**3. Capsular polysaccharide**

Capsular polysaccharides are well known as the major virulence factors of *S. pneumoniae*. Today more than 92 serotypes have been identified based on the different chemical structures of these polysaccharides [16, 17]. This diversity determines the ability of the serotypes to survive in the bloodstream and very likely the ability to cause invasive disease, especially in

carbohydrate-based vaccines [10].

growing *S. pneumoniae* bacteria.

**3. Capsular polysaccharide** 

**2. Streptococcus pneumoniae** 

low avidity. Moreover, no immunological memory is generated and the antigens are poorly immunogenic in infants. Latter characteristic has major implications for development of vaccines against polysaccharide encapsulated bacteria. It was hypothesized that by linking small carbohydrates (oligosaccharides) to a carrier protein, the immunogenic behavior would change to that of a thymus-dependent (TD) antigen. Therefore, the studies of both Goebel [3, 4] and Campbell and Pappenheimer [5], who first isolated the antigenic determinant of *Streptococcus pneumoniae* type 3, were combined and extended. The hapteninhibition studies by Mage and Kabat [6] demonstrated that the antibody-combining site of type 3 pneumococcal polysaccharide consists of two to three cellobiuronic acid units. In the dextran-anti-dextran system extensively studied by Kabat and colleagues [7] the upper size limit of the antibody-combining site appeared to be a hexa- or heptasaccharide and the lower limit was estimated to be somewhat larger than a monosaccharide. Snippe and colleagues [8] proved in 1983 that small synthetic oligosaccharides (tetra- and hexasaccharides) of *S. pneumoniae* type 3 could be transformed into TD antigens by conjugating them to a protein carrier. This opened the way to explore the synthesis and immunogenicity of numerous oligosaccharide-carrier protein conjugates of different pneumococcal serotypes. Those studies culminated in 2004 in the large-scale synthesis and introduction of a synthetic oligosaccharide vaccine for *Haemophilus influenzae* type b for use in humans in Cuba [9]. The recent exploration of gold nanoclusters coated with synthetic oligosaccharides and peptides as a vaccine are a promising platform towards the development of fully synthetic

*Streptococcus pneumoniae* (*S. pneumoniae* or pneumococcus) is a leading cause of bacterial pneumonia, meningitis, and sepsis in children worldwide. It is estimated that 1.6 million people die from these infections each year, of whom one million are children [11, 12]. *S. pneumoniae* are lancet-shaped, gram-positive, and alpha-hemolytic bacteria that colonize the mucosal surfaces of the upper respiratory tract [13]. Three major surface layers can be distinguished from the inside to the outside: the plasma membrane, the cell wall, and the capsule (Fig. 1) [14]. The cell wall consists of a triple-layered peptidoglycan backbone that anchors the capsular polysaccharide, the cell wall polysaccharide, and also various proteins such as pneumococcal surface protein A (pspA) and hyluronate lyase (Hyl) (Fig. 1). The capsule is the thickest layer, completely concealing the inner structures of exponentially

Capsular polysaccharides are well known as the major virulence factors of *S. pneumoniae*. Today more than 92 serotypes have been identified based on the different chemical structures of these polysaccharides [16, 17]. This diversity determines the ability of the serotypes to survive in the bloodstream and very likely the ability to cause invasive disease, especially in the respiratory tract [14, 16]. Recently, new *S. pneumoniae* serotypes have been identified, e.g. serotype 6C [17], 6D [18, 19], and 11E [20]. Capsular polysaccharides are large polymers (0.5- 2x106 Da), composed of multiple repeating units of up to eight sugar residues [14]. The capsular polysaccharides are generally synthesized by the Wzx/Wzy-dependent pathway, except for type 3 and 37 which are synthesized by the synthase pathway [21, 22] (Fig. 2). In the synthase pathway capsule is produced through processive transferase activity [23, 24].

**Figure 1. Schematic structure of** *S. pneumoniae*. StrA=sortase A. Hyl=hyluronate lyase. PavA=pneumococcal adhesion and virulence. Eno=enolase. NanA=neuraminidase. PsrP=pneumococcal serine-rich repeat protein. LytA=autolysin. LTA=lipoteichoic acid. PspA=pneumococcal surface protein A. PspC=pneumococcal surface protein C. PiaA/PiuA=pneumococcal iron acquisition and uptake. PsaA=pneumococcal surface antigen A. (Adopted from van der Poll, T. and Opal, S.M. [15] and de Velasco, E.A. et al [14])

Many studies have demonstrated that antibodies directed against the capsular polysaccharide are essential for protection against pneumococcal disease [25-27]. However, the native capsular polysaccharides are well-known thymus-independent type-2 (TI-2) antigens that lack T-helper epitopes and therefore mainly induce IgM antibodies, and to a lesser degree IgG [28]. The TI-2 characteristics of polysaccharides can be altered by conjugation of polysaccharide to a protein carrier (glycoconjugate) resulting in a switch to an anti-polysaccharide antibody response with characteristics of a T-cell-dependent response. This is reflected by the generation of memory B and T cells and the induction of high titers of anti-polysccharide IgG antibodies after booster immunization [29].

It should be noted that not all polysaccharides behave as TI-2 antigens. Zwitterionic polysaccharides such as *S. pneumoniea* type 1 polysaccharide: [3)--AATGal-(14)--D-Gal*p*A-(13)--D-Gal*p*A-(1]n with a right-handed helix with repeated zwitterionically charged grooves elicit potent T cell responses *in vivo* and *in vitro* [30, 31].

**Figure 2. Representation of the Wzx/Wzy-dependent pathway for biosynthesis of CPS 9A** (Adopted from Bentley. S.D. et al [21]). Representation of the Wzx/Wzy-Dependent Pathway Pictured is a hypothetical model for capsule biosynthesis in *S. pneumoniae* based on a mixture of experimental evidence and speculation.


6. Wzd/Wze complex translocates mature CPS to the cell surface and may be responsible for the attachment to peptidoglycan.
