a t o g r a p h y w a s p e r f o r m e d as p r e v i o u s l y d e s c r i b e d ( C u a t r e c a s a s a n d A n f i n s e n , 1971; H o n d a et al., 1981a). 10 m g s a m p l e s of t h e p u r e t o x i n s CT, H-LT, o r P-LT, w e r e e a c h c o u p l e d to a p p r o x i m a t e l y I g m of c y a n o g e n b r o m i d e - a c t i v a t e d S e p h a r o s e 4B ( P h a r m a c i a F i n e C h e m i c a l s , Inc.). E a c h of t h r e e g o a t a n t i s e r a , a n t i c h o l e r a g e n o i d , a n t i - H - L T , a n d anti-P-LT, w a s a p p l i e d to a n i m m u n o a f f i n i t y c o l u m n o n w h i c h t h e h o m o l o g o u s t o x i n w a s c o u p l e d . E l u t i o n w i t h 0.5 M NaCI p l u s 0.2 M g l y c i n e - H C l , p H 2.7 y i e l d e d s p e c i f i c h o m o l o g o u s a n t i b o d y p r e p a r a t i o n s , w h i c h w e r e c a p t u r e d i n 4 v o l u m e s of T E A N buffer, p H 7.9 (Geary et al., 1982), a n d t h e n c o n c e n t r a t e d b y m e m b r a n e u l t r a f i l t r a t i o n a n d l y o p h i l i z e d . S a m p l e s of e a c h s e r u m w e r e a l s o a p p l i e d to i m m u n o a f f i n i t y c o l u m n s o n w h i c h t h e h e t e r o l o g o u s t o x i n s w e r e b o u n d . T h e f l o w t h r o u g h ( a d s o r b e d sera) c o n t a i n e d i m m u n o g l o b u l i n s d i r e c t e d against unique antigenic determinants not found on the bound toxin. The eluted i m m u n o g l o b u l i n r e c o g n i z e d c o m m o n d e t e r m i n a n t s o n all t h e t o x i n s .
RESULTS A l t h o u g h t h e r e is s o m e v a r i a t i o n a m o n g r e p e a t e d a s s a y s , t h e d a t a i n T a b l e 1 s h o w that sera directed against CT antigens neutralized H-LT considerably more effectively t h a n t h e y n e u t r a l i z e d P-LT. F o r e x a m p l e , t h e U.S. S t a n d a r d C h o l e r a A n t i t o x i n a n d
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the goat anticholeragenoid or anticholeragen sera (G5156-11) neutralized H-LT 70-90% as effectively as they neutralized CT. The same sera neutralized P-LT only 5-28% as effectively as they neutralized CT. Some anticholera sera, e.g., horse anticholeragen (E1006-A-21) and rabbit anticholeragen B subunit, appeared to neutralize H-LT better than the homologous toxin. These differences, which were at most twofold, may be attributable to experimental variability, or they could reflect fundamental differences in the availability or number of common antigenic sites recognized by the particular antisera. With the exceptions of the horse anticholeragen and the rabbit anticholera A subunit, each of the sera against cholera toxin antigens neutralized the porcine LT much less effectively than they neutralized CT or H-LT, with the relative potencies vs. P-LT ranging from 3-19%. The rabbit anticholera A subunit serum, although of lower titer than the other sera tested, neutralized both H-LT and P-LT to a similar degree, suggesting that exposed determinants of the A subunits of these three toxins may be more closely related than the B subunits. The higher relative potency of the horse anticholeragen for P-LT may thus reflect its anti-A activity. Although the goat anti-H-LT was more effective against the homologous toxin, it was relatively equally potent against both CT and P-LT (43% and 34%, respectively). Its content of anti-subunit A activity is not yet known. Antiserum against the porcine-LT was relatively ineffective against both CT and H-LT (12% and 21%, respectively), even though it had been shown to contain precipitating antibody against P-LT subunit A (Clements et al., 1980). Interestingly, some variation in cross-neutralization of cholera toxin from two different strains of V. cholerae was also revealed by this study. For example, the U.S. Standard cholera goat antitoxin and the horse anticholeragenoid neutralized CT isolated from strain 3083 only 60% and 50% as effectively as they neutralized CT from strain 569B. The S.S.V.I. cholera antitoxin was even less effective in neutralizing 3083 CT (6% when compared to 569B CT). In contrast, rabbit anti-3083-choleragenoid was equally effective for neutralization of 569B CT and 3083 CT. The heterologous antisera (anti-H-LT, or anti-P-LT) neutralized both CTs to approximately the same titer. The immunological specificities of selected goat serum preparations were examined in Ouchterlony double diffusion tests (Figure 1). The unchromatographed sara (Figure 1, A1, B1, C1) recognized all three toxins with typical spur formation, indicating the presence of unique antigenic determinants in the homologous systems. Similar spurring was also seen when immunoaffinity purified sera were reacted with heterologous toxins (Figure 1, A2, B2, C2}. In each case, the immunoadsorbed serum did not recognize the toxin bound to the column. Anti-H-LT serum, eluted from a choleragen column (A4), gave a line of identity with all three toxins. However, the anti-H-LT serum, which had been adsorbed with CT (A3}, recognized H-LT and, to a lesser degree, P-LT. Thus, H-LT and P-LT have common antigenic determinants which are not present on CT. The CT-adsorbed anti-P-LT serum (A5} recognized PLT more strongly than it recognized H-LT. Thus, even though the LTs have shared determinants not present on CT, they each also have unique specificities. The choleragen-eluted anti-P-LT (A6) precipitated CT and H-LT but gave a barely visible line of precipitation with P-LT. Anti-CT adsorbed with H-LT (B3) recognized CT but only gave a minimal reaction with P-LT while the eluted antibody (B4} recognized all the antigens similarly. Anti-P-LT adsorbed with H-LT (B5) recognized CT very weakly but still reacted with P-LT. The eluted serum (B6) was weakly reactive with all the toxins. These observations further indicate that CT and H-LT are more closely related antigenically than are CT and P-LT. Further, anticholeragenoid, adsorbed with P-LT
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FIGURE 1. CT, H, and P denote choleragen, human and porcine LT respectively. A1 = whole anticholeragenoid; A2 = immunopurified anticholeragenoid; A3 = anti-H-LT adsorbed with choleragen; A4 -- anti-H-LT eluted from choleragen; A5 = anti-P-LT adsorbed with choleragen; A6 = anti-P-LT eluted from choleragen. B1 = whole anti-H-LT; B2 = immunopurified antiH-LT; B3 = anticholeragenoid adsorbed with H-LT; B4 = anticholeragenoid eluted from antiH-LT; B5 = anti-P-LT adsorbed with H-LT; B6 = anti-P-LT eluted from H-LT. C1 = whole anti-P-LT; C2 = immunopurified anti-P-LT; C3 = anticholeragenoid adsorbed with P-LT; C4 -- anticholeragenoid eluted from P-LT; C5 = anti-H-LT adsorbed with P-LT; C6 = anti-H-LT eluted from P-LT. (Faint bands, such as those between A6 and P, B3 and CT and P, B5 and CT, and around B6, did not reproduce well in the photographs.)
(C3), still r e c o g n i z e d t h e o t h e r t o x i n s strongly. T h u s , m u c h of the a n t i b o d y is d i r e c t e d against c o m p o n e n t s w h i c h are n o t a v a i l a b l e o n P-LT a l t h o u g h , o n e l u t i o n (C4), t h e a d s o r b e d a n t i b o d i e s are s t r o n g l y r e a c t i v e w i t h all t h r e e toxins. A d s o r p t i o n of a n t i - H - L T w i t h P-LT (C5) left a c t i v i t y against b o t h CT a n d H-LT, a n d t h e e l u t e d a n t i b o d y w a s again r e a c t i v e w i t h all t h r e e t o x i n s (C6).
Immunological Differences Among Cholera/Coli
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Neutralization studies with these immunospecific sera (Table 2) yielded conclusions similar to those derived from the Ouchterlony assays. Adsorption of goat anticholeragenoid serum with either H-LT or P-LT removed most of the homologous antibody activity, but left nearly original titers of neutralizing activity against the other toxins. The eluted antibody was effective in neutralization of all three toxins. Similarly, with anti-H-LT, a single adsorption with the heterologous toxins removed most of the neutralizing activity for the individual adsorbing toxin but left residual homologous and heterologous activity. In the case of adsorption with P-LT, 10% of the activity against H-LT remained, but the titer against CT was only slightly affected. Similar trends were observed with the anti-P-LT although, because of the relatively low cross-neutralization activity, the results were less striking. DISCUSSION
The present data confirm and extend previous reports on both the immunologic relatedness as well as the immunologic differences among members of the cholera/ coli family of ADP-ribosylating, adenylate cyclase-activating, heat-labile enterotoxins. Ouchterlony-type precipitin analyses and neutralization tests with specifically immunoaffinity purified and adsorbed antisera, indicate that the pure enterotoxins (CT, H-LT, and P-LT) examined all share common antigens and, at the same time, have their own unique antigenic determinants. The results also reinforce previous observations (Finkelstein et al., 1974b; Vasil et al., 1974) which suggested that there may be some antigenic differences even in cholera toxins isolated from different strains of V. cholerae. The data also indicate that enterotoxin-type specific antibodies may be more important in neutralization, and possibly also in protection, than has previously been recognized. For example, as shown in Table 2, adsorption of anticholeragenoid with H-LT removes most of its neutralizing activity against H-LT (as expected), but leaves a third of its activity against CT. Similarly, adsorption of anti-H-LT with CT leaves about a third of the homologous activity. The observations are compatible in general with the operational model depicted in Figure 2. CT, H-LT, and P-LT are each depicted as having common determinants which are shared with the other two (Figure 2, C). Each has unique determinants (Figure 2 (clear areas)). Each may also share determinants with one, but not the other toxin (Figure 2, A, B, and D), and choleragan and H-LT appear to be more closely related to each other than P-LT is to each of the others. The observed immunologic differences probably reflect differences in the amino acid composition of the three toxins (Geary et al., 1982), as well as differences in the conformation of the toxin molecules and availability of antigenic sites. However, it should also be recognized that the model is somewhat simplistic in that it does not take into account the fact that the toxins may have multiple unique and specific antigenic determinants which may be recognized, by antibody, following some immunizing regimens in some animal species, but not in others. An example of this point is the Swiss Serum and Vaccine Institute cholera antitoxin (Table 1), which has a high titer of neutralizing activity against the homologous toxin, but relatively low activity against cholera enterotoxin from strain 3083 (and the other LTs), while other sara, e.g., U.S. standard cholera goat antitoxin and horse anticholeragenoid, exhibit different patterns. It will therefore be of interest to apply the absolute specificity of hybridomaderived monoclonal antibodies to the identification of the common and the unique antigenic determinants of each of the subunits of the various toxin species. Such
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'.'.'.'.'.'.~ i::i:!:i:i:i:i::.:. .. . • e°° e°o°e e°°° o•
CT
iiiiiiiiiiiiiiiiiiiii P--LT
A-@ B-© C - i
D-@ FIGURE Z. Artistic representation of the distribution of unique (clear areas) and common (shaded areas) antigenic determinants of CT, H-LT, and P-LT. A = antigen(s) shared by CT and H-LT, exclusively; B = antigen(s) shared by CT and P-LT, exclusively; C = antigen(s) common to all three toxins; D = antigen(s} shared by P-LT and H-LT exclusively.
sera w i l l be useful in r a p i d detection and identification of these enter•toxins. It will also be i m p o r t a n t to d e t e r m i n e the full extent of the antigenic and structural drift in the cholera/coli family of heat-labile enter•toxins. These observations also have implications for the d e v e l o p m e n t of b r o a d - s p e c t r u m antitoxic i m m u n i t y . For example, specific protective i m m u n i t y against a cholera e n t e r • t o x i n m a y not be as effective against E. coil LT (or, for that matter, cholera e n t e r • t o x i n s from other strains of V. chalerae) as was p r e v i o u s l y generally presumed. The results m a y also lead to the d e v e l o p m e n t of synthetic antigens w h i c h will be capable of affording equal protection against all the m e m b e r s of the family.
We appreciate the technical assistance of Mr Dennis Palmer in the preparation of Y-1 adrenal cell cultures. This work was supported in part by U.S. Public Health Service grants AI-16776 and AI-17312 (to R.A.F.) under the U.S.-Japan Cooperative Medical Science Program from the National Institute of Allergy and Infectious Diseases.
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ADDENDUM Following s u b m i s s i o n of this paper, Robb et al. (Infect I m m u n 38:267, 1982) reported the isolation of a total of seven hybridoma cell lines w h i c h produced m o n o c l o n a l antibodies against cholera toxin. Of these, five were stated to b i n d to the A s u b u n i t s of CT, P-LT, and H-LT a n d had slight neutralizing activity against CT. The other two b o u n d to the B s u b u n i t s of CT, but not P-LT or H-LT, a n d had higher neutralizing activity against CT than did the anti-A m o n o c l o n a l sera. Neutralizing activity against the E. coli LTs was not tested. As far as they go, their results are i n accord with the present observations.
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