Vol. 14, No. 1
bmCTION AND IMMUNrrY, July 1976, p. 106-113 Copyright © 1976 American Society for Microbiology
Printed in U.S.A.
Immunological and Biochemical Studies of Meningococcal C Polysaccharides Isolated by Diethylaminoethyl Chromatography MICHAEL A. APICELLA
Division ofInfectious Diseases, Department of Medicine, State University of New York at Buffalo, Buffalo, New York 14215 Received for publication 6 January 1976
Meningococcal C polysaccharides isolated from the broth supernatant of C,+ and C,_ strains by a combination of Sepharose 4B and diethylaminoethyl-52 chromatography are antigenically identical to the group C antigen isolated by the Cetavlon procedure. Immunizations result in the induction of precipitating, hemagglutinating, and bactericidal antibodies in rabbits and sheep and hemagglutinating and bactericidal antibodies in gerbils. Selective destruction of C,_ polysaccharides with neuraminidase has demonstrated that these antibodies are directed against the C polysaccharide. Neuraminidase digestions of the C,_ polysaccharides resulted in release of up to 84% of the antigen as N-acetylneuraminic acid. C,+ polysaccharides proved to be more resistant to neuraminidase, requiring greater concentrations of enzyme and longer intervals of exposure for digestion. Chemical analysis of the the C,+ and C,_ polysaccharides indicated that they contain less than 0.5% protein and less than 0.5% nucleic acid.
Vaccination of human subjects with meningococcal capsular C polysaccharide provides protection from disease related to this serogroup in an adult population (11). But the same vaccine used so effectively in adults is a poor immunogen in children under the age of 2 years, as its protective value has not been proven (13, 20). This is important. The majority of serious meningococcal disease in this country occurs in children under the age of 2 years. But vaccinating young children with polysaccharides presents a number of potential problems, including the induction of either low or high dose-immune tolerance. For reasons not clearly understood, the capsular C polysaccharide used as adult vaccine has proven to be immunogenic only in humans (13). This limits any studies of immunogenicity, relative protective value of different vaccine preparations, or dose response to measurements made only
glutinating (HA), and precipitating antibodies. Using this preparation, many questions concerning the vaccination of young children could possibly be answered through animal models, prior to human immunization. MATERIALS AND METHODS
Organisms. Neisseria meningitidis group C strains S-608 and MP-20 were used as a source of capsular C antigens. Strain S-608, obtained from Harry A. Feldman of the State University of New York in Syracuse, produces a C, capsular polysaccharide. Strain MP-20, obtained from our own collection, produces a C,+ capsular antigen. C,+ and C, polysaccharide are two antigenically and biochemically distinct types of group C capsular antigens (2). Both contain the serogroup C determinant. The C,+ polysaccharides contain an additional subgroup determinant and are relatively resistant to digestion by neuraminidase, whereas the Cl polysaccharides lack this determinant but are very susceptible after human vaccination. to digestion by this enzyme. Both strains were preWe previously reported on a method for the served in defibrinated rabbit blood and frozen at - 70 production of capsular C polysaccharide which C and lyophilized in Mueller-Hinton broth in glass resulted in a preparation that is immunogenic ampoules. For antigen production the frozen orgain several animal species (24). In the follow- nisms were defrosted and streaked heavily on trypagar plates containing 5% defibrinated ing report we describe a modification of that tose blood blood and grown overnight at 35 C in a moist procedure which can be adapted to methods of sheep with 5% CO2. large-scale production and which is similar incubator Production of broth cultures and initial concenchemically and identical antigenically to the tration of the polysaccharide. One liter of Muellerpresent adult vaccine. The antigen isolated by Hinton broth in a 2-liter screw-topped flask was this method is immunogenic in several ani- inoculated with a loopful of organisms from an 18-h mal species and induces bactericidal, hemag- plate of freshly reconstituted group C meningococci. 106
VOL. 14, 1976
Twelve flasks were grown overnight in a G-25 shaking incubator (New Brunswick Scientific Co., Inc.) at 35 C at 150 rpm. In turn, the flasks were subcultured to confirm cultural purity and were maintained aseptically at 4 C for 48 h to insure killing of the meningococci. Those bottles, which were culturally and biochemically affirmed to contain meningococci, were pooled. The capsular C polysaccharide was concentrated from the broth supernatant by precipitation with ammonium sulfate (540 g/liter). The precipitate was then collected by centrifugation at 2,500 x g in a continuous-flow centrifuge (Ivan Sorvall, Inc.), and the polysaccharide was resolubilized by dialysis against multiple changes of distilled water. After dialysis, this polysaccharide-rich material was adjusted to pH 11 by the addition of 1.0 N NaOH. (The final concentration was approximately 0.04 N NaOH.) The pH was maintained at 11 for 1 h, retitrated to pH 7 with 0.1 N HCl, and then centrifuged at 20,000 x g to remove insoluble debris prior to molecular sieve chromatography. For comparative analysis, capsular C polysaccharide was isolated by the Cetavlon method of Gotschlich et al. (14) and by the method of Robinson and Apicella (24) using strain MP-20. Chromatographic procedures. Molecular sieve chromatography was performed by an upward flow over a Sepharose 4B (Pharmacia Fine Chemicals, Inc.) column (100 by 2.5 cm) in phosphate-buffered saline. (The void volume [KJ] of the column was 150 ml, and the bed volume [Kt] was 330 ml. Sample size was 10 ml and the elution volume was 12 ml/tube.) Ion exchange chromatography of the group C capsular polysaccharide was performed over a diethylaminoethyl-52 (Whatman Inc.) column (10 by 2 cm) using an ionic-strength gradient elution. The gradient was produced in a Varigrad mixer (Buchler Instruments Div., Nuclear-Chicago Corp.) using 0.02 M K2HPO4-KH2PO4 (pH 6.5) in the first chamber, 0.2 M K2HPO4-KH2PO4 (pH 6.5) in the second, and 2.0 M NaCl in the third. The volume of buffer in each well was 50 ml. Twenty-five milligrams of lyophilized antigen from the Sepharose 4B chromatography was then reconstituted in *10 ml of starting buffer and appled to the column. (Column flow rate was 20 ml/h, and the elution volume was 10 ml/ tube.) The ionic-strength gradient was measured with a Serfass conductivity bridge (Arthur H. Thomas Co.) using a dip conductivity cell with a K value of 1.0. This method of C polysaccharide isolation will be referred to as the DEAE method. Immunological methods. A hemagglutination inhibition (HAI) system specific for the group C polysaccharide was performed as previously described (2). Immunodiffusion in gel was performed using 1% Noble agar in 0.05 M barbital (pH 8.5) (21), whereas counterimmunoelectrophoresis (CEP) was performed using the method of Alter et al. (1). A microtiter bactericidal assay was employed which is a modification of the method of Frasch and Chapman (9) but incorporates the same ratio of reactants as Roberts' bactericidal system (23). (This method used sterile, flat-bottomed microtiter plates [Linbro Scientific] with a well volume of 400 ,lA.) The inoculum size used in the system was approximately 5 x 103 organisms/ml. Antisera were serially diluted in 25
MENINGOCOCCAL C POLYSACCHARIDE
107
,ul of fresh normal rabbit serum, the complement source, which had been diluted(1:2) with saline. The normal rabbit serum was previously absorbed with nontypable meningococci using Roberts' method (23). Hemolytic complement activity after absorption was greater than 100 Mul/ml by standard assay. Meningococci were grown in Mueller-Hinton broth to a concentration of 107 organisms/ml and diluted in saline containing 0.1% bovine serum albumin to a final concentration of 4 x 104 organisms/ml. Twenty-five microliters of organisms was added to each well, followed by 100 ,lM of saline-bovine serum albumin. An identical sequence was prepared and contained diluted antisera in heat-inactivated rabbit serum at 56 C. This served as the control from which the percentage of killing in each antiserum dilution was calculated. Plates were sealed and shaken at 180 rpm for 1 h at 37 C. After incubation the contents of each well were diluted with an equal volume (150 ,ul) of saline-bovine serum albumin and mixed by aspiration with a sterile Pasteur pipette, and a 25-Il aliquot was removed to a predried tryptose blood agar plate. These were incubated overnight at 35 C and subsequently counted with a Bactonic colony counter (New Brunswick Scientific Co., Inc.). Meningococci used in these studies were kept frozen in defibrinated rabbit blood until ready for use and were afterwards transferred no more than three times. Criteria for the presence of bactericidal antibody were established by the method of Reller et al. (22). The tolerance limits (8) for strain survival under control conditions were established and set to include 95% of the control population with 95% confidence. (For killing to be considered significant [i.e., due to the antibody] the percentage of strain survival must be less than the tolerance limits for that particular strain.) For strain S-608, used in these studies, killing by antisera must be greater than 60%. Animal immunizations. C,+ and C, polysaccharides produced by the DEAE method and the Cetavlon procedure were used to immunize male New Zealand rabbits. Three animals were immunized with each DEAE preparation, and two with each Cetavlon antigen. Immunization schedules were identical, with each animal receiving subcutaneously in the posterior cervical region 1 mg of polysaccharide in 1 ml of phosphate-buffered saline emulsified in an equal volume of complete Freund adjuvant. At 3 and 4 weeks after the first injection, the animals were given 10 Mug of antigen intravenously and bled for antibody 1 week after the last injection. Mongolian gerbils 6 to 10 weeks old were immunized with a single intraperitoneal injection of either 0.05, 0.5, or 5.0 Mg of capsular polysaccharide per 100 g of body weight, the antigen being administered in 0.1 ml of phosphate-buffered saline. The gerbils were then bled for antibody 28 days after the initial immunization. Antigens produced by the DEAE method and by the Cetavlon procedure were used as immunogens. Two sheep were immunized on a schedule similar to that described for rabbits, but 10 mg of capsular polysaccharide was given subcutaneously in the posterior cervical region in complete Freund adjuvant
108
INFECT. IMMUN.
APICELLA
and the intravenous dose was 50 zg. Antigens pre- Quantitative analysis of these antigens for O-acetyl pared by the Cetavlon method were not given to groups was performed by the Hestrin method using acetylcholine (Sigma Chemical Co.) as standard in sheep. Enzymatic digestions. Digestion of the C1+ and concentrations from 0.5 to 5 jumol/ml (16). Analysis C, polysaccharides with Clostridium perfringens for ribonucleic acid (RNA) was performed by absorbneuraminidase (Worthington Biochemicals Corp.) ancy at 260 nm (29) using yeast RNA (Worthington was performed as previously described (2). The per- Biochemicals Corp.) as standard. Analysis for decentage ofrelease of free sialic acid was measured by oxyribonucleic acid (DNA) was performed using the diphenylamine assay (7) with Escherichia coli DNA the thiobarbituric acid assay (26). Chemical studies. Analysis of column eluates for (Worthington Biochemicals Corp.) as standard. protein was performed by 280-nm absorption. Samples of purified C polysaccharide were studied for RESULTS protein content by ion exchange chromatography for The broth supernatant of overnight cultures amino acids on a Beckman 120 C amino acid analyzer (25). Prior to analysis, samples were hydro- was the source of the capsular polysaccharide. lyzed in vacuo at 110 C in 6 N HCI for 24 and 48 h. Prior to chromatographic procedures, the anti-
128
1.4 1.2
1.0
GIOP C
HiAl ,*
I*,
0i/
I
160 2 40 160
\-
0.6
11I
32 16 8 240
Elutiom
40'0
320
O.D. 280 rnm
0.8
480
0.4 0.2
560
Volum in ml
FIG. 1. Sepharose 4B elution ofcapsular C polysaccharide from strain S-608. The hatched area represents tubes containing antigen as detected by the group C-specific HAI and the dotted line represents the results of 280-nm absorption. Pooling of the antigen peak was from 156 to 240 ml. The arrow indicates the position of the void volume of the column, as determined by blue dextran 2,000 elution. 5
2
GROP C
.4
HAI
8 .24
/-\~~~~~~0 .1o
jA*~~~~~~~~A
-3
300
400
500
Elution Voluze in ml
FIG. 2. DEAE-52 chromatography of capsular C polysaccharide S-608 after Sepharose 4B chromatography. The hatched area represents tubes containing antigen as detected by group C-specific HAI; the dotted line represents the results of 280-nm absorption; and the solid line represents the conductance values (K) in each tube. Pooling of the antigen peak was from 170 to 240 ml.
VOL. 14, 1976
MENINGOCOCCAL C POLYSACCHARIDE
109
FIG. 3. Counterelectrophoresis of capsular C polysaccharide S-608 before (set A) and after (set B) neuraminidase digestion. Antiserum 19 used in this study was generated by antigen S-608. The first row of set A contains antigen S-608 in serial, twofold dilutions running from left to right. The initial concentration of antigen is 30 pg/ml. The first row of set B is identical to that of row A, except that the antigen has been digested with 0.1 p1 of neuraminidase per mg ofantigen for 18 h. Each well in the second row of sets A and B contains antiserum 19 diluted (1:2) with 0.05 M barbital at a pH of 8.6.
gen was concentrated and partially purified, as outlined in Materials and Methods. Molecular sieve chromatography was performed over Sepharose 4B, resulting in the separation of C polysaccharide from the bulk of broth and organism proteins. The antigen eluted in a broad peak beginning at the void volume of the column (Fig. 1). Pooling of the capsular antigen was performed as indicated, and the sarnple was dialyzed exhaustively against distilled water and lyophilized. Fig. 2 shows the typical elution pattern of a Cl_ polysaccharide after gradient elution from a DEAE-52 column. The polysaccharide elutes at a conductance of 2.2 x 104 to 2.4 x 104 gmho. Analysis of absorbancy at 280 nm revealed the presence of two peaks; the first eluted at a conductance of 104 Mnho and the second at 4.2 x 104 ,umho. Several ionicstength gradients were tested, and the shape of the gradient had little effect on the polysaccharide elution. The gradient shown in Fig. 2 illustrates the best separation of capsular polysaccharide from protein contaminants: approximately 7 mg of capsular antigen was isolated from each liter of processed broth supernatant.
Studies with C,+ polysaccharide from strain MP-20 revealed that it had the same elution characteristics as the C,_ polysaccharides. C polysaccharides isolated by this method were then studied in a series of immunochemical and physiocochemical systems to determine: (i) their immunogenicity in different animal species; (ii) their chemical and immunological characteristics; and (iii) their relationship with C polysaccharides isolated by other methods. Immunogenicity studies were performed in rabbits, sheep, and gerbils. Four of six rabbits immunized with polysaccharide isolated by the DEAE method developed precipitating antibody as measured by immunodiffusion and CEP. All six had HA antibody peak titers ranging from 1:32 to 1:256 when matched against cells coated with homologous antigen. Bactericidal titers were present in all animals and ranged from 1:40 to 1:640. None of the animals immunized with polysaccharide produced by the Cetavlon method had detectable antibody in these same systems. Fig. 3 shows a CEP plate containing C,_ polysaccharide isolated from S-608 reacting
110
INFECT. IMMUN.
APICELLA
TABLE 1. Percentage of strain S-608 killed by antiserum 19 Antibody titer Antiserum 19 absorptionl 1:20 1:40 1:80 1:160 96 100 100 0 0 21 90 100 81
96 Unabsorbed S-608 capsular antigen0 23 8-608 capsular antigen 48 with neuraminidase di-
1:320 1:640 100 18 11
15 5 21
gesteda a Antiserum 19 was absorbed with 500 ,ug of antigen S-608 per ml.
with a homologous antiserum. A single precipitin line can be seen between the upper set of antigen-antibody wells. A prozone characteristic of this test, was noted at higher antigen concentrations. In the lower set of wells the antigen, which is neuraminidase sensitive, had been digested with this enzyme prior to CEP. The precipitin line (present in the upper set of wells) is absent, indicating that the precipitating antibody induced by the antigen preparations is directed against the neuraminidasesensitive C,_ polysaccharide, rather than against a contaminant in the preparation. Table 1 shows the results of bactericidal studies performed with rabbit antiserum induced to capsular Cl polysaccharide from strain S-608. As can be seen, bactericidal antibodies are present in this antiserum and these can be absorbed with capsular C, polysaccharide. Absorption of the antiserum with neuraminidase-digested Cl antigen causes a one-dilution fall in the bactericidal titer, which probably relates to the absorption of some antibody by the small quantities of undigested antigen remaining in the preparation after the enzyme treatment. Table 2 shows the results of immunization without adjuvant in young gerbils immunized with antigens isolated by the DEAE method. Bactericidal antibody can be induced with a single injection of capsular antigen. This response was dose related, with no evidence of antibody production at antigen concentrations below 0.5 ug/100 g of body weight. With 5.0 ,ug/100 g of body weight doses, 17 to 20 animals developed bactericidal antibody and 13 of 20 developed HA antibody titers. Studies of these sera by CEP failed to demonstrate the presence of precipitating antibody. HA, precipitating, and bactericidal antibodies could not be detected in an identical experiment with gerbils immunized with capsular polysaccharides isolated by the Cetavlon method. Two sheep were hyperimmunized with C,+ and C,_ polysaccharides emulsified in Freund adjuvant. Antisera from both animals contained bactericidal, HA, and precipitating antibodies. Fig. 4 is an immunodiffusion plate us-
ing sheep antiserum to C0+ antigen from strain MP-20 and compares capsular C,+ polysaccharide from strain MP-20 as it is isolated by three different methods. Single precipitin lines are present with each preparation; they fuse in compete immunological identity. HAI studies were performed using the C,+ sheep antiserum and ovine erythrocytes coated with C polysaccharide produced by the Cetavlon method. Inhibition of this system was quantitatively identical (< 7 > 3.5 ug/ml) when either Cetavlon-isolated C1+ polysaccharide or C,+ polysaccharide produced by the method described in this report was used. Table 3 shows the results of neuraminidase digestion of a C0_ and C,+ capsular polysaccharide at different concentrations of enzyme. The Cl_ antigen is readily digested by this enzyme at all concentrations, with up to 84% of the dry weight of the antigen released as free N-acetylneuraminic acid in an 18-h exposure to 0.1 unit/ mg of antigen. Enzymatic digestions occur rapidly, as demonstrated by the complete loss of antigenicity in the serogroup C HAI within 15 min after the onset of enzyme treatment. The C0+ antigen was relatively resistant to neuraminidase. Low concentrations of enzyme (0.01 unit/mg of antigen) had little effect on the antigen. At longer intervals of exposure (72 h) with enzyme concentrations of 0.1 unit/mg of antigen, up to 34% of the dry weight of the antigen was released as free N-acetylneuraminic acid. This level of exposure of enzyme to the antigen resulted in complete loss of antigenicity in the serogroup HAI system. Cl and C0+ polysaccharides were analyzed for the presence of O-acetyl groups by Hestrin analysis. Polysaccharide concentrations of 50 mg/ml were studied, but the level of O-acetyl groups was below 0.5 ,mol/50 mg of C, or Cl+ polysaccharide. The protein content of the polysaccharides was determined by ion exchange chromatography for amino acids after hydrolysis. Total protein content of C, and C0+ polysaccharides was less than 0.5% of the dry weight of antigen. An TABLE 2. Immunization ofgerbils with DEAE C1 polysaccharide S-608 Dose
Group
(gg/
100 g of
Results of immunizing animals with antibody Animals (no. developing antibody/ total in group) (no.)
body wt)
HA
Control I II III
0 0.05 0.5 5.0
10 10 10 20
0/10 0/10 2/10 13/20
CEP
Bactericidal
0/10 0/10 0/10 0/10 0/10 2/10 0/20 17/20
VOL. 14, 1976
MENINGOCOCCAL C POLYSACCHARIDE ::?;
zF.|;i
bmCTION AND IMMUNrrY, July 1976, p. 106-113 Copyright © 1976 American Society for Microbiology
Printed in U.S.A.
Immunological and Biochemical Studies of Meningococcal C Polysaccharides Isolated by Diethylaminoethyl Chromatography MICHAEL A. APICELLA
Division ofInfectious Diseases, Department of Medicine, State University of New York at Buffalo, Buffalo, New York 14215 Received for publication 6 January 1976
Meningococcal C polysaccharides isolated from the broth supernatant of C,+ and C,_ strains by a combination of Sepharose 4B and diethylaminoethyl-52 chromatography are antigenically identical to the group C antigen isolated by the Cetavlon procedure. Immunizations result in the induction of precipitating, hemagglutinating, and bactericidal antibodies in rabbits and sheep and hemagglutinating and bactericidal antibodies in gerbils. Selective destruction of C,_ polysaccharides with neuraminidase has demonstrated that these antibodies are directed against the C polysaccharide. Neuraminidase digestions of the C,_ polysaccharides resulted in release of up to 84% of the antigen as N-acetylneuraminic acid. C,+ polysaccharides proved to be more resistant to neuraminidase, requiring greater concentrations of enzyme and longer intervals of exposure for digestion. Chemical analysis of the the C,+ and C,_ polysaccharides indicated that they contain less than 0.5% protein and less than 0.5% nucleic acid.
Vaccination of human subjects with meningococcal capsular C polysaccharide provides protection from disease related to this serogroup in an adult population (11). But the same vaccine used so effectively in adults is a poor immunogen in children under the age of 2 years, as its protective value has not been proven (13, 20). This is important. The majority of serious meningococcal disease in this country occurs in children under the age of 2 years. But vaccinating young children with polysaccharides presents a number of potential problems, including the induction of either low or high dose-immune tolerance. For reasons not clearly understood, the capsular C polysaccharide used as adult vaccine has proven to be immunogenic only in humans (13). This limits any studies of immunogenicity, relative protective value of different vaccine preparations, or dose response to measurements made only
glutinating (HA), and precipitating antibodies. Using this preparation, many questions concerning the vaccination of young children could possibly be answered through animal models, prior to human immunization. MATERIALS AND METHODS
Organisms. Neisseria meningitidis group C strains S-608 and MP-20 were used as a source of capsular C antigens. Strain S-608, obtained from Harry A. Feldman of the State University of New York in Syracuse, produces a C, capsular polysaccharide. Strain MP-20, obtained from our own collection, produces a C,+ capsular antigen. C,+ and C, polysaccharide are two antigenically and biochemically distinct types of group C capsular antigens (2). Both contain the serogroup C determinant. The C,+ polysaccharides contain an additional subgroup determinant and are relatively resistant to digestion by neuraminidase, whereas the Cl polysaccharides lack this determinant but are very susceptible after human vaccination. to digestion by this enzyme. Both strains were preWe previously reported on a method for the served in defibrinated rabbit blood and frozen at - 70 production of capsular C polysaccharide which C and lyophilized in Mueller-Hinton broth in glass resulted in a preparation that is immunogenic ampoules. For antigen production the frozen orgain several animal species (24). In the follow- nisms were defrosted and streaked heavily on trypagar plates containing 5% defibrinated ing report we describe a modification of that tose blood blood and grown overnight at 35 C in a moist procedure which can be adapted to methods of sheep with 5% CO2. large-scale production and which is similar incubator Production of broth cultures and initial concenchemically and identical antigenically to the tration of the polysaccharide. One liter of Muellerpresent adult vaccine. The antigen isolated by Hinton broth in a 2-liter screw-topped flask was this method is immunogenic in several ani- inoculated with a loopful of organisms from an 18-h mal species and induces bactericidal, hemag- plate of freshly reconstituted group C meningococci. 106
VOL. 14, 1976
Twelve flasks were grown overnight in a G-25 shaking incubator (New Brunswick Scientific Co., Inc.) at 35 C at 150 rpm. In turn, the flasks were subcultured to confirm cultural purity and were maintained aseptically at 4 C for 48 h to insure killing of the meningococci. Those bottles, which were culturally and biochemically affirmed to contain meningococci, were pooled. The capsular C polysaccharide was concentrated from the broth supernatant by precipitation with ammonium sulfate (540 g/liter). The precipitate was then collected by centrifugation at 2,500 x g in a continuous-flow centrifuge (Ivan Sorvall, Inc.), and the polysaccharide was resolubilized by dialysis against multiple changes of distilled water. After dialysis, this polysaccharide-rich material was adjusted to pH 11 by the addition of 1.0 N NaOH. (The final concentration was approximately 0.04 N NaOH.) The pH was maintained at 11 for 1 h, retitrated to pH 7 with 0.1 N HCl, and then centrifuged at 20,000 x g to remove insoluble debris prior to molecular sieve chromatography. For comparative analysis, capsular C polysaccharide was isolated by the Cetavlon method of Gotschlich et al. (14) and by the method of Robinson and Apicella (24) using strain MP-20. Chromatographic procedures. Molecular sieve chromatography was performed by an upward flow over a Sepharose 4B (Pharmacia Fine Chemicals, Inc.) column (100 by 2.5 cm) in phosphate-buffered saline. (The void volume [KJ] of the column was 150 ml, and the bed volume [Kt] was 330 ml. Sample size was 10 ml and the elution volume was 12 ml/tube.) Ion exchange chromatography of the group C capsular polysaccharide was performed over a diethylaminoethyl-52 (Whatman Inc.) column (10 by 2 cm) using an ionic-strength gradient elution. The gradient was produced in a Varigrad mixer (Buchler Instruments Div., Nuclear-Chicago Corp.) using 0.02 M K2HPO4-KH2PO4 (pH 6.5) in the first chamber, 0.2 M K2HPO4-KH2PO4 (pH 6.5) in the second, and 2.0 M NaCl in the third. The volume of buffer in each well was 50 ml. Twenty-five milligrams of lyophilized antigen from the Sepharose 4B chromatography was then reconstituted in *10 ml of starting buffer and appled to the column. (Column flow rate was 20 ml/h, and the elution volume was 10 ml/ tube.) The ionic-strength gradient was measured with a Serfass conductivity bridge (Arthur H. Thomas Co.) using a dip conductivity cell with a K value of 1.0. This method of C polysaccharide isolation will be referred to as the DEAE method. Immunological methods. A hemagglutination inhibition (HAI) system specific for the group C polysaccharide was performed as previously described (2). Immunodiffusion in gel was performed using 1% Noble agar in 0.05 M barbital (pH 8.5) (21), whereas counterimmunoelectrophoresis (CEP) was performed using the method of Alter et al. (1). A microtiter bactericidal assay was employed which is a modification of the method of Frasch and Chapman (9) but incorporates the same ratio of reactants as Roberts' bactericidal system (23). (This method used sterile, flat-bottomed microtiter plates [Linbro Scientific] with a well volume of 400 ,lA.) The inoculum size used in the system was approximately 5 x 103 organisms/ml. Antisera were serially diluted in 25
MENINGOCOCCAL C POLYSACCHARIDE
107
,ul of fresh normal rabbit serum, the complement source, which had been diluted(1:2) with saline. The normal rabbit serum was previously absorbed with nontypable meningococci using Roberts' method (23). Hemolytic complement activity after absorption was greater than 100 Mul/ml by standard assay. Meningococci were grown in Mueller-Hinton broth to a concentration of 107 organisms/ml and diluted in saline containing 0.1% bovine serum albumin to a final concentration of 4 x 104 organisms/ml. Twenty-five microliters of organisms was added to each well, followed by 100 ,lM of saline-bovine serum albumin. An identical sequence was prepared and contained diluted antisera in heat-inactivated rabbit serum at 56 C. This served as the control from which the percentage of killing in each antiserum dilution was calculated. Plates were sealed and shaken at 180 rpm for 1 h at 37 C. After incubation the contents of each well were diluted with an equal volume (150 ,ul) of saline-bovine serum albumin and mixed by aspiration with a sterile Pasteur pipette, and a 25-Il aliquot was removed to a predried tryptose blood agar plate. These were incubated overnight at 35 C and subsequently counted with a Bactonic colony counter (New Brunswick Scientific Co., Inc.). Meningococci used in these studies were kept frozen in defibrinated rabbit blood until ready for use and were afterwards transferred no more than three times. Criteria for the presence of bactericidal antibody were established by the method of Reller et al. (22). The tolerance limits (8) for strain survival under control conditions were established and set to include 95% of the control population with 95% confidence. (For killing to be considered significant [i.e., due to the antibody] the percentage of strain survival must be less than the tolerance limits for that particular strain.) For strain S-608, used in these studies, killing by antisera must be greater than 60%. Animal immunizations. C,+ and C, polysaccharides produced by the DEAE method and the Cetavlon procedure were used to immunize male New Zealand rabbits. Three animals were immunized with each DEAE preparation, and two with each Cetavlon antigen. Immunization schedules were identical, with each animal receiving subcutaneously in the posterior cervical region 1 mg of polysaccharide in 1 ml of phosphate-buffered saline emulsified in an equal volume of complete Freund adjuvant. At 3 and 4 weeks after the first injection, the animals were given 10 Mug of antigen intravenously and bled for antibody 1 week after the last injection. Mongolian gerbils 6 to 10 weeks old were immunized with a single intraperitoneal injection of either 0.05, 0.5, or 5.0 Mg of capsular polysaccharide per 100 g of body weight, the antigen being administered in 0.1 ml of phosphate-buffered saline. The gerbils were then bled for antibody 28 days after the initial immunization. Antigens produced by the DEAE method and by the Cetavlon procedure were used as immunogens. Two sheep were immunized on a schedule similar to that described for rabbits, but 10 mg of capsular polysaccharide was given subcutaneously in the posterior cervical region in complete Freund adjuvant
108
INFECT. IMMUN.
APICELLA
and the intravenous dose was 50 zg. Antigens pre- Quantitative analysis of these antigens for O-acetyl pared by the Cetavlon method were not given to groups was performed by the Hestrin method using acetylcholine (Sigma Chemical Co.) as standard in sheep. Enzymatic digestions. Digestion of the C1+ and concentrations from 0.5 to 5 jumol/ml (16). Analysis C, polysaccharides with Clostridium perfringens for ribonucleic acid (RNA) was performed by absorbneuraminidase (Worthington Biochemicals Corp.) ancy at 260 nm (29) using yeast RNA (Worthington was performed as previously described (2). The per- Biochemicals Corp.) as standard. Analysis for decentage ofrelease of free sialic acid was measured by oxyribonucleic acid (DNA) was performed using the diphenylamine assay (7) with Escherichia coli DNA the thiobarbituric acid assay (26). Chemical studies. Analysis of column eluates for (Worthington Biochemicals Corp.) as standard. protein was performed by 280-nm absorption. Samples of purified C polysaccharide were studied for RESULTS protein content by ion exchange chromatography for The broth supernatant of overnight cultures amino acids on a Beckman 120 C amino acid analyzer (25). Prior to analysis, samples were hydro- was the source of the capsular polysaccharide. lyzed in vacuo at 110 C in 6 N HCI for 24 and 48 h. Prior to chromatographic procedures, the anti-
128
1.4 1.2
1.0
GIOP C
HiAl ,*
I*,
0i/
I
160 2 40 160
\-
0.6
11I
32 16 8 240
Elutiom
40'0
320
O.D. 280 rnm
0.8
480
0.4 0.2
560
Volum in ml
FIG. 1. Sepharose 4B elution ofcapsular C polysaccharide from strain S-608. The hatched area represents tubes containing antigen as detected by the group C-specific HAI and the dotted line represents the results of 280-nm absorption. Pooling of the antigen peak was from 156 to 240 ml. The arrow indicates the position of the void volume of the column, as determined by blue dextran 2,000 elution. 5
2
GROP C
.4
HAI
8 .24
/-\~~~~~~0 .1o
jA*~~~~~~~~A
-3
300
400
500
Elution Voluze in ml
FIG. 2. DEAE-52 chromatography of capsular C polysaccharide S-608 after Sepharose 4B chromatography. The hatched area represents tubes containing antigen as detected by group C-specific HAI; the dotted line represents the results of 280-nm absorption; and the solid line represents the conductance values (K) in each tube. Pooling of the antigen peak was from 170 to 240 ml.
VOL. 14, 1976
MENINGOCOCCAL C POLYSACCHARIDE
109
FIG. 3. Counterelectrophoresis of capsular C polysaccharide S-608 before (set A) and after (set B) neuraminidase digestion. Antiserum 19 used in this study was generated by antigen S-608. The first row of set A contains antigen S-608 in serial, twofold dilutions running from left to right. The initial concentration of antigen is 30 pg/ml. The first row of set B is identical to that of row A, except that the antigen has been digested with 0.1 p1 of neuraminidase per mg ofantigen for 18 h. Each well in the second row of sets A and B contains antiserum 19 diluted (1:2) with 0.05 M barbital at a pH of 8.6.
gen was concentrated and partially purified, as outlined in Materials and Methods. Molecular sieve chromatography was performed over Sepharose 4B, resulting in the separation of C polysaccharide from the bulk of broth and organism proteins. The antigen eluted in a broad peak beginning at the void volume of the column (Fig. 1). Pooling of the capsular antigen was performed as indicated, and the sarnple was dialyzed exhaustively against distilled water and lyophilized. Fig. 2 shows the typical elution pattern of a Cl_ polysaccharide after gradient elution from a DEAE-52 column. The polysaccharide elutes at a conductance of 2.2 x 104 to 2.4 x 104 gmho. Analysis of absorbancy at 280 nm revealed the presence of two peaks; the first eluted at a conductance of 104 Mnho and the second at 4.2 x 104 ,umho. Several ionicstength gradients were tested, and the shape of the gradient had little effect on the polysaccharide elution. The gradient shown in Fig. 2 illustrates the best separation of capsular polysaccharide from protein contaminants: approximately 7 mg of capsular antigen was isolated from each liter of processed broth supernatant.
Studies with C,+ polysaccharide from strain MP-20 revealed that it had the same elution characteristics as the C,_ polysaccharides. C polysaccharides isolated by this method were then studied in a series of immunochemical and physiocochemical systems to determine: (i) their immunogenicity in different animal species; (ii) their chemical and immunological characteristics; and (iii) their relationship with C polysaccharides isolated by other methods. Immunogenicity studies were performed in rabbits, sheep, and gerbils. Four of six rabbits immunized with polysaccharide isolated by the DEAE method developed precipitating antibody as measured by immunodiffusion and CEP. All six had HA antibody peak titers ranging from 1:32 to 1:256 when matched against cells coated with homologous antigen. Bactericidal titers were present in all animals and ranged from 1:40 to 1:640. None of the animals immunized with polysaccharide produced by the Cetavlon method had detectable antibody in these same systems. Fig. 3 shows a CEP plate containing C,_ polysaccharide isolated from S-608 reacting
110
INFECT. IMMUN.
APICELLA
TABLE 1. Percentage of strain S-608 killed by antiserum 19 Antibody titer Antiserum 19 absorptionl 1:20 1:40 1:80 1:160 96 100 100 0 0 21 90 100 81
96 Unabsorbed S-608 capsular antigen0 23 8-608 capsular antigen 48 with neuraminidase di-
1:320 1:640 100 18 11
15 5 21
gesteda a Antiserum 19 was absorbed with 500 ,ug of antigen S-608 per ml.
with a homologous antiserum. A single precipitin line can be seen between the upper set of antigen-antibody wells. A prozone characteristic of this test, was noted at higher antigen concentrations. In the lower set of wells the antigen, which is neuraminidase sensitive, had been digested with this enzyme prior to CEP. The precipitin line (present in the upper set of wells) is absent, indicating that the precipitating antibody induced by the antigen preparations is directed against the neuraminidasesensitive C,_ polysaccharide, rather than against a contaminant in the preparation. Table 1 shows the results of bactericidal studies performed with rabbit antiserum induced to capsular Cl polysaccharide from strain S-608. As can be seen, bactericidal antibodies are present in this antiserum and these can be absorbed with capsular C, polysaccharide. Absorption of the antiserum with neuraminidase-digested Cl antigen causes a one-dilution fall in the bactericidal titer, which probably relates to the absorption of some antibody by the small quantities of undigested antigen remaining in the preparation after the enzyme treatment. Table 2 shows the results of immunization without adjuvant in young gerbils immunized with antigens isolated by the DEAE method. Bactericidal antibody can be induced with a single injection of capsular antigen. This response was dose related, with no evidence of antibody production at antigen concentrations below 0.5 ug/100 g of body weight. With 5.0 ,ug/100 g of body weight doses, 17 to 20 animals developed bactericidal antibody and 13 of 20 developed HA antibody titers. Studies of these sera by CEP failed to demonstrate the presence of precipitating antibody. HA, precipitating, and bactericidal antibodies could not be detected in an identical experiment with gerbils immunized with capsular polysaccharides isolated by the Cetavlon method. Two sheep were hyperimmunized with C,+ and C,_ polysaccharides emulsified in Freund adjuvant. Antisera from both animals contained bactericidal, HA, and precipitating antibodies. Fig. 4 is an immunodiffusion plate us-
ing sheep antiserum to C0+ antigen from strain MP-20 and compares capsular C,+ polysaccharide from strain MP-20 as it is isolated by three different methods. Single precipitin lines are present with each preparation; they fuse in compete immunological identity. HAI studies were performed using the C,+ sheep antiserum and ovine erythrocytes coated with C polysaccharide produced by the Cetavlon method. Inhibition of this system was quantitatively identical (< 7 > 3.5 ug/ml) when either Cetavlon-isolated C1+ polysaccharide or C,+ polysaccharide produced by the method described in this report was used. Table 3 shows the results of neuraminidase digestion of a C0_ and C,+ capsular polysaccharide at different concentrations of enzyme. The Cl_ antigen is readily digested by this enzyme at all concentrations, with up to 84% of the dry weight of the antigen released as free N-acetylneuraminic acid in an 18-h exposure to 0.1 unit/ mg of antigen. Enzymatic digestions occur rapidly, as demonstrated by the complete loss of antigenicity in the serogroup C HAI within 15 min after the onset of enzyme treatment. The C0+ antigen was relatively resistant to neuraminidase. Low concentrations of enzyme (0.01 unit/mg of antigen) had little effect on the antigen. At longer intervals of exposure (72 h) with enzyme concentrations of 0.1 unit/mg of antigen, up to 34% of the dry weight of the antigen was released as free N-acetylneuraminic acid. This level of exposure of enzyme to the antigen resulted in complete loss of antigenicity in the serogroup HAI system. Cl and C0+ polysaccharides were analyzed for the presence of O-acetyl groups by Hestrin analysis. Polysaccharide concentrations of 50 mg/ml were studied, but the level of O-acetyl groups was below 0.5 ,mol/50 mg of C, or Cl+ polysaccharide. The protein content of the polysaccharides was determined by ion exchange chromatography for amino acids after hydrolysis. Total protein content of C, and C0+ polysaccharides was less than 0.5% of the dry weight of antigen. An TABLE 2. Immunization ofgerbils with DEAE C1 polysaccharide S-608 Dose
Group
(gg/
100 g of
Results of immunizing animals with antibody Animals (no. developing antibody/ total in group) (no.)
body wt)
HA
Control I II III
0 0.05 0.5 5.0
10 10 10 20
0/10 0/10 2/10 13/20
CEP
Bactericidal
0/10 0/10 0/10 0/10 0/10 2/10 0/20 17/20
VOL. 14, 1976
MENINGOCOCCAL C POLYSACCHARIDE ::?;
zF.|;i
