Vol. 24, No. 3
INFECTION AND IMMUNITY, June 1979, p. 774-779 0019-9567/79/06-0774/06$02.00/0
Antigenic Specificity of Neutralizing Antibody to Cholera Toxin JOHNNY W. PETERSON,`* KELLY E. HEJTMANCIK,' DAIEL E. MARKEL,' JOHN P. CRAIG,2
AND
ALEXANDER KUROSKY3
Department of Microbiology' and Department of Human Genetics and Biological Chemistry,3 The University of Texas Medical Branch, Galveston, Texas 77550; and Department of Microbiology and Immunology, Downstate Medical Center, State University of New York, Brooklyn, New York 112032
Received for publication 13 March 1979
Selected rabbit antisera to cholera toxin antigens and convalescent cholera patient sera were analyzed using the permeability factor neutralization test and two sensitive in vitro serological assays specific for cholera toxin, cholera toxin A subunit, and cholera toxin B subunit. The results indicated that antisera to cholera toxin contained toxin-neutralizing activity as well as antibodies specific for both the A subunit and B subunit. It was clearly established that antisera to B subunit, devoid of significant anti-A subunit activity, neutralized the vascular permeability activity of cholera toxin. Antisera to A subunit contained neutralizing antibodies and antibodies to both A and B subunits. Absorption with B subunit removed both the toxin-neutralizing and anti-B subunit activities, while the anti-A activity was unaffected. Neutralizing antibody titers of rabbits immunized with B subunit were also observed to be significantly higher than neutralizing antibody titers of sera from A subunit-immunized rabbits, despite the overall similarity in anti-B subunit titers as determined by passive hemagglutination and radioimmunoassay of sera from the two groups of rabbits. Anti-a chain sera neither neutralized cholera toxin nor possessed significant antitoxin or anti-B subunit titers as determined by passive hemagglutination and radioimmunoassay. The anti-a chain sera contained high levels of antibody specific for A subunit, which is consistent with the hypothesis that the a chain is part of the A subunit structure. In contrast, the -y chain was not shown to be antigenic. Sera from convalescent cholera patients possessed toxin-neutralizing antibody as well as passive hemagglutination and radioimmunoassay antibody against both A and B subunits. Antibodies specific for cholera toxin arise following natural infection with Vibrio cholerae (2, 5, 14), and in response to immunization of humans (13) and experimental animals (12) with cholera toxoids (15). A variety of serological assays have been used to quantitate the serum antitoxin levels by either measuring toxin-neutralizing capacity or merely detecting toxin-antibody binding reactions. There is considerable uncertainty as to the nature of the determinants on the toxin molecule that are responsible for eliciting neutralizing antibodies in human and animal sera (1, 12). In contrast, the chemical structure of the toxin is better defined (8). It is composed of two subunits designated A and B. The A subunit, which is responsible for activation of adenylate cyclase, consists of two polypeptides referred to as the a (24,000 daltons) and y (9,700 daltons) chains. The B subunit is responsible for binding the toxin to tissue ganglioside (GM,) and is composed of an aggregate of 774
four to six /3 chains (9,700 daltons). To further define the antigenic structure of the toxin, this study was initiated to examine various antisera to cholera toxin and its purified components by the passive hemagglutination (PHA) test, radioimmunoassay (RIA), and permeability factor (PF) neutralization test. These procedures have allowed us to determine the component of cholera toxin that is responsible for eliciting and reacting with neutralizing antibody. MATERIALS AND METHODS Preparation of antigens. Cholera toxin was purified from fermenter cultures of V. cholerae 569B grown in asparagine-glucose medium (10) and characterized as described previously (6, 8). The A and B subunits of cholera toxin were separated by gel filtration on a column of Bio-Gel P-60 acrylamide eluted with 0.2 M sodium formate buffer (pH 3.5) containing 5.2 M guanidine-hydrochloride (9). The A subunit preparation was then treated with 2-mercaptoethanol and alkylated with iodoacetamide prior to chromatog-
VOL. 24, 1979
NEUTRALIZING ANTIBODY TO CHOLERA TOXIN
raphy on the same Bio-Gel P-60 column in the presence of 5.2 M guanidine-hydrochloride to obtain the constituent a and y polypeptide chains. Guanidine was removed from each preparation by dialysis against tris(hydroxymethyl)aminomethane buffer (pH 7.5) (4). Preparation of antisera. Antiserum specific for cholera toxin was obtained by immunization of four rabbits with multiple subcutaneous doses of purified cholera toxin (50 fug) emulsified in Freund complete adjuvant (6). Serum samples obtained over a period of 6 months were pooled and distributed into aliquots prior to storage at -20oC. Three groups of five rabbits each were immunized with A subunit, B subunit, a chain, or y chain emulsified in Freund complete adjuvant (D. E. Markel, K. E. Hejtmancik, J. W. Peterson, F. Martin, and A. Kurosky, submitted for publication). Briefly, rabbits were immunized with an initial dose of 200 .g of the respective antigen preparation, which was administered by multiple-site injection by the intraperitoneal, intramuscular, and subcutaneous routes. Serum samples were taken from the rabbits every 3 weeks and stored at -20'C. A booster dose of 200 jig was administered by multiple-site injections whenever antibody titers began to decrease, as determined by PHA with toxinsensitized erythrocytes (5). A 1-ml sample of each antiA subunit serum was absorbed with purified B subunit by the addition of 50 jig of B subunit contained in 60 p1 of tris(hydroxymethyl)aminomethane buffer. This amount of B subunit was twice the concentration necessary to prevent formation of precipitin lines when the anti-A subunit sera were tested against B subunit using Ouchterlony double-gel diffusion (Markel et al., submitted for publication). Convalescent sera from cholera patients were obtained from the Cholera Research Laboratory in Dacca, Bangladesh. Serological titrations. Identical sets of sera specific for cholera toxin and its components were assayed by each of three basic tests. The PHA test for cholera antitoxin (5) was performed as previously described using chicken erythrocytes sensitized with cholera toxin, A subunit, or B subunit. In each assay system, antibody titers were expressed in units per milliliter based on the provisional standard Swiss Serum and Vaccine Institute serum [EC3-(A-2/67)-B], which has been assigned a value of 4,470 antitoxin units per ml
(3).
The sera were also examined by the RIA method of Hejtmancik et al. employing "nI-labeled cholera toxin, A subunit, or B subunit (6). Antibody titers were expressed in units per milliliter based on a pooled rabbit antiserum to cholera toxin (8/13/73) that had been standardized by the PF neutralization test against the Swiss Serum and Vaccine Institute stan-
dard. Whereas the above assays measured antibody levels specific for toxin, A subunit, or B subunit, respectively, none measured the neutralizing potency of the selected sera. Therefore, the PF neutralization test originally described by Craig (1) was selected to measure the functional significance of antibodies to the separate regions of the cholera toxin molecule. The test, performed as described previously, measured the capacity
775
of each serum to prevent the increase in permeability of the small blood vessels of the skin of adult rabbits induced by cholera toxin (2).
RESULTS Table 1 depicts the general format of analysis used throughout this study. All sera were assayed for toxin-neutralizing antibody by the PF neutralization test, and total antibody to cholera toxin, A subunit, and' B subunit was measured by the PHA test and RIA. Rabbit antiserum to cholera toxin contained both toxin-neutralizing antibodies and antibodies specific for both the A and B subunits of cholera toxin. In contrast, each of the serological assays revealed that normal rabbit and human sera contained no significant levels of antibody to cholera toxin. When successive blood samples of two rabbits immunized with B subunit were examined by the same assays (Table 2), we observed that toxin-neutralizing antibody titers correlated well with PHA and RIA cholera antitoxin titers. The anti-B subunit assays also' revealed titers comparable to the neutralizing and anti-cholera toxin (PHA and RIA) levels, whereas antibody to A subunit was undetectable by RIA'and of low titer by PHA. To determine if anti-A subunit sera contained neutralizing antibody, anti-A subunit sera were tested in the same assays before and after absorption with B subunit (Table 3). Successive serum samples from A subunit-immunized rabbits contained significant levels of toxin-neutralizing antibody, but virtually all of the neutralizing activity was removed from each serum upon absorption of 1-ml samples of the anti-A sera with 50 ,ug of B subunit. On closer inspection it is evident that the anti-A subunit sera contained high levels of anti-B subunit antibody activity that was also removed by absorption with B subunit. After the removal of the anti-B subunit activity, antibody to A subunit remained elevated but toxin-neutralizing activity was lost. The antitoxin titer of antisera to A subunit was virtually eliminated by absorption with B subunit, as determined by the PHA test with toxinsensitized erythrocytes. The levels of antibody to B subunit (PHA and RIA) observed with sera of A and B subunitimmunized rabbits were remarkably similar, as shown in Tables 2 and 3. Despite the similar levels of antibody to B subunit, the neutralizing antibody titers of the two groups of animals were quite different. Table 4 presents a comparison of serum titer ratios of PHA and RIA titers to the B subunit with anti-PF titers of rabbits immunized with the A or B subunit. The PHA anti-B titer/PF titer ratio was at least 10-fold higher in sera of A subunit-immuiized rabbits than in sera
776
PETERSON ET AL.
INFECT. IMMUN.
TABLE 1. Serological analysis ofpooled cholera antitoxin compared to normal rabbit serum Rabbit Serum Specificity
Neutralization Titer
(AU/ml)
Anti-Cholera Toxin Anti-A Subunit Titer Anti-B Subunit Titer Titer (AU/ml)
PF
PHA/RIA
PHA/RIA
PHA/RIA
Pooled Antiserum to Cholera toxin
790
2235/810*
140/810*
4470/810*
Pooled normal serum
INFECTION AND IMMUNITY, June 1979, p. 774-779 0019-9567/79/06-0774/06$02.00/0
Antigenic Specificity of Neutralizing Antibody to Cholera Toxin JOHNNY W. PETERSON,`* KELLY E. HEJTMANCIK,' DAIEL E. MARKEL,' JOHN P. CRAIG,2
AND
ALEXANDER KUROSKY3
Department of Microbiology' and Department of Human Genetics and Biological Chemistry,3 The University of Texas Medical Branch, Galveston, Texas 77550; and Department of Microbiology and Immunology, Downstate Medical Center, State University of New York, Brooklyn, New York 112032
Received for publication 13 March 1979
Selected rabbit antisera to cholera toxin antigens and convalescent cholera patient sera were analyzed using the permeability factor neutralization test and two sensitive in vitro serological assays specific for cholera toxin, cholera toxin A subunit, and cholera toxin B subunit. The results indicated that antisera to cholera toxin contained toxin-neutralizing activity as well as antibodies specific for both the A subunit and B subunit. It was clearly established that antisera to B subunit, devoid of significant anti-A subunit activity, neutralized the vascular permeability activity of cholera toxin. Antisera to A subunit contained neutralizing antibodies and antibodies to both A and B subunits. Absorption with B subunit removed both the toxin-neutralizing and anti-B subunit activities, while the anti-A activity was unaffected. Neutralizing antibody titers of rabbits immunized with B subunit were also observed to be significantly higher than neutralizing antibody titers of sera from A subunit-immunized rabbits, despite the overall similarity in anti-B subunit titers as determined by passive hemagglutination and radioimmunoassay of sera from the two groups of rabbits. Anti-a chain sera neither neutralized cholera toxin nor possessed significant antitoxin or anti-B subunit titers as determined by passive hemagglutination and radioimmunoassay. The anti-a chain sera contained high levels of antibody specific for A subunit, which is consistent with the hypothesis that the a chain is part of the A subunit structure. In contrast, the -y chain was not shown to be antigenic. Sera from convalescent cholera patients possessed toxin-neutralizing antibody as well as passive hemagglutination and radioimmunoassay antibody against both A and B subunits. Antibodies specific for cholera toxin arise following natural infection with Vibrio cholerae (2, 5, 14), and in response to immunization of humans (13) and experimental animals (12) with cholera toxoids (15). A variety of serological assays have been used to quantitate the serum antitoxin levels by either measuring toxin-neutralizing capacity or merely detecting toxin-antibody binding reactions. There is considerable uncertainty as to the nature of the determinants on the toxin molecule that are responsible for eliciting neutralizing antibodies in human and animal sera (1, 12). In contrast, the chemical structure of the toxin is better defined (8). It is composed of two subunits designated A and B. The A subunit, which is responsible for activation of adenylate cyclase, consists of two polypeptides referred to as the a (24,000 daltons) and y (9,700 daltons) chains. The B subunit is responsible for binding the toxin to tissue ganglioside (GM,) and is composed of an aggregate of 774
four to six /3 chains (9,700 daltons). To further define the antigenic structure of the toxin, this study was initiated to examine various antisera to cholera toxin and its purified components by the passive hemagglutination (PHA) test, radioimmunoassay (RIA), and permeability factor (PF) neutralization test. These procedures have allowed us to determine the component of cholera toxin that is responsible for eliciting and reacting with neutralizing antibody. MATERIALS AND METHODS Preparation of antigens. Cholera toxin was purified from fermenter cultures of V. cholerae 569B grown in asparagine-glucose medium (10) and characterized as described previously (6, 8). The A and B subunits of cholera toxin were separated by gel filtration on a column of Bio-Gel P-60 acrylamide eluted with 0.2 M sodium formate buffer (pH 3.5) containing 5.2 M guanidine-hydrochloride (9). The A subunit preparation was then treated with 2-mercaptoethanol and alkylated with iodoacetamide prior to chromatog-
VOL. 24, 1979
NEUTRALIZING ANTIBODY TO CHOLERA TOXIN
raphy on the same Bio-Gel P-60 column in the presence of 5.2 M guanidine-hydrochloride to obtain the constituent a and y polypeptide chains. Guanidine was removed from each preparation by dialysis against tris(hydroxymethyl)aminomethane buffer (pH 7.5) (4). Preparation of antisera. Antiserum specific for cholera toxin was obtained by immunization of four rabbits with multiple subcutaneous doses of purified cholera toxin (50 fug) emulsified in Freund complete adjuvant (6). Serum samples obtained over a period of 6 months were pooled and distributed into aliquots prior to storage at -20oC. Three groups of five rabbits each were immunized with A subunit, B subunit, a chain, or y chain emulsified in Freund complete adjuvant (D. E. Markel, K. E. Hejtmancik, J. W. Peterson, F. Martin, and A. Kurosky, submitted for publication). Briefly, rabbits were immunized with an initial dose of 200 .g of the respective antigen preparation, which was administered by multiple-site injection by the intraperitoneal, intramuscular, and subcutaneous routes. Serum samples were taken from the rabbits every 3 weeks and stored at -20'C. A booster dose of 200 jig was administered by multiple-site injections whenever antibody titers began to decrease, as determined by PHA with toxinsensitized erythrocytes (5). A 1-ml sample of each antiA subunit serum was absorbed with purified B subunit by the addition of 50 jig of B subunit contained in 60 p1 of tris(hydroxymethyl)aminomethane buffer. This amount of B subunit was twice the concentration necessary to prevent formation of precipitin lines when the anti-A subunit sera were tested against B subunit using Ouchterlony double-gel diffusion (Markel et al., submitted for publication). Convalescent sera from cholera patients were obtained from the Cholera Research Laboratory in Dacca, Bangladesh. Serological titrations. Identical sets of sera specific for cholera toxin and its components were assayed by each of three basic tests. The PHA test for cholera antitoxin (5) was performed as previously described using chicken erythrocytes sensitized with cholera toxin, A subunit, or B subunit. In each assay system, antibody titers were expressed in units per milliliter based on the provisional standard Swiss Serum and Vaccine Institute serum [EC3-(A-2/67)-B], which has been assigned a value of 4,470 antitoxin units per ml
(3).
The sera were also examined by the RIA method of Hejtmancik et al. employing "nI-labeled cholera toxin, A subunit, or B subunit (6). Antibody titers were expressed in units per milliliter based on a pooled rabbit antiserum to cholera toxin (8/13/73) that had been standardized by the PF neutralization test against the Swiss Serum and Vaccine Institute stan-
dard. Whereas the above assays measured antibody levels specific for toxin, A subunit, or B subunit, respectively, none measured the neutralizing potency of the selected sera. Therefore, the PF neutralization test originally described by Craig (1) was selected to measure the functional significance of antibodies to the separate regions of the cholera toxin molecule. The test, performed as described previously, measured the capacity
775
of each serum to prevent the increase in permeability of the small blood vessels of the skin of adult rabbits induced by cholera toxin (2).
RESULTS Table 1 depicts the general format of analysis used throughout this study. All sera were assayed for toxin-neutralizing antibody by the PF neutralization test, and total antibody to cholera toxin, A subunit, and' B subunit was measured by the PHA test and RIA. Rabbit antiserum to cholera toxin contained both toxin-neutralizing antibodies and antibodies specific for both the A and B subunits of cholera toxin. In contrast, each of the serological assays revealed that normal rabbit and human sera contained no significant levels of antibody to cholera toxin. When successive blood samples of two rabbits immunized with B subunit were examined by the same assays (Table 2), we observed that toxin-neutralizing antibody titers correlated well with PHA and RIA cholera antitoxin titers. The anti-B subunit assays also' revealed titers comparable to the neutralizing and anti-cholera toxin (PHA and RIA) levels, whereas antibody to A subunit was undetectable by RIA'and of low titer by PHA. To determine if anti-A subunit sera contained neutralizing antibody, anti-A subunit sera were tested in the same assays before and after absorption with B subunit (Table 3). Successive serum samples from A subunit-immunized rabbits contained significant levels of toxin-neutralizing antibody, but virtually all of the neutralizing activity was removed from each serum upon absorption of 1-ml samples of the anti-A sera with 50 ,ug of B subunit. On closer inspection it is evident that the anti-A subunit sera contained high levels of anti-B subunit antibody activity that was also removed by absorption with B subunit. After the removal of the anti-B subunit activity, antibody to A subunit remained elevated but toxin-neutralizing activity was lost. The antitoxin titer of antisera to A subunit was virtually eliminated by absorption with B subunit, as determined by the PHA test with toxinsensitized erythrocytes. The levels of antibody to B subunit (PHA and RIA) observed with sera of A and B subunitimmunized rabbits were remarkably similar, as shown in Tables 2 and 3. Despite the similar levels of antibody to B subunit, the neutralizing antibody titers of the two groups of animals were quite different. Table 4 presents a comparison of serum titer ratios of PHA and RIA titers to the B subunit with anti-PF titers of rabbits immunized with the A or B subunit. The PHA anti-B titer/PF titer ratio was at least 10-fold higher in sera of A subunit-immuiized rabbits than in sera
776
PETERSON ET AL.
INFECT. IMMUN.
TABLE 1. Serological analysis ofpooled cholera antitoxin compared to normal rabbit serum Rabbit Serum Specificity
Neutralization Titer
(AU/ml)
Anti-Cholera Toxin Anti-A Subunit Titer Anti-B Subunit Titer Titer (AU/ml)
PF
PHA/RIA
PHA/RIA
PHA/RIA
Pooled Antiserum to Cholera toxin
790
2235/810*
140/810*
4470/810*
Pooled normal serum
