| Popis: |
High mortality rates of bacterial pneumonia and increased antibiotic resistance are major reasons to develop novel vaccine strategies against Streptococcus pneumoniae. S. pneumoniae serotype 3 (ST3) is one of the most frequent serotypes isolated from patients with invasive pneumococcal diseases, even though it is included in the routine immunization schedule. To improve the immunogenic properties of ST3, synthetic antigenic tetrasaccharide based on capsular polysaccharide repeating units have been conjugated to a carrier protein and used as a vaccine candidate. The study illustrates the principle of proper optimization of variable aspects of vaccine formulation, such as dosage, adjuvant, and carrier protein. Use of highly pure and well-characterized synthetic oligosaccharide allowed to significantly decreased the dosage of antigen while maintaining sufficient protection shown by in-vitro opsonophagocytic killing assay. In defiance of the general notion “the more, the better”, the higher dose did not improve the protective effect of immunization but could even diminish the final success of the vaccination. In order to improve the immunogenicity of semi-synthetic ST3-glycoconjugates, several commercially available adjuvants were used and incorporated into biodegradable poly-lactic-acid (PLA) microparticles. Screening experiments in mice yielded promising results for agonists of TLR7/8, namely resimiquimod (R848) and bacterial RNA, as well as MPLA, a TRIF-biased TLR4 agonist used in several commercial vaccines. The carrier protein derived from S.pneumoanie can serve as “double-action bullet”, being both a carrier essential for glycan presentation and an additional vaccine antigen providing broader protection, so-called "additional valency". Hence, ST3-tetrasaccharide was conjugated to pneumolysin and PspA protein and vaccine were evaluated in-vivo in the mouse and swine model. The mouse study showed that ST3-tetrasaccharide pneumolysin conjugates decrease the bacteria load in blood and lungs as well as reduce the disease severity in mice challenged with S.pneumoniae serotype 3. Additionally, the synthetic oligosaccharide conjugated to pneumolysin and PspA inhibited colonization of the nasopharynx after infection with bacteria. The immunization of piglets provides the first evidence for the immunogenicity of the synthetic glycoconjugate vaccine in a swine model. The generated antibodies were able to kill pneumococci and neutralize the toxic effect of pneumolysin in-vitro. However, the protective activity of the glycoconjugate vaccines in the swine in-vivo infection model has to be further investigated. The study presented in the thesis combined a series of innovations, which enhance the efficacy and applicability of glycoconjugate vaccines and help to further clarify the principles of anti-carbohydrate- and anti-bacterial immunity. |
