Identification of Neutralizing Epitopes of TOSV: A Mouse Model

Emerging and re-emerging viral infections have been an important public health problem in recent years. The Bunyavirales order is one of the largest groups of segmented negative-sense single-stranded RNA viruses, which includes many pathogenic strains that cause severe human diseases. Toscana virus (TOSV), a Phlebovirus belonging to the Phenuiviridae family, is considered an emergent pathogen associated with acute neurological disease, such as meningitis, meningoencephalitis and encephalitis, occurring in the Mediterranean countries (principally Italy, Spain, France) during the summer months (Valassina et al., 2003 a; Valassina et al., 2003 b; Charrel et al., 2005; Sanbonmatsu –Gàmez et al., 2009; Cusi et al., 2010). The mechanisms of protection against natural infection of Phlebovirus are not known, however it is supposed that a virus neutralizing antibody response against the viral glycoproteins could be needed to block the first stages of infection. Neutralizing antibody responses are critical components of the host defense against viral infections and are recognized as a key element in the protective immune response against infection elicited by many prophylactic vaccines (Burton et al., 2012; Corti et al., 2013). In this setting, we focused our attention on TOSV glycoproteins (Gn and Gc). In a previous work, Prof. Cusi's group was able to localize three neutralizing epitopes on Gn glycoprotein using human mAbs obtained by immortalization of B cells from a subject infected with TOSV. It was postulated that these 3 aminoacid sequences, separated in the primary structure of the protein, had probably a neutralizing activity in the tridimensional structure, in which peptides 1- 2 could be part of a conformational epitope without peptide 3; which instead, is necessary to strengthen the neutralizing activity and hinder the virus replication, by blocking the binding of Gn to the receptor. The present study was aimed to confirm in vivo the immunogenic efficacy of these three epitopes in various combinations in mouse model and to test if the mice serum obtained show any cross reactivity with other members of Phlebovirus (such as SFNV, PUNV, SNVF, CYPR) and eventually, to evaluate if these cross-reactive sera are also neutralizing against other Phlebovirus. Moreover, these results could be used to design epitopes that can serve as potential targets for the production of epitope-based diagnostics and vaccines against TOSV and other related Phlebovirus. The study also developed monoclonal antibodies against TOSV NSs protein by immortalization of human B cells by a modified methodology. These monoclonals could be used for better understanding the role of NSs in viral infection of the host and eventually in passive immunization.