RESUMEN
The role of T cell receptor (TCR) diversity in infectious disease susceptibility is not well understood. We use a systems immunology approach on three cohorts of herpes zoster (HZ) patients and controls to investigate whether TCR diversity against varicella-zoster virus (VZV) influences the risk of HZ. We show that CD4+ T cell TCR diversity against VZV glycoprotein E (gE) and immediate early 63 protein (IE63) after 1-week culture is more restricted in HZ patients. Single-cell RNA and TCR sequencing of VZV-specific T cells shows that T cell activation pathways are significantly decreased after stimulation with VZV peptides in convalescent HZ patients. TCR clustering indicates that TCRs from HZ patients co-cluster more often together than TCRs from controls. Collectively, our results suggest that not only lower VZV-specific TCR diversity but also reduced functional TCR affinity for VZV-specific proteins in HZ patients leads to lower T cell activation and consequently affects the susceptibility for viral reactivation.
Asunto(s)
Herpes Zóster , Herpesvirus Humano 3 , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T , Humanos , Herpes Zóster/inmunología , Herpes Zóster/virología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Activación de Linfocitos/inmunología , Herpesvirus Humano 3/inmunología , Femenino , Persona de Mediana Edad , Masculino , Linfocitos T CD4-Positivos/inmunología , Anciano , Adulto , Epítopos de Linfocito T/inmunologíaRESUMEN
The varicella-zoster virus (VZV) infects over 95% of the population. VZV reactivation causes herpes zoster (HZ), known as shingles, primarily affecting the elderly and immunocompromised individuals. However, HZ can also occur in otherwise healthy individuals. We analyzed the immune signature and risk profile in HZ patients using a genome-wide association study across different UK Biobank HZ cohorts. Additionally, we conducted one of the largest HZ HLA association studies to date, coupled with transcriptomic analysis of pathways underlying HZ susceptibility. Our findings highlight the significance of the MHC locus for HZ development, identifying five protective and four risk HLA alleles. This demonstrates that HZ susceptibility is largely governed by variations in the MHC. Furthermore, functional analyses revealed the upregulation of type I interferon and adaptive immune responses. These findings provide fresh molecular insights into the pathophysiology and the activation of innate and adaptive immune responses triggered by symptomatic VZV reactivation.
RESUMEN
With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons.
Asunto(s)
Varicela , Herpes Zóster , Células Madre Pluripotentes Inducidas , Infección por el Virus de la Varicela-Zóster , Humanos , Herpesvirus Humano 3 , Técnicas de Cocultivo , Replicación Viral/fisiología , Neuronas , Macrófagos , Interferones , Antivirales , Inmunidad InnataRESUMEN
Herpesviruses hijack the MHC class I (MHC I) and class II (MHC II) antigen-presentation pathways to manipulate immune recognition by T cells. First, we illustrate herpes simplex virus-1 (HSV-1) and varicella-zoster virus (VZV) MHC immune evasion strategies. Next, we describe MHC-T cell interactions in HSV-1- and VZV- infected neural ganglia. Although studies on the topic are scarce, and use different models, most reports indicate that neuronal HSV-1 infection is mainly controlled by CD8+ T cells through noncytolytic mechanisms, whereas VZV seems to be largely controlled through CD4+ T cell-specific immune responses. Autologous human stem-cell-derived in vitro models could substantially aid in elucidating these neuroimmune interactions and are fit for studies on both herpesviruses.
Asunto(s)
Herpes Simple , Herpes Zóster , Herpesvirus Humano 1 , Humanos , Herpesvirus Humano 3/fisiología , Herpesvirus Humano 1/fisiología , GangliosRESUMEN
Varicella-zoster virus (VZV) infection of neuronal cells and the activation of cell-intrinsic antiviral responses upon infection are still poorly understood mainly due to the scarcity of suitable human in vitro models that are available to study VZV. We developed a compartmentalized human-induced pluripotent stem cell (hiPSC)-derived neuronal culture model that allows axonal VZV infection of the neurons, thereby mimicking the natural route of infection. Using this model, we showed that hiPSC-neurons do not mount an effective interferon-mediated antiviral response following VZV infection. Indeed, in contrast to infection with Sendai virus, VZV infection of the hiPSC-neurons does not result in the upregulation of interferon-stimulated genes (ISGs) that have direct antiviral functions. Furthermore, the hiPSC-neurons do not produce interferon-α (IFNα), a major cytokine that is involved in the innate antiviral response, even upon its stimulation with strong synthetic inducers. In contrast, we showed that exogenous IFNα effectively limits VZV spread in the neuronal cell body compartment and demonstrated that ISGs are efficiently upregulated in these VZV-infected neuronal cultures that are treated with IFNα. Thus, whereas the cultured hiPSC neurons seem to be poor IFNα producers, they are good IFNα responders. This could suggest an important role for other cells such as satellite glial cells or macrophages to produce IFNα for VZV infection control.
Asunto(s)
Varicela , Herpes Zóster , Células Madre Pluripotentes Inducidas , Interferón-alfa , Neuronas , Humanos , Herpesvirus Humano 3/fisiología , Células Madre Pluripotentes Inducidas/virología , Interferón-alfa/inmunología , Neuronas/virología , Células CultivadasRESUMEN
Despite the considerable impact of stroke on both the individual and on society, a neuroprotective therapy for stroke patients is missing. This is partially due to the current lack of a physiologically relevant human in vitro stroke model. To address this problem, we have developed a luminescent human iPSC-derived neurospheroid model that enables real-time read-out of neural viability after ischemia-like conditions. We subjected 1- and 4-week-old neurospheroids, generated from iPSC-derived neural stem cells, to 6 h of oxygen-glucose deprivation (OGD) and measured neurospheroid luminescence. For both, we detected a decrease in luminescent signal due to ensuing neurotoxicity, as confirmed by conventional LDH assay and flow cytometric viability analysis. Remarkably, 1-week-old, but not 4-week-old neurospheroids recovered from OGD-induced injury, as evidenced by their reduced but overall increasing luminescence over time. This underscores the need for more mature neurospheroids, more faithfully recapitulating the in vivo situation. Furthermore, treatment of oxygen- and glucose-deprived neurospheroids with the pan-caspase inhibitor Z-VAD-FMK did not increase overall neural survival, despite its successful attenuation of apoptosis, in a human-based 3D environment. Nevertheless, owing to its three-dimensional organization and real-time viability reporting potential, the luminescent neurospheroids may become readily adopted in high-throughput screens aimed at identification of new therapeutic agents to treat acute ischemic stroke patients.
Asunto(s)
Células Madre Pluripotentes Inducidas , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Apoptosis , Supervivencia Celular/fisiología , Glucosa , Humanos , Luminiscencia , Oxígeno/efectos adversosRESUMEN
Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization worldwide each year and there is presently no licensed vaccine to prevent severe RSV infections. Two major RSV glycoproteins, attachment (G) and fusion (F) protein, regulate viral replication and both proteins contain potential glycosylation sites which are highly variable for the G protein and conserved for the F protein among virus isolates. The RSV F sequence possesses five N-glycosylation sites located in the F2 subunit (N27 and N70), the p27 peptide (N116 and N126) and the F1 subunit (N500). The importance of RSV F N-glycosylation in virus replication and immunogenicity is not yet fully understood, and a better understanding may provide new insights for vaccine development. By using a BAC-based reverse genetics system, recombinant viruses expressing F proteins with loss of N-glycosylation sites were made. Mutant viruses with single N-glycosylation sites removed could be recovered, while this was not possible with the mutant with all N-glycosylation sites removed. Although the individual RSV F N-glycosylation sites were shown not to be essential for viral replication, they do contribute to the efficiency of in vitro and in vivo viral infection. To evaluate the role of N-glycosylation sites on RSV F antigenicity, serum antibody titers were determined after infection of BALB/c mice with RSV expressing the glycomutant F proteins. Infection with recombinant virus lacking the N-glycosylation site at position N116 (RSV F N116Q) resulted in significant higher neutralizing antibody titers compared to RSV F WT infection, which is surprising since this N-glycan is present in the p27 peptide which is assumed to be absent from the mature F protein in virions. Thus, single or combined RSV F glycomutations which affect virus replication and fusogenicity, and which may induce enhanced antibody responses upon immunization could have the potential to improve the efficacy of RSV LAV approaches.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/fisiología , Virus Sincitial Respiratorio Humano/patogenicidad , Proteínas Virales de Fusión/metabolismo , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Células Gigantes/virología , Glicosilación , Humanos , Inmunización , Inmunogenicidad Vacunal , Ratones Endogámicos BALB C , Mutación , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/patología , Virus Sincitial Respiratorio Humano/crecimiento & desarrollo , Virus Sincitial Respiratorio Humano/inmunología , Células Vero , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunología , Replicación ViralRESUMEN
Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hRSV) remains a major public health priority. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. The protein conserves 5 N-glycosylation sites, two of which are located in the F2 subunit (N27 and N70), one in the F1 subunit (N500) and two in the p27 peptide (N116 and N126). To study the influence of the loss of one or more N-glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with plasmids encoding RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titres were observed following immunization with F N116Q. Moreover, RSV A2-K-line19F challenge of mice that had been immunized with mutant F N116Q DNA was associated with lower RSV RNA levels compared with those in challenged WT F DNA immunized animals. Since p27 is assumed to be post-translationally released after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this glycan can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates.