Although you’re probably not a virologist, chances are you know a thing or two about herpes simplex virus (HSV), which is estimated to infect roughly two-thirds of the world’s population and causes oral and genital herpes. If you’re lucky, you might have asymptomatic HSV and not even realize it. If you’re unlucky, you might have what is often described as a debilitating disease, with frequent, painful flare-ups that impact quality of life and may make you more susceptible to acquiring other viral infections like HIV. While several antivirals exist to control HSV infection, these medications are not cures; after infecting you, HSV quietly gains access to the innervating nerve network and stays as an episome in sensory neurons, where it can evade both antivirals and your immune system indefinitely (‘herpes is forever’). Not only are these antivirals not effective against this latent HSV reservoir, but they also aren’t a perfect treatment for flare-ups, either: they must be taken consistently at large doses, often fail to adequately control symptoms, and become ineffective in an increasing number of patients who are developing resistance. Clearly, we need to do better.
According to Dr. Jia Zhu, an associate professor in the Fred Hutch Vaccine and Infectious Disease Division who has dedicated her career to studying HSV, the key to designing better HSV treatments is a better understanding of what got us to our current armory of antivirals. “While HSV exists in the latent state in sensory neurons, our previous work has shown that innervating nerve endings can release virus from these neurons directly to epithelial cells. Productive viral replication (that can be targeted by antivirals) primarily occurs in the basal layer of epithelial cells,” begins Zhu. This model of HSV partitioning between latent, neuron-residing and replicating, epithelial-residing populations explains why the sores and ulcers characteristic of herpes tend to appear in barrier epithelial tissues like the oral mucosa and genital skin. “While this is a complex process facilitated by many different cell types that collectively structure your epidermal tissue” continues Zhu, “the antivirals we use to treat HSV today were developed using in vitro culture of Vero cells and fibroblasts. It’s perhaps not surprising that these antivirals show sub-optimal performance in HSV infections in patients.”
As ever, what we know is fundamentally constrained by the models we study. In the case of HSV, the Zhu lab is on a mission to modernize our models to better understand how HSV antivirals work and how to make them better. Their recent study was spearheaded by postdoctoral scholars Dr. Ian Hayman of the Zhu lab and Dr. Tori Ellison of the Ferrer lab at the National Center for Advancing Translational Sciences, who are experts in biofabrication using 3D-printing technologies. By using a specialized 3D printer to deposit fibroblasts into specially-designed culture vessels, adding keratinocytes on top of this layer of fibroblasts, and incubating the cells in several different media formulations, the group was able to produce what they term ‘3D-bioprinted human skin equivalents’—small organoids that can be manufactured en masse and closely resemble human skin, with dermal tissue (consisting mainly of fibroblasts) and well-stratified layers of epidermal tissue (consisting mainly of keratinocytes).
To further simulate different modes of HSV infection, the team devised two variants of this organoid system: a submerged infection model, in which epidermal cells grow in a monolayer submerged in culture media to which HSV is added (this would simulate initial infection of HSV through a break in the skin, for example), and an air-liquid interface (ALI) model, in which the organoids are repositioned to the surface of the culture media, which induces stratification in the air-exposed epidermal tissue—in this ALI model, HSV was added to the media side of the tissue (i.e. from ‘underneath the epidermis’), better mimicking an HSV flare-up originating from latent reservoirs. This organoid system was used to screen the potency of 738 medicinal compounds—including both novel and FDA-approved medications—against experimental HSV infection. By using a recombinant HSV that expressed green fluorescent protein and fibroblasts with a red fluorescent protein, the group was able to use high-content fluorescent microscopy to track both the on-target effects of these drugs (i.e. how well do they eliminate the green HSV signal?) and their off-target effects on host tissue (i.e. how much do they reduce the red fibroblast signal?).
Right away, the team was surprised by what their screens found. Not only did they uncover nearly 20 antiviral compounds that potently suppressed HSV infection with minimal host-cell toxicity, but they also found striking cell type-specific differences in the potency of novel and existing HSV antivirals. Among these was a drug called acyclovir—the current standard of care for treating HSV infection. “We originally used acyclovir as a positive control to screen novel candidate antivirals,” notes Hayman, “but we were shocked to find that acyclovir was at least an order of magnitude less effective in the submerged model (in which HSV mainly infected keratinocytes) than in the ALI model (in which HSV mainly infected fibroblasts). We confirmed these results using patient-derived monolayer cultures of fibroblasts and keratinocytes and showed that the doses of acyclovir required to effectively suppress HSV in keratinocytes were higher than the maximal serum concentrations of acyclovir reported in patients taking the drug.” Dr. Zhu adds, “considering that keratinocytes are the major skin cell type in which HSV replicates in patients, the fact that acyclovir isn’t all that potent at suppressing HSV in this cell type may explain why it isn’t always effective in treating HSV flare-ups!”
Looking forward, the team is excited to follow up on some of their top antiviral candidates and keep developing their organoid models, whose utility in deciphering cell type-specific differences in antiviral effectiveness is now clear. “We’re particularly excited at the prospect of using patient-derived cells to 3D-print the next generation of these skin organoids,” note Hayman and Zhu, “because this would allow us to incorporate patient-specific biology into the drug discovery pipeline, and ensure that the drugs we are spending time and money to test are actually showing effectiveness in the cellular environments they will eventually be used in.”
The spotlighted work was funded by the National Institutes of Health and the Cure Acceleration Network Program.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium members Drs. Youyi Fong, Anna Wald, and Lawrence Corey contributed to this study.
Ellison, S. T., Hayman, I., Derr, K., Derr, P., Frebert, S., Itkin, Z., Shen, M., Jones, A., Olson, W., Corey, L., Wald, A., Johnston, C., Fong, Y., Ferrer, M., & Zhu, J. (2024). Identification of potent HSV antivirals using 3D bioprinted human skin equivalents.
Science Spotlight writer David Sokolov is a graduate student in the Sullivan Lab at the Fred Hutch. He studies how cancer cells modify their metabolism to facilitate rapid proliferation and accommodate tumor-driving mitochondrial defects. He's originally from the east coast and has bachelors' and masters' degrees from West Virginia University. Outside of the lab, you'll find him enjoying the outdoors, playing music, or raising composting worms in his front yard.
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