Periodic Reporting for period 1 - Divir (Deciphering the role of host immunity in development of herpes virus meningitis)
Okres sprawozdawczy: 2023-01-01 do 2024-12-31
Viruses are responsible for a wide range of severe diseases, including influenza, hepatitis, and COVID-19, and represent a persistent global health burden. An individual’s susceptibility to viral infection is influenced by multiple factors—host genetics, viral properties, and environmental conditions—yet the underlying mechanisms remain poorly understood.
In humans, infections with any of the nine known human herpesviruses can result in outcomes ranging from asymptomatic to life-threatening. Alphaherpesviruses typically cause lesions on the skin or mucosal surfaces, but in certain cases, they can lead to serious neurological complications such as encephalitis and meningitis. The reasons why some individuals develop severe central nervous system (CNS) complications following alphaherpesvirus infections or viral reactivation from latency are still largely unknown.
The primary objective of this project is to identify host factors that contribute to the pathological outcomes of neurotropic herpes simplex virus type 2 (HSV-2) infections, and to elucidate their physiological roles in the human antiviral immune response. Studying patients with rare genetic mutations that result in extreme clinical responses to infection offers a powerful approach to uncover fundamental insights into the immune system.
In humans, infections with any of the nine known human herpesviruses can result in outcomes ranging from asymptomatic to life-threatening. Alphaherpesviruses typically cause lesions on the skin or mucosal surfaces, but in certain cases, they can lead to serious neurological complications such as encephalitis and meningitis. The reasons why some individuals develop severe central nervous system (CNS) complications following alphaherpesvirus infections or viral reactivation from latency are still largely unknown.
The primary objective of this project is to identify host factors that contribute to the pathological outcomes of neurotropic herpes simplex virus type 2 (HSV-2) infections, and to elucidate their physiological roles in the human antiviral immune response. Studying patients with rare genetic mutations that result in extreme clinical responses to infection offers a powerful approach to uncover fundamental insights into the immune system.
We have access to whole-exome sequencing (WES) data from a unique cohort of 50 patients diagnosed with HSV-2 meningitis, collected from hospitals in Sweden and Denmark. In the Swedish cohort, exome sequences from 15 patients have been successfully analyzed to identify potential inborn errors of immunity and loss-of-function mutations. The WES analysis revealed several point mutations, including frameshift deletions and nucleotide substitutions leading to altered amino acid sequences.
Genetic analysis conducted as planned in Divir identified a number of potentially pathogenic mutations in patients with HSV-2 meningitis. Preliminary findings suggest that these patients carry mutations affecting key antiviral signaling pathways, including autophagy, protein ubiquitination, cell cycle regulation, and type I interferon (IFN) induction.
The identified mutations were confirmed by Sanger sequencing. Comprehensive clinical data and medical histories have been recorded for the selected patients. Peripheral blood mononuclear cells (PBMCs) and fibroblasts were isolated from these individuals for downstream functional studies.
We conducted a series of functional assays to evaluate the proinflammatory and antiviral responses of patient-derived PBMCs and fibroblasts following infection with HSV-2 and other control viruses, as well as stimulation with synthetic immune agonists. These experiments focused on assessing the activation and integrity of the signaling pathways predicted to be defective based on WES results.
Patient cells were infected with HSV-2 and other viruses, after which we measured expression levels of IFNs and inflammatory cytokines at both the RNA and protein levels. We also assessed cell viability and activation of relevant immune signaling pathways. To establish a causal link between the identified mutations and the clinical phenotype, we reintroduced the wild-type allele into patient fibroblasts using lentiviral gene delivery. Additionally, we introduced disease-associated alleles into healthy control cells to determine whether they induce a similar disease phenotype and impaired antiviral response.
Beyond identifying signaling defects, we also investigated the underlying molecular mechanisms responsible for the immune dysfunction in these patients. To do this, we employed a range of model systems, including genome-edited HEK293T cells, knockout neuronal cell lines, and induced pluripotent stem cell (iPSC)-derived microglia and neurons, using CRISPR/Cas9 gene editing. We also employed siRNA-mediated gene silencing in microglial cells to replicate patient-specific mutations.
Genetic analysis conducted as planned in Divir identified a number of potentially pathogenic mutations in patients with HSV-2 meningitis. Preliminary findings suggest that these patients carry mutations affecting key antiviral signaling pathways, including autophagy, protein ubiquitination, cell cycle regulation, and type I interferon (IFN) induction.
The identified mutations were confirmed by Sanger sequencing. Comprehensive clinical data and medical histories have been recorded for the selected patients. Peripheral blood mononuclear cells (PBMCs) and fibroblasts were isolated from these individuals for downstream functional studies.
We conducted a series of functional assays to evaluate the proinflammatory and antiviral responses of patient-derived PBMCs and fibroblasts following infection with HSV-2 and other control viruses, as well as stimulation with synthetic immune agonists. These experiments focused on assessing the activation and integrity of the signaling pathways predicted to be defective based on WES results.
Patient cells were infected with HSV-2 and other viruses, after which we measured expression levels of IFNs and inflammatory cytokines at both the RNA and protein levels. We also assessed cell viability and activation of relevant immune signaling pathways. To establish a causal link between the identified mutations and the clinical phenotype, we reintroduced the wild-type allele into patient fibroblasts using lentiviral gene delivery. Additionally, we introduced disease-associated alleles into healthy control cells to determine whether they induce a similar disease phenotype and impaired antiviral response.
Beyond identifying signaling defects, we also investigated the underlying molecular mechanisms responsible for the immune dysfunction in these patients. To do this, we employed a range of model systems, including genome-edited HEK293T cells, knockout neuronal cell lines, and induced pluripotent stem cell (iPSC)-derived microglia and neurons, using CRISPR/Cas9 gene editing. We also employed siRNA-mediated gene silencing in microglial cells to replicate patient-specific mutations.
Herpes simplex virus 2 (HSV-2) meningitis is a rare neurological condition resulting from recurrent infection with the neurotropic HSV-2 virus in the central nervous system (CNS). Despite its clinical relevance, the pathogenesis of HSV-2 meningitis, along with its underlying genetic and immunological basis, remains poorly understood.
This study contributes to advancing our knowledge of infection immunology and CNS pathogenesis. Previously, our group identified defects in autophagy-related genes in patients with HSV-2 meningitis and demonstrated that autophagy exerts a direct, cell-autonomous antiviral function. Notably, this antiviral activity was independent of the type I interferon (IFN) cytokine system, distinguishing it from what has been described in herpes simplex encephalitis (HSE), which is caused by HSV-1. In HSE, numerous genetic etiologies involving components of the type I IFN signaling pathway have been identified through our work and that of others.
Type I IFN expression is typically induced upon sensing viral nucleic acids by pattern recognition receptors (PRRs), which signal through adaptor proteins and the kinases IKKε and TBK1. These kinases activate the transcription factors IRF3 and IRF7, driving the expression of IFN genes. However, it remains unclear whether—and how—components of the type I IFN pathway play a role in protecting against HSV-2 meningitis, or what specific molecular and cellular mechanisms are involved.
In this study, we report HSV-2 meningitis patients carrying heterozygous loss-of-function mutations in the IKBKE confirming the involvement of type I interferon in antiviral defense.
This study contributes to advancing our knowledge of infection immunology and CNS pathogenesis. Previously, our group identified defects in autophagy-related genes in patients with HSV-2 meningitis and demonstrated that autophagy exerts a direct, cell-autonomous antiviral function. Notably, this antiviral activity was independent of the type I interferon (IFN) cytokine system, distinguishing it from what has been described in herpes simplex encephalitis (HSE), which is caused by HSV-1. In HSE, numerous genetic etiologies involving components of the type I IFN signaling pathway have been identified through our work and that of others.
Type I IFN expression is typically induced upon sensing viral nucleic acids by pattern recognition receptors (PRRs), which signal through adaptor proteins and the kinases IKKε and TBK1. These kinases activate the transcription factors IRF3 and IRF7, driving the expression of IFN genes. However, it remains unclear whether—and how—components of the type I IFN pathway play a role in protecting against HSV-2 meningitis, or what specific molecular and cellular mechanisms are involved.
In this study, we report HSV-2 meningitis patients carrying heterozygous loss-of-function mutations in the IKBKE confirming the involvement of type I interferon in antiviral defense.