HYBRIDOMA Volume 11, Number 6, 1992 Mary Ann Liebert, Inc., Publishers
Bactericidal Activity of Two IgG2a Murine Monoclonal Antibodies with Distinct Fine Specificities for Group B Neisseria meningitidis Capsular Polysaccharide CHRISTIAN M. HURPIN,1 EDGARDO D. CAROSELLA,1 and PIERRE-ANDRÉ CAZENAVE2
immunology Research Department, Pasteur Mérieux Serums et Vaccins, 1541 avenue Marcel Mérieux, 69280 Marcy l'Etoile, France 2Immunology Department, Institut Pasteur, 75724 Paris, France ABSTRACT To analyze the fine specificity of the protective IgG response for the caps ule of group ß Neisseria meningitidis (Men B) induced after immunization with live bacteria, two specific lgG2a monoclonal antibodies (mAb) have been generated from hyperimmunized Balb/c and NZB mice (101C11 and 30H12). They specifically recognize in direct and competitive binding assays the capsular polysaccharides of Men B and Escherichia coli k1 on condition that the length of the polysaccharidic chain is sufficient to make a conformational structure (more than 15 monomers of a (2 --> 8) linked N-acetyl neuraminic acid). They do not interact with group A and group C Neisseria meningitidis polysaccharides in ELISA. A chemical derivative of the Men B polysaccharide, the Npropionylated Men B polysaccharide, considered as mimicking a unique bactericidal epitope on the surface of Men B is recognized by 101C11 but not by 30H12. The two mAb have, in vitro, a specific bactericidal activity against live Men B which do not seem serotype specific. Moreover, the killing of Men B mediated by 30H12 can be neutralized by an anti-idiotypic mAb (216F11) generated from A/J mice, immunized with polymerized 30H12. These data show that at least two distinct bactericidal epitopes exist on the surface of the Men B
capsule.
INTRODUCTION
Among invasive gram negative bacteria, group B Neisseria meningitidis (MenB) and Escherichia coli K. (E. coli K1) strains often produce fatal infections. The capsules of these two bacteria, whicn are immunologically and chemically identical (homopolymer of a (2 -> 8 ) linked N-acetylneuraminic acid) (1), represent one of the most important pathogenicity factors (2, 3). Antibodies to the capsular polysaccharide are known to play a major protective role against the extension of the infection (4). In the cases of MenB and E. coli K1, the absence of significative induction of bactericidal antibodies due to the low immunogenicity of the capsule promotes the dissemination of the bacteria and precludes the use of the purified capsular polysaccharide (PS) as a vaccinal
agent.
Several considerations have been suggested to explain this poor immunogenicity : structural similarity to the host tissues (5), sensitivity to neuraminidases (6), structural instability of the PS which adopts by itself a conformational structure different from the one present on the surface of the live bacteria (7, 8). To develop a potent vaccine against group B Neisseria meningitidis two dimensional nuclear -
-
-
677
magnetic resonance studies have recently been performed to characterize the three dimensional structure of the a 2-8 linked sialic acid polymer (9). An other strategy concerns the analysis of the fine specificity of the well identified bactericidal antibody population for group B Neisseria meningitidis in order to select Important determinants on the PS. In our study we were interested in the fine characterization of the specificity of the bactericidal IgG for the Men B capsule, induced after immunization with live bacteria. Although bactericidal IgG antibodies represent a very low part of the total antibody response, it could represent finally the unique long term protective population (10, 11) against live Men B. So we have generated and characterized for the first time two lgG-2a mAb specific for the capsule of MenB and E. coli K1 which have bactericidal activity. The first one comes from the fusion of hyperimmunized Balb/c mice splenocytes with murine myeloma cells whereas the second one is issuing from hyperimmunized NZB mice. These two mAbs recognize two different bactericidal epitopes present on the MenB capsule from their analytical features which are presented here. MATERIALS AND METHODS
Polysaccharides. Group A Neisseria meningitidis (MenA) PS, MenB PS and O-acetylated group C Neisseria meningitidis (MenC) PS were purified by Pasteur-Mérieux Serums & Vaccins, Marcy l'Etoile. Colominic acid (lot 96F0063) and sialic acid were obtained from Sigma Chemical Co, Saint-Louis. N-propionylated form of MenB PS was prepared as previously described (12). The PS purified from E.coli K1 capsules was tested for the absence of O-acetylated group (13). Purified Lipo Oligosaccharides (L.O.S.) from MenB (strain M986) was a kindly gift of Frasch CE. (Center for Biologies Evaluation and Research, US food and Drug Administration, Bethesda, Maryland).
The molecular size of colominic acid, E. coll K1 PS, MenB PS and its N-proplonylated form was estimated by two methods : i) CL-4B sepharose or S300 gel permeation chromatography (14). The molecular size was expressed by the Kd (partition coefficient) and calculated using the formula Kd Ve-Vo =
Vt-Vo
where Vo of the PS.
was
the void volume, Vt the total volume of the column and Ve the elution volume
ii) quantification of sialic acids and reducing sugars Sialic acid content was determined by the resorcinol method of Svennerholm (15). The reducing sugar concentration was measured by a modification of the Park-Johnson ferricyanide submicromethod (16, 17). Number of monomers per molecule was calculated from the ratio: sialic acid concentration (ug/ml) reducing sugar concentration (/ig/ml) Protein and DNA impurities were respectively quantified by the Lowry method (18) and by -
absorbance at 260
nm
and found lower than 1%
(wt/wt).
Bacterial strains. The capsulated strains of Neisseria meningitidis used in this study were M986 (B : 2a ; P1.2), H44/76 (B : 15 ; P1.7.16) and C11. These strains were kindly provided, respectively by Frasch CE. (U.S. Food and Drug Administration, Bethesda, Maryland), Poolman J.T. (RIVM Bilthoven) and Gotschich E.C (Rockefeller University, New York, New
York).
-
Production of anti MenB polysaccharide monoclonal antibodies (mAbi). Six week old NZB mice (C.N.R.S., Orléans, France) or eight week old Balb/c mice (Iffa Credo, L'Arbresle, France) were immunized with live capsulated bacteria (M986 strain of Neisseria meningitidis group B). To have a maximal number of capsulated bacteria, they were subcultured, once, on Muller Hinton gelose supplemented with 2°/oo glucose for 6 h at 37° C in a humid 5% C02 atmosphere. Colonies were resuspended in physiologic saline buffer supplemented with 1°/oo trypton. The suspension was then diluted to have the concentration of bacteria required for the immunization. Balb/c mice were given intraperitoneally two injections of 108 live bacteria in a 4 week interval. Eight weeks after the second immunization they were boosted by intraperitoneal injection of 2.108 bacteria. NZB mice were given intraperitoneally eight weekly injections of live bacteria (three injections of 108 bacteria followed by five injections of 2.108 bacteria). In all cases, the primary injection was given with complete
678
Freund's adjuvant (CFA). Three days after the final immunization, the mice were splenectomized and the spleen cells were fused with X63 Ag 8.653 myeloma cells as previously described (19). The positive hybrids were cloned by limiting dilution. The isotypes of the mAb were tested by Ouchterlony precipitation (20). For the production of ascites fluids, hybridoma cells were Injected intraperitoneally into Pristane-pretreated Balb/cA-Nude mice (Iffa-Credo). The purification of mAb from a concentrated culture supernatant was performed on protein A-sepharose CI-4B chromotography (21). The degree of purity of the antibodies was tested by SDS-PAGE and chromatography on superóse 12HR (Pharmacia) and was found
over
than 95%.
ELISA for the screening of mAbi: Men B ELISA. 10 mg methylated placental albumine (Pasteur-Mérieux Serums & Vaccins) was dissolved in 1 ml purified water. 200 /¿I of this solution was added drop by drop to 4 ml MenB PS solution (0,5 mg/ml purified water). After a 20 min stirring period, the mixture was diluted 20 times in PBS (coating solution). Microwell plates (Dynateck) were sensitized with 100 /¿I of the coating solution for 3 h at 37° under rotative stirring followed by 3 washings with PBS/0,3% BRIJ 35 (Sigma) (washing buffer). After blocking the unspecific sites with PBS/10% BSA (Sigma) for 2 h (37°, rotative stirring) 100 ¡A of hybridoma culture supernatants or diluted immune murine sera (in PBS/0,3% BRIJ 35/1% BSA) were added to microwells and incubated for 1 h (37°, rotative stirring). After 3 with washings, 100 iA of an optimal concentration of antimurine IgG,A,M serum labelled alcaline phosphatase (Zymed) were distributed in the different microwells. After 2 h of incubation (37°, rotative stirring) and washings, each well received 100 ¡A of 1 mg/ml Paranitrophenyl phosphate in 0,1 M diethanolamine HCI buffer (pH 9,8) containing 3 10"4 M MgCl2. The reaction was stopped with 10 /J of NaOH 5N and the light absorbance at 405 nm was measured using a molecular device ELISA reader. =
-
ELISA for characterization of mAbi. *Direct binding assays on microwell plates coated with different antigenic solutions. ELISA procedures were performed like MenB ELISA but the preparations of the coating solutions were different. The MenA PS and MenC PS coating solutions were prepared as the protocol described for the MenB PS coating solution except that they were lastly diluted 100 times in PBS. To sensitize microwell plates with N-propionylated MenB PS, the coating solution was obtained as follows: to 1 ml N-propionylated MenB PS solution (250 ¿ig/ml) was added drop by drop 50 pi methylated placental albumine (2 mg/ml). After a 20 min stirring period, the coating solution was diluted 10 times in 10"2 M carbonate buffer pH 9,6 before use. The coating solution of MenB L.O.S. was prepared as previously described (22). 10 mM MgCI2 were added to all buffers and washing solutions used for the ELISA (22). Competitive binding assays on microwell plates coated with MenB PS. Increasing concentrations of competitors (sialic acid, colominic acid, E. coli K1 PS, MenC PS, MenB PS, N-propionylated MenB PS, L.O.S. from MenB) in PBS/0,3% BRIJ 35/1% BSA (110 u\) were added to 110 ¡A of an optimal dilution of the mAb tested either in purified or ascites fluid form (the highest dilution of the antibody preparation that still shows the maximum optical density value in MenB ELISA was determined as optimal in competitive binding assays). After 1 h incubation at 37°, 100 ^l of each sample was added to microwell plates coated with MenB PS. The assay was then performed like the MenB ELISA's one. =
Production and characterization of anti-idiotypic monoclonal antibody (mAb2) to mAbi. Polymerized mAbi (30H12) was used as immunogen to generate mAb2 : 2 ml of purified 30H12 (2 mg/ml in PBS) was dialyzed overnight in 0,1 M potassium phosphate buffer (pH 6,8), polymerized with 30 ^l 1% glutaraldehyde (Sigma) for 1 h (20°C, rotative stirring) and next dialyzed overnight in PBS.8-10 week old A/J mice (Institut Pasteur, Paris, France) were used to obtain mAb2. They were given intraperitoneally four injections with a 3 =
week period between each injection. The primary injection was given with CFA, the others with incomplete Freund's adjuvant (IFA). Three days after the final immunization, the spleen cells were fused with X63 Ag 8.653 myeloma cells (19). Positive hybrids were screened by ELISA as follows: Microwell plates were sensitized with 100 ¿J/well of 10 ^g/ml purified 30H12 in PBS (pH 7,4) overnight at + 4°C After 3 washings in PBS/0,5% Tween20 and blocking of the unspecific sites with PBS/10% BSA, 100 ¡A of hybridoma culture supernatants were added to the antibody-coated wells and incubated for 1 h at 37°C. After 3 washings, 100 >A of an =
-
679
a mixture of antimurine IgGi, lgG2b, lgG3, IgM sera labelled with phosphatase (Caltag) were distributed in the microwells. The revelation of the assay was performed as described above. Positive culture supernatants were further tested for their ability to inhibit the binding of 3OH12 to the MenB PS coated microwells. -110 /il of culture supernatants were preincubated 1 h at 37° with 110 iA of an optimal dilution of the purified 30H12 (see above for its determination). The samples were further tested by following the procedure of MenB ELISA Hybrids which were positive in the two tests, were cloned and the concentrated culture supernatant purified by anion exchange chromatography on Q Fast Flow sepharose (Pharmacia) with a gradient of NaCI (23). The degree of purity of the mAb2 was checked and found over than 95%. Bactericidal assays. The colonies of different strains of Neisseria meningitidis (M986, H 44/76, C11) after overnight growth on Muller Hinton gelose supplemented with 2°/oo glucose, were resuspended in Muller Hinton broth supplemented with 2°/oo glucose and incubated for 3 h at 37°C under constant shaking. This period corresponds to the logarithmic phase of bacterial growth. For the assays, all the dilutions were performed in Dulbecco buffer. To 200 /J of increasing dilutions of purified mAbi set in macrowells (LINBRO) were successively added 100 /J of an optimal dilution of New born rabbit complement and immediately after 100 ¡A of 0,7.103 bacteria/ml in logarithmic phase of growth. After a 30 min incubation period at 37°C under constant rotative shaking, 1 ml of melted agarose diluted at 15°/oo in Muller Hinton broth (w/v) and supplemented with 1% decomplemented foal serum was distributed in each well. After solidification, the plates were incubated at 37°C overnight under 5% C02 and the colonies were counted the next day. The bactericidal titer was expressed as the inverse of the highest dilution of the purified mAb tested in duplicate, giving more than 50% of killing of capsulated bacteria as compared to the complement control. Positive control (polyclonal sera of hyperimmunized rabbits) and negative control (non related purified lgG2a mAb) have been introduced in each test. In competitive bactericidal assays, 100 ¡A of an optimal dilution of purified mAbi was preincubated 6h at + 4°C with 100 ¡A of serial dilutions of purified mAb2 ¡n macrowells. The assay was then performed as described above.
optimal
concentration of
alcaline
.
RESULTS Generation of lgG2a mAb anti MenB PS (mAbi) in Balb/c and NZB mice. In a preliminary study we analyzed by MenB ELISA the immunological response to the MenB PS obtained after immunization of NZB and Balb/c mice with 10° or 107 live bacteria. The titers of specific IgG and IgM were respectively 7 and 8 times higher in sera of NZB mice immunized with 108 bacteria (data not shown). On the other hand no increasing level of specific IgG or IgM was observed in the sera of Balb/c mice when they were immunized with 108 bacteria and the titer of specific IgG remained very low (data not shown). Since the immunological response better in NZB mice immunized with 108 bacteria we used this dose to generate mAbs in NZB mice. The mAbs screened by MenB ELISA were almost exclusively of IgM isotype (table 1). However we have obtained 2 clones secreting lgG2a mAb ; the first one (30H12) derivated from the immunized NZB mice splenocytes and the second one (IOIC11) derivated from immmunized Balb/c mice splenocytes (table 1).
was
Balb/c and
TABLE 1
Hybrids From Mice Immunized With Live Group B Neisseria Meningitidis (M986) Origine of splenocytes
Number of hybrids Number of positive Number of positive tested in MenB hybrids * hybrids secreting _ELISA_IgG mAb 1 597 16 1 NZB mice 2 2 0 611 *
Fusion number
Balb/c mice_3_379_48_1_ Hybrids were considered as positive when the optical density of the supernatants tested in MenB ELISA were 3 times higher than the background (optical density (O.D.) > 0,3). 680
The two
weight.
lgG2a
mAbi interact with
a
2-8 Linked sialic acid
polymers
of
high molecular
To determine the type of interaction between the mAbi and the polysialic acids, different polymers of a2-8 linked N-acetylneuraminic acid have been tested in a competitive binding assay. The molecular size of the 3 polymers tested have been determined by two methods (see materials and methods). Colominic acid is only 15 monomer units long while E. coli K1 PS and Men B PS are respectively 269 and 217 monomer units long. As shown in fig. 1 the results obtained for the 2 mAbi are comparable: MenB PS and E. coli K1 PS interacts with the two mAbi in a similar manner.
-t25
50
100
200
250
500 1000
25
Competitor(ug/ml)
co
50
100
200
250
mpeli tor (ug/ml)
FIGURE 1. Binding Inhibition of 30H12 or 101C11 to MenB PS by Different «2-8 Linked Sialic Acid Polymers. Increasing Concentrations of either MenB PS ( - - ), E. Coli -o- ) were K1 PS ( ), Colominic Acid (-a- ) or Sialic Acid ( Tested as Competitive Inhibitors, the Percentages of Inhibition were Calculated Using the Formula: % Inhibition: O.Dx -O.Dbg Ux100 I O.Dmax-O.Dbg) in which O.Dx was the Average O.D of the Experimental well with Inhibitor, O.Dbg was the Average Background O.D and O.Dmax was the Average Maximal Binding without Inhibitor.
pi -[
As few as 10 /¿g/ml ¡s enough to inhibit the binding (more than 50%) of the antibody to the MenB PS coated solid phase. On the other hand more than 500 /¿g/ml of low molecular weight colominic acid is required to inhibit at the same level the binding of the two mAbi to the same specific solid phase. Sialic acid does not interact with mAbi at any concentration
(fig. 1).
The mAbi do not interact with the MenA and MenC PS. The specificity of the mAbi after purification on protein A sepharose chromatography has been tested by direct and competitive binding assays. No significant cross reactions between mAbi and the MenA and MenC PS has been observed (fig. 2, 3). Likewise, LO.S. from MenB do not interact with these antibodies (fig 2,3).
N-propionvlated MenB PS interferes in the binding of 101 Cn to the MenB PS but does not interact with 30H12. N-propionylated MenB PS is a chemical structure considered as mimicking a unique bactericidal epitope on the surface of MenB (24, 25). Only antibodies which recognize this epitope exhibit a significant bactericidal activity against group B Neisseria meningitidis (24). So, it is of a particular interest in testing the interaction between the two mAbi and this molecule. In a direct binding assay, IOIC11 interacts weakly with the N-propionylated MenB PS bound to the solid phase (fig.2). In a competitive binding assay which is a more sensitive test, the level of inhibition of the 101C11 binding to the MenB PS reaches 43% with 100 ug/m\ N-propionylated MenB PS. On the other hand 500 ug/ml of this product (the highest concentration tested) is unable to prevent significantly the binding of 30H12 to Men B PS (the inhibition percentage is lower than 10%) (fig. 3). To quantify more precisely the degree of cross reactivity of the two mAbs towards Men B PS and N-proprionylated Men B PS, 50% inhibition concentration values (IC50) of each compound have been calculated from the different competitive inhibition curves. In the case 681
5
11
22
45
90
30H12(ug/ml)
FIGURE 2.
180
11
360
22
4S
90
1 01 C11
18s
37°
740
(ug/ml)
Of 30H12 or 101C11 to Different Antigens. Increasing Concentrations 101C11 were Tested by ELISA on Microwell Plates Sensitized with Different Antigenic Solutions: MenB PS ( -d- ), MenA PS (-a- ), MenC PS (-o- ), MenB LO.S. ( - - ) or N-Proprionylated MenB PS (-•-). To Validate Coating, Positive Controls such as Specific Polyclonal Sera were Included in each Test.
Binding
of either 30H12
or
Competltor(ug/ml)
competitor! ug/ml)
FIGURE 3. Binding Inhibition of 30H12 or 101C11 to MenB PS by Increasing Concentrations of Either MenB PS (-O- ), MenC PS (-D- ), MenB L.O.S. ( - - ) or NPropionylated MenB PS (-•- ). The Percentages of Inhibition were Calculated Using the Same Formula as in Figue 1. The Results are Expressed as Mean + S.D (n=3). of the 101C11 mAb, IC50 of Men B PS and N-proprionylated Men B PS are respectively 31 and 140 /¿g/ml and the degree of the cross reactivity of this mAb obtained from the ratio: IC50of/WenBPS_ x 100 is 22%. IC50 of N-proprionylated Men B PS In the case of the 30H12 mAb, IC50 of Men B PS and N-proprionylated Men B PS are respectively 23 Mg/ml and much more than 500 jug/ml and the degree of cross reactivity is less than 5%. Specific bactericidal activity (in vitro) oí the mAbi 3OH12 and 101C11. The mAb have been tested against two strains of Neisseria meningitidis group B (B : 15 ; P1.16 and B : 2a ; P1.2) and one strain of Neisseria meningitidis group C (C11). The specific bactericidal activity of the two mAbi is clearly demonstrated from the results shown in table 2. To block specifically the bactericidal activity of the mAbi we produced and characterized an IgGi anti idiotypic monoclonal antibody (mAb2), 216F11, against the mAbi 30Hi2. Ion exchange chromatography-purified 216F11 binds to 30H12 when tested in a direct binding
682
TABLE 2
Bactericidal
Activity Of The MAb 30H12 And 101C11 Against Group B And Group C
_Neisseria Meningitidis
Identity of the strain tested C11 (B:2a ; P1.2) H44/76 (B:15 ; P1.7, 16) < 256 ( 25 ug/ml) 512(12,5/tg/ml) 4(> 160
Bactericidal Activity of Two IgG2a Murine Monoclonal Antibodies with Distinct Fine Specificities for Group B Neisseria meningitidis Capsular Polysaccharide CHRISTIAN M. HURPIN,1 EDGARDO D. CAROSELLA,1 and PIERRE-ANDRÉ CAZENAVE2
immunology Research Department, Pasteur Mérieux Serums et Vaccins, 1541 avenue Marcel Mérieux, 69280 Marcy l'Etoile, France 2Immunology Department, Institut Pasteur, 75724 Paris, France ABSTRACT To analyze the fine specificity of the protective IgG response for the caps ule of group ß Neisseria meningitidis (Men B) induced after immunization with live bacteria, two specific lgG2a monoclonal antibodies (mAb) have been generated from hyperimmunized Balb/c and NZB mice (101C11 and 30H12). They specifically recognize in direct and competitive binding assays the capsular polysaccharides of Men B and Escherichia coli k1 on condition that the length of the polysaccharidic chain is sufficient to make a conformational structure (more than 15 monomers of a (2 --> 8) linked N-acetyl neuraminic acid). They do not interact with group A and group C Neisseria meningitidis polysaccharides in ELISA. A chemical derivative of the Men B polysaccharide, the Npropionylated Men B polysaccharide, considered as mimicking a unique bactericidal epitope on the surface of Men B is recognized by 101C11 but not by 30H12. The two mAb have, in vitro, a specific bactericidal activity against live Men B which do not seem serotype specific. Moreover, the killing of Men B mediated by 30H12 can be neutralized by an anti-idiotypic mAb (216F11) generated from A/J mice, immunized with polymerized 30H12. These data show that at least two distinct bactericidal epitopes exist on the surface of the Men B
capsule.
INTRODUCTION
Among invasive gram negative bacteria, group B Neisseria meningitidis (MenB) and Escherichia coli K. (E. coli K1) strains often produce fatal infections. The capsules of these two bacteria, whicn are immunologically and chemically identical (homopolymer of a (2 -> 8 ) linked N-acetylneuraminic acid) (1), represent one of the most important pathogenicity factors (2, 3). Antibodies to the capsular polysaccharide are known to play a major protective role against the extension of the infection (4). In the cases of MenB and E. coli K1, the absence of significative induction of bactericidal antibodies due to the low immunogenicity of the capsule promotes the dissemination of the bacteria and precludes the use of the purified capsular polysaccharide (PS) as a vaccinal
agent.
Several considerations have been suggested to explain this poor immunogenicity : structural similarity to the host tissues (5), sensitivity to neuraminidases (6), structural instability of the PS which adopts by itself a conformational structure different from the one present on the surface of the live bacteria (7, 8). To develop a potent vaccine against group B Neisseria meningitidis two dimensional nuclear -
-
-
677
magnetic resonance studies have recently been performed to characterize the three dimensional structure of the a 2-8 linked sialic acid polymer (9). An other strategy concerns the analysis of the fine specificity of the well identified bactericidal antibody population for group B Neisseria meningitidis in order to select Important determinants on the PS. In our study we were interested in the fine characterization of the specificity of the bactericidal IgG for the Men B capsule, induced after immunization with live bacteria. Although bactericidal IgG antibodies represent a very low part of the total antibody response, it could represent finally the unique long term protective population (10, 11) against live Men B. So we have generated and characterized for the first time two lgG-2a mAb specific for the capsule of MenB and E. coli K1 which have bactericidal activity. The first one comes from the fusion of hyperimmunized Balb/c mice splenocytes with murine myeloma cells whereas the second one is issuing from hyperimmunized NZB mice. These two mAbs recognize two different bactericidal epitopes present on the MenB capsule from their analytical features which are presented here. MATERIALS AND METHODS
Polysaccharides. Group A Neisseria meningitidis (MenA) PS, MenB PS and O-acetylated group C Neisseria meningitidis (MenC) PS were purified by Pasteur-Mérieux Serums & Vaccins, Marcy l'Etoile. Colominic acid (lot 96F0063) and sialic acid were obtained from Sigma Chemical Co, Saint-Louis. N-propionylated form of MenB PS was prepared as previously described (12). The PS purified from E.coli K1 capsules was tested for the absence of O-acetylated group (13). Purified Lipo Oligosaccharides (L.O.S.) from MenB (strain M986) was a kindly gift of Frasch CE. (Center for Biologies Evaluation and Research, US food and Drug Administration, Bethesda, Maryland).
The molecular size of colominic acid, E. coll K1 PS, MenB PS and its N-proplonylated form was estimated by two methods : i) CL-4B sepharose or S300 gel permeation chromatography (14). The molecular size was expressed by the Kd (partition coefficient) and calculated using the formula Kd Ve-Vo =
Vt-Vo
where Vo of the PS.
was
the void volume, Vt the total volume of the column and Ve the elution volume
ii) quantification of sialic acids and reducing sugars Sialic acid content was determined by the resorcinol method of Svennerholm (15). The reducing sugar concentration was measured by a modification of the Park-Johnson ferricyanide submicromethod (16, 17). Number of monomers per molecule was calculated from the ratio: sialic acid concentration (ug/ml) reducing sugar concentration (/ig/ml) Protein and DNA impurities were respectively quantified by the Lowry method (18) and by -
absorbance at 260
nm
and found lower than 1%
(wt/wt).
Bacterial strains. The capsulated strains of Neisseria meningitidis used in this study were M986 (B : 2a ; P1.2), H44/76 (B : 15 ; P1.7.16) and C11. These strains were kindly provided, respectively by Frasch CE. (U.S. Food and Drug Administration, Bethesda, Maryland), Poolman J.T. (RIVM Bilthoven) and Gotschich E.C (Rockefeller University, New York, New
York).
-
Production of anti MenB polysaccharide monoclonal antibodies (mAbi). Six week old NZB mice (C.N.R.S., Orléans, France) or eight week old Balb/c mice (Iffa Credo, L'Arbresle, France) were immunized with live capsulated bacteria (M986 strain of Neisseria meningitidis group B). To have a maximal number of capsulated bacteria, they were subcultured, once, on Muller Hinton gelose supplemented with 2°/oo glucose for 6 h at 37° C in a humid 5% C02 atmosphere. Colonies were resuspended in physiologic saline buffer supplemented with 1°/oo trypton. The suspension was then diluted to have the concentration of bacteria required for the immunization. Balb/c mice were given intraperitoneally two injections of 108 live bacteria in a 4 week interval. Eight weeks after the second immunization they were boosted by intraperitoneal injection of 2.108 bacteria. NZB mice were given intraperitoneally eight weekly injections of live bacteria (three injections of 108 bacteria followed by five injections of 2.108 bacteria). In all cases, the primary injection was given with complete
678
Freund's adjuvant (CFA). Three days after the final immunization, the mice were splenectomized and the spleen cells were fused with X63 Ag 8.653 myeloma cells as previously described (19). The positive hybrids were cloned by limiting dilution. The isotypes of the mAb were tested by Ouchterlony precipitation (20). For the production of ascites fluids, hybridoma cells were Injected intraperitoneally into Pristane-pretreated Balb/cA-Nude mice (Iffa-Credo). The purification of mAb from a concentrated culture supernatant was performed on protein A-sepharose CI-4B chromotography (21). The degree of purity of the antibodies was tested by SDS-PAGE and chromatography on superóse 12HR (Pharmacia) and was found
over
than 95%.
ELISA for the screening of mAbi: Men B ELISA. 10 mg methylated placental albumine (Pasteur-Mérieux Serums & Vaccins) was dissolved in 1 ml purified water. 200 /¿I of this solution was added drop by drop to 4 ml MenB PS solution (0,5 mg/ml purified water). After a 20 min stirring period, the mixture was diluted 20 times in PBS (coating solution). Microwell plates (Dynateck) were sensitized with 100 /¿I of the coating solution for 3 h at 37° under rotative stirring followed by 3 washings with PBS/0,3% BRIJ 35 (Sigma) (washing buffer). After blocking the unspecific sites with PBS/10% BSA (Sigma) for 2 h (37°, rotative stirring) 100 ¡A of hybridoma culture supernatants or diluted immune murine sera (in PBS/0,3% BRIJ 35/1% BSA) were added to microwells and incubated for 1 h (37°, rotative stirring). After 3 with washings, 100 iA of an optimal concentration of antimurine IgG,A,M serum labelled alcaline phosphatase (Zymed) were distributed in the different microwells. After 2 h of incubation (37°, rotative stirring) and washings, each well received 100 ¡A of 1 mg/ml Paranitrophenyl phosphate in 0,1 M diethanolamine HCI buffer (pH 9,8) containing 3 10"4 M MgCl2. The reaction was stopped with 10 /J of NaOH 5N and the light absorbance at 405 nm was measured using a molecular device ELISA reader. =
-
ELISA for characterization of mAbi. *Direct binding assays on microwell plates coated with different antigenic solutions. ELISA procedures were performed like MenB ELISA but the preparations of the coating solutions were different. The MenA PS and MenC PS coating solutions were prepared as the protocol described for the MenB PS coating solution except that they were lastly diluted 100 times in PBS. To sensitize microwell plates with N-propionylated MenB PS, the coating solution was obtained as follows: to 1 ml N-propionylated MenB PS solution (250 ¿ig/ml) was added drop by drop 50 pi methylated placental albumine (2 mg/ml). After a 20 min stirring period, the coating solution was diluted 10 times in 10"2 M carbonate buffer pH 9,6 before use. The coating solution of MenB L.O.S. was prepared as previously described (22). 10 mM MgCI2 were added to all buffers and washing solutions used for the ELISA (22). Competitive binding assays on microwell plates coated with MenB PS. Increasing concentrations of competitors (sialic acid, colominic acid, E. coli K1 PS, MenC PS, MenB PS, N-propionylated MenB PS, L.O.S. from MenB) in PBS/0,3% BRIJ 35/1% BSA (110 u\) were added to 110 ¡A of an optimal dilution of the mAb tested either in purified or ascites fluid form (the highest dilution of the antibody preparation that still shows the maximum optical density value in MenB ELISA was determined as optimal in competitive binding assays). After 1 h incubation at 37°, 100 ^l of each sample was added to microwell plates coated with MenB PS. The assay was then performed like the MenB ELISA's one. =
Production and characterization of anti-idiotypic monoclonal antibody (mAb2) to mAbi. Polymerized mAbi (30H12) was used as immunogen to generate mAb2 : 2 ml of purified 30H12 (2 mg/ml in PBS) was dialyzed overnight in 0,1 M potassium phosphate buffer (pH 6,8), polymerized with 30 ^l 1% glutaraldehyde (Sigma) for 1 h (20°C, rotative stirring) and next dialyzed overnight in PBS.8-10 week old A/J mice (Institut Pasteur, Paris, France) were used to obtain mAb2. They were given intraperitoneally four injections with a 3 =
week period between each injection. The primary injection was given with CFA, the others with incomplete Freund's adjuvant (IFA). Three days after the final immunization, the spleen cells were fused with X63 Ag 8.653 myeloma cells (19). Positive hybrids were screened by ELISA as follows: Microwell plates were sensitized with 100 ¿J/well of 10 ^g/ml purified 30H12 in PBS (pH 7,4) overnight at + 4°C After 3 washings in PBS/0,5% Tween20 and blocking of the unspecific sites with PBS/10% BSA, 100 ¡A of hybridoma culture supernatants were added to the antibody-coated wells and incubated for 1 h at 37°C. After 3 washings, 100 >A of an =
-
679
a mixture of antimurine IgGi, lgG2b, lgG3, IgM sera labelled with phosphatase (Caltag) were distributed in the microwells. The revelation of the assay was performed as described above. Positive culture supernatants were further tested for their ability to inhibit the binding of 3OH12 to the MenB PS coated microwells. -110 /il of culture supernatants were preincubated 1 h at 37° with 110 iA of an optimal dilution of the purified 30H12 (see above for its determination). The samples were further tested by following the procedure of MenB ELISA Hybrids which were positive in the two tests, were cloned and the concentrated culture supernatant purified by anion exchange chromatography on Q Fast Flow sepharose (Pharmacia) with a gradient of NaCI (23). The degree of purity of the mAb2 was checked and found over than 95%. Bactericidal assays. The colonies of different strains of Neisseria meningitidis (M986, H 44/76, C11) after overnight growth on Muller Hinton gelose supplemented with 2°/oo glucose, were resuspended in Muller Hinton broth supplemented with 2°/oo glucose and incubated for 3 h at 37°C under constant shaking. This period corresponds to the logarithmic phase of bacterial growth. For the assays, all the dilutions were performed in Dulbecco buffer. To 200 /J of increasing dilutions of purified mAbi set in macrowells (LINBRO) were successively added 100 /J of an optimal dilution of New born rabbit complement and immediately after 100 ¡A of 0,7.103 bacteria/ml in logarithmic phase of growth. After a 30 min incubation period at 37°C under constant rotative shaking, 1 ml of melted agarose diluted at 15°/oo in Muller Hinton broth (w/v) and supplemented with 1% decomplemented foal serum was distributed in each well. After solidification, the plates were incubated at 37°C overnight under 5% C02 and the colonies were counted the next day. The bactericidal titer was expressed as the inverse of the highest dilution of the purified mAb tested in duplicate, giving more than 50% of killing of capsulated bacteria as compared to the complement control. Positive control (polyclonal sera of hyperimmunized rabbits) and negative control (non related purified lgG2a mAb) have been introduced in each test. In competitive bactericidal assays, 100 ¡A of an optimal dilution of purified mAbi was preincubated 6h at + 4°C with 100 ¡A of serial dilutions of purified mAb2 ¡n macrowells. The assay was then performed as described above.
optimal
concentration of
alcaline
.
RESULTS Generation of lgG2a mAb anti MenB PS (mAbi) in Balb/c and NZB mice. In a preliminary study we analyzed by MenB ELISA the immunological response to the MenB PS obtained after immunization of NZB and Balb/c mice with 10° or 107 live bacteria. The titers of specific IgG and IgM were respectively 7 and 8 times higher in sera of NZB mice immunized with 108 bacteria (data not shown). On the other hand no increasing level of specific IgG or IgM was observed in the sera of Balb/c mice when they were immunized with 108 bacteria and the titer of specific IgG remained very low (data not shown). Since the immunological response better in NZB mice immunized with 108 bacteria we used this dose to generate mAbs in NZB mice. The mAbs screened by MenB ELISA were almost exclusively of IgM isotype (table 1). However we have obtained 2 clones secreting lgG2a mAb ; the first one (30H12) derivated from the immunized NZB mice splenocytes and the second one (IOIC11) derivated from immmunized Balb/c mice splenocytes (table 1).
was
Balb/c and
TABLE 1
Hybrids From Mice Immunized With Live Group B Neisseria Meningitidis (M986) Origine of splenocytes
Number of hybrids Number of positive Number of positive tested in MenB hybrids * hybrids secreting _ELISA_IgG mAb 1 597 16 1 NZB mice 2 2 0 611 *
Fusion number
Balb/c mice_3_379_48_1_ Hybrids were considered as positive when the optical density of the supernatants tested in MenB ELISA were 3 times higher than the background (optical density (O.D.) > 0,3). 680
The two
weight.
lgG2a
mAbi interact with
a
2-8 Linked sialic acid
polymers
of
high molecular
To determine the type of interaction between the mAbi and the polysialic acids, different polymers of a2-8 linked N-acetylneuraminic acid have been tested in a competitive binding assay. The molecular size of the 3 polymers tested have been determined by two methods (see materials and methods). Colominic acid is only 15 monomer units long while E. coli K1 PS and Men B PS are respectively 269 and 217 monomer units long. As shown in fig. 1 the results obtained for the 2 mAbi are comparable: MenB PS and E. coli K1 PS interacts with the two mAbi in a similar manner.
-t25
50
100
200
250
500 1000
25
Competitor(ug/ml)
co
50
100
200
250
mpeli tor (ug/ml)
FIGURE 1. Binding Inhibition of 30H12 or 101C11 to MenB PS by Different «2-8 Linked Sialic Acid Polymers. Increasing Concentrations of either MenB PS ( - - ), E. Coli -o- ) were K1 PS ( ), Colominic Acid (-a- ) or Sialic Acid ( Tested as Competitive Inhibitors, the Percentages of Inhibition were Calculated Using the Formula: % Inhibition: O.Dx -O.Dbg Ux100 I O.Dmax-O.Dbg) in which O.Dx was the Average O.D of the Experimental well with Inhibitor, O.Dbg was the Average Background O.D and O.Dmax was the Average Maximal Binding without Inhibitor.
pi -[
As few as 10 /¿g/ml ¡s enough to inhibit the binding (more than 50%) of the antibody to the MenB PS coated solid phase. On the other hand more than 500 /¿g/ml of low molecular weight colominic acid is required to inhibit at the same level the binding of the two mAbi to the same specific solid phase. Sialic acid does not interact with mAbi at any concentration
(fig. 1).
The mAbi do not interact with the MenA and MenC PS. The specificity of the mAbi after purification on protein A sepharose chromatography has been tested by direct and competitive binding assays. No significant cross reactions between mAbi and the MenA and MenC PS has been observed (fig. 2, 3). Likewise, LO.S. from MenB do not interact with these antibodies (fig 2,3).
N-propionvlated MenB PS interferes in the binding of 101 Cn to the MenB PS but does not interact with 30H12. N-propionylated MenB PS is a chemical structure considered as mimicking a unique bactericidal epitope on the surface of MenB (24, 25). Only antibodies which recognize this epitope exhibit a significant bactericidal activity against group B Neisseria meningitidis (24). So, it is of a particular interest in testing the interaction between the two mAbi and this molecule. In a direct binding assay, IOIC11 interacts weakly with the N-propionylated MenB PS bound to the solid phase (fig.2). In a competitive binding assay which is a more sensitive test, the level of inhibition of the 101C11 binding to the MenB PS reaches 43% with 100 ug/m\ N-propionylated MenB PS. On the other hand 500 ug/ml of this product (the highest concentration tested) is unable to prevent significantly the binding of 30H12 to Men B PS (the inhibition percentage is lower than 10%) (fig. 3). To quantify more precisely the degree of cross reactivity of the two mAbs towards Men B PS and N-proprionylated Men B PS, 50% inhibition concentration values (IC50) of each compound have been calculated from the different competitive inhibition curves. In the case 681
5
11
22
45
90
30H12(ug/ml)
FIGURE 2.
180
11
360
22
4S
90
1 01 C11
18s
37°
740
(ug/ml)
Of 30H12 or 101C11 to Different Antigens. Increasing Concentrations 101C11 were Tested by ELISA on Microwell Plates Sensitized with Different Antigenic Solutions: MenB PS ( -d- ), MenA PS (-a- ), MenC PS (-o- ), MenB LO.S. ( - - ) or N-Proprionylated MenB PS (-•-). To Validate Coating, Positive Controls such as Specific Polyclonal Sera were Included in each Test.
Binding
of either 30H12
or
Competltor(ug/ml)
competitor! ug/ml)
FIGURE 3. Binding Inhibition of 30H12 or 101C11 to MenB PS by Increasing Concentrations of Either MenB PS (-O- ), MenC PS (-D- ), MenB L.O.S. ( - - ) or NPropionylated MenB PS (-•- ). The Percentages of Inhibition were Calculated Using the Same Formula as in Figue 1. The Results are Expressed as Mean + S.D (n=3). of the 101C11 mAb, IC50 of Men B PS and N-proprionylated Men B PS are respectively 31 and 140 /¿g/ml and the degree of the cross reactivity of this mAb obtained from the ratio: IC50of/WenBPS_ x 100 is 22%. IC50 of N-proprionylated Men B PS In the case of the 30H12 mAb, IC50 of Men B PS and N-proprionylated Men B PS are respectively 23 Mg/ml and much more than 500 jug/ml and the degree of cross reactivity is less than 5%. Specific bactericidal activity (in vitro) oí the mAbi 3OH12 and 101C11. The mAb have been tested against two strains of Neisseria meningitidis group B (B : 15 ; P1.16 and B : 2a ; P1.2) and one strain of Neisseria meningitidis group C (C11). The specific bactericidal activity of the two mAbi is clearly demonstrated from the results shown in table 2. To block specifically the bactericidal activity of the mAbi we produced and characterized an IgGi anti idiotypic monoclonal antibody (mAb2), 216F11, against the mAbi 30Hi2. Ion exchange chromatography-purified 216F11 binds to 30H12 when tested in a direct binding
682
TABLE 2
Bactericidal
Activity Of The MAb 30H12 And 101C11 Against Group B And Group C
_Neisseria Meningitidis
Identity of the strain tested C11 (B:2a ; P1.2) H44/76 (B:15 ; P1.7, 16) < 256 ( 25 ug/ml) 512(12,5/tg/ml) 4(> 160
