APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1976, p. 234-242 Copyright ©D 1976 American Society for Microbiology
Vol. 31, No. 2 Printed in U.S.A.
Toxoid of Clostridium botulinum Type F: Purification and Immunogenicity Studies CHARLES L. HATHEWAY' Biologic Products Division, Bureau of Laboratories, Michigan Department of Public Health, Lansing, Michigan 48914
Received for publication 23 September 1975
Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purified by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography, or (iii) diethylaminoethylcellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography. Toxin purities as reflected by specific activity were 1.83 x 10', 9.8 x 10', and 2.0 x 107 mouse 50% lethal doses (LD5,,)/mg of N, respectively, for toxins purified by the three methods. The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days. Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin. The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered. The toxoid prepared from the most highly purified toxin (method [iii]) conferred the highest immunity in guinea pigs at a given dose level. A relation between serum antitoxin level and resistance to challenge was observed. At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10) mouse LD50 of type F botulinal toxin. A dose of 3.75 ,ug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids. Clostridium botulinum type F was first iso- toxin per ml in dialysis cultures (15). The data lated from liver paste on the Danish island indicate that the toxin was unstable under conLangeland by Moller and Scheibel (11). The ditions of continued incubation or storage at organism was designated type F after further 4 C. studies by Dolman and Murakami (4). One outHoldeman and Smith (7) grew the type F break of type F botulism in this country has Langeland strain in chemically defined media been reported by Midura et al. (9). At least four to determine the nutritional requirements. isolations of the type F organism not associated They found that the maximum toxin level in with botulism have been made in the United synthetic media was only 1/10 that obtained States. Eklund and Poysky isolated it from with complex media. Howell (Ph.D. thesis, West Coast marine sediments (5); Craig and Univ. of North Carolina, Chapel Hill, 1969) Pilcher recovered it from Columbia River sal- found that toxin levels in stationary liquid type mon (3); Walls found it in crabs taken from F cultures diminished rapidly if incubation was Chesapeake Bay (19); and Wertz et al. have iso- continued beyond the time that maximum lated it from a North Dakota stream (21). The toxicity occurred, but in biphasic media (agar/ level of toxin produced by type F cultures has liquid) cultures or in a dialysis system the toxin been generally much lower than that of the level was stable. Of the 12 type F isolates availother types. Rymkiewicz separated three colo- able to him for testing, he found that the strain nial variants of the Langeland strain and re- isolated by Walls (19) produced the highest ported differences in biochemical, morphologi- toxin levels (6 x 10' mouse 50% lethal doses cal, and toxigenic properties (14). One of these ILD..,I/ml in dialysis culture). variants (S/3) reportedly yielded a phenomenal The only reported immunogenicity studies on level of 2 x 1012 mouse minimal lethal doses of type F toxoids were based on crude antigens, i.e., formalinized culture supernatant fluids ' Present address: Enterobacteriology Branch, Bacteriology Division, Bureau of Laboratories, Center for Disease and culture filtrates. Mikhailova (10) immuControl, Atlanta, Ga. 30333. nized rabbits initially with type F culture fil234
VOL. 31, 1976
trate toxoid and then gave booster injections with either crude toxin or crude toxoid. She found that the antitoxin titers were significantly higher in the serum of the animals receiving the toxin than in those receiving toxoid. Rymkiewicz (16) used similar crude toxoids of the Polish S/3 variant for studying quantitative response to measured doses. Trembowler et al. (18) tested the response to crude type F toxoid in combination with five other botulinal toxoids (types A, B, C, D, and E). They reported no significant difference in response whether the toxoid was given alone or in combination. Botulinal toxoids of types A, B, C, D, and E have been made for human immunization from purified toxins (1) and are available for the protection of high-risk laboratory personnel. However, at present no toxoid is available in this country for protection against the type F toxin. In this report we describe the preparation of partially purified type F toxoids and their immunogenicity in laboratory animals. MATERIALS AND METHODS Organism. The proteolytic 8GF strain of C. botulinum, which Walls (19) isolated from a Chesapeake Bay crab, was chosen as the source of toxin. V. R. Dowell, Jr., of the Center for Disease Control, Atlanta, Ga., provided the strain. Medium. The medium contained 17% (vol/vol) corn steep liquor (Anheuser-Busch), 1% (vol/vol) glycerine, and 0.12% (wt/vol) CaCl2 2H2O and was adjusted to pH 7.4 with sodium hydroxide before autoclaving. Media in 10-liter volumes were autoclaved for 3 h at 121 C. Culturing method. Culture by dialysis was performed in 3-gallon (ca. 11.4-liter) bottles at 35 C according to the method of Sterne and Wentzel (17) as described by Cardella et al. (2) for growing C. botulinum type D. Each bottle contained 9 liters of medium, into which was suspended an intussuscepted dialysis sac (Visking cellophane tubing of approximately 12.7-cm flat width before opening). The bottles were autoclaved with the sacs empty, and 1 liter of sterile saline was then added to each. Five milliliters of an 18- to 24-h thioglycolate broth culture was then inoculated into the dialysis sac. LD,,, determinations. Dilutions of culture samples and purification fractions were made in gelatin phosphate buffer (0.2% gelatin, 0.4% Na2HPO4, pH 6.5). Each dilution was tested in a group of four 18to 22-g mice; 0.2 ml was injected intraperitoneally (i.p.) into each mouse. All deaths occurring within 96 h were recorded, and the 50% end point was calculated according to the Reed and Muench method (13) and the number of LD.,,, of toxin per milliliter was found by multiplying by 5, since each mouse received only 0.2 ml. Purification of toxin by ammonium sulfate and ethanol precipitation. Toxin from dialysis cultures of the 8GF strain in corn steep medium was purified according to a method used for type E toxin
TYPE F BOTULINAL TOXOID
235
(6), except that it was not activated with trypsin. The contents of the dialysis sac(s) in one or several bottles were removed, the sac was washed out, and the volume was brought to 1,500 ml with distilled water. Dry ammonium sulfate was added to a concentration of 50% (wt/vol) to precipitate toxin. The next day, or several days later, the supernatant was drawn off and the precipitate was packed by centrifugation in an International PR2 centrifuge at 750 x g. The precipitate was suspended in 750 to 1,000 ml of water, and CaCl2 was added to 0.075 M. At this point the pH was 6.0 to 6.3. The suspension was filtered through paper pulp on a Buchner funnel. The filtrate was adjusted to pH 5.0 with 1 N HCl and was precipitated by adding ethanol to a concentration of 25% while the temperature was lowered to -5 C. The precipitate was collected by centrifugation. It was then dissolved in 0.2 M sodium succinate, pH 6.0, dialyzed against distilled water, and then dialyzed to equilibrium with an equal volume of 0.4 M sodium succinate, pH 6.0. Purification of toxin by DEAE-cellulose chromatography. The method described by Yang and Sugiyama (23) was used for chromatographing type F toxin on 0-diethylaminoethyl (DEAE)-cellulose. The toxin was precipitated from the culture by adding 1.5 volumes of saturated ammonium sulfate solution (room temperature) to 1 volume of whole culture. The precipitate was collected by centrifugation and dialyzed against 0.07 M phosphate buffer, pH 6.0 (starting buffer), with numerous changes until the outer dialysate was negative for ammonium ions when tested with Nessler reagent. The solution containing toxin was then clarified by centrifugation. DEAE-cellulose (72 g of Cellex-D, Bio-Rad Laboratories, Richmond, Calif.) was cycled in 0.1 to 0.2 N sodium hydroxide and hydrochloric acid, washed several times with starting buffer, and then put into a column (4.7 by 30 cm) and washed again with 5- to 10-bed volumes of buffer. After each,run the cellulose was removed from the column and regenerated by washing in water, followed by two cycles of the 0.1 to 0.2 N alkali and acid, and then washed thoroughly with buffer. The toxin solution was applied to the column at the rate of 0.1 g of material (estimated by ultraviolet light absorbance) (20) per g of DEAE-cellulose. Twenty-milliliter fractions were collected. The column was washed with about 700 ml of starting buffer, after which the ultraviolet absorbance of the effluent was less than 0.3. Then, elution was begun with 0.15 M NaCl in starting buffer. After the recovery of most of the toxin in the 0.15 M eluate, elution was continued with 1 M NaCl in starting buffer, which released very much ultraviolet-absorbing material. Further purification by CM-cellulose chromatography. Some of the toxin recovered from DEAEcellulose columns was chromatographed on 0carboxymethyl (CM)-cellulose. The procedure used was an early developmental stage of the method reported by Yang and Sugiyama (23) (personal communication, H. Sugiyama, 1971). A 4.7- by 9.5-cm column containing 20 g of CM-cellulose (Cellex-
236
HATHEWAY
CM, Bio-Rad Laboratories) was prepared with the 0.1 N acid- and 0.1 N base-washed cellulose and equilibrated against 0.02 M sodium citrate buffer, pH 5.0. DEAE-purified toxin directly from the column (the pooled 0.15 M NaCl eluate) was diluted with an equal volume of distilled water and adjusted to pH 5.0 with 1 N HCI before it was added to the CM column. The column was loaded at the rate of about 15 mg of protein per g of cellulose. The column was washed with 0.02 M citrate buffer, pH 5.0. The toxin was eluted with 0.02 M citrate buffer, pH 5.6. Further elution with 0.02 M citrate buffer, pH 6.0, released very little additional material. Detoxification of partially purified toxin and innocuity test. The partially purified toxins were sterilized by filtration through membrane filters (0.22- or 0.45-am pore size; Millipore Corp.). Formalin was added to 0.30 or 0.45% concentration (vol/ vol), and the solutions were incubated at 35 C until they were nontoxic for guinea pigs. Samples of the formalin-toxin mixtures were taken after 1 or 2 weeks of incubation and dialyzed against 10 volumes of 0.2 M succinate buffer to lower the formaldehyde content. Three guinea pigs were each injected with 5 ml of the dialyzed solution and observed daily for 21 days. If death or signs of toxicity occurred in any of the injected animals, the formalin-toxin mixures were incubated for an additional 7 to 10 days and retested. After a successful innocuity test, the toxoid solution was stored in a cold room at 4 C. Immunogenicity testing. The fluid toxoids were combined with an equal volume of Holt's 7/8 aluminum phosphate adjuvant (14 mg of AlPO4 per ml) (22). Dilutions for testing of the adsorbed toxoid were made with 7/8 AlPO4 suspensions in saline containing 7 mg/ml to keep the adjuvant concentration constant. Guinea pigs weighing 300 to 400 g [Michigan Department of Public Health Stock designated MDH:SR(A)] were injected subcutaneously with 1 ml of adsorbed toxoid. Thirty days later, the guinea pigs were bled by cardiac puncture and aliquot pools were made from the sera for each toxoid dilution tested. The sera of at least four animals were included in a pool. Three days later, guinea pigs showing no ill effects from the trauma of bleeding (at least nine in each case) were challenged with 10) mouse i.p. LD7,,, of type F botulinal toxin contained in 1 ml by i.p. injection. The guinea pigs were observed for 10 days, deaths were recorded, and the survival ratio (number of survivors/number injected) was determined for each group. Four unimmunized guinea pigs served as controls each time. The control animals usually died within 24 h of challenge, and, with one exception, all of them died within the 10-day observation period. The antitoxin level of each serum pool was determined by titration against a test dose of type F toxin. One milliliter of toxin containing approximately 40 mouse i.p. LD.-,, was mixed with 1 ml of serum or diluted serum and incubated for 1 h at room temperature, and then 0.2 ml of the mixture was injected i.p. into each of four 18- to 22-g mice (MDPH strain). Deaths were recorded for 4 days, and the 50% neutralization end point of each serum
APPL. ENVIRON. MICROBIOL.
was calculated. Five twofold dilutions, chosen to bracket the end point, were used. The 1-ml test dose of toxin was neutralized by approximately 0.0025 antitoxin units. In each test an antitoxin standard was also titrated against the test toxin. The 50% end point of the antitoxin standard, calculated in units per milliliter, was equal to the unitage of the test sera at their end point dilution. This unitage multiplied by the dilution factor gives the antitoxin units per milliliter (,u/ml in figures) of the undiluted test sera. All end points were estimated by the method of Reed and Muench (13). The antitoxin standard, obtained from the Center for Disease Control, contained 8 international units per ml when reconstituted with 50% glycerol. All type F antitoxin levels in these studies were calculated in relation to this standard. Decantation of adsorbed toxoid. A portion of one toxoid (no. 115) prepared by ammonium sulfateethanol precipitation was combined with Holt's 7/8 aluminum phosphate adjuvant and incubated for several hours at 35 C and then 3 days at room temperature with occasional shaking; the adsorbed toxoid was centrifuged, the supernatant fluid comprising 80% of the total volume was removed, and the original volume was restored by adding saline. The total nitrogen of the adsorbed toxoid was diminished by 40%, and residual formaldehyde was greatly reduced. The decanted toxoid, designated as 115 d, was tested for immunogenicity. Hexavalent combination. Two type F toxoids (no. 115 d and 124) were combined with botulinal toxoids of types A, B, C, D, and E to determine if the combination with the other antigens altered the response to the type F component. In addition, the effect of adding a sixth component to a pentavalent set was determined by using toxoid no. 124. Toxoids of the other botulinal toxins (A-E) were manufactured by the Michigan Department of Public Health under contract with the Center for Disease Control for use in human immunization. The toxoid combinations were prepared with the A, C, D, and E components in the amounts prescribed by Cardella (1), but the type B toxoid was added to twice the prescribed amount. The combined preparations contained 7 mg of aluminum phosphate per ml. The antitoxin responses to types A through E botulinal toxoids were measured essentially as for type F above, according to the procedure described by Cardella (1). Parallel titrations of the World Health Organization antitoxin standards were done simultaneously. Resistance to challenge of guinea pigs immunized with hexavalent and pentavalent toxoids was tested by injecting (i.p.) separate groups of animals with 10' mouse LD,,, of the respective toxins and recording 10-day survivor ratios (number of survivors/number injected). Nitrogen and protein determinations. The nitrogen content of purified toxin solutions and toxoids was determined by the micro-Kjeldahl method (8). Nitrogen and protein contents of some fractions in chromatography experiments were estimated by absorbance of ultraviolet light by using a Beckman DU spectrophotometer. The absorbance at 280 nm in a 1cm cell of a solution of DEAE-purified toxin mul-
VOL. 31, 1976
TYPE F BOTULINAL TOXOID
tiplied by the factor 0.142 gives the concentration in milligrams of N per milliliter. The factor for CMpurified preparations is about the same (0.145).
Starting Buffer
015M NaCl
lIOM
NaCl
I
RESULTS
Culturing and toxin production. The average toxicity of 13 different cultures of strain 8GF in corn steep medium was 7.9 x 105 LD-,,,/ml (range, 2.5 x 105 to 1.6 x 10; median, 8.0 x 10"). Peak toxicity occurred between 7 and 20 days and usually did not diminish with continued incubation. Toxin was harvested after 17 to 20 days. Toxin purification. The data from four ammonium sulfate-ethanol purification runs are given in Table 1. The specific toxic activity of the purified material ranged from 1.26 x i0' to 2.5 x 10' mouse i.p. LDl,58mg of N. DEAE-cellulose chromatography yielded toxin of greater purity. Figure 1 shows the details of a typical run, afid Table 2 shows the data for six runs. Recovery was estimated in absorbance units by multiplying optical densities by volumes. The absorbance recovered from the column was about 50% of that applied. The first peak consisting of unadsorbed material contained about 2.5% of the absorbance and less than 1% of the toxicity of the applied toxin. The second peak, eluted by 0.15 M NaCl, also contained about 2.5% of the absorbance but up to 71% of the total applied toxicity (in five or six peak fractions). The third peak, eluted by 1.0 M NaCl, contained about 45% of the absorbance; less than 1% of the toxic activity applied to the column was found in the 100-ml pool of the most highly absorbing fractions of this peak (pool III). In each case the purified toxin came from pool II, with a range of specific activities of 7.1 x 106 to 1.31 x 107 mouse LD,5,/mg of N. The average purity of the DEAEpurified toxin is more than fivefold greater than the ammonium sulfate-ethanol-purified toxin. The recovery of active toxin in the pools ranged from 30 to 70%. The unrecovered toxin was lost either by irreversible absorption or by inactivation. Data for two runs of DEAE-purified toxin on
237
I
Pool I F9
Pool II M
E 0
cx
O0
0
40
80 120 Fraction No.
160
200
FIG. 1. DEAE-cellulose chromatography of type F toxin of C. botulinum,
run no.
122-2. Column, 4.7 were col-
by 30 cm, 72 g of Cellex-D; 20-ml fractions lected.
CM-cellulose are given in Table 3. The specific activity increased more than twofold in each case, with 21 and 50% recoveries. The loss of toxin in this process is largely due to failure of the toxin to absorb. Figure 2 shows the elution pattern (ultraviolet absorbance) of fractions versus fraction number. It appears that a portion of the toxin was retarded rather than adsorbed, because after 440 ml of diluted DEAE toxin was added the effluent became toxic. The rate of application was a conservative 14 mg of protein per g of cellulose. The manufacturer recommends 100 mg/g of cellulose. The conditions used in this procedure were not optimum for adsorption of toxin. Nevertheless, the pH 5.6 eluates contained type F toxin with the highest specific activity in these studies. The amount of loss of toxin by leaking or overloading was not quantitated. In six trials there was never much material eluted by the pH 6 citrate buffer. Table 4 shows the purity of toxin obtained by the three methods. Detoxification. Three toxins prepared by
TABLE 1. Purification of type F botulinal toxin by ammonium sulfate and ethanol precipitation
(NH4)2SO4 precipitate-
Culture
Ethanol precipitate
CaC!. extract
Run no.
Vol (ml)
108 109 115 116
300 750
1,700 1,800
Total LD5,
4.8 6.62 8.6 2.3
x
1O"
x
108
x x
10" 10'
Total LDs,,
2.82 4.85 1.19 1.33
x 108
x
108
x x
10" 10"
%
Total LDS,,
58.8 73 100 5.8
3.1 X 107 2.48 x 108 1.98 x 108 2.68 x 10"
%
LD.,,/ml
6.5 1.41 x 1037.5 1.13 x 106 6 x 105 23 11.7 6.3 x 10-
mg of N oml N/ LDs /mg of N 0.112
1.26 x 10'i
0.45
2.5 x 106
0.29 0.424
2.07 x 106 1.48 x 106
238
APPL. ENVIRON. MICROBIOL.
HATHEWAY
TABLE 2. DEAE-cellulose purification of type F botulinal toxin Run no.
Determinationa 122-1
122-2
122-3
124-1
124-2
125
1.41 x 106 5.64 x 108 45.0 3.1 x 104
1.41 x 106 5.08 x 108 45.0 3.1 x 104
1.26 x 10'; 3.66 x 108 45.0 2.8 x 104
2.82 x 106 7.75 x 108 62.5 4.44 x 104
2.82 x 106 7.75 x 108 62.5 4.44 x 104
2.52 x 10i 8.8 x 108 54.7 4.6 x 104
3.0 x 106 1.95 1.5 x 104
Vol. 31, No. 2 Printed in U.S.A.
Toxoid of Clostridium botulinum Type F: Purification and Immunogenicity Studies CHARLES L. HATHEWAY' Biologic Products Division, Bureau of Laboratories, Michigan Department of Public Health, Lansing, Michigan 48914
Received for publication 23 September 1975
Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purified by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography, or (iii) diethylaminoethylcellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography. Toxin purities as reflected by specific activity were 1.83 x 10', 9.8 x 10', and 2.0 x 107 mouse 50% lethal doses (LD5,,)/mg of N, respectively, for toxins purified by the three methods. The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days. Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin. The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered. The toxoid prepared from the most highly purified toxin (method [iii]) conferred the highest immunity in guinea pigs at a given dose level. A relation between serum antitoxin level and resistance to challenge was observed. At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10) mouse LD50 of type F botulinal toxin. A dose of 3.75 ,ug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids. Clostridium botulinum type F was first iso- toxin per ml in dialysis cultures (15). The data lated from liver paste on the Danish island indicate that the toxin was unstable under conLangeland by Moller and Scheibel (11). The ditions of continued incubation or storage at organism was designated type F after further 4 C. studies by Dolman and Murakami (4). One outHoldeman and Smith (7) grew the type F break of type F botulism in this country has Langeland strain in chemically defined media been reported by Midura et al. (9). At least four to determine the nutritional requirements. isolations of the type F organism not associated They found that the maximum toxin level in with botulism have been made in the United synthetic media was only 1/10 that obtained States. Eklund and Poysky isolated it from with complex media. Howell (Ph.D. thesis, West Coast marine sediments (5); Craig and Univ. of North Carolina, Chapel Hill, 1969) Pilcher recovered it from Columbia River sal- found that toxin levels in stationary liquid type mon (3); Walls found it in crabs taken from F cultures diminished rapidly if incubation was Chesapeake Bay (19); and Wertz et al. have iso- continued beyond the time that maximum lated it from a North Dakota stream (21). The toxicity occurred, but in biphasic media (agar/ level of toxin produced by type F cultures has liquid) cultures or in a dialysis system the toxin been generally much lower than that of the level was stable. Of the 12 type F isolates availother types. Rymkiewicz separated three colo- able to him for testing, he found that the strain nial variants of the Langeland strain and re- isolated by Walls (19) produced the highest ported differences in biochemical, morphologi- toxin levels (6 x 10' mouse 50% lethal doses cal, and toxigenic properties (14). One of these ILD..,I/ml in dialysis culture). variants (S/3) reportedly yielded a phenomenal The only reported immunogenicity studies on level of 2 x 1012 mouse minimal lethal doses of type F toxoids were based on crude antigens, i.e., formalinized culture supernatant fluids ' Present address: Enterobacteriology Branch, Bacteriology Division, Bureau of Laboratories, Center for Disease and culture filtrates. Mikhailova (10) immuControl, Atlanta, Ga. 30333. nized rabbits initially with type F culture fil234
VOL. 31, 1976
trate toxoid and then gave booster injections with either crude toxin or crude toxoid. She found that the antitoxin titers were significantly higher in the serum of the animals receiving the toxin than in those receiving toxoid. Rymkiewicz (16) used similar crude toxoids of the Polish S/3 variant for studying quantitative response to measured doses. Trembowler et al. (18) tested the response to crude type F toxoid in combination with five other botulinal toxoids (types A, B, C, D, and E). They reported no significant difference in response whether the toxoid was given alone or in combination. Botulinal toxoids of types A, B, C, D, and E have been made for human immunization from purified toxins (1) and are available for the protection of high-risk laboratory personnel. However, at present no toxoid is available in this country for protection against the type F toxin. In this report we describe the preparation of partially purified type F toxoids and their immunogenicity in laboratory animals. MATERIALS AND METHODS Organism. The proteolytic 8GF strain of C. botulinum, which Walls (19) isolated from a Chesapeake Bay crab, was chosen as the source of toxin. V. R. Dowell, Jr., of the Center for Disease Control, Atlanta, Ga., provided the strain. Medium. The medium contained 17% (vol/vol) corn steep liquor (Anheuser-Busch), 1% (vol/vol) glycerine, and 0.12% (wt/vol) CaCl2 2H2O and was adjusted to pH 7.4 with sodium hydroxide before autoclaving. Media in 10-liter volumes were autoclaved for 3 h at 121 C. Culturing method. Culture by dialysis was performed in 3-gallon (ca. 11.4-liter) bottles at 35 C according to the method of Sterne and Wentzel (17) as described by Cardella et al. (2) for growing C. botulinum type D. Each bottle contained 9 liters of medium, into which was suspended an intussuscepted dialysis sac (Visking cellophane tubing of approximately 12.7-cm flat width before opening). The bottles were autoclaved with the sacs empty, and 1 liter of sterile saline was then added to each. Five milliliters of an 18- to 24-h thioglycolate broth culture was then inoculated into the dialysis sac. LD,,, determinations. Dilutions of culture samples and purification fractions were made in gelatin phosphate buffer (0.2% gelatin, 0.4% Na2HPO4, pH 6.5). Each dilution was tested in a group of four 18to 22-g mice; 0.2 ml was injected intraperitoneally (i.p.) into each mouse. All deaths occurring within 96 h were recorded, and the 50% end point was calculated according to the Reed and Muench method (13) and the number of LD.,,, of toxin per milliliter was found by multiplying by 5, since each mouse received only 0.2 ml. Purification of toxin by ammonium sulfate and ethanol precipitation. Toxin from dialysis cultures of the 8GF strain in corn steep medium was purified according to a method used for type E toxin
TYPE F BOTULINAL TOXOID
235
(6), except that it was not activated with trypsin. The contents of the dialysis sac(s) in one or several bottles were removed, the sac was washed out, and the volume was brought to 1,500 ml with distilled water. Dry ammonium sulfate was added to a concentration of 50% (wt/vol) to precipitate toxin. The next day, or several days later, the supernatant was drawn off and the precipitate was packed by centrifugation in an International PR2 centrifuge at 750 x g. The precipitate was suspended in 750 to 1,000 ml of water, and CaCl2 was added to 0.075 M. At this point the pH was 6.0 to 6.3. The suspension was filtered through paper pulp on a Buchner funnel. The filtrate was adjusted to pH 5.0 with 1 N HCl and was precipitated by adding ethanol to a concentration of 25% while the temperature was lowered to -5 C. The precipitate was collected by centrifugation. It was then dissolved in 0.2 M sodium succinate, pH 6.0, dialyzed against distilled water, and then dialyzed to equilibrium with an equal volume of 0.4 M sodium succinate, pH 6.0. Purification of toxin by DEAE-cellulose chromatography. The method described by Yang and Sugiyama (23) was used for chromatographing type F toxin on 0-diethylaminoethyl (DEAE)-cellulose. The toxin was precipitated from the culture by adding 1.5 volumes of saturated ammonium sulfate solution (room temperature) to 1 volume of whole culture. The precipitate was collected by centrifugation and dialyzed against 0.07 M phosphate buffer, pH 6.0 (starting buffer), with numerous changes until the outer dialysate was negative for ammonium ions when tested with Nessler reagent. The solution containing toxin was then clarified by centrifugation. DEAE-cellulose (72 g of Cellex-D, Bio-Rad Laboratories, Richmond, Calif.) was cycled in 0.1 to 0.2 N sodium hydroxide and hydrochloric acid, washed several times with starting buffer, and then put into a column (4.7 by 30 cm) and washed again with 5- to 10-bed volumes of buffer. After each,run the cellulose was removed from the column and regenerated by washing in water, followed by two cycles of the 0.1 to 0.2 N alkali and acid, and then washed thoroughly with buffer. The toxin solution was applied to the column at the rate of 0.1 g of material (estimated by ultraviolet light absorbance) (20) per g of DEAE-cellulose. Twenty-milliliter fractions were collected. The column was washed with about 700 ml of starting buffer, after which the ultraviolet absorbance of the effluent was less than 0.3. Then, elution was begun with 0.15 M NaCl in starting buffer. After the recovery of most of the toxin in the 0.15 M eluate, elution was continued with 1 M NaCl in starting buffer, which released very much ultraviolet-absorbing material. Further purification by CM-cellulose chromatography. Some of the toxin recovered from DEAEcellulose columns was chromatographed on 0carboxymethyl (CM)-cellulose. The procedure used was an early developmental stage of the method reported by Yang and Sugiyama (23) (personal communication, H. Sugiyama, 1971). A 4.7- by 9.5-cm column containing 20 g of CM-cellulose (Cellex-
236
HATHEWAY
CM, Bio-Rad Laboratories) was prepared with the 0.1 N acid- and 0.1 N base-washed cellulose and equilibrated against 0.02 M sodium citrate buffer, pH 5.0. DEAE-purified toxin directly from the column (the pooled 0.15 M NaCl eluate) was diluted with an equal volume of distilled water and adjusted to pH 5.0 with 1 N HCI before it was added to the CM column. The column was loaded at the rate of about 15 mg of protein per g of cellulose. The column was washed with 0.02 M citrate buffer, pH 5.0. The toxin was eluted with 0.02 M citrate buffer, pH 5.6. Further elution with 0.02 M citrate buffer, pH 6.0, released very little additional material. Detoxification of partially purified toxin and innocuity test. The partially purified toxins were sterilized by filtration through membrane filters (0.22- or 0.45-am pore size; Millipore Corp.). Formalin was added to 0.30 or 0.45% concentration (vol/ vol), and the solutions were incubated at 35 C until they were nontoxic for guinea pigs. Samples of the formalin-toxin mixtures were taken after 1 or 2 weeks of incubation and dialyzed against 10 volumes of 0.2 M succinate buffer to lower the formaldehyde content. Three guinea pigs were each injected with 5 ml of the dialyzed solution and observed daily for 21 days. If death or signs of toxicity occurred in any of the injected animals, the formalin-toxin mixures were incubated for an additional 7 to 10 days and retested. After a successful innocuity test, the toxoid solution was stored in a cold room at 4 C. Immunogenicity testing. The fluid toxoids were combined with an equal volume of Holt's 7/8 aluminum phosphate adjuvant (14 mg of AlPO4 per ml) (22). Dilutions for testing of the adsorbed toxoid were made with 7/8 AlPO4 suspensions in saline containing 7 mg/ml to keep the adjuvant concentration constant. Guinea pigs weighing 300 to 400 g [Michigan Department of Public Health Stock designated MDH:SR(A)] were injected subcutaneously with 1 ml of adsorbed toxoid. Thirty days later, the guinea pigs were bled by cardiac puncture and aliquot pools were made from the sera for each toxoid dilution tested. The sera of at least four animals were included in a pool. Three days later, guinea pigs showing no ill effects from the trauma of bleeding (at least nine in each case) were challenged with 10) mouse i.p. LD7,,, of type F botulinal toxin contained in 1 ml by i.p. injection. The guinea pigs were observed for 10 days, deaths were recorded, and the survival ratio (number of survivors/number injected) was determined for each group. Four unimmunized guinea pigs served as controls each time. The control animals usually died within 24 h of challenge, and, with one exception, all of them died within the 10-day observation period. The antitoxin level of each serum pool was determined by titration against a test dose of type F toxin. One milliliter of toxin containing approximately 40 mouse i.p. LD.-,, was mixed with 1 ml of serum or diluted serum and incubated for 1 h at room temperature, and then 0.2 ml of the mixture was injected i.p. into each of four 18- to 22-g mice (MDPH strain). Deaths were recorded for 4 days, and the 50% neutralization end point of each serum
APPL. ENVIRON. MICROBIOL.
was calculated. Five twofold dilutions, chosen to bracket the end point, were used. The 1-ml test dose of toxin was neutralized by approximately 0.0025 antitoxin units. In each test an antitoxin standard was also titrated against the test toxin. The 50% end point of the antitoxin standard, calculated in units per milliliter, was equal to the unitage of the test sera at their end point dilution. This unitage multiplied by the dilution factor gives the antitoxin units per milliliter (,u/ml in figures) of the undiluted test sera. All end points were estimated by the method of Reed and Muench (13). The antitoxin standard, obtained from the Center for Disease Control, contained 8 international units per ml when reconstituted with 50% glycerol. All type F antitoxin levels in these studies were calculated in relation to this standard. Decantation of adsorbed toxoid. A portion of one toxoid (no. 115) prepared by ammonium sulfateethanol precipitation was combined with Holt's 7/8 aluminum phosphate adjuvant and incubated for several hours at 35 C and then 3 days at room temperature with occasional shaking; the adsorbed toxoid was centrifuged, the supernatant fluid comprising 80% of the total volume was removed, and the original volume was restored by adding saline. The total nitrogen of the adsorbed toxoid was diminished by 40%, and residual formaldehyde was greatly reduced. The decanted toxoid, designated as 115 d, was tested for immunogenicity. Hexavalent combination. Two type F toxoids (no. 115 d and 124) were combined with botulinal toxoids of types A, B, C, D, and E to determine if the combination with the other antigens altered the response to the type F component. In addition, the effect of adding a sixth component to a pentavalent set was determined by using toxoid no. 124. Toxoids of the other botulinal toxins (A-E) were manufactured by the Michigan Department of Public Health under contract with the Center for Disease Control for use in human immunization. The toxoid combinations were prepared with the A, C, D, and E components in the amounts prescribed by Cardella (1), but the type B toxoid was added to twice the prescribed amount. The combined preparations contained 7 mg of aluminum phosphate per ml. The antitoxin responses to types A through E botulinal toxoids were measured essentially as for type F above, according to the procedure described by Cardella (1). Parallel titrations of the World Health Organization antitoxin standards were done simultaneously. Resistance to challenge of guinea pigs immunized with hexavalent and pentavalent toxoids was tested by injecting (i.p.) separate groups of animals with 10' mouse LD,,, of the respective toxins and recording 10-day survivor ratios (number of survivors/number injected). Nitrogen and protein determinations. The nitrogen content of purified toxin solutions and toxoids was determined by the micro-Kjeldahl method (8). Nitrogen and protein contents of some fractions in chromatography experiments were estimated by absorbance of ultraviolet light by using a Beckman DU spectrophotometer. The absorbance at 280 nm in a 1cm cell of a solution of DEAE-purified toxin mul-
VOL. 31, 1976
TYPE F BOTULINAL TOXOID
tiplied by the factor 0.142 gives the concentration in milligrams of N per milliliter. The factor for CMpurified preparations is about the same (0.145).
Starting Buffer
015M NaCl
lIOM
NaCl
I
RESULTS
Culturing and toxin production. The average toxicity of 13 different cultures of strain 8GF in corn steep medium was 7.9 x 105 LD-,,,/ml (range, 2.5 x 105 to 1.6 x 10; median, 8.0 x 10"). Peak toxicity occurred between 7 and 20 days and usually did not diminish with continued incubation. Toxin was harvested after 17 to 20 days. Toxin purification. The data from four ammonium sulfate-ethanol purification runs are given in Table 1. The specific toxic activity of the purified material ranged from 1.26 x i0' to 2.5 x 10' mouse i.p. LDl,58mg of N. DEAE-cellulose chromatography yielded toxin of greater purity. Figure 1 shows the details of a typical run, afid Table 2 shows the data for six runs. Recovery was estimated in absorbance units by multiplying optical densities by volumes. The absorbance recovered from the column was about 50% of that applied. The first peak consisting of unadsorbed material contained about 2.5% of the absorbance and less than 1% of the toxicity of the applied toxin. The second peak, eluted by 0.15 M NaCl, also contained about 2.5% of the absorbance but up to 71% of the total applied toxicity (in five or six peak fractions). The third peak, eluted by 1.0 M NaCl, contained about 45% of the absorbance; less than 1% of the toxic activity applied to the column was found in the 100-ml pool of the most highly absorbing fractions of this peak (pool III). In each case the purified toxin came from pool II, with a range of specific activities of 7.1 x 106 to 1.31 x 107 mouse LD,5,/mg of N. The average purity of the DEAEpurified toxin is more than fivefold greater than the ammonium sulfate-ethanol-purified toxin. The recovery of active toxin in the pools ranged from 30 to 70%. The unrecovered toxin was lost either by irreversible absorption or by inactivation. Data for two runs of DEAE-purified toxin on
237
I
Pool I F9
Pool II M
E 0
cx
O0
0
40
80 120 Fraction No.
160
200
FIG. 1. DEAE-cellulose chromatography of type F toxin of C. botulinum,
run no.
122-2. Column, 4.7 were col-
by 30 cm, 72 g of Cellex-D; 20-ml fractions lected.
CM-cellulose are given in Table 3. The specific activity increased more than twofold in each case, with 21 and 50% recoveries. The loss of toxin in this process is largely due to failure of the toxin to absorb. Figure 2 shows the elution pattern (ultraviolet absorbance) of fractions versus fraction number. It appears that a portion of the toxin was retarded rather than adsorbed, because after 440 ml of diluted DEAE toxin was added the effluent became toxic. The rate of application was a conservative 14 mg of protein per g of cellulose. The manufacturer recommends 100 mg/g of cellulose. The conditions used in this procedure were not optimum for adsorption of toxin. Nevertheless, the pH 5.6 eluates contained type F toxin with the highest specific activity in these studies. The amount of loss of toxin by leaking or overloading was not quantitated. In six trials there was never much material eluted by the pH 6 citrate buffer. Table 4 shows the purity of toxin obtained by the three methods. Detoxification. Three toxins prepared by
TABLE 1. Purification of type F botulinal toxin by ammonium sulfate and ethanol precipitation
(NH4)2SO4 precipitate-
Culture
Ethanol precipitate
CaC!. extract
Run no.
Vol (ml)
108 109 115 116
300 750
1,700 1,800
Total LD5,
4.8 6.62 8.6 2.3
x
1O"
x
108
x x
10" 10'
Total LDs,,
2.82 4.85 1.19 1.33
x 108
x
108
x x
10" 10"
%
Total LDS,,
58.8 73 100 5.8
3.1 X 107 2.48 x 108 1.98 x 108 2.68 x 10"
%
LD.,,/ml
6.5 1.41 x 1037.5 1.13 x 106 6 x 105 23 11.7 6.3 x 10-
mg of N oml N/ LDs /mg of N 0.112
1.26 x 10'i
0.45
2.5 x 106
0.29 0.424
2.07 x 106 1.48 x 106
238
APPL. ENVIRON. MICROBIOL.
HATHEWAY
TABLE 2. DEAE-cellulose purification of type F botulinal toxin Run no.
Determinationa 122-1
122-2
122-3
124-1
124-2
125
1.41 x 106 5.64 x 108 45.0 3.1 x 104
1.41 x 106 5.08 x 108 45.0 3.1 x 104
1.26 x 10'; 3.66 x 108 45.0 2.8 x 104
2.82 x 106 7.75 x 108 62.5 4.44 x 104
2.82 x 106 7.75 x 108 62.5 4.44 x 104
2.52 x 10i 8.8 x 108 54.7 4.6 x 104
3.0 x 106 1.95 1.5 x 104
