- Email: [email protected]
Fractionation studies on the antigenic nature of Toxoplasma gondii
EXPERIMENTAL
6, 449-458 (1957)
PARASITOLOGY
ORIGINAL * Fractionation
PAPERS *
Studies on the Antigenic Toxoplasma gondii
William Department
RESEARCH * * *
A. Hook
of Bacteriology,
and
TIniversity
John
E. Faber,
of Maryland,
(Submitted for publication,
Nature
of
Jr.
College Park,
Maryland
26 October 1956)
Cutchins and Warren (1956) showed that antibodies could be induced in guinea pigs by antigens prepared from killed toxoplasma. Using formaldehyde-killed and sonic-fragmented toxoplasma these authors demonstrated that vaccines could be prepared which imparted immunity as well as elicited antibodies detectable by complement fixation and the Sabin-Feldman (1948) dye test. Because the mechanisms of these two tests differ as well as some of the characteristics of the antibodies produced (Sabin, 1949), it was thought possible that some differences might be demonstrable between antigenic components of toxoplasma responsible for these antibodies. An effort was made to fractionate toxoplasma into protein and carbohydrate components and to investigate their antigenic activity in vitro and in vivo. MATEHIAL~
AND METHODS
A subline’ of the RH strain (Sabin, 1941) of ToxopZusma go&ii was used in this work. Serial passagesat 6 day intervals were made by interperitoneal inoculation of 10 albino mice each with 0.1 ml of a lo-” dilution of peritoneal exudate from the previous passage. To harvest toxoplasmas for fractionation, large numbers of mice were inoculated interperitoneally and tapped from the same site. An average of 0.5 ml of exudate containing between 15,000,OOOand 19,000,OOO toxoplasmas per ml was usually obtained. Toxoplasmas and accompanying cellular material from the exudate were washed twice with 0.85 % saline and 1 Obtained from Dr. E. C. Cutchins, National Maryland. 449
Institut,es of Health, Bethesda,
450
HOOK
AND
FABER
resuspended to original exudate volume. This material was freeze dried and stored at -20°C until used. Approximately 1 gram of dried organisms were obtained in this manner. Although the quantity of toxoplasmas was thus limited, the fractionation experiments in this work were performed at least twice. A soluble, standard complement king antigen for comparative purposes was prepared from toxoplasmas obtained from the peritoneal exudate of infected mice by a method similar to that described by Sabin (1949) and modified by Cutchins and Warren (1956). Toxoplasmas were washed and resuspended to original exudate volume with 0.85% saline and fragmented by sonic vibrations at a frequency of 9 kc for 30 minutes in a water-cooled transducer of a sonic oscillator2. The suspension containing ruptured cells was then centrifuged at 8000 g for 30 minutes at 4°C to remove cellular debris, The supernatant constituted the antigen and was stored at -2O’C. Standardization of the antigen was performed by cross titrating with immune rabbit serum of known titer. The complement fixing activity of standard antigens and of the carbohydrate and protein fractions prepared in this study was tested by a procedure similar to that described by Warren and Russ (1948). Cross titrations were made of test antigens with a pooled serum from six adult guinea pigs bled 30 days after interperitoneal inoculation with a sub-lethal dose of live toxoplasmas. The complement fixing titer of this serum was 1:512. Negative sera were obtained from uninoculated guinea pigs. The Sabin-Feldman dye test for toxoplasma antibodies was performed according to a procedure outlined by Frenkel and Friedlander (1951). Dye test positive sera for controls and for the antibody neutralization experiment were obtained from adult rats that had been intraperitoneally inoculated with live toxoplasmas and bled 15 days later. All sera collected for complement fixation and dye tests were first inactivated at 56°C for 30 minutes. Ammonium Sulfate Precipitation
of Protein
Sonic-fragmented toxoplasmas were precipitated with increasing concentrations of ammonium sulfate to isolate the fraction most active as a complement fixing antigen and, if possible, to identify a fraction p Manufactured
ment, Waltham
by Raytheon Manufacturing 54, Massachusetts.
Co., Medical
Products
Depart-
ANTIGENIC
NATURE
OF TOXOPLASMA
451
with the dye test. Preliminary experiments showed that the pH range 7 to 8 was optimal for carrying out the precipitation procedure. A phosphate buffered (M/100) saline suspension of dried toxoplasmas containing 25 mg/ml dry weight was made and vibrated at 9 kc for 45 minutes. In separate experiments the vibrated material was in one case first centrifuged to remove cellular debris and in the other the cellular debris was not removed. Sufficient ammonium sulfate was added to obtain fractions at 5, 10, 20, 30, 70, and 100% of saturation. This scheme is outlined in Fig. 1. Precipitation was carried out at 0°C by slowly adding dry ammonium sulfate with constant stirring. Precipitates that, formed after standing at 4°C for several hours mere removed Freeze-dried toxoplasma cells resuspended 25 mg/ml in butfered saline at pH i * Remove cellular debris
Sonic Vibrate 9 kc, 45 minutes
- --
Precipitate---
I ~5% saturated ammonium sulfate I
Resuspend, dialyze in buffered saline pH 7 I I Dye-test C.-F. test neutralization Precipitate !
___----
Dialyze in buffered saline pH 7
Supernatan----I I I
C.-L. test
Dye-test neutralization
30% saturated ammonium sulfate
Resuspend, dialyze in buffered saline pH 7 C.-F. test
Superhatant
I Dye-test nelItralization
-
----Dialyze saline I C.-F. test
Similarly saturated
in buffered pH 7 Dye-test neutralization
for 70 and 100% ammonium sulfate
* Two experiments performed. When cellular debris was not removed, no precipitates were obtained with 10 or 20% saturated ammonium sulfate. When cellular debris was removed, no precipitates were obtained with 5,10,2O, or 100% saturated ammonium sulfate. FIG. 1.
Scheme for obtaining
ammonium
sulfate
fractions.
452
HOOK
AND
FABER
by centrifugation and resuspended to approximately one-fourth original volume in buffered saline at pH 7. Supernatants and resuspended precipitates were dialysed at 4°C in buffered saline at pH 7. Complement fixation tests were run on these precipitates and supernatants to determine their antigenic activity. In order to study the relationship of protein material thus obtained to the dye test antibody, neutralization of dye test antibodies was attempted. Neutralization was carried out by incubating equal quantities of positive rat serum with each ammonium sulfate fraction (from fractionation in which cellular debris was not removed) at 4°C for 24 hours. After this time, mixtures were centrifuged to remove any precipitates that had formed and the supernatants tested for drop in dye-test titer. Extraction of Carbohydrate Fractions
Extraction of antigenic, protein-free carbohydrate material from sonic-fragmented toxoplasmas was attempted with trichloroacetic acid and cold ethanol fractionation (Martin, 1953, see Figure 2). Precipitates were obtained at 0°C with 50 and 75 per cent ethanol and were resuspended in 5 ml. 0.85 per cent saline. To determine whether carbohydrate material obtained by the above procedure would react in vitro as a complement fixing antigen or would neutralize dye test antibodies, complement fixation tests and dye test serum neutralizations were performed as above with protein fractions. In vivo Studies
In order to examine the ability of some of the carbohydrate and protein materials to elicit antibodies in guinea pigs, vaccines were prepared. Protein-free, carbohydrate material obtained by trichloroacetic acid treatment of toxoplasmas (point B, Fig. 2) was reduced in volume 75% by freeze-drying and preserved with 1: 10,000 merthiolate. This mixture was combined with an equal quantity of adjuvant prepared in the manner described by Freund et al. (1948). Toxoplasma protein known to be active as a complement fixing antigen, obtained by treatment of fragmented cells with 30 % saturated ammonium sulfate was used as follows to prepare a vaccine. The 30% saturated precipitate was resuspended to approximately one-fourth original volume, dialysed against buffered saline at pH 7 and preserved with 1: 10,000 merthiolate. This suspension was combined with an equal quantity of Freund adjuvant. Ten adult guinea pigs were each subcutaneously inoculated with 1 ml
ANTIGENIC
NATURE
OF
453
TOXOPLASMA
Freeze-dried toxoplasmas resuspended 25 mg/ml in distilled water I Sonic Vibrate 9 kc, 45 minutes I Remove protein with lo’% w/v trichloroacetic acid I Supernatant
Discard sediment
Neutralize to pH 7
Dialyze in water
I B IIJ
I Neutralize to pH 7 I
25% Ethanol
No Precipitate
I Supernatant 1 50% Ethanol
Precipitate I Resuspend in saline I
i
Supernatant I
177 C
75% Ethanol
Precipitate I Resuspend in saline I
FIG. 2. Scheme for obtaining
carbohydrate
fractions.
454
HOOK AND FABER
of the appropriate vaccine and were heart-bled after 1, 2, 3, 4, and 6 weeks had elapsed. Sera were stored at -20°C until complement fixation and dye tests were performed. Control Experiments
The peritoneal fluid of Toxoplasma-infected mice contains, in addition to toxoplasmas, white blood cells and perhaps other foreign material not removable by washing with saline. Therefore, it was felt that attempts should be made to determine whether sera used in the above experiments would fix complement with exudates and peritoneal tissue from mice not inoculated with toxoplasmas. Tissue from the abdominal walls of normal albino mice was ground in a mortar and suspended to approximately a 10% suspension with 0.85% saline. In addition, peritoneal exudate production was induced in another group of mice by intraperitoneal inoculation of 0.2 ml of Freund adjuvant. A viscous exudate was collected 3 days later and diluted with saline washings of the peritoneum to yield a cloudy suspension. Cross titration of these tissues and exudate antigens with strongly positive sera from Toxoplasma-infected guinea pigs showed that no non-specific fixation of complement occurred. RESULTS
A preliminary experiment showed that sonic vibrations at 9 kc for 30 to 45 minutes were effective in releasing complement fixing antigen from saline suspended toxoplasmas. Vibration for as long as 75 minutes did not appreciably destroy antigenicity. Protein Fractions
Fractional precipitation of fragmented toxoplasmas with ammonium sulfate at concentrations of 5, 10, 20, 30, 70, and 100% of saturation indicated that maximum precipitation of antigenic material occurred at 30% of saturation regardless of whether cellular debris was [or was not] removed before precipitation. When cellular debris was removed before fractional precipitation, precipitates were obtained only with 30 and 70 % saturated ammonium sulfate. This 30% saturated precipitate had an antigenic titer of 1:s while the 70 % precipitate titered at 1: 2. Precipitation of sonic fragmented toxoplasmas without first removing cellular debris also yielded precipitates with 30 and 70% saturated ammonium sulfate which had antigenic titers of 1: 8 and 1: 1 respectively.
ANTIGENIC
NATURE
455
OF TOXOPLASMA
TABLE
I
Complement-Fixing Antigenicity of Ammonium Sulfate Fractions of Sonically Fragmented Toxoplasmas both with and without Removal of Cellular Debris after Vibration Complement-fixation Antigen (ammonium sulfate fraction)
titers*
Cellular debris removed
Cellular debris included
5% Sat. precipitate 5% Sat. supernatant
None obtained 1:s
1:2 1:16
30% Sat. precipitate 30% Sat. supernatant
1:8 I:2
1:s 1:2
70% Sat. precipitate 70% Sat. supernatant
1:2 0
I:1 0
100~0 Sat. precipitate 100~o Sat. supernatant
None obtained -
0 0
* Reciprocal of antigen dilution yielding 50% inhibition tion with .verum dilutions of 1:64. 1: 128, 1:256, and 1:512.
of hemolysia (2 plus reaction) in cross titra.
In addition, a copious precipitate was obtained with 5% saturated ammonium sulfate and its removal yielded a clear supernatant. A slight precipitate formed at 100% of saturation. The 5 % precipitate, probably containing much of the cellular debris, had a titer of 1:2 while the precipitate formed at full saturation was inactive. No precipitates were obtained with 10 or 20% ammonium sulfate in either part of this experiment. Complement fixing titers of these fractions are shown in Table I. Ammonium sulfate fractions (as was the standard antigen) were in some casesanticomplementary in low dilutions. Anticomplementary activity could be reduced by heating undiluted antigen to 50°C for 30 minutes. Such treatment did not reduce complement fixing activity with positive sera. All complement fixing activity was destroyed by heating antigen to 100°C for 30 minutes. Carbohydrate Fractions
Removal of protein from sonic-fragmented toxoplasmas by trichloroacetic acid yielded a material which gave a positive anthrone reaction, indicating the presence of a carbohydrate, and a negative biuret test. Samples of this carbohydrate were tested for ability to act as comple-
456
HOOK
AND
FABER
ment-fixing antigens both before and after fractional precipitation with cold ethanol, but in neither case was activity detected. Precipitates were obtained with 50 and 75 %, but not with25 % ethanol. Carbohydrate material neutralized to pH 7.0 but not precipitated with ethanol (point B, Fig. 2) was found to be anticomplementary in low dilutions. DiaIysis of this material eliminated the anticomplementary factor but it did not reveal any complement-fixing activity. Neutralization
of Dye-Test Antibodies
It was possible to neutralize dye-test antibodies with the 30% saturated ammonium sulfate precipitate obtained in the experiment where cellular debris was not removed after fragmentation (Table II). A reduction of dye-test titer from 1:64 to 1: 16 was accomplished. It appeared that the ability to neutralize dye-test antibodies was related to complement fixing activity. Dye-test antibodies were not neutralized with carbohydrate material from toxoplasmas. Antibody Production in Guinea Pigs Inoculated with Toxoplasma Protein and Carbohydrate
Carbohydrate material obtained by trichloroacetic acid treatment of toxoplasmas, followed by neutralization to pH 7 (point B, Fig. 2) and TABLE Neutralization
of Dye-Test
Neutralizing
antigens
Saline (control)
l
II
Antibodies with Ammonium of Sonically Fragmented Toxoplasmas
Sulfate
Dye-test titer’
1:64
5% Precipitate 5% Supernatant
1:64 1:32
30% Precipitate 30% Supernatant
I:16 1:64
700/, Precipitate 70% Supernatant
1:64 1:64
100% Precipitate 100% Supernatant
1:64 1:64
Titer expressed aa reciprocal of dilution.
Fractions
rlNTIGENIC
NATURE
OF
457
TOXOPLASMA
TABLE III Complement-Fixing and Dye-Test Titers Induced in Guinea Pigs Inoculated with Protein from Sonically Fragmented Toxoplasmas plus Adjwant I
; Complement-fixation Guinea pig No.
Vaccine
‘Toxoplasma protein 30% saturated ammonium sulfate precipitate
* Titer expressed m--No serum. a/c-Anticomplementntry
ad reciprocal
Days
after
A3 A4 A5 A8 A9
titers’
Dye-test
inoculation
Days
0 0 0 0
0 0 0 0
after
64 64 16 64
titer’ inoculation
64 Died 64 64 16 16 64 64
16 16 16 16 -
of dilution.
serum.
combined with Freund adjuvant did not produce either dye test or complement fixing antibodies. Toxoplasma protein obtained by precipitation of fragmented cells with 30 % saturated ammonium sulfate was capable of eliciting dye-test and complement-fixing antibodies. Antibody response to this vaccine in combination with adjuvant is shown in Table III. Combined with adjuvant, this protein vaccine induced maximum complement-fixing titers in most cases, ranging between 1: 16 and 1: 32 and a maximum dye test titer of 1:64. Dye-test titers, which were detectable 2 weeks after inoculation, could be demonstrated 6 weeks later. A protein vaccine not combined with adjuvant produced lower complement fixing and dye-test titers than did the same vaccine with adjuvant. It is of interest to note the relatively low dye-test titers detected. In animals immunized or experimentally infected by Cutchins and Warren (1956), dye-test titers were commonly much higher than those encountered in the current work. This might indicate that our antigen was partially inactivat’ed by the fractionation procedure. SUMMARY
Fractionation of Tozoplaasma gondii suggested that both dye-test and complement-fixing antigenicity were related to protein material obtained from sonically fragmented cells by precipitation with 30% saturated ammonium sulfate at pH 7.0. This protein material was capable of
458
HOOK AND FABER
neutralizing dye-test antibodies, acting as a complement-fixing antigen, and producing both complement-fixing and dye-test antibodies in guinea pigs. Carbohydrate from sonically fragmented toxoplasmas showed no antigenic activity in relation to the complement fixation or dye tests. REFERENCES CUTCHINS, E. C., AND WARREN, J. 1956. Immunity patterns in the guinea pig and rat following Toxoplasma infection and vaccination with killed Toxoplasma. Am, J. Trap. Med. Hyg. 6, 197-209. FRENBEL, J. K., AND FRIEDLANDER, S. 1951. Toxoplasmosis. Pathology of neonatal disease. Pathogenesis, diagnosis and treatment. Public Health Service Pub. No. 141, Govt. Printing Office, Washington, D. C. 105 pp. FREUND, J., THOMSON, K. J., HOUOH, H. B., SOMMER, H. E., AND PISANI, T. M. 1948. Antibody formation and sensitization with the aid of adjuvants. J. Immunol. 80.383-398. MARTIN, D. S. 1953. Serologic studies on North American blastomycosis. Studies
with soluble antigens from untreated and sonic treated yeast-phase cells of Blastomyces dermatitidis. J. Immunol. 71,192~291. SABIN, A. B. 1941. Toxoplasmic encephalitis in children. J. A. M. A. 116,801~807. SABIN, A. B. 1949. Complement fixation test in toxoplasmosis and persistence of antibody in human beings. Pediatrics 4,443453. SABIN, A. B., AND FELDMAN, H. A. 1948. Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoon parasite (Tozoplasma). Science 108, 669-663. WARREN, J., AND Russ, S. B. 1948. Cultivation of toxoplasma in embryonated egg. An antigen derived from chorioallantoic membrane. Proc. Sot. Ezptl. Biol. Med. 67, 85-89.
6, 449-458 (1957)
PARASITOLOGY
ORIGINAL * Fractionation
PAPERS *
Studies on the Antigenic Toxoplasma gondii
William Department
RESEARCH * * *
A. Hook
of Bacteriology,
and
TIniversity
John
E. Faber,
of Maryland,
(Submitted for publication,
Nature
of
Jr.
College Park,
Maryland
26 October 1956)
Cutchins and Warren (1956) showed that antibodies could be induced in guinea pigs by antigens prepared from killed toxoplasma. Using formaldehyde-killed and sonic-fragmented toxoplasma these authors demonstrated that vaccines could be prepared which imparted immunity as well as elicited antibodies detectable by complement fixation and the Sabin-Feldman (1948) dye test. Because the mechanisms of these two tests differ as well as some of the characteristics of the antibodies produced (Sabin, 1949), it was thought possible that some differences might be demonstrable between antigenic components of toxoplasma responsible for these antibodies. An effort was made to fractionate toxoplasma into protein and carbohydrate components and to investigate their antigenic activity in vitro and in vivo. MATEHIAL~
AND METHODS
A subline’ of the RH strain (Sabin, 1941) of ToxopZusma go&ii was used in this work. Serial passagesat 6 day intervals were made by interperitoneal inoculation of 10 albino mice each with 0.1 ml of a lo-” dilution of peritoneal exudate from the previous passage. To harvest toxoplasmas for fractionation, large numbers of mice were inoculated interperitoneally and tapped from the same site. An average of 0.5 ml of exudate containing between 15,000,OOOand 19,000,OOO toxoplasmas per ml was usually obtained. Toxoplasmas and accompanying cellular material from the exudate were washed twice with 0.85 % saline and 1 Obtained from Dr. E. C. Cutchins, National Maryland. 449
Institut,es of Health, Bethesda,
450
HOOK
AND
FABER
resuspended to original exudate volume. This material was freeze dried and stored at -20°C until used. Approximately 1 gram of dried organisms were obtained in this manner. Although the quantity of toxoplasmas was thus limited, the fractionation experiments in this work were performed at least twice. A soluble, standard complement king antigen for comparative purposes was prepared from toxoplasmas obtained from the peritoneal exudate of infected mice by a method similar to that described by Sabin (1949) and modified by Cutchins and Warren (1956). Toxoplasmas were washed and resuspended to original exudate volume with 0.85% saline and fragmented by sonic vibrations at a frequency of 9 kc for 30 minutes in a water-cooled transducer of a sonic oscillator2. The suspension containing ruptured cells was then centrifuged at 8000 g for 30 minutes at 4°C to remove cellular debris, The supernatant constituted the antigen and was stored at -2O’C. Standardization of the antigen was performed by cross titrating with immune rabbit serum of known titer. The complement fixing activity of standard antigens and of the carbohydrate and protein fractions prepared in this study was tested by a procedure similar to that described by Warren and Russ (1948). Cross titrations were made of test antigens with a pooled serum from six adult guinea pigs bled 30 days after interperitoneal inoculation with a sub-lethal dose of live toxoplasmas. The complement fixing titer of this serum was 1:512. Negative sera were obtained from uninoculated guinea pigs. The Sabin-Feldman dye test for toxoplasma antibodies was performed according to a procedure outlined by Frenkel and Friedlander (1951). Dye test positive sera for controls and for the antibody neutralization experiment were obtained from adult rats that had been intraperitoneally inoculated with live toxoplasmas and bled 15 days later. All sera collected for complement fixation and dye tests were first inactivated at 56°C for 30 minutes. Ammonium Sulfate Precipitation
of Protein
Sonic-fragmented toxoplasmas were precipitated with increasing concentrations of ammonium sulfate to isolate the fraction most active as a complement fixing antigen and, if possible, to identify a fraction p Manufactured
ment, Waltham
by Raytheon Manufacturing 54, Massachusetts.
Co., Medical
Products
Depart-
ANTIGENIC
NATURE
OF TOXOPLASMA
451
with the dye test. Preliminary experiments showed that the pH range 7 to 8 was optimal for carrying out the precipitation procedure. A phosphate buffered (M/100) saline suspension of dried toxoplasmas containing 25 mg/ml dry weight was made and vibrated at 9 kc for 45 minutes. In separate experiments the vibrated material was in one case first centrifuged to remove cellular debris and in the other the cellular debris was not removed. Sufficient ammonium sulfate was added to obtain fractions at 5, 10, 20, 30, 70, and 100% of saturation. This scheme is outlined in Fig. 1. Precipitation was carried out at 0°C by slowly adding dry ammonium sulfate with constant stirring. Precipitates that, formed after standing at 4°C for several hours mere removed Freeze-dried toxoplasma cells resuspended 25 mg/ml in butfered saline at pH i * Remove cellular debris
Sonic Vibrate 9 kc, 45 minutes
- --
Precipitate---
I ~5% saturated ammonium sulfate I
Resuspend, dialyze in buffered saline pH 7 I I Dye-test C.-F. test neutralization Precipitate !
___----
Dialyze in buffered saline pH 7
Supernatan----I I I
C.-L. test
Dye-test neutralization
30% saturated ammonium sulfate
Resuspend, dialyze in buffered saline pH 7 C.-F. test
Superhatant
I Dye-test nelItralization
-
----Dialyze saline I C.-F. test
Similarly saturated
in buffered pH 7 Dye-test neutralization
for 70 and 100% ammonium sulfate
* Two experiments performed. When cellular debris was not removed, no precipitates were obtained with 10 or 20% saturated ammonium sulfate. When cellular debris was removed, no precipitates were obtained with 5,10,2O, or 100% saturated ammonium sulfate. FIG. 1.
Scheme for obtaining
ammonium
sulfate
fractions.
452
HOOK
AND
FABER
by centrifugation and resuspended to approximately one-fourth original volume in buffered saline at pH 7. Supernatants and resuspended precipitates were dialysed at 4°C in buffered saline at pH 7. Complement fixation tests were run on these precipitates and supernatants to determine their antigenic activity. In order to study the relationship of protein material thus obtained to the dye test antibody, neutralization of dye test antibodies was attempted. Neutralization was carried out by incubating equal quantities of positive rat serum with each ammonium sulfate fraction (from fractionation in which cellular debris was not removed) at 4°C for 24 hours. After this time, mixtures were centrifuged to remove any precipitates that had formed and the supernatants tested for drop in dye-test titer. Extraction of Carbohydrate Fractions
Extraction of antigenic, protein-free carbohydrate material from sonic-fragmented toxoplasmas was attempted with trichloroacetic acid and cold ethanol fractionation (Martin, 1953, see Figure 2). Precipitates were obtained at 0°C with 50 and 75 per cent ethanol and were resuspended in 5 ml. 0.85 per cent saline. To determine whether carbohydrate material obtained by the above procedure would react in vitro as a complement fixing antigen or would neutralize dye test antibodies, complement fixation tests and dye test serum neutralizations were performed as above with protein fractions. In vivo Studies
In order to examine the ability of some of the carbohydrate and protein materials to elicit antibodies in guinea pigs, vaccines were prepared. Protein-free, carbohydrate material obtained by trichloroacetic acid treatment of toxoplasmas (point B, Fig. 2) was reduced in volume 75% by freeze-drying and preserved with 1: 10,000 merthiolate. This mixture was combined with an equal quantity of adjuvant prepared in the manner described by Freund et al. (1948). Toxoplasma protein known to be active as a complement fixing antigen, obtained by treatment of fragmented cells with 30 % saturated ammonium sulfate was used as follows to prepare a vaccine. The 30% saturated precipitate was resuspended to approximately one-fourth original volume, dialysed against buffered saline at pH 7 and preserved with 1: 10,000 merthiolate. This suspension was combined with an equal quantity of Freund adjuvant. Ten adult guinea pigs were each subcutaneously inoculated with 1 ml
ANTIGENIC
NATURE
OF
453
TOXOPLASMA
Freeze-dried toxoplasmas resuspended 25 mg/ml in distilled water I Sonic Vibrate 9 kc, 45 minutes I Remove protein with lo’% w/v trichloroacetic acid I Supernatant
Discard sediment
Neutralize to pH 7
Dialyze in water
I B IIJ
I Neutralize to pH 7 I
25% Ethanol
No Precipitate
I Supernatant 1 50% Ethanol
Precipitate I Resuspend in saline I
i
Supernatant I
177 C
75% Ethanol
Precipitate I Resuspend in saline I
FIG. 2. Scheme for obtaining
carbohydrate
fractions.
454
HOOK AND FABER
of the appropriate vaccine and were heart-bled after 1, 2, 3, 4, and 6 weeks had elapsed. Sera were stored at -20°C until complement fixation and dye tests were performed. Control Experiments
The peritoneal fluid of Toxoplasma-infected mice contains, in addition to toxoplasmas, white blood cells and perhaps other foreign material not removable by washing with saline. Therefore, it was felt that attempts should be made to determine whether sera used in the above experiments would fix complement with exudates and peritoneal tissue from mice not inoculated with toxoplasmas. Tissue from the abdominal walls of normal albino mice was ground in a mortar and suspended to approximately a 10% suspension with 0.85% saline. In addition, peritoneal exudate production was induced in another group of mice by intraperitoneal inoculation of 0.2 ml of Freund adjuvant. A viscous exudate was collected 3 days later and diluted with saline washings of the peritoneum to yield a cloudy suspension. Cross titration of these tissues and exudate antigens with strongly positive sera from Toxoplasma-infected guinea pigs showed that no non-specific fixation of complement occurred. RESULTS
A preliminary experiment showed that sonic vibrations at 9 kc for 30 to 45 minutes were effective in releasing complement fixing antigen from saline suspended toxoplasmas. Vibration for as long as 75 minutes did not appreciably destroy antigenicity. Protein Fractions
Fractional precipitation of fragmented toxoplasmas with ammonium sulfate at concentrations of 5, 10, 20, 30, 70, and 100% of saturation indicated that maximum precipitation of antigenic material occurred at 30% of saturation regardless of whether cellular debris was [or was not] removed before precipitation. When cellular debris was removed before fractional precipitation, precipitates were obtained only with 30 and 70 % saturated ammonium sulfate. This 30% saturated precipitate had an antigenic titer of 1:s while the 70 % precipitate titered at 1: 2. Precipitation of sonic fragmented toxoplasmas without first removing cellular debris also yielded precipitates with 30 and 70% saturated ammonium sulfate which had antigenic titers of 1: 8 and 1: 1 respectively.
ANTIGENIC
NATURE
455
OF TOXOPLASMA
TABLE
I
Complement-Fixing Antigenicity of Ammonium Sulfate Fractions of Sonically Fragmented Toxoplasmas both with and without Removal of Cellular Debris after Vibration Complement-fixation Antigen (ammonium sulfate fraction)
titers*
Cellular debris removed
Cellular debris included
5% Sat. precipitate 5% Sat. supernatant
None obtained 1:s
1:2 1:16
30% Sat. precipitate 30% Sat. supernatant
1:8 I:2
1:s 1:2
70% Sat. precipitate 70% Sat. supernatant
1:2 0
I:1 0
100~0 Sat. precipitate 100~o Sat. supernatant
None obtained -
0 0
* Reciprocal of antigen dilution yielding 50% inhibition tion with .verum dilutions of 1:64. 1: 128, 1:256, and 1:512.
of hemolysia (2 plus reaction) in cross titra.
In addition, a copious precipitate was obtained with 5% saturated ammonium sulfate and its removal yielded a clear supernatant. A slight precipitate formed at 100% of saturation. The 5 % precipitate, probably containing much of the cellular debris, had a titer of 1:2 while the precipitate formed at full saturation was inactive. No precipitates were obtained with 10 or 20% ammonium sulfate in either part of this experiment. Complement fixing titers of these fractions are shown in Table I. Ammonium sulfate fractions (as was the standard antigen) were in some casesanticomplementary in low dilutions. Anticomplementary activity could be reduced by heating undiluted antigen to 50°C for 30 minutes. Such treatment did not reduce complement fixing activity with positive sera. All complement fixing activity was destroyed by heating antigen to 100°C for 30 minutes. Carbohydrate Fractions
Removal of protein from sonic-fragmented toxoplasmas by trichloroacetic acid yielded a material which gave a positive anthrone reaction, indicating the presence of a carbohydrate, and a negative biuret test. Samples of this carbohydrate were tested for ability to act as comple-
456
HOOK
AND
FABER
ment-fixing antigens both before and after fractional precipitation with cold ethanol, but in neither case was activity detected. Precipitates were obtained with 50 and 75 %, but not with25 % ethanol. Carbohydrate material neutralized to pH 7.0 but not precipitated with ethanol (point B, Fig. 2) was found to be anticomplementary in low dilutions. DiaIysis of this material eliminated the anticomplementary factor but it did not reveal any complement-fixing activity. Neutralization
of Dye-Test Antibodies
It was possible to neutralize dye-test antibodies with the 30% saturated ammonium sulfate precipitate obtained in the experiment where cellular debris was not removed after fragmentation (Table II). A reduction of dye-test titer from 1:64 to 1: 16 was accomplished. It appeared that the ability to neutralize dye-test antibodies was related to complement fixing activity. Dye-test antibodies were not neutralized with carbohydrate material from toxoplasmas. Antibody Production in Guinea Pigs Inoculated with Toxoplasma Protein and Carbohydrate
Carbohydrate material obtained by trichloroacetic acid treatment of toxoplasmas, followed by neutralization to pH 7 (point B, Fig. 2) and TABLE Neutralization
of Dye-Test
Neutralizing
antigens
Saline (control)
l
II
Antibodies with Ammonium of Sonically Fragmented Toxoplasmas
Sulfate
Dye-test titer’
1:64
5% Precipitate 5% Supernatant
1:64 1:32
30% Precipitate 30% Supernatant
I:16 1:64
700/, Precipitate 70% Supernatant
1:64 1:64
100% Precipitate 100% Supernatant
1:64 1:64
Titer expressed aa reciprocal of dilution.
Fractions
rlNTIGENIC
NATURE
OF
457
TOXOPLASMA
TABLE III Complement-Fixing and Dye-Test Titers Induced in Guinea Pigs Inoculated with Protein from Sonically Fragmented Toxoplasmas plus Adjwant I
; Complement-fixation Guinea pig No.
Vaccine
‘Toxoplasma protein 30% saturated ammonium sulfate precipitate
* Titer expressed m--No serum. a/c-Anticomplementntry
ad reciprocal
Days
after
A3 A4 A5 A8 A9
titers’
Dye-test
inoculation
Days
0 0 0 0
0 0 0 0
after
64 64 16 64
titer’ inoculation
64 Died 64 64 16 16 64 64
16 16 16 16 -
of dilution.
serum.
combined with Freund adjuvant did not produce either dye test or complement fixing antibodies. Toxoplasma protein obtained by precipitation of fragmented cells with 30 % saturated ammonium sulfate was capable of eliciting dye-test and complement-fixing antibodies. Antibody response to this vaccine in combination with adjuvant is shown in Table III. Combined with adjuvant, this protein vaccine induced maximum complement-fixing titers in most cases, ranging between 1: 16 and 1: 32 and a maximum dye test titer of 1:64. Dye-test titers, which were detectable 2 weeks after inoculation, could be demonstrated 6 weeks later. A protein vaccine not combined with adjuvant produced lower complement fixing and dye-test titers than did the same vaccine with adjuvant. It is of interest to note the relatively low dye-test titers detected. In animals immunized or experimentally infected by Cutchins and Warren (1956), dye-test titers were commonly much higher than those encountered in the current work. This might indicate that our antigen was partially inactivat’ed by the fractionation procedure. SUMMARY
Fractionation of Tozoplaasma gondii suggested that both dye-test and complement-fixing antigenicity were related to protein material obtained from sonically fragmented cells by precipitation with 30% saturated ammonium sulfate at pH 7.0. This protein material was capable of
458
HOOK AND FABER
neutralizing dye-test antibodies, acting as a complement-fixing antigen, and producing both complement-fixing and dye-test antibodies in guinea pigs. Carbohydrate from sonically fragmented toxoplasmas showed no antigenic activity in relation to the complement fixation or dye tests. REFERENCES CUTCHINS, E. C., AND WARREN, J. 1956. Immunity patterns in the guinea pig and rat following Toxoplasma infection and vaccination with killed Toxoplasma. Am, J. Trap. Med. Hyg. 6, 197-209. FRENBEL, J. K., AND FRIEDLANDER, S. 1951. Toxoplasmosis. Pathology of neonatal disease. Pathogenesis, diagnosis and treatment. Public Health Service Pub. No. 141, Govt. Printing Office, Washington, D. C. 105 pp. FREUND, J., THOMSON, K. J., HOUOH, H. B., SOMMER, H. E., AND PISANI, T. M. 1948. Antibody formation and sensitization with the aid of adjuvants. J. Immunol. 80.383-398. MARTIN, D. S. 1953. Serologic studies on North American blastomycosis. Studies
with soluble antigens from untreated and sonic treated yeast-phase cells of Blastomyces dermatitidis. J. Immunol. 71,192~291. SABIN, A. B. 1941. Toxoplasmic encephalitis in children. J. A. M. A. 116,801~807. SABIN, A. B. 1949. Complement fixation test in toxoplasmosis and persistence of antibody in human beings. Pediatrics 4,443453. SABIN, A. B., AND FELDMAN, H. A. 1948. Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoon parasite (Tozoplasma). Science 108, 669-663. WARREN, J., AND Russ, S. B. 1948. Cultivation of toxoplasma in embryonated egg. An antigen derived from chorioallantoic membrane. Proc. Sot. Ezptl. Biol. Med. 67, 85-89.