The genomic integration of HIV into cells results in long-term persistence of virally infected cell populations. This integration event acts as a heritable mark that can be tracked to monitor infected cells that persist over time. Previous reports have documented clonal expansion in people and have linked them to proto-oncogenes; however, their significance or contribution to the latent reservoir has remained unclear. Here, we demonstrate that a directed pattern of clonal expansion occurs in vivo, specifically in gene pathways important for viral replication and persistence. These biological processes include cellular division, transcriptional regulation, RNA processing, and posttranslational modification pathways. This indicates preferential expansion when integration events occur within genes or biological pathways beneficial for HIV replication and persistence. Additionally, these expansions occur quickly during unsuppressed viral replication in vivo, reinforcing the importance of early intervention for individuals to limit reservoir seeding of clonally expanded HIV-infected cells.
Kevin G. Haworth, Lauren E. Schefter, Zachary K. Norgaard, Christina Ironside, Jennifer E. Adair, Hans-Peter Kiem
The nasal mucosa is an important component of mucosal immunity. Immunogenic particles in inspired air are known to activate the local nasal mucosal immune system and can lead to sinonasal inflammation; however, little is known about the effect of this activation on the lung immune environment. Here, we showed that nasal inoculation of murine coronavirus (CoV) in the absence of direct lung infection primes the lung immune environment by recruiting activated monocytes (Ly6C+ inflammatory monocytes) and NK cells into the lungs. Unlike infiltration of these cells into directly infected lungs, a process that requires type I IFN signaling, nasally induced infiltration of Ly6C+ inflammatory monocytes into the lungs is IFN-I independent. These activated macrophages ingested antigen and migrated to pulmonary lymph nodes, and enhanced both innate and adaptive immunity after heterologous virus infection. Clinically, such nasal-only inoculation of MHV-1 failed to cause pneumonia but significantly reduced mortality and morbidity of lethal pneumonia caused by severe acute respiratory syndrome CoV (SARS-CoV) or influenza A virus. Together, the data indicate that the nose and upper airway remotely prime the lung immunity to protect the lungs from direct viral infections.
Xiaoyang Hua, Rahul Vijay, Rudragouda Channappanvar, Jeremiah Athmer, David K. Meyerholz, Nitin Pagedar, Stephen Tilley, Stanley Perlman
Type 1 diabetes (T1D) is a chronic disease characterized by an autoimmune-mediated destruction of insulin-producing pancreatic β cells. Environmental factors such as viruses play an important role in the onset of T1D and interact with predisposing genes. Recent data suggest that viral infection of human islets leads to a decrease in insulin production rather than β cell death, suggesting loss of β cell identity. We undertook this study to examine whether viral infection could induce human β cell dedifferentiation. Using the functional human β cell line EndoC-βH1, we demonstrate that polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA that mimics a byproduct of viral replication, induces a decrease in β cell–specific gene expression. In parallel with this loss, the expression of progenitor-like genes such as SOX9 was activated following PolyI:C treatment or enteroviral infection. SOX9 was induced by the NF-κB pathway and also in a paracrine non–cell-autonomous fashion through the secretion of IFN-α. Lastly, we identified SOX9 targets in human β cells as potentially new markers of dedifferentiation in T1D. These findings reveal that inflammatory signaling has clear implications in human β cell dedifferentiation.
Masaya Oshima, Klaus-Peter Knoch, Marc Diedisheim, Antje Petzold, Pierre Cattan, Marco Bugliani, Piero Marchetti, Pratik Choudhary, Guo-Cai Huang, Stefan R. Bornstein, Michele Solimena, Olivier Albagli-Curiel, Raphael Scharfmann
Dengue virus (DENV) is the most prevalent mosquito-borne virus causing human disease. Of the 4 DENV serotypes, epidemiological data suggest that DENV-2 secondary infections are associated with more severe disease than DENV-4 infections. Mass cytometry by time-of-flight (CyTOF) was used to dissect immune changes induced by DENV-2 and DENV-4 in human DCs, the initial targets of primary infections that likely affect infection outcomes. Strikingly, DENV-4 replication peaked earlier and promoted stronger innate immune responses, with increased expression of DC activation and migration markers and increased cytokine production, compared with DENV-2. In addition, infected DCs produced higher levels of inflammatory cytokines compared with bystander DCs, which mainly produced IFN-induced cytokines. These high-dimensional analyses during DENV-2 and DENV-4 infections revealed distinct viral signatures marked by different replication strategies and antiviral innate immune induction in DCs, which may result in different viral fitness, transmission, and pathogenesis.
Rebecca E. Hamlin, Adeeb Rahman, Theodore R. Pak, Kevin Maringer, Ignacio Mena, Dabeiba Bernal-Rubio, Uma Potla, Ana M. Maestre, Anthony C. Fredericks, El-ad D. Amir, Andrew Kasarskis, Irene Ramos, Miriam Merad, Ana Fernandez-Sesma
Marie-Astrid Vernet, Stéphanie Reynard, Alexandra Fizet, Justine Schaeffer, Delphine Pannetier, Jeremie Guedj, Max Rives, Nadia Georges, Nathalie Garcia-Bonnet, Aboubacar I. Sylla, Péma Grovogui, Jean-Yves Kerherve, Christophe Savio, Sylvie Savio-Coste, Marie-Laure de Séverac, Philippe Zloczewski, Sandrine Linares, Souley Harouna, Bing M’Lebing Abdoul, Frederic Petitjean, Nenefing Samake, Susan Shepherd, Moumouni Kinda, Fara Roger Koundouno, Ludovic Joxe, Mathieu Mateo, Patrick Lecine, Audrey Page, Tang Maleki Tchamdja, Matthieu Schoenhals, Solenne Barbe, Bernard Simon, Tuan Tran-Minh, Christophe Longuet, François L’Hériteau, Sylvain Baize
Flow cytometry is utilized extensively for cellular analysis, but technical limitations have prevented its routine application for characterizing virus. The recent introduction of nanoscale fluorescence-activated cytometric cell sorting now allows analysis of individual virions. Here, we demonstrate staining and sorting of infectious HIV. Fluorescent antibodies specific for cellular molecules found on budding virions were used to label CCR5-tropic Bal HIV and CXCR4-tropic NL4.3 HIV Env-expressing pseudovirions made in THP-1 cells (monocyte/macrophage) and H9 cells (T cells), respectively. Using a flow cytometer, we resolved the stained virus beyond isotype staining and demonstrated purity and infectivity of sorted virus populations on cells with the appropriate coreceptors. We subsequently sorted infectious simian/human immunodeficiency virus from archived plasma. Recovery was approximately 0.5%, but virus present in plasma was already bound to viral-specific IgG generated in vivo, likely contributing to the low yield. Importantly, using two broadly neutralizing HIV antibodies, PG9 and VRC01, we also sorted virus from archived human plasma and analyzed the sorted populations genetically and by proteomics, identifying the quasispecies present. The ability to sort infectious HIV from clinically relevant samples provides material for detailed molecular, genetic, and proteomic analyses applicable to future design of vaccine antigens and potential development of personalized treatment regimens.
Thomas Musich, Jennifer C. Jones, Brandon F. Keele, Lisa M. Miller Jenkins, Thorsten Demberg, Thomas S. Uldrick, Robert Yarchoan, Marjorie Robert-Guroff
Epstein-Barr virus (EBV) infects B cells and ~95% of adults are infected. EBV glycoprotein gp42 is essential for entry of virus into B cells. EBV gp42 binds to the β1 chain of HLA-DQ, -DR, and -DP on B cells, and uses these molecules for infection. To investigate if certain HLA-DQ alleles are associated with EBV seronegativity, we recruited ~3,300 healthy adult blood donors, identified 106 EBV-seronegative individuals, and randomly selected a control group of EBV-seropositive donors from the donor pool. A larger than expected proportion of EBV-seronegative subjects were HLA-DQ β1 *04/*05 and *06/*06, and to a lesser extent, *02/*03, compared with the control group, while a larger than expected portion of EBV-seropositive persons were HLA-DQ β1 *02/*02. We examined the ability of EBV gp42 to bind to different HLA-DQ molecules using human and mouse cells stably expressing these alleles. EBV gp42 bound less effectively to cells expressing HLA-DQ β1 *04/*05, *06/*06, or *03/*03 than to cells expressing HLA-DQ β1 *02/*02. These data are consistent with our observations of increased EBV seronegativity with DQ β1 *04/*05 or *06/*06 alleles. These findings emphasize the importance of a single genetic locus (HLA-DQ β1) to influence infectivity with EBV.
Qingxue Li, Wei Bu, Erin Gabriel, Fiona Aguilar, Yo Hoshino, Hiroko Miyadera, Christoph Hess, Ronald L. Hornung, Amitava Roy, Jeffrey I. Cohen
The second-generation HIV-1 integrase strand transfer inhibitor (InSTI) dolutegravir (DTG) has had a major impact on the treatment of HIV-1 infection. Here we describe important but previously undetermined pharmacodynamic parameters for DTG. We show that the dose-response curve slope, which indicates cooperativity and is a major determinant of antiviral activity, is higher for DTG than for first-generation InSTIs. This steepness does not reflect inhibition of multiple steps in the HIV-1 life cycle, as is the case for allosteric integrase inhibitors and HIV-1 protease inhibitors. We also show that degree of independence, a metric of interaction favorability between antiretroviral drugs, is high for DTG and nucleoside reverse transcriptase inhibitors. Finally, we demonstrate poor selective advantage for HIV-1 bearing InSTI resistance mutations. Selective advantage, which incorporates both the magnitude of resistance conferred by a mutation and its fitness cost, explains the high genetic barrier to DTG resistance. Together, these parameters provide an explanation for the remarkable clinical success of DTG.
Sarah B. Laskey, Robert F. Siliciano
Viral hepatitis remains a global health challenge despite recent progress in the development of more effective therapies. Although virus-specific CD8+ and CD4+ T cell responses are essential for viral clearance, it remains largely unknown what regulates T cell–mediated viral clearance. Thus, a better understanding of the regulation of anti-viral T cell immunity would be critical for the design of more effective therapies for viral hepatitis. Using a model of adenovirus-induced hepatitis, here we showed that adenoviral infection induced recruitment of Ly6Chi monocytes to the liver in a CCR2-dependent manner. These recruited Ly6Chi monocytes suppressed CD8+ and CD4+ T cell responses to adenoviral infection, leading to a delay in viral clearance. In vivo depletion of Ly6Chi monocytes markedly enhanced anti-viral T cell responses and promoted viral clearance. Mechanistically, we showed that induction of iNOS and the production of NO by Ly6Chi monocytes are critical for the suppression of T cell responses. In addition, a contact-dependent mechanism mediated by PD-1 and PD-L1 interaction is also required for T cell suppression by Ly6Chi monocytes. These findings suggest a critical role for Ly6Chi monocytes in the regulation of T cell immunity in viral hepatitis and may provide new insights into development of more effective therapies for treating viral hepatitis based on targeting the immunosuppressing monocytes.
Jiangao Zhu, Huiyao Chen, Xiaopei Huang, Songfu Jiang, Yiping Yang
Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4–/– mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4–/– mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design.
Giridhar Murlidharan, Andrew Crowther, Rebecca A. Reardon, Juan Song, Aravind Asokan
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