Evaluation of the antiviral effects of HCMV-targeting drugs in differentiating neurons and cerebral organoids.

Human cytomegalovirus (HCMV) is a beta-herpesvirus widespread among humans. It usually gives asymptomatic infections in immunocompetent individuals, while it can cause severe diseases in immunocompromised patients. More importantly, congenital CMV infection (cCMV) is the leading viral cause of congenital birth defects, with clinical manifestations primarily affecting the central nervous system. Given the strict species-specificity of the virus and the difficulty in developing accurate in vitro models fully recapitulating HCMV pathogenesis, currently, there are no approved drugs or vaccines for cCMV. The aim of this study was to exploit a 2D dynamic model of neurogenesis (iNeurons) and 3D cerebral organoids (COs) to evaluate whether the treatment with approved and investigational antiviral drugs (Ganciclovir, Letermovir, and OZ418) could be effective in inhibiting HCMV replication. Differentiating neural progenitor cells were infected with HCMV TB4-UL83-EYFP and treated with different concentrations of the antivirals. After 14 days of differentiation, iNeurons were fixed and stained for βIII-tubulin to evaluate the possible rescue of the morphogenetic changes induced by the infection, i.e., the reduction in neurite outgrowth. On the other hand, a 3D platform of cerebral organoids that closely mimics the human developing brain was produced and optimized to investigate the effects of HCMV infection and the potential recovery after drug treatment. Thirty-day-old cerebral organoids were infected with two strains of HCMV, i.e., TB4-UL83-EYFP and the clinical strain VR1814 and treated for 21 days with antiviral drugs. Then, the antiviral effects on infected organoids were evaluated by 1) analyzing the reduction of intracellular virus production and the release of infectious viral particles in the supernatant of infected organoids upon drug treatment; 2) evaluating over time, through live confocal microscopy, the EYFP fluorescent signal as a readout of viral infection; 3) assessing, by means of qPCR expression analysis, whether different innate antiviral response markers could be rescued upon antiviral treatment. Finally, an independent set of organoids was treated with the drugs after viral infection was well established, to better resemble the in vivo situation and examine the drugs efficacy also in this setting. In the 2D model system, antiviral treatment was able not only to reduce viral replication but also to exert a neuroprotective effect on iNeurons, as evidenced by the significant restoration of neurite outgrowth and preservation of neuronal morphology. On the other hand, 3D cerebral organoids enabled the investigation of viral spread dynamics and revealed inter-strain differences in viral replication kinetics, together with a robust activation of innate immune and pro-inflammatory responses within infected organoids. The evaluation of antiviral compounds in COs demonstrated a strong antiviral activity for both Ganciclovir and Letermovir, which were highly effective in suppressing viral replication, even when administered post-infection. Notably, Letermovir exhibited a broader capacity to normalize the dysregulated immune responses. Overall, this study provides comprehensive evidence that human pluripotent stem cell-derived neural models represent powerful and physiologically relevant systems to dissect the neuropathogenesis of cCMV in the developing brain and to evaluate potential therapeutic interventions. Furthermore, these preliminary findings may help explain the heterogeneous clinical outcomes observed in cCMV-affected newborns based on the infecting clinical strain and may also point to differential responses to antiviral therapies.