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Background: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. Method: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. Results: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. Discussion: The way forward towards a subunit vaccine against Usutu virus infection is discussed.

Dengue fever is one of the most wide-spread vector-borne diseases in the world. Although dengue-associated mortality is low, morbidity and economic impact are high. Current licensed vaccines are limited and mediate only partial protection, thus a cost-effective vaccine with improved efficacy is strongly needed. In this work, recombinant dengue serotype 1 E protein was produced in E. coli, inclusion bodies were isolated and the E protein solubilized in urea and purified using an immobilized metal chelate affinity column. The protein was refolded by dialysis in order to obtain virus-like particles (VLPs). Particle assembly was confirmed using size-exclusion chromatography, dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy and stimulated emission depletion fluorescence (STED) microscopy. Particle diameter was strongly dependent on temperature, pH, buffer salt composition, and addition of L-arginine. Particles were stable in carbonate buffer at pH 9.5 and higher at 4 °C and did not aggregate during short-term temperature increase up to 55 °C. However, on basis of the above analyses, especially the results of DLS, TEM and STED, it was concluded that the particles obtained did not have an optimal virus-like structure and were therefore designated “virus-sized particles” (VSPs) rather than VLPs. Immunization of rabbits with the particles did not induce neutralizing antibodies, despite the recognition of the native virus by rabbit antibodies. As the titers against the immunogen were much higher than against the (heat-inactivated) virus, it is assumed that the conformation of the particles at the time of immunization was not optimal. Studies are currently underway to improve the quality of the E protein virus-sized particles towards true virus-like particles in order to optimize its potential as a dengue vaccine candidate.