**5.3. Booster immunization either with either neoglycoconjugate or native polysaccharide**

We investigated further the immune response to a neoglycoconjugate of Pn14PS (GC) on the outcome of sustained immunity to *S. pneumoniae* type 14 in a mouse model after the booster injection with either (GC) or native Pn14PS (PS) [85]. We found, as we expected, that the amount of specific IgG antibodies against Pn14PS increased substantially when a GC booster was given to mice previously primed with the same GC [85]. The induced antibodies were capable to opsonise *S. pneumoniae* type 14. Boosting with PS following a primary conjugate vaccine injection did not result in IgG antibody formation to Pn14PS (Table 1).

In order to explain these phenomena we investigated how a booster immunization with a GC or PS affects the cell-mediated immune response by measuring the production profile of a panel of cytokines [85]. We observed a high level of IL-5 in serum after a booster injection with GC (GC-GC or GC-GC-GC). Boosting with PS did not result in the induction of IL-5 nor of any of the other tested cytokines (Table 1; GC-PS and GC-PS-PS). We conclude that induction of the cytokine IL-5 in serum is an early sign of a successful booster immunization and is a prerequisite for the production of specific anti-polysaccharide IgG antibodies [85]. In-vitro spleen cell cultures were also used to investigate the effect of a booster injection on activation of memory T cells. IL-5 which well known Th2 cytokines, were evoked by the GC in spleen cell cultures of mice previously primed and boosted with the same GC [85]. In


conclusion, the inability of polysaccharide to boost primed mice might be due to the incapability to induce the cytokines.

1Five mice per group were immunized with a CRM-neoglycoconjugate (GC), a synthetic branched tetrasaccharide of Pn14PS that is conjugated to a CRM197 protein. Booster doses containing either a GC (GC-GC and GC-GC-GC) or a native polysaccharide of Pn14PS (PS) (GC-PS, GCGC-PS, and GC-PS-PS) were injected at Weeks 5 and 10. 2ELISA was employed to measure specific anti-Pn14PS IgG antibodies, and expressed as the log10 of the sera dilution 3 Cytokine levels in sera from mice receiving booster injection. Sera were collected on Day 1 after the primary immunization 4Splenocytes were isolated 7 days after the first booster injection. Spleen cells were cultured in vitro and stimulated with CRM-neoglycoconjugate and supernatants were collected 72 h after culture initiation.

**Table 1.** Effect of booster immunization either with with either the same neoglycoconjugate or a native polysaccharide (Adopted from Safari, D. el at [85] with permission)

## **5.4. Improvement of anti-Pn14PS antibodies level by coadjuvant administration**

The immunogenicity of neoglycoconjugate was increased with adjuvant coadministration [73, 86]. We set out to investigate in a mouse model the effect of adjuvant coadministration i.e. Quil-A, MPL, DDA, CpG and Alum on both the antibody- and cell-mediated immune response against a neoglycoconjugate as reported by Safari et al [87]. In the absence of adjuvant, immunization with neoglycoconjugate leads after a booster merely to IgG1 antibodies against PnP14PS. Coadministration of adjuvant had multiple effects: a diversified anti-Pn14PS IgG antibody response (also other IgG subclasses than IgG1 were evoked), an enhanced avidity and increased opsonic activity of these antibodies [87]. We found that next to Quil-A also DDA as a single dose or in combination with CpG had similar effects on the diversification of eliciting a broader variety of anti-Pn14PS IgG antibody subclasses. Meanwhile, CpG or alum on their own showed in majority IgG1 antibodies after booster immunization in a same pattern as in non adjuvant groups [87]. Compared to other adjuvants, codelivered Quil-A strongly improved the antibody avidity and enhanced the phagocytosis of *S. pneumoniae* type 14 [87].

### **6. Future researches**

In this review, synthetic oligosaccharide-protein conjugates are proven to be effective vaccines in mice model. A logical next step would be a feasibility and immunogenicity study in human volunteers. Before that, a study should be started with synthetic oligosaccharide-protein conjugates for at least the pneumococcal serotypes 1, 4, 5, 9V and 18C and should even have been completed, because the minimal epitopes for these polysaccharides are still unknown.

The Future of Synthetic Carbohydrate Vaccines: Immunological Studies on *Streptococcus pneumoniae* Type 14 627

To improve the immunogenicity of oligosaccharide-protein conjugates co-delivery of adjuvants are required. As an alternative to the addition of adjuvants, studies should be initiated to direct oligosaccharide-protein conjugates to dendritic cells by incorporation of specific ligands. Targeting to and activation of dendritic cells by TLR5 is a possibility to be explored.
