The Complex World of Polysaccharides
622
all, pneumococcal serotypes (Fig. 1). Protection induced by the proteins should be serotype-
independent 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 8
th
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 O-
antigen 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
The Future of Synthetic Carbohydrate Vaccines: Immunological Studies on Streptococcus pneumoniae Type 14 623
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 cross-
reactive material of diphteria toxin (CRM
197
) protein [60] and most recently overlapping
oligosaccharide varying from tri- to dodecasaccharides related to polysaccharide type 14
conjugated to CRM
197
protein [73, 74].
5. Immunogenicity of synthetic oligosaccharide based vaccines
This review focuses on the S. pneumoniae type 14 capsular polysaccharide (Pn14PS) which
consists of biosynthetic repeating units of the tetrasaccharide {6)-[-D-Galp-(1→4)-]-D-
GlcpNAc-(13)--D-Galp-(1→4)--D-Glcp-(1→}n [75] (Fig. 3).
Figure 3.
A branched tetrasaccharide repeating unit of S. pneumoniae type 14 capsular polysaccharide
(A) and its nomenclature symbol (B): filled circle = glucose (Glc); open circle = galactose (Gal), and filled
square = N-acetylglucosamine (GlcNAc)
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 (CRM
197
), 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 CRM
197
. 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 CRM
197
-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].