Human Polymorphonuclear Cells Support Zika Virus to Cross Endothelial Monolayer and Access Bloodstream.

The rapid spread of new outbreaks of human infection caused by Zika virus (ZIKV) has raised many global concerns since 2016. Despite the increasing knowledge of this virus, data on the pathogenesis of ZIKV are still missing. In particular, it is still unknown how the virus crosses the endothelial monolayer and gets access to the bloodstream. In the present work, we used human umbilical vein endothelial cells (HUVECs) as a model to study ZIKV infection in vitro. We demonstrated that HUVECs are an optimal reservoir for viral replication, as they were able to sustain ZIKV infection up to two weeks, without showing a cytopathic effect. In order to evaluate the integrity of endothelial monolayer, immunofluorescence was performed on mock-infected or ZIKV-infected cells ± peripheral blood mononuclear cells (PBMCs) or polymorphonuclear cells (PMN), 48 h p.i., by using an anti-VE-Cadherin antibody, a major adherence protein that maintains the integrity of intercellular junctions. In addition to infection, we noted that the presence of some components of the immune system, such as PMNs, played an important role in altering the endothelial monolayer in cell junctions, suggesting that presence at the site of infection probably promotes the spread of ZIKV in vivo in the bloodstream.

Infectious Toscana Virus in Seminal Fluid of Young Man Returning from Elba Island, Italy.

We report detecting infectious Toscana virus in the seminal fluid of a 25-year-old man from Italy returning from Elba Island. The presence of infectious virus in human semen adds Toscana virus to the long list of viruses detected in this genital fluid and indicates a potential for sexual transmission.

Hyperimmune plasma in three immuno-deficient patients affected by non-severe, prolonged COVID-19: a single-center experience.

BACKGROUND: Convalescent plasma (CP) and hyperimmune plasma (HP) are passive immunotherapies consisting in the infusion of plasma from recovered people into infected patients. Following pre-existing evidence in many other viral diseases, such as SARS, MERS and Ebola, CP and HP have also been proposed for the treatment of COVID-19. Nevertheless, due to the lack of large, well-designed, clinical trials, no clear-cut guidelines exist about what subtype of patient CP and HP should be administered to. CASE PRESENTATION: We have reported the cases of 3 patients, all immunosuppressed and affected by non-severe, prolonged COVID-19. They were treated with HP, whose neutralizing titer was higher than 1/80. The first patient was a 55-year-old male, who had undergone lung transplant. He was under therapy with Tacrolimus and developed non-neutralizing antibodies against SARS-CoV2. The second patient was a 77-year-old female, affected by follicular lymphoma. She had tested positive for SARS-CoV2 after 6 months. The third was a 60-year-old patient, affected by chronic leukemia. He did not develop antibodies after 2-month disease. All 3 patients received HP and had tested negative for SARS-CoV2 within 2 weeks. CONCLUSION: Despite encouraging initial data, no strong evidence exist in support of CP and HP to treat COVID-19. In our experience, although limited due to the reduced number of patients, we found a good safety and efficacy of HP in 3 immuno-deficient subjects. Further data are needed in order to assess whether this subtype of patients may particularly benefit from passive immunization.

Hepatitis C Virus Core Antigen (HCVAg): an affordable assay to monitor the efficacy of treatment in DAAs era.

Hepatitis C virus (HCV) Core Antigen (HCVAg) and HCV-RNA were tested in 962 plasma/serum samples from 180 patients during Direct Antiviral Agents (DAAs) treatment and at follow-up. One hundred and eighty individuals were included: 71% carried advanced fibrosis and 43% were treatment-experienced. A Sustained Virological Response (SVR) was achieved in 166/180 (92%) individuals: 96/102 (94.1%) na ve and 70/78 (89.7%) treatment-experienced (p=0.20). The baseline median levels of HCV-RNA and HCVAg were not significantly different between individuals achieving SVR (5.92 x 105 IU/mL, IQR 5.4-6.4, and 3,417 fmol/L, 2,900-3,795) and those without SVR (6.06 x 105 IU/mL, 5.63-6.57, and 3,391 fmol/L, 2,828-4,077). The HCV-RNA vs. HCVAg assays results showed a fair correlation with an overall moderate qualitative agreement (kappa=0.52). Among treatment-failed individuals, at failure 100% of the assays results were positive for both techniques, with HCV-RNA median value 3.09 x 105 IU/mL (2.10-29.09) and HCVAg median value 1570.28 fmol/L (360.15-9317.67). Undetectable HCV-RNA at EOT showed sensitivity 54%, specificity 100%, negative predictive value (NPV) 93% and positive predictive value (PPV) 100%. Undetectable HCVAg at EOT showed sensitivity 74%, specificity 100%, NPV 97% and PPV 100%. The operative and economic advantages of the HCVAg support the alternative use of HCVAg to monitor DAAs treatment outcome.

How long can SARS-CoV-2 persist in human corpses?

We report the finding of the SARS-CoV-2 genome in the corpse of an exhumed infected person, one month after her death. The viral gene targets were still present in her lungs and heart, however, the virus was no longer alive. Infectious risks from human corpses should be considered.

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

BACKGROUND: Our 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. METHODS: Patients 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. RESULTS: Of 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. CONCLUSIONS: This 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.

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.

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.

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 T(reg)s, 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.

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.

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.

Immunological characterization of respiratory syncytial virus N protein epitopes recognized by human cytotoxic T lymphocytes.

Virus-specific cytotoxic T lymphocytes (CTLs) are crucial for the control of respiratory syncytial virus (RSV) infection. This study has identified CTL epitopes of the RSV N protein in healthy subjects. We screened the primary structure of the N protein for HLA-A 0201-binding amino acid consensus motifs, identifying three peptides designated as N-RSV1, N-RSV2, and N-RSV3. These peptides were used to generate CTL lines by stimulating human HLA-A 02.01 peripheral blood mononuclear cells (PBMCs) in vitro. These CTL lines were then characterized by performing CTL chromium release assays and IFN-gamma secretion detection by intracellular cytokine staining. N-RSV1 and N-RSV3 peptides elicited the strongest cytolytic activity against RSV-infected cells and they could be useful epitopes for the analysis of CTL responses to RSV and for understanding immune-induced disease pathogenesis.

5-fluorouracil-based chemotherapy enhances the antitumor activity of a thymidylate synthase-directed polyepitopic peptide vaccine.

BACKGROUND: Thymidylate synthase (TS), a key enzyme in DNA synthesis, is often overexpressed in cancer cells. Some chemotherapeutic agents, such as 5-fluorouracil (5-FU), act by inhibiting TS expression. We evaluated whether a novel 28-amino acid multiepitope peptide, TS/PP, that contains the sequences of three TS-derived epitopes with binding motifs for HLA-A(*)02.01 could induce a TS-directed cytotoxic T-lymphocyte (CTL) response with antitumor activity. METHODS: TS/PP peptide immunologic activity in CTL lines derived from human leukocyte antigen (HLA)-A(*)02.01+ peripheral blood mononuclear cells (PBMCs) was tested in the presence of interleukin-2 and autologous TS/PP peptide-loaded dendritic cells. Immunologic and antitumor activities of TS/PP and its toxicity were also evaluated in vivo in HLA-A(*)02.01 transgenic (HHD) mice that were vaccinated with TS/PP, control, or TS-peptide cocktail and treated with or without 5-FU chemotherapy. The mice were also inoculated subcutaneously with TS-expressing EL-4/HHD lymphoma cells to assess immune response against these tumor cells. RESULTS: TS/PP-specific CTL lines showed a TS-multiepitopic specificity and were able to kill TS+/HLA-A(*)02.01+ breast and colon carcinoma cells. The killing ability against target cells previously exposed to sublethal doses of 5-FU was statistically significantly greater than against untreated target cells (43.5% versus 26.5% at 25/1 effector to target ratio [Difference {diff} = 17.0]; 95% confidence interval [CI] = 12.6 to 20.4) for MDA-MB-231 breast carcinoma cells and 73.5 versus 48.5 (diff = 25.0; 95% CI = 16.2 to 33.8) for the SW-1463 colon carcinoma cells. HHD mice vaccinated with TS/PP manifested a TS-peptide-specific CTL response with no sign of autoimmunity or toxicity. Furthermore, treatment of these mice with 5-FU delayed or prevented the occurrence of tumors formed by inoculation with autologous (TS+)EL-4/HHD lymphoma cells. CONCLUSIONS: The multiepitopic TS/PP vaccine induces a tumor-specific immune response in mice and is especially potent when used in combination with 5-FU-based chemotherapy.

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.

Efficient delivery of DNA to dendritic cells mediated by influenza virosomes.

In an attempt to enhance the immunological efficacy of DNA-based vaccines, we have investigated a new biological means for delivering target gene DNA directly to professional antigen presenting cells (APC), such as the dendritic cells (DC), which are ultimately responsible for the antigen presentation and the primary activation of the immune system. For this purpose we investigated influenza virosomes (IRIV) with assembled DNA as a possible biological carrier for targeting the APC in vivo and in vitro. By cytofluorimetric analysis of the draining lymph nodes of Balb/c mice which had received (by intranasal (in.) administration) FITC-labeled DNA assembled with IRIV, we detected a significant labeled DNA uptake in a subset of lymph node deriving cells expressing DC surface markers. Subsequent mRNA analysis of these lymph nodes showed that the trans-gene delivered by the virosomes was effectively expressed as mRNA. Finally, a further cytofluorimetric analysis performed on human DC-enriched-PMBC, infected in vitro with labeled DNA/IRIV lead to the conclusion that the majority of APC (DC, B lymphocytes and CD16+ cells) are able to incorporate the labeled DNA transported by the construct. These findings suggest that the virosome is an efficient delivery system for testing infectious, as well as anti-cancer, DNA-based vaccine research.