The complex world of polysaccharides edited by Desiree Nedra Karunaratne

 

The Complex World of Polysaccharides 

624 

 

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 CRM

197

 protein and the immunogenicity of those conjugates were studies in a mouse model. 

Mice were immunized with polysaccharide type 14 conjugated to CRM

197

 (CRM

197

-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).  

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The Future of Synthetic Carbohydrate Vaccines: Immunological Studies on Streptococcus pneumoniae Type 14  625 

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 phagocytosis-

promoting 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 

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 (α23)-linked 

sialic acid N-acetylneuraminic acid (Neu5Ac) in their side chains: {→4)-β-D-Glcp-(1→6)-[±α-

Neu5Ac-(23)-β-D-Galp-(14)-]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 (α23)-linked sialic acid, as described previously [83, 84]. 

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 

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The Complex World of Polysaccharides 

626 

conclusion, the inability of polysaccharide to boost primed mice might be due to the 

incapability to induce the cytokines. 

 

Immunization

1

 

IgG titer 

(Log

10

)

2

 

Level of Cytokine IL-5 (pg/ml) 

In serum

3

After stimulation

4

 

GC-GC 

2.18±0.22 1022.3±275.2 

571.2±20.0 

GC-PS  0.34±0.47 0.3±0.5 

66.1±0.4 

GC-GC-GC 3.02±0.17 2700.4±112.3 

1172.8±7.1 

GC-PS-PS 0.0  0.0 

664.9±221. 

Saline  0.0 6.9±1.1 

0.0 

1

Five mice per group were immunized with a CRM-neoglycoconjugate (GC), a synthetic branched tetrasaccharide of 

Pn14PS that is conjugated to a CRM

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 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. 

2

ELISA 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 

4

Splenocytes 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. 

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