Heterogeneity in immunodominant outer membrane proteins has been
proposed as a significant factor in the failure of an NTHi infection to induce
immune protection against subsequent infections. This study has examined
the vaccine potential of three outer membrane proteins in an attempt to
identify conserved regions that could be targeted by an immune response
after vaccination. The three proteins investigated were: TbpB, P5 and P48
(HI0164). The optimal route of immunisation in clearing a bolus inoculum
of NTHi to the lung in the rat has been shown to be a combination of gut
sensitisation with a respiratory boost and this regime was used in the present
study.
A panel of NTHi isolates was assessed to determine the frequency with
which strains were able to bind transferrin and thus be targeted by a TbpBspecific
immune response. A high proportion of strains was able to bind
transferrin with similar frequencies in isolates associated with infection and
those from normal throat swabs. A protocol was developed to purify
nonlipidated recombinant TbpB from NTHi using a glutathione-Stransferase
(GST)-rTbpB fusion protein and Glutathione-Sepharose affinity
chromatography. Mucosal-directed immunisation with rTbpB significantly
enhanced clearance of an NTHi challenge to the lung, however, whilst
rTbpB-specific antibodies were cross-reactive on Western immunoblots, the
cross-reactivity was variable in both transferrin binding inhibition assays and
bactericidal activity. This suggested that the rTbpB-specific humoral response
would be variable in the recognition of heterologous NTHi isolates.
The secondary structure of P5 has been controversial with several reports
suggesting that P5 was a fimbrin protein composed of coiled coils. In this
present study the interstrain variation in P5 amongst isolates from diverse
anatomical sites, as well as computer prediction methods and
spectrophotometric analysis, generated a model of P5 based on the
homologous E. coli protein, OmpA. This model suggested a B-barrel
conformation with no evidence of coiled coils. Synthetic peptides
corresponding to conserved regions of P5 that were thought to be surface
exposed, as well as a region (H3) with some homology to a protective epitope
in the P. aeruginosa protein, OprF, were then combined with a
"promiscuous" T cell epitope from the measles virus F protein (MVF) and
used for immunisation studies. Whilst variable protection was seen with the
peptides, the MVF/H3 peptide was the most efficacious of the antigens
assessed in this study in enhancing clearance of NTHi. This occurred in the
absence of detectable peptide- or PS-specific antibody leading to the
suggestion that cell mediated responses may have played an important role
in enhancing clearance in this model. The highly conserved nature of the
region in P5 represented by the H3 peptide suggests that further study should
be focused on this peptide as a potential NTHi vaccine candidate.
The last antigen, P48, is homologous to a A. pleuropneumoniae antigen,
AopA, which has been proposed to have potential as a vaccine component
against pleuropneumonia in pigs. Sequence analysis of the gene encoding
P48 from several isolates showed that this protein was well conserved.
Recombinant P48 was purified from a GST-rP48 fusion protein and used for
immunisation, which also conferred significant protection. However,
immunisation with rP48 was not as efficacious as immunisation with the
MVF/H3 peptide. Whilst immunisation with rP48 induced high antibody
titres, no bactericidal activity could be detected indicating that bactericidal
antibody had not contributed to the observed clearance. In addition, the rP48-
specific serum IgG was predominantly of the IgG2a isotype suggesting that
Thl cell mediated responses had been induced by immunisation with rP48.
| Identifer | oai:union.ndltd.org:ADTP/219439 |
| Date | January 1998 |
| Creators | Webb, Dianne, n/a |
| Publisher | University of Canberra. Applied Science |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | ), Copyright Dianne Webb |
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