Identification of potent HSV antivirals using 3D bioprinted human skin equivalents.

Herpes simplex virus (HSV) infection has worldwide public health concerns and lifelong medical impacts. The standard therapy, acyclovir, has limited efficacy in preventing HSV subclinical virus shedding, and drug resistance occurs in immunocompromised patients, highlighting the need for novel therapeutics. HSV infection manifests in the skin epidermal layer, but current drug discovery utilizes Vero cells and fibroblasts monolayer cultures, capturing neither relevance nor tissue environment. To bridge the gap, we established 3D bioprinted human skin equivalents that recapitulate skin architecture in a 96-well plate format amenable for antiviral screening and preclinical testing. Screening a library of 738 compounds with broad targets and mechanisms of action, we identified potent antivirals, including most of the known anti-HSV compounds, validating the translational relevance of our assay. Acyclovir was dramatically less potent for inhibiting HSV in keratinocytes compared to donor-matched fibroblasts. In contrast, antivirals against HSV helicase/primase or host replication pathways displayed similar potency across cell types and donor sources in 2D and 3D models. The reduced potency of acyclovir in keratinocytes, the primary cell type encountered by HSV reactivation, helps explain the limited benefit acyclovir and its congeners play in reducing sexual transmission. Finally, we demonstrated that our 3D bioprinted skin platform can integrate patient-derived cells, facilitating the incorporation of variable genetic backgrounds early into drug testing. Thus, these data indicate that the 3D bioprinted human skin equivalent assay platform provides a more physiologically relevant approach to identifying potential antivirals for HSV-directed drug development.