Jembrana disease is an acute and severe disease of Bali cattle (Bos javanicus)
endemic in Indonesia that is caused by a bovine lentivirus designated Jembrana
disease virus (JDV). Previous studies have demonstrated that it is possible to
induce a protective immunity against the disease by immunisation with a crude
whole virus vaccine prepared from the tissues of infected cattle. This vaccine has
been demonstrated to ameliorate the clinical signs of disease resulting from
exposure to virus infection but a safer vaccine amenable to commercial
production techniques is required.
JDV, like all lentiviruses, encodes a transcriptional trans-activator Tat protein that
is encoded from one or both of two exons of the tat gene. Tat is particularly
essential for virus replication and it was hypothesised that the induction of an
immune response in cattle against JDV Tat may effect protection against virus
infection. Investigations were therefore conducted on JDV Tat to provide basic
information on the protein that would enable it to be further investigated as a
potential immunogen for incorporation into vaccines for the control of Jembrana
disease.
Analysis of tat transcripts obtained from tissues of cattle infected with three
strains of JDV suggested that, during the acute clinical disease, Tat produced at
this stage of the disease process was translated from the first coding exon only.
Nucleotide variation in this exon, which would have translated into amino acid
variations in the Tat protein, was evident especially between strains from
geographically different regions of Indonesia. There was; however, conservation
of the essential functional domains of cysteine-rich, core and basic regions, which
suggested immunity to a single Tat protein might protect against infection by
heterologous strains. Subsequent studies on Tat reported in the thesis therefore
concentrated on the protein encoded by tat exon 1 of a single strain of JDV. The exon 1 of tat was cloned into the pGEX vector and recombinant Tat
expressed in Escherichia coli. Methods for the purification of the expressed
protein were developed. Immunogenicity of the recombinant protein was initially
demonstrated by inoculation of the protein into a sheep which developed a high
titred specific antibody response. Antibodies induced by this recombinant protein
recognised native Tat proteins produced by three JDV strains in Bali cattle and
provided a valuable reagent for the subsequent detection of Tat in vitro and in
vivo.
Aspects of the antibody response to Tat were determined in cattle that had been
infected naturally or experimentally with JDV, and compared with the levels of
antibody to the immunodominant capsid protein. Tat antibodies were detected in
23 % of 128 Bali cattle from Jembrana disease-endemic areas of Indonesia; in all
these cattle, evidence of previous virus infection had been demonstrated by
detection of antibody to the JDV capsid protein by Western blot analysis. In cattle
experimentally infected with JDV, low levels of serum antibody to Tat were
detected by Western blot in the first month post-infection but the levels of
antibody then decreased; levels of antibody to the JDV capsid protein increased
over the 6-month observation period following infection. The detection of Tatantibody
soon after the acute clinical disease suggested that this protein is
secreted extracellularly during JDV infection in cattle. In contrast to the antibody
response to Tat in JDV-infected cattle, an apparently greater antibody response
to Tat was induced by injection of recombinant Tat in Bali cattle. The strong
antibody response resulting from inoculation of the recombinant Tat and low
levels of Tat antibody in animals that had been naturally or experimentally
infected with virus suggested there might be a conformational difference in the
recombinant and native Tat protein and that the native protein was a poor
immunogen, or that the levels of Tat in infected cattle were too low to induce a
strong antibody response. As an alternative means of inducing an immune response to JDV Tat, perhaps
one associated with a greater cell-mediated rather than an antibody response, a
candidate tat DNA vaccine was produced by insertion of tat exon 1 into a DNA
vaccine vector. Transfection of this naked DNA plasmid into mammalian cells
induced the expression of a functional Tat protein which maintained antigenicity.
The results suggested this construct merits further animal studies attempting to
induce a protective immune response against Jembrana disease in cattle. A
method of assaying the trans-acting function of Tat was also developed which will
have application for quality control procedures for large-scale production of tat
DNA vaccine.
| Identifer | oai:union.ndltd.org:ADTP/221766 |
| Date | January 2006 |
| Creators | ssetiyan@gmail.com, Surachmi Setiyaningsih |
| Publisher | Murdoch University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.murdoch.edu.au/goto/CopyrightNotice, Copyright Surachmi Setiyaningsih |
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