**5.1. Identification of the minimal structure of oligosaccharide capable in evoking anti-Pn14PS antibodies.**

It was reported that a synthetic branched tetrasaccharide, corresponding to a single structural repeating unit of Pn14PS conjugated to the cross-reactive material of diptheria toxin (CRM197), was found to induce anti-polysaccharide type 14 antibodies by Mawas, F. et al [74]. We continued to investigate further how small the minimal structure in Pn14PS can be and still produce specific antibodies against native polysaccharide type 14 [73]. 16 overlapping oligosaccharide fragments of Pn14PS were synthesized as described previously [76-79] and were conjugated to the protein carrier CRM197. The mice immunization studies were performed to investigate the immunogenicity of the neoglycoconjugates. We found that the fragments with a linear and/or incomplete branched structure did not elicit specific antibodies against native Pn14PS (Fig. 4: JJ118, JJ42, JJ141, DM65, JJ153, JJ9, JJ6 and DM35) [73]. High titer of anti-Pn14PS IgG antibodies was observed when the complete branched structure fragments, conjugated to the protein carrier were used in the mouse model (Fig. 4: JJ1, DM66, DM36, ML1, ML2, and CRM197-Pn14PS as a positive control), excepted for JJ5 and JJ10 which elicited low titer of anti-Pn14PS antibodies.

We also tested the phagocytic capacity of mice sera by human polymorph nuclear cells and a mouse macrophage cell line. We found that the sera containing antibodies against Pn14PS were also capable of promoting the phagocytosis of *S. pneumoniae* type 14. Conjugates that did not evoke specific antibodies against polysaccharide type 14 also did not display phagocytic capacity [73].

**Figure 4. Level of anti-Pn14PS antibodies and schematic structure of overlapping synthetic oligosaccharide fragments of Pn14PS** (Adopted from Safari et al 2008 [73]). The oligosaccharides were conjugated to CRM197 protein and the immunogenicity of those conjugates were studies in a mouse model. Mice were immunized with polysaccharide type 14 conjugated to CRM197 (CRM197-Pn14PS) as a positive control. Enzyme-linked immunosorbent assay was employed to measure specific anti-Pn14PS IgG antibodies after the booster immunization. Antibody titers were expressed as the log10 of the dilution Filled circle = glucose (Glc); open circle = galactose (Gal), and filled square = N-acetylglucosamine (GlcNAc).

In conclusion, the present study has shown that the branched trisaccharide Glc-(Gal-)GlcNAc is the core structure inducing Pn14PS-specific antibodies and that the neighboring galactose at the non-reducing end significantly contributes to the induction of phagocytosispromoting antibodies [73]. Our study provides evidence that the branched tetrasaccharide Gal-Glc-(Gal-)GlcNAc is a prime candidate for a synthetic oligosaccharide conjugate vaccine against infections caused by *S. pneumoniae* type 14 [73].

## **5.2. Relationship between polysaccharide of Pn14PS and GBSIII**

624 The Complex World of Polysaccharides

**Figure 4. Level of anti-Pn14PS antibodies and schematic structure of overlapping synthetic oligosaccharide fragments of Pn14PS** (Adopted from Safari et al 2008 [73]). The oligosaccharides were conjugated to CRM197 protein and the immunogenicity of those conjugates were studies in a mouse model. Mice were immunized with polysaccharide type 14 conjugated to CRM197 (CRM197-Pn14PS) as a positive control. Enzyme-linked immunosorbent assay was employed to measure specific anti-Pn14PS IgG antibodies after the booster immunization. Antibody titers were expressed as the log10 of the dilution Filled circle = glucose (Glc); open circle = galactose (Gal), and filled square = N-acetylglucosamine (GlcNAc).

We also determined the minimal epitope in group B streptococcus type III polysaccharide (GBSIIIPS), using both a panel of anti-Pn14PS mouse sera and sera of humans vaccinated with either Pn14PS or GBSIIIPS as reported by Safari et al [80]. Native Pn14PS is structurally related to and has cross-reactivity with GBSIIIPS [81]. The branched structures of Pn14PS and GBSIIIPS differ only in the absence (in Pn14PS) or presence (in GBSIIIPS) of the (α23)-linked sialic acid N-acetylneuraminic acid (Neu5Ac) in their side chains: {→4)-β-D-Glcp-(1→6)-[±α-Neu5Ac-(23)-β-D-Galp-(14)-]β-D-GlcpNAc-(1→3)-β-D-Galp-(1→}n [82]. We reported that type-specific Pn14PS antibodies which recognize the branched structure of Pn14PS have a low affinity for the native GBSIIIPS and do not promote opsonophagocytosis of GBSIII, however desialylation of GBSIIIPS, however, resulted in dramatically higher affinity of anti-Pn14PS antibodies in mice when GBSIIIP was treated by nurimindase (desialylation) [80]. These results revealed that GBSIII bacteria are protected from binding of antibodies against Pn14PS by a residue of (α23)-linked sialic acid, as described previously [83, 84].
