Virology
Abstract
In humans, lymph nodes are the primary site of measles virus (MeV) replication. To understand the immunological events that occur at this site, we infected human lymphoid tissue explants using a pathogenic strain of MeV that expresses GFP. We found that MeV infected 5%–15% of cells across donors. Using single-cell RNA-Seq and flow cytometry, we found that while most of the 29 cell populations identified in the lymphoid culture were susceptible to MeV, there was a broad preferential infection of B cells and reduced infection of T cells. Further subsetting of T cells revealed that this reduction may be driven by the decreased infection of naive T cells. Transcriptional changes in infected B cells were dominated by an interferon-stimulated gene (ISG) signature. To determine which of these ISGs were most substantial, we evaluated the proteome of MeV-infected Raji cells by mass spectrometry. We found that IFIT1, IFIT2, IFIT3, ISG15, CXCL10, MX2, and XAF1 proteins were the most highly induced and positively correlated with their expression in the transcriptome. These data provide insight into the immunological events that occur in lymph nodes during infection and may lead to the development of therapeutic interventions.
Authors
Joshua A. Acklin, Aum R. Patel, Andrew P. Kurland, Shu Horiuchi, Arianna S. Moss, Emma J. DeGrace, Satoshi Ikegame, Jillian Carmichael, Shreyas Kowdle, Patricia A. Thibault, Naoko Imai, Hideki Ueno, Benjamin Tweel, Jeffrey R. Johnson, Brad R. Rosenberg, Benhur Lee, Jean K. Lim
Abstract
HIV infection cannot be cured due to the persistence of a reservoir of latently infected cells. Furthermore, virally suppressed individuals experience chronic immune activation from ongoing low-level viral expression. Drugs that inhibit HIV transcription and/or reactivation of latent HIV have been proposed as a strategy to reduce HIV-associated immune activation and/or to achieve a functional cure. We evaluated 26 small molecules, both previously reported drugs and new drug candidates, for their ability to act as “latency promoting/silencing agents (LPAs)” that can reduce or prevent HIV expression after T cell activation. Using a panel of RT-ddPCR assays, we measured the progression through HIV transcription and pinpointed the step at which each of those drugs inhibited HIV transcription, with and without prior activation. While some drugs primarily inhibited one or two steps in HIV reactivation, other drugs (CDK inhibitors, splicing inhibitors, tanespimycin, and triptolide) inhibited multiple stages of HIV transcription and blocked the production of supernatant viral RNA. Dinaciclib, AZD4573, and pladienolide B also appeared to inhibit HIV splicing in unstimulated PBMC. By selecting drugs with known mechanisms of action, we specifically identified cellular factors and pathways that may be involved in regulation of HIV expression. These drugs/targets deserve further study in strategies aimed at reducing HIV-associated immune activation or achieving a functional cure.
Authors
Julie Janssens, Peggy Kim, Sun Jin Kim, Adam Wedrychowski, Gayatri N. Kadiyala, Peter W. Hunt, Steven G. Deeks, Joseph K. Wong, Steven A. Yukl
Abstract
The skin at the site of HSV-2 reactivation is enriched for HSV-2–specific T cells. To evaluate whether an immunotherapeutic vaccine could elicit skin-based memory T cells, we studied skin biopsies and HSV-2–reactive CD4+ T cells from PBMCs by T cell receptor (TCR) β chain (TRB) sequencing before and after vaccination with a replication-incompetent whole-virus HSV-2 vaccine candidate (HSV529). The representation of HSV-2–reactive CD4+ TRB sequences from PBMCs in the skin TRB repertoire increased after the first vaccine dose. We found sustained expansion after vaccination of unique, skin-based T cell clonotypes that were not detected in HSV-2–reactive CD4+ T cells isolated from PBMCs. In one participant, a switch in immunodominance occurred with the emergence of a TCR αβ pair after vaccination that was not detected in blood. This TCRαβ was shown to be HSV-2 reactive by expression of a synthetic TCR in a Jurkat-based NR4A1 reporter system. The skin in areas of HSV-2 reactivation possessed an oligoclonal TRB repertoire that was distinct from the circulation. Defining the influence of therapeutic vaccination on the HSV-2–specific TRB repertoire requires tissue-based evaluation.
Authors
Emily S. Ford, Alvason Z. Li, Kerry J. Laing, Lichun Dong, Kurt Diem, Lichen Jing, Koshlan Mayer-Blackwell, Krithi Basu, Mariliis Ott, Jim Tartaglia, Sanjay Gurunathan, Jack L. Reid, Matyas Ecsedi, Aude G. Chapuis, Meei-Li Huang, Amalia S. Magaret, Christine Johnston, Jia Zhu, David M. Koelle, Lawrence Corey
Abstract
Influenza poses a persistent health burden worldwide. To design equitable vaccines effective across all demographics, it is essential to better understand how host factors such as genetic background and aging affect the single-cell immune landscape of influenza infection. Cytometry by time-of-flight (CyTOF) represents a promising technique in this pursuit, but interpreting its large, high-dimensional data remains difficult. We’ve developed a new analytical approach iGATE (in-silico gating annotating training elucidating) based on probabilistic support vector machine classification. By rapidly and accurately “gating” tens of millions of cells in silico into user-defined types, iGATE enabled us to track 25 canonical immune cell types in mouse lung over the course of influenza infection. Applying iGATE to study effects of host genetic background, we show that the lower survival of C57BL/6 mice compared to BALB/c is associated with a more rapid accumulation of inflammatory cell types and decreased IL-10 expression. Further, we demonstrate that the most prominent effect of aging is a defective T-cell response, reducing survival of aged mice. Finally, iGATE reveals that the 25 canonical immune cell types exhibit differential influenza infection susceptibility and replication permissiveness in vivo, but neither property varies with host genotype or aging. Software is available at https://github.com/UmichWenLab/iGATE.
Authors
Brett D. Hill, Andrew J. Zak, Sanjeev Raja, Luke F. Bugada, Syed M. Rizvi, Saiful B. Roslan, Hong Nhi Nguyen, Judy Chen, Hui Jiang, Akira Ono, Daniel R. Goldstein, Fei Wen
Abstract
The viral kinetics of documented SARS-CoV-2 infections exhibit a high degree of inter-individual variability. We identified six distinct viral shedding patterns, which differed according to peak viral load, duration, expansion rate and clearance rate, by clustering data from 768 infections in the National Basketball Association cohort. Omicron variant infections in previously vaccinated individuals generally led to lower cumulative shedding levels of SARS-CoV-2 than other scenarios. We then developed a mechanistic mathematical model that recapitulated 1510 observed viral trajectories, including viral rebound and cases of reinfection. Lower peak viral loads were explained by a more rapid and sustained transition of susceptible cells to a refractory state during infection, as well as an earlier and more potent late, cytolytic immune response. Our results suggest that viral elimination occurs more rapidly during omicron infection, following vaccination, and following re-infection due to enhanced innate and acquired immune responses. Because viral load has been linked with COVID-19 severity and transmission risk, our model provides a framework for understanding the wide range of observed SARS-CoV-2 infection outcomes.
Authors
Katherine Owens, Shadisadat Esmaeili, Joshua Schiffer
Abstract
Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we find that during the first 24 h of infection, CHIKV RNA accumulates in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response, including recruitment of myeloid cells to the LN, was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, we find that antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.
Authors
Cormac J. Lucas, Ryan M. Sheridan, Glennys V. Reynoso, Bennett J. Davenport, Mary K. McCarthy, Aspen L. Martin, Jay R. Hesselberth, Heather D. Hickman, Beth A.J. Tamburini, Thomas E. Morrison
Abstract
Nipah virus (NiV), a bat-borne paramyxovirus, results in neurological and respiratory diseases with high mortality in humans and animals. Developing vaccines is crucial against the diseases. Previous few studies focused on fusion (F) protein alone as the immunogen. Numerous NiV strains have been identified, including two representative strains from Malaysia (NiV-M) and Bangladesh (NiV-B), which differ significantly from the other. In this study, an F protein sequence with the potential to prevent different NiV strain infections was designed by bioinformatics analysis after an in-depth study of NiV sequences in GenBank. Then, a chimpanzee adenovirus vector vaccine and a DNA vaccine were developed. High levels of immune responses were detected by AdC68-F, pVAX1-F and a prime-boost strategy (pVAX1-F/AdC68-F) in mice. After high titers of humoral responses were induced, the hamsters were challenged by the lethal NiV-M and NiV-B strains, respectively. It was reassuring that the vaccinated hamsters did not show any clinical signs and survived 21 days after infection with either strain of NiV, and no virus was detected in different tissues either. These results indicated that vaccines provided complete protection against representative strains of NiV infection and had the potential to be developed as a broad-spectrum vaccine for human use.
Authors
Mingqing Lu, Yanfeng Yao, Hang Liu, Xuekai Zhang, Xuejie Li, Yuanhua Liu, Yun Peng, Tong Chen, Yun Sun, Ge Gao, Miaoyu Chen, Jiaxuan Zhao, XiaoYu Zhang, Chunhong Yin, Weiwei Guo, Peipei Yang, Xue Hu, Juhong Rao, Entao Li, Gary Wong, Zhiming Yuan, Sandra Chiu, Chao Shan, Jiaming Lan
Abstract
Optimal lung repair and regeneration is essential for recovery from viral infections including influenza A virus (IAV). We have previously demonstrated that acute inflammation and mortality induced by IAV is under circadian control. However, it is not known if the influence of the circadian clock persists beyond the acute outcomes. Here, we utilize the UK Biobank to demonstrate an association between poor circadian rhythms and morbidity from lower respiratory tract infections including the need for hospitalization and post-discharge mortality; this persists even after adjusting for common confounding factors. Further, we use a combination of lung organoid assays, single cell RNA sequencing (Sc-seq) and IAV infection in different models of clock disruption to investigate the role of the circadian clock in lung repair and regeneration. We show for the first time that lung organoids have a functional circadian clock, and the disruption of this clock impairs regenerative capacity. Finally, we find that the circadian clock acts through distinct pathways in mediating lung regeneration- in tracheal cells via the Wnt/β-catenin pathway and through IL1β in alveolar epithelial cells. We speculate, that adding a circadian dimension to the critical process of lung repair and regeneration will lead to novel therapies and improve outcomes.
Authors
Amruta Naik, Kaitlyn M. Forrest, Oindrila Paul, Yasmine Issah, Utham Kashyap Valekunja, Soon Yew Tang, Akhilesh B. Reddy, Elizabeth J. Hennessy, Thomas G. Brooks, Fatima N. Chaudhry, Apoorva Babu, Michael P. Morley, Jarod A. Zepp, Gregory R. Grant, Garret FitzGerald, Amita Sehgal, G. Scott Worthen, David B. Frank, Edward E. Morrisey, Shaon Sengupta
Abstract
Antiviral immunity often requires CD8+ cytotoxic T lymphocytes (CTLs) that actively migrate and search for virus-infected targets. Regulatory T cells (Tregs) have been shown to suppress CTL responses, but it is not known whether this is also mediated by effects on CTL motility. Here, we used intravital 2-photon microscopy in the Friend retrovirus (FV) mouse model to define the impact of Tregs on CTL motility throughout the course of acute infection. Virus-specific CTLs were very motile and had frequent short contacts with target cells at their peak cytotoxic activity. However, when Tregs were activated and expanded in late-acute FV infection, CTLs became significantly less motile and contacts with target cells were prolonged. This phenotype was associated with development of functional CTL exhaustion. Tregs had direct contacts with CTLs in vivo and, importantly, their experimental depletion restored CTL motility. Our findings identify an effect of Tregs on CTL motility as part of their mechanism of functional impairment in chronic viral infections. Future studies must address the underlying molecular mechanisms.
Authors
Daniela Mittermüller, Lucas Otto, Zoë Long, Andreas Kraus, Alexander Beer, Anja Hasenberg, Gennadiy Zelinskyy, Jaana Westmeier, Kim J. Hasenkrug, Ulf Dittmer, Matthias Gunzer
Abstract
Influenza A virus (IAV) infection is commonly complicated by secondary bacterial infections, leading to increased morbidity and mortality. Our recent work demonstrates that IAV disrupts airway homeostasis, leading to airway pathophysiology resembling cystic fibrosis disease through diminished cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we use human airway organotypic cultures to investigate how IAV alters the airway microenvironment to increase susceptibility to secondary infection with Streptococcus pneumoniae (Spn). We observed that IAV-induced CFTR dysfunction and airway surface liquid acidification is central to increasing susceptibility to Spn. Additionally, we observed that IAV induced profound transcriptional changes in the airway epithelium and proteomic changes in the airway surface liquid in both CFTR dependent and independent manners. These changes correspond to multiple diminished host defense pathways and altered airway epithelial function. Collectively, these findings highlight both the importance of CFTR function during infectious challenge and demonstrate a central role for the lung epithelium in secondary bacterial infections following IAV.
Authors
Erin Y. Earnhardt, Jennifer L. Tipper, Adonis D'Mello, Ming-Yuan Jian, Elijah S. Conway, James A. Mobley, Carlos J. Orihuela, Hervé Tettelin, Kevin S. Harrod
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