Immunogical Study On Synthetic Oligosaccharide-Based Conjugate Vaccines Against Haemophilus Influenzae Type B And Preparation Of Related Monoclonal Antibodies

Haemophilus influenzae (Hi) is an important human pathogenic bacterium, which can cause secondary infections in infants when primary respiratory infections happen. H. influenzae has been divided into capsular and non-capsular bacteria according to its presence of capsule or not. Local infections are usually caused by non-capsular bacterium, whereas severe infections in infants are often caused by the capsular one. The capsular bacterium are divided into a, b, c, d, e, f, and non-sub types according to the structure and antigenicity of the capsular polysaccharides. The strongest pathogen is type b (Hib), which can cause severe meningitis, pneumonia, epiglottitis, pericarditis, otitis media, and so on. The main population infected by Hib is infants under 5 years old, especially those under 2 years old. Antibiotics are commonly used to treat Hib infections, but the recent emergency and rapid increase in drug-resistance, especially multi-drug resistance, have become a serious problem. Consequently, vaccines as an alternative and largely effective strategy to control bacterial infections, including Hib infections, have attracted significant attentions in recent years. In addition, to prevent the invasive infections caused by Hib, early detection and timely treatment are also of great importance. As a result, effective methods for the detection of the bacterium at the beginning of infection are also in urgent need.In recent decades, the application of carbohydrate-based antibacterial vaccines, including vaccines against Hib, has made great contributions to the control of bacterial infections. However, most glycoconjugate vaccines currently in clinical usage have inherent problems because they are composed of natural polysaccharides derived from bacteria that are heterogeneous and easily contaminated and thereby have quality and safety concerns. To deal with the issue, a new type of vaccines composed of synthetic carbohydrate antigens that have well-defined structure and purity has emerged. Based on this concept, a semi-synthetic Hib vaccine was successfully developed by Cuban scientists and marketed, demonstrating the potential of the strategy.Our research project aims at the development of Hib vaccines based on the new strategy and methods for the early diagnosis of Hib infection. In this regard, our group has recently synthesized a number of oligomers of the disaccharide repeating units, including a hepta-, a nona-and an undecasaccharides respectively, of the Hib capsular polysaccharide, that is, polyribosylribitol phosphate, and coupled them with a carrier protein, namely, keyhole limpet hemocyanin (KLH), to form glycoconjugates that are investigated as potential Hib vaccines. This thesis is focused on the immunological evaluations of these Heptasaccharide-, Nonasaccharide-, and Undecasaccharide-KLH conjugates, as well as the development of monoclonal antibodies for Hib detection by using the synthetic conjugates.Immunological studies of the above glycoconjugates were conducted in mouse, which revealed that nona-KLH elicited higher titers of protective IgGl antibodies and robust T cell-mediated immune responses than the other two conjugates, suggesting that longer oligosaccharides were not necessarily better antigens or immunogens. The induced antisera were also shown to recognize and combine to Hib 10211 bacteria.A monoclonal antibody of IgM subtype, which was specific to polyribosylribitol phosphate, the Hib capsular polysaccharide, was prepared from mouse inoculated with the Nonasaccharide-KLH conjugate. This monoclonal antibody showed not only strong and specific binding to Hib bacteria but also obvious complement-dependent bactericidal activity to Hib in vitro.In summary, in this thesis, the immunological properties of the KLH conjugates of three oligomers of the repeating unit of Hib capsular polyribosylribitol phosphate were evaluated to reveal their detailed structure-activity relationship. A nonasaccharide was identified as a promising antigen for the development of functional Hib vaccines. In the meantime, an IgM monoclonal antibody was obtained from mouse inoculated with the semi-synthetic glycoconjugate, which could be useful for early detection or diagnosis of Hib bacteria and detections.