Limitations of acyclovir and identification of potent HSV antivirals using 3D bioprinted human skin equivalents

Abstract

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 manifests in the skin and mucosal epithelium. Here, we found acyclovir significantly less effective in skin-derived keratinocytes than donor-matched fibroblasts. To recapitulate in vivo tissue architecture, we 3D bioprinted human skin equivalents (HSE) in a 96-well plate format amenable for antiviral screening and preclinical testing. We screened a library of 738 compounds with broad targets and mechanisms of action and identified potent antivirals, including 23 known or experimental HSV treatments, validating the translational relevance of our assay. Unlike acyclovir, antivirals against HSV helicase/primase or host replication pathways displayed similar potency across cell types and donor sources in 2D and 3D models. Our 3D bioprinted platform allowed for integrating patient-derived cells and incorporating genetic variability early in drug development. The reduced potency in keratinocytes helps explain the limited benefit acyclovir and its congeners play in reducing sexual transmission. These data indicate that the 3D bioprinted HSE assay platform provides a more physiologically relevant approach to identifying potential antivirals for HSV.

Fig. 1.. Acyclovir potency in donor-derived primary keratinocytes and fibroblasts and in Vero cells.

(A) Punch biopsies from six donors were collected and dissociated by enzymatic and mechanical processes. Vero cells, keratinocytes (B), and fibroblasts (C) were infected with GFP-expressing HSV-1, and live cell images were taken every two hours. (D) Keratinocytes, fibroblasts, and Vero cells were infected with GFP-expressing HSV-1 and then treated with acyclovir at the specified doses. Representative live cell images were taken at the peak of GFP expression. (Scale bar 500μm). (E) Dose-response curve of acyclovir in keratinocyte cultures compared to Vero cells (grey line). (F) Dose-response curve of acyclovir in fibroblast cultures compared to Vero cells (grey line). (G) IC₅₀ values for each donor in each cell type (*** P < 0.001, * P < 0.05, linear mixed model). Data represents averages of six donors; keratinocytes (N = 5 per donor), fibroblasts (N = 3 per donor), and Vero (N = 5) respectively. Error bars represent standard deviation.

Fig. 2.. 3D bioprinted HSE assay development and validation

(A) Dermis equivalents were 3D printed onto the apical side of transwell inserts using the RegenHU 3D Discovery bioprinter (image courtesy of RegenHU). Keratinocytes were pipetted onto the apical surface of the dermis. In the submerged model, the tissues were infected at the apical surface. In the ALI model, tissues were brought to ALI and then infected at the basolateral surface (created with BioRender.com). (B) H&E and IHC images of differentiated ALI tissues. K10 (cyan) and K14 (red) identify keratinocytes in the suprabasal and basal layer of the epidermis respectively (scale bar 50μm) (C) Submerged tissues were infected at various MOI and then imaged at specified times. Fibroblasts express tdTomato (orange) while infected cells express GFP (green) (scale bar 1mm). (D) GFP and tdTomato signal at each MOI and timepoint (N = 6, *** P < 0.001, **** P < 0.0001 by ordinary one-way ANOVA). (E) Maximum projection of infected tissues from the top (column 1) or side (column 2) view. H&E (column 3) and IHC (column 4) staining of infected submerged or ALI models (scale bar 1mm (column 1) or 50μm (column 2, 3, 4).

Fig. 3.. Primary screen of compound library in 3D bioprinted assay platform

(A) Plate layout for compound screen. (B) Representative images from primary screen of submerged and ALI models. Controls are highlighted in boxes with colors corresponding to part (A). (C) Wilcoxon two-sample analysis of GFP intensity (left) and tdTomato intensity (right) of the median of the six negative control wells (HSV-1 + DMSO) across two replicate plates (N =16, * P < 0.05). Bars represent average with error bars as standard deviation. (D) Correlation plots of the two replicates for the primary screen of compounds. The red shaded box represents compounds that were selected as hits for re-testing if they reduced GFP expression (%Activity) by at least 15% in either replicate or model (left submerged, right ALI). Acyclovir was included in the collection and denoted by a cyan circle.

Fig. 4.. Dose response of candidate antivirals in 3D bioprinted assay platform

(A) Correlation plot of Max %Activity (maximum reduction in GFP) vs. Max %Viability (maximum reduction in tdTomato) of 106 ‘hits’ tested in dose response. Top candidate antivirals (50% or greater reduction in GFP) that did not kill over 50% of tdTomato transduced fibroblasts are identified by the red shaded box. (B) Schematic illustrating %Activity dose response profiles of different Concentration-Response Curve classes (CRC). (C) Venn diagram showing divergent and coinciding targets for 41 top candidate antivirals in both submerged and ALI models. (D) Schematic of compounds selection from 738 compounds in the primary screen to 106 ‘hits’ tested in dose-response to 41 selected candidates and 11 top candidates selected to move forward. Of the 41 selected candidates, 23 are current or experimental HSV treatments. (E) Dose response curves of candidate antivirals in “ciclovir” family, known to treat HSV-1, in submerged and ALI models (N = 1).

Fig. 5.. Top candidate antiviral potency, efficacy, and cytotoxicity in 3D bioprinted HSE.

(A) Average dose-response curves for the 11 top candidate antivirals for submerged (blue) and ALI (green) models. (B) IC₅₀ values for each top candidate antiviral compared between submerged (blue) and ALI (green) models. (C) Maximum inhibition for each top candidate antiviral compared between submerged (blue) and ALI (green) models. Statistical significance was determined by linear mixed model (*** P < 0.001, ** P < 0.01, * P < 0.05) for (B) and (C). (D) CC₅₀ dose response curves for 11 top candidate antivirals. All data is plotted as the average of three replicates (N = 3), error bars represent standard deviation.

Fig. 6.. Candidate antiviral potency, efficacy, and cytotoxicity in donor-derived primary keratinocyte and fibroblast monocultures.

(A) Dose-response curves for the 11 top candidate antivirals compared to acyclovir (ACV) (keratinocytes blue and grey, respectively; fibroblasts green and black, respectively). (B) IC₅₀ values for each top candidate antiviral compared between keratinocytes (blue) and fibroblasts (green). Striped bars (FMP, VRD) indicate candidate antivirals that failed to reduce GFP expression by at least 50% consistently. (C) Maximum inhibition for each top candidate antiviral is compared between keratinocytes (blue) and fibroblasts (green). Statistical significance was determined by linear mixed model (*** P < 0.001, ** P < 0.01, * P < 0.05) for (B) and (C). (D) CC₅₀ dose-response curves for all twelve candidate antivirals compared to their respective IC50 to IC80 dose ranges. Keratinocyte data is from 20HPI (grey), while fibroblast data is from 48HPI (red). All data is plotted as the average of three replicates in three distinct donors (N = 9), error bars represent standard deviation.

Fig 7:. Comparison of 3D and 2D models in testing novel antiviral candidates.

(A) Pairwise comparisons of IC₅₀ values for candidate antivirals in the four models tested. (B) Pairwise comparisons of CC₅₀ values for candidate antivirals in the four models tested. (C) Fold change was determined by dividing the IC₅₀ value of each candidate antiviral in keratinocytes by the IC₅₀ of the same candidate antiviral in submerged models. (D) Fold change was determined by dividing the IC₅₀ value of each candidate antiviral in fibroblasts by the IC₅₀ of the same candidate antiviral in ALI models. (E) IC₅₀ values for each candidate antiviral were pooled (keratinocytes and fibroblasts, submerged and ALI), then IC₅₀ values for candidate antivirals in 2D were divided by IC₅₀ values in 3D. (C) (D) (E) Green bars indicate candidate antivirals that were more potent in 3D, while blue bars indicate candidate antivirals that are potent in 2D. Data for 2D (N = 9) and 3D (N = 3) is reported as averages, error bars are standard deviation.

Fig 8:. Generation of 3D bioprinted HSE using adult donor-derived keratinocytes

H&E and IHC staining of commercial neonatal or adult-derived bioprinted HSE in submerged (A) and ALI (B) models. Comparison of calculated IC50 in submerged (C) and ALI (D) models for top candidate antivirals. Neonatal data is plotted as an average of three replicates (N = 3), error bars are standard deviation. Adult data has an N = 1.