Gianni Gori Savellini

Toscana Virus Epidemiology: From Italy to Beyond

Toscana virus (TOSV) is an arthropod-borne virus which is transmitted to humans by Phlebotomus spp sandflies. Infection is the cause of brain injuries, such as aseptic meningitis and meningoencephalitis, in Italy mainly during the summer. More recently some unusual clinical manifestations due to TOSV with severe sequelae, such as ischemic complications and hydrocephalus, have been reported. TOSV represents an important emerging pathogen and its presence is being investigated in several European countries on the Mediterranean basin, including Italy, France, Spain, Portugal and Cyprus. Phylogenetic analysis has distinguished two genotypes of TOSV, A and B; the first is circulating mainly in Italy and the second in Spain, indicating a different geographic distribution possibly related to the vector. This distribution, evolving with the climate, globalization and habitat modification, has implications for the epidemiology of TOSV.

Chemotherapeutic drugs may be used to enhance the killing efficacy of human tumor antigen peptide-specific CTLs.

The effects of anticancer chemotherapy on antigen-specific cytotoxic T lymphocytes (CTLs) are mostly unknown. We tested the effects of cytotoxic drugs such as 5-fluorouracil, gemcitabine, and oxaliplatin on the functional activity of antigen-specific CTL cultures derived from the peripheral blood mononuclear cells of human donors. We found that a biweekly drug-exposure of human HLA-A(*)02.01+ CTLs derived from bulk cultures led to completely different effects if occurring early (day second) or late (day thirteenth) after the in vitro stimulations with the cognate peptides. In the first case, there was a significant CTL inhibition, whereas in the second, there was a marked enhancement of the antigen-specific cytolytic activity. Results of immunocytofluorimetric studies and CTL/natural killer inhibition assays suggested that the latter effect could be related to a more selective drug-mediated inhibition of cohabitant T regulatory (reg) cells. These results were translated in an in vivo therapeutic mouse model where humanized HLA-A(*)02.01 transgenic mice inoculated with EL-4/humanized HLA-A(*)02.01 transgenic mice showed a prolonged survival and the greatest rate of cure when receiving a combined treatment with a thymidylate synthase-specific peptide vaccine and a multidrug chemotherapy regimen administered late after immunization. Tumor samples derived from this group of mice showed a reduced expression of the target thymidylate synthase antigen, a marked reduction of Tregs, and a noteworthy infiltration of C8+ T cells. These results may have clinical implications for the design of new translational anticancer regimens aimed at combining chemotherapy and immunotherapy.

Toscana virus induces interferon although its NSs protein reveals antagonistic activity.

Toscana virus (TOSV) is a phlebotomus-transmitted virus that belongs to the family Bunyaviridae and causes widespread infections in humans; about 30 % of these cases result in aseptic meningitis. In the present study, it was shown that TOSV is an inducer of beta interferon (IFN-β), although its non-structural protein (NSs) could inhibit the induction of IFN-β if expressed in a heterologous context. A recombinant Rift Valley fever virus expressing the TOSV NSs could suppress IFN-β expression in infected cells. Moreover, in cells expressing NSs protein from a cDNA plasmid, IFN-β transcripts were not inducible by poly(I : C). Unlike other members of the family Bunyaviridae, TOSV appears to express an NSs protein that is a weak antagonist of IFN induction. Characterization of the interaction of TOSV with the IFN system will help our understanding of virus-host cell interactions and may explain why the pathogenesis of this disease is mostly mild in humans.

Immune-Reconstituted Influenza Virosome Containing CD40L Gene Enhances the Immunological and Protective Activity of a Carcinoembryonic Antigen Anticancer Vaccine

The correct interaction of a costimulatory molecule such as CD40L with its contrareceptor CD40 expressed on the membrane of professional APCs, provides transmembrane signaling that leads to APC activation. This process can be exploited to significantly improve the efficacy of cancer vaccines and the outcome of a possible cancer vaccine-induced, Ag-specific CTL response. Therefore, we investigated whether a novel intranasal delivery of immune-reconstituted influenza virosomes (IRIV), assembled with the CD40L gene (CD40L/IRIV), could be used to improve protective immunity and the Ag-specific CTL response against carcinoembryonic Ag (CEA) generated with a novel vaccine constituted of IRIV assembled with the CEA gene (CEA/IRIV). Our results suggest that CD40L/IRIV was able to augment CEA-specific CTL activity and CEA-specific protective immunity induced by CEA/IRIV most likely through the induction of a CTL response associated with a Th1 phenotype. In conclusion, we provide evidence that CD40L/IRIV, by acting through the CD40L/CD40 signaling pathway, acts as an immune-adjuvant that could increase the efficacy of a CEA-specific cancer vaccine, which could provide an efficacious new strategy for cancer therapy.

Nucleotide variability of Toscana virus M segment in strains isolated from clinical cases

Toscana virus (TOSV), a member of the Bunyaviridae family, is an important etiologic agent of neurologic infection transmissible to humans by bites of the Phlebotomus spp. In consideration of the variations in the antigenic properties of Bunyaviruses and their potential genetic variability, we analysed a large region (2500nt) of the Toscana virus M segment coding for the non-structural protein (NSm) and the G(N) and G(C) glycoproteins in several strains isolated from patients with meningitis from 1998 to 2004 in the region of Tuscany in Italy. The sequences were compared with the reference strain of Toscana virus isolated from phlebotomus (ISS Phl. 3) and revealed some changes in amino acids, particularly in the G(C) protein, that are probably involved in recognition and binding to the cell receptor. The analyses were aimed at identifying the amino acid changes commonly to all of the clinical isolates potentially related to TOSV virulence.

Immunization with Toscana virus N-Gc proteins protects mice against virus challenge

Toscana virus (TOSV) is an emerging virus, circulating in the Mediterranean area, that is responsible for aseptic meningitis, meningoencephalitis, and encephalitis. The development of a vaccine that could provide complete protection from TOSV infection is needed. In this study we investigated the capacity of TOSV structural proteins, nucleocapsid protein N and the two Gc and Gn glycoproteins, produced as recombinant proteins, in an animal model. In particular, we investigated their role in inducing specific and protective immune responses against virus infection. Mice were immunized intraperitoneally using TOSV antigens singly or in combination. The results show that only the N-Gc combination was able to protect 100% of animals from a lethal challenge with a neurovirulent strain of TOSV. This potential vaccine induces high serum antibody titres with neutralizing activity and it is safe for animals. Moreover, immunization induces a virus specific cell-mediated immune response, in particular a CD8+ T cell response associated with a marked expression of interferon gamma. These results indicate that the N+Gc viral antigen combination could be useful for future development of a vaccine controlling the spread of this emerging virus that could pose a new threat for humans.

Serological and molecular detection of Toscana and other Phleboviruses in patients and sandflies in Tunisia

BackgroundOur aim is to detect the infection by Toscana virus (TOSV) and other Phleboviruses in the sera and cerebro-spinal fluid (CSF) of patients with meningitis in Tunisia. We examined various species of phlebotomus present in Tunisia to determine whether or not a direct relationship exists between cases of meningitis and the viruses circulating in the insect vectors.MethodsPatients with the meningeal syndrome were tested for anti-TOSV IgM and IgG using an indirect Enzyme-Linked Immunosorbent Assay (ELISA) and for the presence of TOSV and other Phleboviruses using a RT-PCR test.An entomological study was carried out using CDC light traps to trap sandflies in different bioclimatic zones of Tunisia. Collected sandflies were tested by RT-PCR for the presence of TOSV and other Phleboviruses and subsequently by viral isolation on Vero cells.ResultsOf 263 patients were tested using ELISA of which 12.16% (n = 32/263) were IgM positive for anti TOSV. Of these 32 patients, 78% (n = 25/32) were IgG positive. 12.86% (n = 18/140) of the CSF samples tested by RT-PCR were positive for the Toscana virus.One CSF sample tested by RT-PCR revealed the presence of Sandfly Fever Sicilian Virus (SFSV). The Punique virus was identified in one sandfly pool.ConclusionsThis study confirms, for the first time, that TOSV is involved in a neurological disorder in North Africa. The incidence of this involvement in Tunisia conforms with observations made in other Mediterranean countries. Moreover, for the first time, a molecular approach was used to detect SFSV in a Tunisian patient displaying neurological symptoms.

Diagnostic tools for Toscana virus infection

Toscana virus (TOSV; Phlebovirus, Bunyaviridae) is an important etiological agent of acute meningitis and meningoencephalitis in Mediterranean countries. Laboratory diagnosis has been carried out in serological studies using ELISA, immunofluorescence and/or neutralization tests that are not influenced by the virus viability; however, in the acute phase of the infection, nucleic acid amplification techniques are the methods of choice to diagnose viral meningitis from cerebrospinal fluid samples. Molecular methods are rapid and sensitive and, unlike traditional methods, such as virus isolation by cell culture, they are not influenced by the viability of the virus in the clinical specimen; however, the RNA integrity is crucial for the success of these methods. Real-time PCR is the most important molecular method used in laboratories worldwide, since it is less time-consuming and it reduces the risk of contamination. Therefore, a sensitive real-time PCR has been developed for diagnosis of suspected cases of TOSV infection either autochthonous and/or imported, since a new lineage of TOSV, divergent from the Italian prototype, has recently been reported in Spain.

Anti-angiogenetic effects of immune-reconstituted influenza virosomes assembled with parathyroid hormone-related protein derived peptide vaccine

BACKGROUND C-IRIV/PTR-4 is a novel anticancer vaccine construct composed of immune-reconstituted influenza virosomes (IRIV) assembled with the PTH-rP derived peptide (PTR)-4, a synthetic CTL epitope with HLA-A(*)02.01 amino acid binding motifs. This peptide is able to generate a human PTH-rP specific CTL response with anti-tumor activity in vitro and in mice. MATERIALS AND METHODS We have investigated the immunological and preventive anti-tumor activity of C-IRIV/PTR-4 compared with the soluble PTR-4 peptide, in HHD mice inoculated with autologous PTH-rP+ tumor cells. RESULTS Peptide vaccination with either a soluble and an IRIV formulation showed similar immunological activity and the ability to purge the tumor tissue of tumor cell clones able to produce the target antigen (PTR-rP). The most efficient protection from tumor growth was however observed in animals vaccinated with C-IRIV/PTR-4 in which an additional IRIV related anti-angiogenetic effect was detected in the tumor tissue. CONCLUSIONS These results confirm the immunological activity of PTR-4 vaccination and suggest a more efficacious therapeutic potential of C-IRIV/PTR-4 against bone metastases and malignancies like breast, prostate and lung which very often over-express PTH-rP.

Seroprevalence of Antibodies to Sandfly Fever Sicilian Virus in a Sample Population in Tuscany, Italy

Toscana virus is the main phlebovirus circulating in Tuscany during the warm season, thus, a seroprevalence study was performed in the same area to estimate the antibody prevalence rates for sandfly fever Sicilian virus (SFSV) that is endemic in the Mediterranean countries. The low seroprevalence observed in this study shows that this virus does not play an important role in the etiology of febrile illness in central Italy.