Development of new synthetic oligosaccharide vaccines : the immunogenicity of oligosaccharide-CRM197 neoconjugates and oligosaccharide/peptide hybrid gold nanoparticles based on the capsular polysaccharide structure of Streptococcus pneumoniae type 14

Abstract

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and sepsis in children worldwide. Nowadays, the antibiotic resistance of S. pneumoniae bacteria has increased worldwide. This makes treatment of S. pneumoniae 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. Currently, two vaccine types are commercially available: a 23-valent pneumococcal polysaccharide vaccine (PPV23) and 7-valent pneumococcal conjugate vaccine (PCV7). The PPV23 vaccine was shown to be moderately effective in young adults but not in young children and elderly and also not in immunocompromised patients. Large scale introduction of PCV7 has resulted in an overall decline in invasive pneumococcal disease (IPD). However IPD caused by the non-vaccine serotypes has increased (replacement disease), highlighting the need for inclusion of additional serotypes in improved vaccine formulations to come. In the search of new candidate pneumococcal conjugate vaccines investigations have been made by many researchers on issues such as covering more serotypes, reducing the effect of serotype replacement and making vaccines for a reasonable price and/or more reliable storing properties for use in the third world. Our group has been working on a conjugate vaccine based on a synthetic carbohydrate (neoglycoconjugate) against S. pneumoniae bacteria. In this thesis, we focus on the immunogenicity of a series of synthetic overlapping oligosaccharide fragments corresponding to S. pneumoniae type 14 polysaccharide (Pn14PS) which were conjugated to CRM197 protein and evaluated in a mouse model. We demonstrated that the branched trisaccharide element Glc-(Gal-)GlcNAc is essential in inducing Pn14PS-specific antibodies and that the neighboring galactose unit at the non-reducing end contributes clearly to the immunogenicity of this epitope. The antibodies in sera obtained from mice immunized with neoglycoconjugates of Pn14PS did not cross-react with other S. pneumoniae polysaccharide serotypes and displayed very low binding activity with group B streptococcus type III polysaccharide. We showed that a neoglycoconjugate booster vaccination is required for the activation of memory cells and the establishment of sustained immunity. A booster with native polysaccharide is ineffective to evoke functional antibodies. We observed that codelivery of Quil-A alone or in combination with MPL had the most dramatic effect on antibody- and cell-mediated immune response to neoglycoconjugate of Pn14PS. We concluded that the branched tetrasaccharide Gal-Glc-(Gal-)GlcNAc is a serious candidate for a synthetic oligosaccharide conjugate vaccine against infections caused by S. pneumoniae type 14. The above mentioned synthetic branched tetrasaccharide was used for the synthesis of hybrid gold nanoparticles [GNPs]. In this thesis, we prepared a series of GNPs with different molar ratios of tetrasaccharide, inert D-glucose, and OVA323-339-peptide in a one-step reaction. Their immunogenicity was evaluated in the mouse model. We found that the presence of the T-cell stimulating peptide OVA323-339 in the hybrid gold nanoparticles was a prerequisite for the induction of specific anti-Pn14PS IgG antibodies. We showed that well designed hybrid gold nanoparticles display a promising starting point towards the development of synthetic oligosaccharide-based vaccines.