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.
