Doherty Institute - Research Publications

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    Macrophage infectivity potentiator protein, a peptidyl prolyl cis-trans isomerase, essential for Coxiella burnetii growth and pathogenesis
    Debowski, AWW ; Bzdyl, NMM ; Thomas, DRR ; Scott, NEE ; Jenkins, CHH ; Iwasaki, J ; Kibble, EAA ; Khoo, CA ; Scheuplein, NJJ ; Seibel, PMM ; Lohr, T ; Metters, G ; Bond, CSS ; Norville, IHH ; Stubbs, KAA ; Harmer, NJJ ; Holzgrabe, U ; Newton, HJJ ; Sarkar-Tyson, M ; Zamboni, DS (PUBLIC LIBRARY SCIENCE, 2023-07)
    Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyse the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria. To date the role of the Mip protein in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. The pipecolic acid derived compounds, SF235 and AN296, which have shown utility in targeting other Mip proteins from pathogenic bacteria, demonstrate inhibitory activities against CbMip. These compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain in axenic culture. Comparative proteomics, in the presence of AN296, revealed alterations in stress responses with H2O2 sensitivity assays validating that Mip inhibition increases the sensitivity of C. burnetii to oxidative stress. In addition, SF235 and AN296 were effective in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for replication and that the development of more potent inhibitors against CbMip is warranted and offer potential as novel therapeutics against this pathogen.
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    Coxiella co-opts the Glutathione Peroxidase 4 to protect the host cell from oxidative stress-induced cell death.
    Loterio, RK ; Thomas, DR ; Andrade, W ; Lee, YW ; Santos, LL ; Mascarenhas, DPA ; Steiner, TM ; Chiaratto, J ; Fielden, LF ; Lopes, L ; Bird, LE ; Goldman, GH ; Stojanovski, D ; Scott, NE ; Zamboni, DS ; Newton, HJ (Proceedings of the National Academy of Sciences, 2023-09-05)
    The causative agent of human Q fever, Coxiella burnetii, is highly adapted to infect alveolar macrophages by inhibiting a range of host responses to infection. Despite the clinical and biological importance of this pathogen, the challenges related to genetic manipulation of both C. burnetii and macrophages have limited our knowledge of the mechanisms by which C. burnetii subverts macrophages functions. Here, we used the related bacterium Legionella pneumophila to perform a comprehensive screen of C. burnetii effectors that interfere with innate immune responses and host death using the greater wax moth Galleria mellonella and mouse bone marrow-derived macrophages. We identified MceF (Mitochondrial Coxiella effector protein F), a C. burnetii effector protein that localizes to mitochondria and contributes to host cell survival. MceF was shown to enhance mitochondrial function, delay membrane damage, and decrease mitochondrial ROS production induced by rotenone. Mechanistically, MceF recruits the host antioxidant protein Glutathione Peroxidase 4 (GPX4) to the mitochondria. The protective functions of MceF were absent in primary macrophages lacking GPX4, while overexpression of MceF in human cells protected against oxidative stress-induced cell death. C. burnetii lacking MceF was replication competent in mammalian cells but induced higher mortality in G. mellonella, indicating that MceF modulates the host response to infection. This study reveals an important C. burnetii strategy to subvert macrophage cell death and host immunity and demonstrates that modulation of the host antioxidant system is a viable strategy to promote the success of intracellular bacteria.
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    CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity
    Gressier, E ; Schulte-Schrepping, J ; Petrov, L ; Brumhard, S ; Stubbemann, P ; Hiller, A ; Obermayer, B ; Spitzer, J ; Kostevc, T ; Whitney, PG ; Bachem, A ; Odainic, A ; van de Sandt, C ; Nguyen, THO ; Ashhurst, T ; Wilson, K ; Oates, CVL ; Gearing, LJ ; Meischel, T ; Hochheiser, K ; Greyer, M ; Clarke, M ; Kreutzenbeck, M ; Gabriel, SS ; Kastenmueller, W ; Kurts, C ; Londrigan, SL ; Kallies, A ; Kedzierska, K ; Hertzog, PJ ; Latz, E ; Chen, Y-CE ; Radford, KJ ; Chopin, M ; Schroeder, J ; Kurth, F ; Gebhardt, T ; Sander, LE ; Sawitzki, B ; Schultze, JL ; Schmidt, SV ; Bedoui, S (NATURE PORTFOLIO, 2023-06)
    Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-β (IFNα/β)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/β or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.
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    Prevention and post-exposure management of occupational exposure to Ebola virus
    Moso, MA ; Lim, CK ; Williams, E ; Marshall, C ; McCarthy, J ; Williamson, DA (ELSEVIER SCI LTD, 2024-02)
    There have been significant advances in the prevention and management of Ebola virus disease (EVD) caused by Zaire Ebola virus (ZEBOV), including the development of two effective vaccines, rVSV-ZEBOV and Ad26.ZEBOV/MVA-BN-Filo. In addition, ZEBOV monoclonal antibodies have become first-line therapy for EVD. However, the 2022-23 outbreak of Sudan Ebola virus (SUDV) in Uganda has highlighted the gap in current therapies and vaccines, whose efficacy is uncertain against non-ZEBOV species. Health-care and laboratory staff working in EVD treatment centres or Ebola virus diagnostic and research laboratories face unique risks relating to potential occupational exposure to Ebola viruses. Given the substantial morbidity and mortality associated with EVD, facilities should have strategies in place to manage occupational exposures, including consideration of post-exposure therapies. In this Review, we discuss currently available evidence for prevention and post-exposure prophylaxis of EVD, including therapies currently under evaluation for SUDV.
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    Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform
    Tumpach, C ; Cochrane, CR ; Kim, Y ; Ong, J ; Rhodes, A ; Angelovich, TA ; Churchill, MJ ; Lewin, SR ; Telwatte, S ; Roche, M (MEDISCRIPT LTD, 2023-06)
    Quantification of intact proviruses is a critical measurement in HIV cure studies both in vitro and in vivo. The widely adopted 'intact proviral DNA assay' (IPDA), designed to discriminate and quantify genetically intact HIV proviruses based on detection of two HIV sequence-specific targets, was originally validated using Bio-Rad's droplet digital PCR technology (ddPCR). Despite its advantages, ddPCR is limited in multiplexing capability (two-channel) and is both labor- and time intensive. To overcome some of these limitations, we utilized a nanowell-based digital PCR platform (dPCR, QIAcuity from Qiagen) which is a fully automated system that partitions samples into nanowells rather than droplets. In this study we adapted the IPDA assay to the QIAcuity platform and assessed its performance relative to ddPCR. The dPCR could differentiate between intact, 5' defective and 3' defective proviruses and was sensitive to single HIV copy input. We found the intra-assay and inter-assay variability was within acceptable ranges (with coefficient of variation at or below 10%). When comparing the performance of the IPDA in ex vivo CD4+ T cells from people with HIV on antiretroviral therapy, there was a strong correlation in the quantification of intact (rs = 0.93; p < 0.001) and 3' defective proviruses (rs = 0.96; p < 0.001) with a significant but less strong correlation for 5' defective proviruses (rs = 0.7; p = 0.04). We demonstrate that the dPCR platform enables sensitive and accurate quantification of genetically intact and defective proviruses similar to the ddPCR system but with greater speed and efficiency. This flexible system can be further optimized in the future, to detect up to 5 targets, enabling a more precise detection of intact and potentially replication-competent proviruses.
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    Intra- and interhost genomic diversity of monkeypox virus
    Taouk, ML ; Steinig, E ; Taiaroa, G ; Savic, I ; Tran, T ; Higgins, N ; Tran, S ; Lee, A ; Braddick, M ; Moso, MA ; Chow, EPF ; Fairley, CK ; Towns, J ; Chen, MY ; Caly, L ; Lim, CK ; Williamson, DA (WILEY, 2023-08)
    The impact and frequency of infectious disease outbreaks demonstrate the need for timely genomic surveillance to inform public health responses. In the largest known outbreak of mpox, genomic surveillance efforts have primarily focused on high-incidence nations in Europe and the Americas, with a paucity of data from South-East Asia and the Western Pacific. Here we analyzed 102 monkeypox virus (MPXV) genomes sampled from 56 individuals in Melbourne, Australia. All genomes fell within the 2022 MPXV outbreak lineage (B.1), with likely onward local transmission detected. We observed within-host diversity and instances of co-infection, and highlight further examples of structural variation and apolipoprotein B editing complex-driven micro-evolution in the current MPXV outbreak. Updating our understanding of MPXV emergence and diversification will inform public health measures and enable monitoring of the virus' evolutionary trajectory throughout the mpox outbreak.
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    Regional Analysis of Intact and Defective HIV Proviruses in the Brain of Viremic and Virally Suppressed People with HIV
    Angelovich, TA ; Cochrane, CR ; Zhou, J ; Tumpach, C ; Byrnes, SJ ; Eddine, JJ ; Waring, E ; Busman-Sahay, K ; Deleage, C ; Jenkins, TA ; Hearps, AC ; Turville, S ; Gorry, PR ; Lewin, SR ; Brew, BJ ; Estes, JD ; Roche, M ; Churchill, MJ (WILEY, 2023-10)
    Here, we provide the first regional analysis of intact and defective HIV reservoirs within the brain. Brain tissue from both viremic and virally suppressed people with HIV (PWH) harbored HIV pol DNA in all regions tested, with lower levels present in basal ganglia and cerebellum relative to frontal white matter. Intact proviruses were primarily found in the frontal white matter but also detected in other brain regions of PWH, demonstrating frontal white matter as a major brain reservoir of intact, potentially replication competent HIV DNA that persists despite antiretroviral therapy. ANN NEUROL 2023;94:798-802.
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    Perturbation of ATG16L1 function impairs the biogenesis of Salmonella and Coxiella replication vacuoles
    Lau, N ; Thomas, DR ; Lee, YW ; Knodler, LA ; Newton, HJ (WILEY, 2022-02)
    Anti-bacterial autophagy, known as xenophagy, is a host innate immune response that targets invading pathogens for degradation. Some intracellular bacteria, such as the enteric pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), utilize effector proteins to interfere with autophagy. One such S. Typhimurium effector, SopF, inhibits recruitment of ATG16L1 to damaged Salmonella-containing vacuoles (SCVs), thereby inhibiting the host xenophagic response. SopF is also required to maintain the integrity of the SCV during the early stages of infection. Here we show disruption of the SopF-ATG16L1 interaction leads to an increased proportion of cytosolic S. Typhimurium. Furthermore, SopF was utilized as a molecular tool to examine the requirement for ATG16L1 in the intracellular lifestyle of Coxiella burnetii, a bacterium that requires a functional autophagy pathway to replicate efficiently and form a single, spacious vacuole called the Coxiella-containing vacuole (CCV). ATG16L1 is required for CCV expansion and fusion but does not influence C. burnetii replication. In contrast, SopF did not affect CCV formation or replication, demonstrating that the contribution of ATG16L1 to CCV biogenesis is via its role in autophagy, not xenophagy. This study highlights the diverse capabilities of bacterial effector proteins to dissect the molecular details of host-pathogen interactions.
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    Assessment of the cobas® HBV RNA investigational assay in the setting of nucleoside analog therapy cessation
    Jackson, K ; Bonanzinga, S ; Edwards, R ; Visvanathan, K ; Li, X ; Hall, S ; Kuchta, A ; Canchola, JA ; Thompson, AJ (WILEY, 2022-12)
    HBV RNA is used as a marker of cccDNA transcription and is applicable in the setting of nucleos(t)ide analog (NA) treatment, which suppresses HBV DNA. Traditional assays for quantification of HBV RNA rely on labor-intensive 3'RACE assays targeting the polyA tail. In this study, the high-throughput Roche cobas®HBV RNA investigational assay was assessed on the Roche cobas® 6800 automated platform. Of 969 samples collected for a NA treatment cessation trial, and tested on the cobas assay, 249 were analyzed for sensitivity, reproducibility, sample type applicability, and results were compared to a RACE-based assay. Results of 97 paired serum and plasma samples demonstrated an excellent correlation of 0.98. However, 14.5% of plasma samples yielded detectable (below the limit of quantification) results, when the paired serum was undetectable, and plasma was shown to yield a statistically significant (p < 0.001) greater mean 0.119 log10 copies/ml. Quantification of 152 samples showed good correlation (0.91) between the cobas and RACE assays. The cobas assay demonstrated superior lower limit of quantification, 10 copies/ml, which resulted in detection of 13.2% more samples than the RACE assay. Reproducibility and linear range of the automated assay were also confirmed. The Roche cobas assay for HBV RNA is sensitive and highly recommended.
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    Financial sustainability planning for immunization services in Cambodia
    Soeung, SC ; Grundy, J ; Maynard, J ; Brooks, A ; Boreland, M ; Sarak, D ; Jenkinson, K ; Biggs, B-A (OXFORD UNIV PRESS, 2006-07)
    The expanded programme of immunization was established in Cambodia in 1986. In 2002, 67% of eligible children were immunized, despite significant health sector and macro-economic financial constraints. A financial sustainability planning process for immunization was introduced in 2002, in order to mobilize national and international resources in support of the achievement of child health objectives. The aim of this paper is to outline this process, describe its early impact as an advocacy tool and recommend additional strategies for mobilizing additional resources for health. The methods of financial sustainability planning are described, including the advocacy strategies that were applied. Analysis of financial sustainability planning results indicates rising programme costs associated with new vaccine introduction and new technologies. Despite this, the national programme has demonstrated important early successes in using financial sustainability planning to advocate for increased mobilization of national and international sources of funding for immunization. The national immunization programme nevertheless faces formidable system and financial challenges in the coming years associated with rising costs, potentially diminishing sources of international assistance, and the developing role of sub-national authorities in programme management and financing.