RESUMEN
Pathogens use virulence factors to inhibit the immune system1. The guard hypothesis2,3 postulates that hosts monitor (or 'guard') critical innate immune pathways such that their disruption by virulence factors provokes a secondary immune response1. Here we describe a 'self-guarded' immune pathway in human monocytes, in which guarding and guarded functions are combined in one protein. We find that this pathway is triggered by ICP0, a key virulence factor of herpes simplex virus type 1, resulting in robust induction of anti-viral type I interferon (IFN). Notably, induction of IFN by ICP0 is independent of canonical immune pathways and the IRF3 and IRF7 transcription factors. A CRISPR screen identified the ICP0 target MORC34 as an essential negative regulator of IFN. Loss of MORC3 recapitulates the IRF3- and IRF7-independent IFN response induced by ICP0. Mechanistically, ICP0 degrades MORC3, which leads to de-repression of a MORC3-regulated DNA element (MRE) adjacent to the IFNB1 locus. The MRE is required in cis for IFNB1 induction by the MORC3 pathway, but is not required for canonical IFN-inducing pathways. As well as repressing the MRE to regulate IFNB1, MORC3 is also a direct restriction factor of HSV-15. Our results thus suggest a model in which the primary anti-viral function of MORC3 is self-guarded by its secondary IFN-repressing function-thus, a virus that degrades MORC3 to avoid its primary anti-viral function will unleash the secondary anti-viral IFN response.
Asunto(s)
Adenosina Trifosfatasas/inmunología , Proteínas de Unión al ADN/inmunología , Modelos Inmunológicos , Factores de Virulencia/inmunología , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Edición Génica , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/patogenicidad , Humanos , Proteínas Inmediatas-Precoces/inmunología , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Monocitos/inmunología , Receptor de Interferón alfa y beta , Proteínas Represoras/deficiencia , Proteínas Represoras/inmunología , Proteínas Represoras/metabolismo , Elementos de Respuesta/genética , Ubiquitina-Proteína Ligasas/inmunologíaRESUMEN
Expression and activity of serum- and glucocorticoid-inducible kinase 1 (SGK1) are associated with many metabolic and inflammatory diseases. In this study, we report that SGK1 promotes alternative macrophage polarization and restrains inflammation in the infectious milieu of the gingiva. Inhibition of SGK1 expression or activity enhances characteristics of classically activated (M1) macrophages by directly activating the transcription of genes encoding iNOS, IL-12P40, TNF-α, and IL-6 and repressing IL-10 at message and protein levels. Moreover, SGK1 inhibition robustly reduces the expression of alternatively activated (M2) macrophage molecular markers, including arginase-1, Ym-1, Fizz1, and Mgl-1. These results were confirmed by multiple gain- and loss-of-function approaches, including small interfering RNA, a plasmid encoding SGK1, and LysM-Cre-mediated sgk1 gene knockout. Further mechanistic analysis showed that SGK1 deficiency decreases STAT3 but increases FoxO1 expression in macrophages under M2 or M1 macrophage-priming conditions, respectively. Combined with decreased FoxO1 phosphorylation and the subsequent suppressed cytoplasmic translocation observed, SGK1 deficiency robustly enhances FoxO1 activity and drives macrophage to preferential M1 phenotypes. Furthermore, FoxO1 inhibition abrogates M1 phenotypes, and STAT3 overexpression results in a significant increase of M2 phenotypes, indicating that both FoxO1 and STAT3 are involved in SGK1-mediated macrophage polarization. Additionally, SGK1 differentially regulates the expression of M1 and M2 molecular markers, including CD68 and F4/F80 and CD163 and CD206, respectively, and protects against Porphyromonas gingivalis-induced alveolar bone loss in a mouse model. Taken together, these results have demonstrated that SGK1 is critical for macrophage polarization and periodontal bone loss, and for the first time, to our knowledge, we elucidated a bifurcated signaling circuit by which SGK1 promotes alternative, while suppressing inflammatory, macrophage polarization.
Asunto(s)
Proteína Forkhead Box O1/inmunología , Proteínas Inmediatas-Precoces/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Factor de Transcripción STAT3/inmunología , Animales , Activación de Macrófagos/inmunología , Ratones , Transducción de Señal/inmunologíaRESUMEN
Following acute infection, herpes simplex virus 1 (HSV-1) lytic cycle viral gene expression is silenced; consequently, lifelong latency in neurons is established. Certain external stimuli that trigger reactivation from latency also activate the glucocorticoid receptor (GR). The synthetic corticosteroid dexamethasone, but not a GR-specific antagonist, increases the frequency of explant-induced reactivation from latency and stimulates productive infection. Furthermore, dexamethasone increases expression of cellular transcription factors in trigeminal ganglionic neurons: for example, SLUG and three Krüppel-like transcription factor (KLF) family members, KLF4, KLF15, and promyelocytic leukemia zinc finger protein (PLZF). Consequently, we hypothesized that stress-induced transcription factors stimulate expression of ICP4, a viral transcriptional regulator required for productive infection. New studies demonstrated that GR and KLF4, PLZF, or SLUG cooperatively transactivate the ICP4 enhancer upstream of a minimal promoter in monkey kidney cells (Vero) and mouse neuroblastoma cells (Neuro-2A). Strikingly, mutagenesis of two KLF4/Sp1 binding sites reduced GR- plus KLF4-, PLZF-, or SLUG-mediated transactivation to basal levels. A consensus enhancer (E)-Box adjacent to a KLF4/Sp1 binding site was also required for GR- and SLUG-, but not KLF family member-, mediated transactivation of the ICP4 promoter. Chromatin immunoprecipitation studies (ChIP) revealed GR and stress-induced transcription factors occupy ICP4 enhancer sequences. Conversely, specific binding was generally reduced in the KLF4/Sp1 mutant. Furthermore, GR and SLUG occupancy of ICP4 enhancer sequences was reduced in the E-Box mutant. Based on these studies, we suggest stressful stimuli can trigger productive infection because GR and specific stress-induced transcription factors activate ICP4 expression.IMPORTANCE Certain stressful stimuli activate the glucocorticoid receptor (GR) and increase the incidence of herpes simplex virus 1 (HSV-1) reactivation from latency. For example, a corticosteroid antagonist impairs productive infection and virus shedding following explant of trigeminal ganglia from latently infected mice. Infected cell protein 4 (ICP4) is the only immediate early viral transcriptional regulator required for productive infection, suggesting stressful stimuli stimulate ICP4 expression. New studies revealed GR and stress-induced transcription factors identified during reactivation from latency, SLUG and three Krüppel-like transcription factor family members (KLF4, KLF15, and promyelocytic leukemia zinc finger protein), cooperatively transactivate the ICP4 enhancer. Two KLF4 consensus binding sites were crucial for cooperative transactivation of the ICP4 enhancer. A consensus enhancer-box also mediated cooperative transactivation of the ICP4 enhancer by GR and SLUG. The ability of GR and stress-induced transcription factors to transactivate ICP4 enhancer activity is predicted to trigger productive infection following stressful stimuli.
Asunto(s)
Herpes Simple , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/inmunología , Receptores de Glucocorticoides/inmunología , Activación Viral , Latencia del Virus , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Regulación Viral de la Expresión Génica , Herpes Simple/inmunología , Herpes Simple/virología , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/inmunología , Ratones , Proteína de la Leucemia Promielocítica con Dedos de Zinc/inmunología , Factores de Transcripción de la Familia Snail/inmunología , Activación Transcripcional , Células VeroRESUMEN
Promyelocytic leukemia (PML) bodies are nuclear organelles implicated in intrinsic and innate antiviral defense. The eponymous PML proteins, central to the self-organization of PML bodies, and other restriction factors found in these organelles are common targets of viral antagonism. The 72-kDa immediate-early protein 1 (IE1) is the principal antagonist of PML bodies encoded by the human cytomegalovirus (hCMV). IE1 is believed to disrupt PML bodies by inhibiting PML SUMOylation, while PML was proposed to act as an E3 ligase for IE1 SUMOylation. PML targeting by IE1 is considered to be crucial for hCMV replication at low multiplicities of infection, in part via counteracting antiviral gene induction linked to the cellular interferon (IFN) response. However, current concepts of IE1-PML interaction are largely derived from mutant IE1 proteins known or predicted to be metabolically unstable and globally misfolded. We performed systematic clustered charge-to-alanine scanning mutagenesis and identified a stable IE1 mutant protein (IE1cc172-176) with wild-type characteristics except for neither interacting with PML proteins nor inhibiting PML SUMOylation. Consequently, IE1cc172-176 does not associate with PML bodies and is selectively impaired for disrupting these organelles. Surprisingly, functional analysis of IE1cc172-176 revealed that the protein is hypermodified by mixed SUMO chains and that IE1 SUMOylation depends on nucleosome rather than PML binding. Furthermore, a mutant hCMV expressing IE1cc172-176 was only slightly attenuated compared to an IE1-null virus even at low multiplicities of infection. Finally, hCMV-induced expression of cytokine and IFN-stimulated genes turned out to be reduced rather than increased in the presence of IE1cc172-176 relative to wild-type IE1. Our findings challenge present views on the relationship of IE1 with PML and the role of PML in hCMV replication. This study also provides initial evidence for the idea that disruption of PML bodies upon viral infection is linked to activation rather than inhibition of innate immunity.
Asunto(s)
Infecciones por Citomegalovirus , Citomegalovirus/fisiología , Proteínas Inmediatas-Precoces , Inmunidad Innata , Proteína de la Leucemia Promielocítica , Replicación Viral , Línea Celular , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/patología , Regulación Viral de la Expresión Génica/inmunología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Mutación , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/inmunología , Sumoilación/inmunología , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
INTRODUCTION: Epstein-Barr virus (EBV/HHV-4) has been implicated in the pathogenesis of multiple sclerosis (MS). This study was conducted to investigate the levels of pro-inflammatory cytokines IL-1ß and IL-6 in healthy EBV carriers and MS patients with prior EBV infection in response to treatment with EBV nuclear antigen 1 (EBNA-1) and replication and transcription activator (BRLF-1/Rta) peptide antigens in whole blood cell culture to assess the cytokine expression across all cells in the peripheral blood. METHODS: Isolated whole blood cells from the included participants were incubated at a concentration of 106 cells/mL with BRLF-1 or EBNA-1. The amount of IL-1ß and IL-6 transcripts were measured with quantitative RT-PCR at day 3 after incubation. MTT assay was conducted to examine cytotoxicity of the peptides and their effect on cell viability. Changes in cytokine expression and cell viability were analyzed using one-way and two-way ANOVA, respectively. RESULTS: Ten MS patients and ten healthy donors were enrolled in the study. Treatment with the peptide antigens resulted in increased cytokines expression in both MS patients and healthy subjects. Furthermore, IL-1ß levels were higher in MS patients compared to healthy EBV carriers. MTT assay revealed no significant difference in cell viability between the two groups. DISCUSSION: The higher levels of IL-1ß in response to EBV antigens in MS patients may reflect the host neuroinflammatory environment and support the notion that immune response against EBV has a role as an aggravating factor in the progression of MS by contributing to the neuroinflammatory cascade.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Antígenos Nucleares del Virus de Epstein-Barr , Proteínas Inmediatas-Precoces , Esclerosis Múltiple , Transactivadores , Citocinas/metabolismo , Infecciones por Virus de Epstein-Barr/complicaciones , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4 , Humanos , Proteínas Inmediatas-Precoces/inmunología , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Transactivadores/inmunologíaRESUMEN
Type I IFNs play an important role in innate immunity against viral infections by inducing the expression of IFN-stimulated genes (ISGs), which encode effectors with various antiviral functions. We and others previously reported that HSV type 2 (HSV-2) inhibits the synthesis of type I IFNs, but how HSV-2 suppresses IFN-mediated signaling is less understood. In the current study, after the demonstration of HSV-2 replication resistance to IFN-ß treatment in human epithelial cells, we reveal that HSV-2 and the viral protein ICP22 significantly decrease the expression of ISG54 at both mRNA and protein levels. Likewise, us1 del HSV-2 (ICP22-deficient HSV-2) replication is more sensitive to IFN-ß treatment, indicating that ICP22 is a vital viral protein responsible for the inhibition of type I IFN-mediated signaling. In addition, overexpression of HSV-2 ICP22 inhibits the expression of STAT1, STAT2, and IFN regulatory factor 9 (IRF9), resulting in the blockade of ISG factor 3 (ISGF3) nuclear translocation, and mechanistically, this is due to ICP22-induced ubiquitination of STAT1, STAT2, and IRF9. HSV-2 ICP22 appears to interact with STAT1, STAT2, IRF9, and several other ubiquitinated proteins. Following further biochemical study, we show that HSV-2 ICP22 functions as an E3 ubiquitin protein ligase to induce the formation of polyubiquitin chains. Taken together, we demonstrate that HSV-2 interferes with type I IFN-mediated signaling by degrading the proteins of ISGF3, and we identify HSV-2 ICP22 as a novel E3 ubiquitin protein ligase to induce the degradation of ISGF3. Findings in this study highlight a new mechanism by which HSV-2 circumvents the host antiviral responses through a viral E3 ubiquitin protein ligase.
Asunto(s)
Herpes Genital/inmunología , Herpesvirus Humano 2/inmunología , Proteínas Inmediatas-Precoces/inmunología , Interferón beta/inmunología , Transducción de Señal/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Proteínas Virales/inmunología , Antivirales/inmunología , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células HeLa , Herpesvirus Humano 1/inmunología , Humanos , Inmunidad Innata/inmunología , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT2/inmunología , Ubiquitinación/inmunologíaRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic etiological factor for Kaposi's sarcoma and primary effusion lymphoma in immunocompromised patients. KSHV utilizes two immune evasion E3 ubiquitin ligases, namely K3 and K5, to downregulate the expression of antigen-presenting molecules and ligands of natural killer (NK) cells in the host cells through an ubiquitin-dependent endocytic mechanism. This allows the infected cells to evade surveillance and elimination by cytotoxic lymphocytes and NK cells. The number of host cell molecular substrates reported for these ubiquitin ligases is limited. The identification of novel substrates for these ligases will aid in elucidating the mechanism underlying immune evasion of KSHV. This study demonstrated that K5 downregulated the cell surface expression of l-selectin, a C-type lectin-like adhesion receptor expressed in the lymphocytes. Tryptophan residue located at the centre of the E2-binding site in the K5 RINGv domain was essential to downregulate l-selectin expression. Additionally, the lysine residues located at the cytoplasmic tail of l-selectin were required for the K5-mediated downregulation of l-selectin. K5 promoted the degradation of l-selectin through polyubiquitination. These results suggest that K5 downregulates l-selectin expression on the cell surface by promoting polyubiquitination and ubiquitin-dependent endocytosis, which indicated that l-selectin is a novel substrate for K5. Additionally, K3 downregulated l-selectin expression. The findings of this study will aid in the elucidation of a novel immune evasion mechanism in KSHV.
Asunto(s)
Herpesvirus Humano 8/enzimología , Proteínas Inmediatas-Precoces/inmunología , Selectina L/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virología , Ubiquitina-Proteína Ligasas/inmunología , Proteínas Virales/inmunología , Regulación hacia Abajo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/inmunología , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/genética , Evasión Inmune , Células Asesinas Naturales/inmunología , Selectina L/inmunología , Sarcoma de Kaposi/inmunología , Ubiquitina-Proteína Ligasas/genética , Proteínas Virales/genéticaRESUMEN
ND10 nuclear bodies, as part of the intrinsic defenses, impose repression on incoming DNA. Infected cell protein 0 (ICP0), an E3 ubiquitin ligase of herpes simplex virus 1 (HSV-1), can derepress viral genes by degrading ND10 organizers to disrupt ND10. These events are part of the initial tug of war between HSV-1 and host, which determines the ultimate outcome of infection. Previously, we reported that ICP0 differentially recognizes promyelocytic leukemia (PML) isoforms. ICP0 depends on a SUMO-interaction motif located at residues 362 to 364 (SIM362-364) to trigger the degradation of PML isoforms II, IV, and VI, while using a bipartite sequence flanking the RING domain to degrade PML I. In this study, we investigated how the SUMO-SIM interaction regulates the degradation of PML II and PML II-associated proteins in ND10. We found that (i) the same regulatory mechanism for PML II degradation was detected in cells permissive or nonpermissive to the ICP0-null virus; (ii) the loss of a single SIM362-364 motif was restored by the presence of four consecutive SIMs from RNF4, but was not rescued by only two of the RNF4 SIMs; (iii) the loss of three C-terminal SIMs of ICP0 was fully restored by four RNF4 SIMs and also partially rescued by two RNF4 SIMs; and (iv) a PML II mutant lacking both lysine SUMOylation and SIM was not recognized by ICP0 for degradation, but was localized to ND10 and mitigated the degradation of other ND10 components, leading to delayed viral production. Taken together, SUMO regulates ICP0 substrate recognition via multiple fine-tuned mechanisms in HSV-1 infection.IMPORTANCE HSV-1 ICP0 is a multifunctional immediate early protein key to effective replication in the HSV-1 lytic cycle and reactivation in the latent cycle. ICP0 transactivates gene expression by orchestrating an overall mitigation in host intrinsic/innate restrictions. How ICP0 coordinates its multiple active domains and its diverse protein-protein interactions is a key question in understanding the HSV-1 life cycle and pathogenesis. The present study focuses on delineating the regulatory effects of the SUMO-SIM interaction on ICP0 E3 ubiquitin ligase activity regarding PML II degradation. For the first time, we discovered the importance of multivalency in the PML II-ICP0 interaction network and report the involvement of different regulatory mechanisms in PML II recognition by ICP0 in HSV-1 infection.
Asunto(s)
Herpesvirus Humano 1/inmunología , Interacciones Huésped-Patógeno/inmunología , Proteínas Inmediatas-Precoces/inmunología , Proteínas Nucleares/inmunología , Proteína de la Leucemia Promielocítica/inmunología , Procesamiento Proteico-Postraduccional , Factores de Transcripción/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Línea Celular Tumoral , Células Epiteliales/inmunología , Células Epiteliales/virología , Regulación de la Expresión Génica , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Mutación , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica/genética , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Proteolisis , Transducción de Señal , Sumoilación , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Neurotropic Alphaherpesvirinae subfamily members such as bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) establish and maintain lifelong latent infections in neurons. Following infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia (TG) are an important site for latency. Certain external stressors can trigger reactivation from latency, in part because activation of the glucocorticoid receptor (GR) stimulates productive infection and promoters that drive expression of key viral transcriptional regulators. The Akt serine/threonine protein kinase family is linked to maintaining latency. For example, Akt3 is detected in more TG neurons during BoHV-1 latency than in reactivation and uninfected calves. Furthermore, Akt signaling correlates with maintaining HSV-1 latency in certain neuronal models of latency. Finally, an active Akt protein kinase is crucial for the ability of the HSV-1 latency-associated transcript (LAT) to inhibit apoptosis in neuronal cell lines. Consequently, we hypothesized that viral and/or cellular factors impair stress-induced transcription and reduce the incidence of reactivation triggered by low levels of stress. New studies demonstrate that Akt1 and Akt2, but not Akt3, significantly reduced GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, the HSV-1 infected cell protein 0 (ICP0) promoter, and the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Akt3, but not Akt1 or Akt2, significantly enhanced neurite formation in mouse neuroblastoma cells, which correlates with repairing damaged neurons. These studies suggest that unique biological properties of the three Akt family members promote the maintenance of latency in differentiated neurons.IMPORTANCE External stressful stimuli are known to increase the incidence of reactivation of Alphaherpesvirinae subfamily members. Activation of the glucocorticoid receptor (GR) by the synthetic corticosteroid dexamethasone (DEX) stimulates bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) reactivation. Furthermore, GR and dexamethasone stimulate productive infection and promoters that drive expression of viral transcriptional regulators. These observations lead us to predict that stress-induced transcription is impaired by factors abundantly expressed during latency. Interestingly, activation of the Akt family of serine/threonine protein kinases is linked to maintenance of latency. New studies reveal that Akt1 and Ak2, but not Akt3, impaired GR- and dexamethasone-mediated transactivation of the BoHV-1 immediate early transcription unit 1 and HSV-1 ICP0 promoters. Strikingly, Akt3, but not Akt1 or Akt2, stimulated neurite formation in mouse neuroblastoma cells, a requirement for neurogenesis. These studies provide insight into how Akt family members may promote the maintenance of lifelong latency.
Asunto(s)
Herpes Simple/inmunología , Infecciones por Herpesviridae/inmunología , Interacciones Huésped-Patógeno/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Células Receptoras Sensoriales/virología , Animales , Bovinos , Diferenciación Celular , Línea Celular Tumoral , Herpes Simple/genética , Herpes Simple/patología , Herpes Simple/virología , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/inmunología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/inmunología , Interacciones Huésped-Patógeno/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Ratones , Neuritas/inmunología , Neuritas/ultraestructura , Neuritas/virología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/inmunología , Células Receptoras Sensoriales/inmunología , Células Receptoras Sensoriales/patología , Transducción de Señal , Activación Transcripcional/inmunología , Ganglio del Trigémino/inmunología , Ganglio del Trigémino/patología , Ganglio del Trigémino/virología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunologíaRESUMEN
The T cell receptor (TCR) repertoire is an essential component of the CD8 T-cell immune response. Here, we seek to investigate factors that drive selection of TCR repertoires specific to the HLA-A2-restricted immunodominant epitope BRLF1109-117 (YVLDHLIVV) over the course of primary Epstein Barr virus (EBV) infection. Using single-cell paired TCRαß sequencing of tetramer sorted CD8 T cells ex vivo, we show at the clonal level that recognition of the HLA-A2-restricted BRLF1 (YVL-BR, BRLF-1109) epitope is mainly driven by the TCRα chain. For the first time, we identify a CDR3α (complementarity determining region 3 α) motif, KDTDKL, resulting from an obligate AV8.1-AJ34 pairing that was shared by all four individuals studied. This observation coupled with the fact that this public AV8.1-KDTDKL-AJ34 TCR pairs with multiple different TCRß chains within the same donor (median 4; range: 1-9), suggests that there are some unique structural features of the interaction between the YVL-BR/MHC and the AV8.1-KDTDKL-AJ34 TCR that leads to this high level of selection. Newly developed TCR motif algorithms identified a lysine at position 1 of the CDR3α motif that is highly conserved and likely important for antigen recognition. Crystal structure analysis of the YVL-BR/HLA-A2 complex revealed that the MHC-bound peptide bulges at position 4, exposing a negatively charged aspartic acid that may interact with the positively charged lysine of CDR3α. TCR cloning and site-directed mutagenesis of the CDR3α lysine ablated YVL-BR-tetramer staining and substantially reduced CD69 upregulation on TCR mutant-transduced cells following antigen-specific stimulation. Reduced activation of T cells expressing this CDR3 motif was also observed following exposure to mutated (D4A) peptide. In summary, we show that a highly public TCR repertoire to an immunodominant epitope of a common human virus is almost completely selected on the basis of CDR3α and provide a likely structural basis for the selection. These studies emphasize the importance of examining TCRα, as well as TCRß, in understanding the CD8 T cell receptor repertoire.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Proteínas Inmediatas-Precoces/inmunología , Epítopos Inmunodominantes/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Citotóxicos/inmunología , Transactivadores/inmunología , Secuencia de Aminoácidos , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Epítopos de Linfocito T/inmunología , Infecciones por Virus de Epstein-Barr/virología , Antígeno HLA-A2/inmunología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) requires sustained extracellular signal-regulated kinase (ERK)-p90 ribosomal S6 kinase (RSK) activation, which is induced by an immediate early (IE) gene-encoded tegument protein called ORF45, to promote the late transcription and translation of viral lytic genes. An ORF45-null or single-point F66A mutation in ORF45 abolishes ORF45-RSK interaction and sustained ERK-RSK activation during lytic reactivation and subsequently results in a significant decrease in late lytic gene expression and virion production, indicating that ORF45-mediated RSK activation plays a critical role in KSHV lytic replication. Here, we demonstrate that a short ORF45-derived peptide in the RSK-binding region is sufficient for disrupting ORF45-RSK interaction, consequently suppressing lytic gene expression and virion production. We designed a nontoxic cell-permeable peptide derived from ORF45, TAT-10F10, which is composed of the ORF45 56 to 76 amino acid (aa) region and the HIV Tat protein transduction domain, and this peptide markedly inhibits KSHV lytic replication in iSLK.219 and BCBL1 cells. Importantly, this peptide enhances the inhibitory effect of rapamycin on KSHV-infected cells and decreases spontaneous and hypoxia-induced lytic replication in KSHV-positive lymphoma cells. These findings suggest that a small peptide that disrupts ORF45-RSK interaction might be a promising agent for controlling KSHV lytic infection and pathogenesis.IMPORTANCE ORF45-induced RSK activation plays an essential role in KSHV lytic replication, and ORF45-null or ORF45 F66A mutagenesis that abolishes sustained RSK activation and RSK inhibitors significantly decreases lytic replication, indicating that the ORF45-RSK association is a unique target for KSHV-related diseases. However, the side effects, low affinity, and poor efficacy of RSK modulators limit their clinical application. In this study, we developed a nontoxic cell-permeable ORF45-derived peptide from the RSK-binding region to disrupt ORF45-RSK associations and block ORF45-induced RSK activation without interfering with S6K1 activation. This peptide effectively suppresses spontaneous, hypoxia-induced, or chemically induced KSHV lytic replication and enhances the inhibitory effect of rapamycin on lytic replication and sensitivity to rapamycin in lytic KSHV-infected cells. Our results reveal that the ORF45-RSK signaling axis and KSHV lytic replication can be effectively targeted by a short peptide and provide a specific approach for treating KSHV lytic and persistent infection.
Asunto(s)
Herpesvirus Humano 8/efectos de los fármacos , Proteínas Inmediatas-Precoces/inmunología , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Línea Celular , Regulación Viral de la Expresión Génica/genética , Genes Virales/genética , Células HEK293 , Infecciones por Herpesviridae/genética , Herpesvirus Humano 8/patogenicidad , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Unión Proteica , Proteínas Quinasas S6 Ribosómicas 90-kDa/inmunología , Virión/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiologíaRESUMEN
Varicella-zoster virus (VZV) is a pathogenic human herpesvirus that causes varicella (chickenpox) as a primary infection following which it becomes latent in ganglionic neurons. Following viral reactivation many years later VZV causes herpes zoster (shingles) as well as a variety of other neurological syndromes. The molecular mechanisms of the conversion of the virus from a lytic to a latent state in ganglia are not well understood. In order to gain insights into the neuron-virus interaction, we studied virus-induced apoptosis in cultures of both highly pure terminally differentiated human neurons and human fetal lung fibroblasts (HFL). It was found that (a) VZV DNA did not accumulate in infected human neurons; (b) VZV transcripts were present at lower levels at all days studied post-infection in neurons; (c) Western blot analysis showed less VZV IE 63 and very little detectable VZV gE proteins in infected neurons compared with HFL; (d) lower levels of the apoptotic marker cleaved Caspase-3 protein were detected in VZV-infected neurons compared with HFL, and higher levels of the known anti-apoptotic proteins Bcl2, Bcl-XL and also the mitochondrial MT-CO2 protein were found in VZV-infected neurons compared with uninfected cells; and (e) both the MT-CO2 protein and VZV IE 63-encoded protein were detected in infected neurons by dual immunofluorescence. These findings showed that neurons are resistant to VZV-induced apoptosis, which may have relevance to the switching of VZV from a lytic to latent ganglionic neuronal infection.
Asunto(s)
Apoptosis/genética , ADN Viral/genética , Herpesvirus Humano 3/genética , Interacciones Huésped-Patógeno/genética , Neuronas/virología , Latencia del Virus/genética , Apoptosis/inmunología , Caspasa 3/genética , Caspasa 3/inmunología , Línea Celular , ADN Viral/inmunología , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/inmunología , Feto , Fibroblastos/inmunología , Fibroblastos/virología , Regulación de la Expresión Génica , Herpesvirus Humano 3/crecimiento & desarrollo , Herpesvirus Humano 3/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Neuronas/inmunología , Especificidad de Órganos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología , Transducción de Señal , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Latencia del Virus/inmunología , Proteína bcl-X/genética , Proteína bcl-X/inmunologíaRESUMEN
Pseudorabies virus (PRV) establishes a lifelong latent infection in swine trigeminal ganglion (TG) following acute infection. Increased corticosteroid levels, due to stress, increases the incidence of reactivation from latency. Muscle injection combined with intravenous deliver of the synthetic corticosteroid dexamethasone (DEX) consistently induces reactivation from latency in pigs. In this study, PRV-free piglets were infected with PRV. Viral shedding in nasal and ocular swabs demonstrated that PRV infection entered the latent period. The anti-PRV antibody was detected by enzyme-linked immunosorbent assay and the serum neutralization test, which suggested that the PRV could establish latent infection in the presence of humoral immunity. Immunohistochemistry and viral genome detection of TG neurons suggested that PRV was reactivated from latency. Viral gene expressions of IE180, EP0, VP16, and LLT-intron were readily detected at 3-h post-DEX treatment, but gB, a γ1 gene, was not detectable. The differentially expressed phosphorylated proteins of TG neurons were analyzed by ITRAQ coupled with LC-MS/MS, and p-EIF2S2 differentially expression was confirmed by western blot assay. Taken together, our study provides the evidence that typical gene expression in PRV reactivation from latency in TG is disordered compared with known lytic infection in epithelial cells.
Asunto(s)
Dexametasona/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Herpesvirus Suido 1/efectos de los fármacos , Seudorrabia/virología , Enfermedades de los Porcinos/virología , Ganglio del Trigémino/efectos de los fármacos , Activación Viral/efectos de los fármacos , Animales , Anticuerpos Antivirales/sangre , Ojo/virología , Glucocorticoides/farmacología , Proteína Vmw65 de Virus del Herpes Simple/genética , Proteína Vmw65 de Virus del Herpes Simple/inmunología , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/inmunología , Herpesvirus Suido 1/patogenicidad , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Inmunidad Humoral/efectos de los fármacos , Cavidad Nasal/virología , Neuronas/efectos de los fármacos , Neuronas/inmunología , Neuronas/virología , Seudorrabia/inmunología , Seudorrabia/patología , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/patología , Ganglio del Trigémino/inmunología , Ganglio del Trigémino/virología , Latencia del Virus/efectos de los fármacos , Esparcimiento de Virus/efectos de los fármacosRESUMEN
Connexin 43 (Cx43) deficiency was found to increase mortality in a mouse model of bacterial peritonitis, and Cx43 is upregulated in macrophages by LPS treatment. In this study, we characterized a novel signaling pathway for LPS-induced Cx43 expression in RAW264.7 cells and thioglycolate-elicited peritoneal macrophages (TGEMs). LPS alone or LPS-containing conditioned medium (CM) upregulated Cx43. Overexpression or silencing of Cx43 led to the enhancement or inhibition, respectively, of CM-induced TGEM migration. This response involved the inducible NO synthase (iNOS)/focal adhesion kinase (FAK)/Src pathways. Moreover, CM-induced migration was compromised in TGEMs from Cx43+/- mice compared with TGEMs from Cx43+/+ littermates. Cx43 was upregulated by a serum/glucocorticoid-regulated kinase 1 (SGK) activator and downregulated, along with inhibition of CM-induced TGEM migration, by knockdown of the SGK gene or blockade of the SGK pathway. LPS-induced SGK activation was abrogated by Torin2, whereas LPS-induced Cx43 was downregulated by both Torin2 and rapamycin. Analysis of the effects of FK506 and methylprednisolone, common immunosuppressive agents following organ transplantation, suggested a link between these immunosuppressive drugs and impaired macrophage migration via the Cx43/iNOS/Src/FAK pathway. In a model of Escherichia coli infectious peritonitis, GSK650349-, an SGK inhibitor, or Torin2-treated mice showed less accumulation of F4/80+CD11b+ macrophages in the peritoneal cavity, with a delay in the elimination of bacteria. Furthermore, following pretreatment with Gap19, a selective Cx43 hemichannel blocker, the survival of model mice was significantly reduced. Taken together, our study suggested that Cx43 in macrophages was associated with macrophage migration, an important immune process in host defense to infection.
Asunto(s)
Movimiento Celular/inmunología , Conexina 43/biosíntesis , Macrófagos/inmunología , Transducción de Señal/inmunología , Animales , Conexina 43/inmunología , Quinasa 1 de Adhesión Focal/inmunología , Quinasa 1 de Adhesión Focal/metabolismo , Regulación de la Expresión Génica/inmunología , Proteínas Inmediatas-Precoces/inmunología , Proteínas Inmediatas-Precoces/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/inmunología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Células RAW 264.7 , Serina-Treonina Quinasas TOR/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Familia-src Quinasas/inmunología , Familia-src Quinasas/metabolismoRESUMEN
BACKGROUND: Human cytomegalovirus (HCMV) infection in glioblastoma multiforme (GBM) is associated with a poor prognosis and may affect the pathogenesis of GBM. In this study, we investigated the role of HCMV-infected astrocytoma cells in impairing the activity of cytotoxic T lymphocytes (CTLs) specific to the HCMV protein. METHODS: CTLs specific to HCMV immediate early (IE)-1 were expanded from peripheral blood mononuclear cells of healthy donors by stimulating CD8+ T lymphocytes with U373MG cells (ATCC HTB-17: male) expressing HCMV IE-1. The death rate of the target and the effector cells was determined by the total count of the remaining respective cells after the interaction of them. RESULTS: The death rate of the target cells by CTLs increased depending on HLA restriction and the effector:target (E:T) ratio. The death rate of effector cells in the HCMV-infected U373MG cell culture was 37.1% on day 4 post-infection. The removal of the culture supernatant from HCMV-infected U373MG cells prior to adding the effector cells increased target cell death from 8.4% to 40.8% at E:T = 1:1, but not at E:T = 3:1. The transfer of cells from a 24-hour co-culture of the HCMV-infected U373MG cells and CTLs to HCMV IE-1-expressing target cells resulted in decreasing the cell death rate of the target cells from 31.1% to 13.0% at E:T = 1:1, but not at E:T = 3:1. HCMV infection of U373MG cells decreases the activity of CTLs specific to HCMV when the number of CTLs is low. CONCLUSION: These results suggest that HCMV could impair CTL activity and facilitate glioblastoma growth unchecked by CTLs.
Asunto(s)
Astrocitoma/patología , Linfocitos T CD8-positivos/inmunología , Citomegalovirus/fisiología , Apoptosis , Astrocitoma/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Células Cultivadas , Técnicas de Cocultivo , Citomegalovirus/metabolismo , Antígenos HLA-A/metabolismo , Humanos , Proteínas Inmediatas-Precoces/inmunología , Proteínas Inmediatas-Precoces/metabolismo , Interferón gamma/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , MasculinoRESUMEN
T cell immunity is essential for the control of cytomegalovirus (CMV) infection after transplantation. We evaluated a CMV-specific peptide-based enzyme-linked immunosorbent spot (ELISPOT) assay to determine whether assay results could predict subsequent CMV events. Adult kidney transplant recipients at 43 centers underwent ELISPOT testing to enumerate interferon gamma (IFN-γ) binding spot-forming units (sfu) after stimulation of cells with an overlapping peptide pool of CMV phosphoprotein 65 (pp65) and immediate early-1 (IE-1) protein at the end of antiviral prophylaxis (EOP) and various time points thereafter. The primary outcome was a CMV event in the first posttransplant year. In 583 kidney transplant recipients (260 seropositive donor [D+]/seronegative recipient [R-] and 277 R+), CMV events occurred in 44 of 368 eligible patients (11.8%) at a median of 227 days (range 92-360) posttransplant. A cutoff value of >40 sfu/2.5 × 105 cells for either IE-1 or pp65 was derived as a threshold for positivity, with a negative predictive value of >97% for CMV events. CMV events were significantly lower in assay positive vs assay negative patients (3.0% vs 19.5%, P < .0001 for pp65). Time to CMV event post-EOP was significantly greater in those with sfu >40 at EOP (P < .0001). In this large, multicenter trial of kidney transplant recipients, we show that an assessment of CMV-specific immunity using a novel ELISPOT assay is able to predict protection from CMV infection.
Asunto(s)
Infecciones por Citomegalovirus/complicaciones , Inmunidad Celular , Fallo Renal Crónico/cirugía , Trasplante de Riñón/efectos adversos , Adulto , Anciano , Antivirales/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Proteínas Inmediatas-Precoces/inmunología , Sistema Inmunológico , Estimación de Kaplan-Meier , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/inmunología , Masculino , Persona de Mediana Edad , Péptidos/química , Valor Predictivo de las Pruebas , Estudios Prospectivos , Curva ROC , Linfocitos T/citología , Resultado del Tratamiento , Proteínas de la Matriz Viral/inmunología , Adulto JovenRESUMEN
We describe a method of rendering polyclonal cytokine-induced killer cells (CIK) specific against cytomegalovirus (CMV), focusing on GMP compliance. Peripheral blood mononuclear cells (PBMNC) are stimulated with pooled CMV peptides pp65 and IE-1 for 16-24â¯h and the reactive T cell subset which up-regulate CD137 is further co-stimulated with anti-CD137, followed by expansion in G-Rex flasks under standard CIK culture condition. This method generates a large number CMV-specific CIK with superior potency compared to published method currently in clinical trials. The cytotoxicity as measured by chromium release assay correlates with the upregulation of CD107a upon peptide re-challenge as measured by flow cytometry. CMV-CIK at maturity consist of mainly late effector memory CD8 T cells and have a skewed TCR repertoire with preferential expansion of a few families. Such CMV-CIK retain their function after freezing and thawing. CMV-CIK thus generated is ready for clinical trial against drug-resistant CMV disease.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Citomegalovirus/inmunología , Proteínas Inmediatas-Precoces/inmunología , Memoria Inmunológica/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células Asesinas Inducidas por Citocinas/citología , Células Asesinas Inducidas por Citocinas/inmunología , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Subgrupos de Linfocitos T/citología , Linfocitos T Citotóxicos/citologíaRESUMEN
Due to their roles in the regulation of programmed cell death and inflammation, the cellular caspase proteases are considered antiviral factors. However, recent studies have revealed examples of proviral functions for caspases. Here, we review a growing body of literature on the role of caspases in promoting the replication of human gammaherpesviruses. We propose that gammaherpesviruses have evolved ways to redirect these enzymes and to use their activation to support viral replication and immune evasion.
Asunto(s)
Caspasas/genética , Células Eucariotas/virología , Gammaherpesvirinae/genética , Proteínas Inmediatas-Precoces/genética , Evasión Inmune/genética , Provirus/genética , Animales , Apoptosis , Caspasas/inmunología , Células Eucariotas/inmunología , Células Eucariotas/metabolismo , Evolución Molecular , Gammaherpesvirinae/inmunología , Gammaherpesvirinae/metabolismo , Regulación de la Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/inmunología , Provirus/inmunología , Provirus/metabolismo , Transducción de Señal , Virión/genética , Virión/inmunología , Virión/metabolismo , Replicación ViralRESUMEN
We previously isolated a herpes simplex virus 1 (HSV-1) mutant, KOS-NA, that carries two nonsynonymous mutations in UL39, resulting in L393P and R950H amino acid substitutions in infected cell protein 6 (ICP6). Our published data studying KOS-NA pathogenesis strongly suggest that one of these ICP6 substitutions expressed from KOS-NA, R950H, severely impaired acute viral replication in the eyes and trigeminal ganglia of mice after inoculation onto the cornea and consequently impaired establishment and reactivation from latency. Because of its significant neuroattenuation, we tested KOS-NA as a potential prophylactic vaccine against HSV-1 in a mouse model of corneal infection. KOS-NA stimulated stronger antibody and T cell responses than a replication-competent ICP0-null mutant and a replication-incompetent ICP8-null mutant optimized for immunogenicity. Immunizations with the ICP0-, ICP8-, and KOS-NA viruses all reduced replication of wild-type HSV-1 challenge virus in the corneal epithelium to similar extents. Low immunizing doses of KOS-NA and the ICP8- virus, but not the ICP0- virus, protected mice against eyelid disease (blepharitis). Notably, only KOS-NA protected almost completely against corneal disease (keratitis) and greatly reduced latent infection by challenge virus. Thus, vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated disease of the eye, even at a very low immunizing dose. These results suggest that KOS-NA may be the foundation of an effective prophylactic vaccine to prevent or limit HSV-1 ocular diseases.IMPORTANCE HSV-1 is a ubiquitous human pathogen that infects the majority of the world's population. Although most infections are asymptomatic, HSV-1 establishes lifelong latency in infected sensory neurons, from which it can reactivate to cause deadly encephalitis or potentially blinding eye disease. No clinically effective vaccine is available. In this study, we tested the protective potential of a neuroattenuated HSV-1 mutant (KOS-NA) as a vaccine in mice. We compared the effects of immunization with KOS-NA to those of two other attenuated viruses, a replication-competent (ICP0-) virus and a replication-incompetent (ICP8-) virus. Our data show that KOS-NA proved superior to the ICP0- and ICP8-null mutants in protecting mice from corneal disease and latent infection. With its significant neuroattenuation, severe impairment in establishing latency, and excellent protective effect, KOS-NA represents a significant discovery in the field of HSV-1 vaccine development.
Asunto(s)
Herpesvirus Humano 1/genética , Vacunas contra Herpesvirus/inmunología , Queratitis Herpética/prevención & control , Proteínas Virales/genética , Animales , Chlorocebus aethiops , Modelos Animales de Enfermedad , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Vacunas contra Herpesvirus/administración & dosificación , Vacunas contra Herpesvirus/genética , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Queratitis Herpética/inmunología , Queratitis Herpética/virología , Ratones , Mutación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Células Vero , Proteínas Virales/inmunología , Latencia del Virus , Replicación ViralRESUMEN
Attenuated poxvirus modified vaccinia Ankara (MVA) is a useful viral-based vaccine for clinical investigation, because of its excellent safety profile and property of inducing potent immune responses against recombinant (r) antigens. We developed Triplex by constructing an rMVA encoding 3 immunodominant cytomegalovirus (CMV) antigens, which stimulates a host antiviral response: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5). We completed the first clinical evaluation of the Triplex vaccine in 24 healthy adults, with or without immunity to CMV and vaccinia virus (previous DryVax smallpox vaccination). Three escalating dose levels (DL) were administered IM in 8 subjects/DL, with an identical booster injection 28 days later and 1-year follow-up. Vaccinations at all DL were safe with no dose-limiting toxicities. No vaccine-related serious adverse events were documented. Local and systemic reactogenicity was transient and self-limiting. Robust, functional, and durable Triplex-driven expansions of CMV-specific T cells were detected by measuring T-cell surface levels of 4-1BB (CD137), binding to CMV-specific HLA multimers, and interferon-γ production. Marked and durable CMV-specific T-cell responses were also detected in Triplex-vaccinated CMV-seronegatives, and in DryVax-vaccinated subjects. Long-lived memory effector phenotype, associated with viral control during CMV primary infection, was predominantly found on the membrane of CMV-specific and functional T cells, whereas off-target vaccine responses activating memory T cells from the related herpesvirus Epstein-Barr virus remained undetectable. Combined safety and immunogenicity results of MVA in allogeneic hematopoietic stem cell transplant (HCT) recipients and Triplex in healthy adults motivated the initiation of a placebo-controlled multicenter trial of Triplex in HCT patients. This trial was registered at www.clinicaltrials.gov as #NCT02506933.