With Glutaraldehyde*

CHOLERA

Experiments with cholera toxin detoxified with glutaraldehyde*

M. SALETTI 1 & A. RICCI 2

Studies on the production, purification, and detoxification of cholera toxin are reported. The toxin was first partially purified with aluminium hydroxide andfurther purification was effected with Bio Gel A-Sm and Sephadex G-75. The toxins were detoxified with glutaraldehyde; the toxoids so obtained, when injected into the skin of rabbits, appeared to have- no residual toxicity. All the toxins were immunogenic. The toxoid partially purified with aluminium hydroxide retained most of its immunizing capacity, but the highly purified toxoids did not retain their capacity to stimulate antibody production in rabbits.

In a previous study (1) partially purified and incubated with 200 ml of broth culture. Incubation concentrated cholera toxin was prepared by the was at 30°C for 18 h with aeration, automatic method of Spyrides & Feeley (2). This toxin, after control of oxygen pressure at 2 kPa,a pH at 7.6, and testing, was detoxified with glutaraldehyde. When stirring at 750 r/min. The vibrios were killed with injected into the skin of rabbits it appeared to have 0.01 % thiomersal. no residual toxicity but retained its ability to induce immunity. Purification of the toxin In the present study the immunizing capacity of The crude toxin, after separation from the vibrios cholera toxin detoxified with glutaraldehyde was by centrifugation, was purified and concentrated controlled at the different stages of purification. with aluminium hydroxide according to the method of Spyrides & Feeley (2). The toxin thus prepared was ultrafiltered by means of an Amicon cell with a MATERIALS AND METHODS PM 20 selective membrane and then separated on a Preparation of the broth culture 5 x 100-cm column of Bio Gel A-Sm. The active peak was then concentrated and transferred to a The highly toxinogenic strain 569 B serotype Inaba double column of Sephadex G-75 according to the of Vibrio cholerae, supplied in 1970 by Dr Feeley of method of Finkelstein & LoSpalluto (4). the National Institute of Health, Bethesda, MD, USA, was used. It was stored in a freeze-dried state Detoxification at 4°C and rehydrated before use. When rehydrated The toxins at the 3 stages of purification (called it was streaked on heart infusion agar at pH 7.4 and toxin aluminium hydroxide, toxin Bio Gel, and toxin incubated at 37°C for 18 h. A suspension was then Sephadex, respectively) were dialysed in phosphate- made in saline solution comprising 4 colonies per ml. buffered saline at pH 7.2 and treated with 0.01 ml Two ml of this suspension was introduced into an of a 25% glutaraldehyde solution per 100 ml of Erlenmeyer flask containing 200 ml of Syncase medi- toxin at a protein concentration of 0.15 mg/ml um (3) and shaken for 7 h at 35°C. (140 mol of glutaraldehyde per mol of toxin). The The cholera toxin was produced in a fermenter optimum amount of glutaraldehyde was established containing 40 litres of Syncase medium, which was in pieliminary tests. Detoxification was carried out at 37°C and the samples were then dialysed in * From Centro Richerche, Istituto Sieroterapico e Vac- phosphate-buffered saline at pH 7.2 and filtered cinogeno Toscano " A. Sclavo ", Siena, Italy. with a Millipore 0.22 filter. 1 Associate Director. 2 Chief, Microbiology Unit. aI mmHg- 1,33x102Pa.

3309 - 633 BULL. WORLD HEALTH ORGAN., Vol. 51, 1974 4* 634 M. SALErTI & A. RICCI

Reference toxin (ph 7.4) were added to an equal volume of reference cholera toxin containing 20 BD per ml. The mixture A toxin partially purified with aluminium hydrox- was incubated at 37°C for 1 h, and 0.1-ml doses were ide and maintained at -80°C was used. then injected intracutaneously in the shaved dorsal area of New Zealand rabbits. After 23 h Evans blue Immunization was injected intravenously and 1 h later the diameter Rabbits were immunized with toxin or toxoid each of blueing was measured. The smallest quantity of at the 3 levels of purity. All the animals received serum capable of reducing the diameter of blueing subcutaneously in the inguinal region 100 ,ug of caused by 1 BD from 7 mm to 4 mm was taken to antigen as protein in Freund's adjuvant. Four weeks represent 1 antitoxin unit. later the rabbits were injected with 100 ,ug of protein in phosphate-buffered saline at pH 7.2 in the same Challenge dose region. After 10 days a blood sample was taken and Previously vaccinated rabbits (13-15) were inocul- a challenge dose given. ated intracutaneously in the shaved dorsal area with challenge doses of 0.1 ml of toxin in concentrations Tests of 3, 9, 27, and 81 BD. After 23 h an intravenous The vibriocidal antibodies were titrated with the injection of Evans blue was given and the diameter technique described by Verwey et al. (5). of blueing was measured 1 h later. Protein was determined by the Lowry micro- method (6). The polysaccharide content of the toxins and toxoids was determined according to the method RESULTS of Winzler (7). Electrophoresis was effected on acryl- amide by the method of Ornstein (8). Haemagglutin- During the stages of purification of the toxin there ation tests were carried out according to the method was a considerable reduction in its polysaccharide of Boyden (9) as modified by Schneibel (10). content, as shown in Table 1. After Sephadex G-75 Ouchterlony-type immunodiffusion tests were per- treatment the polysaccharides completely disap- formed at room temperature in agar with antiserum peared. The BD values did not increase during prepared in rabbits. One hundred ,ug of toxin purified purification. with aluminium hydroxide was injected subcutane- Fig. 1 shows the results of the immunodiffusion ously in the inguinal region. A second injection was tests. The successive stages of purification of the given 4 weeks later. toxins showed a decrease in the number of bands of precipitation. With the toxoids there was a decrease Vascular permneability factor (blueing dose) in band intensity; this may have been caused by a partial alteration in antigenicity owing to glutaral- The vascular permeability factor of the toxin was dehyde treatment. assayed by intracutaneous injections of serial dilu- In Fig. 2 the results of polyacrylamide gel electro- tions of the toxin in a total volume of 0.1 ml into the phoresis of the toxins and toxoids are given. The shaved dorsal area of New Zealand rabbits, accord- successive stages of purification of the toxins showed ing to the technique described by Craig (11, 12). a decrease in the number of electrophoretic bands, After 23 h the animals were given an intravenous leaving only the specific band for the toxin. With the injection of Evans blue and 1 h later the average toxoids there was a diffuse band owing to the diameter of blueing of the skin lesions was measured. formation of polydispersed material. The dilution that gave 7 mm diameter of blueing was Immunization of the rabbits produced the results selected to represent 1 blueing dose (BD) when the shown in Table 2. The rabbits immunized with standard deviation was not greater than 1.1. toxins showed a reduced anti-Inaba vibriocidal activity related to the loss of polysaccharides during Titration ofantitoxin units purification. The toxoid-immunized rabbits showed The protective capacity of the serum of the levels of vibriocidal antibodies similar to those pro- vaccinated rabbits was measured by means of a duced by the toxins. Antitoxin levels were higher in modification of the technique described by Craig the rabbits that received the toxins than in those that (13). Serial 0.30 log dilutions of serum in 0.007 received the toxoids, but there was little difference mol/litre phosphate buffer with 0.01 % gelatin among the values for the 3 toxins. The toxoid A B c ...7. .. 7....7. ... 71.; :3:i l V.z1It _:f " L5wai'S l777 : _

D E F r~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~IT 7 I7 : .4

W. 754-65 WHO 75465

Fig. 1. Results of the immunodiffusion tests. The outer wells contain toxin or toxoid in the various stages of purification. Starting at the left hand side and proceeding clockwise the wells contain dilutions of 1:4 and 1: 2 alternately. A, toxin aluminium hydroxide; B, toxin Bio Gel; C, toxin Sephadex; D, toxoid aluminium hydroxide; E, toxoid Bio Gel; F, toxoid Sephadex. A B C

D E F

Fig. 2. Results of polyacrylamide gel electrophoresis. A, toxin aluminium hydroxide; B, toxin Bio Gel; C, toxin Sephadex; D, toxoid aluminium hydroxide; E, toxoid Bio Gel; F, toxoid Sephadex. GLUTARALDEHYDE DETOXIFICATION OF CHOLERA TOXIN 637

Table 1. Proteins, polysaccharides and blueing doses in samples of cholera toxins and toxoids

Proteins Polysaccharides Percentage of Poec B10 Toxin or toxoid (mg/litre) (mg/litre) p rotei Ag of protein)

toxin aluminium hydroxide 310 60 19.4 1 140 toxin Bio Gel 550 13 2.4 1 000 toxin Sephadex 200 - 0.0 1 060 toxoid aluminium hydroxide 200 40 20.0 - toxoid Bio Gel 540 10 1.9 toxoid Sephadex 200 - 0.0

Table 2. Cholera antitoxin, anti-Inaba vibriocidal, and derived from the toxin partially purified with alu- haemagglutination levels in rabbits immunized with minium hydroxide was still capable of stimulating cholera toxin or toxoid in 2 doses each of 100 pg protein antitoxins although at a slightly lower level than that obtained with the toxins. The toxoids derived from Antivibrio- Antitoxin Haemag- the toxins further purified with Bio Gel and/or units/mla units/ml a glutination a Sephadex did not stimulate antitoxin production. A close relationship was found between the antitoxin toxin aluminium hydroxide 110 000 1 000 350 titre as determined by haemagglutination and that toxin Bio Gel 395 1 280 180 determined by neutralization of the vascular permeability factor. toxin Sephadex <50 1 100 200 Reactions to the challenge dose of cholera toxin toxoid aluminium hydroxide 100 000 720 360 are shown in Table 3. Where there were antitoxins toxoid Bio Gel 79 <20 <8 present there was neutralization of the inoculated toxoid Sephadex <50 <20 <8 toxin and therefore a decrease in the effect caused by saline (control) <50 <20 <8 the vascular permeability factor. In the absence of antitoxins the effect was similar to that obtained in a Geometric mean of 5 rabbits. the control rabbits.

Table 3. Diameter in cm of blueing produced by a challenge dose of cholera toxin in previously vaccinated rabbits (mean of 5 rabbits)

Blueing doses inoculated Toxin or toxoid 3 9 27 81

toxin aluminium hydroxide 1.0 (0.53) a 2.8 (1.12) 4.0 (1.41) 5.2 (1.05) toxin Bio Gel 1.1 (0.58) 3.0 (0.92) 2.2 (1.22) 2.5 (1.12) toxin Sephadex 0.4 (0.45) 3.5 (0.81) 4.2 (1.20) 4.8 (1.07) toxoid aluminium hydroxide 1.1 (0.60) 3.4 (1.20) 4.9 (1.04) 6.2 (1.43) toxoid Bio Gel 7.0 (1.18) 8.4 (1.39) 9.12 (1.24) 12.4 (1.97) toxoid Sephadex 7.4 (1.05) 8.7 (1.10) 1.10 (1.21) 11.5 (1.08) saline (control) 9.4 (1.1) 10.4 (1.22) 11.5 (1.06) 12.8 (1.10)

a Standard deviations are given in parentheses. 638 M. SALETTI & A. RICCI

DISCUSSION taneously. The production of vibriocidal antibodies was proportionate to the polysaccharide content of The purification of cholera toxin with Bio Gel A- the toxin. The values for haemagglutination did not 5m and double column Sephadex G-75 as previously show any significant differences. carried out by Finkelstein & LoSpalluto (4) results in the disappearance of polysaccharide. The toxin prep- Detoxification with glutaraldehyde caused a loss aration reported herein had a specific activity of of toxic activity, which is demonstrated by the 10 BD/,ug; the preparations of Finkelstein & LoSpal- differences in the reactions caused by the vascular luto (16) and Richardson (17) had a specific activity permeability factor. However, only the toxin which of 2 000 BD/,ug. was detoxified after partial purification with alumi- The ratio of mg of protein to BD at the various nium hydroxide retained its immunizing capacity; stages of purification remained more or less constant. the highly purified toxoids completely lost their In effect, during purification there was a separation ability to stimulate the production of antitoxins. of choleragen from the other proteins. This may This shows that treatment of highly purified toxin have been due either to structural changes in the with glutaraldehyde causes such a change in the choleragen with a consequent loss of activity by the antigen that it is no longer able to maintain an cholera toxin, or to the fact that polysaccharides, immunizing effect. which disappear during the later stages of purifica- Glutaraldehyde is clearly unsuitable for detoxify- tion, act as an adjuvant to the action of the toxin. ing cholera toxin that is to be used for tests on man. We are inclined to support the former hypothesis. Further studies will be necessary in order to find a The toxin at all three levels of purity stimulated substance capable of detoxifying highly purified toxin antitoxin production in animals vaccinated subcu- without destroying its immunizing capacity.

RI:SUMIt EXPERIENCES REALISEES AVEC LA TOXINE CHOLE'RIQUE DE'TOXIFIE'E PAR LE GLUTARALDEHYDE

Des recherches ont et6 menees sur la production, la dans la peau du lapin, semblaient n'avoir conserve purification et la detoxication de la toxine cholerique. La aucune toxicit6 residuelle. Avant detoxication, toutes les toxine cholerique a ete preparee en cuve de fermentation toxines possedaient un pouvoir immunisant. Mais, si dans 40 litres de milieu A la Syncase, ensemence avec la l'anatoxine traitee a l'hydroxyde d'aluminium gardait souche 569 B Inaba de V. Cholerae. Apres incubation a une activite immunisante, fuit-elle legerement reduite, les 30°C pendant 24 heures, avec a6ration et agitation i anatoxines hautement purifiees sur Bio Gel A-5 m et 750 r/min, les micro-organismes ont ete tu6s par addition Sephadex G-75 perdaient, dans les conditions de notre de thiomersal. La toxine a d'abord ete partiellement experience, leur capacit6 de susciter des anticorps anti- purifiee par l'hydroxyde d'aluminium, et la purification toxiques. En vue d'eprouver sur l'homme le pouvoir a ete poursuivie at l'aide de Bio Gel A-5 m, puis de immunisant de l'anatoxine cholerique, de nouvelles Sephadex G-75. Les toxines ont ete detoxifiees par le etudes seront necessaires pour decouvrir une substance glutarald6hyde. Les anatoxines ainsi obtenues (hydroxyde capable de detoxifier la toxine tres purifiee tout en main- d'aluminium, Bio Gel A-5 m et Sephadex G-75), injectees tenant son activite immunisante.

REFERENCES

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