Letermovir shows antiviral and neuroprotective effects in differentiating neurons and cerebral organoids mimicking human developing brain

Abstract

Human cytomegalovirus (HCMV) is the leading viral cause of congenital defects. The triggers of viral neuropathogenesis during congenital infection (cCMV) are still unclear, and treatment options are limited. We used both a two-dimensional model of dynamic neurogenesis and cerebral organoids (COs), recapitulating the developing brain in the first trimester of gestation, to investigate the neuropathogenesis induced by HCMV. We also evaluated antiviral and neuroprotective effects of different compounds, both approved, direct-acting drugs and investigational, host-directed antivirals. In differentiating neurons, treatment with direct-acting antivirals blocked HCMV active replication and provided some protection from virus-induced defects. COs exposed to two different strains of HCMV showed viral spread throughout the organoids, dysregulation of key players of neurogenesis, alteration of the tissue cytoarchitecture, and triggering of innate antiviral and pro-inflammatory responses. Inter-strain differences in virus release and growth attenuation were detected in infected COs. Regardless of the strain, treatment with direct-acting antivirals, particularly letermovir, completely abolished HCMV replication, protected COs from virus-induced disorganization of tissue architecture, and dampened innate immune and pro-inflammatory response activation. Importantly, we also demonstrated the efficacy of the antiviral treatment in HCMV-infected COs in blocking an already established infection. This study contributes to shed light on HCMV-induced neuropathogenesis that occurs during congenital infection. Importantly, we demonstrated the neuroprotective effects of letermovir in models of human developing brain, holding promise for its evaluation as a candidate therapeutic agent to ameliorate cCMV-associated neurodevelopmental defects.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-025-06015-x.

Introduction

Human cytomegalovirus (HCMV) is a virus of the TORCH pathogens group and the leading viral cause of congenital defects worldwide [1, 2]. Congenital CMV infection (cCMV), which affects 0.4–1.4% of live newborns at birth, occurs when the virus reaches the fetus via vertical transmission as a consequence of primary infection or non-primary maternal infection (reactivation/reinfection) during pregnancy [1, 2]. In symptomatic subjects, cCMV hallmarks are mainly neurodevelopmental defects that can be detected in fetuses, newborns, and children even years after birth [2–5]. Fetal transmission can also lead to disseminated HCMV infection targeting different organs and severe long-term sequelae, thus representing a global burden for the public health sector, families, and caregivers [6, 7]. A vaccine effective in preventing HCMV infection in both children and adults, especially during pregnancy, is still lacking, although a Phase III clinical trial of an mRNA-based vaccine is ongoing (CMVictory, NCT05085366). The commonly available antivirals target the viral DNA polymerase (ganciclovir, acyclovir, foscarnet, and cidofovir), the viral terminase (letermovir), and the viral kinase (maribavir) [8–10]. Compounds targeting other steps of the viral replication cycle or the host are also being developed [11–18], including the approved anti-parasitic drug nitazoxanide (pregnancy category B) and the anti-protozoan ozonide OZ418 with investigational anti-HCMV activity that were used in this study [19, 20]. Despite viral DNA polymerase inhibitors might cause teratogenic effects [9, 10, 21], the efficacy for cCMV of some of them has been investigated in clinical trials [22–25]. Letermovir is under clinical investigation in a study aimed at measuring its accumulation in amniotic fluid during the second trimester of gestation (CYMEVAL3-STEP1, NCT04732260).

The neuropathogenic mechanisms of cCMV that lead to fetal brain damage are still poorly understood. The obstacles posed by ethical and technical reasons in having access to fetal samples, the lack of animal models that faithfully recapitulate human neurocortex development, as well as the strict species-specificity of HCMV, have been the main factors contributing to the gap in both research on neuropathogenesis and drug development [26]. In recent years, this has changed thanks to experimental models based on stem cell technology, such as two- and three-dimensional (2D and 3D) culture systems derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) [27, 28]. To model cCMV neuropathogenesis and to evaluate antiviral and neuroprotective strategies, different 2D stem cell-based models have been employed [29–33]. Based on the evidence collected so far, neural stem/progenitor cells are a major target during cCMV in vivo and are able to fully sustain HCMV replication [29, 31–37]. Moreover, human cortical neurons are infected by HCMV in vitro and viral infection induces severe functional and structural defects in this cell model [34].

3D models such as human cerebral organoids (COs) recapitulate the brain architecture and the different cell types during fetal brain development, particularly in the first trimester of gestation [27]. As COs closely mimic the structure and transcriptional profile of the fetal developing brain, they represent a faithful model to study congenital diseases of microbial origin [38, 39], including the one caused by HCMV in the developing nervous system [40–47]. So far, all studies have reported similar virus-induced alterations, such as dysregulation of key neurodevelopmental genes, disruption of neuronal differentiation, disruption of organoid architecture, and abnormal Ca²⁺-dependent signaling [40–47]. The morphological changes observed in HCMV-infected COs agree with pathological cCMV signs detected in samples of infected fetal brains, with a preferential tropism for NPCs in the subventricular zone and neurons [34–47].

In this study, we investigated the effects and neuroprotective potential of different antivirals in both a 2D dynamic in vitro model of neurogenesis and in COs to shed light on HCMV-induced neuropathogenesis in the developing brain and to contribute to the development of effective therapeutic options for cCMV.

Materials and methods

Cell lines

The human embryonic stem cell (hESC) line H9 (hPSCReg ID: WAe009-A) was obtained from WiCell Research Institute (Madison, WI), while hESCs H9-NGN2 cells were a kind gift from J. Wade Harper (Harvard Medical School, Boston, MA) and were previously reported [48]. H9 cells were cultured in Geltrex-coated plates (Gibco, Thermo Fisher Scientific) in mTeSR-1 medium (Stem cell technologies). Human Foreskin Fibroblast cells (HFF) were from the American Type Culture Collection (ATCC, HFF-1 SCRC-1041) and were cultured in Dulbecco modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin and 100 mg/mL streptomycin (P/S) (Gibco). Human Aortic Endothelial cells (HAoEC) were from Promocell (C-12271) and were cultured in Endothelial Cell Growth Medium MV (Promocell C-22020) and maintained up to 4 passages using the Detach Kit (Promocell C-41200). All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂.

Viruses

Recombinant HCMV TB4-UL83-EYFP expressing viral pp65 protein (UL83) fused to an enhanced Yellow Fluorescent Protein (EYFP) was previously described [49]. HCMV VR1814, kindly provided by G. Gerna (IRCCS Policlinico San Matteo, Pavia, Italy), was recovered from a cervical swab of a pregnant woman [50]. For both viruses, low-titer cell-free virus stocks were prepared by infecting human aortic endothelial cells (HAoEC, Promocell) to avoid loss of epithelial/endothelial tropism. HAoEC were infected at a multiplicity of infection (MOI) of 0.01, and infected cells were separated from the supernatant when 90% of cytopathic effect was detectable. Cell pellets were first subjected to three cycles of freezing and thawing and then reunited with the cell supernatants. Cellular debris was removed by centrifugation at 5,500 × g for 5 min, and the resulting supernatant was filtered and stored at − 80 °C. Viral titers were determined by standard plaque assay in HFF cells. High-titer viral stocks were obtained with a single round of amplification of HCMV strains in HFF cells, as previously reported [51]. MOI refers to plaque-forming units (PFU) obtained from virus titration in HFF cells.

Compounds

Ganciclovir (GCV) and nitazoxanide (NTZ) were purchased from Merck. Letermovir (LTV) was purchased from Selleck Chemicals. OZ418 was provided by Dr. Jonathan Vennerstrom and was previously described [20].

Differentiation of H9-NGN2 into induced neurons

Differentiation of H9-NGN2 into induced Neurons (iNeu) was obtained as previously described [33, 48]. Briefly, H9-NGN2 were seeded on plates coated with Geltrex in DMEM/F12 (Gibco) supplemented with 1% N2, 1% nonessential amino acids (NEAA), 0.2 µg/ml mouse laminin (all from Gibco), 10 ng/ml brain-derived neurotrophic factor (BDNF), 10 ng/ml NT3 (both from Cell guidance system), 10 µM ROCK Inhibitor (RI, Selleckchem), and 100 U/mL penicillin and 100 mg/mL streptomycin (Pen/Strep, Gibco). The next day, the same medium without RI was refreshed. Two days post-differentiation, the medium was switched to a differentiation medium containing Neurobasal medium, 2% B-27, 1% Glutamax (all from Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco), and BDNF and NT3 at the previous concentrations. Two µg/ml doxycycline (Sigma-Aldrich) was included in the media during the differentiation period to force the expression of Neurogenin 2.

Generation of cerebral organoids

Cerebral organoids (COs) were generated from H9 cells following the protocol by Lancaster et al. [52] with a few modifications. H9 cells growing in mTeSR-1 were detached with Accutase (Gibco) and seeded (in mTeSR-1 supplemented with 50 µM RI) into U-Shaped-Bottom Nunclon™ Sphera™ 96-Well plates (Thermo Fisher Scientific) at a density of 9,000 cells/well. On day 3, mTeSR-1 without RI was replaced. On day 5 of differentiation, the medium was changed to Neural Induction medium composed of DMEM-F12 with 1% N2, 1% GlutaMAX, 1% NEAA, 1 µg/ml heparin (Sigma-Aldrich), and 1% Antibiotic-Antimycotic (Gibco). Neural induction medium was refreshed every other day. On day 11 of differentiation, the neuroepithelial tissues were transferred to Matrigel (Corning™) droplets in cerebral organoid medium (CO medium) composed by DMEM/F12 and Neurobasal medium (1:1), 1% Glutamax, 1% anti-anti, 1% B27, 0.5% N2, 0.5% NEAA, 0.035% of 2-mercaptoethanol (Gibco, diluted 1:100 in DMEM/F12), and 0.025% of human insulin (Sigma-Aldrich). During the initial stages of growth in Matrigel, B27 without retinoic acid was supplemented into the medium, while after the transfer to the orbital shaker, B27 containing retinoic acid (Gibco) was added.

HCMV infection of differentiating H9-NGN2 cells

Differentiating H9-NGN2 cells were detached on day 6 of differentiation and plated at a density of 10⁵ cells/cm² on either 6- or 96-well/plates, depending on the experiment. The next day (i.e., day 7 of differentiation), cells were infected with HCMV TB4-UL83-EYFP at MOI = 0.1 or 1 and treated with different concentrations of test compounds. Uninfected cells treated only with the drugs were included as mock. Tested drug concentrations were 25, 10, 1, 1, 0.1 µM for GCV, NTZ, and OZ418, and 10, 1, 0.1, 0.01 nM for LTV. Plates were incubated for 7 days at 37 °C with half medium change and drug refresh every other day. On day 14 of differentiation, corresponding to 7 days post-infection (dpi), cells were harvested for further analysis.

HCMV infection of cerebral organoids

To determine the average number of cells in one organoid and the multiplicity of infection (MOI) to be used for COs infection experiments, single organoids (n = 5 organoids) at different days of differentiation were washed with PBS and dissociated in 500 µl of 0.05% Trypsin-EDTA for 30 min at 37 °C. During incubation, COs were gently pipetted with a cut 500 µl pipette tip, to facilitate dissociation into single cells. After incubation, 1 ml of DMEM 10% FBS was added to dissociated cells, and these were centrifuged at 1,100 rpm for 5 min. The resulting cell pellet was resuspended and filtered through a 40-µm strainer to remove clumps. Cells were then stained with Trypan Blue and counted. The average cell counts for each subset were used as a reference to calculate the MOI to be used in further experiments. For pilot experiments, COs were infected with HCMV TB4-UL83-EYFP strain at the indicated days of differentiation at MOI = 1, unless otherwise stated. All further infections of 30-day-old organoids with HCMV TB4-UL83-EYFP and VR1814 strains were carried out at MOI = 0.1. Briefly, organoids were harvested with a wide-bore 1,000-µl tip and singularly transferred to a 2-ml screw cap tube. Carried over medium was removed, and viruses were added to each tube at the desired MOI. Tubes were then centrifuged at 1,100 rpm for 30 min at RT and then incubated for 90 min at 37 °C in a humidified atmosphere with 5% CO₂. COs were then transferred into Nunclon Sphera 6-well plates in CO medium and moved to an orbital shaker. Organoids were kept in the orbital shaker for 21 days with medium change twice a week. At 21 dpi, organoids were harvested for further analysis. To evaluate the effects of test antivirals after the onset of infection, a subset of organoids was infected with both strains at MOI = 0.1. At 7 dpi, organoids were randomly divided and treatment with test antivirals was started and continued for 21 days. For each condition, n ≥ 4 organoids were treated as biological replicates. All experiments were repeated for n ≥ 2 independent batches of organoids.

Cell viability assays

On day 6 of differentiation, H9-NGN2 (30,000/well) were plated in 96-well plates coated with Geltrex in differentiation medium with 10 µM RI and incubated overnight at 37 °C. The next day, cells were treated with different concentrations of test compounds for 7 days at 37 °C with half medium/compound refresh every other day. On day 14 of differentiation (i.e., 7 days post-treatment), the effect of test compounds on cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) method as described previously [33]. Cell viability of COs was determined with the CellTiter-Glo^® (Promega). Tissues were dissociated with a 21G gauge needle, transferred with 100 µl of medium to an opaque-walled multiwell plate, and 100 µl of lysis buffer was added to each well. The plate was then mixed vigorously for 15 min to induce cell lysis and incubated at RT for an additional 25 min to stabilize the luminescent signal. Luminescence was recorded on a Victor X2 (PerkinElmer).

Quantification of HCMV progeny

For virus yield reduction assays with test antivirals, HCMV-infected iNeu were subjected to one cycle of freezing and thawing. HCMV titers were determined on fresh HFF monolayers as previously described [53]. The Effective Concentration at half-maximal response (EC₅₀) of test antivirals was determined by nonlinear regression analysis with GraphPad Prism 10.5. The same protocol was followed to quantify the infectious viral progeny, both cell-associated and released in the supernatant of mock-, HCMV-infected, and HCMV-infected and treated COs (see below). Briefly, intracellular infectious viral particles were recovered from infected organoids by dissociating the tissues with a 21G gauge needle into small fragments or single cells. After centrifugation at 1,100 rpm for 5 min, cells were resuspended in 150 µl of serum-free DMEM and subjected to three freeze-thaw cycles. Cells were then centrifuged again, and the released viral progeny was quantified on fresh HFF as previously described [53].

Analysis of gene expression

iNeu (3 × 10⁵ per well) at day 6 of differentiation were plated in 6-well plates coated with Geltrex and incubated overnight at 37 °C. The next day, cells were infected with HCMV at MOI = 1 and treated with 50 µM GCV, 10 nM LTV, 30 µM NTZ, and 30 µM OZ418. Thereafter, half medium was refreshed every other day and at 7 dpi, cells were subjected to RNA isolation with the Total RNA Purification Plus Kit (Norgen Biotek Corp.). After isolation, total RNA was reverse transcribed. For COs, total RNA was isolated at 21 dpi from mock-, HCMV-infected, and HCMV-infected and treated COs by adding 700 µl of lysis buffer to the tissues, followed by mechanical disruption using a syringe with a 21G gauge needle to promote lysis. RNA extraction was then performed using the RNeasy Kit (Qiagen), according to the manufacturer’s instructions. SYBR Green (Thermo Fisher Scientific) chemistry was applied in all reactions in a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). Oligonucleotides used for qPCR reactions are reported in Table S1. Each reaction was performed in duplicate and the results for the target genes were normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2^−ΔΔC_T method.

Cryopreserving and sectioning of COs

Cerebral organoids at 30 days of differentiation were infected with HCMV TB4-UL83-EYFP and VR1814 at MOI = 0.1, and at 21 dpi, organoids were treated with 4% paraformaldehyde (PFA, Thermo Fisher Scientific) in PBS overnight at 4 °C. The next day, the tissues were washed three times with D-PBS and exposed to a gradient of sucrose (10%, 20%, and 30% sucrose solution in D-PBS, 30 min each) to cryopreserve the tissues. The samples were then transferred into cryomolds, and, after the removal of excess liquid, they were included in Optimal Cutting Temperature (OCT) compound (VWR Chemicals). A thermomixer (conditions: 30 min, 300 rpm, RT) was used to stir the cryomolds, making the organoids sink in the solution. When all organoids within the same cryomold were aligned in the same plane, they were snap frozen using dry ice before transferring them to −80 °C. Frozen samples were then sectioned at a thickness of 20 micron using a Leica CM1950 cryostat (Leica), and slides were stored at −20 °C.

Immunofluorescence staining

Immunofluorescence analysis was performed as previously reported [54], with some modifications. Mock-infected, HCMV-infected, and HCMV-infected-treated iNeu at 14 days of differentiation (corresponding to 7 dpi) were fixed with 4% PFA for 20 min at RT, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 15 min, and incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at RT.

Cerebral organoids cryosections were rinsed with PBS for 10 min. The sections were then treated with blocking solution containing bovine serum albumin (BSA, Sigma-Aldrich) 4% in PBS + 0.2% Triton-X100 for 1.5 h at RT. Primary antibodies diluted in blocking solution were added to the sections and incubated overnight at 4 °C. After washing 3 times with PBS, secondary antibodies diluted in blocking solution were applied to the sections for 2 h at RT. Finally, tissue sections were washed again 3 times with PBS and cell nuclei were counterstained with DRAQ5 (diluted 1:2,000 in PBS) before mounting with Fluoromount-G^® (FG) mounting medium (Thermo Fisher Scientific) and further analyzed by confocal microscopy. The list of primary antibodies and of fluorophore-conjugated secondary antibodies used is reported in Table S2.

Confocal imaging and analysis

Images were obtained with a Nikon Eclipse Ti confocal microscope and processed with ImageJ (Fiji) software. Live images were acquired using the Z-stack mode, enabling the capture of multiple focal planes to generate a three-dimensional representation of the sample. ImageJ was used to process images for protein expression analysis in COs. Background subtraction was performed for each image. Expression areas were determined using the threshold function. Fluorescent intensity was then quantified by generating a mask, analyzing the mean brightness of detected particles, and measuring the Integrated Density and Mean Gray Value.

Total neurite length was calculated on iNeu images using ImageJ/Fiji software by setting a threshold, converting the image to binary, performing skeletonization, and then analyzing the skeleton to obtain the total length of neurites. COs areas were measured using ImageJ/Fiji software. The images were converted to 8-bit grayscale, and the threshold function was used. The areas of the organoids were selected and measured using a pixel scale.

Western blotting

Cerebral organoids at 30 days of differentiation were infected with HCMV TB4-UL83-EYFP and VR1814 at MOI = 0.1, and at 21 dpi organoids were harvested. Whole-cell protein extracts were prepared starting from 3 organoids for each condition. Organoids were incubated with Cell Recovery Solution (Corning) to eliminate Matrigel for 1 h at 4 °C. The solution was then removed, and the organoids were washed two times with D-PBS. RIPA buffer (50 mM Tris, 150 mM NaCI, 1% IGEPAL, 0.1% SDS, pH 7.8), containing Complete Protease Inhibitor Cocktail 1× (Roche), was added to organoids and then they were incubated for 10 min on ice. Organoids were then homogenized using a syringe and needle following a 30-min incubation in RIPA buffer on ice. Samples were centrifuged at 13,000 rpm for 15 min at 4 °C and supernatants were collected. Ten µg of total proteins for each condition were fractionated through 8% SDS-PAGE and then transferred to PVDF membranes (BioRad). After blocking with 5% nonfat dry milk in TBS-Tween 0.1%, membranes were incubated overnight at 4 °C with the primary antibodies (Table S2). Immunocomplexes were then detected with different secondary antibodies conjugated to horseradish peroxidase (Table S2).

Statistical analysis

Data analysis was performed with GraphPad Prism version 10.5. Data in bar graphs are presented as mean ± standard error of the mean (SEM) or mean ± standard deviation (SD). Data in box plots and violin plots are presented as described in the figure legends. The exact n value for each experiment and the statistical test used are described in the figure legends.

Results

Antiviral treatment blocks HCMV replication in a dynamic model of neurogenesis in vitro

We previously demonstrated that antiviral treatment with different anti-HCMV drugs effectively inhibits viral replication in two distinct lines of human neural progenitors, which are the main target of HCMV in the developing brain [33]. In this study, we exploited an in vitro model of dynamic neurogenesis in which human embryonic stem cells are forced to differentiate into induced neurons (iNeu) passing through a neural progenitor cell (NPC)-like phase (Fig. 1A) [48]. On day 7 post-differentiation, intermediate NPCs, expressing key markers of neural stem cells, i.e., SOX2, PAX6, and Nestin (Fig. S1A), were infected with HCMV (TB4-UL83-EYFP strain) and let to differentiate into beta-III-tubulin (TUBB3)-expressing neurons (Fig. 1A, Fig. S1B-C). On day 14 post-differentiation, infected EYFP⁺ cells showed a cytomegalic shape, formation of syncytia (Fig. 1B), and a significant reduction in neurite outgrowth (Figs. 1B-C). Infected neurons expressed high levels of viral UL83 mRNA (Fig. 1D), indicating that HCMV replication is sustained during neurogenesis and alters iNeu morphology (Figs. 1B-C).

Fig. 1.

Antiviral treatment blocks HCMV replication in differentiating neurons. (A) In vitro model of dynamic neurogenesis. At day 0, by forcing the expression of Neurogenin 2, H9-NGN2 human embryonic stem cells are differentiated into induced neurons (iNeu) in 14 days passing through a neural progenitor cell (NPC)-like phase at approximately day 7 of differentiation. In this experimental model of cCMV, differentiating cells at the stage of NPCs (7 days post-differentiation) are infected with HCMV (TB4-UL83-EYFP strain) and the effects are assessed at the iNeu stage at day 14 of differentiation. All infections were conducted at MOI = 1, except for the antiviral assays, which were performed at MOI = 0.1. Created in BioRender. (B) Comparison of cell morphology in mock- and HCMV-infected neurons expressing EYFP. The dashed line highlights the formation of syncytia among infected cells. Scale bars: 100 μm. (C) Quantification of total neurite length of mock- and HCMV-infected iNeu. Box plot shows the analysis of n ≥ 10 images for each condition measured from n = 2 independent experiments. The horizontal line in the box plot indicates the median, the box denotes the interquartile range (IQR), and the whiskers beyond the box extend to min and max of the data. Data were analyzed by a Kruskal-Wallis test followed by a Dunn’s test. (****p < 0.0001, versus mock-infected iNeu). (D) Bar graph showing viral UL83 mRNA levels (expressed as delta Ct calculated over the GAPDH housekeeping gene) in mock- and HCMV-infected neurons. Data show the mean ± SEM of n = 6 independent experiments. (E) Concentration-dependent inhibition of HCMV replication upon antiviral treatment with ganciclovir (GCV), letermovir (LTV), and OZ418. Data show the mean ± standard deviation (SD) of n = 3 independent experiments in duplicate and are expressed as % of control (i.e., vehicle-treated HCMV-infected neurons set at 100%). (F) Effects of antiviral treatment with 50 µM GCV, 10 µM LTV, and 30 µM OZ418 on EYFP expression in HCMV-infected neurons (MOI = 1). Scale bars: 100 μm. (G) Violin plots showing the quantitative comparison of EYFP-derived fluorescence signal in HCMV-infected, vehicle-treated iNeu versus HCMV-infected iNeu treated with the indicated antivirals. Data represents the ratio between the EYFP and DRAQ5 fluorescence signals of n = 10 images for each condition from n = 2 independent experiments. The dashed line represents the median, while the dotted lines represent the quartiles. Data were analyzed with a Kruskal-Wallis test followed by Dunnett’s multiple comparison test. (****p < 0.0001, versus HCMV-infected, vehicle-treated iNeu). (H) Bar graph showing the reduction in viral UL83 gene expression upon antiviral treatment in HCMV-infected neurons. Data show the mean ± SEM of n ≥ 3 independent experiments in duplicate and are expressed as fold change calculated with the 2^−ΔΔCT method. Data were analyzed by a Kruskal-Wallis test followed by Dunnet’s multiple comparison test. (****p < 0.0001, versus calibrator sample, i.e., HCMV-infected, vehicle-treated cells). (I) Bar graph showing the reduction in infectious viral progeny production in HCMV-infected neurons upon antiviral treatment expressed as PFU/ml. Data show the mean ± SEM of n ≥ 3 independent experiments in duplicate and were analyzed by a Kruskal-Wallis test followed by Dunnet’s multiple comparison test. (***p < 0.001, **p < 0.01, versus calibrator sample, i.e., HCMV-infected, vehicle-treated cells). (J) Quantification of total neurite length in HCMV-infected, vehicle-treated iNeu and in HCMV-infected iNeu treated with the indicated antivirals. Box plot shows the analysis of n ≥ 10 images for each condition measured from n = 2 independent experiments. The horizontal line in the box plot indicates the median, the box denotes the IQR, and the whiskers beyond the box extend to min and max of the data. Data were analyzed by a Kruskal-Wallis test followed by a Dunn’s test. (*p < 0.05, versus vehicle-treated iNeu)

Next, we tested in this 2D model the effects of two approved direct-acting antivirals (DAA), i.e., GCV and LTV, a DNA polymerase inhibitor and a terminase inhibitor, respectively) and of two investigational, host-directed anti-HCMV drugs, i.e., NTZ and OZ418 [19, 20]. Before assessing the effects of antiviral treatment in the dynamic model, we tested the effects of antivirals on the viability of uninfected cells. Differentiating intermediate NPCs were treated at 7 days post-differentiation with GCV, LTV, NTZ, and OZ418. Cell viability was evaluated after 7 days of treatment at the end of the differentiation. While GCV, LTV, and OZ418 did not show any cytotoxicity at the tested concentrations, NTZ showed significant toxicity and was excluded from further studies (Fig. S2). Subsequently, to determine whether antiviral treatment could block HCMV replication during neurogenesis, we infected differentiating cells with HCMV (MOI = 0.1) at the NPC-like stage (day 7) and treated them with different concentrations of antiviral drugs up to the neuronal stage (at 14 days post-differentiation, Fig. 1A). The infectious viral progeny produced under the different treatment conditions was then quantified. As reported in Fig. 1E, both GCV and LTV inhibited the production of viral progeny compared to the vehicle-treated, infected cells in a concentration-dependent manner. On the other hand, OZ418 was inactive at concentrations up to 25 µM (Fig. 1E). To further confirm the antiviral activity of these drugs during neurogenesis, we proceeded to the determination of pp65-EYFP⁺ cells, viral UL83 expression by qPCR, and infectious viral progeny production. After 7 days of treatment, UL83 expression as well as the number of EYFP⁺ cells were significantly downregulated only in HCMV-infected, GCV-treated cells (Fig. 1F-H). This result was expected based on the late-acting antiviral effect of LTV [33, 55], and on the low activity demonstrated by OZ418. Accordingly, production of infectious viral particles was significantly inhibited upon treatment with GCV and LTV, but OZ418 was ineffective (Fig. 1I). On the other hand, all the test compounds induced a significant rescue of total neurite length (Fig. 1J). Notably, treatment with GCV, LTV, and OZ418 in uninfected iNeu did not affect the length of neurites (Fig. S3). Collectively, these results pointed out that GCV and LTV uphold a favorable Selectivity Index (Table S3) and can efficiently inhibit HCMV replication in cells during neurogenesis, while OZ418 might lose antiviral effect during the differentiation of NPCs to iNeu.

Antiviral treatment partially rescues the expression of marker genes involved in neurogenesis and neuronal differentiation

As we and others have previously demonstrated, HCMV dysregulates the expression of neural stem cell key markers, thus leading to altered cell differentiation, impaired neurogenesis, and potential developmental abnormalities within the neural tissue [29, 31, 33, 35, 56]. We thus sought to analyze whether the expression of key markers of neurogenesis might be affected by HCMV infection during the differentiation of NPCs into iNeu. NPCs were infected at 7 days post-differentiation with HCMV, and at 7 dpi infected cells were harvested for total RNA isolation. The expression of key markers known to be involved in neurogenesis processes, i.e., Peroxisome proliferator-activated receptor γ (PPARγ) and Doublecortin (DCX), and neuron marker TUBB3, was analyzed upon infection. As shown in Fig. 2A, HCMV infection at 7 dpi significantly affected the expression of all three considered genes and led to the upregulation of PPARγ, while downregulating DCX and TUBB3. Overall, these results confirmed that HCMV infection during the differentiation of infected neural progenitor cells into neurons disrupts neurogenesis, possibly contributing to neurological defects in the developing brain.

Fig. 2.

Antiviral treatment partially restores the expression of genes involved in neurogenesis and neuronal differentiation altered by HCMV. (A) Bar graph showing the effects of HCMV infection (TB4-UL83-EYFP strain) of differentiating neurons on the expression of Peroxisome proliferator-activated receptor γ (PPARγ), Doublecortin (DCX), and beta-III-tubulin (TUBB3). mRNA levels are expressed as fold change calculated with the 2^−ΔΔCT method over GAPDH housekeeping gene in mock- or HCMV-infected neurons. Data show the mean ± SEM of n ≥ 6 independent experiments performed in duplicate and were analyzed by a Mann-Whitney test. (***p < 0.001; **p < 0.01, versus HCMV-infected, vehicle-treated neurons). (B) Bar graph showing the effects of antiviral treatment on HCMV-induced dysregulation of gene expression in infected differentiating neurons. mRNA levels are expressed as fold change calculated with the 2^−ΔΔCT method. Data show the mean ± SEM of n ≥ 5 independent experiments performed in duplicate and were analyzed by a Kruskal-Wallis followed by Dunn’s test. (***p < 0.001; versus HCMV-infected, vehicle-treated neurons)

We have also previously demonstrated that the treatment of infected neural stem cells (NSCs) with antiviral compounds can restore HCMV-mediated dysregulation of key markers important to maintain NSCs identity, i.e., SOX2 and Nestin [33]. Here, we sought to evaluate whether the dysregulation of PPARγ, DCX, and TUBB3 caused by HCMV could be restored by antiviral treatment. When we treated infected NPCs with test antivirals for 7 days during differentiation, we detected a restorative effect on the expression of PPARγ, a key promoter of neurogenesis, upon GCV treatment (Fig. 2B). PPARγ upregulation might be an event occurring very early in the replication cycle of HCMV; thus, the late-acting mechanism of LTV likely makes it not able to counteract this dysregulation within the treatment window.

Human cerebral organoids sustain efficient HCMV replication after day 20 of maturation and display typical signs of HCMV infection

To investigate the neuropathogenesis of HCMV infection and the effects of antiviral treatment in a model of early human brain development, we generated unguided human cerebral organoids (COs) following Lancaster’s protocol [52], with few modifications. Differentiating COs demonstrated proper ectodermal differentiation by day 9, forming radialized neuroepithelium with smooth edges and brightened surfaces (Fig. S4A, panels i-ii), which later developed into pseudostratified epithelium (Fig. S4A, panels iii-vi) and neural rosette-like structures positive for PAX6, indicating neuroectodermal specification and active cell proliferation (Fig. S4B). By day 40, COs displayed abundant neuron markers MAP2 and TUBB3, implying advanced neurogenesis and maturation. At the same time, neural rosettes were still present, marked by Nestin expression, reflecting the persistence of neural progenitor cells in the later stages of development (Figs. S4C-D). To evaluate the permissiveness to HCMV infection, COs were infected with HCMV at different stages of maturation, corresponding to different human embryo developmental phases [57]. COs at day 0, 3, 10, 15, 20, and 30 of maturation were exposed to HCMV (TB4-UL83-EYFP, MOI = 1) (Fig. 3A). Infected H9 cells (day 0) showed suboptimal embryoid bodies (EBs) formation and failed to show EYFP⁺ cells, confirming that hESCs are not permissive to HCMV infection (data not shown) [33]. EBs infected on day 3 did not lead to a productive infection, while going through normal cerebral organoid differentiation (Fig. 3A). Infected tissues on day 10 of differentiation displayed few EYFP⁺ cells at 4 dpi, but the number of positive cells did not increase as the organoid grew and matured (Fig. 3B). On the contrary, EYFP expression gradually decreased until disappearing completely (Fig. 3B). COs infected on day 15 and 20 of maturation (4 and 9 days after Matrigel embedding, respectively) showed an increase in EYFP⁺ cells compared to organoids infected at earlier stages, while still not showing high number of infected cells on 21 dpi (Fig. 3A). Finally, one-month-old COs were exposed to HCMV and from 4 dpi a widespread fluorescence signal was observed in these more mature cerebral organoids (Fig. 3A-C). Furthermore, regions of radially organized columnar cells surrounding an empty lumen reminiscent of the neural tube were observed in both mock- and HCMV-infected organoids. However, whereas neural rosettes in mock-infected organoids were characterized by an ordered arrangement of the intermediate filament protein Nestin, which surrounded the radially organized cells, infected organoids displayed an alteration of this pattern showing a disorganized expression of this marker (Fig. 3D). Infected organoids displayed a significant number of viral IEA⁺ (Immediate-Early Antigen-positive) and pp65⁺ (late antigen-positive) cells, demonstrating that the virus could spread throughout the organoid and that infected cells within the organoids were at different stages of HCMV replication (Fig. 3E, left panel). Furthermore, infected cells showed cytomegalic, kidney-shaped nuclei and the formation of syncytia, typical features of HCMV-induced histopathology (Fig. 3E, right panel). Finally, no infectious viral particles were recovered from the culture medium of infected organoids at any dpi (data not shown), indicating a mechanism of cell-to-cell spread of HCMV TB4-UL83-EYFP within the COs.

Fig. 3.

HCMV infection in cerebral organoids efficiently progresses starting from day 20 of maturation and induces typical signs of histopathology. (A) Differences in the progression of infection in COs infected at day 3, 10, 15, 20, and 30 of maturation exposed to HCMV (TB4-UL83-EYFP strain, MOI = 1). All images were taken at 21 days post-infection (dpi). Scale bars: 100 μm. (B) Progression of infection in 10-days-old COs exposed to HCMV (TB4-UL83-EYFP strain, MOI = 1) at the indicated dpi. Scale bars: 100 μm. (C) Progression of infection in 30-days-old COs exposed to HCMV (TB4-UL83-EYFP strain, MOI = 1) at the indicated dpi. Scale bars: 100 μm. (D) Disorganization of intermediate filaments protein Nestin in COs infected with HCMV. Scale bars: 50 μm. (E) Representative images of the analysis of the expression of different viral antigens (IE1/2 and pp65) in COs that indicate spreading of HCMV within tissues. Scale bars: 50 μm. The dashed area indicates a region with typical signs of CMV histopathology

Antiviral treatment blocks active HCMV replication and reduces viral spread in infected cerebral organoids

To investigate the effects of antiviral compounds in blocking HCMV replication, COs at day 30 of maturation were infected with HCMV (TB4-UL83-EYFP, MOI = 0.1) and treated for 21 days with GCV, LTV, OZ418, and NTZ. Uninfected, drug-treated organoids and vehicle-treated, infected COs were included as controls. Live imaging of both mock- and HCMV-infected organoids upon treatment with vehicle or test drugs was performed at different dpi. A comparison of vehicle-treated, HCMV-infected versus mock-infected COs at different dpi showed an increasing number of EYFP⁺ cells over the course of infection, confirming HCMV ability to infect and spread even upon infection at a lower MOI (Fig. 4A). As expected, mock-infected organoids displayed no signs of infection (Fig. 4A). NTZ-treated HCMV-infected organoids did not show any sign of infection but appeared smaller compared to all other infected and treated organoids (Fig. S5A). Interestingly, mock-infected organoids appeared smaller upon NTZ treatment (Fig. S5B), confirming the compound cytotoxicity previously observed in differentiating neurons, and prompting us to exclude NTZ from further studies in COs. Moreover, we determined the cell viability in COs treated with the other drugs. As reported in Fig. S6, treatment with antiviral drugs did not affect the viability of the COs.

Fig. 4.

Antiviral treatment blocks active HCMV replication and reduces HCMV spread in infected COs. (A) Representative images of the progression of HCMV infection (TB4-UL83-EYFP strain, MOI = 0.1) vs. MOCK-infection in 30-days-old COs. Scale bars: 500 μm. (B) Representative images of the effects of antiviral treatment in 30-days-old COs infected with HCMV (TB4-UL83-EYFP strain, MOI = 0.1). Viral replication is indicated by EYFP expression. Scale bars: 500 μm. (C) Violin plots showing the quantitative comparison of EYFP-derived fluorescence signal in HCMV-infected, vehicle-treated COs and in HCMV-infected COs treated with the indicated antivirals. Data represent the fluorescence signals of n = 10 organoids for each condition from n = 2 independent experiments. The dashed line represents the median, while the dotted lines represent the quartiles. Data were analyzed with a Kruskal-Wallis test followed by Dunnett’s multiple comparison test. (****p < 0.0001; **p < 0.01; *p < 0.05, versus HCMV-infected, vehicle-treated COs). (D) Immunoblotting showing the effects of antiviral treatment on the expression of different Immediate-Early (IE1/2), Early (UL44), and Late (UL55) viral proteins. Samples were obtained by pooling n = 3 organoids for each condition. Vinculin expression was analyzed as a loading control. Marker molecular weights in kDa are indicated on the left. V, virus-infected, vehicle-treated COs. (E) Bar graph showing the reduction in intracellular infectious viral progeny (expressed as PFU/ml) produced in HCMV-infected COs upon antiviral treatment with the indicated antivirals. Data show the mean ± SEM obtained from n = 5 organoids lysed independently. (**p < 0.01; versus HCMV-infected, vehicle-treated COs)

GCV, LTV, and OZ418 treatments were highly effective in HCMV-infected COs, resulting in a significant reduction of viral replication and spread (Figs. 4B-C). Mock-infected COs treated with these compounds displayed no obvious morphological differences compared to the vehicle-treated controls (Fig. 4B), confirming the tolerability of the treatment under the conditions tested. All the tested drugs significantly reduced the expression of viral Immediate-Early (IE) antigens, early protein UL44, and the true-late protein UL55 (Fig. 4D), corroborating the effectiveness of antiviral treatment in blocking active replication in HCMV-infected COs. Finally, to confirm that the antiviral treatment blocked the production of intracellular infectious virus and hence viral spread, we quantified infectious viral progeny obtained from lysates of infected COs treated with the different antivirals. As reported in Fig. 4E, treatment with GCV, LTV, and (to a less extent) OZ418 inhibited viral yield in HCMV-infected COs. Collectively, these results indicated that treatment of HCMV-infected COs with antiviral drugs that act with different mechanisms could block active viral replication and significantly limit viral spread throughout tissues and within the COs.

Cerebral organoids infected with HCMV VR1814 strain showed a reduction in size and the release of infectious viral particles

Previous studies describing COs infections with HCMV employed different viral strains and various experimental conditions [40–47]; thus, we sought to investigate whether, in a complex system such as cerebral organoids, the effects of both HCMV infection and antiviral treatment could differ depending on the strain. To this aim, we employed HCMV strain VR1814, originally isolated from a cervical swab of a pregnant woman [50]. COs were exposed to HCMV (VR1814, MOI = 0.1), and analysis was performed at 21 dpi. Infected organoids showed a sustained expression of viral pp65 antigen (Fig. 5A), as well as obliteration of SOX2⁺ and Nestin⁺ neural rosettes from infected regions, compared to COs infected with TB4-UL32-EYFP at the same MOI (Figs. 5A-B). Moreover, both VR1814- and TB4-UL83-EYFP infected COs showed a downregulation/displacement of TUBB3⁺ neurons (Fig. 5C) and a significant upregulation of PPARγ expression, which was more marked in IEA⁺ cells and surprisingly detected also in uninfected neighboring cells (Fig. 5D and E). Another important difference was the ability of the VR1814 strain to significantly reduce the size of infected COs throughout the infection, which was not detected in COs infected with the TB4-UL83-EYFP strain (Figs. 5F and S7). Moreover, unlike HCMV TB4-UL83-EYFP, infection of COs with the VR1814 strain led to the release of infectious viral particles into the cell culture medium of infected organoids starting from 14 dpi, suggesting a different/additional mechanism of viral spread within COs tissues between the two strains (Fig. 5G). Regardless of these differences, as shown in Fig. 5G, the treatment of VR1814-infected COs with GCV and LTV led to a complete inhibition of the release of viral particles and a remarkable reduction upon treatment with OZ418. This result was mirrored by the total abolishment of intracellular infectious viral progeny production (undetectable in lysates of COs infected with HCMV VR1814, Fig. 5H), and inhibition of the expression of different viral antigens within the tissues exerted by GCV and LTV, while in the OZ418-treated COs only a low level of IE antigens was still detectable (Fig. 5I). Collectively, these results suggested that the effects of HCMV infection in COs might differ depending on the strain. However, more importantly, antiviral treatment elicits comparable inhibitory effects toward both HCMV strains and can block active viral replication, infectious viral progeny production, and viral spread.

Fig. 5.

HCMV VR1814 strain productively replicates in COs and reduces organoids size but is blocked by antiviral treatment. (A) Representative images showing the effects of the infection of 30-day-old COs with HCMV VR1814 and TB4-UL83-EYFP strains (MOI = 0.1). Viral replication is associated with loss of neural rosettes and neural stem cell marker SOX2 expression. Scale bar: 50 μm. (B) Representative images showing the effects of HCMV VR1814 infection (MOI = 0.1) on SOX2 and Nestin expression in infected COs. Scale bars: 50 μm. (C) Representative images of the effects of HCMV VR1814 infection (MOI = 0.1) compared to TB4-UL83-EYFP strain (MOI = 0.1) on the cytoarchitecture of COs and on the localization of neuronal marker TUBB3 (TUJ-1). Scale bars: 500 μm. (D) Representative images of the effects of infection with HCMV VR1814 strain (MOI = 0.1) compared to TB4-UL83-EYFP strain (MOI = 0.1) on PPARγ upregulation. Scale bars: 100 μm. (E) Quantification of PPARγ expression in mock- and HCMV-infected COs. Violin plot showing PPARγ signal in different fields (n > 20) taken from n = 2 independent experiments. The dashed line represents the median, while the dotted lines represent the quartiles. Data were analyzed by a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. (****p < 0.0001, versus mock-infected COs). (F) Quantification of the area of mock- and HCMV-infected COs. Box plot showing the area of n ≥ 10 COs for each condition measured from n = 2 independent experiments. The horizontal line in the box plot indicates the median, the box denotes the IQR, and the whiskers beyond the box extend to min and max of the data. Data were analyzed by a Kruskal-Wallis test followed by a Dunn’s test. (*p < 0.05, versus HCMV-infected, vehicle-treated COs). (G) Bar graph showing the reduction in released infectious viral progeny (expressed as PFU/ml) in medium derived from HCMV VR1814-infected COs upon antiviral treatment compared to vehicle-treated, infected COs. Data show the mean ± SEM of n = 3 independent experiments in duplicate. (*p < 0.05, versus vehicle-infected COs) (H) Bar graph showing the reduction in intracellular infectious viral progeny (expressed as PFU/ml) produced in HCMV VR1814-infected COs upon antiviral treatment with the indicated antivirals compared to vehicle-treated, infected COs. Data show the mean ± SEM obtained from n = 5 organoids lysed independently. (**p < 0.01, versus HCMV-infected, vehicle-treated COs). (I) Immunoblotting showing the effects of antiviral treatment on the expression of different viral proteins in VR1814-infected COs. Samples were obtained by pooling n = 3 organoids per condition. Vinculin expression was analyzed as a loading control. Marker molecular weights in kDa are indicated on the left. V, virus-infected, vehicle-treated COs

Antiviral treatment protects cerebral organoids from the detrimental effects of HCMV infection

A key priority in ameliorating cCMV is not only blocking active viral replication but also preventing virus-induced damage in the developing brain. To test the effects of antiviral drugs on rescuing HCMV-induced defects, we infected COs with both strains of HCMV. Upon drug treatment, at 21 dpi we evaluated different parameters of early brain development, i.e., neural rosettes integrity, PPARγ expression, as well as organoid size. HCMV-infected COs treated with GCV, LTV, and OZ418 showed an improved organization of neural rosettes structure, compared to vehicle-treated, infected COs with both strains (Fig. 6A), suggesting a potential protective effect of these compounds on neural development. When we analyzed the effects on the growth attenuation of the COs infected with VR1814 strain, antiviral treatment showed an overall trend of rescue of organoid size compared to vehicle-treated infected COs, although statistical significance was obtained only for LTV-treated, infected COs (Fig. 6B). On the other hand, both GCV and LTV could significantly reduce the upregulation of PPARγ in COs infected with HCMV VR1814, while the same effect resulted significant only in LTV-treated COs infected with TB4-UL83-EYFP strain – although a trend of reduction was appreciable also for GCV and OZ418 (Fig. 6C). Altogether, these findings indicated that antiviral treatment could counteract HCMV-induced developmental brain damage, paving the way for the exploration of new neuroprotective therapeutic candidates for the treatment of cCMV and amelioration of associated clinical manifestations.

Fig. 6.

Antiviral treatment protects cerebral organoids from detrimental effects of HCMV infection. (A) Representative images of the effects of antiviral treatments on Nestin expression in VR1814- and TB4-UL83-EYFP-infected (MOI = 0.1) COs compared to vehicle-treated, infected COs. Viral replication is associated with loss of neural rosettes. The neural stem cell marker Nestin is restored upon antiviral treatment. Scale bar: 500 μm. (B) Quantification of the area of HCMV-infected COs upon the indicated treatment. Box plots showing the area of mock- and VR1814-infected COs (MOI = 0.1) measured for from n ≥ 10 organoids in either the vehicle-treated or compound-treated conditions, each normalized to their specific treated but uninfected condition and measured from n = 2 independent experiments. The horizontal line in the box plot indicates the median, the box denotes the IQR, and the whiskers beyond the box extend to the minimum and maximum of the data. Data were analyzed by a Kruskal-Wallis test followed by Dunn’s test. (*p < 0.05. versus vehicle-treated COs; ***p < 0.001, versus mock-infected COs). (C) Quantification of PPARγ expression in mock- and HCMV-infected COs upon antiviral treatment. Violin plots showing PPARγ signal in VR1814- and TB4-UL83-EYFP-infected COs treated with the indicated antivirals compared to vehicle-treated, mock-infected COs in different fields (n > 20) taken from n = 2 independent experiments. The dashed line represents the median, while the dotted lines represent the quartiles. Data were analyzed by a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. (*p < 0.05; ***p < 0.001; ****p < 0.0001, versus vehicle-treated, mock-infected COs)

Innate immunity and proinflammatory pathways activated in infected cerebral organoids are dampened by antiviral treatment

To investigate the effects of HCMV infection by both strains on the activation of innate immunity and proinflammatory pathways, we analyzed the expression of type I interferon (IFN) genes and of ISG15, as well as the induction of different inflammatory cytokines such as TNF-α, IL-6, IL-8, and CXCL-10. As reported in Fig. 7A, infection with both HCMV strains upregulated both IFN-α and IFN-β genes, but not ISG15. Regarding the upregulation of the cytokines, a significant induction of IL-6 and TNF-α genes was observed upon infection with both strains, while no significant differences were detected in IL-8, and CXCL-10 expression (Fig. 7A).

Fig. 7.

Innate immunity and proinflammatory pathways activated in infected cerebral organoids are dampened by antiviral treatment. (A) Bar graph showing the effects of HCMV infection (TB4-UL83-EYFP and VR1814 strains) of COs on the expression of innate immunity and proinflammatory markers, interferon alpha (IFNA), interferon beta 1 (IFNB1), interleukin 6 (IL6), interleukin 8 (IL8), tumor necrosis factor alpha (TNFα), C-X-C motif chemokine ligand 10 (CXCL10), and interferon-stimulated gene 15 (ISG15). mRNA levels are expressed as fold change calculated with the 2^−ΔΔCT method over GAPDH housekeeping gene in mock- or HCMV-infected COs. Data show the mean ± SEM of n ≥ 4 organoids from independent experiments performed in duplicate and were analyzed by a Mann-Whitney test. (*p < 0.05; versus mock-infected). (B) Bar graph showing the effects of antiviral treatment on HCMV VR1814-induced dysregulation of innate immunity and proinflammatory genes in infected COs. mRNA levels are expressed as fold change calculated with the 2^−ΔΔCT method. Data show the mean ± SEM of n ≥ 4 organoids from independent experiments performed in duplicate and were analyzed by a Kruskal-Wallis followed by Dunn’s test. (**p < 0.01; *p < 0.05; versus HCMV-infected, vehicle-treated COs)

We next evaluated the effects of antiviral treatment. We observed that LTV significantly reduced the upregulation of IFN-α, IFN-β, and IL-6, but did not affect TNF-α expression in COs infected with both strains (Fig. 7B). The same effects, albeit less marked, were observed for GCV (Fig. 7B). Altogether, these results indicated that HCMV infection in COs activates both IFN-mediated innate immunity and inflammatory cytokines, and these effects seem not strain-dependent. Antiviral treatment can dampen the activation of innate immunity-related pathways and the upregulation of IL-6.

Antiviral treatment blocks HCMV spread in COs when started after the establishment of infection

Finally, to further evaluate the translational potential of LTV and its efficacy after the establishment of infection, we infected COs with both TB4-UL83-EYFP and VR1814 strains at MOI = 0.1. At 7 dpi, when the infection was already established, we added either LTV or GCV and prolonged the treatment for additional 21 days. During the treatment, we evaluated the fluorescence signal in TB4-UL83-EYFP-infected-treated COs and titrated the released virus in VR1814-infected and treated COs. At 21 days post-treatment (corresponding to 28 dpi), we quantified the intracellular viral infectious progeny produced under these conditions for both viruses. As reported in Fig. 8A, a comparison of HCMV-infected, vehicle-treated COs versus HCMV-infected COs treated with the indicated antivirals at different dpi showed a decreasing number of EYFP⁺ cells over the course of the treatment, confirming the efficacy of the test compounds in blocking HCMV spread even after the infection is established. In addition, the antiviral treatment with both GCV and LTV completely blocked the production and release of infectious viral particles (Figs. 8B-C), indicating that antiviral treatment might be effective in blocking viral spread also in the case of a previously established infection.

Fig. 8.

Antiviral treatment blocks HCMV spread in COs when started after the establishment of infection. (A) Representative images of the progression of HCMV infection (TB4-UL83-EYFP strain, MOI = 0.1) in 30-days-old COs. Starting from 7 dpi, infected COs were treated with 50 µM GCV and 10 µM LTV for 21 days. The dashed line represents the timing when the treatments started (DTP, days post-treatment). Scale bar: 500 μm. (B) Violin plots showing the quantitative comparison of EYFP-derived fluorescence signal in HCMV-infected, vehicle-treated COs and in HCMV-infected COs treated with the indicated antivirals. Data represent the fluorescence signals of n = 7 organoids for each condition from n = 2 independent experiments. The dashed line represents the median, while the dotted lines represent the quartiles. Data was analyzed with a Kruskal-Wallis test followed by Dunnett’s multiple comparison test. (*p < 0.05, versus HCMV-infected, vehicle-treated COs). (C) Bar graphs showing the reduction at 21 DPT (corresponding to 28 dpi) in intracellular infectious viral progeny (expressed as PFU/ml) produced in COs infected with HCMV TB4-UL83-EYFP upon treatment with the indicated antivirals compared to vehicle-treated, infected COs. Data show the mean ± SEM obtained from n = 7 organoids lysed independently. (**p < 0.01, versus HCMV-infected, vehicle-treated COs). (D) Bar graphs showing the reduction at 21 DPT (corresponding to 28 dpi) in intracellular infectious viral progeny (expressed as PFU/ml) produced in COs infected with HCMV VR1814 upon treatment with the indicated antivirals compared to vehicle-treated, infected COs. Data show the mean ± SEM obtained from n = 7 organoids lysed independently. (*p < 0.05; versus HCMV-infected, vehicle-treated COs). (E) Points and connecting line showing the reduction in released infectious viral progeny (expressed as PFU/ml) in medium derived from HCMV VR1814-infected COs upon antiviral treatment compared to vehicle-treated, infected COs at different time points. Data show the mean ± SEM of n = 7 organoids for each condition from n = 2 independent experiments

Discussion

In this study, we employed a 2D and a 3D model of the human developing brain to investigate the neuropathogenesis induced by HCMV, a member of the TORCH pathogens group, and the effects of pharmacological treatment. Regarding the 2D platform, we used a model of dynamic neurogenesis in vitro [48]. To our knowledge, this model has never been adopted before for both investigating HCMV infection dynamics and evaluating neuroprotective effects of antiviral drugs. In our belief, it could represent a useful tool for future studies on other TORCH infections. So far, indeed, post-mitotic neurons have been mostly employed for HCMV infection [31, 32]. Quite different from that, the model used in our study provided instead a context of dynamic neurogenesis in which neural progenitors are exposed to the virus; consequently, the infection can be followed up to the stage of neurons, possibly mimicking what happens during the spread of virus in the embryonic developing nervous tissue. We found that productive HCMV infection of neural progenitors progresses through neurogenesis and leads to histopathological changes typical of this virus, such as cytomegaly, and significantly alters total neurites outgrowth. Antiviral treatment blocked active viral replication and in this 2D model, DAA (GCV and LTV) were more effective than OZ418, which likely targets a host cellular function [20]. This result deviates from what we previously obtained in NSCs, in which OZ418 demonstrated a remarkable antiviral effect and might be related to the antiviral activity of artemisinin derivatives toward vimentin, a cellular protein with a central role in neurogenesis [33, 58]. Regarding the neuroprotective effects of antiviral treatment in the 2D model, the ability to reduce virus-induced PPARγ upregulation, an effect associated with the inhibition of neurogenesis and detected in congenitally infected fetuses [36], was observed only for GCV. On the other hand, all the drugs failed to restore the expression of DCX and TUBB-3 in iNeu, confirming further that active viral replication might not be the only driver of viral neuropathogenesis in the context of cCMV, as was reported in previous studies by our group and others [33, 44].

We then shifted our investigation on COs, a 3D model that provides a more complete and faithful recapitulation of human developing brain in the first trimester of gestation. Previous studies with COs were very heterogeneous in terms of the stage of maturation related to the time of infection (ranging from the embryonic stage up to infection of 55-day-old organoids), virus strain, MOI, and time-points and protocols for post-infection analysis [40–47]. This is the reason why we initially followed the infection of embryoid bodies and determined that HCMV starts to replicate efficiently in COs only at approximately 15–20 days post-maturation. In contrast, two other viruses of the TORCH pathogens group, namely Zika virus and herpes simplex virus type 1, infect early-stage organoids and block their growth by causing cell death [43]. Moreover, most of the studies with HCMV-infected COs focused on the investigation of the changes in host gene expression induced by the virus [42, 44–46]. With a focus on the translational potential of the results of our study, we evaluated the antiviral and neuroprotective activity of drugs with different mechanisms of action and compared two HCMV strains with tropism for epithelial/endothelial cells at equal experimental conditions (i.e., equal MOI and organoid maturation stage). By comparing the two strains, we observed significant differences in the release of infectious viral particles (pointing out to a difference in spreading mechanism throughout the tissues), in the virus-induced disorganization of the organoid architecture, and in the attenuation of organoid growth. So far, only one study reported the size reduction of COs upon HCMV infection [42], while all the other studies did not. However, the comparison might be biased by different experimental conditions. In this study, COs at the same developmental age were exposed to an equal amount of the two viruses. The differences that we observed are intriguing, but future studies including a larger number of clinical HCMV isolates will be needed to establish whether inter-strain variability might correlate with different clinical outcomes of cCMV. Our model also has some limitations. Indeed, we used unguided COs, which are heterogeneous in terms of cell type representation [59]. This aspect, along with the absence of microglia might have influenced viral replication efficiency and spreading (since not all cell types are permissive to HCMV infection) and innate immunity/inflammation pathways activation. In the future, use of cortical organoids or, better, neuroimmune assembloids including both COs and microglia will allow the study of the interactions between fetal brain nervous and immune system in the context of cCMV and the effects of antiviral treatment. Moreover, we infected COs from 0 up to 30 days of maturation, a time frame reminiscent of the first trimester of gestation [57]. The effects of HCMV infection and antiviral treatment in late-stage organoids might differ. On the other hand, the probability of neurologic sequelae in cCMV is strongly related to the timing of the transplacental transmission, with the highest risk in the periconceptional period [60].

Treatment with GCV, LTV, and OZ418 remarkably blocked active replication of both HCMV strains in COs by reducing viral spreading, viral proteins expression, and infectious virus release (in VR1814-infected COs). So far, COs have been scarcely employed to evaluate therapeutic anti-HCMV strategies. In one study, treatment of HCMV-infected organoids with the viral kinase inhibitor maribavir did not reduce viral spread but had a moderate effect in restoring COs architecture [40]. Neutralizing antibodies that prevent viral entry into COs showed protective effects and restored organoids growth and cortical development [42]. However, a recent study reported that blocking viral entry in COs is not sufficient to stop HCMV-induced dysregulation of gene expression and that neurotrophic factors might be needed to maintain key developmental gene expression [47]. Here, we showed for the first time that antiviral treatment with DAA (particularly with LTV) has neuroprotective potential and restored the organization of neural rosette structures and of TUBB3-expressing neurons. Moreover, treatment with DAA led to an overall rescuing trend on organoid size observed along with the reduced upregulation of PPARγ signaling. This in turn is one of the few pathways that has been mechanistically linked to inhibition of neurogenesis and has been validated in congenitally infected fetal brain samples [36]. Importantly, we demonstrated that antiviral treatment with LTV might be effective also after the establishment of infection, a more realistic scenario that could occur during cCMV. During the revision of this manuscript, a paper reporting the evaluation of LTV and GCV in different neural models, including dorsal forebrain regionalized COs was published [61]. Unfortunately, it might be difficult to compare the results of this study with ours, since the experimental conditions differ in terms of MOI, drug concentrations, time points, and type of cerebral organoids. Nonetheless, also in this study, differences in the pathogenic potential of different HCMV strains were observed.

In conclusion, our study highlights both antiviral and neuroprotective potential of the available anti-HCMV drugs, particularly of letermovir, in models recapitulating the human brain during the first months of development. We demonstrated that blocking active viral replication may be a feasible therapeutic strategy and represents a promising approach not only to reduce virus-induced damage in target cells but also to protect tissue cytoarchitecture, suppress viral spread, and reduce innate immune activation and inflammation. These results pave the way to further evaluation of letermovir as a candidate drug for amelioration of congenital CMV-associated clinical manifestations.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors’ contributions

Conceptualization: BM, AL, MT; methodology: BM, AP, EP, ER, MT; formal analysis: BM, MT; investigation: BM, AP, EP, ER; resources: RB, GP, AL, MT; writing – original draft: BM, MT; writing – review and editing: BM, RB, GP, AL, MT; visualization: BM, AL, MT; supervision: AL, MT.

Funding

This study was supported by University of Padua, Italy (PRID 2022 to M.T.); by Ministero dell’Università e della Ricerca, Italy (PRIN 2022 PNRR - cod. P20222YKP8 and PRIN 2022 - cod. 20223RYYFC to A.L.), by Associazione Italiana per la Ricerca sul Cancro, AIRC, Italy (grant IG 2021 - ID. 25899) to A.L.; by EU funding within the NextGenerationEU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT) to A.L.; and by The National Institute of Allergy and Infectious Diseases (R21AI164033) to R.A.B.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request.

Declarations

Competing interest

The authors declare no competing interests.