Identification of a Neutralizing Epitope on TOSV Gn Glycoprotein

Emerging and re-emerging viral infections have been an important public health problem in recent years. We focused our attention on Toscana virus (TOSV), an emergent neurotropic negative-strand RNA virus of the family. The mechanisms of protection against phlebovirus natural infection are not known; however, it is supposed that a virus-neutralizing antibody response against viral glycoproteins would be useful to block the first stages of infection. By using an improved memory B cell immortalization method, we obtained a panel of human mAbs which reacted with TOSV antigens. We identified three epitopes of TOSV Gn glycoproteins by neutralizing mAbs using synthetic peptide arrays on membrane support (SPOT synthesis). These epitopes, separated in primary structure, might be exposed near one another as a conformational epitope in their native structure. In vivo studies were conducted to evaluate the humoral response elicited in mice immunized with the identified peptides. The results underlined the hypothesis that the first two peptides located in the NH terminus could form a conformational epitope, while the third, located near the transmembrane sequence in the carboxyl terminus, was necessary to strengthen neutralizing activity. Our results emphasize the importance of identifying neutralizing epitopes shared among the various phleboviruses, which could be exploited for the development of a potential epitope-based diagnostic assay or a polyvalent protective vaccine against different phleboviruses.