**1. Introduction**

616 The Complex World of Polysaccharides

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> Studies on synthetic carbohydrates to be used as potential vaccine candidates for polysaccharide encapsulated bacteria were started in the mid-1970s. They were the logical follow-up to studies being performed at that time on the immunogenicity of antigens composed of carrier proteins and synthetic hapten groups. Hapten-carrier complexes were first introduced in immunology by Karl Landsteiner in the early 1900s [1]. He discovered that (i) small organic molecules with a simple structure, such as phenyl arsonates and nitrophenyls, do not provoke antibodies by themselves, but (ii) if those molecules are attached covalently, by simple chemical reactions, to a protein carrier, then antibodies against those small organic molecules are evoked. Since their introduction, these haptencarrier complexes have become excellent tools to elucidate the role of different antigenreactive cells in the immune response [2]. The key players in this immunological process are thymus-derived T cells and bone marrow-derived B cells. The former group of lymphoid cells is responsible for various phenomena of cell-mediated immunity, e.g. delayed hypersensitivity, allograft-, and graft-versus-host reactions, and reacts with specific determinants on the carrier protein (T cell epitopes). The latter group of lymphoid cells (B cells) give rise to the precursors of antibody-secreting cells, and reacts with both the carrier protein and the synthetic haptenic determinants. This results in antibody formation to both the carrier and the hapten.

> The reason to apply the above concepts and techniques to carbohydrate antigens was to address an immunological problem: polysaccharide molecules are classified as so-called thymus-independent (TI) antigens, because they do not require T cells to induce an immune response of B cells. As a result, the antibodies formed are mainly of the IgM class and have a

© 2012 Snippe et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 carbohydrate-based vaccines [10].

## **2. Streptococcus pneumoniae**

*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 growing *S. pneumoniae* bacteria.
