A Preclinical Model for Studying Herpes Simplex Virus Infection

Abstract

Herpes simplex virus (HSV) infections can cause considerable morbidity. Currently, nucleoside analogues such as acyclovir are widely used for treatment. However, HSV infections resistant to these drugs are a clinical problem among immunocompromised patients. To provide more efficient therapy and to counteract resistance, a different class of antiviral compounds has been developed. Pritelivir, a helicase primase inhibitor, represents a promising candidate for improved therapy. Here, we established an HSV-1 infection model on microneedle-pretreated human skin ex vivo. We identified HSV-1-specific histological changes (e.g., cytopathic effects, multinucleated giant cells), down-regulation of nectin-1, nuclear translocation of NF-κB (p65), interferon regulatory factor 3 (IRF3), and signaling of the IFN-inducible protein MxA. Accordingly, this model was used to test the potency of pritelivir compared with the standard drug acyclovir. We discovered that both drugs had a comparable efficacy for inhibiting HSV-1 replication, suggesting that pritelivir could be an alternative therapeutic agent for patients infected with acyclovir-resistant strains. To our knowledge, we present a previously unreported ex vivo HSV-1 infection model with abdominal human skin to test antiviral drugs, thus bridging the gap between in vitro and in vivo drug screening and providing a valuable preclinical platform for HSV research.

Figure 1. Efficient HSV-1 infection in human skin explants.

(a) The upper panel shows hematoxylin and eosin staining of paraffin sections obtained from microneedle-pretreated, 4 day–cultured untreated skin (left) and HSV-1–treated 4 day-cultured skin (HSV-1 either applied into medium [middle] or topically [left]). Scale bars = 50 μm. The lower panel illustrates bigger magnifications (black box). Arrows denote multinucleated giant cells. Necroptotic changes are indicated with an arrowhead. Scale bars = 15 μm. (b) Immunohistochemistry staining of untreated (left) and HSV-1–treated skin (middle: infected area marked by arrows, uninfected area marked by an arrowhead) with an anti-HSV-1 serum. Data are representative from six donors. HSV-1–infected patient skin (right) was used as a positive control. Scale bars = 50 μm. (c) HSV-1 copies/biopsy sample in untreated (i.e., uninfected) and HSV-1–treated groups were analyzed using RT-PCR (left). Results are expressed as mean ± standard error of the mean from nine independent experiments, and donors and were analyzed with a Student t test (Wilcoxon matched-pairs signed rank test). **P < 0.01. HSV-1 replication kinetics in UT and HSV-1–treated skin were examined by measuring copies/biopsy sample with RT-PCR at indicated time points (right). Shown is one representative donor out of two with similar results. (d) Representative Western blot shows expression of HSV-1–specific glyocoproteins in HSV-1–treated skin but not in untreated skin using an anti-HSV-1 serum (n = 3). Ponceau S staining was included as loading control. HSV, herpes simplex virus; RT-PCR, real-time PCR; UT, untreated.

Figure 2. HSV-1 successfully takes over the cellular system in skin cells.

(a) Immunofluorescence double labeling shows nectin-1⁺HSV-1^– epidermal cells in untreated, 4-day–cultured skin. Upon HSV-1 treatment, nectin-1 expression is down-regulated in infected HSV-1⁺ epidermal areas only (inserts). Nuclear translocation of ICP8 and p65 appears in HSV-1–infected skin compared with untreated skin. Representative images with Hoechst nuclear staining are shown from three donors. (b) Immunofluorescence labeling indicates IRF-3 expression and translocation to the nucleus in HSV-1⁺ epidermal cells in HSV-1–treated but not untreated skin at day 4 of culture. No specific cytoplasmic IRF3 staining of any skin cells is observed in untreated skin. A representative merged image with Hoechst nuclear staining is shown from three donors. (c) Immunofluorescence double labeling shows MxA⁺HSV-1⁺ cells in the epidermis and MxA⁺HSV-1⁻ cells in the dermis underneath the HSV-1–infected epidermal region, whereas the uninfected skin is negative. A representative merged image with Hoechst nuclear staining is shown from three donors. Insert shows single labeling of MxA-positive cells. (d) Immunohistochemical staining of 4-day–cultured HSV-1–treated and untreated skin paraffin sections show a similar MxA protein expression pattern (red-brown) compared with immunofluorescence. The insert indicates MxA⁺ cells in the dermis. Shown is one representative staining out of three independent experiments. Scale bars = 50 μm. Scale bars in inserts = 10 μm. HSV, herpes simplex virus.

Figure 3. Epidermal keratinocytes are a primary portal for entry and spread of HSV-1 infection.

(a) Immunofluorescence double labeling shows a few MxA⁺S100⁺ cells (arrowhead), but most MxA⁺ dermal cells do not express this marker (arrow). All MxA⁺ cells express vimentin in HSV-1–treated paraffin sections. Inserts represent higher magnification of indicated staining. (b, c) Immunofluorescence double labeling indicates that HSV-1⁺ cells co-express keratin 5 but not CD207 (arrowhead). HSV-1⁺ cells do not express CD68, CD31, and tryptase. Representative merged images with Hoechst nuclear staining are shown from three donors. Scale bars = 50 μm; scale bars in inserts = 10 μm. HSV, herpes simplex virus.

Figure 4. Pritelivir inhibits HSV-1 replication in human skin.

(a) H&E-stained skin sections show typical HSV-1–specific changes after 4 days of HSV-1 treatment (left), whereas no morphological changes are observed in the presence of pritelivir (middle) or acyclovir (right). (b) Immunohistochemical staining of paraffin sections with an anti HSV-1 serum shows a striking inhibition of HSV-1 infection with pritelivir (middle) and acyclovir (right) compared with the HSV-1–treated group (left). Shown is one representative from six donors. Scale bars = 50 μm. (c) Representative Western blot shows expression of typical HSV-1–specific glyoproteins in HSV-1–infected skin but not in untreated or drug-treated (pritelivir: 3.3 μmol/L, acyclovir: 43.6 μmol/L) skin when analyzed at day 4 of culture. Ponceau S staining is included as loading control. (d) HSV-1 copies/biopsy sample of indicated groups at day 4 of culture were analyzed by RT-PCR. Results are expressed as mean ± standard error of the mean from six to nine independent experiments and donors. *P < 0.01, **P < 0.001. + indicates HSV-1 treated, and − indicates untreated. (e) Skin was infected for 4 days and then shifted to indicated treatment for another 3 days with concentrations as indicated in (Figure 4d). Viral copies were determined with RT-PCR, and percent HSV-1 inhibition is indicated. Results expressed are mean ± standard error of the mean from three independent experiments with three different donors. H&E, hematoxylin and eosin; HSV, herpes simplex virus; RT-PCR, real-time PCR.