rologie i - European Congress of Virology

5 th European Congress of VirologyOncolytic viruses aim to selectively kill cancer cells. To generate cancerspecific oncolytic Herpes simplex viruses (oHSVs), we retargeted HSVto Human Epidermal growth factor Receptor 2 (HER2), overexpressed inovary and breast cancers. The HER2 retargeted HSV (R LM249) exclusivelyinfects cells expressing HER2, and exerts antitumor activity whenadministered i.t. Ablation of disseminated cancer cells in vivo remains amajor hurdle. Here, we report the efficacy of systemically (i.p.) administeredR LM249 against disseminated tumors in mouse models thatrecapitulate tumor spread in ovarian or breast carcinoma patients. Humanovarian carcinoma SK OV 3 cells were implanted i.p. or breast tumor MDAMB 453 cells were injected i.v. in immunodeficient Rag2/;Il2rg/ mice.Repeated i.p. administrations of R LM249 caused a 95% reduction in thetotal weight of peritoneal neoplastic nodules; 60% of treated mice werefree from tumor peritoneal diffusion. R LM249 exhibited also anti metastaticactivity against ovarian metastases and reduced brain metastases.The i.p. administration of the HER 2 retargeted oncolytic HSV effectivelyreduced i.p. ovarian carcinomatosis and inhibited both visceral and distantmetastases.HER2 is expressed also by high grade gliomas (HGGs). HER2 retargetedHSV R LM113 was effective against HGG and lengthened the medial survivaltime of NOD/SCID or immunocompetent mice carrying orthotopicHGGs.Cumulatively, HER2 retargeted HSVs exerted anticancer activity againstperitoneally diffuse breast and ovary cancers, and against gliomas.REF 0132Gene therapy with a replicative HSV vector expressing LIF limits lossof oligodendrocytes and modulates autoimmunity during experimentaldemyelinating diseaseMichaela NYGÅRDAS 1 , Henrik PAAVILAINEN 1 , Nadine MÜTHER 2 ,Claus Henning NAGEL 2 , Matias RÖYTTÄ 3 , Beate SODEIK 2 ,Hukkanen VEIJO 11 Department of Virology, University of Turku, Turku, FINLAND;2 Institute of Virology, Hannover Medical School, Hannover, GERMANY;3 Department of Pathology, Turku, FINLANDHerpes simplex viruses (HSV) have several features making them goodgene therapy vector candidates, especially for the treatment of central nervoussystem (CNS) disease. Experimental autoimmune encephalomyelitis(EAE) is an inflammatory autoimmune disease of the CNS and is theprimary disease model for multiple sclerosis (MS). Demyelination andinflammation are hallmarks of these diseases. Leukemia inhibitory factor(LIF) has neuroprotective properties and limits demyelination and oligodendrocyteloss. Here, we studied the effect on EAE of an HSV 1 vectorexpressing LIF.We constructed BAC derived HSV 1 vectors, deleted of the neurovirulencegene gamma34.5, encoding LIF or a control gene ZeoR. Female SJL miceinduced for EAE, were treated with 10E7 PFU vector intracranially. ClinicalEAE scores were observed daily. Brain, spinal cord and trigeminalganglion (TG) samples were collected on days 9, 14 and 21 p.i. for microscopicalanalysis and for the study of viral and host gene expression withquantitative RT PCR.The HSV LIF significantly alleviated the clinical scores from day 15 p.i.when compared to untreated mice. Virus replication was detected in brain,TGs and spinal cords by virus culture, PCR and IVIS luminometry. Wedetected an increase in oligodendrocytes in the brains of HSV LIF treatedmice during recovery. The HSV LIF therapy also lowered IL 17 and inducedIL 10 and TGF beta mRNA expression in the brains, indicating a shiftfrom Th17 towardsaTregulatory cell response. We acknowledge MartinMesserle and Eva Maria Borst for their advice on BAC mutagenesis.REF 0133Efficient generation gf Human Natural Killer Cells by viral transformationBernhard FLECKENSTEIN 1 , Armin ENSSER 1 , Benjamin VOGEL 1 ,Karin TENNERT 1 , Florian FULL 1,21 Institut für Klinische und Molekulare Virologie, Universitätsklinikum,Friedrich Alexander Universität, Erlangen, GERMANY; 2 Dept. of Microbiologyand Immunobiology, Harvard Medical School, Boston, MA, USAThe short lifespan of human natural killer (NK) cells is one of the majorobstacles in human NK cell research. Few continuous NK like cell linesexist and all except one derive from NK cell leukemia or lymphomapatients.Here we describe the in vitro growth transformation of functional humanNK cells by Herpesvirus saimiri (HVS), a primate rhadinovirus. Infectionof human NK cells was revealed by using a selectable recombinant HVS H2Kk marker virus. This led us to the discovery that HVS infection resultsin continuous expansion of NK cells; these growth transformed NK celllines have a strict requirement for exogenous IL 2, they express effectormolecules and exert cytolytic activity similar to freshly isolated NK cells.HVS transformation of human NK cells can provide large numbers andspecific populations of NK cells for biochemical and functional analysis.Further studies will have to address an eventual therapeutic potential andtheir value in generating cell lines of NK cell deficiencies and in studyingvirus interactions with human innate immunity.REF 0134Lentiviral delivery of multiple short hairpin RNAS: A gene therapyapproach for HIV 1 infectionFrancesca SPANEVELLO 1 , Arianna CALISTRI 1 , Claudia DELVECCHIO 1 , Barbara MANTELLI 1 , Saverio Giuseppe PARISI 1 , GiorgioPALÙ 1 , Marina CAVAZZANA CALVO 2,3 , Cristina PAROLIN 41 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 René Descartes University of Paris, Paris, FRANCE; 3 NeckerChildren’s Hospital, Paris, FRANCE; 4 Department of Biology, Universityof Padova, Padova, ITALYHIV gene therapy holds considerable promise as an alternative or complementarystrategy to drug based antiretroviral therapy for the treatmentof HIV 1 infection. Among the antiviral genes designed to inhibit HIV 1replication, short hairpin RNAs (shRNAs) mediating knockdown of viralgenes or cellular co factors have proven to be effective. In this study, weobtained lentiviral vectors expressing shRNAs targeting the viral genesvif, tat/rev and the cellular gene CCR5. To account for HIV 1 variability,we adopted combinatorial RNA interference approaches based onthe simultaneous expression of the different shRNAs either from distinctpromoters or as an extended shRNA. We tested and compared three differentpromoters for shRNA expression: the human U6, 7SK and H1polymerase III promoters. The safety and the biological activity of thedeveloped vectors were investigated both in cell lines and in human primarycells. Our results indicate that shRNA efficacy is strictly dependenton the employed promoter. Moreover, single promoter activity can differconsiderably among the selected combinatorial RNAi platforms. Ourfindings show that the insertion of up to three promoter/shRNA cassettesinto a single vector did not negatively affect the inhibitory effect of eachindividual shRNA. These results gain insights into the design constrainsand the promoter selection required to express a combination of antiviralshRNAs and confirmed the potential of RNAi to inhibit HIV 1replication.S96 Virologie, Vol 17, supplément 2, septembre 2013