Doherty Institute - Research Publications

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    Public Health Response to Imported Case of Poliomyelitis, Australia, 2007
    Carnie, JA ; Lester, R ; Moran, R ; Brown, L ; Meagher, J ; Roberts, JA ; Thorley, BR (CENTERS DISEASE CONTROL, 2009-11-01)
    Australia, along with 36 other countries in the Western Pacific Region, was declared free of poliomyelitis by the World Health Organization in October 2000. Yet, the persistence of wild poliovirus in the 4 remaining polio-endemic countries-Afghanistan, India, Nigeria, and Pakistan-poses a risk for its importation into all countries declared polio free. We describe the public health response and outcomes resulting from the importation of a wild poliovirus infection in Melbourne, Australia, in July 2007. This response, based on an assessment of the risk for transmission, included offering vaccination with inactivated polio vaccine to the contacts and placing the index patient in isolation and the household contacts in quarantine until consecutive fecal specimens were negative for poliovirus by culture. The experience gained from the polio importation event in Australia may assist other polio-free countries to prepare for, and respond to, a similar event. No secondary clinical cases resulted from this importation.
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    Using ELISPOT to Expose False Positive Skin Test Conversion in Tuberculosis Contacts
    Hill, PC ; Jeffries, DJ ; Brookes, RH ; Fox, A ; Jackson-Sillah, D ; Lugos, MD ; Donkor, SA ; de Jong, BC ; Corrah, T ; Adegbola, RA ; McAdam, KP ; Ratner, A (PUBLIC LIBRARY SCIENCE, 2007-01-31)
    BACKGROUND: Repeat tuberculin skin tests may be false positive due to boosting of waned immunity to past mycobacterial exposure. We evaluated whether an ELISPOT test could identify tuberculosis (TB) contacts with boosting of immunity to non-tuberculous mycobacterial exposure. METHODOLOGY/PRINCIPAL FINDINGS: We conducted tuberculin and ELISPOT tests in 1665 TB contacts: 799 were tuberculin test negative and were offered a repeat test after three months. Those with tuberculin test conversion had an ELISPOT, chest X-ray and sputum analysis if appropriate. We compared converters with non-converters, assessed the probability of each of four combinations of ELISPOT results over the two time points and estimated boosting with adjustment for ELISPOT sensitivity and specificity. 704 (72%) contacts had a repeat tuberculin test; 176 (25%) had test conversion, which increased with exposure to a case (p = 0.002), increasing age (p = 0.0006) and BCG scar (p = 0.06). 114 tuberculin test converters had ELISPOT results: 16(14%) were recruitment positive/follow-up positive, 9 (8%) positive/negative, 34 (30%) negative/positive, and 55 (48%) were negative/negative. There was a significant non-linear effect of age for ELISPOT results in skin test converters (p = 0.038). Estimates of boosting ranged from 32%-41% of skin test converters with increasing age. Three converters were diagnosed with TB, two had ELISPOT results: both were positive, including one at recruitment. CONCLUSIONS/SIGNIFICANCE: We estimate that approximately one third of tuberculin skin test conversion in Gambian TB case contacts is due to boosting of immunity to non-tuberculous mycobacterial exposure. Further longitudinal studies are required to confirm whether ELISPOT can reliably identify case contacts with tuberculin test conversion that would benefit most from prophylactic treatment.
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    Improved single-chain transactivators of the Tet-on gene expression system
    Zhou, X ; Symons, J ; Hoppes, R ; Krueger, C ; Berens, C ; Hillen, W ; Berkhout, B ; Das, AT (BIOMED CENTRAL LTD, 2007-01-19)
    BACKGROUND: The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application. RESULTS: We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used. CONCLUSION: We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used.
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    Evolution of DC-SIGN use revealed by fitness studies of R5 HIV-I variants emerging during AIDS progression
    Borggren, M ; Repits, J ; Kuylenstierna, C ; Sterjovski, J ; Churchill, MJ ; Purcell, DFJ ; Karlsson, A ; Albert, J ; Gorry, PR ; Jansson, M (BMC, 2008-03-27)
    BACKGROUND: At early stages of infection CCR5 is the predominant HIV-1 coreceptor, but in approximately 50% of those infected CXCR4-using viruses emerge with disease progression. This coreceptor switch is correlated with an accelerated progression. However, those that maintain virus exclusively restricted to CCR5 (R5) also develop AIDS. We have previously reported that R5 variants in these "non-switch virus" patients evolve during disease progression towards a more replicative phenotype exhibiting altered CCR5 coreceptor interactions. DC-SIGN is a C-type lectin expressed by dendritic cells that HIV-1 may bind and utilize for enhanced infection of T cells in trans. To further explore the evolution of the R5 phenotype we analyzed sequential R5 isolates obtained before and after AIDS onset, i.e. at the chronic stage and during end-stage disease, with regard to efficiency of DC-SIGN use in trans-infections. RESULTS: Results from binding and trans-infection assays showed that R5 viruses emerging during end-stage AIDS disease displayed reduced ability to use DC-SIGN. To better understand viral determinants underlying altered DC-SIGN usage by R5 viruses, we cloned and sequenced the HIV-1 env gene. We found that end-stage R5 viruses lacked potential N-linked glycosylation sites (PNGS) in the gp120 V2 and V4 regions, which were present in the majority of the chronic stage R5 variants. One of these sites, amino acid position 160 (aa160) in the V2 region, also correlated with efficient use of DC-SIGN for binding and trans-infections. In fitness assays, where head-to-head competitions between chronic stage and AIDS R5 viruses were setup in parallel direct and DC-SIGN-mediated infections, results were further supported. Competitions revealed that R5 viruses obtained before AIDS onset, containing the V2 PNGS at aa160, were selected for in the trans-infection. Whereas, in agreement with our previous studies, the opposite was seen in direct target cell infections where end-stage viruses out-competed the chronic stage viruses. CONCLUSION: Results of our study suggest R5 virus variants with diverse fitness for direct and DC-SIGN-mediated trans-infections evolve within infected individuals at end-stage disease. In addition, our results point to the importance of a glycosylation site within the gp120 V2 region for efficient DC-SIGN use of HIV-1 R5 viruses.
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    Impact of Emerging Antiviral Drug Resistance on Influenza Containment and Spread: Influence of Subclinical Infection and Strategic Use of a Stockpile Containing One or Two Drugs
    McCaw, JM ; Wood, JG ; McCaw, CT ; McVernon, J ; Montgomery, JM (PUBLIC LIBRARY SCIENCE, 2008-06-04)
    BACKGROUND: Wide-scale use of antiviral agents in the event of an influenza pandemic is likely to promote the emergence of drug resistance, with potentially deleterious effects for outbreak control. We explored factors promoting resistance within a dynamic infection model, and considered ways in which one or two drugs might be distributed to delay the spread of resistant strains or mitigate their impact. METHODS AND FINDINGS: We have previously developed a novel deterministic model of influenza transmission that simulates treatment and targeted contact prophylaxis, using a limited stockpile of antiviral agents. This model was extended to incorporate subclinical infections, and the emergence of resistant virus strains under the selective pressure imposed by various uses of one or two antiviral agents. For a fixed clinical attack rate, R(0) rises with the proportion of subclinical infections thus reducing the number of infections amenable to treatment or prophylaxis. In consequence, outbreak control is more difficult, but emergence of drug resistance is relatively uncommon. Where an epidemic may be constrained by use of a single antiviral agent, strategies that combine treatment and prophylaxis are most effective at controlling transmission, at the cost of facilitating the spread of resistant viruses. If two drugs are available, using one drug for treatment and the other for prophylaxis is more effective at preventing propagation of mutant strains than either random allocation or drug cycling strategies. Our model is relatively straightforward, and of necessity makes a number of simplifying assumptions. Our results are, however, consistent with the wider body of work in this area and are able to place related research in context while extending the analysis of resistance emergence and optimal drug use within the constraints of a finite drug stockpile. CONCLUSIONS: Combined treatment and prophylaxis represents optimal use of antiviral agents to control transmission, at the cost of drug resistance. Where two drugs are available, allocating different drugs to cases and contacts is likely to be most effective at constraining resistance emergence in a pandemic scenario.
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    A Small Community Model for the Transmission of Infectious Diseases: Comparison of School Closure as an Intervention in Individual-Based Models of an Influenza Pandemic
    Milne, GJ ; Kelso, JK ; Kelly, HA ; Huband, ST ; McVernon, J ; Montgomery, JM (PUBLIC LIBRARY SCIENCE, 2008-12-23)
    BACKGROUND: In the absence of other evidence, modelling has been used extensively to help policy makers plan for a potential future influenza pandemic. METHOD: We have constructed an individual based model of a small community in the developed world with detail down to exact household structure obtained from census collection datasets and precise simulation of household demographics, movement within the community and individual contact patterns. We modelled the spread of pandemic influenza in this community and the effect on daily and final attack rates of four social distancing measures: school closure, increased case isolation, workplace non-attendance and community contact reduction. We compared the modelled results of final attack rates in the absence of any interventions and the effect of school closure as a single intervention with other published individual based models of pandemic influenza in the developed world. RESULTS: We showed that published individual based models estimate similar final attack rates over a range of values for R(0) in a pandemic where no interventions have been implemented; that multiple social distancing measures applied early and continuously can be very effective in interrupting transmission of the pandemic virus for R(0) values up to 2.5; and that different conclusions reached on the simulated benefit of school closure in published models appear to result from differences in assumptions about the timing and duration of school closure and flow-on effects on other social contacts resulting from school closure. CONCLUSION: Models of the spread and control of pandemic influenza have the potential to assist policy makers with decisions about which control strategies to adopt. However, attention needs to be given by policy makers to the assumptions underpinning both the models and the control strategies examined.
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    A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells
    Burk, U ; Schubert, J ; Wellner, U ; Schmalhofer, O ; Vincan, E ; Spaderna, S ; Brabletz, T (NATURE PUBLISHING GROUP, 2008-06-01)
    The embryonic programme 'epithelial-mesenchymal transition' (EMT) is thought to promote malignant tumour progression. The transcriptional repressor zinc-finger E-box binding homeobox 1 (ZEB1) is a crucial inducer of EMT in various human tumours, and was recently shown to promote invasion and metastasis of tumour cells. Here, we report that ZEB1 directly suppresses transcription of microRNA-200 family members miR-141 and miR-200c, which strongly activate epithelial differentiation in pancreatic, colorectal and breast cancer cells. Notably, the EMT activators transforming growth factor beta2 and ZEB1 are the predominant targets downregulated by these microRNAs. These results indicate that ZEB1 triggers an microRNA-mediated feedforward loop that stabilizes EMT and promotes invasion of cancer cells. Alternatively, depending on the environmental trigger, this loop might switch and induce epithelial differentiation, and thus explain the strong intratumorous heterogeneity observed in many human cancers.
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    Control of viremia and prevention of AIDS following immunotherapy of SIV-infected macaques with peptide-pulsed blood
    De Rose, R ; Fernandez, CS ; Smith, MZ ; Batten, CJ ; Alcantara, S ; Peut, V ; Rollman, E ; Loh, L ; Mason, RD ; Wilson, K ; Law, MG ; Handley, AJ ; Kent, SJ ; Koup, RA (PUBLIC LIBRARY SCIENCE, 2008-05-01)
    Effective immunotherapies for HIV are needed. Drug therapies are life-long with significant toxicities. Dendritic-cell based immunotherapy approaches are promising but impractical for widespread use. A simple immunotherapy, reinfusing fresh autologous blood cells exposed to overlapping SIV peptides for 1 hour ex vivo, was assessed for the control of SIV(mac251) replication in 36 pigtail macaques. An initial set of four immunizations was administered under antiretroviral cover and a booster set of three immunizations administered 6 months later. Vaccinated animals were randomized to receive Gag peptides alone or peptides spanning all nine SIV proteins. High-level, SIV-specific CD4 and CD8 T-cell immunity was induced following immunization, both during antiretroviral cover and without. Virus levels were durably approximately 10-fold lower for 1 year in immunized animals compared to controls, and a significant delay in AIDS-related mortality resulted. Broader immunity resulted following immunizations with peptides spanning all nine SIV proteins, but the responses to Gag were weaker in comparison to animals only immunized with Gag. No difference in viral outcome occurred in animals immunized with all SIV proteins compared to animals immunized against Gag alone. Peptide-pulsed blood cells are an immunogenic and effective immunotherapy in SIV-infected macaques. Our results suggest Gag alone is an effective antigen for T-cell immunotherapy. Fresh blood cells pulsed with overlapping Gag peptides is proceeding into trials in HIV-infected humans.
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    Humans lack iGb3 due to the absence of functional iGb3-synthase: Implications for NKT cell development and transplantation
    Christiansen, D ; Milland, J ; Mouhtouris, E ; Vaughan, H ; Pellicci, DG ; McConville, MJ ; Godfrey, DI ; Sandrin, MS ; Ploegh, HL (PUBLIC LIBRARY SCIENCE, 2008-07-01)
    The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galalpha(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galalpha(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation.
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    Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS
    Sterjovski, J ; Churchill, MJ ; Ellett, A ; Gray, LR ; Roche, MJ ; Dunfee, RL ; Purcell, DF ; Saksena, N ; Wang, B ; Sonza, S ; Wesselingh, SL ; Karlsson, I ; Fenyo, E-M ; Gabuzda, D ; Cunningham, AL ; Gorry, PR (BMC, 2007-12-12)
    BACKGROUND: CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively. RESULTS: Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure. CONCLUSION: Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.