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 focussed 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.
| Identifer | oai:union.ndltd.org:ADTP/218708 |
| Date | January 2004 |
| Creators | Kunthalert, Duangkamol, n/a |
| Publisher | University of Canberra. Health Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | ), Copyright Duangkamol Kunthalert |
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