Immunological and structural characterisation of the nontypeable Haemophilus influenzae vaccine protein, OMP26

  • Duangkamol Kunthalert

    Student thesis: Doctoral Thesis


    Nontypeable Haemophilus influenzas (NTHi) is recognised as a significant human pathogen causing mild to severe respiratory tract infections. At present, no vaccine is available for prevention of infection caused by this pathogen. Several outer membrane proteins (OMPs) of NTHi and its lipooligosaccharide have been investigated as possible vaccine antigens against NTHi infections. Previous investigations in our laboratory have shown that OMP26 from an NTHi 289 strain was able to significantly enhance pulmonary clearance of NTHi in a rat model in which animals were immunised via intestinal Peyer's patches and then boosted intratracheally (Kyd and Cripps,1998; El- Adhami et al.,1999). In recent studies, the OMP26,when used as a parenteral immunogen, was also highly effective at inducing immune responses that led to significantly enhanced clearance of the chinchilla nasopharynx (Kyd et al.,2003). These studies indicate significant potential of the OMP26 as a candidate vaccine antigen and warrant further investigations for development of a vaccine against NTHi. This thesis focused on the immunological and structural characterisation of the NTHi vaccine candidate,OMP26. Peptides of OMP26 were used as tools to localise the immunologically important regions of the OMP26. Two different E. coli expression systems, the GST gene fusion and the 6xHis tagged systems, were employed to construct the OMP26 peptides. It was found in this study that, despite efforts to optimise the system, the GST-fusion protein system failed to produce consistent results for the purification and storage of the OMP26 peptides. In contrast, the 6xHis tagged system exhibited more reliable outcomes in the production of the recombinant OMP26 peptides and the stability of the stored purified peptides. As such, the purified OMP26 peptides from the 6xHis tagged system were chosen to map major regions of immunological significance for the OMP26 protein. The regions of the OMP26 which are involved in the induction of the acquired immune responses have been identified in the present study. Based on the antigen specific lymphocyte proliferation assay, the dominant T cell epitopes for OMP26 were located between amino acid residues 95 and 197 (T3+T4 region). These identified T cell epitopes exhibited the capability of efficient T cell activation, suggesting that the epitopes within the T3+T4 region potentially had the highest affinity for binding to the MHC molecules than did any other OMP26 region. Using two different assay systems, ELISA and BIA,the predominant B cell epitopes of OMP26 were located between amino acid residues 45 and 145 (T2+T3 region). This region was also found to be immunodominant across all animal species tested, and with all immunisation regimens used. Flow cytometry analysis also revealed that these particular epitopes were expressed on the surface of NTHi cells. By integration of the data obtained from these current experimental studies and the computational analysis of the OMP26 sequence, two hypothetical models of the OMP26 were also proposed in this study. The significant outcomes obtained in this thesis provide a better understanding of the specificity of the host immune responses to the OMP26 protein These findings provide great benefit not only for the development of a future NTHi vaccine but for the development of the peptide-based immunodiagnostic reagents as well. These diagnostic reagents will be valuable, in particular, for the evaluation of efficacy of an NTHi vaccine in humans that may include OMP26 or specific conformational structures. Future studies are still required to further define the minimum epitope length required for the B and T cell responses identified in this study. The significance of these responses in immune protection against NTHi infection also requires further investigations. Human immune responses also need to be determined, but this can only be achieved following clinical trial studies.
    Date of Award2004
    Original languageEnglish
    SupervisorJennelle Kyd (Supervisor) & Allan W. Cripps (Supervisor)

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