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ARBOVIRUS SKIN INFECTION MODELLING FOR MORE ACCURATE VIRAL PATHOGENESIS

Periodic Reporting for period 1 - VirIVITES (ARBOVIRUS SKIN INFECTION MODELLING FOR MORE ACCURATE VIRAL PATHOGENESIS)

Reporting period: 2020-10-01 to 2022-09-30

Arthropods, such as mosquitoes, are responsible for the transmission of viruses (arboviruses) to humans, while blood feeding on the skin. These viruses, causing more than a 100 million of symptomatic infections per year, are accountable for over 40 thousand deaths each year worldwide. The ill effects associated to these diseases, are a burden for the patients and for the economy of the affected countries. Furthermore, the geographical distribution of the vectors, and de facto of their associated diseases, is changing owing to convergence of multiple factors including globalization and global warming. Despite advances in public health awareness, little medical advancements have been made. One reason for such slow biomedical progress is the lack of adequate research models. In that sense, one alternative could be the use of organoids. Defined as self-organizing tridimensional cell cultures, organoids have already proven to be useful in the study of human diseases including viral infection. However, their use to study arbovirus skin infection had never been achieved so far.

The overall aim of this research project was to establish an experimental workflow demonstrating that human skin organoids could be used to study arboviruses infection. The first step and aim consisted into the in vitro generation of reconstructed human skins and their characterisation. Once this step validated, the second aim was to determine the experimental conditions in order to have infected mosquitoes probing the engineered tissues. Finally, the last aim was to confirm and explore the infection of the tissues in order to validate the possibility of using skin organoids to study arbovirus cutaneous infection.

At the end of the period allocated to this project, we have demonstrated that human skin organoids can be use in research to explore cutaneous infection by arboviruses delivered through the bite of mosquitoes. We put together an experimental workflow including the generation of human skin equivalents, the infection of mosquitoes with ZIKA and Sindbis viruses and the productive infection of the tissues exposed to the infected mosquitoes. This new research model opens unprecedented venues in the exploration of pathophysiological mechanisms involved in arbovirus skin infection in humans and to test new therapeutic approaches to counter these infections.
Before the beginning of this project, the host laboratory had acquired the know-how to generate human skin equivalents in vitro comprising fibroblasts, sensory neurons and keratinocytes. To further enrich these tissues, we generated macrophages using human blood monocytes and incorporated these cells to the engineered skins. This latter cell addition permitted to implement a first immune defense within the tissue and to unlock possibility to explore a potential role of macrophages in the progression of the infection. Characterization of the obtained tissues by immunofluorescence staining demonstrated expected spatial self-organization of the cells.
Recent studies have demonstrated that many proteins contain in the mosquito saliva favorize the dissemination of the viruses. It was therefore important for us to integrate mosquitoes in the process of our skin organoid infection. We obtained robust infection of mosquitoes with ZIKA virus by micro-injecting the viral particles into these vectors. More than 80% of the infected mosquitoes were able to excrete the viruses through their saliva, validating their infectiveness. Once in contact with the organoids, mosquitoes exhibited an exploratory behaviour, including multiple probings of the tissues. To explore whether these mosquito bites resulted in tissue infection, we aimed to detect the presence of the virus both within the tissues and in the culture supernatants. Through multiple experimental approaches, including quantitative PCR and immunofluorescence, we confirmed a productive infection of the tissues over time. Finally, we used another arbovirus (Sindbis virus) and demonstrated that productive infections of the skin organoids were also achieved in that condition.

To diffuse these ground-breaking results to the scientific community, we are currently preparing a manuscript recapitulating all our major findings and will submit it to a prestigious scientific journal for publication. In addition, I have already presented our results in multiple national and international meetings where they were enthusiastically received. Finally, in order to promote the novelty of our Research activity to the public, I have participated to speed-meetings with college and high-school students in Strasbourg, France.
Arboviral diseases are a major health issue worldwide with an impact on the long-term well-being of the patients while causing an economical burden to the affected countries. Although not an arboviral disease, the recent Covid pandemic has particularly exemplified how rapid spreading viral infections can have poor socio-economic consequences. In that sense, it is alarming to observe that owing to the convergence of multiple factors, including global warming, the mosquitoes carrying the arboviruses are now globally spreading in the Northern countries, including Europe. To face this challenge, new research models are needed to understand the mechanisms that underly the viral infections, particularly in the skin. In that sense, the results we obtained over the time of this project are ground breaking. We demonstrated for the first time that skin organoids, which offer a lot of experimental flexibility, can be combined with infected mosquitoes and used as physiological model to study arboviral infections in humans. Although the socio-economic impact of this discovery is not yet measurable with regard to arboviral infections, we anticipate that our work will be the foundation for finding new preventive and therapeutic approaches to counter these infection in the coming years. In addition, beyond the implications of this project in the virology field, the generation and implementation of tissue-engineered human skins also offers new opportunities in the field of dermatology research. Indeed, the past years have demonstrated that while rodents (the most common models used in research) are useful in fundamental research, they often fall short when it comes to translational research. Therefore alternative models are required to mimic human biology and organoids represent one interesting alternative. However, more efforts are necessary to develop these tools. In that sense, this project has already contributed to the improvement of the tissue-engineering in dermatology, demonstrating that a complex tissue harbouring macrophages and sensory neurons together can be achieved. It makes no doubt that these advancements will have a strong impact on the dermatological research community. Finally, the growing interest in mimicking human tissue also answer to the increasing societal and political pressure to reduce animals in Research, particularly in the European Union. This is particularly exemplified by the banishment of animal use for cosmetic tests. This research project directly reply to this growing demand by completely avoiding the use of any animal models. Interest of cosmetic private compagnies regarding our human skin model has already been proven multiple times and we expect to develop partnerships with some of these compagnies in a near future.
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