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Streptococcus pneumoniae produces capsular polysaccharides that have been found to be important in infective processes, such as pneumonia, meningitis, otitis, and several other infectious diseases that cause considerable morbidity and mortality in children in most countries of the world (11). These polysaccharides are type specific and contain the main antigenic determinant of S. pneumoniae. The bacterium presents a multiplicity of serotypes, and serotype 14 is the predominant serotype in South American countries (2).

Chemical studies have shown that there is a relationship between capsular structure and immunological specificity, and both aspects of the serotype 14 capsular polysaccharide (CPS-14) have been extensively studied (3, 4). This polysaccharide is composed of a tetrasaccharide repeating unit containing D-glucose, N-acetyl-D-glucosamine, and D-galactose (6), as shown in Fig. 1. It is one of the few neutral capsular polysaccharides produced by pneumococcus strains, and several patents (1a, 3a) have been issued for its preparation and purification. Our purification procedure is based on the use of immobilized lectins. Soybean (Glycine max) lectin, which is specific for D-galactose and N-acetyl-D-galactosamine, can recognize the galactosyl residues present in the repeating unit of CPS-14 (8). Purification of CPS-14 by affinity chromatography as described here consists of a one-step procedure in which a commercial immobilized soybean lectin is used.

View larger version (6K): [in this window] [in a new window]   FIG. 1.   Structure of the tetrasaccharide repeating unit of S. pneumoniae type 14 capsular polysaccharide (CPS-16) (6).

Production and purification of CPS-14. CPS-14 was prepared from S. pneumoniae strains obtained from The National Centre for Streptococcus (Alberta, Canada) as described by Lund and Henrichsen (7) in 5-liter flasks containing 2 liters of tryptic soy broth (Difco Laboratories, Detroit, Mich.) and incubated at 37°C for 18 to 24 h. Growth was stopped by adding formaldehyde to a final concentration of 0.2% (wt/vol); the cells were lysed with sodium deoxycholate (0.1%, wt/vol) and then centrifuged. The supernatant was concentrated by ultrafiltration (using a hollow fiber cartridge with a molecular mass cutoff of 10 kDa) to 1/10 the original volume, and it was extensively dialyzed against distilled water and lyophilized.

Specific latex reagent. Rabbits were immunized with serotype 14 aldehyde-inactivated S. pneumoniae bacterial cells by using the immunization protocol of Lund and Henrichsen (7). Peripheral blood was collected, and the immunoglobulin fraction was purified from the serum by salt precipitation with 30% saturated ammonium sulfate. To avoid cross-reactions with the C polysaccharide produced by all serotypes, anti-C antibodies were eliminated by adsorption with a suspension of inactivated cells of a noncapsulated rugose C-mutant S. pneumoniae strain. The antisera were adsorbed onto latex reagent by the method of Battistoni (1). Agglutination of the samples was quantified by a twofold serial dilution assay. The titer was defined as the reciprocal of the highest dilution able to produce visible agglutination.

Protein content. The protein content was determined both spectrophotometrically by using the relationship between absorbance at 280 nm and absorbance at 260 nm (5) and by the bicinchoninic acid (Pierce, Rockford, Ill.) method (10).

¹H-NMR analysis. The ¹H nuclear magnetic resonance (¹H-NMR) spectrum of a sample dissolved in D₂O was determined with a Brucker DPX-400 spectrometer by using standard pulse sequences. Sodium 3-trimethylsilylpropionate-d₄ (_H 0.00) was used as an internal reference.

Sugar analysis. Purified polysaccharide (0.5 mg) was hydrolyzed with 2 M trifluoroacetic acid at 120°C for 1 h. The resulting monosaccharides were analyzed as their alditol acetates by gas-liquid chromatography on an SE-54 fused-silica capillary column by using the following temperature program: 140°C for 3 min, followed by an increase to 240°C at a rate of 3°C min¹. Gas chromatography-mass spectrometry was performed by using the column and conditions mentioned above with a Shimadzu QP-5050 gas chromatograph-mass spectrometer.

Production and affinity purification of CPS-14. The latex reagent was calibrated with a pure sample of S. pneumoniae CPS-14, and the detection limit was found to be 60 ng/ml. This value was used to estimate the amounts of CPS-14 in the culture medium and the culture supernatant after cell lysis, and the data indicated that almost all of the CPS-14 was released into the medium.

Identity and quality of the product. The polysaccharide obtained reacted positively with CPS-14-specific latex reagent, giving a high titer, as indicated in Table 1. Sugar analysis of the product revealed the presence of D-glucose, N-acetyl-D-glucosamine, and D-galactose at relative proportions of 1:1:2, which corresponded to the reported composition of the polysaccharide (6). The ¹H-NMR spectra (Fig. 2) showed the tetrasaccharide repetitive unit having all of the monosaccharides in the anomeric -configuration and the presence of an N-acetyl group, as expected. No leakage of lectin from the column was observed, as indicated by negative hemagglutination (9) performed with the eluted material.

View larger version (12K): [in this window] [in a new window]   FIG. 2.   1H-NMR spectrum of CPS-14 obtained by affinity chromatography.