Experiments with cholera toxin detoxified 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 concentrated cholera toxin was prepared by the method of Spyrides & Feeley (2). This toxin, after testing, was detoxified with glutaraldehyde. When injected into the skin of rabbits it appeared to have no residual toxicity but retained its ability to induce immunity. In the present study the immunizing capacity of cholera toxin detoxified with glutaraldehyde was controlled at the different stages of purification. MATERIALS AND METHODS Preparation of the broth culture The highly toxinogenic strain 569 B serotype Inaba of Vibrio cholerae, supplied in 1970 by Dr Feeley of the National Institute of Health, Bethesda, MD, USA, was used. It was stored in a freeze-dried state at 4°C and rehydrated before use. When rehydrated it was streaked on heart infusion agar at pH 7.4 and incubated at 37°C for 18 h. A suspension was then made in saline solution comprising 4 colonies per ml. Two ml of this suspension was introduced into an Erlenmeyer flask containing 200 ml of Syncase medi- um (3) and shaken for 7 h at 35°C. The cholera toxin was produced in a fermenter containing 40 litres of Syncase medium, which was * From Centro Richerche, Istituto Sieroterapico e Vac- cinogeno Toscano " A. Sclavo ", Siena, Italy. 1 Associate Director. 2 Chief, Microbiology Unit. incubated with 200 ml of broth culture. Incubation was at 30°C for 18 h with aeration, automatic control of oxygen pressure at 2 kPa,a pH at 7.6, and stirring at 750 r/min. The vibrios were killed with 0.01 % thiomersal. Purification of the toxin The crude toxin, after separation from the vibrios by centrifugation, was purified and concentrated 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 PM 20 selective membrane and then separated on a 5 x 100-cm column of Bio Gel A-Sm. The active peak was then concentrated and transferred to a double column of Sephadex G-75 according to the method of Finkelstein & LoSpalluto (4). Detoxification The toxins at the 3 stages of purification (called toxin aluminium hydroxide, toxin Bio Gel, and toxin Sephadex, respectively) were dialysed in phosphate- buffered saline at pH 7.2 and treated with 0.01 ml of a 25% glutaraldehyde solution per 100 ml of toxin at a protein concentration of 0.15 mg/ml (140 mol of glutaraldehyde per mol of toxin). The optimum amount of glutaraldehyde was established in pieliminary tests. Detoxification was carried out at 37°C and the samples were then dialysed in phosphate-buffered saline at pH 7.2 and filtered with a Millipore 0.22 filter. aI mmHg- 1,33x102Pa. 3309 - 633 BULL. WORLD HEALTH ORGAN., Vol. 51, 1974 4* M. SALErTI & A. RICCI Reference toxin A toxin partially purified with aluminium hydrox- ide and maintained at -80°C was used. Immunization Rabbits were immunized with toxin or toxoid each at the 3 levels of purity. All the animals received subcutaneously in the inguinal region 100 ,ug of antigen as protein in Freund's adjuvant. Four weeks later the rabbits were injected with 100 ,ug of protein in phosphate-buffered saline at pH 7.2 in the same region. After 10 days a blood sample was taken and a challenge dose given. Tests The vibriocidal antibodies were titrated with the technique described by Verwey et al. (5). Protein was determined by the Lowry micro- method (6). The polysaccharide content of the toxins and toxoids was determined according to the method of Winzler (7). Electrophoresis was effected on acryl- amide by the method of Ornstein (8). Haemagglutin- ation tests were carried out according to the method of Boyden (9) as modified by Schneibel (10). Ouchterlony-type immunodiffusion tests were per- formed at room temperature in agar with antiserum prepared in rabbits. One hundred ,ug of toxin purified with aluminium hydroxide was injected subcutane- ously in the inguinal region. A second injection was given 4 weeks later. Vascular permneability factor (blueing dose) The vascular permeability factor of the toxin was assayed by intracutaneous injections of serial dilu- tions of the toxin in a total volume of 0.1 ml into the shaved dorsal area of New Zealand rabbits, accord- ing to the technique described by Craig (11, 12). After 23 h the animals were given an intravenous injection of Evans blue and 1 h later the average diameter of blueing of the skin lesions was measured. The dilution that gave 7 mm diameter of blueing was selected to represent 1 blueing dose (BD) when the standard deviation was not greater than 1.1. Titration of antitoxin units The protective capacity of the serum of the vaccinated rabbits was measured by means of a modification of the technique described by Craig (13). Serial 0.30 log dilutions of serum in 0.007 mol/litre phosphate buffer with 0.01 % gelatin (ph 7.4) were added to an equal volume of reference cholera toxin containing 20 BD per ml. The mixture was incubated at 37°C for 1 h, and 0.1-ml doses were then injected intracutaneously in the shaved dorsal area of New Zealand rabbits. After 23 h Evans blue was injected intravenously and 1 h later the diameter of blueing was measured. The smallest quantity of serum capable of reducing the diameter of blueing caused by 1 BD from 7 mm to 4 mm was taken to represent 1 antitoxin unit. Challenge dose Previously vaccinated rabbits (13-15) were inocul- ated intracutaneously in the shaved dorsal area with challenge doses of 0.1 ml of toxin in concentrations of 3, 9, 27, and 81 BD. After 23 h an intravenous injection of Evans blue was given and the diameter of blueing was measured 1 h later. RESULTS During the stages of purification of the toxin there was a considerable reduction in its polysaccharide content, as shown in Table 1. After Sephadex G-75 treatment the polysaccharides completely disap- peared. The BD values did not increase during purification. Fig. 1 shows the results of the immunodiffusion tests. The successive stages of purification of the toxins showed a decrease in the number of bands of precipitation. With the toxoids there was a decrease in band intensity; this may have been caused by a partial alteration in antigenicity owing to glutaral- dehyde treatment. In Fig. 2 the results of polyacrylamide gel electro- phoresis of the toxins and toxoids are given. The successive stages of purification of the toxins showed a decrease in the number of electrophoretic bands, leaving only the specific band for the toxin. With the toxoids there was a diffuse band owing to the formation of polydispersed material. Immunization of the rabbits produced the results shown in Table 2. The rabbits immunized with toxins showed a reduced anti-Inaba vibriocidal ac- tivity related to the loss of polysaccharides during purification. The toxoid-immunized rabbits showed levels of vibriocidal antibodies similar to those pro- duced by the toxins. Antitoxin levels were higher in the rabbits that received the toxins than in those that received the toxoids, but there was little difference among the values for the 3 toxins. The toxoid 634 A ...7. .. 7....7. ... D r~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ : .4 B It f _: " L5wai'S : _ V.z1 E IT 7 I7 c l777 71.; :3:i l F W. 754-65 WHO 75465 Fig. 1. Results of the immunodiffusion tests. The outer wells contain toxin or toxoid in the various stages of purifica- tion. 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. BD 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. A C GLUTARALDEHYDE DETOXIFICATION OF CHOLERA TOXIN 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 haemagglutination levels in rabbits immunized with cholera toxin or toxoid in 2 doses each of 100 pg protein Antivibrio- Antitoxin Haemag- units/mla units/ml a glutination a toxin aluminium hydroxide 110 000 1 000 350 toxin Bio Gel 395 1 280 180 toxin Sephadex <50 1 100 200 toxoid aluminium hydroxide 100 000 720 360 toxoid Bio Gel 79 <20 <8 toxoid Sephadex <50 <20 <8 saline (control) <50 <20 <8 a Geometric mean of 5 rabbits. derived from the toxin partially purified with alu- minium hydroxide was still capable of stimulating antitoxins although at a slightly lower level than that obtained with the toxins. The toxoids derived from the toxins further purified with Bio Gel and/or Sephadex did not stimulate antitoxin production. A close relationship was found between the antitoxin titre as determined by haemagglutination and that determined by neutralization of the vascular perme- ability factor. Reactions to the challenge dose of cholera toxin are shown in Table 3. Where there were antitoxins present there was neutralization of the inoculated toxin and therefore a decrease in the effect caused by the vascular permeability factor. In the absence of antitoxins the effect was similar to that obtained in 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. 637 638 M. SALETTI & A. RICCI DISCUSSION The purification of cholera toxin with Bio Gel A- 5m and double column Sephadex G-75 as previously carried out by Finkelstein & LoSpalluto (4) results in the disappearance of polysaccharide. The toxin prep- aration reported herein had a specific activity of 10 BD/,ug; the preparations of Finkelstein & LoSpal- luto (16) and Richardson (17) had a specific activity of 2 000 BD/,ug. The ratio of mg of protein to BD at the various stages of purification remained more or less constant. In effect, during purification there was a separation of choleragen from the other proteins. This may have been due either to structural changes in the choleragen with a consequent loss of activity by the cholera toxin, or to the fact that polysaccharides, which disappear during the later stages of purifica- tion, act as an adjuvant to the action of the toxin. We are inclined to support the former hypothesis. The toxin at all three levels of purity stimulated antitoxin production in animals vaccinated subcu- taneously. The production of vibriocidal antibodies was proportionate to the polysaccharide content of the toxin. The values for haemagglutination did not show any significant differences. Detoxification with glutaraldehyde caused a loss of toxic activity, which is demonstrated by the differences in the reactions caused by the vascular permeability factor. However, only the toxin which was detoxified after partial purification with alumi- nium hydroxide retained its immunizing capacity; the highly purified toxoids completely lost their ability to stimulate the production of antitoxins. This shows that treatment of highly purified toxin with glutaraldehyde causes such a change in the antigen that it is no longer able to maintain an immunizing effect. Glutaraldehyde is clearly unsuitable for detoxify- ing cholera toxin that is to be used for tests on man. Further studies will be necessary in order to find a substance capable of detoxifying highly purified toxin 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 purification et la detoxication de la toxine cholerique. La toxine cholerique a ete preparee en cuve de fermentation dans 40 litres de milieu A la Syncase, ensemence avec la souche 569 B Inaba de V. Cholerae. Apres incubation a 30°C pendant 24 heures, avec a6ration et agitation i 750 r/min, les micro-organismes ont ete tu6s par addition de thiomersal. La toxine a d'abord ete partiellement purifiee par l'hydroxyde d'aluminium, et la purification a ete poursuivie at l'aide de Bio Gel A-5 m, puis de Sephadex G-75. Les toxines ont ete detoxifiees par le glutarald6hyde. Les anatoxines ainsi obtenues (hydroxyde d'aluminium, Bio Gel A-5 m et Sephadex G-75), injectees dans la peau du lapin, semblaient n'avoir conserve aucune toxicit6 residuelle. Avant detoxication, toutes les toxines possedaient un pouvoir immunisant. Mais, si l'anatoxine traitee a l'hydroxyde d'aluminium gardait une activite immunisante, fuit-elle legerement reduite, les anatoxines hautement purifiees sur Bio Gel A-5 m et Sephadex G-75 perdaient, dans les conditions de notre experience, leur capacit6 de susciter des anticorps anti- toxiques. En vue d'eprouver sur l'homme le pouvoir immunisant de l'anatoxine cholerique, de nouvelles etudes seront necessaires pour decouvrir une substance capable de detoxifier la toxine tres purifiee tout en main- tenant son activite immunisante. REFERENCES 1. SALErri, M. ET AL. Experimental studies in cholera toxin and toxoid; In: Proceedings of the Symposium on Bacterial Vaccines, Zagreb, 27-28 Oct. 1971. Zagreb, Yugoslav Academy of Sciences and Arts, 1972, pp. 75-91. 2. SPYRIDES, G. J. & FEELEY, J. C. Concentration and purification of cholera exotoxin by absorption on aluminum compound gels. Journal of infectious diseases, 121: S96-S99 (1970). 3. FINKELSTEIN, R. A. ET AL. 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