Differential Efficacy of Novel Antiviral Substances in 3D and Monolayer Cell Culture.

Repurposing of approved drugs that target host functions also important for virus replication promises to overcome the shortage of antiviral therapeutics. Mostly, virus biology including initial screening of antivirals is studied in conventional monolayer cells. The biology of these cells differs considerably from infected tissues. 3D culture models with characteristics of human tissues may reflect more realistically the in vivo events during infection. We screened first, second, and third generation epidermal growth factor receptor (EGFR)-inhibitors with different modes of action and the EGFR-blocking monoclonal antibody cetuximab in a 3D cell culture infection model with primary human keratinocytes and cowpox virus (CPXV) for antiviral activity. Antiviral activity of erlotinib and osimertinib was nearly unaffected by the cultivation method similar to the virus-directed antivirals tecovirimat and cidofovir. In contrast, the host-directed inhibitors afatinib and cetuximab were approx. 100-fold more efficient against CPXV in the 3D infection model, similar to previous results with gefitinib. In summary, inhibition of EGFR-signaling downregulates virus replication comparable to established virus-directed antivirals. However, in contrast to virus-directed inhibitors, in vitro efficacy of host-directed antivirals might be seriously affected by cell cultivation. Results obtained for afatinib and cetuximab suggest that screening of such drugs in standard monolayer culture might underestimate their potential as antivirals.

Simplified Bioprinting-Based 3D Cell Culture Infection Models for Virus Detection.

Studies of virus-host interactions in vitro may be hindered by biological characteristics of conventional monolayer cell cultures that differ from in vivo infection. Three-dimensional (3D) cell cultures show more in vivo-like characteristics and may represent a promising alternative for characterisation of infections. In this study, we established easy-to-handle cell culture platforms based on bioprinted 3D matrices for virus detection and characterisation. Different cell types were cultivated on these matrices and characterised for tissue-like growth characteristics regarding cell morphology and polarisation. Cells developed an in vivo-like morphology and long-term cultivation was possible on the matrices. Cell cultures were infected with viruses which differed in host range, tissue tropism, cytopathogenicity, and genomic organisation and virus morphology. Infections were characterised on molecular and imaging level. The transparent matrix substance allowed easy optical monitoring of cells and infection even via live-cell microscopy. In conclusion, we established an enhanced, standardised, easy-to-handle bioprinted 3D-cell culture system. The infection models are suitable for sensitive monitoring and characterisation of virus-host interactions and replication of different viruses under physiologically relevant conditions. Individual cell culture models can further be combined to a multicellular array. This generates a potent diagnostic tool for propagation and characterisation of viruses from diagnostic samples.

A novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells.

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non-small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix-based cell culture model and compared to the respective monolayer culture. 3D-cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell-cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100-fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy-to-handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo-like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments.

Inhibition of poxvirus spreading by the anti-tumor drug Gefitinib (Iressa).

The threat of smallpox virus as a bioterrorist weapon is raising international concerns again since the anthrax attacks in the USA in 2001. The medical readiness of treating patients suffering from such infections is a prerequisite of an effective civil defense system. Currently the only therapeutic option for the treatment of poxvirus infections relies on the virostatic nulceosid analog cidofovir, although severe side effects and drug resistant strains have been described. A growing understanding of poxvirus pathogenesis raises the possibility to explore other appropriate targets involved in the viral replication cycle. Poxvirus encoded growth factors such as the Vaccinia Growth Factor (VGF) stimulate host cells via the Epidermal Growth Factor Receptor (EGFR) and thereby facilitate viral spreading. In this study we could visualize for the first time the paracrine priming of uninfected cells for subsequent infection by orthopoxviruses directly linked to EGFR phosphorylation. Since EGFR is a well known target for anti-tumor therapy small molecules for inhibition of its tyrosine kinase (TK) activity are readily available and clinically evaluated. In this study we analyzed three different EGFR receptor tyrosine kinase inhibitors for inhibition of orthopoxvirus infection in epithelial cells. The inhibitor shown to be most effective was Gefitinib (Iressa) which is already approved as a drug for anti-tumor medication in the USA and in Europe. Thus Gefitnib may provide a new therapeutic option for single or combination therapy of acute poxvirus infections, acting on a cellular target and thus reducing the risk of viral resistance to treatment.