Optimizing environmental safety and cell-killing potential of oncolytic Newcastle Disease virus with modifications of the V, F and HN genes.

Newcastle Disease Virus (NDV) is an avian RNA virus, which was shown to be effective and safe for use in oncolytic viral therapy for several tumour malignancies. The presence of a multi basic cleavage site (MBCS) in the fusion protein improved its oncolytic efficacy in vitro and in vivo. However, NDV with a MBCS can be virulent in poultry. We aimed to develop an NDV with a MBCS but with reduced virulence for poultry while remaining effective in killing human tumour cells. To this end, the open reading frame of the V protein, an avian specific type I interferon antagonist, was disrupted by introducing multiple mutations. NDV with a mutated V gene was attenuated in avian cells and chicken and duck eggs. Although this virus still killed tumour cells, the efficacy was reduced compared to the virulent NDV. Introduction of various mutations in the fusion (F) and hemagglutinin-neuraminidase (HN) genes slightly improved this efficacy. Taken together, these data demonstrated that NDV with a MBCS but with abrogation of the V protein ORF and mutations in the F and HN genes can be safe for evaluation in oncolytic viral therapy.

Soluble F proteins exacerbate pulmonary histopathology after vaccination upon respiratory syncytial virus challenge but not when presented on virus-like particles.

Respiratory syncytial virus (RSV) fusion (F) protein is suggested to be a protective vaccine target although its efficacy and safety concerns remain not well understood. We investigated immunogenicity, efficacy, and safety of F proteins in a soluble form or on virus-like particle (F-VLP). F VLP preferentially elicited IgG2a antibody and T helper type 1 (Th1) immune responses whereas F protein induced IgG1 isotype and Th2 responses. Despite lung viral clearance after prime or prime-boost and then RSV challenge, F protein immune mice displayed weight loss and lung histopathology and high mucus production and eosinophils. In contrast, prime or prime-boost vaccination of F VLP induced effective protection, prevented infiltration of eosinophils and vaccine- enhanced disease after challenge. This study provides insight into developing an effective and safe RSV vaccine candidate.

Novel Respiratory Syncytial Virus-Like Particle Vaccine Composed of the Postfusion and Prefusion Conformations of the F Glycoprotein.

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and children and represents an important global health burden for the elderly and the immunocompromised. Despite decades of research efforts, no licensed vaccine for RSV is available. We have developed virus-like particle (VLP)-based RSV vaccines assembled with the human metapneumovirus (hMPV) matrix protein (M) as the structural scaffold and the RSV fusion glycoprotein (F) in either the postfusion or prefusion conformation as its prime surface immunogen. Vaccines were composed of postfusion F, prefusion F, or a combination of the two conformations and formulated with a squalene-based oil emulsion as adjuvant. Immunization with these VLP vaccines afforded full protection against RSV infection and prevented detectable viral replication in the mouse lung after challenge. Analyses of lung cytokines and chemokines showed that VLP vaccination mostly induced the production of gamma interferon (IFN-γ), a marker of the Th1-mediated immune response, which is predominantly required for viral protection. Conversely, immunization with a formalin-inactivated RSV (FI-RSV) vaccine induced high levels of inflammatory chemokines and cytokines of the Th2- and Th17-mediated types of immune responses, as well as severe lung inflammation and histopathology. The VLP vaccines showed restricted production of these immune mediators and did not induce severe bronchiolitis or perivascular infiltration as seen with the FI-RSV vaccine. Remarkably, analysis of the serum from immunized mice showed that the VLP vaccine formulated using a combination of postfusion and prefusion F elicited the highest level of neutralizing antibody and enhanced the Th1-mediated immune response.

Use of viral fusogenic membrane glycoproteins as novel therapeutic transgenes in gliomas.

Malignant gliomas are the most common primary brain tumors in adults and, with few exceptions, have a dismal prognosis despite the therapeutic use of surgery, radiation therapy, and chemotherapy. Because CNS gliomas rarely metastasize, they represent an attractive target for gene therapy through local gene delivery. Here we report on the use of two different fusogenic membrane glycoproteins (FMGs), the measles virus proteins F and H (MV-F and MV-H) and a mutated form of the retroviral envelope protein of the gibbon ape leukemia virus (GALV.fus), as a novel class of therapeutic transgenes in gliomas. Transfection of U87 and U118 cells with MV-F and MV-H cDNA or GALV.fus cDNA led in 48 hr to massive syncytial formation followed by cell death. FMG-mediated cytotoxicity in the U87 and U118 cell lines was superior to the cytotoxicity caused by transfection with HSV-tk cDNA followed by ganciclovir (GCV) treatment at all time points. At high-density cell seeding, addition of tumor cells transfected with MV-F and H killed at least 1 log more cells than by HSV-tk + GCV treatment, indicating higher bystander effect. Similar results were obtained with GALV.fus. The mechanism of syncytial death in cultured glioma cell lines was predominantly apoptotic. Transfection of U87 cells with F + H or GALV.fus expression constructs completely suppressed their tumorigenicity. Treatment of established U87 xenografts in nude mice with a combination of F and H adenoviruses at 1:1 ratio led to complete tumor regression, significantly higher antitumor effect, and prolongation of survival as compared with control animals treated with a GFP adenovirus. In summary, the viral fusogenic membrane glycoproteins (GALV and the MV-F + MV-H combination) are potent therapeutic transgenes with potential utility in the gene therapy of gliomas.