Doherty Institute - Research Publications

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    Memory CD4+ T cells that co-express PD1 and CTLA4 have reduced response to activating stimuli facilitating HIV latency.
    Rasmussen, TA ; Zerbato, JM ; Rhodes, A ; Tumpach, C ; Dantanarayana, A ; McMahon, JH ; Lau, JSY ; Chang, JJ ; Gubser, C ; Brown, W ; Hoh, R ; Krone, M ; Pascoe, R ; Chiu, CY ; Bramhall, M ; Lee, HJ ; Haque, A ; Fromentin, R ; Chomont, N ; Milush, J ; Van der Sluis, RM ; Palmer, S ; Deeks, SG ; Cameron, PU ; Evans, V ; Lewin, SR (Elsevier BV, 2022-10-18)
    Programmed cell death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) suppress CD4+ T cell activation and may promote latent HIV infection. By performing leukapheresis (n = 21) and lymph node biopsies (n = 8) in people with HIV on antiretroviral therapy (ART) and sorting memory CD4+ T cells into subsets based on PD1/CTLA4 expression, we investigate the role of PD1 and CTLA 4 in HIV persistence. We show that double-positive (PD1+CTLA4+) cells in blood contain more HIV DNA compared with double-negative (PD1-CTLA4-) cells but still have a lower proportion of cells producing multiply spliced HIV RNA after stimulation as well as reduced upregulation of T cell activation and proliferation markers. Transcriptomics analyses identify differential expression of key genes regulating T cell activation and proliferation with MAF, KLRB1, and TIGIT being upregulated in double-positive compared with double-negative cells, whereas FOS is downregulated. We conclude that, in addition to being enriched for HIV DNA, double-positive cells are characterized by negative signaling and a reduced capacity to respond to stimulation, favoring HIV latency.
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    Assessing the fitness of a dual-antiviral drug resistant human influenza virus in the ferret model
    Stannard, HL ; Mifsud, EJ ; Wildum, S ; Brown, SK ; Koszalka, P ; Shishido, T ; Kojima, S ; Omoto, S ; Baba, K ; Kuhlbusch, K ; Hurt, AC ; Barr, IG (NATURE PORTFOLIO, 2022-09-28)
    Influenza antivirals are important tools in our fight against annual influenza epidemics and future influenza pandemics. Combinations of antivirals may reduce the likelihood of drug resistance and improve clinical outcomes. Previously, two hospitalised immunocompromised influenza patients, who received a combination of a neuraminidase inhibitor and baloxavir marboxil, shed influenza viruses resistant to both drugs. Here-in, the replicative fitness of one of these A(H1N1)pdm09 virus isolates with dual resistance mutations (NA-H275Y and PA-I38T) was similar to wild type virus (WT) in vitro, but reduced in the upper respiratory tracts of challenged ferrets. The dual-mutant virus transmitted well between ferrets in an airborne transmission model, but was outcompeted by the WT when the two viruses were co-administered. These results indicate the dual-mutant virus had a moderate loss of viral fitness compared to the WT virus, suggesting that while person-to-person transmission of the dual-resistant virus may be possible, widespread community transmission is unlikely.
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    Whole genome sequencing for tuberculosis in Victoria, Australia: A genomic implementation study from 2017 to 2020.
    Dale, K ; Globan, M ; Horan, K ; Sherry, N ; Ballard, S ; Tay, EL ; Bittmann, S ; Meagher, N ; Price, DJ ; Howden, BP ; Williamson, DA ; Denholm, J (Elsevier BV, 2022-11)
    BACKGROUND: Whole genome sequencing (WGS) is increasingly used by tuberculosis (TB) programs to monitor Mycobacterium tuberculosis (Mtb) transmission. We aimed to characterise the molecular epidemiology of TB and Mtb transmission in the low-incidence setting of Victoria, Australia, and assess the utility of WGS. METHODS: WGS was performed on all first Mtb isolates from TB cases from 2017 to 2020. Potential clusters (≤12 single nucleotide polymorphisms [SNPs]) were investigated for epidemiological links. Transmission events in highly-related (≤5 SNPs) clusters were classified as likely or possible, based on the presence or absence of an epidemiological link, respectively. Case characteristics and transmission settings (as defined by case relationship) were summarised. Poisson regression was used to examine associations with secondary case number. FINDINGS: Of 1844 TB cases, 1276 (69.2%) had sequenced isolates, with 182 (14.2%) in 54 highly-related clusters, 2-40 cases in size. Following investigation, 140 cases (11.0% of sequenced) were classified as resulting from likely/possible local-transmission, including 82 (6.4%) for which transmission was likely. Common identified transmission settings were social/religious (26.4%), household (22.9%) and family living in different households (7.1%), but many were uncertain (41.4%). While household transmission featured in many clusters (n = 24), clusters were generally smaller (median = 3 cases) than the fewer that included transmission in social/religious settings (n = 12, median = 7.5 cases). Sputum-smear-positivity was associated with higher secondary case numbers. INTERPRETATION: WGS results suggest Mtb transmission commonly occurs outside the household in our low-incidence setting. Further work is required to optimise the use of WGS in public health management of TB. FUNDING: The Victorian Tuberculosis Program receives block funding for activities including case management and contact tracing from the Victorian Department of Health. No specific funding for this report was received by manuscript authors or the Victorian Tuberculosis Program, and the funders had no role in the study design, data collection, data analysis, interpretation or report writing.
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    Phenotypic analysis of the unstimulated in vivo HIV CD4 T cell reservoir
    Neidleman, J ; Luo, X ; Frouard, J ; Xie, G ; Hsiao, F ; Ma, T ; Morcilla, V ; Lee, A ; Telwatte, S ; Thomas, R ; Tamaki, W ; Wheeler, B ; Hoh, R ; Somsouk, M ; Vohra, P ; Milush, J ; James, KS ; Archin, NM ; Hunt, PW ; Deeks, SG ; Yukl, SA ; Palmer, S ; Greene, WC ; Roan, NR (ELIFE SCIENCES PUBLICATIONS LTD, 2020-09-29)
    The latent reservoir is a major barrier to HIV cure. As latently infected cells cannot be phenotyped directly, the features of the in vivo reservoir have remained elusive. Here, we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently infected cells reactivated ex vivo to their original pre-activation states. Our results suggest that, contrary to common assumptions, the reservoir is not randomly distributed among cell subsets, and is remarkably conserved between individuals. However, reservoir composition differs between tissues and blood, as do cells successfully reactivated by different latency reversing agents. By selecting 8-10 of our 39 original CyTOF markers, we were able to isolate highly purified populations of unstimulated in vivo latent cells. These purified populations were highly enriched for replication-competent and intact provirus, transcribed HIV, and displayed clonal expansion. The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies on HIV persistence.
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    Tissue memory CD4+T cells expressing IL-7 receptor-alpha (CD127) preferentially support latent HIV-1 infection
    Hsiao, F ; Frouard, J ; Gramatica, A ; Xie, G ; Telwatte, S ; Lee, GQ ; Roychoudhury, P ; Schwarzer, R ; Luo, X ; Yukl, SA ; Lee, S ; Hoh, R ; Deeks, SG ; Jones, RB ; Cavrois, M ; Greene, WC ; Roan, NR ; Douek, DC (PUBLIC LIBRARY SCIENCE, 2020-04-01)
    The primary reservoir for HIV is within memory CD4+ T cells residing within tissues, yet the features that make some of these cells more susceptible than others to infection by HIV is not well understood. Recent studies demonstrated that CCR5-tropic HIV-1 efficiently enters tissue-derived memory CD4+ T cells expressing CD127, the alpha chain of the IL7 receptor, but rarely completes the replication cycle. We now demonstrate that the inability of HIV to replicate in these CD127-expressing cells is not due to post-entry restriction by SAMHD1. Rather, relative to other memory T cell subsets, these cells are highly prone to undergoing latent infection with HIV, as revealed by the high levels of integrated HIV DNA in these cells. Host gene expression profiling revealed that CD127-expressing memory CD4+ T cells are phenotypically distinct from other tissue memory CD4+ T cells, and are defined by a quiescent state with diminished NFκB, NFAT, and Ox40 signaling. However, latently-infected CD127+ cells harbored unspliced HIV transcripts and stimulation of these cells with anti-CD3/CD28 reversed latency. These findings identify a novel subset of memory CD4+ T cells found in tissue and not in blood that are preferentially targeted for latent infection by HIV, and may serve as an important reservoir to target for HIV eradication efforts.
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    Heterogeneity in HIV and cellular transcription profiles in cell line models of latent and productive infection: implications for HIV latency
    Telwatte, S ; Moron-Lopez, S ; Aran, D ; Kim, P ; Hsieh, C ; Joshi, S ; Montano, M ; Greene, WC ; Butte, AJ ; Wong, JK ; Yukl, SA (BMC, 2019-11-11)
    BACKGROUND: HIV-infected cell lines are widely used to study latent HIV infection, which is considered the main barrier to HIV cure. We hypothesized that these cell lines differ from each other and from cells from HIV-infected individuals in the mechanisms underlying latency. RESULTS: To quantify the degree to which HIV expression is inhibited by blocks at different stages of HIV transcription, we employed a recently-described panel of RT-ddPCR assays to measure levels of 7 HIV transcripts ("read-through," initiated, 5' elongated, mid-transcribed/unspliced [Pol], distal-transcribed [Nef], polyadenylated, and multiply-sliced [Tat-Rev]) in bulk populations of latently-infected (U1, ACH-2, J-Lat) and productively-infected (8E5, activated J-Lat) cell lines. To assess single-cell variation and investigate cellular genes associated with HIV transcriptional blocks, we developed a novel multiplex qPCR panel and quantified single cell levels of 7 HIV targets and 89 cellular transcripts in latently- and productively-infected cell lines. The bulk cell HIV transcription profile differed dramatically between cell lines and cells from ART-suppressed individuals. Compared to cells from ART-suppressed individuals, latent cell lines showed lower levels of HIV transcriptional initiation and higher levels of polyadenylation and splicing. ACH-2 and J-Lat cells showed different forms of transcriptional interference, while U1 cells showed a block to elongation. Single-cell studies revealed marked variation between/within cell lines in expression of HIV transcripts, T cell phenotypic markers, antiviral factors, and genes implicated in latency. Expression of multiply-spliced HIV Tat-Rev was associated with expression of cellular genes involved in activation, tissue retention, T cell transcription, and apoptosis/survival. CONCLUSIONS: HIV-infected cell lines differ from each other and from cells from ART-treated individuals in the mechanisms governing latent HIV infection. These differences in viral and cellular gene expression must be considered when gauging the suitability of a given cell line for future research on HIV. At the same time, some features were shared across cell lines, such as low expression of antiviral defense genes and a relationship between productive infection and genes involved in survival. These features may contribute to HIV latency or persistence in vivo, and deserve further study using novel single cell assays such as those described in this manuscript.
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    As Omicron Takes Hold and Other New Variants Arise, COVID-19 Testing Remains the Universally Agreed Tool to Effect Transition From Pandemic to Endemic State.
    Miller, MB ; Ooi, EE ; Rhoads, DD ; Kulldorff, M ; Anderson, DE ; Lee, H ; Gupta, S ; Mel, K (Frontiers Media SA, 2022)
    The COVID-19 pandemic has caused more than 448 million cases and 6 million deaths worldwide to date. Omicron is now the dominant SARS-CoV-2 variant, making up more than 90% of cases in countries reporting sequencing data. As the pandemic continues into its third year, continued testing is a strategic and necessary tool for transitioning to an endemic state of COVID-19. Here, we address three critical topics pertaining to the transition from pandemic to endemic: defining the endemic state for COVID-19, highlighting the role of SARS-CoV-2 testing as endemicity is approached, and recommending parameters for SARS-CoV-2 testing once endemicity is reached. We argue for an approach that capitalizes on the current public health momentum to increase capacity for PCR-based testing and whole genome sequencing to monitor emerging infectious diseases. Strategic development and utilization of testing, including viral panels in addition to vaccination, can keep SARS-CoV-2 in a manageable endemic state and build a framework of preparedness for the next pandemic.
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    Influenza A(H1N1)pdm09 But Not A(H3N2) Virus Infection Induces Durable Seroprotection: Results From the Ha Nam Cohort( )
    Le, NMH ; Sullivan, SG ; Le, QM ; Khvorov, A ; Hoang, VMP ; Nguyen, LKH ; Pham, QT ; Le, TT ; Carolan, L ; Dang, DA ; Tran, ND ; Bryant, JE ; van Doorn, HR ; Wertheim, HFL ; Horby, P ; Fox, A (OXFORD UNIV PRESS INC, 2022-08-12)
    BACKGROUND: The extent to which influenza recurrence depends upon waning immunity from prior infection is undefined. We used antibody titers of Ha-Nam cohort participants to estimate protection curves and decay trajectories. METHODS: Households (270) participated in influenza-like-illness (ILI) surveillance and provided blood at intervals spanning laboratory-confirmed virus transmission. Sera were tested in hemagglutination inhibition assay. Infection was defined as influenza virus-positive ILI and/or seroconversion. Median protective titers were estimated using scaled-logistic regression to model pretransmission titer against infection status in that season, limiting analysis to households with infection(s). Titers were modelled against month since infection using mixed-effects linear regression to estimate decay and when titers fell below protection thresholds. RESULTS: From December 2008-2012, 295 and 314 participants were infected with H1N1pdm09-like and A/Perth/16/09-like (H3N2Pe09) viruses, respectively. The proportion protected rose more steeply with titer for H1N1pdm09 than for H3N2Pe09, and estimated 50% protection titers were 19.6 and 37.3, respectively. Postinfection titers started higher against H3N2Pe09 but decayed more steeply than against H1N1pdm09. Seroprotection was estimated to be sustained against H1N1pdm09 but to wane by 8-months for H3N2Pe09. CONCLUSIONS: Estimates indicate that infection induces durable seroprotection against H1N1pdm09 but not H3N2Pe09, which could in part account for the younger age of A(H1N1) versus A(H3N2) cases.
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    Multiparameter immunohistochemistry analysis of HIV DNA, RNA and immune checkpoints in lymph node tissue
    Richardson, ZA ; Deleage, C ; Tutuka, CSA ; Walkiewicz, M ; Del Rio-Estrada, PM ; Pascoe, RD ; Evans, VA ; Reyesteran, G ; Gonzales, M ; Roberts-Thomson, S ; Gonzalez-Navarro, M ; Torres-Ruiz, F ; Estes, JD ; Lewin, SR ; Cameron, PU (ELSEVIER, 2022-02-01)
    The main barrier to a cure for HIV is the persistence of long-lived and proliferating latently infected CD4+ T-cells despite antiretroviral therapy (ART). Latency is well characterized in multiple CD4+ T-cell subsets, however, the contribution of regulatory T-cells (Tregs) expressing FoxP3 as well as immune checkpoints (ICs) PD-1 and CTLA-4 as targets for productive and latent HIV infection in people living with HIV on suppressive ART is less well defined. We used multiplex detection of HIV DNA and RNA with immunohistochemistry (mIHC) on formalin-fixed paraffin embedded (FFPE) cells to simultaneously detect HIV RNA and DNA and cellular markers. HIV DNA and RNA were detected by in situ hybridization (ISH) (RNA/DNAscope) and IHC was used to detect cellular markers (CD4, PD-1, FoxP3, and CTLA-4) by incorporating the tyramide system amplification (TSA) system. We evaluated latently infected cell lines, a primary cell model of HIV latency and excisional lymph node (LN) biopsies collected from people living with HIV (PLWH) on and off ART. We clearly detected infected cells that coexpressed HIV RNA and DNA (active replication) and DNA only (latently infected cells) in combination with IHC markers in the in vitro infection model as well as LN tissue from PLWH both on and off ART. Combining ISH targeting HIV RNA and DNA with IHC provides a platform to detect and quantify HIV persistence within cells identified by multiple markers in tissue samples from PLWH on ART or to study HIV latency.
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    Bats and Coronaviruses in the Context of COVID-19.
    Wang, L ; Ahn, M ; Anderson, DE (Chinese Center for Disease Control and Prevention, 2021-02-12)