APMIS 100: 91-94, 1992

Murine monoclonal antibodies against pneumococcal capsular polysaccharide types 4,8,22F and 19A/19F JAN KOLBERG', INGEBORG S. AABERGE', ERIK JANTZEN', MARTINUS LOVIK', GRO LERMARK3 and TORUNN STEEN' 'Department of Immunology, 'Department of Vaccine, 3Department of Bacteriology, National Institute of Public Health, Oslo, Norway

I Kolberg, J., Aaberge, I. S., Jantzen, E., Lervik, M., Lermark, G. & Steen, T. Murine monoclonal antibodies against pneumococcal capsular polysaccharide types 4, 8, 22F and 19A/ 19F. APMIS 100: 91-94, 1992. Monoclonal antibodies (MAbs) were produced against pneumococcal capsular polysaccharides after subcutaneous immunization of BALB /c mice with a 23-valent vaccine (Pneumovax@ N, Merck, Sharp & Dohme). Selected antibodies were tested in ELISA against individual polysaccharides from 23 different pneumococcal types and in a dot blot assay with heat-killed whole bacteria adhered to nitrocellulose paper. Three MAbs (isotype IgM) were found to be specific for types 4, 8 and 22F, respectively, whereas one (isotype IgA) reacted both with 19A and 19F. Very mild acid hydrolysis of the capsular polysaccharides resulted in loss of reaction with the antibodies. Key words: Monoclonal antibodies; Streptococcus pneumoniae; capsular polysaccharides. Jan Kolberg, Department of Immunology, National Institute of Public Health, Geitmyrsveien 75, N-0462 Oslo 4, Norway.

Streptococcus pneumoniue is still a major cause of morbidity and mortality, especially among infants and the elderly. The development of polysaccharide vaccines represents great achievements in the prevention of the disease. The current 23-valent vaccine was based upon the prevalence of types isolated mainly in the U S A . and Europe. The structure of some of these polysaccharides is known ( 1 2). Typing of pneumococci is based on capsular polysaccharide antigens, and they can be divided into 83 different types using commercially available polyclonal antisera. Monoclonal antibodies (MAbs) to capsular antigens would represent valuable tools for studying the structure and immunogenicity of these antigens. The development of monoclonal anticapsular antibodies has been difficult because of the relatively poor immune responses of mice to these antiReceived April 9, I99 1. Accepted June 11, 1991.

gens. MAbs against the type 3 polysaccharides have previously been produced using cells from mice immunized with the type 3 polysaccharides and the plant mitogen Con A (10). Capsular specific MAbs have also been generated by using type 6A and type 19F polysaccharides coupled to sheep erythrocytes (6). Here we report on the production and characterization of MAbs obtained after immunization of mice with the 23-valent vaccine.

MATERIALS AND METHODS A 23-valent pneumococcal vaccine (Pneumovaxn N) and the 23 different polysaccharides included in the vaccine were obtained from Merck, Sharp, and Dohme, Rahway, New Jersey and from the American Type Culture Collection, Rockville, Maryland. Pneumococcal strains of the 23 different capsular types included in the vaccine were isolated from human sources. Typing was performed by the capsular reaction test with rabbit antisera obtained from Sta-


KOLBERG et al.

tens Seruminstitut, Copenhagen, Denmark. Differentiation of types within groups was carried out at Statens Seruminstitut. The mutant strain CSR-SCS 2, purified pneumococcal cell wall polysaccharides and the monoclonal antibody HAS against phosphorylcholine were obtained from Dr J ~ r g e nHenrichsen, Statens Seruminstitut, Copenhagen. Before the first fusion seven-week-old BALB/c mice were injected subcutaneously with 12 pg of the vaccine in saline. Thirty-three weeks later and four days prior to the cell fusion the animals received 6 pg of the vaccine. Before the second fusion sevenweek-old mice were immunized subcutaneously twice with 6 pg of the vaccine with a one week interval. Forty-four weeks after the last injection and four days prior to the fusion the mice were given 10 pg of 19F polysaccharides. Spleen cells were fused with NSO myeloma by standard techniques. Selected hybrids were cloned (one cell per well). A kit (No. 93-6550) from Zymed Lab. Inc., U.S.A. was used for isotyping. Enzyme-linked immunosorbent assays (ELISA) were performed with microtitre wells coated directly with the different pneumococcal polysaccharides (1). Gamma irradiated microtest plates no. 269787 and Immuno Plates I from Nunc, Denmark were used. Washed wells were incubated with 0.05 ml phosphatebuffered saline, pH 7.4 (PBS), with 0.02% NaN,, containing 0.12 pg of the 23-valent vaccine or 0.025 pg of the individual vaccine components. The plates were incubated for two h at 37"C, then stored at 4°C. The wells were incubated for two h at 37°C with cell culture supernatants or ascitic fluids diluted in PBS with 3% bovine serum albumin. After washing alkaline phosphatase-conjugated goat antibodies against mouse immunoglobulins (Sigma, No. A-0162, dilution 1:500) were added. The enzyme substrate pnitrophenyl phosphate, 1 mg/ml in 10% diethanolamine buffer (pH 9.8) containing 10 mM MgC12, was added to washed plates. Specific rabbit antisera from Statens Seruminstitut, Copenhagen, against each type in the vaccine were used as controls for the antigen coating in ELISA. For dot blot assays pneumococci were cultivated overnight at 37°C in Todd-Hewitt broth medium. After washing the bacterial cells were resuspended in PBS containing 0.02% NaN,, followed by heating at 56°C for 30 min and incubation at 4°C overnight. The suspensions were then diluted to give an OD of 0.3 at 650 nm in 1 cm cuvettes. Two p1 amounts of the suspensions were spotted onto nitrocellulose membranes from Schleicher & Schuell, Dassel, Germany (0.2 pm). After blocking for 30 min at room temperature with 3% bovine serum albumin in PBS, the strips were incubated for two h at room temperature with each of the MAbs (ascitic fluid diluted 1:1000 in the blocking solution). After washing four times in PBS the strips were incubated for two h at room temperature with peroxidase-conjugated rabbit immunoglobulins to mouse immunoglobulins (Dakopatts, Denmark, diluted 1:lOOO). The substrate solu92

tion consisted of 0.4 mg/ ml 3-amino-9-ethyl-carbazole (previously dissolved in N,N-dimethylformamide) and 0.015% HzOZin 50 mM sodium acetate buffer (pH 5.0). For thin-layer chromatography (TLC) immunoblotting the capsular carbohydrates were hydrolyzed with 0.1 M HCl at 100°C and samples were taken at different periods. The hydrolysates were spotted onto amino-bound silica TLC plates followed by immunostaining as described in (4), except that peroxidaseconjugated rabbit anti-mouse immunoglobulins (see above) were used instead of '251-labelledanti-mouse immunoglobulins. Periodate oxidation of polysaccharides bound to the TLC plates was performed as in (13). The spotted plates were oxidized in the dark for one h with 10 mM sodium periodate in 0.1 M sodium acetate buffer (pH 6.0). The plates were then washed in saline before blocking and immunostaining.


Murine hybridomas secreting antibodies against pneumococcal capsular polysaccharides have

'"iu WAb CS4 D l 0



WAb C I S B8

2*ol 1.5

0.5 1










rn 1



5 ( 7 ~9N110A 12F 1 5 8 18C 19F 22P 31F

Fig. 1. ELISA analyses of MAb binding to the 23 different capsular polysaccharides in the vaccine used as antigen. Values below 0.1 were regarded as negative.


csa PI C84 DlO CS9 Hb







Fig. 2. Dot blot assay showing the binding of the MAbs to heat-killed whole pneumococci representing the 23 vaccine types.

often been difficult to develop ( 5 , 6, 11). Mice immunized intravenously with formalin-killed whole pneumococcal cells were found to generate hybridomas specific for the phosphorylcholine determinant of the cell wall carbohydrates (1 1). Immunization intravenously with capsular polysaccharides gave rise to antibodies against cell wall carbohydrates which have been shown to be present in these preparations (8, l l ) , but capsular-specific antibodies could be obtained by coupling the polysaccharides to erythrocytes (6). In our study capsular-specific MAbs were readily obtained from fusions of splenocytes of mice immunized subcutaneously with the 23valent vaccine, indicating that the route of immunization might be of importance. We used by chance rather long intervals between the primary and secondary injections, and the mice consequently became relatively old. Further experiments are needed to determine the importance of these factors. Supernatants from hybrid cells obtained from the first fusion were initially screened with the

Treatment None


MAb CS9 H6 with type 4 polysaccharldes

MAb CS3 F7 with type 22F polysaccharldes

Fig. 3. Immunostaining before and after periodate treatment of capsular polysaccharides spotted onto silica thin-layer plates.

23-valent capsular polysaccharide vaccine as coating antigen. After retesting against plates with the individual vaccine components pooled into groups, three hybridomas were selected and cloned. These were tested for specificity in ELISA using the 23 different components in the vaccine as antigens (Fig. 1) and by dot blotting with heat-inactivated pneumococci spotted onto nitrocellulose papers (Fig. 2). When an antibody showed a positive reaction against a certain type of pneumococci, tests were performed against nine additional isolates of this type. The three MAbs (CS3 F7, CS4 D10 and CS9 H6, all isotype IgM) were found to be specific for pneumococcal types 22F, 8 and 4,respectively. In the second fusion the ELISA screening was performed only against the type 19F polysaccharide. The selected primary hybridoma giving rise to clone CS19 HI 1 (isotype IgA) was found to react both with 19A and 19F (Figs. 1 and 2). To determine if the four MAbs reacted with bacteria other than the pneumococci, two strains of Haemophilus influenzae and three strains of Streptococcus sanguis were tested by dot blotting and none stained with the antibodies. The specificity of the four selected MAbs was further supported by their non-reactivity in ELISA against purified pneumococcal cell wall polysaccharides as coating antigen and by their lack of staining in dot blotting with the pneumococcal mutant CSR-SCS 2. This strain is reported to carry a small capsule consisting of pneumococcal cell wall polysaccharides (9). Strong reactions were found when the control MAb HAS was used. This antibody is specific for the phosphorylcholine constituent of the pneumococcal cell wall polysaccharides. 93


Some experiments were performed to try to characterize the epitopes by immunostaining of chemically treated capsular polysaccharides bound to thin-layer plates (4). We modified this technique by using a secondary antibody labelled with peroxidase instead of 12’I. This detection method is both less expensive and more convenient than the use of isotopes. Weak acidic treatment at 100°C destroyed the epitopes for the four antibodies. Heating alone had no effect. This result is probably not due to cleavage of glycosidic linkages. The loss of antigenicities is therefore most likely due to removal of essential side groups, or to the epitopes being conformation dependent. It therefore seems difficult to characterize the epitopes for these MAbs without having chemically synthesized unit structures of these pneumococcal polysaccharides. The proposed structures for these polysaccharides (reviewed in 12) show that only the type 4 capsular polysaccharides contain pyrovate acetal groups (2). These are very sensitive to acid hydrolyses (3) and could thus be involved in the binding of the type 4-specific antibody CS9 H6. Furthermore, the repeating unit of type 4 polysaccharides does not contain monosaccharides with vicinal hydroxyl groups (2) and is therefore resistant to periodate. As expected, this treatment did not alter the binding of MAb CS9 H6, which further supported the specificity of the antibody (Fig. 3). The proposed structure of type 22F (7) capsular polysaccharides contains monosaccharides with vicinal hydroxyl groups; in accordance with this the epitope for MAb CS3 F7 was destroyed by periodate oxidation. -


The initial part of this work was performed by Lena Kristiansen and Torunn Steen over a four month period while they were students at Oslo College of Engineering. We thank Svein Flaathen for skilful technical assistance and for helping the students into the field of hybridoma technology.

REFERENCES 1. Aaberge, I. S., Heier, H. E., Hem, E., Giercksky, K-E. & Groeng E-C.: IgM and IgG response to pneumococcal polysaccharide vaccine in normal


individuals and individuals splenectomized due to trauma, Acta path. microbiol. immunol. scand., Sect. C 92: 11-16, 1984. 2. Jones, C.: A novel method for the determination of the stereochemistry of pyruvate acetal substituents applied to the capsular polysaccharide from Streptococcus pneumoniae type 4. Carbohydr. Res. 198: 353-357, 1990. 3. Higginbotham, J. D. & Heidelberger, M.: The specific capsular polysaccharide of Pneumococcus type IV. Carbohydr. Res. 23: 165-173, 1972. 4. Magnani, J. L.: Immunostaining free oligosaccharides directly on thin-layer chromatograms. Analyt. Biochem. 150: 13-17, 1985. 5 . McDaniel, L. S. & Briles, D. E.: Monoclonal antibodies against surface components of Streptococcus pneumoniae, In: Macario A. J. & Conway, E. (Eds.): Monoclonal Antibodies Against Bacteria. Vol. 3, Academic Press Inc., New York 1986, pp. 143-164. 6. Milligan, G. N . & Braley-Mullen, H.: Production and characterization of monoclonal antibodies specific for types 6 and 19 pneumococcal capsular polysaccharides. Molec. Immunol. 26: 299-307, 1989. 7. Richards, J. C. & Perry, M . B.: Application of two dimensional NMR methods to the structural elucidation of complex polysaccharide antigens. The structure of the capsular polysaccharide of Streptococcus pneumoniae type 22 E Adv. Exp. Med. Biol. 228: 597-598, 1988. 8. Sarvas, H., Rautonen, N., Sippinen, S. & Makela, 0.:IgG subclasses of pneumococcal antibodies effect of allotype G2m(n). Scand. J. Immunol. 29: 229-237, 1989. 9. Schijjfman, G., Bornstein, D. L. & Austrian, R.: Capsulation of Pneumococcus with soluble cell wall-like polysaccharide. J. Exp. Med. 134: 600-617, 1971. 10. Schroer, K. R., Kim, K. J., Prescott, B. & Baker, I! J.: Generation of anti-type 111 pneumococcal polysaccharide hybridomas from mice with an Xlinked B-lymphocyte defect. J. Exp. Med. 150: 698-702, 1979. 1. Ssrensen, U.KS., Agger, R., Bennedsen, J. & Henrichsen, J.: Phosphorylcholine determinants in six pneumococcal capsular polysaccharides detected by monoclonal antibody. Infect. Immun. 43: 876-878, 1984. 2. Van Dam, J. E, G., Fleer, A. & Snippe. H.: Immunogenicity and immunochemistry of Streptococcus pneumoniae capsular polysaccharides. Antonie van Leeuwenhoek 58: 1-17, 1990. 13. Woodward, M . 19, Young, U! u!Jr. & Bloodgood, R. A.: Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J. Immunol. Methods 78: 143-153, 1985.

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Monoclonal antibodies (MAbs) were produced against pneumococcal capsular polysaccharides after subcutaneous immunization of BALB/c mice with a 23-vale...
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