http://espace.library.uq.edu.au/ The University of Queensland en Fez http://blogs.law.harvard.edu/tech/rss http://espace.library.uq.edu.au/view/UQ:76519 Objectives: To describe the prevalence and characteristics of isolates of Neisseria gonorrhoeae grown from urine samples that produced negative results with nucleic acid amplification assays (NAA) targeting the cppB gene. Methods: An initial cluster of culture positive, but cppB gene based NAA negative, gonococcal infections was recognised. Urine samples and suspensions of gonococci isolated over 9 months in the Northern Territory of Australia were examined using cppB gene based and other non-cppB gene based NAA. The gonococcal isolates were phenotyped by determining the auxotype/ serovar (A/S) class and genotyped by pulsed field gel electrophoresis (PFGE). Results: 14 (9.8%) of 143 gonococci isolated were of A/S class Pro(-)/Brpyut, indistinguishable on PFGE and negative in cppB gene based, but not other, NAA. Conclusions: This cluster represents a temporal and geographic expansion of a gonococcal subtype lacking the cppB gene with consequent loss of sensitivity of NAA dependent on amplification of this target. Gonococci lacking the cppB gene have in the past been more commonly associated with the PAU(-)/PCU- auxotype, a gonococcal subtype hitherto infrequently encountered in Australia. NAA based on the cppB gene as a target may produce false positive as well as false negative NAA. This suggests that unless there is continuing comparison with culture to show their utility, cppB gene based NAA should be regarded as suboptimal for use either as a diagnostic or supplemental assay for diagnosis of gonorrhoea, and NAA with alternative amplification targets should be substituted. 2007-08-15T06:31:17Z Lum, G. og Freeman, K. og Nguyen, N. L. og Limnios, E. A. og Tabrizi, S. M. og Carter, I. og Chambers, I. W. og Sloots, T. P. og Whiley, D. M. og Garland, S. M. og Tapsall, J. http://espace.library.uq.edu.au/view/UQ:123798 The mechanism by which respiratory syncytial virus (RSV) suppresses T-cell proliferation to itself and other antigens is poorly understood. We used monocyte-derived dendritic cells (MDDC) and CD4 T cells and measured [H-3]thymidine incorporation to determine the factors responsible for RSV-induced T-cell suppression. These two cell types were sufficient for RSV-induced suppression of T-cell proliferation in response to cytomegalovirus or Staphylococcus enterotoxin B. Suppressive activity was transferable with supernatants from RSV-infected MDDC and was not due to transfer of live virus or RSV F (fusion) protein. Supernatants from RSV-infected MDDC, but not MDDC exposed to UV-killed RSV or mock conditions, contained alpha interferon (IFN-alpha; median, 43 pg/ml) and IFN-lambda (approximately 1 to 20 ng/ml). Neutralization of IFN-alpha with monoclonal antibody (MAb) against one of its receptor chains, IFNAR2, or of IFN-lambda with MAb against either of its receptor chains, IFN-lambda R1 (interleukin 28R [IL-28R]) or IL-10R2, had a modest effect. In contrast, blocking the two receptors together markedly reduced or completely blocked the RSV-induced suppression of CD4 T-cell proliferation. Defining the mechanism of RSV-induced suppression may guide vaccine design and provide insight into previously uncharacterized human T-cell responses and activities of interferons. 2008-01-25T16:50:53Z Chi, B og Dickensheets, HL og Spann, KM og Alston, MA og Luongo, C og Dumoutier, L og Huang, JY og Renauld, JC og Kotenko, SV og Roederer, M og Beeler, JA og Donnelly, RP og Collins, PL og Rabin, RL http://espace.library.uq.edu.au/view/UQ:64943 A self-modulating mechanism by the hepatitis C virus (HCV) core protein has been suggested to influence the level of HCV replication, but current data on this subject are contradictory. We examined the effect of wild-type and mutated core protein on HCV IRES- and cap-dependent translation. The wild-type core protein was shown to inhibit both IRES- and cap-dependent translation in an in vitro system. This effect was duplicated in a dose-dependent manner with a synthetic peptide representing amino acids 1-20 of the HCV core protein. This peptide was able to bind to the HCV IRES as shown by a mobility shift assay. In contrast, a peptide derived from the hepatitis B virus (HBV) core protein that contained a similar proportion of basic residues was unable to inhibit translation or bind the HCV IRES. A recombinant vaccinia-HCV core virus was used to examine the effect of the HCV core protein on HCV IRES-dependent translation in cells and this was compared with the effects of an HBV core-recombinant vaccinia virus. In CV-1 and HuH7 cells, the HCV core protein inhibited translation directed by the IRES elements of HCV, encephalomyocarditis virus and classical swine fever virus as well as cap-dependent translation, whereas in HepG2 cells, only HCV IRES-dependent translation was affected. Thus, the ability of the HCV core protein to selectively inhibit HCV IRES-dependent translation is cell-specific. N-terminal truncated (aa 1-20) HCV core protein that was expressed from a novel recombinant vaccinia virus in cells abrogated the inhibitory phenotype of the core protein in vivo, consistent with the above in vitro data. 2007-08-14T19:30:42Z Li, Dongsheng og Takyar, Seyed Taghi og Lott, William B. og Gowans, Eric J. http://espace.library.uq.edu.au/view/UQ:123904 Equine herpesvinus 1 (EHV-1) causes respiratory and neurological disease and abortion in horses. Animals with high frequencies of cytotoxic T lymphocytes (CTL) show reduced severity of respiratory disease and frequency of abortion, probably by CTL-mediated control of cell-associated viraemia. This study aimed to identify CTL epitopes restricted by selected major histocompatibility complex (MHC) class I alleles expressed in the equine leukocyte antigen (ELA) A3 haplotype. Effector CTL were induced from EHV-1-primed ponies and thoroughbreds with characterized MHC class I haplotypes and screened against P815 target cells transfected with selected EHV-1 genes and MHC class I genes. Targets that expressed EHV-1 gene 64 and the MHC B2 gene were lysed by effector CTL in a genetically restricted manner. There was no T-cell recognition of targets expressing either the MHC B2 gene and EHV-1 genes 2, 12, 14, 16, 35, 63 or 69, or the MHC C1 gene and EHV-1 genes 12, 14, 16 or 64. A vaccinia virus vector encoding gene 64 (NYVAC-64) was also investigated. Using lymphocytes from ELA-A3 horses, the recombinant NYVAC-64 virus induced effector CTL that lysed EHV-1-infected target cells; the recombinant virus also supplied a functional peptide that was expressed by target cells and recognized in an MHC-restricted fashion by CTL induced with EHV-1. This construct may therefore be used to determine the antigenicity of EHV-1 gene 64 for other MHC haplotypes. These techniques are broadly applicable to the identification of additional CTL target proteins and their presenting MHC alleles, not only for EHV-1, but for other equine viruses. 2008-01-25T16:55:43Z Kydd, J. H. og Davis-Poynter, N. J. og Birch, J. og Hannant, D. og Minke, J. og Audonnet, J. C. og Antczak, D. F. og Ellis, S. A. http://espace.library.uq.edu.au/view/UQ:123756 Equid herpesvirus 1 (EHV-1) can cause a wide spectrum of diseases ranging from inapparent respiratory infection to the induction of abortion and, in extreme cases, neurological disease resulting in paralysis and ultimately death. It has been suggested that distinct strains of ERV-1 that differ in pathogenic capacity circulate in the field. In order to investigate this hypothesis, it was necessary to identify genetic markers that allow subgroups of related strains to be identified. We have determined all of the genetic differences between a neuropathogenic strain (Ab4) and a nonneuropathogenic strain (V592) of ERV-1 and developed PCR/sequencing procedures enabling differentiation of ERV-1 strains circulating in the field. The results indicate the occurrence of several major genetic subgroups of ERV-1 among isolates recovered from outbreaks over the course of 30 years, consistent with the proposal that distinct strains of EHV-1 circulate in the field. Moreover, there is evidence that certain strain groups are geographically restricted, being recovered predominantly from outbreaks occurring in either North America or Europe. Significantly, variation of a single amino acid of the DNA polymerase is strongly associated with neurological versus nonneurological disease outbreaks. Strikingly, this variant amino acid occurs at a highly conserved position for herpesvirus DNA polymerases, suggesting an important functional role. 2008-01-25T16:49:08Z Nugent, J. og Birch-Machin, I. og Smith, K. C. og Mumford, J. A. og Swann, Z. og Newton, J. R. og Bowden, R. J. og Allen, G. P. og Davis-Poynter, N. http://espace.library.uq.edu.au/view/UQ:173505 Samples from horses experimentally infected with the “large plaque variant (LP3A+)” of equine arteritis virus were analysed. These included 182 nasal swabs collected from day 1 to 14 post-infection (p.i.), and 21 virus isolates obtained from white blood cells of animals that showed a prolonged viraemia between days 30 to 72 p.i. In order to determine the genetic stability of the virus and particularly to characterise the genetic variants found during the prolonged viraemia, partial sequences of open reading frame 5 (ORF5) encoding glycoprotein 5 (GP5) were generated. Viruses with amino acid substitutions in GP5 were used for further amplification and sequencing of a fragment encompassing ORFs 2b, 3, and 4. The ORF5 nucleotide sequences of the virus present in 65 out of 66 nasal swabs were identical to that of the inoculated virus, suggesting that the ORF5 gene of LP3A+ was genetically stable during the first 2 weeks p.i. Contrary to this, a number of mutations were found in the ORF5 of virus isolates obtained from day 30 p.i. The mutations mainly clustered in antigenic neutralization site C within variable region 1 of the GP5 ectodomain. Sequence variability was also identified in ORFs 2b, 3 and 4, with ORF 4 having the highest proportion of non-synonymous changes (4/6). 2009-04-02T14:25:04Z Liu, Lihong og Castillo-Olivares, Javier og Davis-Poynter, Nick J. og Baule, Claudia og Xia, Hongyan og Belak, Sándor http://espace.library.uq.edu.au/view/UQ:160029 The contributions of a set of herpes simplex virus type 1 membrane proteins towards the process of cell-cell fusion were examined with a series of deletion mutants into which a syncytial mutation had been introduced at codon 855 of the glycoprotein B (gB) gene. Analysis of the fusion phenotypes of these recombinant viruses in Vero cells revealed that while gC, gG, US5, and UL43 are dispensable for syncytium formation at both high and low multiplicities of infection, gD, gHgL, gE, gI, and gM were all required for the fusion of cellular membranes. These data confirm that the requirements for virion entry and cell-cell fusion are not identical. gD and gHgL, like gB, are essential for both processes. gG, gI, and gM, on the other hand, are dispensable for virus penetration, yet play a role in cell-to-cell spread by the direct contact route, at least on an SC16 gBANG background. 2009-01-06T18:05:48Z Davis-Poynter, N. og Bell, S. og Minson, T. og Browne, H. http://espace.library.uq.edu.au/view/UQ:182023 2009-09-03T09:05:57Z Sharp, E. L. og Davis-Poynter, N. J. og Farrell, H. E. http://espace.library.uq.edu.au/view/UQ:160653 The thioesterase activity of palmitoyl protein thioesterase (PPT1) and six commercial lipases was measured against the synthetic substrates, S-palmitoyl-N-acetylcysteamine (Ac-Cym-Pal) and S-(18-methyleicosanoyl)-N-acetylcysteamine (Ac-Cym-18-MEA). PPT1 showed good activity against Ac-Cym-Pal but relatively low activity against the longer chain substrate, Ac-Cym-18-MEA. The highest activity was given by Lipolase 100L type EX (Novozyme) and Lipoprotein Lipase (Sigma) with greater than 90% hydrolysis of Ac-Cym-18-MEA within 10 min at pH 7.4. Other lipases to show high levels of thioesterase activity include Lipex 100L (Novozyme), Lipomod 34P (Biocatalysts) and Lipozyme CALB L (Novozyme). Chemical analysis of wool fibre and fabric treated with the above enzymes under optimal conditions showed that there was no hydrolysis of 18-MEA or other covalently bound fatty acids from the fibre surface. No change in the wettability of the fabric surface was observed following enzyme treatments. Scanning electron micrographs of the fabric treated with the most active enzyme, Lipolase 100L type EX, revealed that the surface of the fibres appeared to have a coating that was not removed by extensive extraction. Reasons for the inability of PPT1 and the other esterases to hydrolyse 18-MEA from the wool fibre surface are discussed. 2009-01-14T10:27:16Z Hutchinson, Sally og Evans, David og Corino,Gary og Kattenbelt, Jacqueline http://espace.library.uq.edu.au/view/UQ:72891 The Roche Cobas Amplicor system is widely used for the detection of Neisseria gonorrhoeae but is known to cross react with some commensal Neisseria spp. Therefore, a confirmatory test is required. The most common target for confirmatory tests is the cppB gene of N. gonorrhoeae. However, the cppB gene is also present in other Neisseria spp. and is absent in some N. gonorrhoeae isolates. As a result, laboratories targeting this gene run the risk of obtaining both false-positive and false-negative results. In the study presented here, a newly developed N. gonorrhoeae LightCycler assay (NGpapLC) targeting the N. gonorrhoeae porA pseudogene was tested. The NGpapLC assay was used to test 282 clinical samples, and the results were compared to those obtained using a testing algorithm combining the Cobas Amplicor System (Roche Diagnostics, Sydney, Australia) and an in-house LightCycler assay targeting the cppB gene (cppB-LC). In addition, the specificity of the NGpapLC assay was investigated by testing a broad panel of bacteria including isolates of several Neisseria spp. The NGpapLC assay proved to have comparable clinical sensitivity to the cppB-LC assay. In addition; testing of the bacterial panel showed the NGpapLC assay to be highly specific for N. gonorrhoeae DNA. The results of this study show the NGpapLC assay is a suitable alternative to the cppB-LC assay for confirmation of N. gonorrhoeae-positive results obtained with Cobas Amplicor. 2007-08-15T04:22:21Z Whiley, D. M. og Buda, P. J. og Bayliss, J. og Cover, L. og Bates, J. og Sloots, T. P. http://espace.library.uq.edu.au/view/UQ:131504 Background: Recently, Allander and co-workers reported the discovery of a new human polyomavirus, KI virus, in respiratory secretions from patients with acute respiratory tract infection (ARTI). Objective. We examined 951 respiratory samples collected in Oueensland, Australia, between November 2002 and August 2003 from patients with respiratory infection, for the presence of the KI virus. Results: Twenty-four (2.5%) samples were positive for KI virus with 20 (83%) of these from children younger than 5 years. In six (25%) patients K! was co-detected with another virus. Full genome sequencing of three isolates 3hcws a high degree of conservation between the Queensland isolates and the original isolates reported from Swedish patients. Conclusions: The newly described KI polyomavirus may commonly be found in the respiratory tract of patients with ARTl, particularly children, and results indicate that the virus has global presence. 2008-03-03T14:19:27Z Bialasiewicz, S. og Whiley, D. M. og Lambert, S. B. og Wang, D. og Nissen, M. D. og Sloots, T. P. http://espace.library.uq.edu.au/view/UQ:122812 In November of 1997 an outbreak of highly pathogenic-avian influenza occurred near the town of Tamworth, in northern New South Wales, Australia. The viruses isolated from chickens on two commercial chicken farms were identified as H7N4 viruses, with hemagglutinin cleavage site amino acid sequences of RKRKRG and intravenous pathogenicity indices of 2.52 and 2.90, respectively. A virus with an identical nucleotide sequence, but with an intravenous pathogenicity index of 1.30, was also isolated from cloacal swabs collected from asymptomatic emus kept on a third property. 2008-01-25T17:01:16Z Selleck, PW og Arzey, G og Kirkland, PD og Reece, RL og Gould, AR og Daniels, PW og Westbury, HA http://espace.library.uq.edu.au/view/UQ:132509 Immunogemcity and safety of a novel combined Haemophilus influenae type b-Neisseria meningitidis serogroups C and Y-tetanus-toxoid conjugate vaccine (Hib-MenCY-TT) candidate was evaluated when co-administered with DTPa-HBV-IPV(Pediarix™3) + PCV7(FrevnarTM~) at 2--4-6 months of age. Anti-PRP concenrrations 1.0 p.g/mL were observed in 92.9-98.7%, rSBA-MenC/Y titres ::: 1:8 in >98%, rSBAMenCIY titres :: 1: 128 in >95.8 and >89.9% subjects. PRP and MenC responses were similar to respective controls (ActHIB™5and Menjugate TM6) including for antibody persistence. Response to co-administered vaccines was not impaired. Polysaccharide challenge (PRP, PSC, PSY at 11-14 months of age) evidenced immune memory was induced for Hib, MenC/Y conjugate components. The safety profile of Hib-MenCY-TI was similar to controls. Hib-MenCY-TI administered according to the current US Hib vaccine schedule has the potential to induce protective antibodies against Hib and meningococcal-CY disease in infants and toddlers 2008-03-17T16:40:41Z Nolan, T. og Lambert, S. B. og Roberton, D. og Marshall, H. og Richmond, P. og Stretton, C. og Poolman, J. og Boutriau, D. http://espace.library.uq.edu.au/view/UQ:160142 A glycoprotein encoded by the UL1 gene of herpes simplex virus type 1 (HSV-1) was detected in infected cells with antipeptide sera. The UL1 gene has previously been implicated in virus-induced cell fusion (S. Little and P. A. Schaffer, Virology 112:686-697, 1981). Two protein species, a 30-kDa precursor form and a 40-kDa mature form of the glycoprotein, both of which were modified with N-linked oligosaccharides, were observed. This novel glycoprotein is the 10th HSV-1 glycoprotein to be described and was named glycoprotein L (gL). A complex was formed between gL and gH, a glycoprotein known to be essential for entry of HSV-1 into cells and for virus-induced cell fusion. Previously, it had been reported that gH expressed in the absence of other viral proteins was antigenically abnormal, not processed, and not expressed at the cell surface (U.A. Gompels and A. C. Minson, J. Gen. Virol. 63:4744-4755, 1989; A. J. Forrester, V. Sullivan, A. Simmons, B. A. Blacklaws, G. L. Smith, A. A. Nash, and A. C. Minson, J. Gen. Virol. 72:369-375, 1991). However, gH coexpressed with gL by using vaccinia virus recombinants was antigenically normal, processed normally, and transported to the cell surface. Similarly, gL was dependent on gH for proper posttranslational processing and cell surface expression. These results suggest that it is a hetero-oligomer of gH and gL which is incorporated into virions and transported to the cell surface and which acts during entry of virus into cells 2009-01-08T13:30:31Z Hutchinson, L. og Browne, H. og Wargent, V. og Davis-Poynter, N. og Primorac, S. og Goldsmith, K. og Minson , A. C. og Johnson , D. C. http://espace.library.uq.edu.au/view/UQ:165104 In this study, we determined the function of a novel non-ribosomal peptide synthetase (NRPS) system carried by a streptococcal integrative conjugative element (ICE), ICESe2. The NRPS shares similarity with the yersiniabactin system found in the highpathogenicity island of Yersinia sp. and is the first of its kind to be identified in streptococci. We named the NRPS product 'equibactin' and genes of this locus eqbA-N. ICESe2, although absolutely conserved in Streptococcus equi, the causative agent of equine strangles, was absent from all strains of the closely related opportunistic pathogen Streptococcuszooepidemicus. Binding of EqbA, a DtxR-like regulator, to the eqbB promoter was increased in the presence of cations. Deletion of eqbA resulted in a small-colony phenotype. Further deletion of the irp2 homologue eqbE, or the genes eqbH, eqbl and eqbJ encoding a putative ABC transporter, or addition of the iron chelator nitrilotriacetate, reversed this phenotype, implicating iron toxicity. Quantification of 55Fe accumulation and sensitivity to streptonigrin suggested that equibactin is secreted by S. equi and that the eqbH, eqbl and eqbJ genes are required for its associated iron import. In agreement with a structure-based model of equibactin synthesis, supplementation of chemically defined media with salicylate was required for equibactin production. 2009-02-25T16:59:51Z Heather, Z. og Holden, M. T. G. og Steward, K. F. og Parkhill, J. og Song, L. og Challis, G. L. og Robinson, C. og Davis-Poynter, N. og Waller, A. S. http://espace.library.uq.edu.au/view/UQ:59900 The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents. 2007-08-14T16:08:20Z Netter, HJ og MacNaughton, TB og Woo, WP og Tindle, R og Gowans, EJ http://espace.library.uq.edu.au/view/UQ:76524 A 5'-nuclease real-time reverse transcriptase-polymerase chain reaction assay was developed for the detection of influenza type A and was validated using a range of influenza A subtypes, including avian strains, and 126 nasopharyngeal aspirate samples. The results show the assay is suitable for screening for influenza A infections, particularly in regions where avian strains may be circulating. (c) 2005 Elsevier Inc. All rights reserved. 2007-08-15T06:31:40Z Whiley, David M. og Sloots, Theo P. http://espace.library.uq.edu.au/view/UQ:132538 Infection with equid herpesvirus type 1 (EHV-l) leads to respiratory disease, abortion, and neurologic disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism resulting in an amino acid variation of the EHV-l DNA polymerase (N752/0752) is significantly associated with the neuropathogenic potential of naturally occurring strains. To test the hypothesis that this single amino acid exchange by itself influences neuropathogenicity, we generated recombinant viruses with differing polymerase sequences. Here we show that the N752 mutant virus caused no neurologic signs in the natural host, while the 0752 virus was able to cause inflammation of the central nervous system and ataxia. Neurologic disease induced by the 0752 virus was concomitant with significantly increased levels of viremia (p = 0.01), but the magnitude of virus shedding from the nasal mucosa was similar between the N752 and 0752 viruses. Both viruses replicated with similar kinetics in fibroblasts and epithelial cells, but exhibited differences in leukocyte tropism. last, we observed a significant increase (p < 0.001) in sensitivity of the N752 mutant to aphidicolin, a drug targeting the viral polymerase. Our results demonstrate that a single amino acid variation in a herpesvirus enzyme can influence neuropathogenic potential without having a major effect on virus shedding from infected animals, which is important for horizontal spread in a population. This observation is very interesting from an evolutionary standpoint and is consistent with data indicating that the N752 ONA pol genotype is predominant in the EHV- 1 population, suggesting that decreased viral pathogenicity in the natural host might not be at the expense of less efficient inter-individual transmission. 2008-03-18T13:31:53Z Goodman, L. B. og Loregian, A. og Perkins, G. A. og Nugent, J. og Buckles, E. L. og Mercorelli, B. og Kydd, J. H. og Palu, G. og Smith, K. C. og Osterrieder, N. og Davis-Poynter, N. http://espace.library.uq.edu.au/view/UQ:76414 Vaccine-induced CD8 T cells directed to tumourspecific antigens are recognised as important components of protective and therapeutic immunity against tumours. Where tumour antigens have pathogenic potential or where immunogenic epitopes are lost from tumours, development of subunit vaccines consisting of multiple individual epitopes is an attractive alternative to immunising with whole tumour antigen. In the present study we investigate the efficacy of two DNA-based multiepitope('polytope') vaccines containing murine (H-2(b)) and human (HLA-A* 0201)-restricted epitopes of the E7 oncoprotein of human papillomavirus type 16, in eliciting tumour-protective cytotoxic T-lymphocyte (CTL) responses. We show that the first of these polytopes elicited powerful effector CTL responses ( measured by IFN-gamma ELISpot) and long-lived memory CTL responses ( measured by functional CTL assay and tetramers) in immunised mice. The responses could be boosted by immunisation with a recombinant vaccinia virus expressing the polytope. Responses induced by immunisation with polytope DNA alone partially protected against infection with recombinant vaccinia virus expressing the polytope. Complete protection was afforded against challenge with an E7-expressing tumour, and reduced growth of nascent tumours was observed. A second polytope differing in the exact composition and order of CTL epitopes, and lacking an inserted endoplasmic reticulum targeting sequence and T-helper epitope, induced much poorer CTL responses and failed to protect against tumour challenge. These observations indicate the validity of a DNA polytope vaccine approach to human papillomavirus E7 - associated carcinoma, and underscore the importance of design in polytope vaccine construction. 2007-08-15T06:27:45Z Doan, T. og Herd, K. og Ramshaw, I. og Thomson, S. og Tindle, R. W. http://espace.library.uq.edu.au/view/UQ:173445 The potential for enhanced diagnosis and cotrol of gonococcal infection through the application of advances in molecular medicine is ow being realized. However, the introduction of diagnostic nucleic acid amplification assays (NAATs) revealed some significant limitations of these applications. Resolution of some, but not all, of these problems has led to recommendations for refined testing algorithms and a better recognition and acceptance of the limitations of NAATs. Resource restriction has limited the use of diagnostic NAATs, especially in less-developed countries where disease rates are highest. However, NAATs, especially in less-developed countries where disease rates are highest. However, NAATs have also proved useful in public health approaches to fonorrhea control in all settings. Additional applications including molecular typing of gonococcal to identify and interupt gonococcal transmission chains and definition of antimicrobial resistance patterns in the gonococcus have been proposed. These approaches, especially those for antimicrobial resistance determination, have been less successful for a number of reasons, including their cost and other unresolved issues. 2009-04-02T13:16:19Z Tapsall, J. og David Whiley og Sloots, Theodorus P. http://espace.library.uq.edu.au/view/UQ:160528 The Prince of Wales Hospital, WHO Collaborating Centre for STD & HIV, Microbiology Department, South Eastern Area Laboratory Services, Randwick, Sydney, Australia. j.tapsall@unsw.edu.au The potential for enhanced diagnosis and control of gonococcal infection through the application of advances in molecular medicine is now being realized. However, the introduction of diagnostic nucleic acid amplification assays (NAATs) revealed some significant limitations of these applications. Resolution of some, but not all, of these problems has led to recommendations for refined testing algorithms and a better recognition and acceptance of the limitations of NAATs. Resource restriction has limited the use of diagnostic NAATs, especially in less-developed countries where disease rates are highest. However, NAATs have also proved useful in public health approaches to gonorrhea control in all settings. Additional applications including molecular typing of gonococci to identify and interrupt gonococcal transmission chains and definition of antimicrobial resistance patterns in the gonococcus have been proposed. These approaches, especially those for antimicrobial resistance determination, have been less successful for a number of reasons, including their cost and other unresolved issues. 2009-01-13T12:13:08Z Tapsall. J. og Whiley, D. og Sloots, T. http://espace.library.uq.edu.au/view/UQ:61346 The detection of Neisseria gonorrhoeae by the polymerase chain reaction (PCR) is now recognized as a sensitive and specific method of diagnosing infection by the organism. In this Study 152 urine specimens were examined for N. gonorrhoeae by a real-time PCR method using the LightCycler platform and results were compared to an in-house PCR assay using an ELISA-based detection method. N. gonorrhoeae DNA was detected in 29 (19%) specimens by LightCycler PCR (LC-PCR) and in 31 (20%) specimens by the in house PCR method. The LightCycler assay proved to be specific and 94% sensitive when compared to the in house PCR method. These features combined with the rapid turn-around time for results makes the LC-PCR particularly suitable for the detection of N. gonorrhoeae in a routine clinical laboratory. (C) 2002 Elsevier Science Inc. All rights reserved. 2007-08-14T17:08:00Z Whiley, D. M. og LeCornec, G. M. og Mackay, I. M. og Siebert, D. J. og Sloots, T. P. http://espace.library.uq.edu.au/view/UQ:76532 2007-08-15T06:31:56Z Whiley, D. og Buda, P og Freeman, K. og Prattle, N.I. og Bates, J. og Sloots, T. P. http://espace.library.uq.edu.au/view/UQ:160690 Murine interferon-inducible T-cell alpha chemoattractant (I-TAC) is a potent non-ELR CXC chemokine that predominantly attracts activated T lymphocytes, binds to the receptor CXCR3 and is induced by interferon-ggr (IFN-ggr). We analysed I-TAC expression by reverse transcriptase-polymerase chain reaction during three different virus-infection models in mice, respiratory syncytial virus (RSV), influenza A and vaccinia virus western reserve (VV-WR). In the lungs from mice infected with RSV or influenza A viruses, peak expression of I-TAC coincided with peak viraemia. Surprisingly, there was no expression in the lungs of mice infected with vaccinia, unlike the elevated expression shown previously for other interferon-regulated chemokines, such as Crg2 and Mig. To further investigate the importance of this difference during vaccinia infection in mice, a recombinant virus encoding I-TAC (rVV I-TAC) was generated. Studies in C57BL/6 and Swiss nude mice showed that I-TAC expression caused increased mononuclear cell infiltration and significantly attenuated the VV. Infection of the footpads of naïve or already immune (with VV-WR) mice with either rVV I-TAC or VV-WR demonstrated that I-TAC expression reduced overall inflammation during infection and that this reduction was more pronounced in already immune mice. The data presented here show that I-TAC can have an important role during virus infections and that vaccinia has evolved ways to avoid inducing I-TAC expression. 2009-01-14T14:28:31Z Hamilton, N. H. R. og Mahalingam, S. og Banyer, I. A. og Ramshaw, I. A. og Thomson, S. http://espace.library.uq.edu.au/view/UQ:69777 Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNIA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADM), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type I virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory. 2007-08-15T03:25:07Z Syrmis, M. W. og Whiley, D. M. og Thomas, M. og Mackay, I. M. og Williamson, J. og Siebert, D. J. og Nissen, M. D. og Sloots, T. P. http://espace.library.uq.edu.au/view/UQ:59826 The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER), Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CIM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway. 2007-08-14T16:05:08Z MacKenzie, J. M. og Westaway, E. G. http://espace.library.uq.edu.au/view/UQ:109312 Defective interfering baculoviruses (DIs) lack considerable portions of the genome, interfere with the replication of helper virus, and cause the so-called "passage-effect" during serial passaging in insect cells and in bioreactor configurations. We investigated their origin by (nested) PCR and demonstrated that DIs lacking approximately 43% (d43) of their DNA are present in low-passage Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)-E2 virus stocks and in polyhedra, but not in the authentic AcMNPV isolate obtained prior to passage in cell culture. To investigate whether DIs are rapidly generated de novo in Sf21 insect cells, a genetically homogeneous AcMNPV bacmid was serially passaged, resulting in the generation of d43 DIs within two passages. AT-rich sequences of up to 66 nucleotides of partly unknown origin were found at the deletion junctions in the d43 DI genomes. These data suggest that the rapid generation of DIs is an intrinsic property of baculovirus infection in insect cell culture and involves several recombination steps. (C) 2001 Academic Press. 2007-09-19T16:24:51Z Pijlman, Gorben P. og van den Born, Erwin og Martens, Dirk E. og Vlak, Just M. http://espace.library.uq.edu.au/view/UQ:60442 2007-08-14T16:33:45Z Nissen, M. D. og Walker, J.C. http://espace.library.uq.edu.au/view/UQ:132722 Bordetella pertussis is a significant cause of respiratory illness and an ongoing public health problem. Pertussis polymerase chain reaction (PCR) testing has been widely utilised since 2001, especially in infants. Uncertainty exists as to how long PCR remains positive following symptom onset. Further information on the time frame for pertussis PCR testing would assist diagnosis, epidemiological research and disease control. The Brisbane Southside Population Health Unit (BSPHU) conducted a retrospective analysis of enhanced surveillance data from pertussis notifications between January 2001 and December 2005, in children less than 5 years of age, in the BSPHU reporting area with the aim to determine the possible range of duration of Bordetella pertussis PCR, from symptom onset for this age group .. Of 1,826 pertussis notifications to BSPHU between January 2001 and December 2005, 155 (8.5%) were children under 5 years of age, with 1 15 pertussis PCR positive results. Analysis indicated a range of PCR positivity from day one to day 31 from the onset of catarrhal symptoms with most (84%) being within 21 days from onset of catarrah I symptoms. The range of PCR positivity following onset of paroxysmal cough was from day one to day 38 with most (89%) being within 14 days from the onset of paroxysmal cough. This review of pertussis PCR data in young children showed that PCR positive results generally mirrored the understood length of infectivity with regard to both catarrhal symptoms and paroxysmal cough; namely that PCR positive results were obtained at least 21 days following onset of catarrhal symptoms and at least 14 days following onset of paroxysmal cough. Commun Dis Intel! 2007;31 :202-205. 2008-03-19T14:45:23Z Palmer, C. M. og McCall, B. og Jarvinen, K. og Nissen, M. D. http://espace.library.uq.edu.au/view/UQ:79663 2007-08-15T08:29:35Z Peadon, Elizabeth og Burgner, David og Nissen, Michael og Buttery, Jim og Zurynski, Yvonne og Elliott, Elizabeth og Gold, Michael og Marshall, Helen og Booy, Robert http://espace.library.uq.edu.au/view/UQ:111264 Successive Viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did not show the accumulation of such concatemers. When introduced into SeUCR1 and ScIZD2109 cells, the non-hr ori concatemers generated in Se301 cells were maintained but did not increase. This suggests that the non-hr ori confers a strong selective advantage in Se301 cells, but not or to a lesser extent in the other cell lines. The cell line-specific accumulation of non-hr ori concatemers might be due to a higher intrinsic recombination frequency in Se301 cells and may reflect tissue related differences involving some host cell factor(s). Since non-hr ori concatemers in Se301 cells were more abundant in intracellular than in extracellular viral DNA preparations, episomal replication and the requirement of a minimal DNA size for packaging into micleocapsids is hypothesized. (C) 2003 Elsevier Inc. All rights reserved. 2007-09-19T17:09:52Z Pijlman, G.n P. og Vermeesch, A. M. G. og Vlak, J. M. http://espace.library.uq.edu.au/view/UQ:161603 2009-01-23T10:38:03Z Mackay, I. M. og Areden, K. E. og Nissen, M. D. og Sloots, T. P http://espace.library.uq.edu.au/view/UQ:137082 The use of real-time PCR in microbial diagnostics has increased to the point where it has evolved from a novelty into a mature and essential technology for the field doing so, real-time PCR has driven significant changes in the way we detect microbes. The predominantly phenotype-related methods of culture and antigen detect while still of considerable value, are being supplanted by the detection, characterization and quantification of microbial nucleic acids. Real-time PCR has engender a wider acceptance of the PCR technique due to its improved rapidity, sensitivity, reproducibility and the considerably reduced risk of carry-over contamination. There many fluorogenic chemistries that can detect PCR product as it accumulates but only a few are useful, popular or exciting enough to be the subject of publication in field of microbiology. We review how real-time PCR has come to be; especially the essential role of fluorescence and we criticallyreview the plethora of detection chemistries available to the end user. 2008-05-01T15:28:53Z Mackay, I. M. og Arden, K. E. og Nissen, M. D. og Sloots, T. P. http://espace.library.uq.edu.au/view/UQ:160590 In 1996 a variant lyssavirus was isolated from an insectivorous bat (yellow bellied, sheath tail bat*/Saccolaimus flaviventris ) in Australia. The nucleocapsid protein (N), matrix protein (M), phosphoprotein (P), glycoprotein (G) and polymerase (L) genes of the Australian bat lyssavirus (ABL) insectivorous isolate were compared with that previously described from a frugivorous bat (Pteropus sp.), and showed sequence divergence at both the nucleotide and amino acid sequence level of 20% and 4/12%, respectively. Comparison of deduced protein sequences of ABL isolates from Pteropus and insectivorous bats, showed that viral isolates were homologous and varied by only a few percent. However, these viruses separated into two distinct clades; those isolated from Pteropus or those from Saccolaimus flaviventris bats, when comparisons were made at the nucleotide level. Nucleoprotein sequence comparisons also showed insectivorous isolates to be of the same putative genotype (genotype 7) as that isolated from frugivorous bats. Immediately after the isolation of ABL from an insectivorous bat, the first human case of ABL infection was identified. PCR and sequence analysis done on cerebrospinal fluid, brain and virus isolated from fresh brain tissue of this human case, was consistent with this infection originating from an insectivorous bat. Monoclonal antibody profiling studies of the virus isolated from the human brain tissues supported this conclusion. Sequence comparisons done on the nucleocapsid (N) gene of insectivorous or frugivorous bats showed no geographic associations between isolates but did delineate between the variants of ABL in Australia. 2009-01-13T16:50:38Z Gould, Allan R. og Kattenbelt, Jacqueline A. og Gumley, Sarah G. og Lunt, Ross A. http://espace.library.uq.edu.au/view/UQ:129250 Background: Human rhinoviruses (HRVs) are some of the earliest identified and most commonly detected viruses associated with acute respiratory tract infections (ARTIs) and yet the molecular epidemiology and genomic variation of individual serotypes remains undefined. Objectives: To molecularly characterise a novel HRV and determine its prevalence and clinical impact on a predominantly paediatric population. Study design: Nucleotide sequencing was employed to determine the complete HRV-QPM coding sequence. Two novel real-time RT-PCR diagnostic assays were designed and employed to retrospectively screen a well-defined population of 1244 specimen extracts to identify the prevalence of HRV-QPM during 2003. Results: Phylogenetic studies of complete coding sequences defined HRV-QPM as a novel member the genus Rhinovirus residing within the previously described HRV-A2 sub-lineage. Investigation of the relatively short VP1 sequence suggest that the virus is resistant to Pleconaril, setting it apart from the HRV A species. Sixteen additional HRV-QPM strains were detected (1.4% of specimens) often as the sole microorganism present among infants with suspected bronchiolitis. HRV-QPM was also detected in Europe during 2006, and a closely related virus circulated in the United States during 2004. Conclusions: We present the molecular characterisation and preliminary clinical impact of a newly identified HRV along with sequences representing additional new HRVs. (C) 2007 Elsevier B.V. All rights reserved. 2008-02-18T17:35:18Z McErlean, P. og Shackelton, L. A. og Lambert, S. B. og Nissen, M. D. og Sloots, T. P. og Mackay, I. M. http://espace.library.uq.edu.au/view/UQ:123734 Equine herpesvirus-1 (EHV-1) is a ubiquitous pathogen of horses, which continues to cause respiratory and neurological disease and abortion, despite the widespread use of vaccines. Cell mediated immunity (CMI) is thought to play a major role in protection against infection with EHV-1. The aim of this study was to characterise the virus-specific CMI response in ponies vaccinated with vP1014, a vaccinia-based construct (NYVAC) coding for the immediate early gene (gene 64) of EHV-1. This gene product is a CTL target protein for an equine MHC class I allele expressed on the A3 haplotype. EHV-primed yearling ponies expressing this haplotype were vaccinated once (n = 1), three (n = 1), or four times (n = 2), and one pony was kept as an unvaccinated control. Cytotoxic T lymphocyte (CTL) activity and interferon gamma (IFN-gamma) synthesis were measured before and after vaccination and challenge infection with EHV-1. Multiple immunisations with vP1014 resulted in increased CTL activity and IFN-gamma synthesis specific for EHV-1 compared with unvaccinated or singly vaccinated ponies. The phenotype of EHV-1 specific T-cells synthesising IFN-gamma was also modified by immunisation. In the unvaccinated pony, the predominant population synthesising IFN-gamma after EHV-1 stimulation was CD8 alpha(+). In contrast, multiply vaccinated ponies demonstrated an increased proportion of CD8 alpha(-) T-cells synthesising IFN-gamma. The results demonstrated that vaccination with a NYVAC-based construct coding for gene 64 stimulated CMI. This immune response alone did not protect against challenge infection. However, the study does illustrate that vaccinia-based vaccines can stimulate CMI in the horse and may therefore contribute to protection against disease caused by EHV-1. (c) 2005 Elsevier Ltd. All rights reserved. 2008-01-25T16:48:18Z Paillot, R. og Ellis, S. A. og Daly, J. M. og Audonnet, J. C. og Minke, J. M. og Davis-Poynter, N. og Hannant, D. og Kydd, J. H. http://espace.library.uq.edu.au/view/UQ:161666 2009-01-23T15:03:28Z Mackay, Ian http://espace.library.uq.edu.au/view/UQ:72993 2007-08-14T12:52:12Z Whiley, D. M. og Sloots, T P http://espace.library.uq.edu.au/view/UQ:71507 Despite recent reports of clonal strains of Pseudomonas aeruginosa in cystic fibrosis (CF) units, the need for routine microbiological surveillance remains contentious. Sputum was collected prospectively from productive patients attending the regional paediatric and adult CF units in Brisbane, Australia. All P. aeruginosa isolates were typed using pulsed-field gel electrophoresis. Spirometry, anthropometrics, hospitalisations and antibiotic sensitivity data were recorded. The first 100 sputum samples (first 50 patients at each clinic) harboured 163 isolates of P. aeruginosa. A total of 39 patients shared a common strain (pulsotype 2), 20 patients shared a strain with at least one other patient and 41 patients harboured unique strains. Eight patients shared a strain identical to a previously reported Australian transmissible strain (pulsotype 1). Compared with the unique strain group, patients harbouring pulsotype 2 were younger and had poorer lung function. Treatment requirements were similar in these two groups, as were the rates of multiresistance. In conclusion, 59% of patients harboured a clonal strain, supporting the need for routine microbiological surveillance. In contrast to previously described clonal strains, the dominant pulsotype was indistinguishable from nonclonal strains with respect to both colonial morphology and multiresistance. The clinical significance of clonal strains remains uncertain and requires longitudinal study. 2007-08-15T03:56:28Z O'Carroll, M. R. og Syrmis, M. W. og Wainwright, C. E. og Greer, R. M. og Mitchell, P. og Coulter, C. og Sloots, T. P. og Nissen, M. D. og Bell, S. C. http://espace.library.uq.edu.au/view/UQ:111146 Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved. 2007-09-19T16:51:51Z Wang, Hanzhong og Deng, Fei og Pijlman, Gorben P. og Chen, Xinwen og Sun, Xiulian og Vlak, Just M. og Hu, Zhihong http://espace.library.uq.edu.au/view/UQ:64562 Background: Herpesviruses are a significant cause of human morbidity. Traditional approaches to the identification of these viruses require infectious or at least antigenic virus. Multiplex PCR (mPCR) is capable of simultaneously amplifying a range of targets from a single preparation of nucleic acids and when combined with a suitable detection assay, it is capable of discriminating each of the amplicons. Objectives: Several methods have been described in the literature, however, they lack one or more significant design features required to suitably control a routinely applied nucleic acid amplification assay. We aimed to design a multiplex herpesvirus PCR that could co-amplify eight human herpesvirus targets plus an internal control (IC) molecule in a single tube. Study Design: Primers were designed to target the DNA polymerase genes of each of the human herpesviruses. Synthetic controls were developed to act as templates for the evaluation of assay sensitivity and specificity and for development of an in-house competitive quantitative PCR. Amplicon was discriminated using a simplified enzyme linked amplicon hybridisation assay (ELAHA). Results and Conclusions: For routine diagnostic use we reduced the number of herpesviral targets from 8 to 6 in order to maintain adequate clinical sensitivity. The ELAHA proved more sensitive than agarose get electrophoresis. Additionally, 36 cytomegalovirus positive patients were examined with an in-house quantitative PCR-ELAHA which was developed to confirm that that the mPCR's co-detection limit of 10(2) copy of synthetic template per millitre was relevant for use in detecting virus from clinical samples. The mPCR-ELAHA was then applied to the screening of 174 patient specimens resulting in a specificity of 98% and a sensitivity of 93%. This preliminary study demonstrated that the mPCR-ELAHA was a complete approach to the detection of herpesviruses from a range of clinical samples and disease states. (C) 2003 Elsevier Science B.V. All rights reserved. 2007-08-14T19:14:26Z Mackay, I. M. og Gardam, T. og Arden, K. E. og McHardy, S. og Whiley, D. M. og Crisante, E. og Sloots, T. P. http://espace.library.uq.edu.au/view/UQ:129459 T cell mediated immune responses are induced following interaction of MHC-presented epitope on professional antigen presenting cells such as dendritic cells (DCs) with cognate T cell receptor. Up-regulation of receptor-ligand pairs of costimulatory molecules linking DC to T cell enhances the resulting T cell responses. This 'second signalling' occurs through the B7 molecules CD80/86 expressed by DCs, and importantly through members of the TNF ligand/TNF receptor superfamilies. We have previously shown that co-expression of RANK/RANKL or 41BB-L in addition to tumour antigen in dendritic cells augmented cognate effector and memory tumour antigen-directed cytotoxic T cell responses when the DCs were used to immunise mice. Here, we examined whether co-immunisation with naked plasmid DNAs encoding antigen and these costimulatory molecule(s), would enhance antigen specific T cell responses. We demonstrate that co-immunisation with DNAs encoding tumour antigen and costimulatory molecules failed to enhance antigen-directed CTL responses, or tumour protection, afforded by immunisation with DNA encoding tumour antigen alone. (c) 2007 Elsevier Ltd. All rights reserved. 2008-02-18T14:25:41Z Herd, Karen A. og Wiethe, Carsten og Tindle, Robert W. http://espace.library.uq.edu.au/view/UQ:160517 Interferon gamma (IFN-gamma) and interleukin-4 (IL-4) are not only generated during cell-mediated immunity (CMI) and humoral immunity (HI), but are also generated by innate immune cells in response to pathogenic factors. How these cytokines differentially effect the development of dendritic cell (DC)-associated immunoregulatory properties from progenitor cells during innate immunity is unresolved. To address this we have utilized a homogeneous DC progenitor-like cell line, MTHC-D2, as a model to examine cytokine-induced maturation of DCs. By 6 h IFN-gamma induced genes that are important for antiviral activity and development of CMI, whereas IL-4 induced genes involved in cellular adhesion, uptake of extracellular antigen, suppression of cytotoxic T-cell responses, and that repair the extracellular matrix. By 48 h the cytokine stimulus had induced many properties characteristic of immature DCs; however, these were differentially effected by IFN-gamma and IL-4. IFN-gamma induced the greatest levels of costimulatory/ activation marker expression, and the highest levels of T-cell proliferation, whereas IL-4 induced the greatest levels of phagocytic activity. Stimulation of the cells with CD40 Ab enhanced the levels of costimulatory marker expression and T-cell stimulatory capacity of cells exposed to IFN-gamma, but had little effect on cells exposed to IL-4 in the absence of IFN-gamma. 2009-01-13T11:37:41Z Banyer, J. L. og Halliday, D. C. og Thomson, S. A. og Hamilton, N. H. R. http://espace.library.uq.edu.au/view/UQ:132725 2008-03-19T16:05:51Z Lambert, Stephen B. og Allen, Kelly M. og Druce, Julian D. og Birch, Chris J. og Mackay, Ian M. og Carlin, John B. og Carapetis, Jonathan R. og Sloots, Theo P. og Nissen, Michael D. og Nolan, Terence M. http://espace.library.uq.edu.au/view/UQ:171418 2009-03-23T16:47:18Z Lambert, Stephen B. og Whiley,David M. og O'Neill, Nicholas T. og Andrews, Emily C. og Canavan, Fiona M. og Bletchly, Cheryl og Siebert, David J. og Sloots, Theo P. og Nissen, Michael D. http://espace.library.uq.edu.au/view/UQ:122739 2008-01-25T15:58:20Z Daly, J. M. og Yates, P. J. og Browse, G. og Swann, Z. og Newton, J. R. og Jessett, D. og Davis-Poynter, N. og Mumford, J. A. http://espace.library.uq.edu.au/view/UQ:76523 Aims: To develop and evaluate multiplex PCR assays for Chlamydia trachomatis and Neisseria gonorrhoeae, using real-time and conventional PCR detection methodologies. Methods: Two real-time multiplex PCR assays, using nuclease (TaqMan-AB17500) and hybridisation (LightCycler) probe formats, and a third assay using conventional PCR with solid-phase hybridisation and colour detection, were developed. The porA pseudogene was targeted for N. gonorrhoeae, and the major outer membrane protein gene for C. trachomatis. A total of 145 urogenital specimens were tested in all assays, and the results were compared with the Roche Cobas Amplicor assay. Results: There was little difference in clinical sensitivity and specificity, result discrimination and test cost for the three in-house assays. Our results showed that competitive inhibition of the PCR occurred in some samples that were positive for both organisms. Conclusions: These results highlight the suitability and versatility of three multiplex PCR methods for the detection of C. trachomatis and N. gonorrhoeae. 2007-08-15T06:31:38Z Whiley, David M. og Sloots, Theo P. http://espace.library.uq.edu.au/view/UQ:160683 T cell immunity plays a critical role in controlling HIV-1 viremia, and encoding a limited set of HIV-1 genes within DNA and poxvirus vectors can, when used sequentially, induce high levels of T cell immunity in primates. However, a limited breadth of T cell immunity exposes the host to potential infection with either genetically diverse HIV-1 strains or T cell escape variants of HIV-1. In an attempt to induce maximally broad immunity, we examined DNA and recombinant fowlpox virus (rFPV) vaccines encoding all HIV-1 genes derived from a global HIV-1 consensus sequence, but expressed as multiple overlapping scrambled 30-amino acid segments (scrambled antigen vaccines, or SAVINEs). Three groups of seven pigtail macaques were immunized with sets of DNA and rFPV expressing Gag/Pol antigens only, the whole genome SAVINE antigens, or no HIV-1 antigens and T cell immunity was monitored by ELISpot and intracellular cytokine staining. High levels of cross-subtype HIV-specific T cell immunity to Gag were consistently induced in the seven macaques primed with DNA and rFPV vaccines expressing Gag/Pol as intact proteins. It was, however, difficult to repeatedly boost immunity with further rFPV immunizations, presumably reflecting high levels of anti- FPV immunity. Unfortunately, this vaccine study did not consistently achieve a broadened level of T cell immunity to multiple HIV genes utilizing the novel whole-virus SAVINE approach, with only one of seven immunized animals generating broad T cell immunity to multiple HIV-1 proteins. Further refinements are planned with alternative vector strategies to evaluate the potential of the SAVINE technology. 2009-01-14T14:04:16Z Pamungkas, Joko og De Rose, Robert og Iskandriati, Diah og Noviana, Rachmitasari og Paramastri, Yasmina og Dale, C. Jane og Shoobridge, Maryanne og Medveczky, C. Jill og Ramshaw, Ian A. og Thomson, Scott og Kent, Stephen J. http://espace.library.uq.edu.au/view/UQ:160840 Vaccination against AIDS is hampered by great diversity between human immunodeficiency virus (HIV) strains. Heterologous B-subtype-based simian-human immunodeficiency virus (SHIV) DNA prime and poxvirus boost vaccine regimens can induce partial, T-cell-mediated, protective immunity in macaques. We analyzed a set of DNA, recombinant fowlpox viruses (FPV), and vaccinia viruses (VV) expressing subtype AE HIV type 1 (HIV-1) Tat, Rev, and Env proteins and SIV Gag/Pol in 30 pigtail macaques. SIV Gag-specific CD4 and CD8 T-cell responses were induced by sequential DNA/FPV vaccination, although lower FPV doses, VV/FPV vaccination, and DNA vaccines alone were not as consistently immunogenic. The SHIV AE DNA prime, FPV boost regimens were significantly less immunogenic than comparable B-subtype SHIV vaccination. Peak viral load was modestly (0.4 log10 copies/ml) lower among the AE subtype SHIV-immunized animals compared to controls following the virulent B subtype SHIV challenge. Protection from persistent high levels of viremia and CD4 T-cell depletion was less in AE subtype compared to B subtype SHIV-vaccinated macaques. Gag was highly immunodominant over the other AE subtype SHIV vaccine proteins after vaccination, and this immunodominance was exacerbated after challenge. Interestingly, the lower level of priming of immune responses did not blunt postchallenge Gag-specific recall responses, despite more modest protection. These studies suggest priming of T-cell immunity to prevent AIDS in humans is possible, but differences in the immunogenicity of various subtype vaccines and broad cross-subtype protection are substantial hurdles. 2009-01-15T12:49:20Z De Rose, Robert og Batten, C. Jane og Smith, Miranda Z. og Fernandez, Caroline S. og Peut, Viv og Thomson, Scott og Ramshaw, Ian A. og Boyle, David B og Venturi, Vanessa og Davenport, Miles P. og Kent, Stephen J. http://espace.library.uq.edu.au/view/UQ:132533 The greatest risk of death from pneumonia in childhood is in the neonatal period. It is estimated that pneumonia contributes to between 750000-1.2 million neonatal deaths annually, accounting for 10% of global child mortality. Congenital and neonatal pneumonias are often a difficult disease to Identify and treat, with clinical manifestations often being non-specific. Many of the normal lung defences are compromised in the fetus and neonate, leading to an increased susceptibility to infection. The aetiology and epidemiology of congenital and neonatal pneumonias will depend on the clinical setting and population that the baby belongs to, the stage in the perinatal period, the gestational age of the baby and the definition of pneumonia. Diagnosis, treatment and prevention strategies are therefore also dependent on these factors, and will differ depending on the clinical setting. This review summarizes the current knowledge concerning congenital and neonatal pneumonia worldwide and discusses future directions in the prevention of the disease. 2008-03-18T13:02:18Z Nissen, M. D.