Isolation of type-specific polysaccharide antigen from group B type Ib streptococci.

Abstract

Lancefield et al.(1) have clearly demonstrated that the capsular polysaccharides of group B streptococci are antigenic and that the antibodies are protective in mice except with type III, where no mouse-virulent strain is available. However, a chicken embryo model has been established for type III and the antibody found to be specifically protective (2). Recently, emphasis has been centered on the isolation and characterization of the capsular antigens from various types (3-8). However, inasmuch as the capsule is very tightly (probably covalently) bound to the cell wall, a major problem is to find an extraction method that will give satisfactory yield. Conventional HC1 extraction results in partial destruction of the polysaccharide antigens with the loss of the sialic acid determinant (6-8). Current investigations have concentrated on obtaining larger size capsular polysaccharides that are antigenically intact. Neutral buffer extractions have been used successfully to obtain such molecules from type Ia and type III (3, 5). However, the yield is low, which hinders structural studies as well as more extensive clinical and immunological investigations.In this report, we describe the isolation of type Ib polysaccharide from whole cells by digestion with muralytic enzymes prepared from Streptomyces albus. The composition and immunological properties of the purified type Ib antigen are also discussed. Materials and MethodsStreptococcal Strain and Antisera. Group B type Ib (H36B) streptococci and antisera against various types used in this study were from the collection of Dr. R. C. Lancefield of The Rockefeller University. The preparation of antisera was previously described by Lancefield et al.(1).Growth Conditions. Strain H36B (type Ib) was grown in Todd-Hewitt (Difco Laboratories, Detroit, Mich.) broth containing 0.2% yeast extract and 0.2% glucose. 20 ml of overnight culture was inoculated into 4 liters of broth at 37°C. After overnight growth, the cells were harvested by centrifugation and washed twice with 1/x5 M phosphate buffer pH 8.0. Digestion of Type Ib Cells with Lytic Enzymes from Streptornyces albus.The extracellular enzymes of Streptomyces albus were prepared according to the procedure described by McCarty (9).The titer of lytic activity of each enzyme preparation was assayed using group A streptococci (either strain $43, type 6, or strain T28, type 28) as described (9). The lyric activity of an enzyme preparation was considered satisfactory when a 1:10 (vol/vol in 1/15 M phosphate buffer) enzyme dilution caused, within 30 min incubation at 37°C, a 70-80% decrease in turbidity using a cell suspension with an optical density of 0.50 at 600 nm in a 10 X 75-ram tube. The enzyme concentrations of different preparations were equalized based on this assay.

The type-specific polysaccharides of Streptococcus suis.

“…In S. suis, the polysaccharides are readily detached from the cocci by digestion with lysozyme . Group B (14) . In group B streptococci and in S. suis, individual-type polysaccharides are each composed of four or five different sugars (14-16) of which glucose, galactose, N-acetyl glucosamine, and sialic acid are common to all .…”

“…S. suis type 1 streptococci elicit in pigs antibodies that protect against infection type specifically (5), but this has yet to be confirmed with type 2 . Group B streptococci induce in rabbits antibodies that protect mice type specifically (17) ; some of the group B type-specific polysaccharides appear capable of eliciting antibodies with more than one homologous specificity directed against different parts of the same molecule (14,15) .…”

Protection of mice from experimental infection with type III group B Streptococcus using monoclonal antibodies.

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It is widely believed that antibodies to the group B streptococcal (GBS) typespecific polysaccharides are of major importance in human host defenses against GBS infection. All four of the type-specific polysaccharides elicit antibodies against at least two forms of the antigens: a complete antigen that contains Nacetylneuraminic acid (sialic acid) at the nonreducing ends of the side chains and an incomplete or nonsialated antigen (1)(2)(3)(4). This was first demonstrated for the type II polysaccharide by Lancefield and Freimer (1) when they showed that rabbit antisera directed against either the complete or incomplete antigen would protect mice from a lethal challenge of live type II organisms.

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A Search for New Group-B Streptococcal Serotypes

“…All the initially untypable strains that were shown by the screening lectin-agglutination test to contain sialic acid also possessed one of the new polysaccharides. The possibility cannot be excluded that some of the lectin-negative GBS contain an amount of type-polysaccharide antigen undetectable by our methods; one must bear in mind the many factors that influence the synthesis and detectability of type antigens or their components (Wilkinson, 1975;Baker et al, 1976;Kane and Karakawa, 1977;Kasper et al, 1979;Tai et a/., 1979;Doran et al, 1980). In any event, the extended typing scheme substantially reduces the number of N T strains.…”