Human herpes simplex virus-1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway resulting in impairment of central nervous system (CNS) cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-a/b induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-b protein, thereby also constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3-/- mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-b secretion and ISGs mRNA in induced pluripotent stem cell-derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro, and by which the human central nervous system prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-b immunity, rather than viral recognition triggering an amplification of IFN-a/b production.
Daxing Gao, Michael J. Ciancanelli, Peng Zhang, Oliver Harschnitz, Vincent Bondet, Mary Hasek, Jie Chen, Xin Mu, Yuval Itan, Aurélie Cobat, Vanessa Sancho-Shimizu, Benedetta Bigio, Lazaro Lorenzo, Gabriele Ciceri, Jessica L. McAlpine, Esperanza Anguiano, Emmanuelle Jouanguy, Damien Chaussabel, isabelle Meyts, Michael S. Diamond, Laurent Abel, Sun Hur, Gregory A. Smith, Luigi D. Notarangelo, Darragh Duffy, Lorenz Studer, Jean-Laurent Casanova, Shen-Ying Zhang
Human T cell leukemia virus type 1 (HTLV-1) is mainly transmitted vertically through breast milk. The rate of mother-to-child transmission (MTCT) through formula feeding, although significantly lower than through breastfeeding, is approximately 2.4%–3.6%, suggesting the possibility of alternative transmission routes. MTCT of HTLV-1 might occur through the uterus, birth canal, or placental tissues; the latter is known as transplacental transmission. Here, we found that HTLV-1 proviral DNA was present in the placental villous tissues of the fetuses of nearly half of pregnant carriers and in a small number of cord blood samples. An RNA ISH assay showed that HTLV-1–expressing cells were present in nearly all subjects with HTLV-1–positive placental villous tissues, and their frequency was significantly higher in subjects with HTLV-1–positive cord blood samples. Furthermore, placental villous trophoblasts expressed HTLV-1 receptors and showed increased susceptibility to HTLV-1 infection. In addition, HTLV-1–infected trophoblasts expressed high levels of viral antigens and promoted the de novo infection of target T cells in a humanized mouse model. In summary, during pregnancy of HTLV-1 carriers, HTLV-1 was highly expressed in placental villous tissues, and villous trophoblasts showed high HTLV-1 sensitivity, suggesting that MTCT of HTLV-1 occurs through the placenta.
Kenta Tezuka, Naoki Fuchi, Kazu Okuma, Takashi Tsukiyama, Shoko Miura, Yuri Hasegawa, Ai Nagata, Nahoko Komatsu, Hiroo Hasegawa, Daisuke Sasaki, Eita Sasaki, Takuo Mizukami, Madoka Kuramitsu, Sahoko Matsuoka, Katsunori Yanagihara, Kiyonori Miura, Isao Hamaguchi
Influenza is a significant cause of morbidity and mortality worldwide. Here we show changes in the abundance and activation states of more than 50 immune cell subsets in 35 individuals over 11 time points during human A/California/2009 (H1N1) virus challenge monitored using mass cytometry along with other clinical assessments. Peak change in monocyte, B cell, and T cell subset frequencies coincided with peak virus shedding, followed by marked activation of T and NK cells. Results led to the identification of CD38 as a critical regulator of plasmacytoid dendritic cell function in response to influenza virus. Machine learning using study-derived clinical parameters and single-cell data effectively classified and predicted susceptibility to infection. The coordinated immune cell dynamics defined in this study provide a framework for identifying novel correlates of protection in the evaluation of future influenza therapeutics.
Zainab Rahil, Rebecca Leylek, Christian M. Schürch, Han Chen, Zach Bjornson-Hooper, Shannon R. Christensen, Pier Federico Gherardini, Salil S. Bhate, Matthew H. Spitzer, Gabriela K. Fragiadakis, Nilanjan Mukherjee, Nelson Kim, Sizun Jiang, Jennifer Yo, Brice Gaudilliere, Melton Affrime, Bonnie Bock, Scott E. Hensley, Juliana Idoyaga, Nima Aghaeepour, Kenneth Kim, Garry P. Nolan, David R. McIlwain
Protection of the brain from viral infections involves the type I interferon (IFN-I) system, defects in which renders humans susceptible to herpes simplex encephalitis (HSE). However, excessive cerebral IFN-I levels leads to pathologies, suggesting the need for tight regulation of responses. Based on data from mouse models, human HSE cases, and primary cell culture systems, we here show that microglia and other immune cells undergo apoptosis in the HSV-1-infected brain through a mechanism dependent on the cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway, but independent of IFN-I. HSV-1 infection of microglia induced cGAS-dependent apoptosis at high viral doses, while lower viral doses led to IFN-I responses. Importantly, inhibition of caspase activity prevented microglial cell death and augmented IFN-I responses. Accordingly, HSV-1-infected organotypic brain slices, or mice treated with caspase inhibitor, exhibited lower viral load and improved outcome of infection. Collectively, we identify an activation-induced apoptosis program in brain immune cells which down-modulates local immune responses.
Line S. Reinert, Ahmad S. Rashidi, Diana N. Tran, Georgios Katzilieris-Petras, Astrid K. Hvidt, Mette Gohr, Stefanie Fruhwürth, Chiranjeevi Bodda, Martin K. Thomsen, Mikkel H. Vendelbo, Ahmad Raza Khan, Brian Hansen, Petra Bergström, Lotta Agholme, Trine H. Mogensen, Maria H. Christensen, Jens R. Nyengaard, Ganes C. Sen, Henrik Zetterberg, Georges M.G.M. Verjans, Soren R. Paluden
Background: Viral load surrogate endpoints transformed development of HIV and hepatitis C therapeutics. Surrogate endpoints for cytomegalovirus (CMV)-related morbidity and mortality could advance development of antiviral treatments. While observational data support using CMV viral load (VL) as a trial endpoint, randomized controlled trials (RCT) demonstrating direct associations between virologic markers and clinical endpoints are lacking. Methods: We performed CMV DNA polymerase chain reaction (PCR) on frozen serum samples from the only placebo-controlled RCT of ganciclovir for early treatment of CMV after hematopoietic cell transplantation (HCT). We used established criteria to assess VL kinetics as surrogates for CMV disease or death by weeks 8, 24, and 48 after randomization and quantified antiviral effects captured by each marker. We used ensemble-based machine learning to assess the predictive ability of VL kinetics and performed this analysis on a ganciclovir prophylaxis RCT for validation. Results: VL suppression with ganciclovir reduced cumulative incidence of CMV disease and death for 20 years after HCT. Mean VL, peak VL, and change in VL during the first five weeks of treatment fulfilled the Prentice definition for surrogacy, capturing > 95% of ganciclovir’s effect, and yielded highly sensitive and specific predictions by week 48. In the prophylaxis trial, viral shedding rate satisfied the Prentice definition for CMV disease by week 24. Conclusion: Our results support using CMV VL kinetics as surrogates for CMV disease, provide a framework for developing CMV preventative and therapeutic agents, and support reductions in viral load as the mechanism through which antivirals reduce CMV disease.
Elizabeth R. Duke, Brian D. Williamson, Bhavesh Borate, Jonathan L. Golob, Chiara Wychera, Terry Stevens-Ayers, Meei-Li Huang, Nicole Cossrow, Hong Wan, T. Christopher Mast, Morgan A. Marks, Mary Flowers, Keith R. Jerome, Lawrence Corey, Peter B. Gilbert, Joshua T. Schiffer, Michael Boeckh
BACKGROUND. The T cell responses to the common cold coronaviruses have not been well characterized. Pre-existing T cell immunity to SARS-CoV-2 has been reported, and a recent study suggested that this was due to cross-recognition of the novel coronavirus by T cells specific for the common cold coronaviruses. METHODS. We used the ELISpot assay to characterize the T cell responses against peptide pools derived from the spike protein of three common cold coronaviruses (HCoV-229E, HCoV-NL63, and HCoV-OC43) and SARS-CoV-2 in 21 healthy donors who were seronegative for SARS-CoV-2 and had no known exposure to the virus. An in vitro expansion culture assay was also used to analyze memory T cell responses. RESULTS. We found responses to the spike protein of the three common cold coronaviruses in many donors. We then focused on HCoV-NL63 and demonstrated broad T cell responses to the spike protein and identified 22 targeted peptides. Interestingly, only one subject had a significant response to SARS-CoV-2 spike or nucleocapsid protein in the ELISpot assay. In vitro expansion studies suggested that T cells specific for the HCoV-NL63 spike protein in this subject could also recognize SARS-CoV-2 spike protein peptide pools. CONCLUSIONS. Healthy donors have circulating T cells specific for the spike proteins of HCoV-NL63, HCoV-229E, and HCoV-OC43. T cell responses to SARS-CoV-2 spike and nucleocapsid proteins were present in only one subject and were potentially the result of cross-recognition by T cells specific for the common cold coronaviruses. Further studies are needed to determine whether this influences COVID-19 outcomes.
Bezawit A. Woldemeskel, Abena K. Kwaa, Caroline C. Garliss, Oliver Laeyendecker, Stuart C. Ray, Joel N. Blankson
Inborn errors of TLR3-dependent IFN-α/β- and -λ-mediated immunity in the central nervous system (CNS) can underlie herpes simplex virus 1 (HSV-1) encephalitis (HSE). The respective contributions of IFN-α/β and -λ are unknown. We report a child homozygous for a genomic deletion of the entire coding sequence and part of the 3’UTR of the last exon of IFNAR1, who died from HSE at the age of two years. An older cousin died following vaccination against measles, mumps and rubella at 12 months of age, and another 17-year-old cousin homozygous for the same variant has had other, less severe viral illnesses. The encoded IFNAR1 protein is expressed on the cell surface but is truncated and cannot interact with the tyrosine kinase TYK2. The patient’s fibroblasts and EBV-B cells did not respond to IFN-α2b or IFN-β, in terms of STAT1, STAT2 and STAT3 phosphorylation, or the genome-wide induction of IFN-stimulated genes. The patient’s fibroblasts were susceptible to viruses, including HSV-1, even in the presence of exogenous IFN-α2b or IFN-β. HSE is therefore a consequence of inherited complete IFNAR1 deficiency. This viral disease occurred in natural conditions, unlike those previously reported in other patients with IFNAR1 or IFNAR2 deficiency. This experiment of Nature indicates that IFN-α/β are essential for anti-HSV-1 immunity in the CNS.
Paul Bastard, Jeremy Manry, Jie Chen, Jérémie Rosain, Yoann Seeleuthner, Omar AbuZaitun, Lazaro Lorenzo, Taushif Khan, Mary Hasek, Nicholas Hernandez, Benedetta Bigio, Peng Zhang, Romain Lévy, Shai Shrot, Eduardo J. Garcia Reino, Yoon Seung Lee, Soraya Boucherit, Mélodie Aubart, Rik Gijsbers, Vivien Béziat, Zhi Li, Sandra Pellegrini, Isabelle Meyts, Flore Rozenberg, Nico Marr, Bertrand Boisson, Aurélie Cobat, Jacinta Bustamante, Qian Zhang, Emmanuelle Jouanguy, Laurent Abel, Raz Somech, Jean-Laurent Casanova, Shen-Ying Zhang
Macrophages are main effectors of heme metabolism, increasing transiently in the liver during heightened disposal of damaged or senescent red blood cells (sRBC). Macrophages are also essential in defense against microbial threats, but pathologic states of heme excess may be immunosuppressive. Herein, we uncovered a mechanism whereby an acute rise in sRBC disposal by macrophages led to an immunosuppressive phenotype following intrapulmonary Klebsiella pneumoniae infection characterized by increased extrapulmonary bacterial proliferation and reduced survival from sepsis in mice. The impaired immunity to K. pneumoniae during heightened sRBC disposal was independent of iron acquisition by bacterial siderophores, as K. pneumoniae mutant lacking siderophore function recapitulated findings observed with wildtype strain. Rather, sRBC disposal induced a liver transcriptomic profile notable for suppression of Stat1 and interferon-related responses during K. pneumoniae sepsis. Excess heme handling by macrophages recapitulated STAT1 suppression during infection that required synergistic NRF1 and NRF2 activation but was independent of heme oxygenase-1 induction. Whereas iron was dispensable, the porphyrin moiety of heme was sufficient to mediate suppression of STAT1-dependent responses in human and mouse macrophages and promoted liver dissemination of K. pneumoniae in vivo. Thus, cellular heme metabolism dysfunction negatively regulates the STAT1 pathway with implications in severe infection.
Tolani F. Olonisakin, Tomeka L. Suber, Shekina Gonzalez-Ferrer, Zeyu Xiong, Hernán F. Peñaloza, Rick van der Geest, Yuting Xiong, David O. Osei-Hwedieh, Jesus Tejero, Matthew R. Rosengart, Wendy M. Mars, Daria Van Tyne, Andreas Perlegas, Samuel Brashears, Daniel B. Kim-Shapiro, Mark T. Gladwin, Michael A. Bachman, Eldad A. Hod, Claudette St. Croix, Yulia Y. Tyurina, Valerian E. Kagan, Rama K. Mallampalli, Anuradha Ray, Prabir Ray, Janet S. Lee
The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the urgent need for assays that detect protective levels of neutralizing antibodies. We studied the relationship between anti-spike ectodomain (ECD), anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two in vitro assays using convalescent plasma samples from 68 COVID-19 patients. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers and in vitro VN titer. The probability of a VN titer ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment, was ≥80% when anti-RBD or anti-ECD titers were ≥1:1350. Of all donors, 37% lacked VN titers ≥160. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease VN or IgG titers. Analysis of 2,814 asymptomatic adults found 73 individuals with anti-ECD IgG titers of ≥1:50 and strong positive correlation with anti-RBD and VN titers. Fourteen of these individuals had VN titers ≥1:160, all of which had anti-RBD titer ≥1:1350. We conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titers of ≥1:1350 may provide critical information about protection against COVID-19 disease.
Eric Salazar, Suresh V. Kuchipudi, Paul A. Christensen, Todd Eagar, Xin Yi, Picheng Zhao, Zhicheng Jin, S. Wesley Long, Randall J. Olsen, Jian Chen, Brian Castillo, Christopher Leveque, Dalton Towers, Jason J. Lavinder, Jimmy Gollihar, Jose A. Cardona, Gregory C. Ippolito, Ruth H. Nissly, Ian Bird, Denver Greenawalt, Randall M. Rossi, Abhinay Gontu, Sreenidhi Srinivasan, Indira Poojary, Isabella M. Cattadori, Peter Hudson, Nicole M. Josleyn, Laura Prugar, Kathleen E. Huie, Andrew S. Herbert, David W. Bernard, John M. Dye, Vivek Kapur, James M. Musser
Although broadly protective, stem-targeted Abs against the influenza A virus hemagglutinin (HA) have been well studied, very limited information is available on Abs that broadly recognize the head domain. We determined the crystal structure of the HA protein of the avian H7N9 influenza virus in complex with a pan-H7, non-neutralizing, protective human Ab. The structure revealed a B cell epitope in the HA head domain trimer interface (TI). This discovery of a second major protective TI epitope supports a model in which uncleaved HA trimers exist on the surface of infected cells in a highly dynamic state that exposes hidden HA head domain features.
Jinhui Dong, Iuliia Gilchuk, Sheng Li, Ryan Irving, Matthew T. Goff, Hannah L. Turner, Andrew B. Ward, Robert H. Carnahan, James E. Crowe Jr.
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