INFECTION AND IMMUNITY, Dec. 1991, p. 4555-4561 0019-9567/91/124555-07$02.00/0 Copyright C 1991, American Society for Microbiology
Vol. 59, No. 12
Relation between Structure and Immunologic Properties of the Vi Capsular Polysaccharide SHOUSUN C. SZU,l* XIU-RU LI,' AUDREY L. STONE,2 AND JOHN B. ROBBINS' Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development,' and Intramural Research Program, National Institute of Mental Health,2 Bethesda, Maryland 20892 Received 13 June 1991/Accepted 7 September 1991
The Vi capsular polysaccharide of Salmonella typhi is a linear homopolymer of poly-a(l-4)GalNAcp variably 0 acetylated at the C-3 position. Serum antibodies elicited by this antigen confer protective immunity against typhoid fever. The relation between the immunologic properties and structure of Vi was investigated by carboxyl reduction, 0 deacetylation, and acid hydrolysis. The immunogenicity of Vi was closely related to its degree of 0 acetylation. Partial 0 deacetylation slightly increased immunogenicity; comphWte 0 deacetylation eliminated the immunogenicity of Vi. 0-deacetylated Vi, however, still reacted with antisera prepared by injection of whole bacteria. Carboxyl reduction, in contrast, had a comparatively slight effect upon both the immunogenicity and antigenicity of Vi. Retention levels of antigenicity after acid treatment were greater for both the native and carboxyl-reduced Vi than for the 0-deacetylated product. The Courtauld-Koltun space-ifiling model of a pentamer of Vi demonstrated that the bulky nonpolar 0-acetyls, which protrude in rows on both sides, make up most of the surface. The carboxyls are less exposed and are partially shielded by the 0-acetyls. The molecular model thus provides an explanation for the dominant role of the 0-acetyls, as well as the lesser effect of carboxyl reduction, upon the immunologic properties of Vi. The Vi capsular polysaccharide (hereafter referred to as Vi) is both a virulence factor and a protective antigen of Salmonella typhi, the causative agent of typhoid fever (7). Vi confers virulence by shielding S. typhi against the actions of serum complement. Serum Vi antibodies initiate complement-mediated reactions; the most probable mechanism is phagocytosis and intracellular killing by monocytes of opsonized S. typhi (24). Recently, in two clinical trials, one injection of Vi conferred protection against typhoid fever in areas with high attack rates of this disease (1, 17). The efficacy was related to the immunogenicity of Vi. In this study, we examine the relation between the structure of Vi and its immunologic properties. Vi is a linear homopolymer of a(1--4)-D-GalpANAc variably 0 acetylated at C-3 (10, 16). Fiber X-ray diffractions of poly-a(1-34)-D-GalAp indicated that this polysaccharide could assume the forms characteristic of helices with two- or three-fold symmetry (23). The linear distance between carboxyl groups was about 0.43 nm. Szewczyk and Taylor (26) found that the 0-acetyls are important in the binding of Vi to antibody. Vi, prepared for clinical investigation by being heated in 1 N acetic acid at 100°C for 24 h, did not confer protection against challenge with S. typhi (11). Vi prepared by this method, however, was devoid of both 0-acetyl and N-acetyl groups and had a reduced molecular size (23). Other bacterial polysaccharides (e.g., pneumococcal type 1 and Escherichia coli KI, K13, K20, K23, K93, and K53) have 0-acetyls that are critical for their serologic specificity (2, 4, 5, 8, 22, 32). Negative charges also contribute to the immunologic properties of some bacterial polysaccharides. For example, the spacing of the carboxyls of poly-a(2--*8)NeupNAc (the capsular polysaccharide of group B Neisseria meningitidis and E. coli K1) is critical for the binding of this polymer to antibodies (14). The carboxyls of polysaccharides with less *
Corresponding author. 4555
negative charge densities, such as pneumococcal type 9N, composed of a pentasaccharide containing glucuronic acid, also play an important role in the antigenicity of the polymer (27). We report the effect of chemical modifications of the carboxyl and 0-acetyl groups upon the antigenicity and immunogenicity of Vi. A molecular model of Vi was used to explain these effects. MATERIALS AND METHODS
Reagents. Vi, isolated from Citrobacterfreundii WR7011, contained
Vol. 59, No. 12
Relation between Structure and Immunologic Properties of the Vi Capsular Polysaccharide SHOUSUN C. SZU,l* XIU-RU LI,' AUDREY L. STONE,2 AND JOHN B. ROBBINS' Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development,' and Intramural Research Program, National Institute of Mental Health,2 Bethesda, Maryland 20892 Received 13 June 1991/Accepted 7 September 1991
The Vi capsular polysaccharide of Salmonella typhi is a linear homopolymer of poly-a(l-4)GalNAcp variably 0 acetylated at the C-3 position. Serum antibodies elicited by this antigen confer protective immunity against typhoid fever. The relation between the immunologic properties and structure of Vi was investigated by carboxyl reduction, 0 deacetylation, and acid hydrolysis. The immunogenicity of Vi was closely related to its degree of 0 acetylation. Partial 0 deacetylation slightly increased immunogenicity; comphWte 0 deacetylation eliminated the immunogenicity of Vi. 0-deacetylated Vi, however, still reacted with antisera prepared by injection of whole bacteria. Carboxyl reduction, in contrast, had a comparatively slight effect upon both the immunogenicity and antigenicity of Vi. Retention levels of antigenicity after acid treatment were greater for both the native and carboxyl-reduced Vi than for the 0-deacetylated product. The Courtauld-Koltun space-ifiling model of a pentamer of Vi demonstrated that the bulky nonpolar 0-acetyls, which protrude in rows on both sides, make up most of the surface. The carboxyls are less exposed and are partially shielded by the 0-acetyls. The molecular model thus provides an explanation for the dominant role of the 0-acetyls, as well as the lesser effect of carboxyl reduction, upon the immunologic properties of Vi. The Vi capsular polysaccharide (hereafter referred to as Vi) is both a virulence factor and a protective antigen of Salmonella typhi, the causative agent of typhoid fever (7). Vi confers virulence by shielding S. typhi against the actions of serum complement. Serum Vi antibodies initiate complement-mediated reactions; the most probable mechanism is phagocytosis and intracellular killing by monocytes of opsonized S. typhi (24). Recently, in two clinical trials, one injection of Vi conferred protection against typhoid fever in areas with high attack rates of this disease (1, 17). The efficacy was related to the immunogenicity of Vi. In this study, we examine the relation between the structure of Vi and its immunologic properties. Vi is a linear homopolymer of a(1--4)-D-GalpANAc variably 0 acetylated at C-3 (10, 16). Fiber X-ray diffractions of poly-a(1-34)-D-GalAp indicated that this polysaccharide could assume the forms characteristic of helices with two- or three-fold symmetry (23). The linear distance between carboxyl groups was about 0.43 nm. Szewczyk and Taylor (26) found that the 0-acetyls are important in the binding of Vi to antibody. Vi, prepared for clinical investigation by being heated in 1 N acetic acid at 100°C for 24 h, did not confer protection against challenge with S. typhi (11). Vi prepared by this method, however, was devoid of both 0-acetyl and N-acetyl groups and had a reduced molecular size (23). Other bacterial polysaccharides (e.g., pneumococcal type 1 and Escherichia coli KI, K13, K20, K23, K93, and K53) have 0-acetyls that are critical for their serologic specificity (2, 4, 5, 8, 22, 32). Negative charges also contribute to the immunologic properties of some bacterial polysaccharides. For example, the spacing of the carboxyls of poly-a(2--*8)NeupNAc (the capsular polysaccharide of group B Neisseria meningitidis and E. coli K1) is critical for the binding of this polymer to antibodies (14). The carboxyls of polysaccharides with less *
Corresponding author. 4555
negative charge densities, such as pneumococcal type 9N, composed of a pentasaccharide containing glucuronic acid, also play an important role in the antigenicity of the polymer (27). We report the effect of chemical modifications of the carboxyl and 0-acetyl groups upon the antigenicity and immunogenicity of Vi. A molecular model of Vi was used to explain these effects. MATERIALS AND METHODS
Reagents. Vi, isolated from Citrobacterfreundii WR7011, contained
