Pseudomonas aeruginosa (P. aeruginosa) is a frequently isolated pathogen that
causes septicaemia and chronic respiratory infection. It exhibits a higher
mortality rate than other gram-negative bacteria and the need for effective
immunotherapy is emphasised by the frequency of antibiotic resistance
associated with this organism. Mucosal immunisation with a whole killed cell P.
aeruginosa vaccine has previously demonstrated a significant immune response
in both rodent studies and human trials. This study is a continuation of that
research, with the major goal being the identification of a purified protein antigen
that could form the basis of a mucosal vaccine against P. aeruginosa.
Specifically, the aims of this study were the development of purification
protocols for the isolation of previously untested protein antigens, assessment of
the efficacy of these antigens to enhance bacterial clearance in an animal model
of acute respiratory infection, determination of the immune parameters that are
associated with the resolution of P. aeruginosa respiratory infection and finally,
cloning of an identified antigen which demonstrated vaccine efficacy.
Protocols were established to isolate proteins for use as antigens in immune
response studies. The proteins purified in this study were Pa 13, Azurin, acyl
carrier protein (ACP), Amidase, Aminopeptidase, KatA and Pa70. These proteins
were used to immunise rats by intestinal intra-Peyer's patch (IPP) inoculation and
intratracheal (IT) boost. The immunisation protocol employed was designed to
target mucosal antigen-specific immune responses where the route of
immunisation, Peyer's patch (PP) intestinal inoculation, is akin to the oral
delivery of antigens to the gut-associated lymphoid tissue (96).
Investigations of a previously uncharacterised antigen, Pa60, later identified this
protein as the P. aeruginosa catalase, KatA. This study demonstrated enhanced
bacterial clearance of both homologous and heterologous challenge following
immunisation with KatA. The level of clearance demonstrated by KatA was
promising when compared to that of killed whole cell immunisation. KatA was
cloned and studies with the recombinant protein showed enhanced bacterial
clearance commensurate with that of the native protein.
Immunisations with other proteins identified four additional antigens which
enhanced bacterial clearance; Pa13, Pa40, Pa45 and Pa70. Amino acid sequence
analysis indicated that Pa13 may be a novel protein, whereas Pa40 was
determined to be amidase and Pa45, aminopeptidase. Pa70 was not successfully
sequenced. These proteins were effective in significantly enhancing bacterial
clearance of homologous P. aeruginosa challenge. For KatA, Pa13 and Pa70,
clearance was associated with a marked phagocytic cell recruitment. In contrast,
amidase and aminopeptidase demonstrated clearance with a minimal cellular
response. Proteins; azurin and ACP were non-protective, failing to clear a live P
aeruginosa challenge. Analysis of the antigen-specific responses of these nonprotective
proteins and comparison with those antigens which enhanced bacterial
clearance were used to determine factors that may contribute to the resolution of
an acute pulmonary infection.
The study has demonstrated that mucosal immunisation using purified protein
antigens can enhance the clearance of pulmonary infection with P. aeruginosa. It
has also contributed to the understanding of immune responses to newfound
antigens of P. aeruginosa and identified antigen-specific responses which
confirm their potential as vaccine candidates.
| Identifer | oai:union.ndltd.org:ADTP/219386 |
| Date | January 2001 |
| Creators | Thomas, Linda D., n/a |
| Publisher | University of Canberra. Human & Biomedical Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | ), Copyright Linda D. Thomas |
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