An in vitro herpes infection model for new antiviral therapies
Fraunhofer Institute for Interfacial Engineering and Biotechnology
Infections with herpes simplex virus (HSV) cause the most common skin diseases Herpes simplex virus (HSV) infections are among the most common disorders of the skin. More than 90 percent of the total world population is infected with HSV. The most common manifestation of herpes simplex infection is herpes labialis (cold sores), which is usually caused by virus type 1 (HSV-1). In addition to the characteristic skin lesions, HSV can also cause serious diseases in other organs, such as the cornea (herpes corneae) and the central nervous system (herpes encephalitis, herpes meningitis), which can be fatal. To date, there are no effective cures for the herpes infection. Antivirals, such as nucleoside analogs like acyclovir or its derivatives, are the most often used treatment for HSV. This treatment only alleviates the symptoms and shortens the time of infection, but it does not prevent the reactivation of the virus.
To date, a physiologically adequate in vitro model of the HSV-1 infection has not been established yet to be established. Consequently, drug development and studies of infection mechanisms are still performed using animal models. Therefore, the aim of the Fraunhofer IGB is to establish a 3D herpes infection model which accurately reproduces the in vivo situation.
Challenge of HSV latency
- Figure 2: Infection model during the airlift cultivation.
An important characteristic of all herpes viruses is that it persists in the human body after the treatment of acute symptoms from the first infection. The virus then goes into latency from which it can be reactivated by various factors. After the primary infection of the or skin epithelium, HSV migrates into the cell body of the sensory neurons that innervate the infected region. HSV enters the associated ganglia over the axons of nerve cells and becomes latent within the Ganglion [1, 2]. During the latent phase, there is no virus replication. The viral DNA persists undetected by the immune system of the host, as a circular episome in the nucleus of the ganglion . In order to simulate a herpes infection in vitro, a latent with HSV infected neuronal component must be integrated, which ensures that this latency can be formed. Previously described 3D infection models lack this crucial neuronal latency-forming component.
Establishment of a HSV-1 infection model
For the establishment of a functional HSV-1 infection model, the Fraunhofer IGB has extended its patented and accredited human 3D skin equivalent with the neuronal cell line PC12. The PC12 cells were previously infected with the herpes simplex virus type 1 strain and were integrated into the collagen matrix dermal layer of the skin model (Fig. 1). Additionally, the neural cells were differentiated with NGF (nerve growth factor) and cultured over an extended period. Using a cell-based TCID50 assay and PCR analysis, viral DNA and cell latency was verified and extracellular virus activity was not found.
Infection model with latently infected neuronal cells
- Figure 3: A: Structure of the 3D skin model with the neuronal cell line PC12 (H&F staining), B: PC12 staining, C: Isotype control.
In previous studies, the Fraunhofer IGB has demonstrated the successful development of a functional in vitro HSV-1 infection model. For the first time, we were able to integrate a neural component in the form of a cell line.
Figure 3A shows the successful integration of latently infected PC12 cells within the 3D skin equivalent with HSV-1 neuronal cells. The detection of neuronal PC12 cells was performed using a specific antibody (Fig. 3B). Another specific immunohistochemical examination of skin sections showed no virus activity. First promising results indicate that the virus can be reactivated specifically by UV-B radiation. Through this targeted and specific reactivation of infected herpes simplex virus in the infection model, it is possible to accurately simulate the in vivo environment.
The established and patented in vitro HSV-1 infection model fulfills an important requirement as a test system for biomedical research in the areas of toxicology, immunology and pharmacology.
However, we are not able to test substances under standardized conditions so far, caused by the instable infection and reactivation process of the system. Thus, the infection conditions have to be further refined in order to achieve a reproducible reactivation within the 3D HSV-1 infection model.
Nevertheless, this in vitro 3D infection model which includes quiescent infected neuronal cells can provide a basis either to investigate the molecular mechanisms involved in establishing, maintaining, and controlling latency or to test pharmaceutical products.
In future, we will extend the 3D infection model with an immune component designed to better represent the physiological environment of the native skin. With the integration of PRRs (pattern recognition receptors), we want to investigate the role of the immune receptors in an active HSV-1 infection more closely.
 Lycke, E.; Hamark, B.; Johansson, M.; Krotochwil, A.; Lycke, J.; Svennerholm, B. (1988) Herpes simplex virus infection of the human sensory neuron. An electron microscopy study. Arch Virol. 101(1-2): 87-104
 Topp, K. S.; Meade, L. B.; LaVail, J. H. (1994) Microtubule polarity in the peripheral processes of trigeminal ganglion cells: relevance for the retrograde transport of herpes simplex virus. J Neurosci. 14(1): 318-325
 Decman, V.; Freeman, M. L., Kinchington, P. R.; Hendricks,R. L. (2005) Immune control of HSV-1 latency. Viral Immunol. 18(3): 466-473