Search

A diverse range of viruses have well-established roles as the primary driver of oncogenesis in various haematological malignancies and solid tumours. Indeed, estimates suggest that approximately 1.5 million patients annually are diagnosed with virus-related cancers. The predominant human oncoviruses include Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV), hepatitis B and C viruses (HBV and HCV), human papillomavirus (HPV), human T-lymphotropic virus type 1 (HTLV1), and Merkel cell polyomavirus (MCPyV). In addition, although not inherently oncogenic, human immunodeficiency virus (HIV) is associated with immunosuppression that contributes to the development of AIDS-defining cancers (specifically, Kaposi sarcoma, aggressive B cell non-Hodgkin lymphoma and cervical cancer). Given that an adaptive T cell-mediated immune response is crucial for the control of viral infections, increasing research is being focused on evaluating virus-specific T cell therapies for the treatment of virus-associated cancers. In this Review, we briefly outline the roles of viruses in the pathogenesis of these malignancies before describing progress to date in the field of virus-specific T cell therapy and evaluating the potential utility of these therapies to treat or possibly even prevent virus-related malignancies., (© 2024. Springer Nature Limited.)

Pseudorabies virus (PRV), an α-herpesvirus, induces immunosuppression and can lead to severe neurological diseases. N-methyl-D-aspartate receptor (NMDAR), an important excitatory nerve receptor in the central nervous system, is linked to various nervous system pathologies. The link between NMDAR and PRV-induced neurological diseases has not been studied. In vivo studies revealed that PRV infection triggers a reduction in hippocampal NMDAR expression, mediated by inflammatory processes. Extensive hippocampal neuronal degeneration was found in mice on the 6th day by hematoxylin-eosin staining, which was strongly correlated with increased NMDAR protein expression. In vitro studies utilizing the CCK-8 assay demonstrated that treatment with an NMDAR antagonist significantly heightened the cytotoxic effects of PRV on T lymphocytes. Notably, NMDAR inhibition did not affect the replication ability of PRV. However, it facilitated the accumulation of pro-inflammatory cytokines in PRV-infected T cells and enhanced the transcription of the CD25 gene through the secretion of interleukin-2 (IL-2), consequently exacerbating immunosuppression. In this study, we found that NMDAR has functional activity in T lymphocytes and is crucial for the inflammatory and immune responses triggered by PRV infection. These discoveries highlight the significant role of NMDAR in PRV-induced neurological disease pathogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Manipulation of immune cell functions, independently of direct infection of these cells, emerges as a key process in viral pathophysiology. Chronic infection by Human T-cell Leukemia Virus type 1 (HTLV-1) is associated with immune dysfunctions, including misdirected responses of dendritic cells (DCs). Here, we interrogate the ability of transformed HTLV-1-infected T cells to manipulate human DC functions. We show that exposure to transformed HTLV-1-infected T cells induces a biased and peculiar transcriptional signature in monocyte-derived DCs, associated with an inefficient maturation and a poor responsiveness to subsequent stimulation by a TLR4 agonist. This poor responsiveness is also associated with a unique transcriptional landscape characterized by a set of genes whose expression is either conferred, impaired or abolished by HTLV-1 pre-exposure. Induction of this functional impairment requires several hours of coculture with transformed HTLV-1-infected cells, and associated mechanisms driven by viral capture, cell-cell contacts, and soluble mediators. Altogether, this cross-talk between infected T cells and DCs illustrate how HTLV-1 might co-opt communications between cells to induce a unique local tolerogenic immune microenvironment suitable for its own persistence., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Carcone et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100-10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion., (© 2024 Teixeira et al.)

Human cytomegalovirus (HCMV), a widely prevalent human beta-herpesvirus, establishes lifelong persistence in the host following primary infection. In healthy individuals, the virus is effectively controlled by HCMV-specific T cells and typically exhibits asymptomatic. The T cell immune response plays a pivotal role in combating HCMV infection, while HCMV employs various strategies to counteract it within the host. Previously, we reported that UL23, a tegument protein of HCMV, facilitates viral immune evasion from interferon-gamma (IFN-γ) responses, and it is well known that IFN-γ is mainly derived from T cells. However, the involvement of UL23 in viral immune evasion from T cell-mediated immunity remains unclear. Herein, we present compelling evidence that UL23 significantly enhances viral resistance against T cell-mediated cytotoxicity during HCMV infection from the co-culture assays of HCMV-infected cells with T cells. We found that IFN-γ plays a major role in regulating T cell cytotoxicity mediated by UL23. More interestingly, we demonstrated that UL23 not only regulates the IFN-γ downstream responses but also modulates the IFN-γ secretion by regulating T cell activities. Further experiments indicate that UL23 upregulates the expression and signaling of programmed death ligand 1 (PD-L1), which is responsible for inhibiting multiple aspects of T cell activities, including activation, apoptosis, and IFN-γ secretion, as determined through RNA-seq analysis and inhibitor-blocking experiments, ultimately facilitating viral replication and spread. Our findings highlight the potential role of UL23 as an alternative antagonist in suppressing T cell cytotoxicity and unveil a novel strategy for HCMV to evade T cell immunity., Importance: T cell immunity is pivotal in controlling primary human cytomegalovirus (HCMV) infection, restricting periodic reactivation, and preventing HCMV-associated diseases. Despite inducing a robust T cell immune response, HCMV has developed sophisticated immune evasion mechanisms that specifically target T cell responses. Although numerous studies have been conducted on HCMV-specific T cells, the primary focus has been on the impact of HCMV on T cell recognition via major histocompatibility complex molecules. Our studies show for the first time that HCMV exploits the programmed death ligand 1 (PD-L1) inhibitory signaling pathway to evade T cell immunity by modulating the activities of T cells and thereby blocking the secretion of IFN-γ, which is directly mediated by HCMV-encoded tegument protein UL23. While PD-L1 has been extensively studied in the context of tumors and viruses, its involvement in HCMV infection and viral immune evasion is rarely reported. We observed an upregulation of PD-L1 in normal cells during HCMV infection and provided strong evidence supporting its critical role in UL23-induced inhibition of T cell-mediated cytotoxicity. The novel strategy employed by HCMV to manipulate the inhibitory signaling pathway of T cell immune activation for viral evasion through its encoded protein offers valuable insights for the understanding of HCMV-mediated T cell immunomodulation and developing innovative antiviral treatment strategies., Competing Interests: The authors declare no conflict of interest.

T lymphocytes play a primary role in the adaptive antiviral immunity. Both lymphocytosis and lymphopenia were found to be associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While lymphocytosis indicates an active anti-viral response, lymphopenia is a sign of poor prognosis. T-cells, in essence, rarely express ACE2 receptors, making the cause of cell depletion enigmatic. Moreover, emerging strains posed an immunological challenge, potentially alarming for the next pandemic. Herein, we review how possible indirect and direct key mechanisms could contribute to SARS-CoV-2-associated-lymphopenia. The fundamental mechanism is the inflammatory cytokine storm elicited by viral infection, which alters the host cell metabolism into a more acidic state. This "hyperlactic acidemia" together with the cytokine storm suppresses T-cell proliferation and triggers intrinsic/extrinsic apoptosis. SARS-CoV-2 infection also results in a shift from steady-state hematopoiesis to stress hematopoiesis. Even with low ACE2 expression, the presence of cholesterol-rich lipid rafts on activated T-cells may enhance viral entry and syncytia formation. Finally, direct viral infection of lymphocytes may indicate the participation of other receptors or auxiliary proteins on T-cells, that can work alone or in concert with other mechanisms. Therefore, we address the role of CD147-a novel route-for SARS-CoV-2 and its new variants. CD147 is not only expressed on T-cells, but it also interacts with other co-partners to orchestrate various biological processes. Given these features, CD147 is an appealing candidate for viral pathogenicity. Understanding the molecular and cellular mechanisms behind SARS-CoV-2-associated-lymphopenia will aid in the discovery of potential therapeutic targets to improve the resilience of our immune system against this rapidly evolving virus., (© 2024. The Author(s).)

We introduce a 35-marker imaging mass cytometry (IMC) panel for a detailed examination of immune cell populations and HIV RNA in formalin fixed paraffin embedded (FFPE) human intestinal tissue. The panel has broad cell type coverage and particularly excels in delineating subsets of mononuclear phagocytes and T cells. Markers for key tissue structures are included, enabling identification of epithelium, blood vessels, lymphatics, and musculature. The described method for HIV RNA detection can be generalized to other low abundance RNA targets, whether endogenous or pathogen derived. As such, the panel presented here is useful for high parameter spatial mapping of intestinal immune cells and their interactions with pathogens such as HIV., (© 2024 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited 1 . Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection., (© 2024. The Author(s).)

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles ( sm HIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Research on the host responses to respiratory viruses could help develop effective interventions and therapies against the current and future pandemics from the host perspective. To explore the pathogenesis that distinguishes SARS-CoV-2 infections from other respiratory viruses, we performed a multi-cohort analysis with integrated bioinformatics and machine learning. We collected 3730 blood samples from both asymptomatic and symptomatic individuals infected with SARS-CoV-2, seasonal human coronavirus (sHCoVs), influenza virus (IFV), respiratory syncytial virus (RSV), or human rhinovirus (HRV) across 15 cohorts. First, we identified an enhanced cellular immune response but limited interferon activities in SARS-CoV-2 infection, especially in asymptomatic cases. Second, we identified a SARS-CoV-2-specific 3-gene signature (CLSPN, RBBP6, CCDC91) that was predominantly expressed by T cells, could distinguish SARS-CoV-2 infection, including Omicron, from other common respiratory viruses regardless of symptoms, and was predictive of SARS-CoV-2 infection before detectable viral RNA on RT-PCR testing in a longitude follow-up study. Thereafter, a user-friendly online tool, based on datasets collected here, was developed for querying a gene of interest across multiple viral infections. Our results not only identify a unique host response to the viral pathogenesis in SARS-CoV-2 but also provide insights into developing effective tools against viral pandemics from the host perspective.

Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia/lymphoma (ATLL) and the neuroinflammatory disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax oncoprotein regulates viral gene expression and persistently activates NF-κB to maintain the viability of HTLV-1-infected T cells. Here, we utilize a kinome-wide shRNA screen to identify the tyrosine kinase KDR as an essential survival factor of HTLV-1-transformed cells. Inhibition of KDR specifically induces apoptosis of Tax expressing HTLV-1-transformed cell lines and CD4 + T cells from HAM/TSP patients. Furthermore, inhibition of KDR triggers the autophagic degradation of Tax resulting in impaired NF-κB activation and diminished viral transmission in co-culture assays. Tax induces the expression of KDR, forms a complex with KDR, and is phosphorylated by KDR. These findings suggest that Tax stability is dependent on KDR activity which could be exploited as a strategy to target Tax in HTLV-1-associated diseases., (© 2024. The Author(s).)

During primary varicella zoster virus (VZV) infection, infected lymphocytes drive primary viremia, causing systemic dissemination throughout the host, including the skin. This results in cytokine expression, including interferons (IFNs), which partly limit infection. VZV also spreads from skin keratinocytes to lymphocytes prior to secondary viremia. It is not clear how VZV achieves this while evading the cytokine response. Here, we show that VZV glycoprotein C (gC) binds IFN-γ and modifies its activity, increasing the expression of a subset of IFN-stimulated genes (ISGs), including intercellular adhesion molecule 1 (ICAM1), chemokines and immunomodulatory genes. The higher ICAM1 protein level at the plasma membrane of keratinocytes facilitates lymphocyte function-associated antigen 1-dependent T cell adhesion and expression of gC during infection increases VZV spread to peripheral blood mononuclear cells. This constitutes the discovery of a strategy to modulate IFN-γ activity, upregulating a subset of ISGs, promoting enhanced lymphocyte adhesion and virus spread., (© 2024. The Author(s).)

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses have developed strategies to manipulate the host UPR, but there is a lack of detailed understanding of UPR modulation and its functional significance during HIV-1 infection in the literature. In this context, the current article describes the protocols used in our laboratory to measure ER stress levels and UPR during HIV-1 infection in T-cells and the effect of UPR on viral replication and infectivity. Thioflavin T (ThT) staining is a relatively new method used to detect ER stress in the cells by detecting protein aggregates. Here, we have illustrated the protocol for ThT staining in HIV-1 infected cells to detect and quantify ER stress. Moreover, ER stress was also detected indirectly by measuring the levels of UPR markers such as BiP, phosphorylated IRE1, PERK, and eIF2α, splicing of XBP1, cleavage of ATF6, ATF4, CHOP, and GADD34 in HIV-1 infected cells, using conventional immunoblotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). We have found that the ThT-fluorescence correlates with the indicators of UPR activation. This article also demonstrates the protocols to analyze the impact of ER stress and UPR modulation on HIV-1 replication by knockdown experiments as well as the use of pharmacological molecules. The effect of UPR on HIV-1 gene expression/replication and virus production was analyzed by Luciferase reporter assays and p24 antigen capture ELISA, respectively, whereas the effect on virion infectivity was analyzed by staining of infected reporter cells. Collectively, this set of methods provides a comprehensive understanding of the Unfolded Protein Response pathways during HIV-1 infection, revealing its intricate dynamics.

APOBEC3G (A3G) restricts HIV-1 replication primarily by reducing viral cDNA and inducing G-to-A hypermutations in viral cDNA. HIV-1 encodes virion infectivity factor (Vif) to counteract A3G primarily by excluding A3G viral encapsidation. Even though the Vif-induced exclusion is robust, studies suggest that A3G is still detectable in the virion. The impact of encapsidated A3G in the HIV-1 replication is unclear. Using a highly sensitive next-generation sequencing (NGS)-based G-to-A hypermutation detecting assay, we found that wild-type HIV-1 produced from A3G-expressing T-cells induced higher G-to-A hypermutation frequency in viral cDNA than HIV-1 from non-A3G-expressing T-cells. Interestingly, although the virus produced from A3G-expressing T-cells induced higher hypermutation frequency, there was no significant difference in viral infectivity, revealing a disassociation of cDNA G-to-A hypermutation to viral infectivity. We also measured G-to-A hypermutation in the viral RNA genome. Surprisingly, our data showed that hypermutation frequency in the viral RNA genome was significantly lower than in the integrated DNA, suggesting a mechanism exists to preferentially select intact genomic RNA for viral packing. This study revealed a new insight into the mechanism of HIV-1 counteracting A3G antiviral function and might lay a foundation for new antiviral strategies.

The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including CPSF6, that direct HIV-1 integration into speckle-associated domains in host chromatin. Upon HIV-1 infection, CPSF6 forms puncta in the nucleus. Here, we characterised these CPSF6 puncta further in HeLa cells, T-cells and macrophages and confirmed that integration and reverse transcription are not required for puncta formation. Indeed, we found that puncta formed very rapidly after infection, correlating with the time that CA entered the nucleus. In aphidicolin-treated HeLa cells and macrophages, puncta were detected for the length of the experiment, suggesting that puncta are only lost upon cell division. CA still co-localised with CPSF6 puncta at the latest time points, considerably after the peak of reverse transcription and integration. Intriguingly, the number of puncta induced in macrophages did not correlate with the MOI or the total number of nuclear speckles present in each cell, suggesting that CA/CPSF6 is only directed to a few nuclear speckles. Furthermore, we found that CPSF6 already co-localised with nuclear speckles in uninfected T-cells, suggesting that HIV-1 promotes a natural behaviour of CPSF6.

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging., Competing Interests: The authors declare no conflict of interest.

Importance: HIV-1 infection of T-lymphocytes depends on co-opting cellular transcriptional and translational machineries for viral replication. This requires significant changes in the cellular microenvironment. We have characterized and compared the changes in cellular chromatin structures as well as gene expression landscapes in T cells that are either actively or latently infected with HIV-1. Our results reveal that chromatin accessibility and expression of both protein-coding mRNAs and non-coding lncRNAs are uniquely regulated in HIV-1-infected T cells, depending on whether the virus is actively transcribing or remains in a transcriptionally silent, latent state. HIV-1 latent infection elicits more robust changes in the cellular chromatin organization than active viral infection. Our analysis also identifies the effects of such epigenomic changes on the cellular gene expression and subsequent biological pathways. This study comprehensively characterizes the cellular epigenomic and transcriptomic states that support active and latent HIV-1 infection in an in vitro model of SupT1 cells., Competing Interests: The authors declare no conflict of interest.

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA + cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir., Competing Interests: Conflict of interest V. R., J. H., K. C., and C. J. B. are listed as inventors on a filed patent describing FcγRIIA-specific DARPins., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

While HIV-1 infection of macrophages plays a major role in viral persistence and pathogenesis, how HIV-1 transfers from infected T cells to macrophages remains elusive. In this issue, Mascarau et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202205103) demonstrate how macrophage polarization drives their ability to fuse with HIV-1 infected T cells via the CD81/RhoA-ROCK/Myosin axis., (© 2023 Naghavi.)

Ebola virus disease (EVD) is a complex infectious disease characterized by high inflammation, multiorgan failure, the dysregulation of innate and adaptive immune responses, and coagulation abnormalities. Evidence accumulated over the last 2 decades indicates that, during fatal EVD, the infection of antigen-presenting cells (APC) and the dysregulation of T cell immunity preclude a successful transition between innate and adaptive immunity, which constitutes a key disease checkpoint. In order to better understand the contribution of the APC-T cell crosstalk to EVD pathophysiology, we have developed avatar mice transplanted with human, donor-specific APCs and T cells. Here, we show that the transplantation of T cells and APCs from Ebola virus (EBOV)-naive individuals into avatar mice results in severe disease and death and that this phenotype is dependent on T cell receptor (TCR)-major histocompatibility complex (MCH) recognition. Conversely, avatar mice were rescued from death induced by EBOV infection after the transplantation of both T cells and plasma from EVD survivors. These results strongly suggest that protection from EBOV reinfection requires both cellular and humoral immune memory responses. IMPORTANCE The crosstalk between dendritic cells and T cells marks the transition between innate and adaptive immune responses, and it constitutes an important checkpoint in EVD. In this study, we present a mouse avatar model in which T cell and dendritic cell interactions from a specific donor can be studied during EVD. Our findings indicate that T cell receptor-major histocompatibility complex-mediated T cell-dendritic cell interactions are associated with disease severity, which mimics the main features of severe EVD in these mice. Resistance to an EBOV challenge in the model was achieved via the transplantation of both survivor T cells and plasma.

Viruses have evolved numerous strategies to impair immunity so that they can replicate more efficiently. Among those, the immunosuppressive effects of morbillivirus infection can be particularly problematic, as they allow secondary infections to take hold in the host, worsening disease prognosis. In the present work, we hypothesized that the highly contagious morbillivirus peste des petits ruminants virus (PPRV) could target monocytes and dendritic cells (DC) to contribute to the immunosuppressive effects produced by the infection. Monocytes isolated from healthy sheep, a natural host of the disease, were able be infected by PPRV and this impaired the differentiation and phagocytic ability of immature monocyte-derived DC (MoDC). We also assessed PPRV capacity to infect differentiated MoDC. Ovine MoDC could be productively infected by PPRV, and this drastically reduced MoDC capacity to activate allogeneic T cell responses. Transcriptomic analysis of infected MoDC indicated that several tolerogenic DC signature genes were upregulated upon PPRV infection. Furthermore, PPRV-infected MoDC could impair the proliferative response of autologous CD4 + and CD8 + T cell to the mitogen concanavalin A (ConA), which indicated that DC targeting by the virus could promote immunosuppression. These results shed new light on the mechanisms employed by morbillivirus to suppress the host immune responses. IMPORTANCE Morbilliviruses pose a threat to global health given their high infectivity. The morbillivirus peste des petits ruminants virus (PPRV) severely affects small-ruminant-productivity and leads to important economic losses in communities that rely on these animals for subsistence. PPRV produces in the infected host a period of severe immunosuppression that opportunistic pathogens exploit, which worsens the course of the infection. The mechanisms of PPRV immunosuppression are not fully understood. In the present work, we demonstrate that PPRV can infect professional antigen-presenting cells called dendritic cells (DC) and disrupt their capacity to elicit an immune response. PPRV infection promoted a DC activation profile that favored the induction of tolerance instead of the activation of an antiviral immune response. These results shed new light on the mechanisms employed by morbilliviruses to suppress the immune responses.

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFβ) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFβ to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFβ affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFβ target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFβ to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFβ interaction may be promising in the development of a new AIDS therapeutic strategy.

The mechanisms of colostrum-mediated virus transmission are difficult to elucidate because of the absence of experimental animal models and the difficulties in tissue sample collection from mothers in the peripartum period. Porcine epidemic diarrhea virus (PEDV) is a reemerging enteropathogenic coronavirus that has catastrophic impacts on the global pig industry. PEDV primarily infects neonatal piglets by multiple routes, especially 1- to 2-day-old neonatal piglets. Here, our epidemiological investigation and animal challenge experiments revealed that PEDV could be vertically transmitted from sows to neonatal piglets via colostrum, and CD3 + T cells in the colostrum play an important role in this process. The results showed that PEDV colonizing the intestinal epithelial cells (IECs) of orally immunized infected sows could be transferred to CD3 + T cells located just beneath the IECs. Next, PEDV-carrying CD3 + T cells, with the expression of integrin α4β7 and CCR10, migrate from the intestine to the mammary gland through blood circulation. Arriving in the mammary gland, PEDV-carrying CD3 + T cells could be transported across mammary epithelial cells (MECs) into the lumen (colostrum), as illustrated by an autotransfusion assay and an MECs/T coculture system. The PEDV-carrying CD3 + T cells in colostrum could be interspersed between IECs of neonatal piglets, causing intestinal infection via cell-to-cell contact. Our study demonstrates for the first time that colostrum-derived CD3 + T cells comprise a potential route for the vertical transmission of PEDV. IMPORTANCE The colostrum represents an important infection route for many viruses. Here, we demonstrate the vertical transmission of porcine epidemic diarrhea virus (PEDV) from sows to neonatal piglets via colostrum. PEDV colonizing the intestinal epithelial cells could transfer the virus to CD3 + T cells located in the sow intestine. The PEDV-carrying CD3 + T cells in the sow intestine, with the expression of integrin α4β7 and CCR10, arrive at the mammary gland through blood circulation and are transported across mammary epithelial cells into the lumen, finally leading to intestinal infection via cell-to-cell contact in neonatal piglets. Our study not only demonstrates an alternative route of PEDV infection but also provides an animal model of vertical transmission of human infectious disease.

Production of infectious HIV-1 particles requires incorporation of the viral envelope glycoprotein (Env) at the plasma membrane (PM) of infected CD4 + T cells. Env trafficking to the PM exposes viral epitopes that can be exploited by the host immune system; however, HIV-1 can evade this response by endocytosis of excess Env from the PM. The fate of Env after internalization remains unclear, with evidence suggesting several different vesicular trafficking steps may be involved, including recycling pathways. To date, there have been very few studies documenting the trafficking pathways of native Env in infected T cells. Furthermore, it remains unclear whether there are T-cell-specific endosomal pathways regulating the fate of endocytic Env. Here, we use a pulse-labeling approach with a monovalent anti-Env Fab probe to characterize the trafficking of internalized Env within infected CD4 + T-cell lines, together with CRISPR/Cas9-mediated endogenous protein tagging, to assess the role of host cell Rab GTPases in Env trafficking. We show that endocytosed Env traffics to Rab14 + compartments that possess hallmarks of late endosomes and lysosomes. We also demonstrate that Env can recycle back to the PM, although we find that recycling does not occur at high rates when compared to the model recycling protein transferrin. These results help to resolve open questions about the fate and relevance of endocytosed Env in HIV-infected cells and suggest a novel role for Rab14 in a cell-type-specific late-endosomal/lysosomal trafficking pathway in T cells. IMPORTANCE HIV-1 envelope glycoprotein (Env) evades immune neutralization through many mechanisms. One immune evasion strategy may result from the internalization of excess surface-exposed Env to prevent antibody-dependent cellular cytotoxicity or neutralization. Characterization of the fate of endocytosed Env is critical to understand which vesicular pathways could be targeted to promote display of Env epitopes to the immune system. In this study, we characterize the endocytic fate of native Env, expressed from infected human T-cell lines. We demonstrate that Env is rapidly trafficked to a late-endosome/lysosome-like compartment and can be recycled to the cell surface for incorporation into virus assembly sites. This study implicates a novel intracellular compartment, marked by host-cell Rab14 GTPases, for the sequestration of Env. Therapeutic approaches aimed at mobilizing this intracellular pool of Env could lead to stronger immune control of HIV-1 infection via antibody-dependent cell-mediated cytotoxicity.

Gammaherpesviruses, such as human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68), are species-specific, ubiquitous pathogens that are associated with multiple cancers, including B cell lymphomas. These viruses have a natural tropism for B cells and usurp B cell differentiation to drive a unique and robust polyclonal germinal center response to establish a long-term latent reservoir in memory B cells. The robust polyclonal germinal center response driven by gammaherpesvirus infection increases the risk for B cell transformation. Unsurprisingly, many gammaherpesvirus cancers are derived from germinal center or post-germinal center B cells. The viral and host factors that influence the gammaherpesvirus-driven germinal center response are not clearly defined. We previously showed that host interleukin 17 receptor A (IL-17RA) signaling promotes the establishment of chronic MHV68 infection and the MHV68-driven germinal center response. In this study, we found that T cell-intrinsic IL-17RA signaling recapitulates some proviral aspects of global IL-17RA signaling during MHV68 infection. Specifically, we found that T cell-intrinsic IL-17RA signaling supports the MHV68-driven germinal center response, the establishment of latency in the spleen, and viral reactivation in the spleen and peritoneal cavity. Our study unveils an unexpected finding where the T cell-specific IL-17RA signaling supports the establishment of a latent reservoir of a B cell-tropic gammaherpesvirus. IMPORTANCE Gammaherpesviruses, such as human EBV, establish lifelong infection in >95% of adults and are associated with B cell lymphomas. Gammaherpesviruses usurp the germinal center response to establish latent infection, and the germinal center B cells are thought to be the target of viral transformation. We previously found that global expression of IL-17RA promotes the establishment of chronic MHV68 infection and the MHV68-driven germinal center response. In this study, we showed that T cell-intrinsic IL-17RA signaling is necessary to promote the MHV68-driven germinal center response by supporting CD4 + T follicular helper cell expansion. We also found that T cell-intrinsic IL-17RA signaling contributes to but is not solely responsible for the systemic proviral role of IL-17RA signaling, highlighting the multifaceted function of IL-17RA signaling during MHV68 infection.

The main target cells for Epstein-Barr virus (EBV) infection and persistence are B lymphocytes, although T and NK cells can also become infected. In this paper, we characterize the EBV present in 21 pediatric and adult patients who were treated in France for a range of diseases that involve infection of T or NK cells. Of these 21 cases, 5 pediatric patients (21%) and 11 adult patients (52%) were of Caucasian origin. In about 30% of the cases, some of the EBV genomes contain a large deletion. The deletions are different in every patient but tend to cluster near the BART region of the viral genome. Detailed investigation of a family in which several members have persistent T or NK cell infection by EBV indicates that the virus genome deletions arise or are selected independently in each individual patient. Genome sequence polymorphisms in the EBV in these T or NK cell diseases reflect the geographic origin of the patient and not a distinct type of EBV (the 21 cases studied included examples of both type 1 and type 2 EBV infection). Using virus produced from type 1 or type 2 EBV genomes cloned in bacterial artificial chromosome (BAC) vectors, we demonstrate infection of T cells in cord blood from healthy donors. Our results are consistent with transient infection of some T cells being part of normal asymptomatic infection by EBV in young children. IMPORTANCE EBV contributes to several types of human cancer. Some cancers and nonmalignant lymphoproliferative diseases involving T or NK cells contain EBV. These diseases are relatively frequent in Japan and China and have been shown sometimes to have deletions in the EBV genome in the disease cells. We identify further examples of deletions within the EBV genome associated with T or NK cell diseases, and we provide evidence that the virus genomes with these deletions are most likely selected in the individual cases, rather than being transmitted between people during infection. We demonstrate EBV infection of cord blood T cells by highly characterized, cloned EBV genomes and suggest that transient infection of T cells may be part of normal asymptomatic infection by EBV in young children.

SARS-CoV-2 vaccines pose as the most effective approach for mitigating the COVID-19 pandemic. High-degree efficacy of SARS-CoV-2 vaccines in clinical trials indicates that vaccination invariably induces an adaptive immune response. However, the emergence of breakthrough infections in vaccinated individuals suggests that the breadth and magnitude of vaccine-induced adaptive immune response may vary. We assessed vaccine-induced SARS-CoV-2 T cell response in 21 vaccinated individuals and found that SARS-CoV-2-specific T cells, which were mainly CD4+ T cells, were invariably detected in all individuals but the response was varied. We then investigated differentiation states and cytokine profiles to identify immune features associated with superior recall function and longevity. We identified SARS-CoV-2-specific CD4+ T cells were polyfunctional and produced high levels of IL-2, which could be associated with superior longevity. Based on the breadth and magnitude of vaccine-induced SARS-CoV-2 response, we identified 2 distinct response groups: individuals with high abundance versus low abundance of SARS-CoV-2-specific T cells. The fractions of TNF-α- and IL-2-producing SARS-CoV-2 T cells were the main determinants distinguishing high versus low responders. Last, we identified that the majority of vaccine-induced SARS-CoV-2 T cells were reactive against non-mutated regions of mutant S-protein, suggesting that vaccine-induced SARS-CoV-2 T cells could provide continued protection against emerging variants of concern.

Recent evidence indicates that viral components of the microbiota can contribute to intestinal homeostasis and protection from local inflammatory or infectious insults. However, host-derived mechanisms that regulate the virome remain largely unknown. In this study, we used colonization with the model commensal murine norovirus (MNV; strain CR6) to interrogate host-directed mechanisms of viral regulation, and we show that STAT1 is a central coordinator of both viral replication and antiviral T cell responses. In addition to restricting CR6 replication to the intestinal tract, we show that STAT1 regulates antiviral CD4 + and CD8 + T cell responses and prevents systemic viral-induced tissue damage and disease. Despite altered T cell responses that resemble those that mediate lethal immunopathology in systemic viral infections in STAT1-deficient mice, depletion of adaptive immune cells and their associated effector functions had no effect on CR6-induced disease. However, therapeutic administration of an antiviral compound limited viral replication, preventing virus-induced tissue damage and death without impacting the generation of inflammatory antiviral T cell responses. Collectively, our data show that STAT1 restricts MNV CR6 replication within the intestinal mucosa and that uncontrolled viral replication mediates disease rather than the concomitant development of dysregulated antiviral T cell responses in STAT1-deficient mice. IMPORTANCE The intestinal microbiota is a collection of bacteria, archaea, fungi, and viruses that colonize the mammalian gut. Coevolution of the host and microbiota has required development of immunological tolerance to prevent ongoing inflammatory responses against intestinal microbes. Breakdown of tolerance to bacterial components of the microbiota can contribute to immune activation and inflammatory disease. However, the mechanisms that are necessary to maintain tolerance to viral components of the microbiome, and the consequences of loss of tolerance, are less well understood. Here, we show that STAT1 is integral for preventing escape of a commensal-like virus, murine norovirus CR6 (MNV CR6), from the gut and that in the absence of STAT1, mice succumb to infection-induced disease. In contrast to the case with other systemic viral infections, mortality of STAT1-deficient mice is not driven by immune-mediated pathology. Our data demonstrate the importance of host-mediated geographical restriction of commensal-like viruses.

CD46 is a receptor for human herpesvirus 6A (HHV-6A) and is in some cells also important for infection with HHV-6B. CD46 has several isoforms of which the most commonly expressed can be distinguished by expression of a BC domain or a C domain in a serine-threonine-proline-rich (STP) extracellular region. Using a SupT1 CD46 CRISPR-Cas9 knockout model system reconstituted with specific CD46 isoforms, we demonstrated that HHV-6A infection was more efficient when BC isoforms were expressed as opposed to C isoforms, measured by higher levels of intracellular viral transcripts and recovery of more progeny virus. Although the B domain contains several O -glycosylations, mutations of Ser and Thr residues did not prevent infection with HHV-6A. The HHV-6A infection was blocked by inhibitors of clathrin-mediated endocytosis. In contrast, infection with HHV-6B was preferentially promoted by C isoforms mediating fusion-from-without, and this infection was less affected by inhibitors of clathrin-mediated endocytosis. Taken together, HHV-6A preferred BC isoforms, mediating endocytosis, whereas HHV-6B preferred C isoforms, mediating fusion-from-without. This demonstrates that the STP region of CD46 is important for regulating the mode of infection in SupT1 cells and suggests an epigenetic regulation of the host susceptibility to HHV-6A and HHV-6B infection. IMPORTANCE CD46 is the receptor used by human herpesvirus 6A (HHV-6A) during infection of T cells, but it is also involved in infection of certain T cells by HHV-6B. The gene for CD46 allows expression of several variants of CD46, known as isoforms, but whether the isoforms matter for infection of T cells is unknown. We used a genetic approach to delete CD46 from T cells and reconstituted them with separate isoforms to study them individually. We expressed the isoforms known as BC and C, which are distinguished by the potential inclusion of a B domain in the CD46 molecule. We demonstrate that HHV-6A prefers the BC isoform to infect T cells, and this occurs predominantly by clathrin-mediated endocytosis. In contrast, HHV-6B prefers the C isoform and infects predominantly by fusion-from-without. Thus, CD46 isoforms may affect susceptibility of T cells to infection with HHV-6A and HHV-6B.

Latency is a hallmark of herpesviruses, allowing them to persist in their host without virion production. Acute exposure to hypoxia (below 3% O 2 ) was identified as a trigger of latent-to-lytic switch (reactivation) for human oncogenic gammaherpesviruses (Kaposi's sarcoma-associated virus [KSHV] and Epstein-Barr virus [EBV]). Therefore, we hypothesized that hypoxia could also induce reactivation of Marek's disease virus (MDV), which shares biological properties with EBV and KSHV (notably oncogenic properties), in lymphocytes. Acute exposure to hypoxia (1% O 2 ) of two MDV-latently infected cell lines derived from MD tumors (3867K and MSB-1) induced MDV reactivation. A bioinformatic analysis of the RB-1B MDV genome revealed 214 putative hypoxia response element consensus sequences on 119 open reading frames. Reverse transcriptase quantitative PCR (RT-qPCR) analysis showed five MDV genes strongly upregulated early after hypoxia. In 3867K cells under normoxia, pharmacological agents mimicking hypoxia (MLN4924 and CoCl 2 ) increased MDV reactivation, but to a lower level than real hypoxia. Overexpression of wild-type or stabilized human hypoxia inducible factor 1α (HIF-1α) in MSB-1 cells in normoxia also promoted MDV reactivation. Under such conditions, the lytic cycle was detected in cells with a sustainable HIF-1α expression but also in HIF-1α-negative cells, indicating that MDV reactivation is mediated by HIF-1 in a direct and/or indirect manner. Lastly, we demonstrated by a reporter assay that HIF-1α overexpression induced the transactivation of two viral promoters, shown to be upregulated in hypoxia. These results suggest that hypoxia may play a crucial role in the late lytic replication phase observed in vivo in MDV-infected chickens exhibiting tumors, since a hypoxic microenvironment is a hallmark of most solid tumors. IMPORTANCE Latent-to-lytic switch of herpesviruses (also known as reactivation) is responsible for pathology recurrences and/or viral shedding. Studying physiological triggers of reactivation is therefore important for health to limit lesions and viral transmission. Marek's disease virus (MDV) is a potent oncogenic alphaherpesvirus establishing latency in T lymphocytes and causing lethal T lymphomas in chickens. In vivo , a second lytic phase is observed during the tumoral stage. Hypoxia being a hallmark of tumors, we wondered whether hypoxia induces MDV reactivation in latently infected T lymphocytes, like previously shown for EBV and KSHV in B lymphocytes. In this study, we demonstrated that acute hypoxia (1% O 2 ) triggers MDV reactivation in two MDV transformed T-cell lines. We provide some molecular basis of this reactivation by showing that hypoxia inducible factor 1 (HIF-1) overexpression induces MDV reactivation to an extent similar to that of hypoxia after 24 h. Hypoxia is therefore a reactivation stimulus shared by mammalian and avian oncogenic herpesviruses of different genera.

Background: Multisystem inflammatory syndrome in children (MIS-C) is an acute, febrile, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated syndrome, often with cardiohemodynamic dysfunction. Insight into mechanism of disease is still incomplete., Objective: Our objective was to analyze immunologic features of MIS-C patients compared to febrile controls (FC)., Methods: MIS-C patients were defined by narrow criteria, including having evidence of cardiohemodynamic involvement and no macrophage activation syndrome. Samples were collected from 8 completely treatment-naive patients with MIS-C (SARS-CoV-2 serology positive), 3 patients with unclassified MIS-C-like disease (serology negative), 14 FC, and 5 MIS-C recovery (RCV). Three healthy controls (HCs) were used for comparisons of normal range. Using spectral flow cytometry, we assessed 36 parameters in antigen-presenting cells (APCs) and 29 in T cells. We used biaxial analysis and uniform manifold approximation and projection (UMAP)., Results: Significant elevations in cytokines including CXCL9, M-CSF, and IL-27 were found in MIS-C compared to FC. Classic monocytes and type 2 dendritic cells (DCs) were downregulated (decreased CD86, HLA-DR) versus HCs; however, type 1 DCs (CD11c + CD141 + CLEC9A + ) were highly activated in MIS-C patients versus FC, expressing higher levels of CD86, CD275, and atypical conventional DC markers such as CD64, CD115, and CX3CR1. CD169 and CD38 were upregulated in multiple monocyte subtypes. CD56 dim /CD57 - /KLRG hi /CD161 + /CD38 - natural killer (NK) cells were a unique subset in MIS-C versus FC without macrophage activation syndrome., Conclusion: Orchestrated by complex cytokine signaling, type 1 DC activation and NK dysregulation are key features in the pathophysiology of MIS-C. NK cell findings may suggest a relationship with macrophage activation syndrome, while type 1 DC upregulation implies a role for antigen cross-presentation., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Variants are globally emerging very quickly following pandemic prototypic SARS-CoV-2. To evaluate the cross-protection of prototypic SARS-CoV-2 vaccine against its variants, we vaccinated rhesus monkeys with three doses of prototypic SARS-CoV-2 inactivated vaccine, followed by challenging with emerging SARS-CoV-2 variants of concern (VOCs). These vaccinated animals produced neutralizing antibodies against Alpha, Beta, Delta, and Omicron variants, although there were certain declinations of geometric mean titer (GMT) as compared with prototypic SARS-CoV-2. Of note, in vivo this prototypic vaccine not only reduced the viral loads in nasal, throat and anal swabs, pulmonary tissues, but also improved the pathological changes in the lung infected by variants of Alpha, Beta, and Delta. In summary, the prototypic SARS-CoV-2 inactivated vaccine in this study protected against VOCs to certain extension, which is of great significance for prevention and control of COVID-19., (© 2022. The Author(s).)

HIV Envelope (Env) is the main vaccine target for induction of neutralizing antibodies. Stabilizing Env into native-like trimer (NLT) conformations is required for recombinant protein immunogens to induce autologous neutralizing antibodies(nAbs) against difficult to neutralize HIV strains (tier-2) in rabbits and non-human primates. Immunizations of mice with NLTs have generally failed to induce tier-2 nAbs. Here, we show that DNA-encoded NLTs fold properly in vivo and induce autologous tier-2 nAbs in mice. DNA-encoded NLTs also uniquely induce both CD4 + and CD8 + T-cell responses as compared to corresponding protein immunizations. Murine neutralizing antibodies are identified with an advanced sequencing technology. The structure of an Env-Ab (C05) complex, as determined by cryo-EM, identifies a previously undescribed neutralizing Env C3/V5 epitope. Beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines., (© 2022. The Author(s).)

HPV68 is a common HR-HPV, its persistent infection is closely related with the occurrence of cervical cancer. In this study, 2939 (27.60%, 2939/10650) positive samples were detected, and 174 (5.92%, 174/2939) were HPV68. 150 HPV68 E6-E7 were successful sequenced, 4 non-synonymous mutations were detected in E6, and E7 were 12. N133S non-synonymous mutations of HPV 68 E6 and C67G, T68 A/M of HPV68 E7 are E6, E7 positive selection sites, they all located in the key domains and major motifs of E6/E7 protein, the above amino-acid substitutions changed the protein structure, disturbed the interaction with other protein or cellular factors and make a difference in epitopes affinity, may affect the pathogenicity and adaptability of HPV68 to the environment. The enrichment of HPV68 data is of great significance for understanding the inherent geographical and biological differences of HPV68 in China., (Copyright © 2021. Published by Elsevier Inc.)

A proportion of patients surviving acute coronavirus disease 2019 (COVID-19) infection develop post-acute COVID syndrome (long COVID (LC)) lasting longer than 12 weeks. Here, we studied individuals with LC compared to age- and gender-matched recovered individuals without LC, unexposed donors and individuals infected with other coronaviruses. Patients with LC had highly activated innate immune cells, lacked naive T and B cells and showed elevated expression of type I IFN (IFN-β) and type III IFN (IFN-λ1) that remained persistently high at 8 months after infection. Using a log-linear classification model, we defined an optimal set of analytes that had the strongest association with LC among the 28 analytes measured. Combinations of the inflammatory mediators IFN-β, PTX3, IFN-γ, IFN-λ2/3 and IL-6 associated with LC with 78.5-81.6% accuracy. This work defines immunological parameters associated with LC and suggests future opportunities for prevention and treatment., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The adaptive immune response is a major determinant of the clinical outcome after SARS-CoV-2 infection and underpins vaccine efficacy. T cell responses develop early and correlate with protection but are relatively impaired in severe disease and are associated with intense activation and lymphopenia. A subset of T cells primed against seasonal coronaviruses cross reacts with SARS-CoV-2 and may contribute to clinical protection, particularly in early life. T cell memory encompasses broad recognition of viral proteins, estimated at around 30 epitopes within each individual, and seems to be well sustained so far. This breadth of recognition can limit the impact of individual viral mutations and is likely to underpin protection against severe disease from viral variants, including Omicron. Current COVID-19 vaccines elicit robust T cell responses that likely contribute to remarkable protection against hospitalization or death, and novel or heterologous regimens offer the potential to further enhance cellular responses. T cell immunity plays a central role in the control of SARS-CoV-2 and its importance may have been relatively underestimated thus far., (© 2022. Springer Nature America, Inc.)

Visualization of the herpesvirus genomes during lytic replication and latency is mainly achieved by fluorescence in situ hybridization (FISH). Unfortunately, this technique cannot be used for the real-time detection of viral genome in living cells. To facilitate the visualization of the Marek's disease virus (MDV) genome during all stages of the virus lifecycle, we took advantage of the well-established tetracycline operator/repressor (TetO/TetR) system. This system consists of a fluorescently labeled TetR (TetR-GFP) that specifically binds to an array of tetO sequences. This tetO repeat array was first inserted into the MDV genome (vTetO). Subsequently, we fused TetR-GFP via a P2a self-cleaving peptide to the C-terminus of the viral interleukin 8 (vIL8), which is expressed during lytic replication and latency. Upon reconstitution of this vTetO-TetR virus, fluorescently labeled replication compartments were detected in the nucleus during lytic replication. After validating the specificity of the observed signal, we used the system to visualize the genesis and mobility of the viral replication compartments. In addition, we assessed the infection of nuclei in syncytia as well as lytic replication and latency in T cells. Taken together, we established a system allowing us to track the MDV genome in living cells that can be applied to many other DNA viruses.

A recent comparison of clinical and inflammatory parameters, together with biomarkers of oxidative stress, in patients who died from aggressive COVID-19 and survivors suggested that the lipid peroxidation product 4-hydroxynonenal (4-HNE) might be detrimental in lethal SARS-CoV-2 infection. The current study further explores the involvement of inflammatory cells, systemic vascular stress, and 4-HNE in lethal COVID-19 using specific immunohistochemical analyses of the inflammatory cells within the vital organs obtained by autopsy of nine patients who died from aggressive SAR-CoV-2 infection. Besides 4-HNE, myeloperoxidase (MPO) and mitochondrial superoxide dismutase (SOD2) were analyzed alongside standard leukocyte biomarkers (CDs). All the immunohistochemical slides were simultaneously prepared for each analyzed biomarker. The results revealed abundant 4-HNE in the vital organs, but the primary origin of 4-HNE was sepsis-like vascular stress, not an oxidative burst of the inflammatory cells. In particular, inflammatory cells were often negative for 4-HNE, while blood vessels were always very strongly immunopositive, as was edematous tissue even in the absence of inflammatory cells. The most affected organs were the lungs with diffuse alveolar damage and the brain with edema and reactive astrocytes, whereas despite acute tubular necrosis, 4-HNE was not abundant in the kidneys, which had prominent SOD2. Although SOD2 in most cases gave strong immunohistochemical positivity similar to 4-HNE, unlike 4-HNE, it was always limited to the cells, as was MPO. Due to their differential expressions in blood vessels, inflammatory cells, and the kidneys, we think that SOD2 could, together with 4-HNE, be a potential link between a malfunctioning immune system, oxidative stress, and vascular stress in lethal COVID-19.

The CCCH-type zinc finger antiviral protein (ZAP) can recognize and induce the degradation of mRNAs and proteins of certain viruses, as well as exerting its antiviral activity by activating T cells. However, the mechanism of ZAP that mediates T cell activation during virus infection remains unclear. Here, we found a potential function of ZAP that relieves immunosuppression of T cell induced by avian leukosis virus subgroup J (ALV-J) via a novel signaling pathway that involves norbin-like protein (NLP), protein kinase C delta (PKC-δ), and nuclear factor of activated T cell (NFAT). Specifically, ZAP expression activated T cells by promoting the dephosphorylation and nuclear translocation of NFAT. Furthermore, knockdown of ZAP weakened the reactivity and antiviral response of T cells. Mechanistically, ZAP reduced PKC-δ activity by upregulating and reactivating NLP by competitively binding with viral protein. Knockdown of NLP decreased the dephosphorylation of PKC-δ by ZAP expression. Moreover, we show that knockdown of PKC-δ reduced the phosphorylation levels of NFAT and enhanced its nuclear translocation. Taken together, these data revealed that ZAP relieves immunosuppression caused by ALV-J and mediates T cell activation through the NLP-PKC-δ-NFAT pathway. IMPORTANCE The evolution of the host defense system is driven synchronously in the process of resisting virus invasion. Accordingly, host innate defense factors effectively work to suppress virus replication. However, it remains unclear whether the host innate defense factors are involved in antiviral immune responses against the invasion of immunosuppressive viruses. Here, we found that CCCH-type zinc finger antiviral protein (ZAP) effectively worked in resistance to immunosuppression caused by avian leukosis virus subgroup J (ALV-J), a classic immunosuppressive virus. Evidence showed that ZAP released the phosphatase activity of NLP inhibited by ALV-J and further activated NFAT by inactivating PKC-δ. This novel molecular mechanism, i.e., ZAP regulation of the antiviral immune response by mediating the NLP-PKC-δ-NFAT pathway, has greatly enriched the understanding of the functions of host innate defense factors and provided important scientific ideas and a theoretical basis for research on immunosuppressive viruses and antiviral immunity.

Gamma delta (γδ) T cells play a key role in the innate immune response and serve as the first line of defense against infection and tumors. These cells are defined as tissue-resident lymphocytes in skin, lung, and intestinal mucosa. They are also relatively abundant in the liver; however, little is known about the residency of hepatic γδT cells. By comparing the phenotype of murine γδT cells in liver, spleen, thymus, and small intestine, a CXCR3 + CXCR6 + γδT-cell subset with tissue-resident characteristics was found in liver tissue from embryos through adults. Liver sinusoidal endothelial cells mediated retention of CXCR3 + CXCR6 + γδT cells through the interactions between CXCR3 and CXCR6 and their chemokines. During acute HBV infection, CXCR3 + CXCR6 + γδT cells produced high levels of IFN-γ and adoptive transfer of CXCR3 + CXCR6 + γδT cells into acute HBV-infected TCRδ -/- mice leading to lower HBsAg and HBeAg expression. It is suggested that liver resident CXCR3 + CXCR6 + γδT cells play a protective role during acute HBV infection. Strategies aimed at expanding and activating liver resident CXCR3 + CXCR6 + γδT cells both in vivo or in vitro have great prospects for use in immunotherapy that specifically targets acute HBV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Guan, Hu, Li, Lu and Zhang.)

The HIV-1 envelope (Env) is an essential determinant of viral infectivity, tropism and spread between T cells. Lentiviral Env contain an unusually long 150 amino acid cytoplasmic tail (EnvCT), but the function of the EnvCT and many conserved domains within it remain largely uncharacterised. Here, we identified a highly conserved tryptophan motif at position 757 (W757) in the LLP-2 alpha helix of the EnvCT as a key determinant for HIV-1 replication and spread between T cells. Alanine substitution at this position potently inhibited HIV-1 cell-cell spread (the dominant mode of HIV-1 dissemination) by preventing recruitment of Env and Gag to sites of cell-cell contact, inhibiting virological synapse (VS) formation and spreading infection. Single-molecule tracking and super-resolution imaging showed that mutation of W757 dysregulates Env diffusion in the plasma membrane and increases Env mobility. Further analysis of Env function revealed that W757 is also required for Env fusion and infectivity, which together with reduced VS formation, result in a potent defect in viral spread. Notably, W757 lies within a region of the EnvCT recently shown to act as a supporting baseplate for Env. Our data support a model in which W757 plays a key role in regulating Env biology, modulating its temporal and spatial recruitment to virus assembly sites and regulating the inherent fusogenicity of the Env ectodomain, thereby supporting efficient HIV-1 replication and spread.

Anti-viral immunity continuously declines over time after SARS-CoV-2 infection. Here, we characterize the dynamics of anti-viral immunity during long-term follow-up and after BNT162b2 mRNA-vaccination in convalescents after asymptomatic or mild SARS-CoV-2 infection. Virus-specific and virus-neutralizing antibody titers rapidly declined in convalescents over 9 months after infection, whereas virus-specific cytokine-producing polyfunctional T cells persisted, among which IL-2-producing T cells correlated with virus-neutralizing antibody titers. Among convalescents, 5% of individuals failed to mount long-lasting immunity after infection and showed a delayed response to vaccination compared to 1% of naïve vaccinees, but successfully responded to prime/boost vaccination. During the follow-up period, 8% of convalescents showed a selective increase in virus-neutralizing antibody titers without accompanying increased frequencies of circulating SARS-CoV-2-specific T cells. The same convalescents, however, responded to vaccination with simultaneous increase in antibody and T cell immunity revealing the strength of mRNA-vaccination to increase virus-specific immunity in convalescents., (© 2022. The Author(s).)

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) blunts the broad inflammatory response induced by damage-associated molecular patterns via binding to extracellular high mobility group box 1 and heat shock proteins, as well as regulating the downstream Siglec10-Src homology 2 domain-containing phosphatase 1 pathway. A recent randomized phase III trial evaluating CD24Fc for patients with severe COVID-19 (SAC-COVID; NCT04317040) demonstrated encouraging clinical efficacy., Methods: Using a systems analytical approach, we studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial to discern the impact of CD24Fc treatment on immune homeostasis. We performed high dimensional spectral flow cytometry and measured the levels of a broad array of cytokines and chemokines to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19., Results: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found that patients with severe COVID-19 had systemic hyper-activation of multiple cellular compartments, including CD8 + T cells, CD4 + T cells, and CD56 + natural killer cells. Treatment with CD24Fc blunted this systemic inflammation, inducing a return to homeostasis in NK and T cells without compromising the anti-Spike protein antibody response. CD24Fc significantly attenuated the systemic cytokine response and diminished the cytokine coexpression and network connectivity linked with COVID-19 severity and pathogenesis., Conclusions: Our data demonstrate that CD24Fc rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19., (© 2022. The Author(s).)

Coronavirus disease 2019 (COVID-19) is a global infectious disease caused by the SARS-CoV-2 coronavirus. T cells play an essential role in the body's fighting against the virus invasion, and the T cell receptor (TCR) is crucial in T cell-mediated virus recognition and clearance. However, little has been known about the features of T cell response in convalescent COVID-19 patients. In this study, using 5'RACE technology and PacBio sequencing, we analyzed the TCR repertoire of COVID-19 patients after recovery for 2 weeks and 6 months compared with the healthy donors. The TCR clustering and CDR3 annotation were exploited to discover groups of patient-specific TCR clonotypes with potential SARS-CoV-2 antigen specificities. We first identified CD4 + and CD8 + T cell clones with certain clonal expansion after infection, and then observed the preferential recombination usage of V(D) J gene segments in CD4 + and CD8 + T cells of COVID-19 patients with different convalescent stages. More important, the TRBV6-5-TRBD2-TRBJ2-7 combination with high frequency was shared between CD4 + T and CD8 + T cells of different COVID-19 patients. Finally, we found the dominant characteristic motifs of the CDR3 sequence between recovered COVID-19 and healthy control. Our study provides novel insights on TCR in COVID-19 with different convalescent phases, contributing to our understanding of the immune response induced by SARS-CoV-2.

SARS-CoV-2 has caused morbidity and mortality across the globe. As the virus spreads, new variants are arising that show enhanced capacity to bypass preexisting immunity. To understand the memory response to SARS-CoV-2, here, we monitored SARS-CoV-2–specific T and B cells in a longitudinal study of infected and recovered golden hamsters (Mesocricetus auratus). We demonstrated that engagement of the innate immune system after SARS-CoV-2 infection was delayed but was followed by a pronounced adaptive response. Moreover, T cell adoptive transfer conferred a reduction in virus levels and rapid induction of SARS-CoV-2–specific B cells, demonstrating that both lymphocyte populations contributed to the overall response. Reinfection of recovered animals with a SARS-CoV-2 variant of concern showed that SARS-CoV-2–specific T and B cells could effectively control the infection that associated with the rapid induction of neutralizing antibodies but failed to block transmission to both naïve and seroconverted animals. These data suggest that the adaptive immune response to SARS-CoV-2 is sufficient to provide protection to the host, independent of the emergence of variants.

Enhanced HSV-1 production is found in activated T-lymphocytes, but the mechanism is still unknown. In this paper, the HSV-1 entry step in CD3 + CD4 - CD8 - Jurkat T lymphocytes was investigated. Observation under electron microscopy revealed the level of filopodia formation on the surface of activated Jurkat cells was significantly higher than that of non-activated Jurkat cells especially after adding HSV-1 for 15 min. A significant increase of actin protein was demonstrated in HSV-1 infected, activated Jurkat cells compared to HSV-1 infected, non-activated Jurkat cells. After the cells were treated with 2.5 and 5 µg/mL cytochalasin D, an inhibitor of actin polymerization that causes depolymerization of actin's filamentous form, the actin protein was decreased significantly, resulting in an absence of filopodia formation. In summary, this is the first study revealing that HSV-1 induced filopodia formation through actin polymerization in activated T cells similar to epithelial, mucosal and neuronal cells. This phenomenon supported the virus entry resulting to increased yield of HSV-1 production., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The systemic processes involved in the manifestation of life-threatening COVID-19 and in disease recovery are still incompletely understood, despite investigations focusing on the dysregulation of immune responses after SARS-CoV-2 infection. To define hallmarks of severe COVID-19 in acute disease (n = 58) and in disease recovery in convalescent patients (n = 28) from Hannover Medical School, we used flow cytometry and proteomics data with unsupervised clustering analyses. In our observational study, we combined analyses of immune cells and cytokine/chemokine networks with endothelial activation and injury. ICU patients displayed an altered immune signature with prolonged lymphopenia but the expansion of granulocytes and plasmablasts along with activated and terminally differentiated T and NK cells and high levels of SARS-CoV-2-specific antibodies. The core signature of seven plasma proteins revealed a highly inflammatory microenvironment in addition to endothelial injury in severe COVID-19. Changes within this signature were associated with either disease progression or recovery. In summary, our data suggest that besides a strong inflammatory response, severe COVID-19 is driven by endothelial activation and barrier disruption, whereby recovery depends on the regeneration of the endothelial integrity., (© 2021. The Author(s).)