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.
Keywords: Dengue; Dynamic light scattering; E. coli; Stability; Virus-like particle; Virus-sized particle.
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