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Janne Ruotsalainen Interferon Response as a Challenge and Possibility for Developing Alphavirus Based Oncolytic Virotherapy Publications of the University of Eastern Finland Dissertations in Health Sciences JANNE RUOTSALAINEN Interferon Response as a Challenge and Possibility for Developing Alphavirus Based Oncolytic Virotherapy To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Tietoteknia auditorium (TTA), Kuopio, on Friday, September 26th 2014, at 1 p.m. Publications of the University of Eastern Finland Dissertations in Health Sciences Number 247 Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, Faculty of Health Sciences, University of Eastern Finland Kuopio 2014 Grano Kuopio, 2014 Series Editors: Professor Veli-Matti Kosma, M.D., Ph.D. Institute of Clinical Medicine, Pathology Faculty of Health Sciences Professor Hannele Turunen, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Olli Gröhn, Ph.D. A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O.Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto ISBN (print): 978-952-61-1554-2 ISBN (pdf): 978-952-61-1555-9 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706 III Author’s address: Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences Doctoral Program of Molecular Medicine University of Eastern Finland P.O. Box 1627 FI-70211 KUOPIO FINLAND [email protected] Supervisors: Professor Ari Hinkkanen, Ph.D. Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND Professor Kari Airenne, Ph.D. Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND Reviewers: Professor Ilkka Julkunen, Ph.D. Department of Virology University of Turku TURKU FINLAND Docent Vincenzo Cerullo, Ph.D. Faculty of Pharmacy Division of Biopharmaceutics and Pharmacokinetics University of Helsinki HELSINKI FINLAND Opponent: Cristian Smerdou, Ph.D. Division of Gene Therapy Center for Applied Medical Research (CIMA) University of Navarra PAMPLONA SPAIN IV V Ruotsalainen, Janne Interferon Response as a Challenge and Possibility for Developing Alphavirus Based Oncolytic Virotherapy University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences 247. 2014. 104 p. ISBN (print): 978-952-61-1554-2 ISBN (pdf): 978-952-61-1555-9 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706 ABSTRACT Certain malignancies such as glioblastoma multiforme are still incurable and in others like colorectal carcinoma, the prognosis greatly depends on the time of diagnosis. Oncolytic virotherapy is a novel approach to address these difficult forms of cancer. In addition to direct lysis viruses can induce innate and adaptive immune responses against the tumor or its vasculature. However, the infection can be prematurely halted by neutralizing antibodies or components of the innate immune system, especially the type I interferon response. Semliki Forest virus based oncolytic virus VA7 has previously been proven to be a promising candidate for the treatment of several different forms of cancer showing significant therapeutic power especially against melanoma xenografts. However, only local virus injections were powerful against A549 lung adenocarcinoma xenografts, and tumor resistance to virus was associated with the type I interferon response. Furthermore, rat BT4C gliomas were resistant to VA7 in vivo and the treated animals developed neutralizing antibodies against the virus. The initial aim of this thesis work (I-III) was to investigate whether peripherally administered VA7 could home into intracranial gliomas, when the tumors per se are virus susceptible. U87 gliomas were discovered to be highly sensitive to VA7 infection in vitro and a single intravenous virus injection resulted in the tumor infection and a cure in 16/17 of treated mice (I). Next the role of adaptive immune responses to virotherapy was examined by treating intracranial mouse GL261 gliomas with VA7. Neither intravenous nor local virus injections improved survival even when adaptive immunity was suppressed with cyclophosphamide or rapamycin. Infected GL261 tumor tissue could not be detected and surprisingly, cyclophosphamide administration increased infection of the healthy brain parenchyma. GL261 cells could secrete and respond to type I interferon upon VA7 infection and host-derived interferons likely had an additional role in the resistance as pre-infected GL261 tumors developed with normal kinetics (II). The contributions of innate and adaptive immune responses to VA7 therapy were studied in more detail by utilizing two CT26 mouse colon carcinoma cell clones found to differ dramatically in both their type I IFN responses and their immunogenicities. Intradermal CT26LacZ tumors were immunogenic but they could be eradicated both from Balb/c and SCID mice and VA7 injection resulted in a progressive perivascular tumor infection. When CT26WT and CT26LacZ cells were mixed, as few as 10 % of the resistant CT26WT cells were sufficient to compromise the therapy. Comparative RNA-Seq transcriptome analysis revealed that in contrast to CT26LacZ, the CT26WT cells constitutively expressed several interferon stimulated genes (III). In conclusion, VA7 demonstrated potential in targeting of gliomas and colorectal carcinomas but the treatment efficacy depended primarily on the tumor type I interferon responsiveness. Thus, careful pre-screening will be necessary to indentify the susceptible candidates for alphavirus based oncolytic virotherapy. National Library of Medicine Classification: QZ 380, WI 529, QW 940, QW 168.5.A7, QU 560 Medical Subject Headings: Oncolytic Virotherapy/methods; Oncolytic Viruses; Colorectal Neoplasms/therapy; Glioma/therapy; Immunotherapy; Gene Therapy; Alphavirus/genetics; Semliki forest virus; Interferon Type I/immunology; Mice VI VII Ruotsalainen, Janne Interferonivaste haasteena ja mahdollisuutena Alfaviruspohjaisen onkolyyttisen viroterapian kehittämisessä Itä-Suomen yliopisto, terveystieteiden tiedekunta Itä-Suomen yliopiston julkaisuja. Terveystieteiden tiedekunnan väitöskirjat 247. 2014. 104 s. ISBN (print): 978-952-61-1554-2 ISBN (pdf): 978-952-61-1555-9 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706 TIIVISTELMÄ Jotkut syövät kuten glioblastoma multiforme ovat parantumattomia, ja toisissa kuten paksu- ja peräsuolen syövissä ennuste riippuu diagnoosin varhaisuudesta. Onkolyyttinen viroterapia on uusi lähestymistapa näiden vakavien syöpätyyppien hoidossa. Onkolyyttiset virukset tuhoavat suoraan syöpäkudosta, minkä lisäksi ne voivat herättää potilaan luontaisen ja hankitun immuniteetin kasvainta tai sen verisuonistoa vastaan. Neutraloivat vasta-aineet tai luontainen immuniteetti (etenkin tyypin I interferonivaste) voivat kuitenkin keskeyttää virus-infektion ennenaikaisesti. Semliki Forest virukseen pohjautuva onkolyyttinen virus VA7 on osoittautunut aiemmissa tutkimuksissa lupaavaksi hoitomuodoksi monissa syöpämalleissa ollen erityisen tehokas ihmisen melanooma ksenotransplantaatteja vastaan. Ainoastaan paikalliset virushoidot toimivat kuitenkin A549 keuhkoadenokarsinooma ksenotransplantaattimallissa, jossa resistenssi systeemisesti annetulle virukselle yhdistettiin tyypin I interferonivasteeseen. Rotan BT4C glioomamalli oli myös resistentti ja viroterapiaa saaneiden eläinten verestä löytyi virusvasta-aineita. Tämän väitöskirjan (I-III) ensimmäisen osatyön tavoitteena oli selvittää hakeutuuko VA7 kallon sisäisiin glioomiin, jos kasvaimet sinänsä ovat virukselle herkkiä. U87 glioomien havaittiin olevan hyvin herkkiä VA7 infektiolle in vitro ja yksi virusannos laskimoon johti kasvaimen infektoitumiseen ja 16/17 hiiren paranemiseen (I). Seuraavaksi hankitun immuniteetin vaikutuksia viroterapiaan tutkittiin hoitamalla hiiren kallon sisäisiä GL261 glioomia. Laskimonsisäiset tai paikalliset VA7-injektiot eivät parantaneet selviytymistä, eikä immuniteetin vaimentaminen syklofosfamidilla tai rapamysiinillä parantanut hoitotulosta. VA7 ei infektoitunut GL261 kasvainkudosta, mutta syklofosfamidi tehosti infektiota terveessä aivokudoksessa. Infektoitujen GL261 solujen havaittiin erittävän tyypin I interferonia ja olevan myös herkkiä interferonille. Hiiren tuottamilla interferoneilla oli luultavasti lisävaikutus sillä etukäteen infektoidut GL261 kasvaimet kehittyivät myös normaalisti (II). Viimeisessä osatyössä selvitettiin tarkemmin synnynnäisen ja hankitun immuniteetin vaikutuksia VA7 terapiaan käyttämällä kahta CT26 hiiren paksu- ja peräsuolisyövän solukloonia, joiden havaittiin eroavan merkittävästi immunogeenisyydeltään ja tyypin I interferonivasteeltaan. Nahkansisäiset CT26LacZ kasvaimet olivat immunogeenisiä, mutta ne kyettiin hävittämään sekä Balb/c että SCID hiiristä ja kasvaininfektion huomattiin keskittyvän verisuonien ympärille. 10 % osuus resistenttejä CT26WT soluja riitti heikentämään hoitotehoa merkittävästi, ja CT26WT solujen havaittiin ilmentävän perustasolla monia interferoni-stimuloituvia geenejä. Yhteenvetona VA7 osoittautui lupaavaksi hoitomuodoksi sekä gliooma- että paksu- ja peräsuolen syöpämalleissa, mutta hoitoteho riippui kasvainten tyypin I interferonivasteesta. Havainnot puoltavat seulontamenetelmien kehittämistä niiden kasvaimien löytämiseksi, jotka voisivat olla herkkiä alfaviruspohjaiselle onkolyyttiselle viroterapialle. Luokitus: QZ 380, WI 529, QW 940, QW 168.5.A7, QU 560 Yleinen Suomalainen asiasanasto: syöpätaudit, glioomat, paksusuolisyöpä, peräsuolisyöpä, syöpähoidot, viroterapia, immuunihoito, Semliki Forest –virus VIII IX Acknowledgements The studies described in this thesis were carried out in the Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, Faculty of Health Sciences, University of Eastern Finland. First of all, I want to sincerely thank my main supervisor Professor Ari Hinkkanen for providing me with the opportunity to work in his laboratory on such an exciting research topic. I have been very lucky to have you as a supervisor. Already at the very earliest stages, you have demonstrated through your personal example the necessary elements of research: curiosity, dedication, patience and genuine excitement in the face of scientific discovery, big or small. I have learned that the only route to success in research goes through a series of disappointments and your great sense of humor has made also those easier to cope with. I want to thank also my second supervisor Kari Airenne for the inspiring scientific talks we have had and for the many memorable national and international conference trips we have shared. I want to express my warm gratitude to the pre-examiners of the thesis work Professor Ilkka Julkunen and Docent Vincenzo Cerullo for the careful inspection of the work. In addition, I am truly thankful to Ewen MacDonald for the professional linguistic revision. I express my deepest gratitude to the many people who have been directly involved in this work: Miika Martikainen, Markus Vähä-Koskela, Jari Heikkilä, Minna Niittykoski, Minna Kaikkonen, Seppo Ylä-Herttuala, John Bell, Tuulia Huhtala, Tytti Aaltonen, Ivana Kholova, Olli Gröhn, Johanna Närväinen, Timo Liimatainen, Chantal Lemay, Julie Cox, Naomi De Silva, Agnieszka Kus, Theresa Falls, Jean-Simon Diallo and Fabrice Le Boeuf. Especially I want to thank Miika and Markus. Miika: thank you for putting up with me and my “humor” all these years without completely losing it. I will be forever grateful not only for your invaluable scientific contribution but also for your friendship. Markus: I want to thank you for the crucial impact you had on this work and for the dozens of mind-boggling scientific talks we have had. You are a great person and a great scientist and it has been a privilege to work with you. Irrespective of whether or not the work has been published, I also want acknowledge the contributions of co-authors and colleagues Erkko Ylösmäki, Ale Närvänen, Diana Schenkwein, Emilia Makkonen, Outi Rautsi, Heini Belt, Kirsi Vuorinen, Maartje Van Geenen, Marianna Héllen, Ville Ruottinen, Jari Pokka, Bijay Dhungel, Jakub Kolodziejski, Michal Burmistrz, Justin Zoppe, Seppo Rönkkö, Leena-Sisko Johansson, Kristiina Järvinen, Jukka Seppälä, Karoliina Autio, Marjukka Anttila, Matti Waris, Akseli Hemminki, Ari Kauppinen, Anssi-Pekka Karttunen, Riikka Pellinen and Jarkko Ketolainen. I want to thank Helena Pernu, Hanne Tanskanen, Jouko Mäkäräinen, Seija Sahrio, Jari Nissinen, Laila Kaskela, Mirka Tikkanen, Anneli Miettinen, Anne Martikainen, Joonas Malinen and the whole staff of LAC for the administrative and technical advice and assistance without which this work would have not been possible. I wish to thank all the friends and colleagues from the former Ark Therapeutics where I did my Master’s thesis for the fun and stimulating working environment, which encouraged me to continue to pursue X a scientific career. Thank you Vesa, Taina, Miia, Tytteli, Tiina, Pyry, Hanna, Mikko, AnnMarie, Tommi, Jere, Haritha, Thomas, Roseanne, Anssi, Tarja, the late Riikka, Siiri, Juha and all the rest. I have continued to bother many of you since those times but have never been turned down. My special thanks go to Ann-Marie, who had an important role at a crucial moment when I still had doubts about which path to follow. I want to thank my high school biology teacher, Esa Hämäläinen, whose inspiring way of teaching was the first spark to engage myself with the mysterious world of biosciences. I owe not only my warmest gratitude but also sincerest apologies to my new and long-term friends and relatives alike; Jarkko, Marko, Aleksi, Jussi, Rebekka, Antti, Susanna, Weronika, Magda, Stefanie, Patrick, Sören, Anja, Pyare, Iina, Mikko, Miia, Ville, Heli, Otto, Tuula, Pentti, Jyrki, Tanja, Make and all the others who I have been neglecting especially during the past year. Thank you for not walking out on me. Without friends, life would be miserable. My deepest gratitude goes to my family members, the late Tarja Ruotsalainen, Tauno Lösönen, Juhani Ruotsalainen, Päivi Ruotsalainen, Joonas Lösönen, Jari Ruotsalainen, Katja Ruotsalainen, Miia Tolvanen and Jari Ollikainen. Without your support, I could have not gone through this and I could not imagine myself or my life without your existence and influence. I dedicate greatest gratitude and respect to my late mother Tarja, who was not only a great personality and a loving mother but also encouraged me to keep pushing the boundaries and educate myself to the highest degree. I also want to thank Katja for being the big sister when I needed one and for setting me on the right track at the critical moment. I would have been even more unbearable as a philosopher than I currently am as a bioscientist. Finally, I want to dedicate my heartfelt gratitude to my wife, medical doctor Jenny Schmidt for sticking with me during this process. Without your love, support, encouragement and trust I could have not kept going. I apologize for how little time I have had for you during the last year. I hope time will prove that all of this was worth it. Kuopio, August 2014 Janne Ruotsalainen This work was supported by Doctoral Program in Molecular Medicine, Orion Foundation, Matti and Vappu Maukonen fund, Northern Savo Regional Fund, Academy of Finland, EVO fund, Cancer Society of Finland and the Cancer Center of the University of Eastern Finland XI List of the original publications This dissertation is based on the following original publications: I II III Heikkilä, J., Vähä-Koskela, M., Ruotsalainen, J., Martikainen, M., Stanford, M. McCart, J.,Bell, J., Hinkkanen, A. Intravenously Administered Alphavirus Vector VA7 Eradicates Orthotopic Human Glioma Xenografts in Nude Mice. PLoS One. 6;5(1):e8603, 2010 Ruotsalainen, J.*, Martikainen, M.*, Niittykoski, M., Huhtala, T., Aaltonen, T., Heikkilä, J., Bell, J., Vähä-Koskela, M., Hinkkanen, A. Interferon- sensitivity of tumor cells correlates with poor response to VA7 virotherapy in mouse glioma models. Molecular Therapy. 20(8):1529-39, 2012. Published online 2012 March 20. doi: 10.1038/mt.2012.53. Ruotsalainen, J., Kaikkonen, M., Niittykoski, M., Martikainen, M., Lemay, C., Cox, J., De Silva, N., Kus, A., Falls, T., Diallo, J-S, Le Boeuf, F., Bell, J., YläHerttuala, S., Hinkkanen, A., Vähä-Koskela, M., Clonal Variation in Interferon Response Determines the Outcome of Oncolytic Virotherapy in Mouse CT26 Colon Carcinoma Model, Gene Therapy, in press * Equal contribution Also unpublished data is presented The publications were adapted with the permission of the copyright owners. XII XIII Contents 1 Introduction .................................................................................................................................................. 1 2 Literature review ........................................................................................................................................... 3 2.1 Cancer ....................................................................................................................................................... 3 2.1.1 Hallmarks of cancer ....................................................................................................................... 3 2.1.2 Oncogenesis, tumor microenvironment and cancer stem cell theory......................................... 3 2.1.3 Cancer immunoediting: Sum of immunosculpting and immunosuppression .......................... 5 2.1.4 Cancer in Finland and modern cancer therapies ......................................................................... 7 2.2 Interferons in cell autonomous, innate and adaptive immunity ........................................................... 9 2.3 Alphaviruses........................................................................................................................................... 15 2.3.1 Taxonomy, natural life cycle and pathogenesis of Alphaviruses and Semliki Forest virus ..................................................................... 15 2.3.2 Alphavirus replication cycle ........................................................................................................ 17 2.3.3 Alphaviruses and adaptive immunity ....................................................................................... 19 2.3.4 Alphaviruses and innate- and cell autonomous immunity........................................................ 19 2.4 Oncolytic virotherapy ............................................................................................................................ 23 2.4.1 From the early 20th century to the present day .......................................................................... 23 2.4.2 Oncolytic virotherapy overlaps with gene therapy and immunotherapy of cancer – emerging concept of oncolytic immunovirotherapy ....................................................................... 28 2.4.3 Combination therapy to improve OV efficacy ............................................................................ 37 2.4.4 Targeting of Oncolytic Viruses..................................................................................................... 39 2.4.5 Naturally targeted oncolytic viruses............................................................................................ 43 2.4.6 VA7 oncolytic virus....................................................................................................................... 46 2.4.7 Armed oncolytic viruses............................................................................................................... 47 2.4.8 Safety and ethics of oncolytic virotherapy .................................................................................. 48 3 Aims of the study ........................................................................................................................................ 51 4 Materials ans methods ................................................................................................................................ 53 4.1 Cell lines and mouse strains (I-III) ........................................................................................................ 53 4.2 VA7-EGFP production (I-III) ................................................................................................................. 54 4.3 Determination of Semliki Forest virus infectious titers (I-III).............................................................. 56 4.4 Preparing mice and cells for tumor transplantations (I-III) ................................................................. 56 4.5 SDS-PAGE and western blotting (III).................................................................................................... 57 4.6 SFV detection from tissues by immunohistochemistry (I-III) ............................................................. 58 5 Results .......................................................................................................................................................... 59 5.1 Peripherally administered VA7 homes into orthotopic U87-Fluc glioma xenografts and destroys them (I) ................................................................................................................................... 59 5.2 The VA7 resistance of GL261 mouse gliomas reflects their robust type I IFN signaling (II)............. 60 5.3 Subclone of resistant CT26 colon carcinoma is highly sensitive to VA7 due to weak type I IFN response (III) ........................................................................................................ 64 6 Discussion .................................................................................................................................................... 69 7 Summary and conclusions.......................................................................................................................... 83 References ....................................................................................................................................................... 85 APPENDIX: Original publications I-III XIV XV Abbreviations AAV Ab arbovirus Ad ADCC APC BBB BHK CAR CEA CHIKV CIK CSC CT CTL DAI dsRNA ECM EGF EEEV EGFP EGFR EMT GBM GM-CSF CML CPA HCC HDAC HGF HPV HSV i.c. adeno-associated virus antibody arthropod-borne virus adenovirus antibody-dependent cellular cytotoxicity antigen presenting cell blood brain barrier baby hamster kidney cells coxsackie virus and adenovirus receptor carcinoembryonic antigen chikungunya virus cytokine induced killer cells cancer stem cells computed tomography cytotoxic T lymphocytes DNA-dependent activator of IFN-regulatory factors double strand RNA extracellular matrix epidermal growth factor Eastern equine encephalitis virus enhanced green fluorescent protein epidermal growth factor receptor epithelial-to-mesenchymal transition glioblastoma multiforme granulocyte-macrophage colony-stimulating factor chronic myeloid leukemia cyclophosphamide hepatocellular carcinoma histone deacetylase hepatocyte growth factor human papilloma virus Herpes simplex virus intracranial ICD IFNAR IL i.p. i.t. i.v. IFN IL IPA IRG ISGF3 LAMR Luc mAb MCA MDSC MET miRNA MLV MMP MOG MRI Nab NDV NIS NK-cell NKT-cell NLS nsP SFV TGF TNF VEEV OV pDC immunogenic cell death Interferon-alpha/beta receptor interleukin intraperitoneal intratumoral intravenous interferon interleukin Ingenuity pathway analysis immune-related GTPase Interferon-stimulated gene factor 3 high-affinity laminin receptor luciferase monoclonal antibody 3-methylcholanthrene myeloid derived suppressor cell mesenchymal-to-epithelial transition microRNA murine leukemia virus matrix metalloproteinase myelin oligodendrocyte glycoprotein magnetic resonance imaging neutralizing antibody Newcastle disease virus sodium iodine symporter natural killer cell natural killer T-cell nuclear localization signal nonstructural protein Semliki Forest virus transforming growth factor tumor necrosis factor Venezuelan equine encephalitis virus oncolytic virotherapy plasmacytoid dendritic cell XVI PET PFU p.i. poly I:C PSA p.t.i. RdRp RLR RNAi SCID-X1 SPECT Tc Th TIMP Treg SAV SFV SMI ssRNA STAT SINV VEGF VPA VSV WEEV positron emission tomography plaque forming units post infection Polyinosinic:polycytidylic acid prostate specific antigen post tumor induction RNA-dependent RNA polymerase RIG-I like receptor RNA interference X-linked severe-combined immunodeficiency Single-photon emission computed tomography cytotoxic T-cell T-helper cell tissue inhibitor of metalloproteinases T-regulatory cell salmonid alphavirus Semliki Forest virus small molecule inhibitor single strand RNA Signal Transducer and Activator of Transcription Sindbis virus vascular endothelial growth factor valproic acid vesicular stomatitis virus Western equine encephalitis virus 1 Introduction Due to improved diagnostics and novel therapeutics, the prognoses of many malignancies including breast cancer, prostate cancer, testicular cancer and childhood leukemia have improved dramatically in western countries such as Finland (De Angelis et al. 2014; Pukkala, Sankila, Rautalahti 2011). For example, in Finland in the period 2007-2009, the 5year survival from the most common cancers i.e. prostate (men) and breast (women) were 93% and 89% respectively. Thus, although the overall incidence of cancer has increased, the mortality has decreased. In fact, due to longevity, more than one third of the Finnish population will suffer cancer at some point in their lives (Pukkala, Sankila, Rautalahti 2011). However, the prognoses of certain cancers such as malignant gliomas, lung cancer and pancreatic cancer still remain poor, while prognoses of others such as colon carcinoma rely heavily on the time of diagnosis: locally confined disease can be resected but advanced and metastatic disease is virtually incurable (Brenner, Kloor, Pox 2014). Oncolytic virotherapy (OV) presents a new and promising approach to tackle these devastating diseases. Due to genetic manipulation or their natural tendencies oncolytic viruses replicate preferentially in neoplastic tissue or in the tumor vasculature, and after initial cell lysis, potent antitumor immune responses can be evoked. However, both innate and adaptive immune responses can also be targeted against the virus being thus detrimental to the therapy (Russell, Peng, Bell 2012). Recently, Talimogene laherparepvec (T-VEC) was employed in the treatment of advanced melanomas, and passed as a first oncolytic virus in history a phase III clinical trial, representing a significant milestone in this rapidly developing field (Andtbacka et al. 2013; Pol et al. 2013). VA7 is an oncolytic alphavirus based on the avirulent Semliki Forest virus (SFV) strain that even before the present work (I-III) had displayed potential in orthotopic xenograft models of human osteosarcoma (Ketola et al. 2008) and lung adenocarcinoma (Maatta et al. 2008), and considerable therapeutic power against human melanoma xenografts after systemic delivery (Vaha-Koskela et al. 2006). This dissertation aimed to further explore the possibilities of targeting tumors occurring in difficult body locations such as the brain using an immunocompromised glioma xenograft model (I) and in different immunocompetent mouse glioma models with or without concomitant immunosuppression (II). The specific roles of innate and adaptive immune responses were then further dissected utilizing two mouse colon carcinoma clones with opposing patterns of cell autonomous antiviral signaling and immunogenicity (III). 2 3 2 Literature review 2.1 CANCER 2.1.1 Hallmarks of Cancer Tissue homeostasis and regeneration are strictly regulated processes, where the proliferation of cells is tightly controlled by growth suppressive and apoptotic environmental signals, and where possible mutations, i.e. mistakes in the genome copying process, are rapidly repaired. Cancer is a disease with a single cell origin. During a long, multistep Darwinian selection process, the pre-cancerous cell acquires several biological properties via genetic and epigenetic changes. These properties, or hallmarks, enable the survival and development of the group of dividing cells into multicellular organ called tumor. The tumor contains not only malignant cancer cells but also several types of microenvironmental cells comprising the tumor stroma (Weinberg 2014). According to the six classical hallmarks, malignant cancer cells succeed in sustaining proliferative signaling, evading growth suppressors, activating invasion and metastasis, enabling replicative immortality, inducing angiogenesis and resisting cell death. Two additional, strongly emerging hallmarks of cancer are reprogramming of energy metabolism and evading immune destruction. Two characteristics that enable this development are genomic instability that provides the genetic diversity required for selection and cancer evolution, and inflammation that promotes several hallmark capabilities (Hanahan and Weinberg 2011). Cancer is not a communicable disease apart from two cancers affecting canine species: devil facial tumor disease (DFTD) and canine transmissible venereal tumor (CTVT) and one transmissible sarcoma affecting Syrian hamsters (Murchison et al. 2010). 2.1.2 Oncogenesis, tumor microenvironment and cancer stem cell theory The primary oncogenesis is caused by exposure to mutagens (sometimes classified as initiator and promoter mutagens) followed by a failure in the DNA-damage sensing or repair/apoptosis mechanisms. Occasionally, hereditary factors (e.g. BRCA gene mutations in breast cancer) may contribute to the initial failure in the DNA-damage sensing and repair mechanisms, while in other cases this machinery may be overdriven by massive and repetitive carcinogen exposure as is the case in smoking related lung cancer. Persistent viral infections can also induce cancer due to the virally encoded oncogenes and/or as a result of infection related chronic inflammation. Viruses oncogenic for humans and other vertebrates include members of papilloma/polyoma viruses, herpes viruses, hepatitis viruses and retroviruses, while some adenoviruses are known to be tumorigenic in hamsters (Weinberg 2014). The microenvironment of cancer cells is undergoing constant spatio/temporal change. During malignant development, the microenvironment changes dramatically from the primary location to the invasive front and then again from the hostile environment of the blood circulation to the completely new microenvironments faced by distant 4 metastases. Migration to new anatomical loci demands a major adaptation from the cancer cells. Cancer cells are thus under constant selective pressure and the process from initial oncogenic mutations to widespread deadly metastases typically spans several years. Indeed, in certain malignancies such as breast cancer and melanoma dormant micrometastases are found in distant target organs already in the early phase of the disease, and after therapeutic destruction of the primary tumor, the metastases might erupt decades later (Burrell et al. 2013; Hanahan and Weinberg 2011; Weinberg 2014). In other cancers, such as pancreatic ductal adenocarcinoma, metastasis seems to occur late in the disease progress (Hanahan and Weinberg 2011). This suggests that tissue colonization, i.e. the development of micrometastases into macrometastases, which is the last and typically fatal phase of tumorigenesis, is often the bottleneck in the complex course of the disease. Even the metastatic preference of different neoplasms for certain target organs seems to depend on the susceptibility of different environments to support metastatic outgrowth. The direction of blood flow from the primary anatomical location plays likely also a part in cases, when, for example, liver metastases sprout from primary colon carcinomas. The evolutionary process creates major genetic and epigenetic clonal heterogeneity not only between the primary and metastatic tumors, but also within the primary tumor itself. In fact, the metastasis can be a two-way process and cancer cells migrate also from the metastases back to the primary tumor thus further contributing to the total heterogeneity of the disease (Burrell et al. 2013; Hanahan and Weinberg 2011; Weinberg 2014). Tumors are complex organs colonized in their microenvironment not only by neoplastic cells with varying sets of mutations and stages of differentiation (including cancer stem cells, CSCs), but also with several types of stromal cells. These stromal cells include local and bone marrow (myeloid) derived stem/progenitor cells, fibroblasts, vascular pericytes and endothelial cells and several types of inflammatory immune cells such as macrophages, neutrophils, mast cells, cytotoxic T-lymphocytes (CTLs), regulatory T-cells (T-regs), B-cells, myeloid derived suppressor cells (MDSC) and macrophages. The last two immune cell classes are closely related and often M2 biased and thus tuned for tissue repair, in contrast to the cytotoxic M1 phenotype (Hanahan and Weinberg 2011; Mantovani et al. 2009). CSCs are a subpopulation of neoplastic cells within tumors that express typical stem cell markers, have an enhanced capability to induce tumors in immunocompromised mice and are inherently resistant to chemo- and radiotherapy, possibly due to their slow pace of proliferation. There is recent data suggesting that CSCs are actually cells that have undergone epithelial-to-mesenchymal transition (EMT), epigenetic change in gene regulatory program induced by inflammatory cytokines and growth factors TNF, IL-6, TGF, HGF and EGF. The EMT program has long been recognized as being crucial for cancer cell invasion and metastasis but it is also a normal physiological program in wound healing and embryogenesis initiated by the sensing of hypoxia (Hanahan and Weinberg 2011; Holzel, Bovier, Tuting 2013). In addition to EMT induction, TGF has also many other important roles in tumor progression, for example by exerting immunosuppressive functions (Pickup, Novitskiy, Moses 2013). Epithelial carcinoma cells acquire a mesenchymal (fibroblastic) phenotype in the invasive front of the tumors by activation of EMT gene regulatory program, process characterized by loss of adhesion molecules such as E-cadherin. Cancer cells then invade the blood vessels via proteolytic destruction of 5 extracellular matrix (ECM) and, once in the circulation, form aggregates with platelets. These aggregates physically clog capillary vessels anchoring the cancer cell to its new metastatic location. Cancer cells can reach the circulation also via the lymphatic system. It is noteworthy, that platelets have a second important function in the metastasis cascade as they protect the cancer cells from the immune system during the circulation (Weinberg 2014). In their new metastatic location, the mesenchymal cancer cells can undergo the reverse program, mesenchymal-to-epithelial transition, and stay dormant in this epithelial state for an extended time. Successful tissue colonization is often followed by reactivation of the EMT program followed by further metastatic spawning of new malignant cells. In the mechanistic cancer invasion and metastasis model, the EMT program induced in the cancer cells by inflammation and hypoxia in the tumor microenvironment serves a dual function, first by enabling the cancer cell mobility, and secondly, by creating therapy resistant CSCs. These CSCs, by their very definition, are also capable of regenerating the tumor even after extended time (possibly via transient epithelial phase) once the therapy has ended. Interestingly, very recently the EMT program has been implicated in inflammation-induced phenotypic plasticity and resistance not only against the classical cancer therapies but also targeted therapies and even immunotherapy as a result of changes in the antigenic landscape (Holzel, Bovier, Tuting 2013). 2.1.3 Cancer immunoediting: Sum of immunosculpting and immunosuppression Cancer immunoediting is a process in which an individual’s immune cells (especially Tlymphocytes of adaptive immunity) protect him against tumorigenesis or sculpt the antigenic profile of the tumors by removing cells, which present immunogenic neo-antigens but are incapable of exerting immunosuppression (DuPage et al. 2012; Matsushita et al. 2012). Immunoediting has three phases: elimination (also called cancer immunosurveillance), equilibrium and escape. In the elimination phase, some (occasionally all) transformed cancer cells are recognized and eliminated by the various arms of the host’s immune system. Natural killer (NK) cells can use their NKG2D receptors to identify tumor stress ligands induced by carcinogens, viral infections or genotoxic stress. CD8+ and CD4+ T-cells recognize tumor associated (TAA) and specific (TSA) antigens presented on MHCI and MHCII molecules of dendritic cells, respectively. B-cells can augment the antitumor immune response by secreting anti-tumor antibodies (Abs) against epitopes on the cancer cells. Other, more recently discovered, immune cells possessing antitumor functions include natural killer T-cells (NKT cells) and T cells. In addition to direct tumor killing, these cells can also secrete cytotoxic and immunostimulatory cytokines. Large amount of data has accumulated during the past few years strongly suggesting that cancer immunosurveillance does exist in mice, and most likely, also in humans. For example, mice with varying genetic defects either in their innate- (including type I and type II IFNs) or adaptive immune responses are more prone to developing tumors both spontaneously and after carcinogen or oncovirus challenge (Dunn, Koebel, Schreiber 2006; Weinberg 2014). Furthermore, only a few of the tumors chemically induced in immunocompromised mice grow when implanted into immunocompetent hosts, whereas all of the tumors induced in immunocompetent mice can thrive in their own 6 immunocompetent strain, demonstrating that primary carcinogenesis does not always produce immunogenic cancer cell clones, but when it does, these clones are indeed rapidly eliminated by the host immune system (Shankaran et al. 2001; Weinberg 2014). If some of the tumor cells survive the elimination, equilibrium phase follows where the tumor does not grow in size but remains seemingly dormant via equilibrium of constant cell proliferation and immunodestruction. The third and the final phase, the escape, begins when immunosculpting has produced non-immunogenic tumor clones as a result of mutations/silencing in the antigen expression (if this is redundant for the tumor), antigen processing or the antigen presenting (MHC) machinery. Escape can take place also when tumor clones with immunosuppressive capabilities emerge or the immune system of the host becomes exhausted, the latter marked, for example, by expression of the inhibitory CTLA4 receptor on T-cells (Hodi 2013; Mellman, Coukos, Dranoff 2011). Occasionally the non-immunogenic or immunosuppressive tumor cell variants might be present already early in disease progression, and since these variants are not eliminated, they also omit the equilibrium phase and proceed directly to escape (Dunn, Koebel, Schreiber 2006). Tumors and their stromal cells such as macrophages, regulatory-T cells (Tregs) and myeloid derived suppressor cells (MDSCs) can produce different soluble or membrane bound immunosuppressive factors in the tumor microenvironment including PD-L1, TGF, RCAS1, IDO2, M-CSF, Galectin-1, VEGF-A, adenosine, prostaglandin E2, IL-10 and MICA/MIC-B. MIC-A and MIC-B are soluble NKG2D ligands that disturb both NK and effector T-cell functions. STAT3 transcription factor is the crossing point for numerous oncogenic and immunosuppressive signaling pathways and thus is constitutively activated in many tumors (Mellman, Coukos, Dranoff 2011; Rabinovich, Gabrilovich, Sotomayor 2007; Salih, Holdenrieder, Steinle 2008; Yu, Kortylewski, Pardoll 2007). Very recently, two independent groups reported a new immunosuppressive ligand “V-domain Ig suppressor of T-cell activation” (VISTA), which mimics PD-L1 in function (Le Mercier et al. 2014; Lines et al. 2014). However, in contrast to PD-L1 which is expressed not only by dendritic cells but also by cancer cells, VISTA seems not to be expressed by cancer cells but only by T-reg and myeloid cells in the tumor microenvironment. The immunosuppressive factors expressed by cancer cells and immunosuppressive cells of the tumor microenvironment can act by directly suppressing T-cell and dendritic cell activation and function, or by activating and expanding immunosuppressive myeloidderived mesenchymal stem cells, T-regs and MDSCs. VEGF is a multifunctional immunosuppressant and it has direct effects against T-cell development. In addition, it can cause disturbances in vascular development resulting in aberrant blood vessels, thus hindering the carefully controlled process of effector lymphocyte extravasation (Mellman, Coukos, Dranoff 2011). Some of the immunosuppressive factors are produced in response to hypoxia, a very common state of tumors with abnormal vasculature and immunosuppressive in its own right. Hypoxia induces glycolysis resulting in the generation of lactic acid, which lowers pH in the tumor microenvironment, thus leading to suppression of lymphocyte function. Interestingly, tumors also often use glycolysis and lactic acid fermentation even when oxygen is available perhaps partly for the purpose of macromolecule synthesis, a phenomenon called the Warburg-effect. This deregulation of 7 cellular energetic is one of the emerging hallmarks of cancer (Choi et al. 2013; Hanahan and Weinberg 2011). In addition to the Darwinian selection of antigen escape variants and immunoevasion by direct expression of immunosuppressants, tumors can also avoid attack by the immune system via inflammation induced transient dedifferentiation, a “wound healing” response after therapeutic injury (Holzel, Bovier, Tuting 2013). Finally, some cancers seem to bias the adaptive immunity towards the Th2 polarity representing allergy and neutralizing Ab response type of immunity (mediated by IL-4) to avoid the tumoricidal Th1 response of cellular immunity and opsonizing Abs mediated by IFN- and IL-12 (Abbas, Lichtman, Pallai 2007; Protti and De Monte 2012). The functional role of recently discovered Th17 cells in cancer remains controversial, as these cells seem to possess both protumorigenic and antitumorigenic properties (Ye, Livergood, Peng 2013). Th17 cells are proinflammatory and important mediators of autoimmune diseases. Both immunosuppressive Tregs and immunostimulative Th17 cells need TGF for their differentiation from the naïve CD4+ T-cells, but certain environmental cues such as the inflammatory cytokine IL-6 can skew the delicate balance between inflammation and immune tolerance by triggering the differentiation of naïve CD4+ T-cells into Th17 cells instead of Tregs (Barbi, Pardoll, Pan 2013). Virus-infected cells can secrete IL-6 (Dienz et al. 2012) and IL-6 could be possibly ectopically expressed from oncolytic viruses to induce antitumor immunity. Taken together, the immunosuppressive microenvironment of cancers is very complex and might also vary significantly between different tumors. Some immune cell classes generally considered to be tumor destructive, such as B-cells, NK-cells, NKT-cells, T cells, have been also reported in certain circumstances to secrete IL-10 and thus exert immunosuppressive functions (Mellman, Coukos, Dranoff 2011; Salih, Holdenrieder, Steinle 2008). 2.1.4 Cancer in Finland and modern cancer therapies As a result of advances in contemporary medicine, cancer is no longer an invariably lethal disease. In fact, because of longevity, approximately one third of Finnish population will suffer cancer at some point during their lifetime. Approximately one third of all cancers are estimated to result from the use of tobacco products, which in addition to causing lung cancers are involved in the etiology of carcinomas of mouth, pharynx, kidney, pancreas, esophagus, cervix and uterus, and might contribute also to breast cancer (Pukkala, Sankila, Rautalahti 2011). The prognoses of certain malignancies such as liver, gall bladder, lung and pancreatic carcinomas and glioblastoma multiforme (GBM) remain still poor (Pol et al. 2013; Pukkala, Sankila, Rautalahti 2011). GBM is the most common and most aggressive primary brain malignancy with a median survival of 15 months and 5-year survival of less than 10% (Pol et al. 2013; Pukkala, Sankila, Rautalahti 2011). The DNA-alkylating agent, temozolomide, has displayed some efficacy in combination with standard surgery and chemo/radio therapy. However, patients cannot be cured and they rarely live beyond two years post diagnosis (Maatta et al. 2009; Zemp et al. 8 2010). In other malignancies, such as colorectal carcinoma, the prognosis depends heavily on the time of diagnosis: locally confined carcinoma in situ can be treated by resection, whereas advanced invasive and metastatic disease is considered incurable. Oncolytic virotherapy represents a novel possibility to treat these devastating diseases, and while no virus medicine has been approved for western markets, T-VEC as the first one has recently completed phase III trial for advanced melanoma (see chapter “2.4 Oncolytic Virotherapy”) (Pol et al. 2013; Russell, Peng, Bell 2012). Gene therapy and immunotherapy of cancer are two additional modern technologies being evaluated in clinical trials and these approaches frequently overlap with oncolytic virotherapy (see chapter “2.4.2 Oncolytic virotherapy overlaps with gene therapy and immunotherapy of cancer – emerging concept of oncolytic immunovirotherapy”). After a long and complex process, the first gene therapeutic drug, Glybera (alipogene tiparvovec), was recently approved for clinical use in Europe. Glybera is an adeno-associated viral (AAV) vector engineered to express lipoprotein lipase in muscle to be used in the treatment of the very rare condition of lipoprotein lipase deficiency (YlaHerttuala 2012). Classical cancer therapies comprise treatments such as surgery, radiotherapy, chemotherapeutics and anti-hormonal treatments (Pukkala, Sankila, Rautalahti 2011). Some targeted cancer therapies are already widely in use e.g. small molecule inhibitors (SMIs) and monoclonal antibodies against targets such as Her2/neu, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), CD20, CD52 and CD33. The antibodies antagonize oncogenic pathways and may also act via opsonizing the tumor cells thus causing cellular death or removal by antibody-dependent cellular cytotoxity (ADCC). According to preclinical data these antibodies might also stimulate adaptive immune responses (Mellman, Coukos, Dranoff 2011). Furthermore, the anti-CTLA-4 antibody, ipilimumab, was recently approved for the treatment of metastatic melanoma demonstrating a marked and occasionally sustaining survival benefit for patients in the late stages of the disease (Hodi 2013; Mellman, Coukos, Dranoff 2011). CTLA-4 is expressed on the T-cell surface 2-3 days after activation and its natural role is to down-regulate T-cell activation thus preventing autoimmune damage resulting from prolonged immune attack. CTLA-4 competes with the activating CD28 receptor on Tc- and Th-cells for binding with the B7 costimulatory ligand on antigen presenting cells (APCs) leading to T-cell deactivation (Mak and Saunders 2011). Intriguingly, CTLA-4 ligation is also important for Treg-mediated immunosuppression through a still poorly understood mechanism, and ipilimumab is also hypothesized to block this interaction. A second class of antibodies on the verge of clinical approval target the inhibitory PD-1 - PD-L1(L2) interaction between activated T-cell and APCs (Mellman, Coukos, Dranoff 2011). PD-1 is functionally related to CTLA-4 as they both mediate immune exhaustion after prolonged activation, but PD-L1 and PD-L2 can often be found not only on APC but also tumor membranes adding a layer of specificity for cancer immunotherapy as compared to CTLA-4. Indeed, antibodies against PD-1 have demonstrated efficacy in clinical trials against a variety of cancers with less autoimmune side effects than ipilimumab (Mellman, Coukos, Dranoff 2011). Interestingly, PD-L1 can also induce Treg differentiation together with TGF (Francisco, Sage, Sharpe 2010). 9 A third class of clinically promising immunomodulatory antibodies against cancer are the agonistic CD40 monoclonal antibodies, which stimulate APCs by binding to their CD40 receptors thus activating CTLs. In the absence of T-cells, these antibodies can activate also cytotoxic myeloid cells (Vonderheide and Glennie 2013). Other cancer immunotherapies in clinical use include IFN- and IL-2 treatments for melanoma and renal cell carcinomas, the two cancer types considered to be the most immunogenic, and intravesical injection of bacillus Calmette-Gue´rin used for the treatment of superficial bladder cancer. Interestingly, those melanoma patients who respond best to the IFN therapy are often naturally prone to autoimmune diseases suggesting that the immunity of these patients might be Th1 biased. One type of immunotherapy, though originally not considered to be one, is the allogeneic bone marrow transplantation and infusion of donor lymphocytes, which are used in the treatment of leukemias and lymphomas where “graft-versusleukemia effect” has been found to have major contribution to the therapy. Finally, the first active cancer immunotherapy Provenge (sipuleucel-T) involving ex vivo activated peripheral blood mononuclear cells was approved in 2010 by Food and Drug Administration for the treatment of advanced prostate cancer. Provenge’s mechanism of action remains incompletely understood, and its therapeutic effects are modest, but the approval was granted since the current therapeutic options for advanced prostate cancer are very limited (Mellman, Coukos, Dranoff 2011). In addition to monoclonal antibodies, the emergence of potent SMIs against a variety of natural or mutated kinases, such as Imatinib (Gleevec) for inhibition of BCR-ABL1 mutant fusion kinase in chronic myeloid leukemia (CML), has advanced significantly the treatment of cancer. Unlike chemotherapeutics and radiation which target all dividing cells, these kinase inhibitors are more cancer specific, thus increasing their safety. Nevertheless, as with traditional therapies, cancer cell clones resistant to the inhibitors frequently develop as a result of acquired mutations to the targeted kinases or the cancer cell’s ability to activate alternative signaling routes (Zhang, Yang, Gray 2009). 2.2 INTERFERONS IN CELL AUTONOMOUS, INNATE AND ADAPTIVE IMMUNITY In order to enter a cell, hijack its macromolecular synthesis machinery and to launch a productive infection, viruses first need to breach natural protective barriers such as the skin, mucus, saliva, tears and gastric acid. Once inside the cell, the first branch of immunity that the virus will encounter is cell autonomous immunity, most importantly the interferon (IFN) system (Figure 1). 10 Figure 1. Type I IFN response in vertebrate immunity. Interferons (INFs) are evolutionally conserved cytokines amongst vertebrates, which have broad antimicrobial, immunostimulatory and growth-suppressive functions. Type I IFNs protect against viruses, bacteria and protozoa and act as a link between innate-, adaptive- and cell-autonomous immunity (Abbas, Lichtman, Pallai 2007; MacMicking 2012). Interferon defense system is evolutionally conserved amongst all vertebrates. IFNs have broad antimicrobial and growth-suppressive functions and can stimulate both the innate and adaptive branches of immunity (MacMicking 2012). IFNs are classified according to their receptors into type I (IFN-, IFN-, IFN-, IFN-, IFN-, IFN- and 13 different IFN- subtypes in humans and 14 in mice), type II (IFN-) and type III (4 different IFN- subtypes) interferons. While type I and type III IFNs have separate receptors, i.e. IFNAR and IFNLR, respectively, they share the same downstream signaling pathway involving Signal Transducer and Activator of Transcription STAT1/STAT2 heterodimer phosphorylation by Jak1/Tyk2 kinases resulting in formation of the STAT1/STAT2/IRF9 transcription complex (Honda and Taniguchi 2006; MacMicking 2012; van Pesch et al. 2004). All IFN classes possess documented antiviral activity but to this end, the best characterized are the type I interferons (mainly IFN-). The only type II IFN is IFN- and it signals via IFNGR and STAT1/STAT2 heterodimer. IFN- is important mediator of adaptive immune response, but it is also involved in virus clearance from the central nervous system (CNS) at least during Sindbis and Vaccinia virus infections (Binder and Griffin 2001; Kundig, Hengartner, Zinkernagel 1993; McFadden et al. 2009). Type I IFN seems to be more important than type II IFN in the restriction of Herpes simplex virus (HSV)-I CNS replication (Leib et al. 1999). While type I IFNs can be responded to and secreted by most somatic cells, both the production and response to antiviral type III IFNs seem to be limited to epithelial cells and tissues, especially the lungs. Nevertheless, the similar signal transduction cascades downstream of type I and type III IFN receptors results in induction of the same set of antiviral genes. The limited response to type III IFNs is related to the fact that high levels of the type III IFN receptor are found only in epithelial 11 cells, and the potential mechanisms of epithelial cell and tissue type specific induction of this IFN class are unknown (McFadden et al. 2009). The engagement of pattern recognition receptor (PRR) with pathogen associated molecular patterns (PAMPs) induces the expression and auto/paracrine signaling of type I IFNs. The PRRs which are most important in viral infections can be roughly divided into two classes according to the PAMP type that they detect and the cell types in which they are found: Toll like receptors (TLRs) which detect microbial structural components and nucleic acids, are mostly found in plasma membranes and endosomes of leukocytes whereas the cytosolic pattern recognition receptors (e.g. RIG-I like receptors RLRs) detect foreign nucleic acids and are found in most cell types. The expression patterns of some TLRs vary between species, and whereas TLR9 detecting CpG DNA is expressed in B-cells and several types of phagocytes in mice, it is expressed only in plasmacytoid dendritic cells (pDCs) and B-cells in humans (Hochrein and Wagner 2004; Thompson et al. 2011). In addition to immune cells, TLRs have been detected also in cancer cells (Sato et al. 2009). Furthermore, non-immune cells can detect cytosolic DNA via receptors such as IFI16 or RNA polymerase III, the latter curiously recognizing foreign DNA indirectly by synthesizing RIG-I substrates out of it. However, signaling via these receptors remains incompletely understood and there might well be cell type specific differences (Broz and Monack 2013). RLRs belong to the H box helicase (Asp–Glu–x–Asp/His box) family (Goubau, Deddouche, Reis e Sousa 2013). They are crucial for the detection of double-strand RNA (dsRNA) and single-strand RNA (ssRNA) genomes as well as replication intermediates of RNA-viruses. RLR family has three central members: MDA-5 (melanoma differentiation factor 5), RIG-I (retinoic acid-inducible gene I) and LGP-2 (laboratory of genetics and physiology 2). RLRs signal through MAVS (mitochondrial antiviral signaling, aka IPS-1, CARDIF or VISA) activating IRF3/IRF7 (in homo- or heterodimers), IRF1 and NF-B leading to the expression of type I IFNs and pro-inflammatory cytokines, including chemokines (Broz and Monack 2013; Honda and Taniguchi 2006). In addition to detecting viral RNA, RLRs themselves are also induced by type I IFNs thus participating in a positive-feedback loop (Goubau, Deddouche, Reis e Sousa 2013). Other PRR classes found in the animal kingdom, which are important for innate immunity against pathogens such as viruses include C-type lectin receptors (CLRs) and scavenger receptors found in phagocytes and some epithelial cells (Jiang et al. 2006), and several soluble PRRs such as mannose binding lectins (Dommett, Klein, Turner 2006). Furthermore, in addition to the classic RLRs new members of cytosolic PRRs are constantly discovered (Broz and Monack 2013). Complement is not part of the PRR system per se but not only does it recognize and destroy lectin or antibody opsonized – microbes, it can also target foreign non-self structures unprotected by complement regulatory proteins (Ricklin et al. 2010). Type I IFNs are produced by and act on almost all nucleated cell types. They can inhibit every stage of the virus lifecycle by inducing hundreds of interferon stimulated genes (ISGs) (Goubau, Deddouche, Reis e Sousa 2013; Grabowska and Riemer 2012; Liu et al. 2012; Sadler and Williams 2008; Thomas et al. 2011). Type I interferons signal through common IFNA-receptor (IFNAR) coupled with JAK/STAT, p38 MAPK and PI3K pathways. However, type I IFNs differ to some extent in the physiological responses that they evoke 12 such as potency against different viruses through poorly understood mechanisms. All type I IFNs cause identical conformational changes in their heterodimeric IFNAR1/2 receptor. This leads to receptor autophosphorylation and activation of Tyk2 and Jak1, which are constitutively attached to IFNAR1 and IFNAR2, respectively, and which further activate STAT1 and STAT2 by phosphorylation. The phosphorylated STAT1 and STAT2 heterodimerise and become associated with IRF9 in the cytoplasm or nucleus. The resulting complex Interferon-stimulated gene factor 3 (ISGF3) acts as a transcription factor binding to interferon stimulated response elements (ISRE) found on promoters of many ISGs. ISGF3 is the only factor binding to ISRE elements. Not only the classical STAT1/STAT2 heterodimer but also the STAT5/CrkL dimer can be phosphorylated and then translocated to the nucleus upon INFAR signaling, inducing the expression of certain ISGs. However, in contrast to ISGF3, STAT5/CrkL complex binds to interferon gamma activated site (GAS) rather than ISRE, thus resembling IFN- activated STAT1-homodimer. ISRE and GAS signaling are not nevertheless biologically fully distinct, and many ISGs harbor both elements. Furthermore, type I IFNs can also induce STAT1 homo-dimerization and signaling via GAS (HervasStubbs et al. 2011; MacMicking 2012; Platanias 2005). Finally, STAT1 has been reported to possess some direct antiviral functions against SARS-CoV, independent of type I, II or III IFN signaling (Frieman et al. 2010). The anti-proliferative effects and ISG induction levels seem to correlate positively with receptor binding affinities of different type I IFNs (Platanias 2005; Thomas et al. 2011). In addition to the differences in the quantity also the quality of ISG induction may occasionally vary between different type I IFNs, and IFN- but not IFN- was reported to induce -R1/CXCL11 (Rani et al. 1996). Importantly, as a result of complex and poorly understood cell physiological interactions, ISGs can also have different roles in different cells and responses to different viruses can be dramatically different also in a given cell type (McFadden et al. 2009). IFN- can be divided into early (IFN-4 and IFN-) and late interferons (IFN-non4, i.e. rest of the IFN- classes) according to whether in addition to PRR activation, priming by auto/paracrinic IFNAR signaling (and resulting IRF7 expression) is a prerequisite for their expression. IFN- is the master-regulator of type I IFN response requiring only PRR activation and IRF3-homodimerization for its induction. IFN is transcribed approximately 2h p.i., which is even earlier than the induction of the other early type I IFN, IFN-4, the transcription of which is detectable around 8h p.i. (Lienenklaus et al. 2008). Paracrine signaling of type I IFN primes neighboring cells for an imminent virus attack by induction of effector ISGs and IRF7. This process is fast, and the first signs of phosphorylated STAT1 can be found already 5 minutes after IFN- stimulation (Dempoya et al. 2012). In the case of PAMP detection in the primed cell, the induced IRF7 is activated by phosphorylation and homo-dimerization, or by heterodimerization with IRF3. Phosphorylated IRF7 dimer is then translocated to the nucleus where it induces the rapid transcription not only of early type I IFNs but also of all the late type I IFNs hence resulting in positive-feedback signaling cascade (Figure 2) (Honda and Taniguchi 2006). Interestingly, type I IFN independent STAT1 phosphorylation has also been recently reported (Dempoya et al. 2012). However, STAT1 phosphorylation was not detectable before 8 h after poly I:C induction of IFNAR-deficient U5A cells, a time point which is 5 h later than that found in IFNAR-signaling competent A549 cells. 13 Figure 2. Paracrinic signaling of type I IFNs (modified with permission from Zemp et al. 2010). 1. Nucleic acids of invading microbes such viruses are detected by pathogen recognition receptors (PRRs), such as cytosolic helicases Rig-I and MDA5 in case of SFV. 2. IRF3 is phosphorylated in a process involving Mitochondrial antiviral-signaling protein (MAVS, not shown) and TANK-binding kinase 1 (TBK1). 3. Phosphorylated IRF3 goes through homodimerization (and heterodimerization with IRF7, not shown) and nuclear translocation leading to induction and low level secretion of chemokines (not shown) and first early type I IFN, IFN-. In addition, some interferon stimulated genes (ISGs) are induced already at this stage, including Zinc-Finger Antiviral Protein (ZAP) and Viperin, both shown to have potent antiviral functions against SFV. 4. IFN- is secreted and causes autocrine and paracrine signaling via common heterodimeric type I IFN receptor IFNAR1/IFNAR2 the chains of which are constitutively coupled to TYK2 and JAK1 kinases, respectively (not shown). 5. The kinases phosphorylate STAT1 and STAT2 causing their heterodimerization and attachment with IRF9, forming the ISGF3 complex. 6. ISGF3 translocates to nucleus and induces transcription of multiple (potentially hundreds) ISGs including antiviral effectors such as OAS, PKR, Viperin, Zap, Ifit1/3 or PRRs such as Rig-I. Some of these ISGs such as Mx1 and ISG20 (not shown) are “true-ISGs” that are not induced by activated IRF3 (see point 3.). 7. In addition to antiviral ISGs and PRRs, IRF7 is induced, priming the cell to react to imminent microbial attack. 8. Nucleic acids of invading microbes are detected by upregulated PRRs in the primed cell 9. leading to IRF7 activation by phosphorylation and IRF7 homodimer formation, which 10. translocates to the nucleus to drive the expression of type I IFNs such as IFN- and several classes of IFN- (Honda and Taniguchi 2006; Zemp et al. 2010). The binding of type I IFN to its receptor initiates a complex and incompletely understood signaling process which can occasionally be IFN type and cell type-specific. In addition to STAT1/STAT2 it can involve also several other STATs (STAT 3 to 6) in homo- and heterodimeric complexes. STAT4 and STAT6 mediated signaling seems to be cell type specific taking place primarily in endothelial and lymphoid cells. Furthermore, in addition 14 to prototypic JAK/STAT signaling, type I IFN signal can be mediated via PI3K and p38 MAPK pathways. Type I IFN induced PI3K signaling can regulate also translation in addition to transcription via mammalian target of rapamycin (mTOR). The cross-talk between these different pathways (especially STAT and p38) downstream of IFNA-receptor appears to be crucial for full scale cell autonomous antiviral response (Platanias 2005). Interestingly, despite the shared receptor IFN- and IFN- induced ISGs do not completely overlap even in one given cell line and 20 ISGs, including PKR and Hif-1, were reported to be exclusively or preferentially induced by IFN- (Der et al. 1998). Recently, more light was shed into the mechanisms of type I IFN specificities when IFN- but not IFN- was found to be capable of signaling via single IFNAR1 receptor chain inducing separate set of genes compared to IFN- signaling via full IFNAR1/2 receptor (Kaur and Platanias 2013). Furthermore, while all nucleated cell types are thought to express IFNAR, the magnitude of ISG induction can vary markedly between tissues (Briolat et al. 2014). In some, presumably rare, cases the presence of type I IFN can actually increase virus replication at least in vitro when IFN signaling, while inherently antiviral, happens to stimulate the expression of the virus receptor. This has been reported to take place in porcine reproductive and respiratory syndrome virus (pRRSV) infection of monocytes (Duan, Nauwynck, Pensaert 1997) and porcine circovirus type 2 (pCV2) infection of porcine cell lines (Meerts, Misinzo, Nauwynck 2005). In addition to the different IFNs, TNF which is produced by leukocytes after TLR engagement also possesses the ability to induce the pro-inflammatory transcription factor NF-B promoting cellular antiviral state. Both TNF and IFNs also mediate inflammation and prime antiviral adaptive immune responses. Despite the clear direct antiviral effects of TNF reported already 1980s, it has been nevertheless much less studied in this context than the IFNs. The third class of cytokines that has important antiviral functions is the interleukins, which however, while also having some direct antiviral effects, mostly stimulate antiviral adaptive immunity (McFadden et al. 2009). Finally, IL-6 can induce ISG gene expression via ISRE elements similarly to type I IFNs, but it uses STAT3 instead of ISGF3 (Hubackova et al. 2012). IL-6 is induced in many normal tissues as a result of PRR activation or an inflammatory cytokine signal (including IFNs). This induction is orchestrated by NF-B together with some other transcription factors (Hong, Angelo, Kurzrock 2007). The transcription of IFN- and several other cytokines is controlled by the enhanceosomecomplex comprised of NF-B, phosphorylated IRF3 (and IRF7 especially in type I IFN primed cells), CBP (or p300) and ATF2-JUN (Honda and Taniguchi 2006). The direct effects of type I IFNs on tumors and cellular immunity are remarkably versatile and they can include direct (immunogenic) oncolysis, inhibition of angiogenesis, MHC-I induction on cancer cells and stimulation of NK, DC and T-cell responses (Hervas-Stubbs et al. 2011). Nevertheless, the effects of type I IFNs on adaptive immunity are both time and concentration dependent. Indeed, type I IFNs have at least seemingly immunosuppressive functions in deletion of old memory T-cells and premature activation of naïve, unproliferated T-cells that have not yet met their cognate, MHC-presented antigen (Welsh et al. 2012). Furthermore, high type I IFN concentrations can impair IL-12 production by DCs (Hervas-Stubbs et al. 2011). The existence of multiple different type I IFNs all signaling 15 via a single receptor probably highlights the evolutionary pressure to ensure that the crucial functions of these cytokines are maintained in antiviral innate and adaptive immunity. While type I IFNs seem to exhibit a certain level of overlap and redundancy, many of their specific roles probably still remain undiscovered. Much of the diversity might lie in the tissue-specific induction rather than in cell type-specific response. Indeed, laboratory of Paul Hertzog demonstrated recently that IFN-, a previously uncharacterized type I IFN, has a unique role in the protection of the female genital tract, and in striking contrast to the other known type I IFNs, it is not induced by PRR activation but instead it is constitutively produced by epithelial cells (Fung et al. 2013). Probably for historical reasons the majority of the studies of type I IFN immunostimulatory functions have been conducted using IFN- subtypes, which are also the ones mainly employed for interferon therapy of different neoplasms (mostly melanoma and hematological malignancies) (Hervas-Stubbs et al. 2011). Hence, it remains poorly defined whether the different type I IFN classes differ in their immunoregulatory and anti-cancer efficacy. However, as type I IFN anti-proliferative effects on cells seems to depend on the IFNAR binding affinity (Thomas et al. 2011) and IFN- has been shown to possess higher receptor affinity and ISG induction power than IFN--classes (van Boxel-Dezaire, Rani, Stark 2006), further comparative studies in the context of cancer immunotherapy are highly warranted. 2.3 ALPHAVIRUSES 2.3.1 Taxonomy, natural life cycle and pathogenesis of Alphaviruses and Semliki Forest virus The genus Alphavirus belongs to family Togaviridae and is comprised of more than 40 small (65-70nm), enveloped, icosahedrally shaped positive-sense ssRNA viruses (Jose, Snyder, Kuhn 2009). Alphaviruses are arthropod-borne viruses (arboviruses) that use birds and mammals as amplifying hosts and are typically spread by mosquito vectors (Murray, Rosenthal, Pfaller 2013). In addition, aquatic salmonid alphavirus (SAV) species exist with an unknown transmission vector, although some louse species have been proposed as the vector (Metz et al. 2011). Some alphaviruses are important pathogens of humans and domestic animals such as Old World Chikungunya virus (CHIKV) and Sindbis virus (SINV) and New World viruses Venezuelan- (VEEV), Western- (WEEV), and Eastern equine encephalitic virus (EEEV). The Old World viruses typically cause symptoms such as fever, rash and arthritis, which especially in CHIKV infection can be coupled with long lasting and disabilitating arthralgia. The New World alphaviruses EEEV, VEEV and WEEV can cause deadly encephalitis in horses and occasionally also in man while no vaccines are widely clinically available (Chattopadhyay et al. 2013; Jose, Snyder, Kuhn 2009). Live attenuated, and formalin inactivated vaccines against VEEV exist for use in horses, U.S. military workers and laboratory personnel at risk of infection, but the vaccines have limited safety and immunogenicity in humans (Dupuy et al. 2011; Goldberg March 6, 2014; Paessler and Weaver 2009). 16 Interestingly, even though Semliki Forest virus (SFV) belongs to Old World Alphaviruses which are associated rather with arthritis than neurologic type of pathologies, even the avirulent strain A7(74) and derived vector VA7 are neurotropic in mice. SFV is not considered to be a human pathogen, but some neuropassaged strains such as L10 cause a rapid and fatal CNS infection in mice and other rodents (Bradish, Allner, Maber 1971). SFV4 is a less virulent SFV strain, which replicates efficiently in mouse brain if administered by an intracranial (ic) injection but only occasionally when injected intraperitoneally. This probably reflects the reduced neurotropism of SFV4 as compared for example to the avirulent A7(74) (Fragkoudis et al. 2007). In addition, the avirulent SFV strains cause lethal encephalitis in neonatal mice younger than 12 days while mice older than 14 days are generally protected against the disease, reflecting a poorly understood phenomenon of age-dependent neurovirulence. While decreased membrane biogenesis in mature neurons has been postulated to underlie the reduced infectivity of A7(74) (Fazakerley 2002), it would be important to study in this context the role of the immunological maturation from Th2 to Th1 biased immunity occurring around day 14 postnatal (Lovett-Racke et al. 2000). A7(74) does not replicate in the CNS cells of SCID mice that are suppressed in their adaptive immunity (Amor et al. 1996). However, IFN- the prototypical Th1 cytokine (along with IL-12), is secreted not only by T-cells but also by NKcells, a class of immune cells present also in SCID mice. CNS infection of avirulent SFV strains is known to cause not only a disruption of the blood brain barrier (BBB) but also a transient demyelinating disease resembling multiple sclerosis. The demyelination might result from direct CTL-mediated destruction of infected oligodendrocytes and/or a CTL response triggered by molecular mimicry between SFV envelope proteins with myelin oligodendrocyte glycoprotein (MOG) (Fazakerley 2002; Mokhtarian et al. 1999). Athymic nude mice are protected against A7(74) induced demyelination, despite the reported subclinical CNS persistence of the virus in these mice (Amor et al. 1996). In adult mice, intraperitoneal injection of both the avirulent A7(74) and virulent SFV4 results in high-titer plasma viremia of 107-108 plaque forming units per milliliter (PFU/mL) peaking at day 1. The neurotropic viruses are seeded from blood into perivascular foci in the brains and spinal cord and are undetectable in the blood beyond day 4 as a result of viral clearance by IgM antibodies (Fazakerley 2002; Tuittila et al. 2000). A7(74) infection results in the appearance of perivascular virus antigen deposits in neurons and oligodendrocytes (but not astrocytes) in which they can be detected until the virus is immunologically cleared by d10 post infection (p.i.). However, SFV4 strain continues to replicate causing pancerebral infection and death of the mice within 4-6 days p.i. SFV4 brain titers can reach up to 108 PFU/mL before the death of the mouse, being approximately four log units higher than the respective peak titers of 104 PFU/mL with the avirulent A7(74). The major virulence determinants between the virulent SFV4 and the avirulent A7(74) have been traced to the nonstructural protein 3 (nsP3) gene (Fazakerley et al. 1993; Tuittila et al. 2000). Human SFV infections are common in Africa, where in certain areas up to 25% of the population (67 of the 267 studied cases) has been reported to be seropositive (Adekolu-John and Fagbami 1983). An obvious diagnostic limitation in the neutralization and hemagglutination inhibition assays used is the possibility of false positives as a resulting from cross-reactive antibodies between two antigenically related viruses. Indeed, 17 while 52% (139/267) of the blood samples tested positive for anti-alphavirus antibodies in Kainji Lake Basin, Nigeria, only in one sample the antibodies were monotypic for SFV (Adekolu-John and Fagbami 1983). Similarly in another study, while 33% (41/123) of the tested adult population in Angola possessed neutralizing antibodies against SFV, in a separate test only 11% (7/63) of the samples containing anti-SFV antibodies had no antibodies against Sindbis or Chikungunya (Kokernot et al. 1965). Relatively mild symptoms such as headache, fever, myalgia and arthralgia have been associated with SFV infection (Mathiot et al. 1990). Interestingly, direct intracranial (i.c.) injection of avirulent A7(74) and neurodetargeted SFV4-miRT124 results in the brains almost exclusive infection of oligodendrocytes in white matter tract of corpus callosum (Fazakerley et al. 2006; Ylosmaki et al. 2013). The A7(74) oligodendrocyte infection can be prevented by intraperitoneal (i.p.) injection of the pattern recognition receptor (PRR) agonist, polyinosinic:polycytidylic acid (poly I:C) 3h pre-infection (Fazakerley et al. 2006). The only Alphavirus found in Finland is Sindbis virus, which is spread by late summer ornitophilic Culex and Culiseta mosquito vectors. The virus is the etiologic agent of Pogosta disease causing rash and occasionally arthritis. Pogosta disease emerges typically in seven year intervals for incompletely understood reasons, but this seasonality may be related to population cycling of the postulated amplifying host; grouse (Sane et al. 2010). 2.3.2 Alphavirus replication cycle SFV enters the cells via receptor-mediated endocytosis in clathrin-coated vesicles, but the main receptor remains unknown (Figure 3). Interestingly, cell culture passaged SFV and Sindbis use glycosaminoglycan heparan sulfate (HS) as an attachment receptor, and virus cell culture adaptation to HS binding has been proposed to correlate with the level of neurovirulence (Ryman and Klimstra 2008; Smit et al. 2002). Sindbis virus produced in mosquito cells but not in mammalian cells, was found to infect dendritic cells using C-type lectins as attachment receptors (Klimstra et al. 2003). It is worth speculating whether this curious phenomenon is an evolutionary trait, as infection of skin resident DCs after a mosquito bite is the first step in the natural life cycle of many alphaviruses (Klimstra et al. 2003) and DC tropism probably becomes redundant in the following secondary infection cycles inside the host. Dendritic cell tropism has been reported also for VEE and this has been exploited in cancer immunotherapy (MacDonald and Johnston 2000; Moran et al. 2007). SFV glycoproteins have also been reported to bind these receptors, and this has obvious immunotherapeutic implications for SFV based oncolytic vectors (see chapter “2.4.2 Oncolytic virotherapy overlaps with gene therapy and immunotherapy of cancer – emerging concept of oncolytic immunovirotherapy”) (Froelich et al. 2011). Nevertheless, Schulz and coworkers reported that SFV is incapable of directly infecting DCs but crosspriming is nevertheless induced when DCs phagocytose the infected cells (Schulz et al. 2005). Though speculative, the discrepancy results concerning the infectivity of DCs with SFV might relate to the differences between cell-culture-adapted laboratory virus strains propagated and maintained in different producer cell lines. 18 SFV E1 glycoprotein mediates viral envelope fusion with the endosomal membrane as a result of acidic pH induced conformational change in the envelope glycoproteins, releasing the viral nucleocapsid into the cytosol (Fazakerley 2002; Kielian, Marsh, Helenius 1986; Wahlberg and Garoff 1992). The RNA genome is then released from the capsid via an incompletely understood molecular mechanism that involves attachment to ribosomes (Singh and Helenius 1992; Singh et al. 1997). Since the SFV genomic RNA has a positive polarity, it can immediately function as messenger RNA, initiating the synthesis of viral nonstructural proteins. Soon after infection, the cell becomes non-permissive to other SFV particles by poorly understood mechanism termed superinfection exclusion. The superinfection exclusion seems to take place at multiple levels of cell surface binding, endosomal penetration and capsid uncoating (Singh et al. 1997). The well characterized temporal processing of the replicase complex by nsP2 protease and the resulting block in the synthesis of negative RNA strands have also been proposed to be responsible for this obscure, but presumably an evolutionary important phenomenon (Jose, Snyder, Kuhn 2009). It is unclear how virus-specific the superinfection exclusion is, and intriguingly recently SFV infection of DBT mouse glioma cells has been reported to inhibit Vaccinia virus infection (Vaha-Koskela et al. 2013). Figure 3. Alphavirus replication cycle. (Helenius et al. 1980; Huotari and Helenius 2011; Jose, Snyder, Kuhn 2009; Schwartz and Albert 2010; Singh and Helenius 1992; Spuul et al. 2011) 19 2.3.3 Alphaviruses and adaptive immunity Type I IFNs are crucial for early restriction of alphavirus replication in extraneural tissues (Fragkoudis et al. 2007) and T-cell derived IFN- contributes to non-cytolytic clearance of attenuated alphaviruses from a neuronal subpopulation (Binder and Griffin 2001; Keogh et al. 2003). After the immediate innate responses, a rapid adaptive IgM response develops followed by class-switching to the IgG humoral response which is primarily responsible for clearing of the virus infection from all parts of the CNS (Binder and Griffin 2001; Fazakerley 2002). Thus, the importance of innate and adaptive humoral immune responses in protection and clearance of alphavirus infections is well established. Following infection with the avirulent SFV strain A7(74), SCID mice lacking T- and B-cells develop a chronic peripheral and CNS viremia, leading to neurological symptoms and death even though the virus does not seem to replicate in the CNS cells (Amor et al. 1996). SCID mice infected with A7(74) derived oncolytic virus VA7 also developed neurological symptoms after successful treatment of human melanoma xenografts (Vaha-Koskela et al. 2006). Peripheral infection of attenuated alphaviruses is eliminated in nude mice; this is ascribed to the gradual development of IgM antibodies. However, the A7(74) infection was reported to result in subclinical but persistent CNS infection in nude mice (Amor et al. 1996). Interestingly, and supporting the role of protective IgM response in nude mice, transfer of immune-sera from nude mice disrupts the persistent blood viremia in SCID mice, while E2 specific nonneutralizing monoclonal antibody (mAb) engraftment or IgG rich immune-sera from fully immunocompetent mice clears the viral infection both from the periphery and the CNS (Amor et al. 1996; Fazakerley 2002). This model was recently refined by the laboratory of Diane Griffin, who demonstrated using Sindbis as a model virus that infection of immunocompetent mice resulted in T- and B-cell CNS infiltration by day 3 p.i. and, interestingly, all infectious Sindbis virus was cleared from the CNS by the early IgM response (Metcalf and Griffin 2011). However, a low level of viral RNA persisted for months in the CNS together with constitutive IgG production which probably kept at bay any recurrence of renewed viral infection. Intriguingly, exogenic antibodies can even rescue mice deficient in type I or type II IFN responses from Sindbis infection (Byrnes, Durbin, Griffin 2000) and antibodies have been reported to clear Sindbis infection even in neuronal cultures (Ubol et al. 1995). Apart from the well known pivotal role Th-cells play in B-cell class switching and thus also in production of IgG antibodies against alphaviruses and the role T-cells have as an IFN- source, the innate and adaptive cellular immune responses seem to be secondary to humoral immunity in alphavirus clearance (Griffin et al. 1997). 2.3.4 Alphaviruses and innate- and cell autonomous immunity The exact mechanisms of cell autonomous anti-viral signaling in alphavirus infection remain incompletely understood. However, type I IFN signaling is crucial for the host survival and IFNAR-knockout mice succumb rapidly to peripheral SFV infection (Fragkoudis et al. 2007; Muller et al. 1994). On the other hand, type II IFN plays an important role in non-cytolytic CNS clearance at least in Sindbis virus infection (Binder and Griffin 2001; Lee, Schultz, Griffin 2013). Nevertheless, ependymal cells, meningeal cells and 20 oligodendrocytes were reported to be infected as a result of impaired type I IFN response, suggesting that separate antiviral cytokines may protect neurons and glial cells of CNS (Fragkoudis et al. 2007; Ryman et al. 2000). Interestingly, neither SFV nor VSV infection was lethal in IFNGR-knockout mice, while vaccinia infection killed IFNAR-knockout and IFNGR-knockout mice equally efficiently (Muller et al. 1994). Type I IFN knockout mice are also vulnerable to Sindbis infection and the virulence is further enhanced in type II IFN receptor deficient animals (Gil et al. 2001). Surprisingly, despite the clear role of STAT1 in mediating type I-III interferon responses, STAT1 knockout mice are resistant to Sindbis virus infection reflecting the importance of STAT1independent IFN signaling pathways (Gil et al. 2001). Infection with another alphavirus, Chikungunya (CHIKV) nevertheless resulted in the rapid death of mice with STAT1knockout genotype (Schilte et al. 2010). Remarkably, two transcription factors Zfx and Mga were recently discovered in in vitro and in vivo RNA interference (RNAi) screens to be master regulators of ISG induction by type I IFN, with their knock-down affecting the basal, IFN- and Sindbis-virus induced expression of several crucial components of the type I IFN pathway: RIG-I, STAT1, STAT2, IRF7, IRF9, IFNAR1, IFNAR2, IKK and IFN- (Varble et al. 2013). SFV infection induces a robust secretion of interferons in vitro and in blood and tissues in vivo (Bradish and Allner 1972; Bradish, Allner, Fitzgeorge 1975; Ylosmaki et al. 2013). Blood interferon levels peak 36h p.i. and are absent by 5d p.i. (Bradish, Allner, Fitzgeorge 1975). Virulent SFV strains induce higher blood interferon levels than the avirulent SFV strains (Alsharifi et al. 2005; Fleming 1977). Similarly, virulent Sindbis virus induced higher type I IFN serum levels than the avirulent counterpart (Trgovcich, Aronson, Johnston 1996). Initiation of alphavirus infection clearly depends on the inoculation dose, and while i.p. injection of 30 PFU of L10 was benign in mice, 1000 PFU almost uniformly caused lethal infection if the animals had not first been primed by a high dose i.p. dsRNA injection (Bradish and Titmuss 1981). Cytosolic RLRs are pivotal for the host detection of alphavirus infections such as that by CHIKV (Werneke et al. 2011). In accordance, RIG-I was found to be a powerful SINV inhibitor especially in conjunction with Zinc-Finger Antiviral protein (ZAP) while MDA-5 had a lesser effect (Karki et al. 2012). It is noteworthy that viral RNA can be detected by one or several members of the RLRs depending on the virus-species (Goubau, Deddouche, Reis e Sousa 2013). In the recent study of Nikovov et al. both MDA5 and RIG-I were important for SFV genome detection and IFN- induction. Strikingly, however, these pattern recognition receptors (especially RIG-I) could also sense host cell 5’ppp dsRNA secondary structures generated by viral RNA-dependent RNA-polymerase (RdRp) through transcription of host cell RNA (Nikonov et al. 2013). Nikonov et al. further proposed that this effect correlates with alphavirus virulence as RdRp from avirulent SFV induced more IFN- than the polymerase from the virulent strain, but this was probably due to the reduced JAK/STAT inhibitory power of the used nsP2-NLS mutant. Furthermore, they showed that type I IFN induction resulted in the interruption of SFV mutant replication due to destruction of its positive-sense RNAs. If the mechanism postulated by Nikonov et al. is confirmed by others in the future, it may represent a groundbreaking and completely novel evolutionary tract for cell autonomous pathogen detection, where in addition to microbial structural components and genomes also their 21 action on host RNAs may be detected by PRRs. Whether the transcription of host RNA by viral RdRp postulated by Nikonov et al. is only inadvertent and thus deleterious to the virus or whether it plays a role in the viral lifecycle remains to be shown (Nikonov et al. 2013). One also has to appreciate the difficulty of controlling the IFN- induction experiments, as even plasmid DNA transfections can induce a type I IFN response (Rautsi et al. 2007). Thus, in the future, it will be important to verify the findings of Nikonov and colleagues by alternative methods such as transfection of the viral polymerase proteins. Like most viruses, SFV has developed mechanisms to avoid IFN response and these include rapid shutoff of the host macromolecular synthesis in the infected cells. A portion of the cytoplasmic nsP2 protein of SFV and other Old World alphaviruses is transported to nucleus utilizing the nuclear localization signal (NLS) of the protein. In the nucleus, nsP2 induces ubiquitination and complete degradation of a catalytic subunit of RNA polymerase II, Rpb1, thus interrupting host cell transcription. This occurs in 6 h post infection and thus even before the reported inhibition of STAT1 phosphorylation. nsP2 is an extremely versatile protein and in addition to its role in host cell transcription shutoff it functions as a protease in the replicase complex and has two additional enzymatic activities, a helicase and an RNA 5’ triphosphatase (Akhrymuk, Kulemzin, Frolova 2012). It will be interesting to see, whether the RNA 5’ triphosphatase activity proves to be an additional means for some alphaviruses to evade PRR detection of viral (or host) RNA-intermediates, as short, blunt end and double stranded 5’ triphosphate RNA is the alleged substrate for RIG-I (Schlee and Hartmann 2010). New World alphaviruses have acquired an alternative way to suppress host cell transcription by blocking nuclear pores with a viral capsid protein. Moreover, following SFV infection, PKR-mediated eIF2 phosphorylation, which is one of the prototypical cell autonomous antiviral responses, preferentially blocks the translation of the host cell mRNA, as translation of SFV structural proteins is not dependent on eIF2 due to the complementary viral RNA secondary structures (Akhrymuk, Kulemzin, Frolova 2012; Garmashova et al. 2007). Amongst the alphaviruses at least CHIKV seems to have also some more specific, yet uncharacterized, mechanisms for translational shutdown of infected cells in addition to PKR induction (White et al. 2011). Strikingly, neither of the two classical IFN- response pathways OAS/RNaseL or PKR, is central for SFV-infected host survival based on in vivo knock-out experiments. While PKR seems to be dispensable in vivo, it does possess an anti-SFV function as a cytosolic PRR inducing type I IFNs (Barry et al. 2009). ZAP has been reported to act in concert with several other ISGs to inhibit alphavirus replication, at least in vitro by blocking specifically the translation of viral (but not host) RNA. In fact, viral translation inhibition seems to be a common effector function also for many other ISGs (Schoggins et al. 2011). ZAP is induced by type I and type II IFNs but also by the IRF3 homodimer which is activated by PRR engagement directly after PAMP detection, and thus ZAP cannot be regarded as a “true ISG” (Karki et al. 2012; Wang et al. 2010b). This seems to be the case also for another important anti-alphavirus effector, Viperin (White et al. 2011) (Figure 2). In addition to global transcriptional- and translational shutdown, different alphaviruses are also specifically capable of inhibiting STAT1 and STAT2 phosphorylation by some still incompletely understood mechanisms. This inhibition seems to be mediated either by the 22 nsP1 (Sindbis) or nsP2 genes (SFV) and it correlates with the virulence of SFV and other alphaviruses (Breakwell et al. 2007; Cho et al. 2013; Deuber and Pavlovic 2007; Farmer et al. 2013; Fros et al. 2010; Simmons, Wollish, Heise 2010; Wollish et al. 2013; Yin et al. 2009). Interestingly, the NLS of Semliki Forest virus nsP2 is required for the suppression of type I IFN response, but the effect is independent of host cell gene expression shutoff (Breakwell et al. 2007). Furthermore, the nsP2 gene has recently been reported to have yet another role in the evasion of type I IFN response, at least for CHIKV, by blocking nuclear translocation of STAT1 (Fros et al. 2013). Intriguingly, the prominent neurovirulence factor for SFV lies in nsP3, but the functions of nsP3 in the replicase complex, or possibly as an independent protein are mostly unknown (Tuittila et al. 2000). In view of the important roles of nsP1 and nsP2 in the inhibition of IFN responses and in alphavirus neurovirulence, it will be interesting to determine whether nsP3 serves a similar purpose. EEEV is a highly virulent Alphavirus and it does not induce any marked systemic IFN- production in infected mice. On the contrary, the virulence of VEEV is based on its ability to replicate despite the presence of even high (80000 IU/ml) IFN- concentrations in the serum (Ryman and Klimstra 2008). The systemic type I IFN is probably produced by pDCs (Cao et al. 2008). Very recently, the virulence of VEEV was mapped to specific secondary 5’UTR motifs that functionally mimic 5cap 2'-O methylation of higher eukaryotic mRNAs to prevent inhibition by IFIT1 ISG (Hyde et al. 2014). IFIT1 is a multifunctional ISG, with one of its most prevalent functions being the inhibition of viral mRNA translation (Diamond and Farzan 2013). As an interesting side note, some tumors can stimulate pDC differentiation, which together with MDSCs, inhibits adaptive immune responses by evoking T-cell tolerance (Rabinovich, Gabrilovich, Sotomayor 2007). It is perceivable that in some cases this may, as a secondary effect, prime for rapid attack against oncolytic viruses in cancer patients receiving virotherapy. In summary, alphaviruses have developed tools to avoid IFN- induction and/or signaling most likely due to high evolutionary pressure. These viral mechanisms include universal shutdown of host cell transcription and translation and inhibition of STAT1 phosphorylation and traffic, and for some alphaviruses the ability to replicate “under the radar” without triggering any marked type I IFN induction. Some alphaviruses are also capable of specifically inhibiting host cell translation. The specific mechanisms for these evasive maneuvers remain to be discovered as does the potential role of SFV neurovirulence factor nsP3. MicroRNAs are a newly recognized field of anti-alphavirus innate immunity. Antiviral microRNA expression has very recently been shown to regulate the viral tropism and to increase pathogenicity of EEEV by limiting the virus replication in myeloid lineage cells (Trobaugh et al. 2014). 23 2.4 ONCOLYTIC VIROTHERAPY 2.4.1 From the early 20th century to the present day Oncolytic virotherapy involves targeting cancer, its microenvironment or existing/developing vasculature with replicating (modified or natural) viruses, and is distinct, though overlapping, with gene therapy and the immunotherapy of cancer. The first reports that viruses may be able to eradicate cancer date back to early 20th century (Vaha-Koskela, Heikkila, Hinkkanen 2007) and involved incidental cancer remissions after either natural viral infections or vaccinations. Interestingly, the reported cases were almost exclusively either lymphomas or leukemias. For instance, a patient suffering from chronic leukemia had a remarkable decrease of white blood cells after having “flu-like” symptoms in 1904. Other reported examples of partial and occasionally even complete responses between the period 1950s-1980s included measles virus and vaccine induced remissions of multiple different lymphomas and leukemias and vaccinia virus vaccination-induced remission of leukemia. In addition to hematological malignancies, one of the early reports described Newcastle disease virus (NDV) induced response of Chicken farmer’s gastric cancer after occupational exposure to the virus (Liu, Galanis, Kirn 2007). Furthermore, a patient with cervical cancer experienced a spontaneous remission after immunization with rabies vaccine in 1912. There are several potential reasons why these fortunate responses to vaccinations or natural viral infections were primarily restricted to leukemias and lymphomas. Firstly, because of the hematologic origin of these diseases, leukemia and lymphoma patients are inherently immunosuppressed, which could facilitate systemic delivery of opportunistic viruses and attenuated vaccine strains. Secondly, leukemias and lymphomas are frequent also among younger patients, who are the individuals most likely to be vaccinated or to contract a primary virus infection (Liu, Galanis, Kirn 2007). Finally, at least one of the reported responsive hematologic neoplasms, Burkitt’s lymphoma is caused by Epstein-Barr virus of the Herpesviridae family, which encodes type I IFN antagonizing gene LF2 (Wu et al. 2009). Hence, it is tempting to speculate that genes expressed from oncoviruses rendered some of these tumors to become good targets for viral infections. The reports of spontaneous cancer remissions after viral infections led to a series of patient treatments with non-engineered viruses starting from 1950s. This was the “first generation” of oncolytic viruses and the strains used were typically attenuated through serial in vitro passages. Mumps, measles, West Nile virus, adenoviruses, reovirus, NDV and vaccinia virus were tested with different tumors and different administration routes. Interestingly, vaccinia induced tumor remissions were occasionally complete and long lasting, and could occur at sites distant from those of viral administration. In general, the treatments were well tolerated, with the most common side effects being pain at the virus injection site and “flu-like” symptoms. Nevertheless, approximately 10% of the patients treated with the West Nile virus were diagnosed with encephalitis (Liu, Galanis, Kirn 2007) and a case of severe encephalitis was also reported in one out of four leukemia patients treated with Bunyamwera (Moore 1954). Many other viruses were also tested in both animal models and in humans during the 1950s and 1960s, but the experiments were often poorly controlled 24 and in general, the results remained modest. For example, one patient with an acute leukemia received as many as six different viruses in succession, while in another study 13 different viruses or strains were provided to 57 patients with varying malignancies but no therapeutic effect was observed (Moore 1954; Vaha-Koskela, Heikkila, Hinkkanen 2007). In parallel with the advances in modern biotechnology, since the 1990s virotherapy has been again actively developed to complement the traditional cancer treatment regimens. The second generation of oncolytic viruses was engineered to carry mutations or deletions in one or more viral genes essential for virus replication in the natural target tissue but which were redundant in neoplastic tissue to create conditionally replicating viruses (See chapter: “2.4.4 Targeting of Oncolytic Viruses”). The mutations were often introduced into virulence genes responsible for inhibiting cell autonomous antiviral (and antiproliferative) responses in healthy tissues, responses of which are often already suppressed in tumors. While this approach increased the tumor selectivity of the viruses, on the one hand, and improved their safety on the other, these improvements occasionally came with the cost of reduced viral replication power (Thorne, Hermiston, Kirn 2005)). There are several requirements for virus design that need to be addressed in the construction of a good oncolytic viral agent. First, the virus must be able to efficiently replicate in and destroy the targeted tumor tissue, but it is equally important that the virus spares the normal, healthy tissue from extensive damage. The genetic stability of the viral agent is desirable for both safety and commercial manufacturing reasons. It is also preferable to use non-integrating viruses to avoid the risk of insertional mutagenesis/oncogenesis. By using non-integrating viruses, one can also be more confident that the virus will take part in the lytic cycle, which is usually considered an important part of the anti-tumor effect of oncolytic viruses. Incorporating a safety mechanism (“off switch”) to inactivate the virus if necessary should also be taken into consideration, whenever the virus is not inherently responsive to antiviral agents. Finally, the oncolytic virus needs to be suitable for high-titer manufacturing and purification processes following the “Good Manufacturing Practices” required for clinical studies (Thorne, Hermiston, Kirn 2005). The third generation conditionally replicating oncolytic viruses are deletion mutants “armed” with exogenous genes encoding for example cytotoxic or immunostimulatory proteins increasing the oncolytic efficacy or suicide genes which additionally increase safety by allowing down-regulation of the infection if necessary (O'Shea 2005). Several viral vectors have been used in clinical trials based on e.g. vaccinia virus, adenoviruses, HSV-I, NDV and reovirus type 3 (Vaha-Koskela, Heikkila, Hinkkanen 2007). A list of the current (5/2014) oncolytic virotherapy trials reported in clinicaltrials.gov can be found in Table 1. The top 3 oncolytic viruses involved at the moment in either phase III or advanced phase II trials are reovirus (Reolysin), HSV-I (Talimogene laherparepvec, T-VEC, previously known OncoVEXGM-CSF) and Vaccinia virus (JX-594). T-VEC and JX-594 harbor immunostimulatory granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, while Reolysin is an unmodified reovirus (Donnelly et al. 2013a; Heo et al. 2013; Miest and Cattaneo 2014). The main immunostimulatory function of GM-CSF in the context of oncolytic immunovirotherapy is thought to be the activation of professional antigen presenting cells 25 (Dranoff et al. 1993; Parato et al. 2012). While NK-cell activation has often been listed as one of the effects of GM-CSF (Bartlett et al. 2013; Cerullo et al. 2010) there seems to be no convincing literature to back this claim. Surprisingly, GM-CSF has been recently reported to play a strong immunosuppressive role in some cancers (Bayne et al. 2012; Kohanbash et al. 2013). 26 Table 1. Ongoing oncolytic virotherapy clinical trials. In the table the clinical trial names, phase and therapeutic viruses together with possible co-treatments are listed as reported “Active” or “Recruiting” in clinicaltrials.gov 16.5.2014 27 Replication competent, oncolytic viruses may reach even metastasized tumors and thus be used as a noninvasive means to target tumors in hard-to-reach body locations such as the brain. For example, intravenous administration of replication-competent NDV led to tumor eradication in several patients with multiresistant GBM, with prolonged survival of more than 9 years and high quality of life (Csatary et al. 2004). Different HSV-I mutants, adenoviruses and reovirus constructs have also been clinically tested for treatment of malignant glioma. Additionally, phase I glioma therapy trials have recently been started for modified polio and measles viruses. In a phase I clinical trial, single adenovirus (Ad) DNX2401 injection into GBM tumors as a first line therapy resulted in notable responses, including complete regression of the disease, in many of the 24 treated patients (Pol et al. 2013). Equally impressive results were reported from a phase I GBM trials using local administration of lentivirus Toca511 as the first line treatment in combination with prodrug TocaFC, and in another study where poliovirus PVS-RIPO was used as the second line treatment combined with resection. Remarkably, three of the seven patients who received PVS-RIPO achieved a complete response. In general, administration of the viruses into the brains of patients has been found to be both safe and well tolerated (Pol et al. 2013; Zemp et al. 2010) (Table 1). Several different GM-CSF expressing viruses have proven to be clinically effective against different solid tumors, thus underlining the importance of stimulating the anti-tumor immunity in order to achieve a good therapeutic efficacy. GM-CSF –encoding oncolytic herpesvirus T-VEC has completed phase III clinical trial in patients with unresectable metastatic melanoma, where the virus proved both safe and effective (response rate 26%). As many as 11% of the patients have been reported to experience complete response (disappearance of the disease), and the primary end point of durable response rate was met. However, T-VEC failed to meet its secondary endpoint of improved overall survival, even though a clear beneficial trend (p=0.051) compared to control group was seen (Williams 4th of April 2014). T-VEC is the first oncolytic virus to have completed phase III trial, giving hope to obtain the first approved oncolytic virus in Western World (Andtbacka et al. 2013; Pol et al. 2013). The phase III melanoma trial design provoked some ethical debate, as the control group received a subcutaneous injection of GM-CSF instead of standard care. T-VEC has shown some efficacy also in phase I/II trial of stage III/IVA squamous cell carcinoma of the head and neck (Sivendran et al. 2010). Curiously, one phase III trial (ClinicalTrials.gov identifier: NCT01161498) for this malignancy was suddenly terminated in early July 2011 “to permit significant modification of clinical trial design mandated by the changing therapeutic landscape for patients with SCCHN” (Regan 29th of July 2011). Vaccinia virus JX-594 expressing GM-CSF has also completed phase I and phase II clinical trials and demonstrated promising efficacy in the treatment of primary and metastatic liver cancers (Breitbach et al. 2010). In a randomized phase II dose-finding trial, the overall survival of the high-dose group compared to low-dose group was significantly better (Donnelly et al. 2013a; Heo et al. 2013; Koski et al. 2009). A phase IIb trial is ongoing and even though the official results remain to be published, according to preliminary information, JX-594 failed to meet the primary endpoint of improved overall survival (Sheridan 4th of September, 2013). 28 GM-CSF-encoding oncolytic adenoviruses Ad5-D24-GM-CSF and Ad5/3-D24-GM-CSF have also been tested in patients in the treatment of several different solid tumors. These viruses were reported to induce antitumoral immunity and to exert antitumoral effects some of which were complete, but the therapy effects on overall survival were not reported. A phase I/II clinical trial with Ad5/3-D24-GMCSF combined with low-dose metronomic cyclophosphamide to reduce T-regs is ongoing (Cerullo et al. 2010; Koski et al. 2010). Finally, after promising results in a phase II trial, an unmodified, oncolytic reovirus Reolysin has entered phase III trial in patients with head and neck carcinoma for use in combination with chemotherapy. In the phase II trial one complete response and six partial responses were seen in 17 patients who received intravenous Reolysin in combination with paclitaxel and carboplatin treatment. In addition, two patients experienced major clinical responses (complete resolution of skin nodules). The safety profile was in general good but one patient experienced sepsis, not a surprising toxicity considering the immunosuppressive nature of the chemotherapeutic agents being combined with a replicative virus (Breitbach et al. 2010; Karapanagiotou et al. 2012). 2.4.2 Oncolytic virotherapy overlaps with gene therapy and immunotherapy of cancer – emerging concept of oncolytic immunovirotherapy Gene therapy, i.e. (mostly virus vector mediated) transfer of therapeutic genes, has proven very promising for the treatment of monogenic diseases but the results in cancer treatment have remained modest (Brenner et al. 2013). Cancer is nevertheless the most frequently chosen disease for gene therapy trials and 64.4% (1186/1843) of all gene therapy trials completed or ongoing before 2012 concerned cancer (oncolytic virotherapy trials included) (Ginn et al. 2013). The concepts of gene therapy of cancer, immunotherapy of cancer and oncolytic virotherapy are overlapping, and while viral gene transfer vectors or replicons are often used to deliver immunostimulatory genes or cancer antigens to tumors or immunecells (Lam et al. 2013), the same genes and antigens can often be incorporated into replicative oncolytic viruses (Russell, Peng, Bell 2012). For example, Rommelfanger et al. recently used the mouse B16 melanoma model and combined systemic VSV carrying human melanoma antigen to adoptive cell transfer with encouraging results (Rommelfanger et al. 2012). A completely novel paradigm was introduced, when instead of selected TAA or TSA, a cDNA library prepared from healthy human prostate was cloned into VSV and injected systemically into mice to successfully treat murine prostate tumors (Kottke et al. 2011). The human prostate cDNA library worked significantly better than mouse cDNA library demonstrating that “altered self” epitopes can be more immunogenic as compared to self epitopes, and it will be interesting to determine in the future whether this holds true when single, universal TAAs such as survivin or telomerase are used as vaccines. Interestingly, the treated mice experienced no autoimmune symptoms and it was postulated that this was because of lack of inflammatory signals in the prostate. However, Kottke and coworkers used a subcutaneous prostate tumor model and thus it remains unclear whether the primary organ inflammation caused by orthotopic tumors would be sufficient to induce autoinflammatory disease in this therapeutic context. 29 Cell death can cause tolerance or be immunogenic depending on the type and location of the dying cell and the factors it releases or expresses on its surface during the death process (Griffith and Ferguson 2011). The effective maturation of dendritic cells resulting in successful presentation of tumor antigens to T-cells in lymph-nodes requires PAMP detection (or other maturation stimulus) upon engulfment of the dying cells, and a lack of the maturation stimulus can promote tolerance (T-cell deletion, anergy or T-reg induction) instead of antitumor immunity (Mellman, Coukos, Dranoff 2011). Oncolytic viruses kill tumor cells directly through apoptosis and necrosis, but they have also been shown to induce adaptive antitumor immune responses via immunogenic cell death (ICD) (Bartlett et al. 2013; Boozari et al. 2010; Donnelly et al. 2013b; Miyamoto et al. 2012; Workenhe and Mossman 2014). Different oncolytic viruses have been reported to cause all four major types of cell death, apoptosis, necrosis, autophagy and pyroptosis, and often the death of a cell population is a mixture of these processes (Bartlett et al. 2013; Fink and Cookson 2005). Alphaviruses kill cells primarily by apoptosis evoked by nsP2 of Old World viruses and the capsid protein of New World viruses, while also the envelope glycoproteins are proapoptotic (Barry et al. 2010; Jose, Snyder, Kuhn 2009). SFV-infected cancer cells have been reported to die both via p53 independent apoptotic (Glasgow et al. 1998; Murphy et al. 2000; Murphy, Sheahan, Atkins 2001; Smyth et al. 2005) and non-apoptotic pathways (Vaha-Koskela et al. 2006). Death by non-apoptotic pathways might result from the fact that varying apoptotic signaling pathways become silenced in the course of tumorigenesis and thus the death of these cells may resemble the necrotic mode of death of alphavirusinfected apoptosis-resistant mosquito cells (Jose, Snyder, Kuhn 2009). The mode of death of oligodendrocytes (Fazakerley et al. 2006; Glasgow et al. 1997) and that of adult neurons infected by neurovirulent alphavirus strains remain incompletely understood as both necrosis and apoptosis have been documented, whereas immature neurons that are susceptible also to avirulent strains die by apoptosis (Glasgow et al. 1997; Griffin 2005; Nargi-Aizenman and Griffin 2001). SFV infected cells accumulate autophagosomes and this effect is viral glycoprotein-dependent, but seems to be a bystander phenomenon as the disruption of autophagy has no effect on the viral replication (Eng et al. 2012). According to a very recent study SFV induced apoptosis of healthy mammalian cell lines depends on the PRR signaling mediator MAVS (aka CARDIF, VISA, IPS-1) that activates caspases 8 and 3, and interestingly is independent of the simultaneous presence of IFN- or even IRF3/7 signaling (El Maadidi et al. 2014). As a side note, since all dead cells are often called necrotic in pathological terminology, the inflammatory “necrotic death process” resulting in the release of cellular contents into the extracellular space is occasionally termed oncosis (Fink and Cookson 2005). Caspase-independent necrosis and autophagy, and caspase-1-dependent pyroptosis are all different types of ICD, whereas caspase-dependent apoptosis can be either immunogenic or tolerogenic. In addition to oncolytic viruses, also some other cancer therapies such as anthracyclines, oxaliplatin, cyclophosphamide (CPA), EGFR-specific antibodies and UV irradiation can promote ICD. The common denominator for ICD is endoplasmic reticulum (ER) stress sensed by unfolded protein response (UPR) causing the expression and release of danger (or damage) associated molecular patterns (DAMPs) calreticulin, ATP and HMGB1. ER-stress results in calreticulin traffic to the cell surface (PRR binding “eat me” signal for APCs), ATP release 30 (“find me” signal) and HMGB1 release, which in its fully reduced or disulphide-bond possessing states (but not in the oxidized state) stimulates DC maturation, Th1 cell expansion and works as a chemoattractant (Bartlett et al. 2013; Workenhe and Mossman 2014). Importantly, apoptosis induced by replicative SFV (but not by replicon) was reported to involve an ER-stress response evoked by envelope glycoproteins (Barry et al. 2010). During their replicative cycle oncolytic viruses trigger multiple pattern-sensing mechanisms resulting in the secretion of different cytokines such as type I IFNs and leading to ER-stress. Virus-induced inflammation, in turn, summons and activates antigenpresenting cells, which prime antiviral adaptive immunity. Since virus infection-induced inflammation is highly immunogenic in nature and optimally occurs only in cancer cells, oncolytic viruses may be able to break tolerance against weak tumor-associated (self) antigens (TAA) and thus unmask those tumor cells otherwise hidden from immune surveillance. The immunogenicity can be further boosted by deleting viral immunosuppressive genes and adding stimulatory cytokines such as IL-12, IL-18, IL-2, GM-CSF, CD40L, B7.1 and CCL5, or PRR agonists such as CpG DNA-elements or a variety of heat shock proteins. Heat shock proteins are especially interesting immunostimulants as not only do they act as direct DAMPs on cell surface or upon release, but they can also bind tumor antigens thus enhancing their presentation (Bartlett et al. 2013). Indeed, depending on the virus and tumor model, the oncolytic efficacy may rely almost exclusively on such bystander immune responses against the tumor rather than lytic virus replication (Cheema et al. 2013; Galivo et al. 2010; Prestwich et al. 2009; Wongthida et al. 2010). However, in other occasions, the initial tumor debulking by the replicative virus is followed by eradication of residual disease by T-cells of activated adaptive immune system (Huang, Guo, Hwang 2012; Naik et al. 2012). While the initial debulking by lytic replication cycle and/or inflammation induced innate immune destruction has been demonstrated in animal models, the debulking function remains to be demonstrated clinically (Russell, Peng, Bell 2012). Occasionally, efficient tumor infection and destruction in preclinical models does not seem to require any adaptive immune responses at all (Lun et al. 2005). In a preclinical settings, oncolytic virotherapy has been successfully combined with different kind of immunotherapies such as adoptive T-cell transfer (Rommelfanger et al. 2012), DC-vaccination (Woller et al. 2011) and agonistic or antagonistic monoclonal antibodies against immune-checkpoint regulators (Dias et al. 2012; John et al. 2012; Quetglas et al. 2012a; Zamarin et al. 2014). The multitude of different successful oncolytic immunovirotherapy approaches suggests that live viruses are indeed powerful adjuvants, and the infection-related inflammation can induce Th1 type of antitumoral immunity. Thus, the viral infection can counteract the tolerogenic and anti-inflammatory Th2 type of immunity, which is typical for the tumor microenvironment and resembles a physiological wound healing process. Finally, classical chemotherapeutics and radiotherapy have been successfully combined with OV. For instance, in a recent study, doxycycline treatment was noted to augment the tumor-specific replication of oncolytic vaccinia virus. Doxycycline also boosted anti-cancer immunity by enhancing and stabilizing the expression of the MIC stress ligand on cancer cells making them more attractive targets for adoptive cell transfer of cytokine induced killer cells (CIK) (Tang et al. 2013). Intriguingly, the best result was obtained when CIK cells were used as a carrier for oncolytic vaccinia virus. Similarly, a 31 combination of oncolytic HSV-1 with an ICD inducer mitoxantrone was recently successfully employed to break immunotolerance against TUBO breast cancer in syngeneic mice (Workenhe et al. 2013). Alphaviruses have been reported to replicate preferentially in tumor cells. This can result from, firstly, from defects in the cancer cell type I IFN induction or secondly, and as growing body of evidence suggests more importantly, from defects in the type I IFN responsiveness of cancer cells. In addition to neoplastic cells, also most of the healthy stromal cells can produce type I IFNs, and thus any defects cancer cells may have in their type I IFN induction are probably not sufficient to make them vulnerable to viral infections (Huang, Guo, Hwang 2012). Alphavirus mediated oncolysis can stimulate antitumor immunity (Huang, Guo, Hwang 2012) and by using SFV replicons, this effect has been shown to be greatly enhanced when immunostimulatory cytokines such as IFN- (Quetglas et al. 2012b) or IL-12 were incorporated into the virus (Rodriguez-Madoz, Prieto, Smerdou 2005; Rodriguez-Madoz et al. 2009; Rodriguez-Madoz et al. 2014). It is uncertain whether SFV can induce cross-priming and cytotoxic T-cell responses by directly infecting DCs. It has been shown that a lentivirus vector pseudotyped with SFV glycoproteins gained dendritic cell tropism by enabling transduction via c-type lectin receptor (Froelich et al. 2011), but Schulz and colleagues could not achieve efficient DC infection with the SFV replicon (Schulz et al. 2005). Nevertheless, SFV was able to induce efficient TLR3 mediated activation of DCs and cross-priming of cytotoxic T-cells after phagocytosis of viral dsRNAcontaining cells (Schulz et al. 2005). The controversial data regarding direct infection of DCs by alphaviruses might stem from the fact that alphaviruses can change their tropism as a result of adaptation to cell culture conditions (Smit et al. 2002). Furthermore, only Sindbis viruses produced in mosquito cell lines but not in mammalian cells were reported to be able to use c-type lectins as their receptor and infect DCs (Klimstra et al. 2003). Interestingly, TLR5 stimulating bacterial Flagellin was recently found to increase both Th1 and Th2 adaptive responses when cloned into an alphavirus replicon (Knudsen et al. 2013). SFV infection per se is also known to induce the global activation of B- and T-cells of mouse in a type I IFN-dependent manner (Alsharifi et al. 2005) and thus SFV infection may not only stimulate local immune responses in the tumor microenvironment but also have broader immunostimulatory effects. IL-12 gene transfer and expression from oncolytic viruses, vectors or replicons, is not a straight-forward process as the cytokine is a heterodimer and it is systemically toxic to animals. However, strategies have been developed to localize the expression to tumor beds (Pan et al. 2012). Notably, SFV-IL-12 was reported to be a more potent immunotherapy vector than adenovirus with the same cytokine insert in two separate syngeneic rodent tumor models. The authors speculated that this presumably resulted firstly from the lower immunogenicity of SFV as compared to adenovirus making multiple intratumoral injections possible, secondly from SFV replicon induced apoptosis and thirdly due to a more efficient IL-12 production from the SFV replicon (Rodriguez-Madoz et al. 2009). Replicons and fully replicative viruses might be a better option for cancer immunotherapy than the classical vectors, which are often based on adeno- or lentiviruses or DNA-plasmids (Matrai, Chuah, VandenDriessche 2010; Volpers and Kochanek 2004) as the classical viral vectors do not replicate their genome inside the cells, possibly resulting in poorer gene 32 expression and reduced vector induced cell death. In addition, unlike many other viruses such as myxomavirus (Ogbomo et al. 2013), alphaviruses are not known to suppress adaptive immune responses. Indeed, in a very recent study, the SFV-IL-12 vector proved to be slightly more efficient than the same cytokine expressed from a plasmid against genetic mouse hepatocellular carcinoma (HCC) model, even though higher and longer term IL-12 expression was obtained with the plasmid. The authors proposed that SFV was superior because it induced direct tumor cell apoptosis and provoked an immunogenic type I IFN response. Furthermore, short term expression was postulated to increase the safety when highly toxic cytokines such as IL-12 were used (Rodriguez-Madoz et al. 2014). This study also confirmed that SFV-IL-12 has efficacy also against antigenically heterogeneous forms of cancer, a question that had remained unanswered after the only transient remissions seen previously in a woodchuck HCC model (Rodriguez-Madoz et al. 2009). Recently, also HSV-IL-12 showed a remarkable efficacy as compared to an unmodified virus in a challenging mouse glioma stem cell model (GSC) demonstrating a multimodal anti-cancer action consisting of direct killing of both GSCs and differentiated cancer cells, antiangiogenesis and T-reg reduction (Cheema et al. 2013). While both the unmodified and IL-12-encoding viruses were able to directly kill cancer cells, the antiangiogenic and T-cell mediated bystander effects remained dependent on the ectopically expressed IL-12. While SFV infection is immunogenic, one has to keep in mind that a productive viral infection combined with the interferon signaling, or even the plain nsP2 protein of SFV derived oncolytic viruses can dramatically change the host cell gene expression (Akhrymuk, Kulemzin, Frolova 2012; Sadler and Williams 2008). While this might be beneficial for the purposes of inflicting direct cell death, it inevitably also changes the antigenic profile of the infected tumor cells compared to distant uninfected cancer cells, possibly biasing the adaptive immune responses towards infection-related antigens. Granot et al., recently presented an interesting immunotherapy approach to circumvent such pitfalls and treated LacZ transduced CT26.CL25 tumors with a Sindbis replicon expressing LacZ (Granot, Yamanashi, Meruelo 2014). While their replicon could not actually infect the tumors, LacZ was expressed in the draining lymph nodes, and immunotherapy resulted in an efficient tumor eradication and cross-protection against CT26WT “immune-escape” variants via epitope spreading. Second obvious caveat to the use of replicative viruses for induction of cytotoxic cellular and opsonizing humoral (i.e. Th1 biased) immune responses relates to the fact that viral infections often trigger a Th2 neutralizing antibody response (Abbas, Lichtman, Pallai 2007). This can possibly be overcome by adding exogenous Th1 cytokines or ligands such IL-12 (Cheema et al. 2013) or soluble CD40L (Pesonen et al. 2012) into the therapeutic viruses. However, the identification of soluble CD40L in the serum of cancer patients has been recently implicated in immunosuppression (Huang et al. 2012). The beneficial and detrimental roles of innate, adaptive and cell autonomous immune responses in the context of oncolytic virotherapy remain incompletely understood and may depend on the tumor model, therapy virus, administration route and timing used. At least the complement system (Ikeda et al. 2000; Wakimoto et al. 2002) and neutralizing antibodies (Nabs)(Ilett et al. 2009) have been reported to inhibit oncolytic viruses. Notably, virus neutralizing antibody responses can occasionally be very fast rather than slow and adaptive. Preimmune IgM antibodies were reported to be able to neutralize oncolytic HSV- 33 1 and very recently also VSV, and the neutralization was further enhanced when complement proteins were present (Ikeda et al. 1999; Tesfay et al. 2014). In addition to direct neutralization effects, complement proteins, antibodies, different coagulation factors and possibly also other serum proteins can also opsonize viruses by sequestrating them to allow their destruction by phagocytotic macrophages in the spleen and Kupffer cells in the liver (Russell, Peng, Bell 2012). The successful preclinical and clinical approaches to evading the viral neutralization and sequestration have included the use of tumor homing cell carriers such as dendritic cells, T-cells (Ilett et al. 2009), mesenchymal stem cells (GarciaCastro et al. 2010) and irradiated myeloma cells (Liu, Russell, Peng 2010), pharmacological ablation or saturation of antibodies, complement and phagocytes (Haisma and Bellu 2011; Ikeda et al. 1999; Ikeda et al. 2000; Koski et al. 2009; Magge et al. 2013; Shashkova et al. 2008; Ziegler et al. 2002), shielding the therapeutic virus with polymers (Eto et al. 2008; Fisher and Seymour 2010) and serotype switching (Sarkioja et al. 2008). Priming of T-cell immunity is frequently observed in oncolytic virotherapy and often considered as essential for the therapeutic efficacy (Naik et al. 2012), but complete and long lasting tumor regressions after virotherapy have been documented also in athymic animal models (Lun et al. 2005). The distinct contributions of CD4+ T-helper cells and CD8+ cytotoxic T-cells for oncolytic virotherapy remain equivocal and while both classes have been reported to be necessary (Kottke et al. 2010), occasionally the CD4+ cell population seems to be redundant (Diaz et al. 2007; Rommelfanger et al. 2012; Sobol et al. 2011). Neutrophils were reported to be the main effector in OV when VSV was used in CT26LacZ mouse colorectal tumor model (Breitbach et al. 2007) and neutrophils played an important role also in the efficacy together with CD4+ and CD8+ T-cells when HSV-1 was combined with the immunogenic cell death inducer, mitoxandrone (Workenhe et al. 2013). Most intriguingly, depletion of any of the CD8+, CD4+ or NK-cell classes impaired the efficacy in a mouse melanoma model when reovirus was combined with an intratumoral VEGF pulse to infect and destroy cancer vasculature (Kottke et al. 2010). However, only CD8+ and NK-cells but not CD4+ cells were recently reported to be necessary for VSV immunovirotherapy in a mouse melanoma model where the tumor antigen expressed from VSV was combined with adoptive cell transfer (Rommelfanger et al. 2012). Similarly, CD8+ T-cells, NK-cells and neutrophils but not CD4+ T-cells were necessary when an immunostimulatory agonistic anti-4-1BB antibody was combined with vaccinia virotherapy in the murine AT-3 breast carcinoma model (John et al. 2012). In a recent study, it was noted that in addition to the CD8+ T-cells that were induced against TAAs via epitope spreading, also CD8+ cells against the oncolytic HSV-1 viral epitopes were important for tumor control in a murine breast cancer model (Sobol et al. 2011). By, using tumor antigen transgenic and thus tolerized mice, adaptive antiviral immune responses were reported to be therapeutically important together with adaptive responses elicited against tumor antigen (Sobol et al. 2011). However, while the authors showed that some level of antitumor effects could be achieved also in the tolerized mice, they failed to provide direct evidence to support their claim for a beneficial in vivo role for antiviral T-cells as they did not exclude the possibility of the presence of secondary tumor antigens against which the mice were not tolerized. In line with these results, Gürlevic et al. 34 reported that while p53 and telomerase promoter targeting by oncolytic adenovirus enhanced tumor specificity, dramatically lowered the off-target liver toxicity and inhibited adaptive antiviral immune responses, surprisingly the non-targeted control viruses were equally potent in eliciting antitumor immune-responses (Gurlevik et al. 2010). This report is one of the few examining the effects of induced antiviral adaptive responses in oncolytic virotherapy efficacy, and contradicts the concept that off-target replication of oncolytic viruses would reduce the likelihood of on-target antitumor immune responses. Similarly, on one occasion robust anti-SFV immune responses were reported to be beneficial instead of detrimental for the efficacy of the therapy, and pre-immunization of mice with the virus resulted in tumor inflammation and improved virotherapy efficacy in CT26 tumor model (Smyth et al. 2005). The role of IFN- (Naik et al. 2012; Quetglas et al. 2012b) and NK-cells (AlvarezBreckenridge et al. 2012b; Bhat and Rommelaere 2013; Cheema et al. 2013; Diaz et al. 2007) is context dependent as they can potentially either augment (via an antitumor immune response), hamper (via an antiviral immune response) or be irrelevant/counterbalancing to the efficacy of oncolytic virotherapy. Type I IFNs are cytotoxic but their direct therapeutic effect against cancer cells can be suppressed as a result of the acquired apoptosis resistance common for malignancies. Intriguingly, Beug et al. demonstrated very recently that oncolytic VSV or viral RNA/DNA mimetics induced durable cures both in an immunocompetent and xenograft mouse tumor models mediated by IFN- response, but the therapeutic efficacy required pharmacological suppression of inhibitor of apoptosis (IAP) proteins (Beug et al. 2014). Not much is known about the role of NKT-cells, T cells, CD4+ or CD8+ T-regs or MDSCs in the context of OV. Unexpectedly, in the study of Diaz and colleagues, depletion of T-regs was reported to hamper VSV mediated oncolytic virotherapy as it stimulated immunity against the therapeutic virus (Diaz et al. 2007). Similar results were obtained when oncolytic virus infection was combined with DC vaccination. T-reg depletion resulted in an antibody response against the virus and, furthermore, to a compensatory induction of MDSCs (Woller et al. 2011). Both direct and indirect evidence exist that macrophages/microglia are often detrimental to virotherapy, presumably because they terminate the infection too early by host cell phagocytosis (Fulci et al. 2006; Fulci et al. 2007; Lun et al. 2010; Spurrell et al. 2014), but also by secreting antiviral IFN- (Liu et al. 2013). However, macrophages are beneficial at least in certain antibody therapies where they, in addition to directly phagocytosing opsonized cancer cells, also prime anti-tumor T-cell responses (Tseng et al. 2013). Type I and type III IFNs have been recently proven to be crucial for the efficacy of oncolytic VSV against B16 melanomas (Wongthida et al. 2011). Oncolytic virotherapy has also been applied successfully in combination with rapamycin, cyclophosphamide (CPA) and different HDAC inhibitors to reduce tumor type I IFN signaling, and some of these combinations simultaneously stimulated adaptive antitumor immune responses (see chapter “2.4.3 Combination therapy to improve OV efficacy”). Despite the reported positive contributions of adaptive antiviral responses, there is however a high amount of evidence suggesting that heterologous prime-boost tumor vaccination strategies, where the adjuvant virus is changed or replaced with a plasmid vector, while the virus-encoding tumor antigen is kept the same, work better than 35 homologous vaccinations with a single virus (Bartlett et al. 2013). This was the case in a study where Vaccinia virus was combined with SFV, with or without the expression of model tumor antigens, and in a second study where adenovirus was combined with vaccinia in Syrian hamster model (Tysome et al. 2012; Zhang et al. 2010). Interestingly, in the latter study, the order of viruses applied was crucial and injection of adenovirus before vaccinia was a superior strategy over the reverse sequence, and resulted in T-celldependent eradication of the tumors. Importantly, these heterologous regimes did not reduce the amounts of Nabs or total antibodies as compared to homologous prime-boost regimen. In another investigation, an SFV-replicon encoding natural P1A tumor antigen worked well together with adenovirus in heterologous prime-boost setting conferring protection against murine P815 mastocytoma. SFV worked best when it was used for priming. Interestingly, SFV-replicon proved to be more powerful in the induction of antitumor immunity than adenovirus-vectors upon homologous prime-boost vaccination, and while the quantity of anti-tumor CD8+ T-cells was higher after adenovirus vaccination, the quality was better with SFV (Naslund et al. 2007). While in the homologous prime-boost regimens the vector neutralization probably plays a role resulting in a reduced efficacy in some models and with certain viruses, it seems clear that xenogenic viral antigens can also easily become immunodominant in a homologous setting especially when weak tumorassociated self antigens are targeted. Indeed, Bridle and colleagues used the mouse B16 melanoma model with oncolytic adenovirus and VSV carrying natural TAAs and demonstrated that VSV as a single agent biased CD4+ and CD8+ T-cell response against viral antigens. However, the use of heterologous prime-boost strategy directed the response towards the tumor antigen resulting in impressive treatment efficacy in the difficult metastatic setting (Bridle et al. 2009). Interestingly, the investigators used dopachrome tautomerase of human origin (hDCT) as the target antigen, further supporting the concept of using “altered self” antigens in immunotherapy. However, the use of xenogenic antigens to disrupt immunotolerance inherently carries the risk that the resulting peptides presented on MHCs will be immunologically too different to induce efficient cross-protection. Thus the cross-immunogenicity of each individual antigen needs to be vigorously validated before advancing to clinical trials. Although heterologous prime-boost strategies seem to work better than homologous approaches when they are compared side-by-side, sequential infections with heterologous, natural pathogens have been also shown to be immunosuppressive by impairing memory T-cell function (Welsh et al. 2012). Xenogenic human tyrosinase-related protein-1 (hTRP-1) expressed from regular DNA plasmid protected mice against B16 melanoma in homologous vaccination regime better than the mouse equivalent in two separate studies (Leitner et al. 2003; Weber et al. 1998). Intriguingly, in the latter study, also mTRP-1 conferred protection when it was expressed from Sindbis replicon instead of a plasmid, a result that was also obtained independently by another group using alphavirus VEE replicon (Goldberg et al. 2005). As a side note, Leitner et al., utilized a “layered” technology where the Sindbis replicon was expressed under a CMV promoter from a DNA plasmid, which was then delivered by gene gun. The layered plasmid-replicon technology probably increases the safety of the vector, as the emergence of fully replicative viruses becomes impossible when alphavirus structural proteins are not needed in the production process. mTRP-1 vaccination was however efficient when expressed from the plasmid in un-tolerized TRP-1 double knock-out mice, 36 elaborating that normal mice displayed tolerance against this tumor-associated self antigen and alphaviral vectors were superior over conventional plasmids in unmasking the immune response against it. Curiously, of the four plasmid/replicon combinations expressing mTRP-1/hTRP1 only the conventional plasmid expressing hTRP1 induced an autoimmune disease, vitiligo. This was not, however, due to the xenogenic nature of the antigen, as vaccinia virus bearing the mTRP-1 insert, while protective against B16, induced the same autoimmune symptoms. Leitner and colleagues used RNase L knock-out mice to demonstrate that the better immunogenicity of the replicon compared to a plasmid was not related to higher antigen expression but, the cell autonomous antiviral response induced by the viral “danger signals”, such as dsRNA (Leitner et al. 2003). Virus derived PAMPs can thus work as an adjuvant enhancing immune responses in a dual manner: on one hand by directly engaging with PRRs of professional APCs (Mellman, Coukos, Dranoff 2011) and on the other hand by inducing an immunogenic type I IFN response in situ. This response causes not only ICD but also induction of MHC-I expression and stimulation of dendritic cells, NK-cells and T-cells (Hervas-Stubbs et al. 2011). As the knockout of the prototypical apoptotic effector RNase L had such a marked effect on the immunogenicity of Sindbis vector (Leitner et al. 2003), this suggests that the most important of the many immunostimulative effects mediated by type I IFN response after alphavirus infection might be the induction of ICD. This hypothesis was later further supported by Leitner and colleagues reporting that Sindbis vector expressing a melanoma antigen was less immunogenic when transfected together with anti-apoptotic Bcl-X(L) even though the expression of the antigen (and antibodies against it) was increased (Leitner et al. 2004). Curiously, another study using the same gene gun delivery approach obtained opposite results, and incorporation of Bcl-X(L) improved the immunogenicity of SFV replicon vaccine against HPV-transduced TC-1 tumors (Kim et al. 2004). One interesting, though purely speculative hypothesis for this discrepancy, is that the primary dermal target cells for the TAA expression in the latter study could have been DCs, and thus the longer survival of these cells enabled enhanced antigen presentation in the lymph nodes. Subsequent to these studies, the importance of cell autonomous immunity and especially type I IFNs for alphavirus mediated antitumor immune response has been corroborated by Leitner and others (Leitner et al. 2006; Ljungberg et al. 2007). In type I IFN receptor knockout mice the T-cell mediated efficacy of alphavirus replicon-based tumor vaccination was abolished, simultaneously causing an increase in Th2 type antibodies against the TAAs (Leitner et al. 2006). Finally, one recent study did not find any correlation between type I IFN induction and immunogenicity of four different alphavirus replicons (Maruggi et al. 2013). In addition to apoptosis, type I IFN signaling has been recently coupled with the induction of autophagy in several cancer cell lines (Schmeisser, Bekisz, Zoon 2014). The oncolytic measles virus was also found to induce selective autophagy to mitigate the cytosolic PRR response in cancer cells (Xia et al. 2014). Intriguingly, in one report, the antitumor efficacy of SFV replicon with ectopic HPV antigen expression depended on the route of immunization, with fewer viruses being needed when i.v. or i.m. routes were used compared to i.p. or s.c. injections (Daemen et al. 2004). It would have been informative to add an intradermal site to the comparison, as it has occasionally been reported to be more immunogenic than subcutaneous route (Bonnotte et al. 2003). However, in the phase I 37 clinical trial of CEA encoding vaccinia against metastatic adenocarcinoma both routes had equal safety and immunogenicities (Conry et al. 1999). The site of initial inoculation is presumably less important for replicative viruses assuming that the tumor antigen is stable and remains in the progeny viruses. Alphaviruses have also been used with some promising preclinical results in vaccination against TAAs of mastocytoma, mammary and prostate tumors, as well as against the antigens of oncogenic viruses. These approaches have been reviewed in detail elsewhere (Quetglas et al. 2010). 2.4.3 Combination therapy to improve OV efficacy Humoral innate- (IFNs, complement, TNF) and adaptive (antibodies) immune responses can seriously hamper the efficacy of virotherapy by clearing the therapy virus too rapidly thus preventing successful infection of tumor tissue. Pre-existing or vehicle-induced antibodies can prevent the virus from even reaching the tumor cells (Kirn, Martuza, Zwiebel 2001). IFN- plays a crucial role in restricting the replication of SFV and other alphaviruses (Fragkoudis et al. 2007; Grieder and Vogel 1999; Ryman et al. 2000) and thus only tumors that are defective in this response can be efficiently targeted. In viral infection, IFN- can be produced systemically by pDCs (Cao et al. 2008) and locally by all other nucleated cell types such as, the CNS cells, tumor infiltrating leukocytes and even by the cancer cells themselves. Some tumors have acquired mutations that render them incapable of producing and/or responding to IFN-, thus enabling tumor specific virus replication, but even in those cases, neutralizing antibodies (Nabs) remain a potential hurdle (McFadden et al. 2009; Zemp et al. 2010). A large variety of different pharmaceuticals has been tested in combination with oncolytic viruses with the intention of lowering the innateand adaptive immune responses against the virus and/or stimulating the immunity against the tumors. Cyclophosphamide (CPA) has been used in different immunocompetent tumor models in combination with oncolytic HSV (Fulci et al. 2006; Ikeda et al. 1999; Ikeda et al. 2000; Wakimoto et al. 2004), reovirus (Qiao et al. 2008), vaccinia (Lun et al. 2009) and VSV (Willmon et al. 2011). The immunomodulatory effects of CPA are pleiotropic (Bartlett et al. 2013). CPA can aid virus replication by reducing neutralizing antibody levels (Ikeda et al. 1999; Qiao et al. 2008) and inhibiting innate immune responses such as antiviral cytokines, complement and phagocytes (Fulci et al. 2006; Ikeda et al. 1999; Ikeda et al. 2000; Qiao et al. 2008; Wakimoto et al. 2004). Furthermore, low dose CPA can promote anti-tumor immunity by depleting regulatory T-cells, stimulating memory T-cell proliferation and sensitizing tumors to TRAIL-dependent CD8+ T-cell-mediated tumor destruction (Koski et al. 2010; Schiavoni et al. 2000; van der Most et al. 2009). Proliferation of memory T-cells may be, at least in part, a result of the induction of type I IFN, and upregulation of IFN- production as a response to CPA treatment has also been reported by other groups (van der Most et al. 2009). Controversially, CPA has also been found to lower the production of antiviral cytokines, including IFN- (Wakimoto et al. 2004). Thus, the cytokine milieu related effects of CPA remain elusive and may depend on the dosage and timing. Importantly, the oncolytic viruses themselves exert complex effects on immunity and occasionally the introduction of CPA does not produce synergistic but antagonistic effects on the 38 therapeutic efficacy (Willmon et al. 2011). Remarkably, the anticancer immunotherapeutic effects of CPA have been recently reported to rely on Gram-positive intestinal bacteria which translocate into secondary lymphoid organs in response to CPA (Viaud et al. 2013). Along with CPA, rapamycin is another immuno-regulator commonly used in combination with OV. Rapamycin has been shown to suppress both tumor and host IFN- responses, thus in optimal circumstances allowing more efficient oncolytic virus replication and tumor killing (Zemp et al. 2010). Rapamycin has been utilized in combination with multiple oncolytic viruses such as VSV (Alain et al. 2010), myxoma (Lun et al. 2010; Lun et al. 2007) and vaccinia (Lun et al. 2010; Lun et al. 2009). Rapamycin was shown to have also direct antineoplastic effects, and interestingly it has been tested also in glioblastoma clinical trials (Cloughesy et al. 2008). Rapamycin is an inhibitor of mTORC1 that can stimulate type I IFN production through phosphorylation of its two target proteins, 4E-BPs and S6K1/2. The main mechanism of rapamycin synergy with OV is likely related to its capability to suppress the systemic production of IFN- by pDCs (Cao et al. 2008), which then aids virus replication in semi-IFN--sensitive tumors (Alain et al. 2010). Valproic acid (VPA) belongs to the class of histone deacetylase (HDAC) inhibitors and along with CPA and rapamycin, is also commonly exploited pharmaceutical in combination with OV. HDAC inhibitors have been shown to act synergistically with OV at least by inhibiting IFNresponses and potentiating the virus-induced cancer cell apoptosis (Nguyen et al. 2008). VPA was shown to suppress NK-cell mediated early clearance of HSV (AlvarezBreckenridge et al. 2012a). Surprisingly, it was demonstrated that VPA may also boost antigen targeted immunity while simultaneously decreasing autoimmunity and the adaptive responses against viral vector (Bridle et al. 2013). Modulation of VEGF-A signaling in combination with replicative oncolytic VSV and reoviruses has been reported to be a powerful therapeutic approach in B16, B16ova and B16-VEGF mouse melanoma models, resulting in long term antitumoral effects even in B16ova tumors where reovirus replication per se was poor (Kottke et al. 2010). The rationale behind this approach was to induce a VEGF-A burst in the tumors by administering VEGFA (in the case of VEGF-A non-expressing B16 tumors) or by first administering and then withdrawing VEGF-A inhibitor (in the case of VEGF-A expressing B16-VEGF tumors). Both of these strategies caused a rebound in the VEGF-A levels and the induced VEGF-A burst allowed tumor-associated endothelial cells transiently to support viral replication. This led to CD4+, CD8+ and NK-cell-mediated immune attack on tumor vasculature resulting in long-term regressions. Intriguingly, co-administration of VEGF-R/PDGF-R inhibitor Sunitinib with OV resulted also in synergistic effects, which, however, were discovered to result from Sunitinib off-target inhibition of both PKR and RNaseL (Jha et al. 2013). Classical chemotherapeutic and radiotherapeutic approaches have been successfully combined with OV to achieve synergy, for example by enhancing tumor-specific viral replication or inducing enhanced antitumor immune responses. Many of these approaches have been reviewed elsewhere (Ottolino-Perry et al. 2010). Finally, oncolytic viruses have been successfully combined not only with different pharmaceuticals, but also with other oncolytic viruses. At least vaccinia and VSV infections seem to act synergistically (Le Boeuf et al. 2010), while vaccinia and SFV do not have this property (Vaha-Koskela et al. 2013). 39 2.4.4 Targeting of Oncolytic Viruses The ultimate goal is of oncolytic virotherapy is to develop safe viruses, which after systemic administration will specifically home to primary and metastatic tumors and destroy them or their vasculature by lytic replication and/or stimulation of antitumor immunity. There are multiple strategies involving a plethora of different constructs to achieve tumor specificity. These strategies include deletion-, transcription-, receptor-, translation-, directed evolution-, cell carrier- and microRNA -targeting of oncolytic viruses. From each of these a few representative examples will be described. Deletion targeting to create conditionally replicating viruses Viral genes are constantly under evolutionary pressure in the changing environment, and unlike higher organisms, viruses do not usually carry redundant genetic material inside their spatially limited virions. Viral genes have evolved to suppress extrinsic and intrinsic apoptotic pathways (for example, degradation of TNF-receptor or p53) (Benedict and Ware 2001; Querido et al. 2001), disrupt DNA-damage signaling (e.g. MRE11) (Martin and Berk 1999), to prevent innate- (Muster et al. 2004) and adaptive immune responses (Andersson et al. 1985), to stimulate nucleotide pool synthesis (in non-dividing cells) (McCart et al. 2001), to induce cellular proliferation by Rb inactivation or direct E2F activation (O'Connor and Hearing 2000) and to activate growth factor receptors (McCart et al. 2001), translational machinery (O'Shea et al. 2005) and histone acetyl transferases (HATs) (Howe et al. 1990). These viral genes are often redundant in cancer cells, which have already acquired mutations and epigenetic changes that deregulate cell survival, proliferation, macromolecular synthesis and immune-evasion (O'Shea 2005). Hence, by deleting one or more of the respective viral genes, one can suppress virus replication in normal healthy cells without significantly hampering the replication in neoplastic cells. Creating conditionally replicating alphaviruses by targeted deletions is not considered possible as the viral genomes are small and most of the viral genes are indispensable for productive infection. Transcriptional targeting A common approach to achieve tumor selective replication of a DNA virus is to place one or more of the essential viral genes under the control of a tumor-specific promoter or a tissue-specific promoter overexpressed in the tumor (Dorer and Nettelbeck 2009; Thorne, Hermiston, Kirn 2005). The examples of tumor- and tissue-specific promoters to target viruses such as HSV-1 and especially different adenovirus serotypes include albumin or alpha-fetoprotein promoters for HCC, calponin promoter for sarcoma, probasin and prostate specific antigen (PSA) promoters for prostate cancer and hypoxia response elements for multiple different cancers. While transcriptional targeting of oncolytic agents has shown promise in terms of specificity, the novel promoters are often weaker in comparison to native viral promoters. An excellent example of a multifaceted transcriptional targeting approach was reported by Gürlevic and colleagues, who designed a highly cancer-specific adenovirus Ad-p53T by 40 exploiting two ubiquitous cancer cell characteristics: expression of telomerase and a suppressed p53 signaling (Gurlevik et al. 2010). As p53 is expressed mostly in noncancerous cells, a viral repressor was placed under the control of the p53 promoter and furthermore, the autologous adenoviral early genes promoter was replaced with the human telomerase promoter. As opposed to the unmodified control virus, the engineered construct exhibited no high virus dosage related hepatotoxicity, and importantly, the promoter modifications did not compromise the oncolytic efficacy in a syngeneic murine cancer model. However, as p53 was retrovirally knocked down in the targeted CMT64 small cell lung cancer cells, the specificity of Ad-p53T remains to be shown in natural tumors. In addition, radiation inducible promoters such as the survivin-promoter have been incorporated into oncolytic viruses to achieve tumor specific replication and transgene expression in conjunction with radiotherapy (Nandi et al. 2008). As eukaryotic cells do not express RdRp, transcriptional targeting is generally not applicable to RNA-viruses such as Semliki Forest virus. An elegant way to circumvent this limitation was described by Guan et al., who engineered a hybrid vector comprising a helper-dependent adenovirus encapsulated SFV replicon driven by alpha-fetoprotein promoter and the IL-12 transgene expressed from SFV subgenomic promoter. The construct was HCC-specific and demonstrated high in vivo efficacy without causing any liver toxicity (Guan et al. 2006). Targeting virus entry In addition to manipulating viral replication, it is possible to achieve retargeting already one step earlier by modifying the viral cell surface attachment. The viral surface proteins of one serotype can be replaced by those of another or even with proteins from a completely different virus, a well established process termed pseudotyping. Oncolytic viruses which work well in selected tumor cell lines, may fail to target the actual tumors in clinical trials, because of lack of expression of viral receptors and co-receptors in the tumors. For instance, adenovirus serotype 5 (Ad5), which has been commonly used in gene therapy and oncolytic virotherapy for GBM, uses its fiber protein knob domain to bind Coxsackie virus and adenovirus receptor (CAR) on the cell surface. However, CAR expression is in general low in tumors and especially in GBM, and this seriously impairs the oncolytic use of the vector (Kim et al. 2003; Nandi and Lesniak 2009). Several successful approaches have been undertaken to retarget the adenovirus tropism towards more prominent epicellular structures in gliomas and other tumors. These include pseudotyping Ad5 with Ad3 fiber knob to create Ad5/3 chimeras (Zheng et al. 2007), incorporation of the RGD motif into the knob to target integrins (Dmitriev et al. 1998) and oncogenic FGFR1 receptor targeting by chemical conjugation of FGF2-ligand to the adenovirus capsid (Wang et al. 2005). One has to bear in mind that chemical conjugation as well as chemical shielding against immune-clearance using substances such as polyethylene glycol (PEG) (Arudchandran et al. 1999; Eto et al. 2008) are probably more suitable for viral vector and replicon mediated uses than for oncolytic virotherapy applications, as these modifications do not pass after the first infection cycle to the budding progeny virions. Stable, genetic retargeting to a commonly overexpressed receptor EGFR has been achieved both for an 41 oncolytic adenovirus (van Beusechem et al. 2003) and more recently for an oncolytic HSV (Uchida et al. 2013). Recently, as a good example of a rationale genetic engineering approach, measles fusion protein and single chain antibodies have been exploited to create a novel and, in theory, universal virus retargeting method. In this approach, the single chain antibody engineered to virus surface first binds to its specific epitope on cancer cell surface, and then the measles fusion protein mediates fusion of viral and cellular membranes leading to virus entry. The measles system has been utilized to retarget a variety viral vectors and replicative viruses to cancer-associated molecules such as EGFR, folate receptor, prostate membrane-specific antigen (Ayala-Breton et al. 2012) and CD133 CSC marker (Bach et al. 2013). The single chain antibody has also been replaced successfully with designed ankyrin repeat protein, in an effort to retarget lentiviral vector to HER2/neu (Munch et al. 2011). With respect to alphavirus vectors, the laboratory of Daniel Meruelo has shown the Sindbis virus replicon to be amenable to retargeting to cancer cells, both by direct genetic engineering of the envelope towards LH/CG receptors of choriocarcinoma cells (Sawai and Meruelo 1998) and ligation of IgG-binding domain of protein A to achieve targeting towards a variety of cell surface molecules when combined with specific monoclonal antibodies (Iijima et al. 1999; Ohno et al. 1997). The impediment to applying similar strategies to SFV is its unknown receptor. Translational targeting One other, though a relatively uncommon approach to achieve tumor-selective virus replication is the translational stabilization of viral mRNAs in a cancer-specific manner. Expression of COX2, a proto-oncogene and an inflammatory mediator has been associated with poor prognosis in many cancer types (Lee, Myung, Song 2013). COX2 is activated by cytokines and growth factors via the Ras/p-MAPK pathway and at least part of the activation can be attributed to stabilization by 3’UTR of its mRNA. Tumor specific stabilization of an oncolytic adenovirus mRNA was accomplished by ligating the 3’UTR of COX2 to the adenovirus early gene, E1A mRNA (Ahmed et al. 2003). Tumor targeting at the level of mRNA stabilization has been achieved also with poliovirus. Poliovirus has an inherent ability to target many tumors including GBM that are rich in its receptor Necl-5 (Goetz and Gromeier 2010; Pol et al. 2013). However, the inherent neuropathogenicity of the virus would normally limit its use in oncolytic virotherapy. As CNS specific translation of poliovirus non-capped mRNA depends on a cell type-specific IRES element, its replacement with complementary sequence from human rhinovirus type 2 resulted in abrogation of neurovirulence while replication in malignant cells was retained (Goetz and Gromeier 2010). According to the preliminary results from an ongoing phase I clinical trial, poliovirus PVS-RIPO has proven to be very promising in the treatment of incurable GBM (Pol et al. 2013). While potentially amenable, translational targeting of oncolytic alphaviruses has not been tested. Some alphaviruses such as EEEV, VEEV and WEEV have a high replication power, but their therapeutic use is limited because of their neurovirulence (Jose, Snyder, Kuhn 2009). 42 Targeting with microRNA technology Different RNAi pathways, especially those involving miroRNAs (miRNAs), are known to be dysregulated in cancer. Some miRNAs are oncogenic and thus overexpressed in malignancies, while others have tumor suppressor functions and are thus silenced (Baer, Claus, Plass 2013). So far, work has concentrated on incorporating target sequences of tissue specific miRNAs into the genomes of oncolytic viruses. This approach was recently successfully adapted to prevent adenovirus hepatotoxicity by addition of miR122 target sites (Ylosmaki et al. 2008; Ylosmaki et al. 2013) and the neuropathogenicity of the virulent SFV4 strain by including miR124 targets in the viral genome (Ylosmaki et al. 2013). In addition, miRNA detargeting of alphaviral replicon has been reported (Kamrud et al. 2010). SFV and other alphavirus vectors might be especially suitable for miRNA based targeting since RNAi is an integral part of antiviral defense against SFV infections in mosquito cells (Siu et al. 2011). Remarkably, high tumor specificity was recently reported by Sugio and colleagues for telomerase promoter-driven adenovirus harboring different target sequences for miRNAs that were under expressed in cancer cells without compromising oncolytic efficacy (Sugio et al. 2011). In this approach, liver detargeting was also accomplished by addition of miR-122a target sequence to the virus genome. Targeting with cell carriers Solid tumors are complex organs that attract several types of non-neoplastic cells to their stroma such as myeloid derived stem/progenitor cells and many types of immune cells (Hanahan and Weinberg 2011). Several laboratories have taken advantage of these tumor stromal cells as carriers for oncolytic viruses by utilizing their inherent tumor homing capabilities and the protection which they provide against antiviral immunity in the circulation. Recent examples of successful application of this approach to improve OV include CIK loaded and CCL5 expressing vaccinia virus utilized in murine MC38 colorectal cancer model (Sampath et al. 2013), neural stem cells loaded with adenovirus in the treatment of murine glioma xenografts (Kim et al. 2013), VSV loaded MDSCs in a metastatic MCA26 murine colon carcinoma model (Eisenstein et al. 2013) and hypoxia promoterdriven-adenovirus delivered by macrophages in a murine prostate cancer xenograft model (Muthana et al. 2013). Targeting by directed evolution A novel and innovative oncolytic virus targeting and potentiating approach was introduced by Hermiston laboratory, which used a “directed evolution” strategy. Pools of different adenovirus serotypes were passaged in human tumor cell lines (colon, breast, pancreatic, prostate) to enable viral recombination and then the most potent variants were selected after subsequent rounds of limiting dilution and repassaging. The resulting virus ColoAd1, a Ad3/Ad11p virus chimera was found to be 2–3 log units more potent and colon carcinoma selective than ONYX-015, one of the clinically most advanced oncolytic adenoviruses (Kuhn et al. 2008). 43 2.4.5 Naturally targeted oncolytic viruses Cancers, per definition, consist of malignant cells that show an abnormally high rate of proliferation and capability to resist cell death. In order to drive unregulated proliferation, cancer cells induce overactive biosynthesis of different macromolecules such as nucleic acids. Since viruses also need these building blocks for the synthesis of progeny particles it is not surprising that metabolically active malignant cells in general support viral replication better than quiescent healthy cells (Le Boeuf et al. 2013; Parato et al. 2012). While most oncolytic viruses used to date have required manipulation in order to achieve a sufficiently cancer-specific infection, a number of essentially unmodified viruses have also been used to treat cancer. These viruses inherently replicate better in transformed cells and thus can be defined as “naturally targeted oncolytic viruses”. Receptor preference Several tumor types have been reported to overexpress poliovirus receptor Necl-5, but due to the well-known neurovirulence of poliovirus additional modifications are necessary and thus poliovirus cannot be considered as a naturally targeted oncolytic virus (Goetz and Gromeier 2010). Sindbis virus uses high-affinity laminin receptor (LAMR) for its entry into mammalian cells, and the receptor has a known role in tumorigenesis. The unoccupied form of the receptor has been reported to be over-expressed in numerous cancer types and thus for long it was thought to account for tumor preference of Sindbis virus (Tseng et al. 2004). This view was, however, recently contradicted by Huang and colleagues, who claimed that cancer defects in type I IFN response rather than the levels of viral receptor, is the primary determinant of Sindbis tumor tropism (Huang, Guo, Hwang 2012). While the relative importance of these two factors might be disputable, it seems conceivable that both the number of Sindbis receptors on cancer cells and the intactness of cell autonomous antiviral responses contribute to the tumor infectivity, albeit at different stages. Defects in apoptosis signaling of cancer The fact that most tumors have defects in their apoptosis mechanisms makes them also sensitive to some viruses, as programmed cell death is a fundamental part of antiviral defense. This seems to hold true at least for NDV. The replication of NDV was documented to occur preferentially in malignant cells with defective apoptosis responses, and interestingly, tumor targeting did not seem to depend on type I IFN defects (Mansour, Palese, Zamarin 2011). However, some controversy regarding the tumor specificity remains, as NDV is not insensitive to the effects of IFN (Buijs et al. 2014). Interestingly, in this very recent study by Buijs et al., 11 different human pancreatic adenocarcinoma cell lines were tested for their IFN signaling and while major differences were observed in IFN induction upon NDV infection, IFN pretreatment inhibited replication of the virus in all tested cell lines. Defects in neoplastic type I IFN signaling Since the IFN system and ISGs have an important role in growth restriction and apoptosis, the responsible genes are occasionally deleted or epigenetically silenced in tumors. For example, IRF1, a transcription factor important in immunity against several viruses, including alphaviruses (Karki et al. 2012; Schoggins et al. 2011) is a well-known tumor 44 suppressor acting in concert with p53 (Tanaka et al. 1996). As a secondary effect, silencing of one or more of these genes can compromise tumor antiviral defense partially or even completely, rendering them as vulnerable targets for IFN sensitive oncolytic viruses such as VSV (Naik et al. 2012), myxomavirus (Lun et al. 2010) and reovirus (Wilcox et al. 2001). Interestingly, simple overexpression of one of the IFNA-receptor chains, IFNAR2c, can reintroduce the antiproliferative effects of type I IFN to many cancer cell lines (Wagner et al. 2004). According to one recent study, a large number of type I IFN receptors was needed for the antiproliferative effects of type I IFN while clearly smaller number was adequate for the antiviral response (Levin, Harari, Schreiber 2011). Different replicative alphaviruses especially those based on Semliki Forest- and Sindbis viruses have been used as naturally targeted oncolytic viruses. Sindbis replication preference in tumors has been reported to result from defects some cancers have in their type I IFN production and responsiveness (Huang, Guo, Hwang 2012). It is understandable that while defects in type I IFN production of cancer cell lines might impair the antiviral defense in vitro, they do not necessarily guarantee successful tumor targeting in vivo, as several tumor-resident nontransformed cell types such as macrophages are potent type I IFN sources (Liu et al. 2013). Hence, tumors that have defects in their type I IFN response are better candidates for the naturally targeted oncolytic viruses. Interestingly, U87 cells seem to have defects both in the response and induction of type I IFN (Wollmann, Robek, van den Pol 2007). There are multiple pathways which may lead to aberrant antiviral signaling in cancer. Suppressors of cytokine signaling (SOCS) proteins are negative-feedback regulators of type I IFN signaling. SOCS1 is a well-known tumor suppressor in the SOCS family of proteins, which is silenced by hypermethylation in many cancers, but intriguingly, induced in melanomas (Davey, Heath, Starr 2006). Melanoma is responsive and thus commonly chosen target malignancy for oncolytic virotherapy in clinical trials (Mastrangelo et al. 1999; Russell, Peng, Bell 2012). This may be at least partly due to the known immunogenicity of melanomas, a fact supporting immuno-virotherapeutic approaches. In addition, tumors do also inherently differ in their potency of launching cell autonomous antiviral attack. Indeed, some melanomas escape interferon therapy by suppressing the expression of STAT1 (Pansky et al. 2000; Wong et al. 1997; Wong et al. 1998). While the role of reduced STAT1 expression in melanoma IFN resistance has been contradicted (Chawla-Sarkar et al. 2002) it is nevertheless clear that melanomas do develop resistance to IFN therapy and, hence, the escape variants could be attractive targets for type I IFN sensitive oncolytic viruses. In addition to melanomas, also other tumors deregulate type I interferon response in order to evade adaptive antitumor immunity, and this seems to be true at least for xenoantigenic and thus for immunogenic human papillomavirus (HPV) induced tumors. HPV encodes several proteins that inhibit the interferon response, apoptosis and MHC presentation, all of which serve both the virus and tumor survival, and inadvertently, make the tumors good targets for oncolytic virotherapy (Grabowska and Riemer 2012; Le Boeuf et al. 2012). Cells infected with Epstein-Barr virus, the etiologic agent in Hodgkins’- and Burkitt's lymphomas and in some nasopharyngeal carcinomas, have also been reported to be resistant to type I IFN induced apoptosis (Ruf et al. 2005; Wu et al. 2009). Strikingly, serious IFN response defects have also been found in a number of human lung adenocarcinomas and prostate cancers (Dunn, Koebel, Schreiber 2006). Some human prostate tumors have been found to 45 be extremely sensitive to VA7-mediated oncolysis resulting in a total eradication of implanted xenografts (Miika Martikainen, personal communication). STAT1 induction has been recently implicated in the development of tumor resistance to genotoxic stress (radiation and chemotherapy), and radioresistant tumors concomitantly acquired resistance to the type I IFN sensitive HSV-1 mutant. Hence, the antiviral response profile of heavily pretreated tumors, as is the case in most oncolytic virotherapy trials, may be inherently unfavorable, proposing a paradigm shift in favor of selecting patients with malignant but early stage tumors for entry into clinical trials (Khodarev et al. 2004; Khodarev, Roizman, Weichselbaum 2012). Since overactive IFN/STAT pathway has also been implicated in inflammation-mediated tumorigenesis of gastric cancer, hepatocellular carcinoma and colorectal carcinoma, the patient selection for oncolytic virotherapy trials, especially with type I IFN sensitive viruses, should be based on careful diagnostic screening for tumor antiviral responses, and herein some tumor types might be inherently more sensitive to virus replication. An ideal screening approach (but applicable only for small scale trials) would be based on functional screenings, which always measure the objective outcome (cancer cell death) regardless of the underlying genotype. These screens could involve ex vivo infections of live tumor slice cultures or freshly prepared primary cells pretreated with type I IFN. Nevertheless, for the purposes of large scale personalized medicine, high throughput screens of applicable tumor resistance markers need to be developed. As an example, constitutive high level expression of OAS and MxA ISGs has been proposed as good candidate marker for tumor resistance against a type I IFN sensitive oncolytic VSV (Moerdyk-Schauwecker et al. 2013). Most viruses are sensitive to type I IFN and at least HSV, reovirus, VSV, poliovirus, influenza- and adenoviruses replicate more efficiently in constitutively Ras-activated tumors that often have defects both in the induction and response to type I IFNs (Christian et al. 2012; Donnelly et al. 2013a; Park et al. 2010). Nevertheless, sometimes the defects are virus-specific, and activation of the same CUG2-Ras pathway that mediated the susceptibility to reovirus also conferred resistance to another type I IFN sensitive virus, VSV through STAT1-OASL2 mediated signaling (Malilas et al. 2013). This apparent discrepancy may stem from differences in the antiviral effects of OASL2 against reovirus and VSV. At least the signaling pathways leading to the defects differ, and although the constitutively activated Ras-MEK pathway is considered to be responsible for the IFN defects facilitating VSV replication (Noser et al. 2007) Ras-p38 signaling mediates these defects for reovirus (Park et al. 2010). Interestingly, the tumor specificity of parvovirus (Paglino, Andres, van den Pol 2014), myxomavirus (Zemp et al. 2013) and possibly NDV (Mansour, Palese, Zamarin 2011) seem to be type I IFN independent. It has become evident that established cancer cell lines vary in their permissiveness to oncolytic viruses. This permissiveness may depend particularly on cancer cell autonomous IFN- antiviral defense capacity, especially when unmodified viruses such as VA7 are used. However, in contrast to clonal cell lines, naturally occurring cancer displays significant intratumoral and patient-to-patient heterogeneity. Thus, while it has been occasionally possible to dissect the mechanisms responsible for the diminished antiviral defenses in defined cell lines, reliable extrapolation of these findings to the clinical setting 46 has proved challenging. Furthermore, while it has been recently estimated in Bell’s laboratory that 65-70% of tumors are unable to produce or respond to type I IFN (Forbes et al. 2013), occasionally the pre-treatment experiments have been carried out using lower affinity IFN- instead of the de facto early interferon IFN- (Stojdl et al. 2000) or concentrations of type I IFN as low as 5 U/ml (Stojdl et al. 2003). 2.4.6 VA7 oncolytic virus SFV is an Alphavirus with a high replication efficiency and broad host range. Fully replication competent VA7 virus is based on the apathogenic and neurotropic SFV strain A7(74) and the virus harbors a multiple cloning site for expression of ectopic therapeutic and immunostimulative genes (Vaha-Koskela et al. 2003). Despite the high mutational rate of RNA-viruses during replication, the multiple cloning site of VA7 was shown to be rather stable, and 60% of plaques obtained from CNS homogenates after VA7-EGFP infection were fluorescent (Vaha-Koskela et al. 2003). VA7 has proven to be safe in immunocompetent hosts and powerful in several xenograft mouse models such as orthotopic human A2058 melanoma (Vaha-Koskela et al. 2006), K7M3 osteosarcoma (Ketola et al. 2008) and A549 lung cancer (Maatta et al. 2007; Maatta et al. 2008). VA7 has been shown to be capable of homing to the tumors even in operatively difficult body locations such as bone (Ketola et al. 2008). Since high viremia is an integral part of the lifecycle of arthropod borne alphaviruses such as SFV, they are likely especially suitable for targeting widely-spread metastatic disease. Furthermore, inherently neurotropic SFV is also well suited for targeting primary and secondary brain tumors. SFV is not endemic in the Western world, reducing the likelihood of pre-existing neutralizing immunity against the vector (Mathiot et al. 1990). Subcutaneous A2058 human melanoma xenografts were very sensitive to VA7 irrespective of the route of administration (i.v., i.p., or i.t.), but the total eradication of tumors was not achieved due to the presence of physical barriers caused by tumor compartmentalization and the emergence of virus resistant cancer cell clones (Vaha-Koskela et al. 2006). The type I IFN responsiveness of the A2058 tumors was not analyzed in the study. VA7 has demonstrated in vivo efficacy also in certain prostate tumor xenografts, which according to preliminary results also seem to have defects in their type I IFN response (Miika Martikainen, personal communication). However, testing the VA7 safety and efficacy in immunocompetent animal models is necessary as immunocompromised xenograft models do not exhibit the tumor microenvironment highly populated with lymphocytes found in many human tumors (Zemp et al. 2010) and also lack the components of antiviral and antitumoral adaptive immunity. SFV-based replicative and non-replicative (replicon) vectors have been utilized widely in oncolytic immunovirotherapy (Quetglas et al. 2010). However, both local and systemic administration of VA7 vector elicited MxA protein activation in subcutaneous A549 xenografts, indicative of IFN- induced responses against the virus. This likely contributed to the poor result with the systemic VA7 delivery as only after intratumoral 47 injections large virus-positive areas were visible in immunohistochemistry 16 hours post infection (Maatta et al. 2007). In line with these results, systemic application of VA7 did not improve survival in the orthotopic A549 lung cancer model, whereas local VA7 injection prolonged the survival of mice significantly (Maatta et al. 2008). 2.4.7 Armed oncolytic viruses A variety of different transgenes have been incorporated into oncolytic viruses to boost the efficacy of virotherapy, to allow noninvasive imaging and occasionally also to improve safety of the treatment. As previously described, some of these transgenes are designed to stimulate antitumor immunity (see chapter “2.4.2 Oncolytic virotherapy overlaps with gene therapy and immunotherapy of cancer – emerging concept of oncolytic immunovirotherapy”) (Naik et al. 2012). Other pre-clinical and clinical strategies include incorporation of cytosine deaminase/thymidine kinase double suicide transgenes into replicative adenovirus to treat prostate cancer (Freytag et al. 2002), adenoviruses releasing anti-angiogenic (Thorne et al. 2006) or ECM degrading (Kim et al. 2006) factors for tumor vasculature targeting and enhanced penetration respectively, sodium iodine symporter (NIS) expressing adeno-, VSV- and measles viruses combined with radioiodide for treatment and/or SPECT/CT imaging of several cancer types (Naik et al. 2012; Opyrchal et al. 2012; Trujillo et al. 2013). Interestingly, both matrix metalloproteinase (MMP) (Schafer et al. 2012) and MMP-inhibitor tissue inhibitor of metalloproteinases (TIMP) (Yang et al. 2011) expressing viruses have exhibited enhanced oncolytic efficacy. The former was thought to be a result of improved viral penetration thus resembling hyaluronidase co-treatment (Ganesh et al. 2008) and the latter due to direct tumor suppression. It will be interesting to determine in the future, whether TIMP expression from oncolytic viruses works partly through immuno-stimulation, as MMP activity has been linked to immunosuppression via stress ligand cleavage (Tang et al. 2013). A novel systems biology approach was recently introduced in the Bell laboratory, which created oncolytic viruses expressing type I IFN soluble receptor antagonists based on mathematical modeling (Le Boeuf et al. 2013). The antagonists were produced late in the viral life cycle, so the initial tumor specificity relied on the presence of a hospitable macromolecular environment in the metabolically hyperactive tumors. This approach was shown to work in vitro and a similar approach has been demonstrated to enhance the in vivo efficacy of oncolytic HSV (Fu et al. 2012). Finally, viruses engineered to express somatostatin (McCart et al. 2004) have been used to enable positron emission tomography (PET) visualization, luciferase and enhanced green fluorescence protein (EGFP) (Msaouel et al. 2009) to enable optical imaging, and the transferrin receptor (Weissleder et al. 2000) and ferritin (Iordanova et al. 2013) to the purpose of magnetic resonance imaging (MRI) of the infection. 48 2.4.8 Safety and ethics of oncolytic virotherapy A wide variety of different oncolytic viruses have been tested in clinical trials and the safety profile, even with the highest viral doses, has been excellent in comparison with classical cancer therapeutics. Nevertheless, careful preclinical and phase I clinical safety assessment are necessary for each new construct. Since viruses typically have restricted species tropism and thus the infection can potentially cause dramatically differing effects in different hosts, pre-clinical safety assessments should be carried out using more than one animal species. Other safety factors which need to be considered include the potential toxicity of the virustransgene combination, viral evolution, risk of horizontal transmission and the prevalence of antiviral medication, vaccinations and pre-existing immunity in the population (Miest and Cattaneo 2014; Russell, Peng, Bell 2012). The demand for careful and impartial biosafety assessments has been spurred especially by two cases where gene therapy resulted in patient fatalities and serious harm. The first case involved an 18 years old patient suffering from a rare genetic disease ornithine transcarbamylase deficiency resulting in defective protein catabolism and accumulation of toxic levels of ammonia. On September 17th 1999, he participated in a gene therapy trial, where the defective enzyme was to be introduced into his liver by non-replicative adenovirus 5-mediated gene transfer. The patient died from systemic inflammatory response syndrome, “cytokine storm”, resulting in multiple organ failure 98h after hepatic artery infusion of the vector (Raper et al. 2003). While all law suits were settled quietly, the investigators were accused of failing to notify the patient about the deaths that had occurred in experimental animals, the serious side effects that other patients receiving the same treatment had experienced and the extent of financial and academic conflict of interest of the principal investigators in the study. Furthermore, the study protocol was violated and the patient had received a higher than approved dose of the vector, and was not excluded from the trial even though his ammonia levels were above the accepted threshold (Wilson 2010). Most of the viral vectors and oncolytic viruses induce potent innate and adaptive immune responses, and these responses may be exacerbated if, as in this case, the patient presents acquired immunity against the vector already prior to treatment, or if the administration protocol is long enough for these memory responses to develop. The trial in question was phase I dose escalation study, and the patient was receiving the highest, ultimately fatal dose of the non-replicative vector. The dosing of replicative oncolytic viruses is likely to increase as viral manufacturing technologies develop thus raising new safety concerns, which will need to be addressed (Russell, Peng, Bell 2012). Furthermore, careful regulation of the levels of replicative viruses is deemed to be more challenging than dosing of classical pharmaceuticals or non-replicative vectors, and it is conceivable that the original input dose may not always correlate linearly with the level of the resulting viremia. Recently, high levels of mortality and morbidity of experimental mice due to cytokine storm were reported when oncolytic VSV was combined with the TLR agonist LPS (Rommelfanger et al. 2013). A second well documented case of gene therapy-related major adverse events emerged from two independent clinical trials aimed to treat X-linked severe-combined immunodeficiency (SCID-X1) by gamma (onco)retroviral transfer of the missing IL-2 49 receptor -gene (Hacein-Bey-Abina et al. 2008; Howe et al. 2008). While the gene therapy could cure most of the 20 infants suffering from the debilitating genetic disease, five of the patients developed leukemia, of which four were chemotherapeutically treatable (Wang et al. 2010a). The trigger for the multistep oncogenesis was proposed to be the activation of LMO2 gene due to inherent enhancer activity originating from the LTR-regions of the murine leukemia virus (MLV) gene transfer vector, which was stably integrated upstream of the proto-oncogene. To reduce the likelihood of insertional oncogenesis, in the thirdgeneration of self-inactivating lentiviral vectors the viral LTR regions are designed to be automatically inactivated; lentiviral vectors also have potentially safer insertional profile than oncoretroviral vectors (Rothe, Modlich, Schambach 2013; Schambach et al. 2013). The concerns of insertional oncogenesis may be especially prevalent when replicative oncolytic viruses capable of genomic integration such as MLV are used, and the risks have to be carefully balanced against the potential benefits achieved in the treatment of pre-existing malignancies (Perez et al. 2012). Some immunomodulatory cytokines such as interleukin-4 (IL-4) seem to be exceptionally dangerous when expressed from oncolytic viruses, resulting in uncontrollable virus replication (Chen et al. 2011; Jackson et al. 2001). These recombinant viruses have to be used with an appropriate biosafety containment level, and the cross-species reactivity of the used transgenes has to be carefully studied to limit the risk for human population. The ectopic inclusion of one of the multiple viral genes targeted against IFN response (Devasthanam 2014) to create recombinant viruses with improved oncolytic efficacy poses obvious safety concerns, but surprisingly so far the safety of the published constructs has been good (Fu et al. 2012; Zamarin et al. 2009). Combining SFV with certain Th1 type immunosuppressants such as linomide (Peltoniemi et al. 2002), CPA (Bradish, Allner, Fitzgeorge 1975), aurothiolates (Scallan and Fazakerley 1999), cyclosporine (Fazakerley and Webb 1987) and tunicamycin (Singh et al. 1987) has been reported to increase neurovirulence by presently incompletely understood mechanisms. Low dose CPA is a commonly used additive in OV preclinical and clinical regimes (see chapter “2.4.3 Combination therapy to improve OV efficacy”). While several mechanisms such as depletion of antigen presenting cells (Peltoniemi et al. 2002) and induction of neuronal membrane synthesis (Fazakerley 2002; Scallan and Fazakerley 1999) have been proposed to lie behind the increased neurovirulence, it will be interesting to determine whether depletion of T- and NK-cell derived IFN- plays any role. IFN- is known to protect neurons against SFV infection, and the latter cell class is present also in SCID mice (Keogh et al. 2003). Interestingly, the colloidal gold compound, myocrisin, was reported to increase A7(74) neurovirulence to L10 levels by unknown mechanism, with highly increased viral titers found also in macrophages (Oaten, Jagelman, Webb 1980). SFV is not considered to be a significant human pathogen, and the occasionally observed seropositivity among laboratory workers supports concept of low virulence of SFV in humans (Willems et al. 1979). However, one case of fatal encephalitis after accidental infection of a laboratory worker with SFV Osterrieth strain has been reported (Fazakerley 2002; Willems et al. 1979). The scientist had been suffering from purulent bronchitis for one year before the incident, and thus was possibly immunosuppressed. Finally, SFV infection in mice is known to cause transient autoimmune demyelinating disease (Fazakerley 2002; 50 Mokhtarian et al. 1999), and malformations or death in fetal mice (Mabruk et al. 1989) but neither of these sequelae have been reported in SFV infected humans. 51 3 Aims of the study The general aim of this thesis project was to explore the applicability of Semliki Forest virus VA7 in the targeting and treatment of malignant gliomas and colorectal carcinoma in vitro and in experimental mouse models. In addition, the original aims were to elucidate the roles of host innate and adaptive immune responses in the therapeutic outcome. The specific aims of the three parts (I-III) of the thesis were as follows: (I) (II) (III) To study whether VA7 after systemic administration would be able to home to orthotopic U87-Fluc glioma xenografts, establish productive infection and prolong survival of immunocompromised mice. To investigate whether VA7 virotherapy would prolong survival of immunocompetent or immunocompromised mice bearing orthotopic, syngeneic GL261 and CT-2A mouse gliomas, and to explore the cell autonomous antiviral signaling of virus-infected U87-Fluc, GL261 and CT-2A glioma cell lines. To characterize two clones of CT26 mouse colon carcinoma differing in their immunogenicity and type I interferon responsiveness upon infection and to utilize the two-clone model in the analysis of the roles of innate and adaptive immunity in VA7-mediated oncolysis. 52 53 4 Materials and methods Several different techniques of biotechnology, molecular biology and virology were applied in this thesis work in vitro, in vivo and ex vivo. The cell lines (Table 2) and mouse strains (Table 3) used and their sources are listed, and some of the techniques are presented at a general level where greater details can be found in the corresponding publications (I-III). 4.1. CELL LINES AND MOUSE STRAINS (I-III) Table 2. Cell lines used in the study. The names of the cell lines are in alphabetical order, their origins and host species and the type of work they were used in are described. The cells were cultured in 5% CO2 in a humidified atmosphere using cell culture media described in the respective publications (I-III). Cell line From Description A172 ATCC human glioma in vitro I BHK-21 ATCC Thomas Seyfried, Boston College, MA baby hamster kidney in vitro I, II, III C57BL/6 glioma in vitro, in vivo II Balb/c colon carcinoma in vitro, in vivo, ex vivo II CT-2A CT26WT ATCC CT26.CL25 ATCC Balb/c colon carcinoma passaged from previous by Balb/c colon carcinoma John Bell Daniel Silbergeld, University Balb/c glioma of Washington, WA CT26LacZ DBT in vitro II II I, II in vitro in vitro, in vivo, ex vivo I human glioma in vitro I African green monkey kidney in vitro II, III Balb/c breast carcinoma in vitro Figure 6A G26-24 Chicago, Glyn Dawson MBA-13 Aimo Salmi, University of Turku U87 ATCC C57BL/6 oligodendroglioma SV40 transformed mouse brain cells positive for CNPase oligodendrocyte marker* human glioma U87-Fluc established human glioma U251 Jarmo Wahlfors, University of Kuopio * (Vähä-Koskela 2007) II in vitro C57BL/6 glioma 4T1 in vitro in vitro, in vivo, ex vivo Figure 6A,B Geza Safrany, NRIRR ATCC Pirkko Härkönen, University of Turku Ref. in vitro, in vivo, ex vivo in vitro, in vivo GL261 VERO(B) Used II I, II 54 Table 3. Mouse strains used in the present study. The names of the mouse strains, their breeders and phenotypes are described. All mice were adult (4w-10w) females. Strain Breeder Phenotype Balb/cOlaHsd Foxn1 -/- Harlan athymic (defective T-cell immunity) I athymic (defective T-cell immunity) II nu B6.Cg/NTac-Foxn1 NE10 Taconic Ref. C57BL/6JOlaHsd House bred in Kuopio immunocompetent C57BL/6 mice II JaxC57BL/6J House bred in Kuopio immunocompetent C57BL/6 mice Figure 6B Balb/cAnNCrl Charles River III Fox Chase SCID Charles River immunocompetent Balb/c mice Prkdcscid/scid mutation causing defective T- and B-cell immunity III 4.2 VA7-EGFP PRODUCTION (I-III) VA7-EGFP plasmid (Figure 4) was heat shock transformed into XL1-Blue chemicompetent bacterial cells and cultured in LB-Ampicillin (50 μg/ml) solution for 16 hours at +37oC. Plasmid DNA was extracted using High-Speed Plasmid Miniprep Kit (GeneAid) and eluted in heated elution buffer according to the manufacturer’s instructions. To enable successful in vitro transcription, the plasmid was linearised with SmaI-restriction enzyme. The linear plasmid was purified using chloroform extraction (without phenol) to remove all protein impurities, especially RNase. The volume of the DNA digest was topped to 100 μl with m.q. H2O and then mixed with 10 μl of 3M NaAc (pH 5.2). A volume of 110 μl of chloroform was then mixed into the solution and the mixture was shortly centrifuged (1 min, full speed). Only half of the aqueous phase was recovered to prevent chloroform carryover. DNA was precipitated with absolute EtOH (2.5 x the sample volume) at -20oC for 1 hour followed by 15 min, +4oC, 16 000 g centrifugation, washing the pellet with 70% cold EtOH (5min, 16 000 g, +4oC), air drying and dissolving the DNA pellet into 15 μl of m.q. H2O. The linearised and purified VA7-EGFP DNA was then used for in vitro transcription by mMESSAGE mMACHINE SP6-kit (Ambion) and the synthesized viral RNA was LiCl precipitated according to the manufacturer’s instructions. A total of 500 000 BHK-21 cells were plated onto 6-well plates in High Glucose (4500 mg/L) DMEM supplemented with 5% FCS, 2mM L-glutamine, Penicillin-streptomycin and 25mM HEPES, and 2 μg of the viral RNA was transfected per well using TransIT-mRNA kit (Mirus Bio) according to the manufacturer’s instructions. The transfection success was verified by observing the cytopathic effect and fluorescence by microscopy, and the primary virus supernatant was collected from the cells 48 hours after transfection and stored at -70oC until used for secondary infections. 55 BHK-21 cells were cultured in a large (175 cm2) tissue culture flask until approximately 90% confluence and infected with the primary virus in 20 ml of fresh cell culture medium (the same composition as in the primary virus productions). The secondary virus preparation was collected at 24 h post infection, before the the appearance of massive cytopathic effect. The cell debris was pelleted by centrifugation and removed, the supernatant was sterile filtered using 0,2 μm filter and then the secondary virus was divided into 0,5-1 ml aliquots and stored at -70oC. Figure 4. Vector map of VA7-EGFP plasmid with XmaI/SmaI linearization site. NSP=non structural protein, Opal=opal stop codon, 26S=sub-genomic promoter, C=capsid, E=envelope genes, 6K=structural gene, MCS=multiple cloning site, EGFP=enhanced green fluorescent protein AmpR= ampicillin resistance. XmaI (5’ overhang) and SmaI (blunt) linearization site. In addition to the linearization site, the unique BamHI and AvrII enzyme sites for MCS cloning along with some commonly used sites for restriction enzyme analysis are presented. 56 4.3 DETERMINATION OF SEMLIKI FOREST VIRUS INFECTIOUS TITERS (I-III) The ability of VA7 to replicate in different cells and tissues in vitro and in vivo was measured by plaque assay. 200 000 VERO(B) cells were seeded in low glucose (1000 mg/L) DMEM supplemented with 5% FCS, 2mM L-glutamine, penicillin-streptomycin and 25mM HEPES to 12-well plates one day prior to virus infectivity titrations. Tenfold virus dilutions were prepared in cold VERO(B) cell medium and 400 μl of dilutions from 1/104 to 1/109 were applied as duplicates to the VERO(B) monolayers without removing the old warm medium. The plates were immediately moved back to the +37oC incubator. The plates were kept in the incubator for longer than 30 minutes to allow all of the virus particles enter the cells, but for less than 2 hours to ensure that budding of new virions had not begun. During the incubation period 0,4% agarose was prepared in +42oC VERO(B) medium from preprepared and autoclaved 4% low melting temperature agarose stock melted in boiling water. The 0,4% agarose was stored at +42oC water bath until the end of the incubation. The plates were taken from the incubator and the medium replaced with the warm agarose that was left to polymerase for 10 minutes in room temperature before returning the plates to the incubator. The final virus incubation under the agarose layer was carried out for 48 hours. Agarose was then removed and the cell monolayers stained with Crystal Violet solution for 5 minutes and additional color was washed away with water. The plaques were then counted manually against light. 4.4 PREPARING MICE AND CELLS FOR TUMOR TRANSPLANTATIONS (IIII) The cancer cells were cultured in large (175 cm2) tissue culture flasks at +37oC, 5% CO2 in a humidified atmosphere until a sufficient amount of cells were acquired. The cells were washed with warm DPBS and trypsinized. Trypsin was inactivated with 10 ml of +4oC Opti-MEM (Life Technologies), and from this point on the cells were stored on ice all of the time. The cells were pelleted in 14 ml Falcon tube by centrifugation (+4oC, 1000 RPM, 4 min) and most of the supernatant was aspirated away. The cells were re-suspended in the remaining volume of Opti-MEM and the total volume of cell/supernatant mixture was measured with a pipette. The cells were first diluted 1/100 in DPBS and then 1/2 in Trypan Blue to measure the viability. Cells in the resulting 1/200 dilution were counted using Bürker chamber, and based on total cell count, the required amount of +4oC Opti-MEM was added to reach the target cell concentration in the transplantation solution (10 000 cells/5μl, 50 000 cells/5μl or 100 000 cells/5μl for intracranial glioma inductions, and 300 000 cells/50μl for intradermal CT26 implantations). Mice were pharmacologically anesthetized for the intradermal tumor transplantations, and the anesthesia was combined with isoflurane gas anesthesia to allow the more invasive intracranial tumor inductions. 1.2% isoflurane concentration was used with air flow of 200 – 250 ml/min. Isoflurane dose was increased when remaining foot pad reflexes were 57 observed and decreased if the mouse had shortness of breath. The chemical anesthesia cocktail diluted in sterile saline contained medetomidine (1mg/kg) and ketamine analgesics (75 mg/kg), and the anesthetic was administered intraperitoneally. Analgesia was supplemented with subcutaneous, saline diluted carprofen (5 mg/kg) administered immediately after the transplantation together with intraperitoneal injection of the antisedative drug atipamezole (1 mg/kg), also diluted in saline. The eyes of anesthetized mice were kept moist with sterile saline, and the furless athymic “nude” mice were protected against anesthesia related hypothermia by using tissue blankets and hot water bottles in the cages. The weight of the mice was monitored daily and the mice were euthanized if there was >20% weight loss or any signs of severe distress such as neurological symptoms. Tumor development was measured using small animal MRI in the case of intracranial gliomas, or with a caliper instrument in intradermal tumors. Pain caused by high tumor burden was medicated with subcutaneous injections of carprofen (5 mg/kg) diluted in saline and buprenorphine (0.1 mg/kg) diluted in sterile water. The intradermal tumor transplants were administered as a bolus using 1 ml syringes with solid 30G needles to the shaved flanks of the mice. For the purpose of intracranial injections the mouse scalp was disinfected with 70% EtOH followed by scalpel incision on the immediate right side of the head midline, an area free of large blood vessels. A hole was manually drilled through the skull using 21G needle 2 mm to the right of bregma (II). Later the protocol was refined and the hole was moved 1 mm anterior, in order to avoid injection to the ventricles (Figure 6B). A Hamilton syringe with a lock plunger was vertically attached to a stereotactic apparatus or laboratory lift bench and lowered to a 3 mm depth utilizing needle stopper made out of cut 10 μl pipette tip. The needle (opening towards the right hemisphere) was lowered into the brain, kept still for 1 minute and the cells were slowly injected in 5 μl volume by moving slowly the lock screw plunger. The needle was kept still for an additional 1 minute after the injection to avoid reflux, the skull surface was wiped with damp 70% EtOH swab and the skin wound sutured. Finally, the mice were antisedated and 1 ml of sterile saline was injected intraperitoneally to prevent dehydration. 4.5 SDS-PAGE AND WESTERN BLOTTING (III) The CT26 cells for SDS-PAGE and western blotting were cultured at least in duplicate in 10 cm plates. After the cells had received the virus and interferon treatments for the predetermined time periods, they were washed twice with ice cold DPBS. Then as adapted (with modifications) from the protocol by Simmons et al., (Simmons, Wollish, Heise 2010), 500 μl ice-cold radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1% Igepal, 0.5% deoxycholate, 0.1% SDS, Complete Mini protease inhibitor cocktail tablets [ref. 11836170001, Roche], phosphatase inhibitor cocktail [ref. 04906845001, Roche]) was added to the plates, the cells were scraped into microcentrifuge tubes and kept on ice for more than 5 minutes. The cell debris was centrifuged and the total protein content was measured using commercially available assay (Biorad, cat. 500-006) according to the manufacturer’s instructions. Cell lysates were stored in -70oC until needed 58 and then boiled in 1x loading buffer (10x stock: 0.45M Tris-HCl, pH 6.5, 0.5M DDT 10% SDS, Bromophenol Blue, <50% glycerol) before running in 10% SDS-PAGE and wet transferring to Hybond-ECL membranes (RPN2020D, GE Healthcare). The anti-OAS rabbit polyclonal antibody was a generous gift from Ilkka Julkunen, (Helsinki, Finland) and used at 1:5000 dilution with 1h incubation at room temperature. Rabbit polyclonal antibody detecting total STAT1 (BD transduction laboratories, cat. 610119) was used in 1:4000 dilution (RT, 1h). Rabbit polyclonal anti STAT1-P (Tyr701) was purchased from Cell Signaling Technology (cat. 91715) and used in 1:5000 dilution (O/N, +4oC). Mouse monoclonal anti -actin (C4) used in 1:3000 dilution (O/N, +4oC) was purchased from Santa Cruz Biotechnology (cat. sc-47778). Secondary detections were carried out with simultaneous incubation of anti-mouse-cy3 (-Actin detection) and anti-rabbit-cy5 fluorophore-coupled antibodies using Amersham ECL Plex Western blotting system combined with Typhoon scanner. 4.6 SFV DETECTION FROM TISSUES BY IMMUNOHISTOCHEMISTRY (IIII) The VA7 infection pattern in tumors and different healthy tissues was examined post mortem by immunohistochemistry. The mice were sacrificed with CO2 and perfused with PBS (I, III) or 4% formalin (II). The tumors, tissues and organs of interest were immersed in PBS diluted in 4% paraformaldehyde for 24 hours (+4oC), after which they were moved into 70% ethanol (+4oC) until embedded into paraffin and sliced into 7-μm sections with a microtome. The sections were lifted on SuperFrost Plus glass slides (Menzel-Gläser) and incubated in +37oC for overnight to ensure proper attachment of tissue sections. Then the sections were deparaffinized and rehydrated by dipping into the following solution series: 2 x xylene (3 min each), 2 x 100% EtOH (3 min each), 2 x 96% EtOH (3 min each), 1 x 70% EtOH (3 min) and PBS (1 min). Then the slices were subjected to antigen retrieval by boiling in 10mM trisodium citrate buffer (pH 6) for 20 minutes. The slices were allowed to cool down and endogenous peroxidase activity was quenched by keeping the slices in methanol containing 0.3% H2O2. The slices were washed 3 x 5 minutes in PBS and blocked in normal goat serum (from Vectastain ABC kit, Vector Laboratories, Burlingame, CA) diluted in PBS for 30 minutes. The slices were incubated for 60 minutes at room temperature in primary rabbit polyclonal anti-SFV antibody diluted 1/4000 in PBS. Then the slices were washed 3 x 5 minutes in PBS and incubated with biotin coupled Vectastain secondary antibody for 30 minutes at room temperature, followed by 3 x 5 minutes PBS washes and 30 minutes of ABC reagent incubation at room temperature according to the manufacturer’s instructions. The slices were washed again for 3 x 5 minutes in PBS and finally DAB (SigmaFast, ref. D4293) stained for a few minutes until a brown color was visible. Staining was terminated by washing the slices under tap water, and slices dehydrated by running the multistep rehydration series at the beginning of protocol in the reverse order (70% EtOH Æ xylene). The slices were left to dry for a few minutes and finally mounted using a few drops of Permount (Fischer Scientific, ref. SP15-500) mounting medium and a glass cover slip. The cover slip was left to attach overnight at room temperature before microscopy. 59 5 Results 5.1 PERIFERALLY ADMINISTERED VA7 HOMES INTO ORTHOTOPIC U87FLUC GLIOMA XENOGRAFTS AND DESTROYS THEM (I) While VA7 Semliki Forest virus had previously demonstrated potency in several xenograft cancer models (Ketola et al. 2008; Maatta et al. 2008; Vaha-Koskela et al. 2006), it was deemed of interest to investigate whether as a neurotropic virus, it was able to target orthotopic gliomas. Three different human glioma cell lines, namely U87, U251 and A172 were tested for their VA7-EGFP sensitivity by infecting the cells with various MOIs and measuring the cell viability in the Calcein AM assay at 96 h p.i. While all of the cells were infectable and died when the high MOI=10 was used, U87 cells were clearly most sensitive towards low virus doses, i.e. even MOI=0.01 infection resulted in almost complete cell death at the 96 h p.i. time point (I, Fig. 1a). With the MOI=1 infection, all the cell lines expressed abundantly SFV structural proteins E1, E2 as well as the pE2-E3 precursor protein p62 (I, Fig. 1c) (Uchime, Fields, Kielian 2013). Due to its potential for virotherapeutic targeting in vitro, the U87 cell line was chosen for further in vivo studies. Subsequently, the U87 cell line expressing firefly luciferase (Fluc) was created by lentiviral (MOI=25) transduction (I, Fig. S1D). Importantly, the transgene insertion did not influence U87-Fluc cell proliferation or virus sensitivity compared to the parental U87 line (I, Fig. S1B, C). U87-Fluc tumors were first implanted subcutaneously into Balb/c OlaHsd Foxn1 -/- nude mice followed by intravenous VA7-EGFP injection. While the virus homed into the small (average size 96.5 mm3) subcutaneous tumors, completely eliminating them, the large tumors (average size 442 mm3) were more refractory (I, Fig. 3). Eradication of half of the large subcutaneous U87-Fluc tumors required three intravenous virus injections at 7 day intervals (I, Fig. 2). However, even the large tumors that did survive the therapy despite the three virus injections appeared to be more necrotic than control tumors when examined histologically with live Fluc positive cells being found mostly in the tumor rim areas (I, Fig. 3B, C). Furthermore, explant cell lines derived from the surviving tumors had remained infectable (I, Fig. S3). According to histological evaluation carried out for samples collected at d3 post intravenous VA7-EGFP injection (I, Fig. 6) and biodistribution monitoring using ectopic renilla luciferase expression from VA7 (VA7-Rluc) during the first survival experiment (I, Fig. S4), intravenous VA7 injection led to the infection of intracranial U87-Fluc gliomas. This infection resulted in tumor elimination and survival of 16/17 mice. The survival experiment was carried out twice and mice were monitored on the first occasion for 120 days post implantation after receiving either VA7-EGFP (n=5) (I, Figs. 4, 5b) or VA7-Rluc (n=6) injections (I, Fig. 5a, b) and on the second time for 90 days after VA7-Rluc injection (n=6) (I, Fig. S6). In the first experiment, one VA7-Rluc treated mouse (mouse 5) showed residual tumor-derived bioluminescence at the last time point imaged (I, Fig 5a), and a 60 cluster of tumor cells was detected in the brain post mortem by anti-Fluc IHC staining (I, Fig. 5c). In the first survival experiment 3/6 of the mice (including mouse 5) in the VA7-Rluc treatment group were let to grow close the bioluminescence endpoint (approximately one week before the appearance of neurological symptoms) before the intravenous virus injection. In the second survival experiment, 1/6 (mouse 1) of the orthotopic tumors escaped the virotherapy (I, Fig. S6). Infective VA7-Rluc could not be recovered from the brains, heart, liver or the spleen after the 90 days monitoring period of the second survival experiment (I, Table 1) and the organs revealed no pathological changes in hematoxylineosin staining apart from occasional wound healing or scarring seen at the presumable tumor induction sites (I, Figs., S5, S7). Similarly, while all of the above organs of the infected naïve mice contained low (max 4.7 x 104 PFU/g) viral titers at day 3 p.i., infectious VA7-Rluc could be recovered only from the brains on day 6 (average virus titers of 2 x 104 PFU/g) and from none of the organs on day 9 (I, Table 1). No pathological changes were seen in these organs of the infected naïve mice at day 3 p.i (I, Fig. S7) and infected mice did not experience weight loss as compared to PBS controls during the 48 day follow up (I, Fig. S2). 5.2 THE VA7 RESISTANCE OF GL261 MOUSE GLIOMAS REFLECTS THEIR ROBUST TYPE I IFN SIGNALING (II) Spurred by the encouraging results in the U87-Fluc glioma xenograft model, it was decided to proceed to test the oncolytic potential of VA7 in the GL261 and CT-2A-Fluc syngeneic mouse glioma models. Both of the cell lines were infected and lysed in vitro, but neither i.v. nor i.c. VA7 injections resulted in tumor infection in vivo and no therapeutic benefit was obtained (II, Fig. 1). Intracranial VA7 injection into GL261 tumors led to infection of the corpus callosum (II, Fig. 2d). Immunosuppression with CPA or rapamycin did not improve the GL261 tumor infectivity or mouse survival following VA7 i.v. injections (II, Fig. 2) though both immunosuppressing agents did reduce the amount of virus neutralizing antibodies (II, Table 1). Importantly, CPA markedly potentiated the infection of healthy brain tissue (II, Fig. 2e). IHC data revealed that the GL261 tumors were actually more resistant in vivo than the healthy brain parenchyma and the spread of the virus was often halted at the border of tumors (II, Fig. 2d). This occurred even after CPA facilitated pancerebral infection (II, Fig. 2e). Explant cells isolated from the GL261 tumors were also infectable (II, Fig. 4c). To exclude the possibility that some remaining adaptive antiviral immune responses had been responsible for the virus neutralization, intracranial GL261 tumors were next implanted and treated in athymic mice. The GL261 tumors were resistant also in B6 nude mice and thus neither i.v. nor i.c. VA7 injections improved survival (II, Fig. 3c). Intracranial VA7 injection resulted in infection of the corpus callosum also in nude mice (II, Fig. 3a, b). After exclusion of a central role for adaptive immunity in the therapeutic outcome, the role of cell autonomous type I IFN response was investigated. A dose of 100 U/ml IFN- pretreatment inhibited VA7 replication efficiently both in GL261 and CT-2A cells, and IFN was shown to work equally well when added 30 minutes or 90 minutes after the virus 61 application to GL261 cells (II, Fig. 4a) but was ineffective 4h after virus administration (II, Fig. 4b). In order to test the hypothesis that therapeutic susceptibility to VA7 infection depended on tumor IFN- sensitivity it was decided to treat GL261 cells in parallel with virotherapy amenable U87-Fluc cells (I) with human and mouse IFN- followed by VA7 infection. Mouse, but not human IFN- blocked infection in GL261 cells, even though a 5 fold higher concentration of human IFN- was applied (100U/ml vs 500 U/ml). IFN- from neither species had any observable effect on U87-Fluc cells (II, Fig. 4d), whereas human IFN- markedly restricted VA7 infection in a patient biopsy-derived primary glioma cell line. Interestingly, despite the clear inhibition caused by human IFN- pretreatment of the primary glioma cells, VA7 infection often escaped at later time points (Figure 5). GL261 cells seemed to be especially sensitive to the effects of mouse IFN- as the pretreatment did not completely block VA7 infection in mouse G26-24 oligodendroglioma and mouse 4T1 mammary carcinoma cell lines (Figure 6A). Furthermore, a tendency towards improved survival was observed after two intracranial VA7 injections in the orthotopic G26-24 glioma model (Figure 6B). Figure 5. Primary human glioma cells were susceptible to VA7-EGFP infection. Primary human glioma cells were VA7-EGFP infected (MOI=0.01 or MOI=1) with or without 100 U/ml or 1000 U/ml hIFN- pretreatment. The cytopathic effect was monitored with phase contrast microscopy (upper panels) and the infection with fluorescence microscopy (lower panels). All pretreatments were carried out in duplicates with similar results. While both MOIs resulted in efficient infection and there were only a few viable cells left at the 72 h p.i. time point, especially the higher dose hIFN- treatment conferred marked protection of the cells. The lower dose of hIFN- could not always prevent the infection, which spread to monolayer cells and spheres. While also the spheres were readily infected, the cytopathic effect was not seen in them before the last 144h time point. 62 GL261 cells were autonomous in their IFN- production (II, Fig. 5b) and response (II, Fig. 5 a) upon VA7 infection as shown by IFN- ELISA and OAS western blot analysis, respectively. Interestingly, the D51 VSV mutant induced more IFN- than VA7 in both GL261 and DBT mouse glioma cell lines; in the latter cell line VA7 caused no detectable IFN- secretion. Confluent GL261 cultures were found to be more resistant to VA7 infection than subconfluent cultures especially when a low MOI was used (II, Fig. 5c). In the mixed confluence assay, the confluent centers of individual wells were resistant while the subconfluent culture rims remained infectable (II, Fig. 5d). The confluent cultures were also more efficient IFN- producers (II, Fig. 5e). CT-2A (II, Fig. S1) and U87-Fluc (II, Fig. 5f) cultures remained susceptible to infection regardless of the seeded cell number. U87-Fluc cultures contained spheres that could sustain the infection for a prolonged time before cell lysis (II, Fig. 6). Similar results were obtained with VA7-EGFP infection of primary glioma cells (Figure 5). Finally, an in vivo antiviral assay was performed where two separate cultures of GL261 cells were infected with VA7-EGFP and one of them was used for intracranial tumor implantation while the other was left in the incubator. While the in vitro culture was efficiently infected, the growth of intracranial tumors was unperturbed as compared to uninfected controls (II, Fig. 7). 63 Figure 6. mIFN- blocks VA7-EGFP infection efficiently in GL261 mouse astrocytoma cells but only partially in 4T1 mouse breast carcinoma and G26-24 mouse oligodendroglioma cells. A. 4T1 and G26-24 cells were efficiently infected with both virus doses tested (MOI=1 and MOI=0.01), but the infection of GL261 cells was incomplete when the lower MOI value was used. The cytopathic effect was monitored by phase contrast microscopy (upper panels) and the infection by fluorescence microscopy (lower panels). With respect to the untreated cells G26-24 were the most sensitive to infection, and a massive cytopathic effect was visible already at 24h p.i. even with the lower virus dose. 100U/ml mIFN- pretreatment efficiently prevented the infection of GL261 cells but only partially that of 4T1 and G26-24 cells. Confluent 4T1 cultures were also efficiently infected. The treatments were carried out in duplicate with similar results. B. C57BL/6 mice received intracranial G26-24 oligodendrogliomas and two i.c. VA7-EGFP injections. The first injection was given 6 days after tumor transplantation and the second injection one week after the first one. Treatment group displayed a tendency towards improved survival (P=0.0753, Log-rank (Mantel-Cox) Test). 64 5.3 SUBCLONE OF RESISTANT CT26 COLON CARCINOMA IS HIGHLY SENSITIVE TO VA7 DUE TO WEAK TYPE I IFN RESPONSE (III) To test whether VA7 virotherapy could be functional in an immuno-competent host presuming that the tumors per se would be susceptible, different syngeneic mouse cancer cell lines were screened for their type I IFN sensitivity. It was soon discovered that two clones of CT26 mouse colon carcinoma, namely CT26WT and CT26LacZ dramatically differed in their VA7 sensitivities. Productive VA7-EGFP infection in vitro was faster in the CT26LacZ cells and the supernatant titers peaked at 24h p.i., one day earlier than in CT26WT cultures (III, Fig. 1a). VA7 infection also killed CT26LacZ cells significantly more efficiently than CT26WT cells as revealed in alamar blue assay at 48 h p.i., especially when low MOI, 0.05, 0.1 or 0.5, were used (III, Fig. 1b). This difference reflected also in the in vivo susceptibility of intradermal CT26WT and CT26LacZ tumors, and while the CT26LacZ tumors were efficiently eradicated, VA7 therapy had no detectable effect on CT26WT tumor growth as compared to sham treatment (III, Fig. 1c). Moreover, VA7-EGFP infection at MOI=0.01 resulted in a massive infection and cell death of CT26LacZ cultures despite 1000 U/ml IFN- pretreatment, whereas the same IFN- concentration prevented infection and death of the CT26WT cells even at MOI=1 (III. Fig. 1d). Nevertheless, at 1000 U/ml IFN- some CT26WT cells could be infected, whereas in the parallel GL261 culture the infection was completely blocked (Figure 7). To investigate whether IFN- depletion was sufficient to confer CT26WT cells susceptible to VA7-EGFP infection, CT26LacZ and CT26WT cells were first plated under agarose layer and then VA7-EGFP was added with or without anti-IFN- or anti-panIFN- antibodies into a well stamped through the agarose. While CT26LacZ cells were successfully infected even without antibody treatment, the CT26WT cells could be successfully infected only when anti-IFN- antibody was used (III, Fig. 2a). The ELISA assay revealed that both cell lines secreted IFN- in response to the infection (III, Fig. 2b). The secreted interferons were also biologically active, and conditioned supernatants collected from both of the cell lines protected CT26WT, but not CT26LacZ cells against VA7-EGFP infection (III, Fig. 2c). VA7EGFP infection was found to be self-limiting in CT26WT but not in CT26LacZ cultures when a very low infectious dose of 10 PFU was used. Pretreatment at 100 U/ml IFN- inhibited virus replication weakly also in the CT26LacZ cells in this setting (III, Fig. 2d). Combining the Jak1/Jak2 inhibitor ruxolitinib with IFN- resulted in successful infection of both cultures, and interestingly pre-treating the CT26WT cells with a combination of ruxolitinib and IFN- (100 U/ml) before virus inoculation resulted in a more prominent infection as compared to the pretreatment with ruxolitinib alone (III, Fig.S2). Intriguingly, transfection of CT26LacZ cells with either the STAT1 or the LacZ overexpression plasmid prevented the 10 PFU VA7-EGFP infections (III, Fig. S4A). In addition to the phenotypic change in the infectivity, transfection of either of the plasmids modified also the gene expression of CT26LacZ cells to resemble that of CT26WT as shown by STAT1 and, even more so, by OAS1 expression in western analysis (III, Fig. S4B). 65 Figure 7. CT26LacZ mouse colon carcinoma cells have diminished mIFN- response compared to CT26WT cells, but interferon pretreatment (1000 U/ml) inhibits VA7-EGFP infection most efficiently in GL261 mouse glioma cells. The cytopathic effect was observed with phase contrast microscopy (upper panels) and the infection with fluorescence microscopy (lower panels). All samples were done in triplicate with similar results. 66 To shed more light on the mechanisms responsible for the dramatic difference in the antiviral response between CT26WT and CT26LacZ, both the uninfected and VA7-EGFP infected cell lines were subjected to genome wide RNA-Seq transcriptome analysis. Surprisingly, CT26WT cells constitutively expressed several PRRs such as RIG-I and Zbp1 (DAI), type I IFN signaling mediators such as IRF7, IRF9 and STAT1, and ISGs such as PKR, Viperin, Ifit1/3 and ISG15/20 (III, Fig. 3a). In line with these data, “Activation of IRF by Cytosolic Pattern Recognition Receptors” and “Interferon Signaling” were the top 2 canonical pathways revealed in Ingenuity IPA analysis, when the transcriptomes of uninfected CT26WT and CT26LacZ cells were compared (III, Table 1). Some of the constitutively overexpressed interferon stimulated genes in CT26WT cells such as Mx1, IRF7 and STAT1 were true ISGs which, according to current understanding, are induced from the low basal level only by interferon signaling and not directly by PRR/IRF3. However, the latter two genes have been evaluated in human cells and the possibility of species-related differences remains (Andersen et al. 2008; Grandvaux et al. 2002; Muller et al. 1994). Furthermore, some of the true-ISGs such as Mx1 observed to be constitutively expressed in CT26WT cells are not normally expressed at all at basal levels without interferon stimulation (Muller et al. 1994). While IFN- transcripts could not be detected at basal level in CT26LacZ cells, very weak IFN- mRNA expression was found in CT26WT cells. It is unclear whether any IFN- protein was produced from this very low basal transcription, but if this were to be the case, the amounts were too small to be detected by ELISA (unpublished data). As an interesting side note, ISG20 is an RNase and a known alphavirus inhibitor (Ryman and Klimstra 2008) and type I IFN has been recently proposed to inhibit SFV infection by destroying its positive sense RNAs (Nikonov et al. 2013). Infection did not cause transcriptional changes in the CT26WT cells, but the levels of inflammatory and antiviral genes Atf3, Cxcl1/11, Fos, IFN- and IL-6 were induced in the CT26LacZ cells. The expression of STAT1, OAS1 and OAS3 was confirmed at the protein level (III. Fig. 3b). Strikingly, instead of expected STAT1 and OAS induction in CT26WT cells upon VA7 infection or after IFN- treatment, the levels of these ISGs were actually transiently decreased together with STAT1 phosphorylation 3h after IFN- stimulation. In order to test for tumor infectivity in vivo, Balb/c mice carrying intradermal CT26WT or CT26LacZ tumors were sacrificed at 24h, 48h and 96h post intratumoral VA7-EGFP injection and the tumors were subjected to IHC using anti-SFV antibody (III, Fig. 4a). SFV could not be found in the CT26WT tumors at any time point, whereas a progressive infection pattern was observed in the CT26LacZ tumors. The initial infection site was detected around the needle cavity 24 h p.i., whereas perivascular virus localization coinciding with moderate and heavy central necrosis was evident 48 h p.i. and 96 h p.i., respectively. In agreement with the histology, live tumor slices obtained from the CT26LacZ tumors, but not from the CT26WT tumors could be efficiently infected ex vivo (III, Fig. 4b). Intradermally implanted CT26LacZ tumors grew better in immune-compromised SCID mice as compared to their immunocompetent counterparts, but one i.t. VA7-EGFP injection eradicated the tumors completely from both groups (III, Fig. 4c, Figure 8). Furthermore, when CT26WT and CT26LacZ tumors were implanted into separate flanks of Balb/c mice, either at the same time (not shown) or when the CT26WT cells were implanted 8 days later than the CT26LacZ cells, the VA7-EGFP mediated destruction of CT26LacZ did not prevent 67 or even retard the CT26WT tumor growth (III, Fig. 5a, b). However, when CT26WT tumor implantation was performed approximately two months after CT26LacZ clearance, 2/4 mice rejected the challenge (III, Fig. 5c). Finally, in order to create tumors which would be heterogenic in their type I IFN response, CT26WT and CT26LacZ cells were mixed in varying proportions and then infected with VA7-EGFP in vitro and in the Balb/c model in vivo. The survival time of the infected in vitro cultures correlated linearly with the proportion of CT26WT cells (III, Fig. 6a), whereas as few as 10% of these cells was enough to reduce the in vivo survival from 100% to approximately 16% (III, Fig. 6b). Figure 8. The growth curves of individual intradermal CT26LacZ tumors (from III, Fig.4C). One intratumoral VA7-EGFP injection destroyed the flank tumors in both Balb/c (B) and SCID mice (D). While the sham treated tumors grew aggressively in SCID mice (C), the growth was erratic in Balb/c mice (A). 68 69 6 Discussion Before the present work was initiated (I-III), VA7 had already shown potential in different subcutaneous and orthotopic cancer models (see Literature Review chapter “VA7 oncolytic virus”). Human A2058 melanoma xenografts in SCID mice were especially amenable to the treatment, and even peripheral VA7 administration had resulted in remarkable tumor regressions (Vaha-Koskela et al. 2006). However, the remissions remained transient and relapses were found to result from tumor compartmentalization and a few virus resistant cancer cells. The emergence of resistant cancer cell clones was observed also in VA7-treated human squamous cell lung cancer cell line SW-900 in vitro (Maatta et al. 2008). Furthermore, transient, though statistically significant, tumor growth retardation was seen in orthotopic rat BT4C gliomas (Maatta et al. 2007). Despite the transient remissions, survival of the rats was not improved even when VA7 was combined with the immunosuppressive agent dexamethasone, a result at least partly suggested to stem from the detected Nab responses against the therapeutic virus. Interestingly, only intratumoral or local injection of the virus resulted in any therapeutic benefits in subcutaneous and orthotopic A549 nude mouse models, respectively (Maatta et al. 2007; Maatta et al. 2008). It was speculated that IgM type Nabs, known to develop also in nude (but not in SCID) mice, could be responsible for the discrepancy between the therapeutic efficacy of the local and systemic virus administration routes, as A2058 xenografts in SCID mice were highly prone also to i.v. and even i.p. VA7 injections. However, it is likely that the kinetics of the type I IFN response also had a role, as A549 cells are known both to produce and respond to type I IFN in response to virus infection (Rautsi et al. 2007), and IFN- can rescue GL261 mouse glioma cells even when applied 90 minutes post infection (II, Figure 4a). It is therefore plausible that in contrast to intratumoral injection, peripheral infection route caused gradually developing VA7 viremia resulting in a slow and focal delivery of the virus into the A549 tumors thus providing the cancer cells with more time to react to the infection by type I IFN signaling. Furthermore, while it is known that IFN- is not species cross-reactive between mouse and human, and thus this interferon class produced by the healthy mouse cells such as pDCs in blood presumably has little effect on human A549 tumors, some IFN- classes might exhibit more cross-species reactivity (Alexenko, Ealy, Roberts 1999). VA7 administration i.v. delayed the induction of MxA in the subcutaneous A549 tumors as compared to i.t. injection (Maatta et al. 2007). MxA is a good biomarker of the type I IFN response since in contrast to many other ISGs, it is not induced directly by viral infection but requires signaling through IFNAR, i.e. it is a true ISG (Holzinger et al. 2007). Systemically administered VA7 showed high safety and remarkable efficacy even against large orthotopic U87-Fluc glioma xenografts (I). Even though VA7 destroyed 16/17 of i.c. U87-Fluc tumors regardless of the original tumor size and small s.c. tumors were equally amenable, only half of the large subcutaneous tumors could be destroyed. This required increase of VA7 dosing up to three injections (I, Figure 2). The reason for the relative resistance of these large tumors remained elusive as no virus resistant U87 populations were observed which was in contrast to the earlier results with A2058 melanoma xenografts 70 (Vaha-Koskela et al. 2006). One possibility is that some tumor cells survived the limited time span of infection in a semi-adherent state in the large necrotic areas (I, Fig. 3) out of the reach of blood perfusion and, thus, the virus. Fully transformed malignant cells do not have a contact inhibition and in addition can thrive in an anchorage independent manner for example in pleural effusions without committing anoikis (Basak et al. 2009; Hanahan and Weinberg 2011; Weinberg 2014). It is also possible that when the U87-Fluc tumors grew over a certain threshold size, the number of CSC spheres became too high. These spheres were readily infected with VA7 but they died rather slowly in comparison to monolayers (II, Fig.6A). A similar result was obtained with primary human glioma samples (Figure 5). Furthermore, as CSC formation is thought to be a dynamic and reversible process (Hanahan and Weinberg 2011; Weinberg 2014), then the absence of virus-resistant populations in the escaped tumors would have not been surprising. Though speculative, clues from bioluminescence data obtained from two mice suggested that innate immune responses may have played a role in the remarkable efficacy against the orthotopic U87-Fluc tumors. For instance, on one occasion (I, Fig. S4, mouse 1) the luminescence signal from the tumor disappeared 24h p.i., then returned to be briefly visible between the 24 d and 42 d time points, to finally disappear again at the final imaging time point of 101 days p.i. As day 6 p.i. was the last time point when infectious virus could be recovered from the brain (I, Table 1), it is unlikely that tumor re-infection would have accounted for this phenomenon. The tumor growth kinetics of mouse number 5 (I, Fig. S4) was presumably similar, judging from the long term survival of the mouse (> 4 months) despite the recurrence of tumor-derived bioluminescence at the final imaging point 64 days p.i. These data highlight the power of preclinical bioluminescence imaging. Similar to single-photon emission computed tomography (SPECT), bioluminescence imaging is very sensitive and can readily detect even a few target cells. It is superior in this respect to the traditional methods such as MRI or CT, which typically have a good resolution but only mediocre sensitivity. However, optical imaging has the drawback that it requires xenogenic reporter enzyme expression by the target cells, which are likely to be immunogenic in immunocompetent hosts (Murakami et al. 2003). Interestingly, a similar pattern of initial tumor regression and transient remission followed by short relapse before final elimination was observed in one untreated Balb/c control mouse that received immunogenic intradermal CT26LacZ tumor (Figure 8, mouse 7). The hypothesis of involvement of innate immunity in the eradication of U87-Fluc tumors after VA7 infection will need to be confirmed in the future by systematic antibody (or genetic) depletion studies of different innate immune cell subclasses and their combinations, especially macrophages, neutrophils and NK-cells. Another question which would be interesting, albeit difficult to answer, is whether U87 tumors with defects in their type I IFN response (II) would be equally sensitive to VA7 in a fully immunocompetent host, or whether the efficacy would require pharmacological inhibition of adaptive responses such as Nabs. Though perhaps counterintuitive, it is conceivable that in vivo VA7 semi-permissive tumors such as DBT or G26-24 mouse gliomas (Vaha-Koskela et al. 2013 and Figure 6, respectively) could become responsive to the virotherapy when the neutralizing adaptive responses had been temporally attenuated. In such a hypothetical model, the initial immunosuppression would be combined with virus injections to achieve partial tumor infection, which would then be followed by immunemediated tumor destruction when immunosuppression was released. As a side note, G26- 71 24 is an oligodendroglioma (Lo Cicero et al. 2011) and tumors of this cellular origin could be good targets for A7(74) SFV mediated virotherapy, since the virus naturally infects perivascular brain oligodendrocytes and neurons, but not astrocytes (Fazakerley et al. 1993). Another important question although likely beyond any feasible means of investigation, is how well the U87 cell line available from ATCC which is one of the most extensively studied human glioblastomas, represents the original tumor isolated from a 44 old Caucasian male in the late 1960s (Ponten 2014). In the framework of this dissertation and the field of oncolytic virotherapy in general, it would be especially interesting to know if the documented defects in type I IFN induction and response in U87 cells (I, (Wollmann, Robek, van den Pol 2007)) were an early aberration and thus a characteristic for the whole tumor, or appeared later as a clonal trait as seems to be the case for CT26LacZ cells derived from the VA7 resistant CT26WT line (III). It will be also crucial to explore in more detail the nature of these defects in the future, as it seems that also U87 cells can respond to type I IFN treatment under certain circumstances thus limiting viral infection (Alain et al. 2010 and Minna Niittykoski, personal communication). Similarly IFN- pretreatment can weakly inhibit VA7-EGFP virus replication also in CT26LacZ cultures when minuscule virus amounts were used (III, Fig.2D). Hence, the type I IFN signaling defects seen in U87 and CT26LacZ cells are possibly more quantitative/epigenetic than qualitative/mutational in their nature. In support of this hypothesis, type I IFN treatment was shown to induce ISG15 expression in U87 cells, but the cells were not successful in mounting antiviral state against VSV infection (Wollmann, Robek, van den Pol 2007). This demonstrates that the signaling circuits beginning from IFNAR do exist also in the U87 cells though rewired or suppressed. Curiously, U87 cells seem to be handicapped both in type I IFN induction and response (Wollmann, Robek, van den Pol 2007). Although only the latter of these defects is sufficient for efficient tumor infection, defects in the type I IFN induction become relevant in xenograft models. As IFN- shows only limited cross-species reactivity, the host derived IFN- may not protect the xenograft tumors (II). For example, VERO green monkey kidney cells used for VA7 titration (II, III) and baby hamster kidney cells (BHK) used for VA7 production (II, III), are both defective in type I IFN induction while the response to these cytokines seem to be normal (Juang et al. 1998; MacDonald et al. 2007). Indeed, BHK cells have been shown to make extremely sensitive xenograft targets for virotherapy (Tseng et al. 2004). Finally, VA7 proved to be safe even in athymic Balb/c mice. No viral induced pathology could be detected in VA7-treated U87 tumor-bearing or in naïve mice (I, Fig. S7), and the virus did not persist in the brains of infected control mice beyond day 6 p.i. (I, Table 1). In line with these results, B6 nude mice did not develop any symptoms of virus encephalitis, and no obvious virus induced pathologies could be seen in the brains (II, Fig. 3). Interestingly, a persisting subclinical A7(74) infection of nude mouse CNS has been reported earlier (Amor et al. 1996) and the discrepancy might stem from the documented attenuating nsp3 mutation that had occurred during construction of the plasmid prA7(74) later used for construction of VA7 (Tuittila and Hinkkanen 2003). As an interesting side note, U87 cultures typically retained their “net-like” morphology (II, Fig. 4d) even when at confluence, while the freezing process in DMSO containing medium occasionally (but not invariably) caused these cells to adapt to a monolayer growth pattern possibly reflecting epigenetic gene regulatory switch in the transcriptional program (unpublished 72 observations). DMSO has long been known to be able to induce cell differentiation (Tsiftsoglou and Sartorelli 1979). VA7 failed to improve survival of C57BL/6J mice carrying orthotopic GL261 glioma tumors even when immunosuppressive agents rapamycin or CPA was applied (II). Rapamycin is thought to improve OV efficacy by reducing the amount of systemic type I IFN produced by pDCs and hence to promote the infection of tumors, which are semi-competent in their type I IFN response. However, if the tumors have managed to retain much of their IFN signaling intact, this partial aid of rapamycin might be insufficient to confer therapeutic benefit for the IFN sensitive viruses. Similar results were obtained when VA7 was combined with another type I IFN signaling inhibitor valproic acid (VPA) (Zemp et al. 2010) in orthotopic GL261 tumor model (unpublished data). However, the experiment was carried out twice and while VPA-VA7 combination resulted in a significant survival advantage in the first pilot study, the encouraging result could not be repeated in a larger scale experiment. Both CPA and rapamycin decreased the level of circulating Nabs (II, Table 1) and unexpectedly CPA dramatically enhanced VA7 replication only in normal brain tissue (II, Figure 2). The neurologic symptoms in the GL261 bearing mice treated with the CPA/VA7 combination developed around d10 p.i., at the same time with the symptoms of mice that received VA7 monotherapy resulting in no viral encephalitis. Hence, despite enhanced viral CNS replication after CPA treatment, the high tumor burden was probably the reason for the neurological symptoms requiring euthanasia of this cohort. The SFV-CPA combination has been investigated once previously, and depending on the dosing schedule, suppression of T- or B-cells was speculated to account for the increased virulence (Bradish, Allner, Fitzgeorge 1975). A high dose CPA is widely immunosuppressive (Huyan et al. 2011; Lacki et al. 1997) and in the future it will be interesting to determine whether the depletion of IFN- secreting T-cells and NK-cells has any role in the observed increased neurovirulence, a mechanism which could potentially also explain age-dependent neurovirulence (see Literature review, Chapter “2.3.1 Taxonomy, natural life cycle and pathogenesis of Alphaviruses and Semliki Forest virus”). Surprisingly, VA7 replication could not be detected in any of the GL261 tumors at any time point, while the dissociated cell explants resumed their infectivity. GL261 cells both produced and responded to IFN-, aborting VA7 infection even when 100 U/ml of IFN- was applied as late as 90 minutes p.i., although, not beyond 4h p.i. Though speculation, it is possible that the ISGs responsible for the termination of VA7 infection can target only the steps occurring before this 4h p.i time point in the alphavirus lifecycle, for instance coinciding with the switch from negative to positive strand RNA-synthesis taking place between 4-6 h p.i. (Sawicki and Sawicki 1980; Sawicki et al. 1981a; Sawicki et al. 1981b). However, as earlier discussed, many ISGs, including known anti-alphavirus effectors ZAP and IFIT1, attack viral translation, making it difficult to understand why the block would occur before bulk translation of viral proteins from the genomic and subgenomic RNAs had been initiated. Indeed, an alternative plausible explanation for the temporal threshold of IFN- effects could be the early effects of nsP2 induced host cell transcriptional shutdown, a process known to be fully implemented around 6 h p.i. (Akhrymuk, Kulemzin, Frolova 2012). Whatever the underlying mechanism, GL261 cells seem to be especially resistant to VA7 infection and they can abort the infection in response to IFN- more efficiently than 73 the 4T1 mouse breast carcinoma cells or G26-24 mouse oligodendroglioma cells (Figure 6A) or CT26WT mouse colon carcinoma cells (Figure 7). GL261 and CT26WT tumors were resistant to VA7 in vivo (II and III), as were also the 4T1 tumors (Markus Vähä-Koskela, personal communication). However, two i.c. injections of VA7 displayed a tendency of improved survival in G26-24 model (Figure 6B), which was not nevertheless statistically significant. Considering the remarkable in vitro sensitivity of G26-24 cells, it could be fruitful to test multiple i.c. virus injections in this model. Intriguingly, i.c. injection of VA7 led to infection of the corpus callosum, both in the immunocompetent and athymic mice (II, Figs. 2d, 3a, b). Infection with parental A7(74), SFV replicon and even with a neuron-detargeted SFV4 resulted in the same infection pattern of the white matter tract of the corpus callosum and these cells have been reported to be oligodendrocytes (Fazakerley et al. 2006; Ylosmaki et al. 2013). If mice are administered with poly I:C i.p. 3h before the i.c. infection, the corpus callosum remains protected. As peripheral administration of both avirulent and virulent SVF strains reportedly results also in infection of neurons, the observed dramatic oligodendrocytic infection upon intracerebral administration warrants further studies. It will be interesting to examine whether this phenomenon relates to a differential intracerebral fluid kinetics resulting from virus administration induced inflammation, which could open access to oligodendrocyte dense corpus callosum (Laule et al. 2004). Alternatively oligodendrocytes and possibly also other cells of glial origin such as GL261 might be inherently more responsive to endocrine type I IFN than neurons, and the latter cell types might respond better to type II IFN. If this proves to be the case, it could also explain why several immunosuppressive Th1/IFN- inhibitors increase alphavirus infection in neurons (see chapter “2.4.8 Safety and Ethics of Oncolytic Virotherapy”). Whatever the underlying mechanism, SFV tropism for the oligodendrocytes of corpus callosum might be exploitable for gene therapy purposes in this brain region, especially as lentiviral vectors can be readily pseudotyped with SFV glycoproteins (Froelich et al. 2011). While defects in type I IFN induction may disable the antiviral defense of cancer cells in vitro, this is not necessarily sufficient for in vivo tumor targeting because there are additional type I IFN sources in the tumor microenvironment (Liu et al. 2013). These findings are consistent with the present results, and while VA7 infection of DBT mouse glioma did not lead to detectable IFN induction (II, Fig.5b) these tumors were refractory in vivo (Vaha-Koskela et al. 2013). However, minute VA7 infection of DBT brain tumor tissue has occasionally been seen in immunohistochemical staining (Vaha-Koskela et al. 2013) but no signs of any infected cells were detected here in the GL261 brain tumors. On the contrary, VA7 infection often terminated just at the border of parenchyma-to-malignant tissue (II, Fig2d, e, Fig.3a, b). Further support for the importance of extratumoral type I IFN sources in virus clearance was obtained when VA7 pre-infected GL261 cells were found to form tumors with comparable kinetics to uninfected cells. Control cells left in the incubator were efficiently infected (II, Fig. 7) demonstrating that once full and instantaneous infection of tumors has been reached, the autonomous type I IFN production alone is not able to protect the cells from cytolytic infection. However, it is important to realize that human tumors which have lost autonomous type I IFN induction might falsely appear to be virus susceptible when analyzed in nude/SCID mouse models. When these human tumors are 74 implanted into the mouse IFN milieu, they may de facto become deficient in their IFN response as the key early player, IFN- is not species cross-reactive (II). If the tumors cannot even partly compensate for the lack of exogenous IFN- by their own type I IFN production, they may be artificially rendered vulnerable to viral infections. While both CPA and rapamycin treatments reduced the quantity of circulating Nabs, GL261 tumors were treated in athymic B6 mice to exclude the possibility of any remaining adaptive antiviral immune responses. Tumor infectivity or therapeutic efficacy was not improved even by direct intracranial virus injection (II, Fig. 3) further confirming that innate rather than adaptive responses inhibited VA7 infection in the GL261 tumors. Interestingly, confluent GL261 cultures were markedly more resistant than sub-confluent cultures to VA7 infection at low MOI in vitro (II, Fig. 5c). This result could not be explained only by the higher amounts of type I IFN in the supernatants of confluent cultures, as the difference in infectivity remained in an assay where tissue culture plates had confluent centers surrounded by sparse rim areas (Fig.5d). Interestingly, unlike confluent GL261 cultures (II, Fig.5C, D), confluent 4T1 cultures were not virus resistant (Figure 6A) thus resembling CT-2A cells and VERO(B) cells (II, Fig.S1). Defective type I IFN induction probably explains at least the sensitivity of the confluent VERO(B) cultures (Juang et al. 1998) and possibly also the CT-2A cells (II, data not shown). The exact mechanism for the notable resistance of confluent GL261 cultures warrants further studies, and mechanisms such as basolateral receptor location and antiviral signaling via gap-junctions may be possible. It is also conceivable that cell-to-cell signaling of confluent in vitro GL261 cultures activates the same virus hostile (possibly astrocytic) gene expression pattern present in the respective in vivo tumors. Indeed, the conditions of both confluence and senescence have been reported to induce ISGs, including STAT1 (Perou et al. 1999). Furthermore, type I IFN gene program was recently shown to be activated when mixed cultures were created out of glioblastoma cells and nervous tissue (Nayernia et al. 2013). Importantly, this effect was not observed for glioblastoma or nervous monocultures, or for mixed cultures with MCF-7 breast carcinoma cells. Indeed, thes heterotypic interactions between gliomas and their stromas might explain the notable discrepancy observed between the in vitro and in vivo infectivity of GL261 gliomas (II). Supporting the spatio-temporal plasticity of GL261 cells, the suppression of infectivity in vivo was reversible, and sparse explant cultures isolated from the tumors were again infectable with VA7 (II, Fig.4c). The potential role of cell culture medium also remains to be investigated in the future, as the RPMI medium used for CT-2A cells has been reported to support baculovirus, adenovirus and lentivirus transductions clearly better than DMEM used for GL261 cells (Mahonen et al. 2010). The number of seeded cells did not markedly affect the infectivity of U87-Fluc cells, but these cells do not typically form confluent monolayers (II, Fig. 5f). Two clones derived from the same CT26 murine colon carcinoma cell line were here found to differ both in terms of their autonomous antiviral defense and their immunogenicity, which had important implications on VA7 mediated oncolytic virotherapy (III). It was discovered that CT26LacZ cells differ dramatically in their IFN- responsiveness in comparison to their parental cell line CT26WT, and this difference directly translated into a difference in the therapeutic efficacy in vivo (III, Fig. 6B). Due to the presence of the xenogenic LacZ antigen, CT26LacZ tumors were highly immunogenic in 75 immunocompetent Balb/c mice (Figure 8, III Fig. 4C). It was first hypothesized that CT26LacZ tumor infection might stimulate robust anti-tumor adaptive immune responses, which could be responsible to their eradication. However, VA7 proved equally powerful against CT26LacZ tumors in immunosuppressed SCID mice demonstrating that the virusinduced tumor destruction occurs independently of adaptive immunity (III, Fig. 4C). VA7 infection caused efficient production of IFN- both in CT26WT and CT26LacZ cell clones, indicating that also CT26LacZ has intact PRR machinery (III, Fig. 2B). In contrast, 1000 U/ml IFN- pretreatment inhibited VA7 replication in CT26WT cells while having only a minor (but not a nonexistent) effect on CT26LacZ cells, where VA7 continued to replicate even after 10 000 U/ml IFN- pretreatment (III, Fig. 1D and S1). Interestingly, some CT26WT cells were infected despite 1000 U/ml IFN- treatment, whereas the same treatment completely blocked VA7 infection in GL261 cells (Figure 7). GL261 cells seem to be exceptionally poised both in their type I IFN production and response and even one log lower IFN- concentration applied 90 minutes after virus was shown here to be sufficient to arrest the infection (II, Fig. 4). However, the in vivo outcome for CT26WT tumors was the same as in the GL261 tumor model, and again no virus antigen could be detected by IHC in CT26WT tumor tissue (III, Fig.4A). In line with these data, also ex vivo infection of live CT26WT tumor slices was poor (III, Fig.4B). SFV viremia is followed by virus penetrance into the tumors, and if the tumor is susceptible, the initial focal plaques start to rapidly expand, resulting in a widely spread intratumoral infection. Interestingly, the initial infection is often focal, regardless of the route of administration ((Maatta et al. 2007; Vaha-Koskela et al. 2006), II, Fig. 6, III, Fig. 4A). This suggests that though the i.t. VA7 administration route has been occasionally found to be more efficient than systemic virus injection (Maatta et al. 2007), even after direct intratumoral injection, much of the virus might come to the tumor from the blood. In an attempt to dissect the mechanisms underlying the apparent incongruity between the in vitro and in vivo infectivity of CT26WT tumors, the focal in vivo infection pattern was mimicked in vitro by casting an agarose overlay on CT26WT and CT26LacZ cultures followed by VA7 injection into a stamped hole in the middle of the culture plate well. In this focal infection model, VA7 replicated efficiently only in the CT26LacZ cells. However, the infection in the CT26WT cultures could be rescued by adding anti-IFN- antibody (III, Fig. 2A). The importance of type I IFN signaling in the termination of the infection was confirmed in an experiment, where CT26LacZ and CT26WT cells were infected in parallel with very small 10 PFU amount of VA7-EGFP. The infection was productive in CT26LacZ cultures but self limiting in CT26WT cells. The infection of CT26WT cells could be rescued using the JAK/STAT inhibitor ruxolitinib, and interestingly, the infection was further enhanced when combined with 100 U/ml IFN- treatment (III, Fig. 2D, Fig. S2). Remarkably, when CT26LacZ cells were transfected with either the STAT1 or LacZ expression plasmids, infection with 10 PFU of VA7-EGFP was inhibited (III, Fig. S4A). Furthermore, the expression of OAS1 was observed to increase to the level seen in CT26WT cells with both transfections (III, Fig.S4B). LacZ plasmid transfection induced also STAT1 expression to the levels seen in unstimulated CT26WT cells, whereas STAT1 expression was higher after STAT1 expression plasmid transfection as expected. This experiment strongly 76 suggested that the diminished type I IFN responsiveness in the CT26LacZ cells had been attributable to plastic epigenetic mechanisms rather than to irreversible mutations at the genomic level. The exact PRRs responsible for responding to transfections remain to be identified, and both cell lines express at least the DNA sensors, cGAS and IFI16 (unpublished observations), but CT26WT cells transcribe significantly more DAI (Zbp1) than CT26LacZ cells do (III, Fig. 3A) (Lam, Stein, Falck-Pedersen 2014). Presently, it is unclear whether transfection itself is sufficient to induce these changes, or whether transfection of any RNA or any non-coding DNA would cause similar effects. However, non-coding DNA has been shown to induce ISGs such as STAT1 in several cancer cell lines in a type I IFN-dependent manner (Li et al. 2005). Importantly, the induction was reported to occur independently of the DNA type (plasmid or PCR-amplified) or the method used for transfection (Lipofectamine, electroporation or CaPO4), and furthermore the DNA or the transfection reagent on their own did not evoke any induction. Interestingly, it is documented that while OAS can be activated directly by IRF3, STAT1 does not respond to IRF3 overexpression, suggesting that despite its un-stimulated basal expression, STAT1 is induced as a true ISG (Grandvaux et al. 2002). The hypothesis that the increase in OAS1 and STAT1 levels of CT26LacZ cells upon plasmid transfection is type I IFN-mediated thus receives further support. It is, however, possible, that in addition to a type I IFN mediated action, direct IRF3 activation may have played an additional role in ISG induction seen in CT26LacZ cells after plasmid transfections, as the change in OAS1 levels was more prominent than the change seen in STAT1 (III, Fig. S4B). VA7 replicated efficiently in CT26LacZ tumors, and after initial infection evident around the i.t. injection site, the infection was found to localize perivascularly (III, Fig. 4A). The perivascular infection coincided with massive necrosis of the surrounding tumor parenchyma. Thus, secondary tumor destruction resulting from compromised blood flow probably contributed to the therapeutic efficacy as has been documented previously with VSV in the CT26LacZ tumor model (Breitbach et al. 2007). VSV induced vascular shutdown was found to be mediated by neutrophils attracted to the inflammation site and interestingly, the VA7-infected CT26LacZ cells expressed CXCL1 and CXCL11 chemokines (III, Fig. 3A). Importantly, rather than infection of the thin endothelial layer previously suggested to underlie the efficacy of oncolytic vaccinia, VSV and reovirus after transient VEGF/FGF “pulsing”(Breitbach et al. 2013; Kottke et al. 2010), profound VA7 infection of perivascular CT26LacZ tumor parenchyma was seen beginning already at 48h p.i (III, Fig. 4). Though the target cells are different, the susceptibilities of tumor vasculature and parenchyma to viral infection may be mechanistically similar, and indeed, RNA-Seq data revealed statistically significant over-expression of both FGF- and PDGF-receptors in CT26LacZ tumors (unpublished observations). In perfect agreement, both FGFR and PDGFR are known to signal using Ras as a second messenger, and activated Ras/MEK signaling has been demonstrated to suppress IFN-induced transcription (Brooks, Kilgour, Smith 2012; Christian et al. 2012; Ostman and Heldin 2007). There was a constitutively high transcription of numerous genes of inflammation-, PRRand IFN-signaling pathways observed in CT26WT- but not in CT26LacZ cells in the genome wide analysis and some of these differences were confirmed at the protein level (III, Fig.3). Comparative IPA pathway analysis of the transcriptomes of CT26WT and 77 CT26LacZ control cells further corroborated this observation, i.e. IRF activation by cytosolic PRRs and interferon signaling were the top 2 canonical pathways differentially regulated between the cell lines (III, Table 1). Several of the genes (or their human counterparts) expressed at high levels in CT26WT cells have been reported to have alphavirus inhibitory activity either alone, or in synergism with ZAP. These included many well-known mediators and effectors of antiviral signaling such as ISG15, ISG20, Viperin, Ifi47, RIG-I, PKR, IRF7, Ifitm3, MDA5, STAT1, IRF9, Ifi44l and OAS3 (Schoggins et al. 2011). In addition to STAT1 phosphorylation, type I IFNs also induce STAT1 gene expression as a positivefeedback mechanism, and also the unphosphorylated STAT1 has been reported to translocate into nucleus driving long-term expression of some ISGs (Cheon and Stark 2009). Strikingly, the majority of those ISGs reported to be induced by unphosphorylated STAT1 were also constitutively expressed in CT26WT cells including, OAS1, OAS2, OAS3, STAT1, IFI44, IFI44L, IFIH1 (MDA5), IFI35, IFIT3, Mx1, IRF7 and IFIT1 (p56) (III, Fig. 3A). This suggests that the constitutive ISG expression in these cells may be driven by the high level expression of unphosphorylated STAT1. IRF3 which is known to be expressed constitutively and not induced by type I IFN, was expressed at equal levels in CT26WT and CT26LacZ cells (Honda and Taniguchi 2006). Constitutive expression of OAS and MxA in human tumor cells has been proposed as a marker for tumor resistance against an IFN sensitive oncolytic virus VSV (MoerdykSchauwecker et al. 2013). Human MxA has been shown to have antiviral functions against SFV in transgenic mice (Hefti et al. 1999) and it is induced in human xenograft tumors upon VA7 therapy (Maatta et al. 2007). Indeed, the high expression of Mx1 and Mx2 genes as well as of the different OAS genes was detected in the virus resistant CT26WT cells (III, Fig. 3A). Mouse Mx1 and Mx2 genes are both antiviral ISGs, but Mx2 is non-functional in all commonly used laboratory mouse strains due to the presence of an insertional mutation resulting in a translational frameshift (Jin et al. 1999). The fact that in addition to actual effectors, high basal levels of transcription of even Mx2 along with some IFN inducible pseudogenes were found in CT26WT cells favors a ubiquitous, non-specific explanation for the antiviral state in these cells. Furthermore, in addition to mouse Mx1 (as well as human MxA), at least human IRF7 and ISG20 genes have been shown to be true ISGs, i.e. induced only by interferon signaling and not directly via the PRR/IRF3 route (Grandvaux et al. 2002; Holzinger et al. 2007; Muller et al. 1994). Thus it is possible that the constitutive expression of many of the antiviral genes in the CT26WT cells was actually attributable to the very low level IFN- transcription seen in these cells (unpublished observations), which may be responsible for auto/paracrinic signaling and upregulation of multiple ISGs. A similar effect has been reported to protect human hepatocytes from viral infection (Tsugawa et al. 2014) and MEFs from malignant transformation (Abbas, Lichtman, Pallai 2007; Adekolu-John and Fagbami 1983; Chen et al. 2009). Furthermore, low, but constitutive IFN- expression has been reported to play a role in priming of bronchial epithelial cells to withstand influenza virus attack (Hsu et al. 2012). The low constitutive type I IFN expression by tumors or stromal cells has also been reported earlier, and very recently this mechanism was found to account for the resistance of some tumors against oncolytic VSV (Liu et al. 2013; Tsai et al. 2011). However, it remains to be determined whether any IFN- protein was produced from the very low level basal transcription in CT26WT cells, as it was not detectable by ELISA (unpublished observations). Low levels of IFNs may be consumed by the cells and 78 thus IFN- production is often detected indirectly by its ability to induce true ISGs. A high level of ISG15 expression has been found to protect tumors from IFN mediated apoptosis (Tisserand et al. 2011), and elevated expression of ISG15 was seen in CT26WT cells, potentially explaining the lack of IFN- treatment-induced cytotoxicity (III, Fig. 3A). It would be worthwhile to examine whether ISG15 inhibition could re-introduce the natural cytotoxicity of type I IFNs in CT26WT cells, a potentially powerful therapeutic effect recently attributed to IAP antagonists (Beug et al. 2014). Finally, the basal type I IFN expression and priming has been shown to enhance the effects of other antiviral cytokines such as IFN- by increasing the expression of STAT1 (Gough et al. 2010). Indeed, it was possible to detect higher constitutive expression of STAT1 in CT26WT cells compared to CT26LacZ cells (III, Fig.3), and anti-IFN- antibody markedly enhanced VA7 replication in the CT26WT cells in the focal infection assay under agarose overlay (III, Fig. 2A). The cell autonomous virus resistance mechanism in CT26WT tumors may be similar to that reported recently by Malilas et al. for VSV in NIH3T3 cells (Malilas et al. 2013), as a similar high constitutive expression pattern of STAT1 and OASL2 genes was seen also in the CT26WT cells (III, Fig. 3A). If high STAT1 mediated OASL2 expression is also revealed to account for the CT26WT virus resistance, it will be interesting to determine whether it can also be attributed to the high activity of the CUG2 gene, as CUG2 expression was approximately twice higher in CT26WT control cells compared to CT26LacZ controls (unpublished observations). Intriguingly, higher levels of both constitutive and IFN- induced expression of several STAT1 dependent ISGs were recently reported by Cho et al. to be responsible for the positive sense RNA-virus resistance in granular cell neurons as compared to virus-permissive cortical neurons (Cho et al. 2013). In addition to many other ISGs, exactly the same antiviral gene signature of IRGs reported by Cho and colleagues (Irgm1, Irgm2, Igtp, Ifi47 and Tgtp1) was also expressed in CT26WT cells (III, Fig. 3A). Cho and coworkers also presented preliminary data suggesting that epigenetic regulation might be responsible for the differences in IFN/STAT1 signatures in different neuronal subtypes resulting in differential susceptibility to RNA-virus infection. As an interesting side note, Cho and colleagues also discovered that VEEV was resistant to most of the ISGs that had potent antiviral effects against the other positive sense RNA-viruses tested, a result consistent with other reports concerning the insensitivity of pathogenic VEEV to the antiviral effects of type I IFN (Ryman and Klimstra 2008). Amongst the several antiviral genes expressed at constitutively high levels in CT26WT cells, there were two crucial downstream mediators of IFNAR signaling, IRF9 and STAT1, which together with STAT2, form the ISGF3 complex. Reduced signaling by this complex could explain the stoichiometric rather than absolute defects in the type I IFN response seen in CT26LacZ cells, further proposing that a global difference in antiviral gene signatures rather than single mutations underlie the virus resistance of CT26WT tumors. Indeed, an increase in the expression of IRF9 and one of the type I IFN receptor chains, IFNAR2, have been shown recently to correlate with anti-alphaviral state of mature neuronal cells (Farmer et al. 2013). The known fact that IFN- binding to its receptor induces not only phosphorylation of STAT1 protein but also an increase in its transcription level (Varble et al. 2013) poses an obvious caveat to the data interpretation as the observed reduction in STAT1 transcript and protein levels in CT26LacZ cells (III, Fig. 3) could be either the result 79 of or the cause of the type I IFN signaling defects in these cells. Reduced STAT1 expression, as also observed in some melanomas (Wong et al. 1997) may benefit the CT26LacZ tumor survival by reducing its immunogenicity. However, STAT1 induction has been implicated in the development of tumor resistance to genotoxic stress (radiation and chemotherapy) and radioresistant tumors concomitantly developed resistance to the type I IFN sensitive HSV-1 mutant (Khodarev et al. 2004; Khodarev, Roizman, Weichselbaum 2012). Hence, the antiviral response profile of heavily pretreated tumors, as in most oncolytic virotherapy trials, may be inherently unfavorable. Furthermore, as the majority of preclinical rodent cancer models in use are created by exposing the animals to high dosages of mutagens such as 3-methylcholanthrene (MCA), there is a risk that some of these tumors inherently express high levels STAT1 and are thus virus resistant. Thus, more natural genetic and spontaneous cancer models should be favored, which would also better display the heterogeneity seen in human tumors (Rodriguez-Madoz et al. 2014). Data from IFN- pretreatment-, single-foci expansion assay-, RNA-Seq- and western blot analysis and results from in vivo and ex vivo infection experiments together argue in favor of a model, where CT26WT (but not CT26LacZ) cells constitutively reside in a semi-primed but inactivated antiviral state. It is notable that the same antiviral state could be induced in CT26LacZ cells by transfection of plasmid DNA (III, Fig. S4) but not with IFN- treatment (III, Fig. 3B). In this model, focal infections in the CT26WT tumors in vivo lead to rapid IFN secretion and final paracrine priming (i.e. increase in the PRR and ISG levels) of the neighboring uninfected tumor cells, which upon PRR engagement were then capable of activating the ISGs by phosphorylation and aborting the infection. Interestingly, this resembles the regulation of poliovirus tropism, in which tissues expressing constitutively high levels of PRRs and ISGs, such as RIG-I, PKR and OAS (all upregulated also in CT26WT cells) have been found to be non-permissive for poliovirus replication (IdaHosonuma et al. 2005). These events in CT26WT tumors are difficult to capture by standard in vitro infection techniques in dissociated cell cultures without agarose cover as any secreted IFN- is immediately diluted into the culture medium. The dilution possibly also contributed to the absence of VA7 infection-induced transcriptional changes in CT26WT cells in RNA-Seq analysis (III, Fig. 3A) and to the absence of STAT1 or OAS protein induction or STAT1 phosphorylation in western analysis (III, Fig. 3B). In support of this hypothesis, treatment with a high dose exogenous IFN- induced STAT1 phosphorylation in CT26WT cells (III, Fig. 3B). Very small (10 PFU) amounts of VA7-EGFP in CT26WT cultures could mimic the in vivo resistance of these tumors (III, Fig. 2D, Fig. S2, Fig. S4A), but it remains to be determined if a small scale focal infection could also induce adequate signaling changes, which could be then detected at mRNA or protein levels. VA7 infection caused efficient eradication of CT26LacZ tumors also from SCID mice, demonstrating that adaptive immune responses did not play any major role at least in the acute therapeutic phase (III, Fig. 4C). Long term follow-up of the SCID mice was not possible, as they gradually develop neurological symptoms upon VA7 infection (VahaKoskela et al. 2006). Indeed, the infected SCID mice had to be eventually sacrificed due to ataxic symptoms appearing roughly at day 16 post infection, when the tumors had been already completely destroyed by the infection. Notably, whereas the CT26LacZ tumors grew aggressively in the SCID mice, tumor development in the immunocompetent Balb/c 80 mice was erratic (Figure 8). Some of the CT26LacZ tumors grew fairly quickly, whereas others disappeared quite soon after i.t. saline injection. Tumor sizes in the third group were increasing and decreasing spontaneously until finally reaching the criteria for sacrifice of the animals. Strikingly, the transplanted CT26LacZ tumor of Balb/c mouse number 7 disappeared after sham treatment for 9 days until aggressive growth resumed again, tumor reached a large size, and then spontaneously disappeared again without recurrence. A similar growth pattern of the CT26LacZ tumors in immunocompetent Balb/c mice was seen when tumors were transplanted for histological analyses (III, Fig. 4A), whereas all of the CT26WT tumors grew aggressively (unpublished observations). This pronounced immunogenicity of the CT26LacZ tumors was somewhat unexpected, as the growth pattern has been previously reported by Dr Restifo, the creator of the two cell lines, to resemble that of CT26WT tumors (Wang et al. 1995). The observed discrepancy possibly derives from different routes of transplantation, i.e. subcutaneous used by Dr. Restifo and the intradermal route used here; the latter has been reported to be more immunogenic due to activation of resident skin dendritic cells (Bonnotte et al. 2003). Double-flank and rechallenge experiments where the CT26LacZ and CT26WT cancer cells were implanted into separate flanks of Balb/c mice either simultaneously, or the latter after CT26LacZ infection, were carried out to determine if cross-immunity against CT26WT tumors could be induced upon eradication of the virus-susceptible CT26LacZ tumors. CT26LacZ tumor infections induced protective immunity against a delayed CT26WT rechallenge in 50% of the treated mice, while the eradication of CT26LacZ tumors did not influence the CT26WT outgrowth in the double-flank experiments upon simultaneous tumor induction (III, Fig. 5). After infection, CT26LaZ cells are capable of secreting efficiently at least IFN- (III, Fig. 2B), an early type I IFN that can be induced by IRF3 independently of positive feedback signaling via IFNAR (Honda and Taniguchi 2006). As type I IFNs are generally considered as being necessary for evoking antigen-specific T-cell responses in alphavirus based vaccination protocols (Leitner et al. 2006) and CT26WT and CT26LacZ cells have been reported to express shared tumor antigens (Kershaw et al. 2001) it was surprising to observe the lack of any cross-protective immunity against CT26WT tumors inflicted by cure of the LacZ expressing tumors in double flank- (III, Fig. 5) and clone-mixing (III, Fig. 6B) experiments. In the latter experiment as few as 10% of CT26WT cells in the implantation mixture was sufficient to seriously compromise the efficacy of the therapy. Hence, the dominant anti-tumor immune response generated during SFV oncolysis was likely targeted at LacZ rather than against any of the previously identified and shared tumor antigens (Castle et al. 2014; Kershaw et al. 2001). LacZ is a xenoantigen and due to its alien origin and redundancy for tumor cell survival it might function in the clonal-mix setting as an immune defense “decoy” diverting immune responses against the dominant but ultimately ineffective target and lead to escape of CT26WT cells modeling antigen loss variants. Immunoediting of cancer cell clones is known to occur in patients undergoing antigen-specific immunotherapies, and malignant cell clones that have undergone epigenetic/genetic suppression of the antigen or antigen-presenting machinery are positively selected (DuPage et al. 2012). Tumors and their stroma can also be directly immunosuppressive or evade immune attack by inflammation-induced transient cellular dedifferentiation, resulting in an altered antigen landscape (Holzel, Bovier, Tuting 2013; Mellman, Coukos, Dranoff 2011). Indeed, transcription of several immunosuppressive 81 genes was detected in the CT26 lines including PD-L1, TGF1, STAT3, RCAS1, IDO2, MCSF and strikingly high levels of Galectin-1 (unpublished observations) (Rabinovich, Gabrilovich, Sotomayor 2007). While oncolytic virotherapy may optimally be able to elicit broad-spectrum anti-tumor immune responses through epitope spreading, these responses may occasionally be biased against only a subset of immune-dominant epitopes. The number of shared epitopes between the virus sensitive and resistant clones may also be an important factor. Notably, in the present genome-wide transcriptome analysis between the CT26WT and CT26LacZ cells it was found out that 10% of all the sequenced mRNAs were differentially transcribed at the basal level, some of these potentially representing important epitope targets for immunotherapy. Moreover, viral infection per se can also cause dramatic changes in gene expression, potentially driving further apart the epitomes of the infected and uninfected cancer cell clones. Tumors defective in the type I IFN response such as CT26LacZ whose transcriptome reacts only mildly during the early phase of the infection (III, Fig. 3A) might be optimal immuno-virotherapeutic targets also in this regard. 82 83 7 Summary and conclusions In conclusion, VA7 Semliki Forest virus showed oncolytic potential in murine models of glioma and colorectal carcinoma, but the therapeutic efficacy was strictly dependent on the type I interferon responsiveness of the tumors (I-III). In the future it will be necessary to develop fast and reliable methods for tumor prescreening in order to direct the therapy to those patients who will most likely benefit from alphavirus-based oncolytic virotherapy. Functional tests such as focal ex vivo infections of sliced live tumor tissue or dissociated cultures with and without type I IFN pretreatments, are likely to yield the most reliable information, especially if tumors (and possible metastases) are sampled in thorough manner. In these tests, the type I IFN class and concentration should be chosen carefully, with the high affinity early IFN- possibly being a better choice than any of the late IFN- subclasses. Historically, a concentration as low as 5 U/ml of type I IFN has been used in these screens to identify cancer cell lines with diminished responsiveness to type I IFN (Stojdl et al. 2000). As a comparison, the minimum IFN concentration that efficiently blocked VA7 replication in CT26WT cells was 1000U/ml of IFN-. Nevertheless, this level of cell autonomous antiviral signaling was sufficient to seriously hamper the in vivo efficacy of oncolytic virotherapy (III). Interestingly, in a recent analysis the majority of primary human melanoma samples were shown to have defects in their type I IFN response as compared to healthy melanocytes. While recombinant IFN- was used instead of IFN-, the tested interferon concentration of 100 U/ml was rather high (Wollmann et al. 2013). Functional tests are nevertheless too laborious and time consuming for routine large scale clinical applications. Hence, high throughput applications based on cutting edge technologies such as single cell genome and transcriptome sequencing (Navin et al. 2011) that are able to account tumor heterogeneity could prove invaluable, presuming that appropriate resistance and susceptibility markers are discovered. The detection of constitutive transcription of interferon stimulative genes such as OAS and MxA may be a good surrogate marker for the anticipated virus resistance (III). 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Janne Ruotsalainen Interferon Response as a Challenge and Possibility for Developing Alphavirus Based Oncolytic Virotherapy In this study, the possibilities of targeting malignant glioma with oncolytic virotherapy were explored in different syngeneic- and xenograft mouse-models. The specific roles of innate and adaptive immune responses were examined using two mouse colon carcinoma clones with opposing patterns of cell autonomous antiviral signaling and immunogenicity. Publications of the University of Eastern Finland Dissertations in Health Sciences isbn 978-952-61-1554-2
