Doherty Institute - Research Publications

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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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    Maintaining a social license to operate for wastewater-based monitoring: The case of managing infectious disease and the COVID-19 pandemic
    Cooper, B ; Donner, E ; Crase, L ; Robertson, H ; Carter, D ; Short, M ; Drigo, B ; Leder, K ; Roiko, A ; Fielding, K (ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2022-10-15)
    Wastewater monitoring as a public health tool is well-established and the SARS-CoV-2 (COVID-19) pandemic has seen its widespread uptake. Given the significant potential of wastewater monitoring as a public health surveillance and decision support tool, it is important to understand what measures are required to allow the long-term benefits of wastewater monitoring to be fully realized, including how to establish and/or maintain public support. The potential for positive SARS-CoV-2 detections to trigger enforced, community-wide public health interventions (e.g., lockdowns and other impacts on civil liberties) further emphasises the need to better understand the role of public engagement in successful wastewater-based monitoring programs. This paper systematically reviews the processes of building and maintaining the social license to operate wastewater monitoring. We specifically explore the relationship between different stakeholder communities and highlight the information and actions that are required to establish a social license to operate and then prevent its loss. The paper adds to the literature on social license to operate by extending its application to new domains and offers a dynamic model of social license to help guide the agenda for researcher and practitioner communities.
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    Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts
    Telwatte, S ; Martin, HA ; Marczak, R ; Fozouni, P ; Vallejo-Gracia, A ; Kumar, GR ; Murray, V ; Lee, S ; Ott, M ; Wong, JK ; Yukl, SA (ACADEMIC PRESS INC ELSEVIER SCIENCE, 2022-05)
    The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.
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    Organizational responses to the COVID-19 pandemic in Victoria, Australia: A qualitative study across four healthcare settings
    McGuinness, SL ; Josphin, J ; Eades, O ; Clifford, S ; Fisher, J ; Kirkman, M ; Russell, G ; Hodgson, CL ; Kelsall, HL ; Lane, R ; Skouteris, H ; Smith, KL ; Leder, K (FRONTIERS MEDIA SA, 2022-09-29)
    OBJECTIVE: Organizational responses that support healthcare workers (HCWs) and mitigate health risks are necessary to offset the impact of the COVID-19 pandemic. We aimed to understand how HCWs and key personnel working in healthcare settings in Melbourne, Australia perceived their employing organizations' responses to the COVID-19 pandemic. METHOD: In this qualitative study, conducted May-July 2021 as part of the longitudinal Coronavirus in Victorian Healthcare and Aged Care Workers (COVIC-HA) study, we purposively sampled and interviewed HCWs and key personnel from healthcare organizations across hospital, ambulance, aged care and primary care (general practice) settings. We also examined HCWs' free-text responses to a question about organizational resources and/or supports from the COVIC-HA Study's baseline survey. We thematically analyzed data using an iterative process. RESULTS: We analyzed data from interviews with 28 HCWs and 21 key personnel and free-text responses from 365 HCWs, yielding three major themes: navigating a changing and uncertain environment, maintaining service delivery during a pandemic, and meeting the safety and psychological needs of staff . HCWs valued organizational efforts to engage openly and honesty with staff, and proactive responses such as strategies to enhance workplace safety (e.g., personal protective equipment spotters). Suggestions for improvement identified in the themes included streamlined information processes, greater involvement of HCWs in decision-making, increased investment in staff wellbeing initiatives and sustainable approaches to strengthen the healthcare workforce. CONCLUSIONS: This study provides in-depth insights into the challenges and successes of organizational responses across four healthcare settings in the uncertain environment of a pandemic. Future efforts to mitigate the impact of acute stressors on HCWs should include a strong focus on bidirectional communication, effective and realistic strategies to strengthen and sustain the healthcare workforce, and greater investment in flexible and meaningful psychological support and wellbeing initiatives for HCWs.
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    Combination Immune Checkpoint Blockade Enhances IL-2 and CD107a Production from HIV-Specific T Cells Ex Vivo in People Living with HIV on Antiretroviral Therapy
    Chiu, CY ; Chang, JJ ; Dantanarayana, A ; Solomon, A ; Evans, VA ; Pascoe, R ; Gubser, C ; Trautman, L ; Fromentin, R ; Chomont, N ; McMahon, JH ; Cameron, PU ; Rasmussen, TA ; Lewin, SR (AMER ASSOC IMMUNOLOGISTS, 2022-01-01)
    In people with HIV (PWH) on antiretroviral therapy (ART), immune dysfunction persists, including elevated expression of immune checkpoint (IC) proteins on total and HIV-specific T cells. Reversing immune exhaustion is one strategy to enhance the elimination of HIV-infected cells that persist in PWH on ART. We aimed to evaluate whether blocking CTL-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell Ig domain and mucin domain 3 (TIM-3), T cell Ig and ITIM domain (TIGIT) and lymphocyte activation gene-3 (LAG-3) alone or in combination would enhance HIV-specific CD4+ and CD8+ T cell function ex vivo. Intracellular cytokine staining was performed using human PBMCs from PWH on ART (n = 11) and expression of CD107a, IFN-γ, TNF-α, and IL-2 was quantified with HIV peptides and Abs to IC. We found the following: 1) IC blockade enhanced the induction of CD107a and IL-2 but not IFN-γ and TNF-α in response to Gag and Nef peptides; 2) the induction of CD107a and IL-2 was greatest with multiple combinations of two Abs; and 3) Abs to LAG-3, CTLA-4, and TIGIT in combinations showed synergistic induction of IL-2 in HIV-specific CD8+ and CD107a and IL-2 production in HIV-specific CD4+ and CD8+ T cells. These results demonstrate that the combination of Abs to LAG-3, CTLA-4, or TIGIT can increase the frequency of cells expressing CD107a and IL-2 that associated with cytotoxicity and survival of HIV-specific CD4+ and CD8+ T cells in PWH on ART. These combinations should be further explored for an HIV cure.
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    Induction of Interferon-Stimulated Genes Correlates with Reduced Growth of Influenza A Virus in Lungs after RIG-I Agonist Treatment of Ferrets
    Schwab, LSU ; Londrigan, SL ; Brooks, AG ; Hurt, AC ; Sahu, A ; Deng, Y-M ; Moselen, J ; Coch, C ; Zillinger, T ; Hartmann, G ; Reading, PC ; Heise, MT (AMER SOC MICROBIOLOGY, 2022-08-24)
    Intracellular RIG-I receptors represent key innate sensors of RNA virus infection, and RIG-I activation results in the induction of hundreds of host effector genes, including interferon-stimulated genes (ISGs). Synthetic RNA agonists targeting RIG-I have shown promise as antivirals against a broad spectrum of viruses, including influenza A virus (IAV), in both in vitro and mouse models of infection. Herein, we demonstrate that treatment of a ferret airway epithelial (FRL) cell line with a RIG-I agonist rapidly and potently induced expression of a broad range of ISGs and resulted in potent inhibition of growth of different IAV strains. In ferrets, a single intravenous injection of RIG-I agonist was associated with upregulated ISG expression in peripheral blood mononuclear cells and lung tissue, but not in nasal tissues. In a ferret model of viral contact transmission, a single treatment of recipient animals 24 h prior to cohousing with IAV-infected donors did not reduce virus transmission and shedding but did result in reduced lung virus titers 6 days after treatment. A single treatment of the IAV-infected donor animals also resulted in reduced virus titers in the lungs 2 days later. Thus, a single intravenous treatment with RIG-I agonist prior to infection or to ferrets with an established IAV infection can reduce virus growth in the lungs. These findings support further development of RIG-I agonists as effective antiviral treatments to limit the impact of IAV infections, particularly in reducing virus replication in the lower airways. IMPORTANCE RIG-I agonists have shown potential as broad-spectrum antivirals in vitro and in mouse models of infection. However, their antiviral potential has not been reported in outbred animals such as ferrets, which are widely regarded as the gold standard small animal model for human IAV infections. Herein, we demonstrate that RIG-I agonist treatment of a ferret airway cell line resulted in ISG induction and inhibition of a broad range of human influenza viruses. A single intravenous treatment of ferrets also resulted in systemic induction of ISGs, including in lung tissue, and when delivered to animals prior to IAV exposure or to animals with established IAV infection treatment resulted in reduced virus replication in the lungs. These data demonstrate the effectiveness of single RIG-I treatment against IAV in the ferret model and highlight the importance of future studies to optimize treatment regimens and delivery routes to maximize their ability to ameliorate IAV infections.
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    Hepatitis B Virus Flares After Nucleot(s)ide Analogue Cessation Are Associated With Activation of Toll-Like Receptor Signaling Pathways
    Al Hall, S ; Burns, GS ; Mooney, BJ ; Millen, R ; Morris, R ; Vogrin, S ; Sundararajan, V ; Ratnam, D ; Levy, MT ; Lubel, JS ; Nicoll, AJ ; Strasser, S ; Sievert, W ; Desmond, P ; Ngu, MC ; Angus, P ; Sinclair, M ; Meredith, C ; Matthews, G ; Revill, PA ; Jackson, K ; Littlejohn, M ; Bowden, S ; Locarnini, SA ; Thompson, AJ ; Visvanathan, K (OXFORD UNIV PRESS INC, 2022-12-28)
    BACKGROUND: We evaluated the patterns of peripheral Toll-like receptor (TLR) signaling activity and the expression of TLRs and natural killer (NK) cell activation in a cohort of patients experiencing severe hepatitis flares after stopping nucleot(s)ide analogues (NAs) therapy. METHODS: Samples were collected longitudinally from patients with chronic hepatitis B who were enrolled in a prospective study of NA discontinuation. Patients experiencing hepatitis flares were compared with patients with normal alanine aminotransferase. Peripheral blood mononuclear cells (PBMCs) were stimulated with TLR ligands and cytokine secretion in the cell culture supernatant measured. Expression of TLR2/4, NKG2D, NKp46, and triggering receptor expressed on myeloid cells 1 (TREM-1) on monocytes, NK, and NK-T cells was measured. RESULTS: Seventeen patients with severe reactivation hepatitis flares were compared to 12 nonflare patients. Hepatitis flares were associated with increased activity of TLR2-8 and TLR9 signaling in PBMCs at the time of peak flare compared to baseline. Hepatitis flares were also associated with (1) upregulation of TLR2 and (2) TREM-1 receptor expression on NK. There were no differences at baseline between flare patients and nonflare patients. CONCLUSIONS: Hepatitis flares off NA therapy have a significant innate inflammatory response with upregulation of TLR signaling on peripheral monocytes and TLR2 and TREM-1 expression on NK cells. This implicates the innate immune system in the immunopathogenesis of hepatitis B flares.
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    Influenza virus infection history shapes antibody responses to influenza vaccination
    Auladell, M ; Hoang, VMP ; Le, TQM ; Tseng, Y-Y ; Carolan, L ; Wilks, S ; Pham, QT ; Price, D ; Nguyen, TD ; Nguyen, LKH ; Le, TT ; Nguyen, THT ; Tran, TKH ; Nguyen, TND ; Vu, TNB ; Khvorov, A ; Hensen, L ; Tran, ND ; Kedzierska, K ; Dang, DA ; Wertheim, H ; Boyd, SD ; Good-Jacobson, KL ; Smith, D ; Barr, I ; Sullivan, S ; van Doorn, HR ; Fox, A (NATURE PORTFOLIO, 2022-02)
    Studies of successive vaccination suggest that immunological memory against past influenza viruses may limit responses to vaccines containing current strains. The impact of memory induced by prior infection is rarely considered and is difficult to ascertain, because infections are often subclinical. This study investigated influenza vaccination among adults from the Ha Nam cohort (Vietnam), who were purposefully selected to include 72 with and 28 without documented influenza A(H3N2) infection during the preceding 9 years (Australian New Zealand Clinical Trials Registry 12621000110886). The primary outcome was the effect of prior influenza A(H3N2) infection on hemagglutinin-inhibiting antibody responses induced by a locally available influenza vaccine administered in November 2016. Baseline and postvaccination sera were titrated against 40 influenza A(H3N2) strains spanning 1968-2018. At each time point (baseline, day 14 and day 280), geometric mean antibody titers against 2008-2018 strains were higher among participants with recent infection (34 (29-40), 187 (154-227) and 86 (72-103)) than among participants without recent infection (19 (17-22), 91 (64-130) and 38 (30-49)). On days 14 and 280, mean titer rises against 2014-2018 strains were 6.1-fold (5.0- to 7.4-fold) and 2.6-fold (2.2- to 3.1-fold) for participants with recent infection versus 4.8-fold (3.5- to 6.7-fold) and 1.9-fold (1.5- to 2.3-fold) for those without. One of 72 vaccinees with recent infection versus 4 of 28 without developed symptomatic A(H3N2) infection in the season after vaccination (P = 0.021). The range of A(H3N2) viruses recognized by vaccine-induced antibodies was associated with the prior infection strain. These results suggest that recall of immunological memory induced by prior infection enhances antibody responses to inactivated influenza vaccine and is important to attain protective antibody titers.
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    Secreted hepatitis B virus splice variants differ by HBV genotype and across phases of chronic hepatitis B infection
    Maslac, O ; Wagner, J ; Sozzi, V ; Mason, H ; Svarovskaia, J ; Tan, S ; Gaggar, A ; Locarnini, S ; Yuen, L ; Littlejohn, M ; Revill, PA (WILEY, 2022-08)
    Chronic hepatitis B (CHB) is characterized by progression through different phases of hepatitis B virus (HBV) infection and disease. Although not necessary for HBV replication, there is increasing evidence that HBV splice variants are associated with liver disease progression and pathogenesis. However, there have been no studies till date on the frequency or diversity of splice variants for different HBV genotypes across the phases of CHB. Next generation sequencing data from 404 patient samples of HBV genotype A, B, C or D in Phase I, Phase II or Phase IV of CHB was analysed for HBV splice variants using an in house bioinformatics pipeline. HBV splice variants differed in frequency and type by genotype and phase of natural history. Splice variant Sp1 was the most frequently detected (206/404, 51% of patients), followed by Sp13 (151/404 37% of patients). The frequency of variants was generally highest in Phase II (123/165, 75% of patients), a phase typically associated with enhanced immune activation, followed by Phase I (69/99, 70% of patients). Splice variants were associated with reduced hepatitis B e antigen (HBeAg) levels and statistically reduced likelihood of achieving HBsAg loss (functional cure) in Phase II patients for Sp1 and Sp13 (p = .0014 and .0156, respectively). The frequency of HBV splice variants in patient serum differed markedly by HBV genotype and phase of CHB natural history. The increased levels of HBV splice variants detected in CHB phase II patients compared with the higher replicative Phase I in particular warrants further investigation.