**4. Development of pneumococcal vaccines**

620 The Complex World of Polysaccharides

evidence and speculation.

phosphate).

1. Non-housekeeping nucleotide sugar biosynthesis.

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

**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

2. The initial transferase (WchA in this case) links the initial sugar as a sugar phosphate (Glc-P) to a membrane-associated lipid carrier (widely assumed to be undecaprenyl

3. Glycosyl transferases sequentially link further sugars to generate repeat unit. 4. Wzx flippase transports the repeat unit across the cytoplasmic membrane. 5. Wzy polymerase links individual repeat units to form lipid-linked CPS.

charged grooves elicit potent T cell responses *in vivo* and *in vitro* [30, 31].

Although the first pneumococcal vaccines, including the application of the principle of conjugate vaccination, were already initiated in the beginning of the previous century, most of these developments stopped when antibiotics were introduced. Existing vaccines were even withdrawn from the market. By now, in many parts of the world, the antibiotic resistance of *S. pneumoniae* bacteria has increased: America [32, 33], Africa [34], Europe [35, 36], Asia [37-39], and Australia [40]. This makes treatment of pneumococcal infections more difficult and stresses the importance of the development of effective vaccines as a strategy to reduce morbidity and mortality caused by *S. pneumoniae* infection worldwide.

## **4.1. Pneumococcal polysaccharide-based vaccines.**

Currently two vaccine types against *S. pneumoniae* are commercially available: a pneumococcal polysaccharide vaccine (PPV) and a pneumococcal conjugate vaccine (PCV) [41]. The first multivalent pneumococcal polysaccharide vaccine (PPV) contains 23 purified capsular polysaccharides (25 µg of each capsule type; Pneumovax®, PPV23: 1, 2, 3, 4, 5, 6B, 7, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15F, 18C, 19A, 19F, 20, 22F, 23F, 33F ) which is licensed for use in adults and children older than 2 years of age [42]. This vaccine was shown to be moderately effective in young adults [43] but not in young children [44] and elderly [45] and also not in immunocompromised patients, e.g HIV infected people [46, 47].

In early 2000, a polysaccharide-protein conjugate vaccine targeting seven pneumococcal serotypes was licensed in the United States for use in young children (Prevnar, PCV7: 4, 6B, 9V, 14, 18C, 19F, 23F). The polysaccharides are conjugated to the non-toxic cross reactive material from diphtheria toxin, CRM197 and each dose contains 2µg of each capsule type, except for 6B, for which 4 µg is included in every vaccine dose[48]. The PCV7 vaccine produces a significant effect regarding prevention of invasive pneumococcal disease in children younger than 24 months (based on a meta-analysis of published data from trials on pneumococcal vaccine) [49]. Large scale introduction of PCV7 has resulted in an overall decline in infectious pneumococcal disease (IPD). However, IPD caused by the non-vaccine serotypes serotypes 1, 19A, 3, 6A, and 7F has increased (replacement disease), highlighting the need for inclusion of these serotypes in future improved vaccine formulations [50]. Apart from the CRM197 based PCV7, several new candidate pneumococcal conjugate vaccines have been developed to cover more serotypes with different protein carriers and most of them are in clinical trials, such as PCV10 vaccine (1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F) [51, 52] and PCV13 vaccine (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) [53].

#### **4.2. Pneumococcal protein-based vaccines**

An alternative vaccine strategy focuses on the use of pneumococcal surface-associated proteins which are to be assumed to elicit protection in all age groups against all, or nearly all, pneumococcal serotypes (Fig. 1). Protection induced by the proteins should be serotypeindependent and possibly cheaper and thus within reach of developing countries [54]. Currently, several surface pneumococcal proteins are investigated as a candidate vaccine against *S. pneumoniae* infection with single or combination of recombinant proteins, such as pspA family fusion protein [55]; pneumolysis and pspA1/pspA2 combined [56]. Recently new candidate protein antigens were discussed at the 8th International Symposium on Pneumococci and Pneumococcal Diseases at Iguaçu Falls, Brazil (2012), phtD (pneumococcal histidin triad protein D) and PcpA (pneumococcal choline binding protein A) [57].

## **4.3. Pneumococcal synthetic oligosaccharide-based vaccines**

The current polysaccharide conjugate vaccines are based on natural polysaccharides, purified form bacterial cultures. Synthetic oligosaccharide–protein conjugates (neoglycoconjugate), involving functional mimics of the natural polysaccharide antigens have emerged as an attractive option [58]. The advantages of neoglycoconjugates are well-defined chemical structures (chain length, epitope conformation, and carbohydrate/protein ratio) as well as a lack of the impurities present in polysaccharides obtained from bacterial cultures [59, 60].

The chemical synthesis of oligosaccharide fragments however is complex. According to the sequence in the natural polysaccharide, monosaccharide residues have to be linked in such a way that they form an oligosaccharide with the required stereospecificity (epitope). Various methodologies and strategies for synthesis of carbohydrates have successfully been used for production of experimental neoglycoconjugates, as reviewed by Kamerling [16]. In 2001, the first automated synthesis of oligosaccharides was reported by Plante, O.J. et al [61].

Neoglycoconjugates have been prepared for saccharides of different microorganisms. In 2004, Verez Bencomo et al., reported the large-scale synthesis and the introduction of a synthetic oligosaccharide vaccine for *Haemophilus influenzae* type b for use in humans in Cuba [9]. The immunogenicity of the synthetic oligosaccharide fragment of the O-specific polysaccharide (O-PS) of *Vibrio cholera* O1, serotype Ogawa, conjugated to bovine serum albumin has been investigated in a mouse model [62, 63]. A multimeric bivalent synthetic hexasaccharide fragment of the O-specific polysaccharide of *Vibrio cholera* O1, serotype Ogawa, in combination with Inaba:1 or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant were prepared and conjugated to recombinant tetanus toxin H(C) fragment as protein carrier [64]. The immunogenicity of synthetic oligosaccharides mimicking the Oantigen of the *Shigella flexneri* 2a lipopolysaccharide (LPS) was also investigated in mice [65, 66]. Immunization of mice with synthetic hexasaccharide of glycosylphosphatidylinositol malarial toxin conjugated to a protein carrier was reported to protect the mice from an otherwise lethal dose of malaria parasites [67]. A fully synthetic carbohydrate-based antitumor candidate vaccine for the common T-synthase was recently reported [68].

Meanwhile we and other groups have been working on improving the immunogenicity of neoglycoconjugates against different *S. pneumoniae* serotypes in animal models: Di-, tri-, and tetrasaccharides related to polysaccharide type 17F conjugated to keyhole limpet hemocyanin (KLH) protein[69, 70] and tri- and tetrasaccharides related to type 23 conjugated to KLH protein [71]; Di-, tri-, and tetrasaccharides related to type 6B conjugated to KLH protein [72]; Di-, tri-, and tetrasaccharide related to type 3 conjugated to the crossreactive material of diphteria toxin (CRM197) protein [60] and most recently overlapping oligosaccharide varying from tri- to dodecasaccharides related to polysaccharide type 14 conjugated to CRM197 protein [73, 74].
