One of the major challenges of vaccine development is the conservation of immunogenicity
and protective efficacy through the stages of design, production, formulation and delivery.
The critical issue is that how and in what form an antigen is taken up by antigen presenting
cells for proteolytic processing and presentation to the immune system bound to MHC can
have dramatic effects on the activation of Th cells to drive clonal responses and induction of
immunological memory.
Nontypeable Haemophilus influenzae (NTHi) is a pathogenic commensal of the human
respiratory tract that causes diseases with enormous socioeoconomic burdens. There is no
licensed vaccine, although the potential for vaccination with outer membrane components to
reduce the incidence of disease caused by NTHi has recently been demonstrated in clinical
trials. The issue of immunomodulation was explored in this thesis in the context of the further
evaluation of a leading NTHi vaccine candidate, the outer membrane protein OMP26. The
efficacy of recombinant OMP26 (rOMP26) against NTHi challenge has been previously
demonstrated in mice, rats and chinchillas. In rats, efficacy was shown to be restricted to the
precursor form (containing the signal peptide) and not the mature form of rOMP26. The
immunodulatory effects of changes to the rOMP26 structure were further investigated in this
thesis. A range of structural variants of rOMP26 were constructed in view of reducing
extraneous plasmid-derived sequence from the antigen and to introduce a unique cysteine
residue as a potential conjugate site for multivalent vaccine development (Chapter 2). It was
demonstrated that minor structural changes to rOMP26 such as the addition, deletion,
modification or relative positioning of a single amino acid or bulky group, designed to
increase the efficiency of production or introduce (cysteine) conjugation sites, altered the
expression of the protein in E. coli and the immunogenicity in Balb/C mice. Furthermore, in
contradiction to the published report (El-Adhami et al. 1999) and a new study in rats (Chapter
3), there was no positive effect of the signal peptide in mice, with precursor and mature forms
of rOMP26 equally immunogenic (Chapter 2). Following confirmation of the need to retain
the signal peptide for the immunogenicity of rOMP26 in rats, a precursor form (rOMP26VTAL)
in which the conserved n-region of the signal peptide was deleted, and shown to reduce the
efficiency of the cleavage of the signal peptide by signal peptidase during protein overexpression
in E. coli (Chapter 3). Not only did this deletion result in an increase the yield and
stability of the purified precursor protein, but rOMP26VTAL was highly immunogenic and
enhanced the clearance of NTHi from the lungs of challenged rats. The potential for signal
peptides to be exploited as an immune-enhancing moiety in a proteinaceous vaccine is
discussed.
Following the development of rOMP26VTAL as a production optimised variant of rOMP26, the
next step was to test the feasibility of rOMP26VTAL as a component of a multivalent vaccine
(Chapter 4). Two chimeras were constructed with LB1(f)2,1,3, a trivalent synthetic B-cell
epitope from the extracellular loop 3 region of the P5 fimbrin protein of NTHi, positioned at
the N- or C-terminus of rOMP26VTAL. The solubility of rOMP26VTAL was affected by the
fusion, with both chimera constructs expressed only in the insoluble fraction, thus requiring a
denaturing protocol for purification. Although rLB1(f)2,1,3-OMP26VTAL was expressed and
purified as a more stable protein and in greater yield than rOMP26VTAL-LB1(f)2,1,3, the
relative positioning of the fusion was important and rOMP26VTAL-LB1(f)2,1,3 was significantly
more immunogenic in rats than rLB1(f)2,1,3-OMP26VTAL. In addition, rOMP26VTALLB1(
f)2,1,3, but not rLB1(f)2,1,3-OMP26VTAL induced a significant degree of bacterial clearance
following pulmonary challenge with NTHi, in levels comparable to the highly efficacious
rOMP26VTAL construct.
In the third part of the thesis, bacterial ghosts were evaluated as a novel mucosal delivery
technology for rOMP26VTAL and rOMP26VTAL-LB1(f)2,1,3, (Chapter 5). To mimic the natural
presentation of OMP26 and P5 fimbrin antigens on the cell surface of NTHi, an OmpA�
sandwich fusion surface display system was developed for the outer membrane expression of
the OMP26 constructs in E. coli ghosts. Following gut immunisation, but not intranasal
immunisation even when co-administered with the cholera toxin�derived adjuvant CTA1-DD,
bacterial ghosts were successful at presenting OMP26VTAL and rOMP26VTAL-LB1(f)2,1,3 to the
immune system for the induction of enhanced clearance of NTHi in the rat pulmonary
challenge model. Although this study was the first to demonstrate enhanced bacterial
clearance induced by heterologous antigens expressed in the outer membrane of bacterial
ghosts, future studies with ghosts will require optimisation of the expression levels of the
OmpA� fusion proteins possibly to avoid cross-reactive responses related to high doses of
ghosts in the inoculum.
This thesis presents data that both supports the further evaluation of rOMP26 constructs for
clinical trials, and has demonstrated the significant effects of structural changes, method of
production and delivery system can have on the immunogenicity of a candidate vaccine. Such
knowledge will contribute to and provide some new approaches for enhancing the efficiency
of vaccine development against a range of diseases including those caused by NTHi.
Major Outcomes:
1. Demonstration that the immunogenicity of rOMP26 antigen constructs is affected by
structural modifications and their positioning within the construct, and by the delivery
system.
2. Development of rOMP26VTAL, an rOMP26 construct with the KNIAK sequence
deletion of the signal peptide n-region. This protein retains the immunogenicity and
protective efficacy of rOMP26, but is produced with reduced cleavage of the signal
peptide, resulting in higher yields and a stable protein. Lacks extraneous plasmidderived
multiple cloning site sequence, and is produced in high yield as a stable
protein.
3. Construction of a NTHi rOMP26VTAL-LB1(f)2,1,3 chimera antigen that induced
enhanced clearance of NTHi in an acute pulmonary challenge model in rats.
4. Development of an OmpA� surface display system for the expression of rOMP26
antigen constructs in the outer membrane of E. coli/bacterial ghosts
5. Bacterial ghosts were successful as delivery vehicles for rOMP26 candidate vaccine
constructs when delivered in the gut.
| Identifer | oai:union.ndltd.org:ADTP/219571 |
| Date | January 2007 |
| Creators | McGrath, John Francis, n/a |
| Publisher | University of Canberra. Health Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | ), Copyright John Francis McGrath |
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