Maraba Virus as a Potent Oncolytic Vaccine Vector

Transcription

1 Oncolytics originl rticle The Americn Society of Gene & Cell Therpy Mr Virus s Potent Oncolytic Vccine Vector Jonthn G Pol 1, Ling Zhng 1, Byrm W Bridle 1,2, Kyle B Stephenson 1,3, Julien Rességuier 1, Stephen Hnson 1, Ln Chen 1, Ntsh Kzdhn 1, Jonthn L Brmson 1, Dvid F Stojdl 4, Yonghong Wn 1 nd Brin D Lichty 1 1 McMster Immunology Reserch Center, Deprtment of Pthology nd Moleculr Medicine, McMster University, Hmilton, Ontrio, Cnd; 2 Ontrio Veterinry College, University of Guelph, Toronto, Ontrio, Cnd; 3 Centre for Innovtive Cncer Reserch, Ottw Hospitl Reserch Institute, Ottw, Ontrio, Cnd; 4 Children s Hospitl of Estern Ontrio Reserch Institute, Ottw, Ontrio, Cnd The rhdovirus Mr hs recently een chrcterized s potent oncolytic virus. In the present study, we engineered n ttenuted Mr strin, defined s MG1, to express melnom-ssocited tumor ntigen. Its ility to mount n ntitumor immunity ws evluted in tumor-free nd melnom tumor-ering mice. Alone, the MG1 vccine ppered insufficient to prime detectle dptive immunity ginst the tumor ntigen. However, when used s oosting vector in heterologous prime-oost regimen, MG1 vccine rpidly generted strong ntigen-specific T-cell immune responses. Once pplied for treting syngeneic murine melnom tumors, our oncolytic prime-oost vccintion protocol involving Mr MG1 drmticlly extended medin survivl nd llowed complete remission in more thn 2% of the nimls treted. This work descries Mr virus MG1 s potent vccine vector for cncer immunotherpy displying oth oncolytic ctivity nd remrkle ility to oost dptive ntitumor immunity. Received 11 April 213; ccepted 1 Octoer 213; dvnce online puliction 1 Decemer 213. doi:1.138/mt INTRODUCTION Oncolytic viruses (OVs) specificlly infect, replicte in nd kill mlignnt cells, leving norml tissues unffected. Severl OVs hve reched dvnced stges of clinicl evlution for the tretment of vrious neoplsms. 1 Dt from clinicl trils nd preclinicl models hve demonstrted tht these virl gents lone or in comintion with stndrd cncer therpies hold gret promise for improved therpeutic efficcy. 2 4 We hve previously reported tht nturlly ttenuted vesiculr stomtitis virus (VSVΔM51), prototypicl rhdovirus, is compelling oncolytic gent due to its sfety profile nd the lck of pre-existing neutrlizing ntiodies in humn popultions, prcticl prolem ssocited with severl other OV pltforms. 5 8 To expnd our current rry of sfe nd potent OVs from the Rhdoviride, we hve recently identified severl new vesiculoviruses tht disply strong oncolytic ctivities. 9,1 Specificlly, the Mr virus showed the rodest oncotropism in vitro nd specific genetic modifictions were shown to drmticlly improved its tumor selectivity nd reduced its virulence in norml cells. In vivo, this ttenuted strin, clled MG1, demonstrted potent ntitumor ctivity in xenogrft nd syngeneic tumor models in mice, with superior therpeutic efficcy thn VSVΔM51. 9 Dt ccumulted over the pst severl yers hs reveled tht ntitumor efficcy of OVs not only depends on their direct oncolysis ut my lso depend on their ility to stimulte ntitumor immunity. 11 This immune-medited tumor control seems to ply criticl role in the overll efficcy of OV therpy. Indeed, tumor-specific dptive immune cells cn ptrol the tissues nd destroy tumor cells tht hve een missed y the OV. Moreover, their memory comprtment cn prevent tumor recurrence. Vrious strtegies hve een developed to improve OV-induced ntitumor immunity. 12 Some groups hve geneticlly engineered OV expressing immunostimultory cytokines. An herpes simplex nd vccini virus expressing grnulocyte-mcrophge colonystimulting fctor hve respectively reched phse III nd IIB of the clinicl evlution for cncer therpy while VSV expressing interferon (IFN)-β hs just entered phse I. 1 Another strtegy, defined s oncolytic vccine, consists of expressing tumor ntigen from the OV. Previously, we nd others hve demonstrted tht VSV could lso e used s cncer vccine vector. 13,14 When pplied in n heterologous prime-oost setting to tret murine melnom model, our VSV oncolytic vccine not only induced n incresed tumor-specific immunity ut lso concomitnt reduction in ntivirl dptive immunity. As result, the therpeutic efficcy ws drmticlly improved with n increse of oth medin nd long-term survivls. 13 Given tht oth VSV nd Mr re clssified s vesiculoviruses, we hypothesized tht Mr MG1 could shre the vccine properties of VSV. In the present study, we demonstrted tht oncolytic Mr MG1 cn e used s n oncolytic vccine pltform. While unle to prime detectle responses ginst melnom-ssocited ntigen, Mr MG1 vccine displyed potent ility to oost pre-existing tumor-specific CD4 + nd CD8 + T-cell immunity. When pplied to the tretment of syngeneic murine melnom tumor models, Mr MG1-medited recll immuniztion resulted in drmtic extension of the medin survivl with complete remission in more thn 2% of the nimls treted. Together with our previous study involving VSV, 13 this work confirmed tht vesiculoviruses cn e potent vccine vectors for cncer immunotherpy displying oth oncolytic ctivity nd remrkle ility to oost dptive cell immunity. Correspondence: Brin D Lichty, McMster Immunology Reserch Centre, Deprtment of Pthology nd Moleculr Medicine, McMster University, 128 Min Street West, Hmilton, Ontrio, L8S 4K1, Cnd. E-mil: lichty@mcmster.c 42 vol. 22 no. 2, fe. 214

2 The Americn Society of Gene & Cell Therpy Mr Virus s Potent Oncolytic Vccine Vector RESULTS Chrcterizing the oncolytic ctivity of Mr MG1 in vitro nd its oncotropism in vivo in the murine B16-F1 melnom model The oncolytic property of Mr virus hs recently een chrcterized in five melnom-derived cell lines from the NCI-6 pnel. 9 However, the B16-F1 metsttic melnom cell line, used s the tumor model in the present study, ws not included. Before evluting the therpeutic efficcy of Mr MG1 in tumorering mice, we evluted its ility to lyse B16-F1 cells in vitro. For this purpose, monolyer culture of B16-F1 melnom cells ws infected with t multiplicity of infection of.1 nd cell viility nd virl repliction were mesured. By visulizing green fluorescent protein (GFP) expression, Mr infection ws noticele t 12 hours nd spred through the whole culture within 24 hours (Figure 1). A urst of virus production ws chieved within 2 hours (Figure 1), long with clernce of the B16-F1 cell popultion (Figure 1c nd visile field on Figure 1). These dt demonstrted tht Mr MG1 ws le to infect, replicte in nd kill B16-F1 melnom cells. We susequently evluted the ility of Mr virus to selectively infect B16-F1 melnom cells in vivo. Immunocompetent mice ering B16-F1 lung metstses, s well s tumor-free controls, were inoculted intrvenously (i.v.) with. Infectious prticles were quntified in the lungs nd in the secondry lymphoid orgns (spleen nd inguinl lymph nodes) t 24 hours nd 48 hours fter virus injection (Figure 2). Mr MG1 ws detected t high titers in the spleen 24 hours fter inocultion (5 log plque-forming units (pfu)/g tissue) ut clered from the orgn y 48 hours (<2 log pfu/g, Figure 2). The kinetic of splenic virus clernce ws similr in oth tumor-free nd tumorering nimls (Figure 2). MG1 ws rrely detectle in the lymph nodes (<2 log pfu/g, Figure 2c), independently of the presence of tumor, suggesting poor distriution or rpid clernce of Mr in the lymph nodes. While MG1 ws clered from the lungs of tumor-free mice t 48 hours, sustntil level of virus ws still persisting t tht time in the mice with B16-F1 lung metstses (Figure 2d, men ± SD = 3.1 log pfu/g ± 1.84 t 48 hours). Anlysis of the MG1 titer t erly timepoint fter infection reveled lrge virus uptke in the lungs which ws significntly higher in presence of metstses: 4.26 log pfu/g ±.4 in tumor-free controls versus 5.84 log pfu/g ±.19 in tumorering mice t 2 hours (Figure 2d). In the lungs with metstses, MG1 titer incresed y 8 times etween 2 nd 12 hours, reching 6.75 log pfu/g ±.18 t 12 hours, efore progressively decresing (Figure 2d). In contrst, in tumor-free lungs, the MG1 lod did not increse nd displyed quick clernce (Figure 2d). These oservtions illustrte the ility of MG1 to selectively trget nd replicte t the tumor site. Mr lone is insufficient to improve the therpeutic outcome nd to induce ntitumor immunity Although Mr MG1 could lyse B16-F1 cells in vitro nd replictes t the tumor site in vivo, i.v. dministrtion of t its highest tolerle dose (1 9 pfu Brun et l.) 9 did not result 8 hours 12 hours 24 hours 36 hours GFP Visile Titer (pfu/ml) Hours fter trnsduction Hours fter trnsduction c % Viility No virus Figure 1 Mr MG1 infects, replictes in nd kill B16-F1 melnom cells in vitro. A monolyer culture of murine B16-F1 melnom cells ws incuting with Mr t multiplicity of infection of.1. () Mr virus infects B16-F1 cells nd spred cross the culture (GFP signl) leding to complete cell popultion clernce within 36 hours (visile field). () Repliction of Mr illustrted y virl titers (pfu/ ml) in B16-F1 culture superntnt. (c) Popultion of vile B16-F1 cells, monitored y MTT ssy, in sence or presence of Mr. (,c) Curves from representtive experiment relized in triplicte where ech timepoint is illustrted s men ± SD. GFP, green fluorescent protein; pfu, plque-forming units. Moleculr Therpy vol. 22 no. 2 fe

3 Mr Virus s Potent Oncolytic Vccine Vector The Americn Society of Gene & Cell Therpy in n improvement of survivl in 5-dy-old B16-F1 lung metsttic model, compred to untreted control nimls (Figure 3). This result ws not entirely surprising s previous studies hve shown tht B16 cells re responsive to type I IFNs. Considering the sensitivity of ttenuted VSV to such response, 15 it ws likely tht type I IFNs my lso protect tumor cells from Mr MG1 oncolysis. We vlidted the hypothesis in vitro y demonstrting tht Mr MG1 ws unle to kill B16-F1 cells tht hve een pre-exposed to IFN-β (Supplementry Figure S1). As consequence, the induction of the interferon response following Mr MG1 repliction t the tumor site my limit the extent of its oncolytic ction. One wy to roden ntitumor spectrum nd ctivity of oncolytic therpy is to increse the ntitumor immunity induced during oncolysis. To this end, we engineered Mr MG1 to express the humn form of the melnom-ssocited ntigen dopchrome tutomerse (termed, see Supplementry Figure S2, for the cloning strtegy nd chrcteriztion of trnsgene expression) tht is highly homologous to its murine counterprt expressed y B16-F1 cells. We postulted tht overexpression of dopchrome tutomerse (DCT) y the Mr vector during oncolysis might enhnce ntigen-specific CD8 + T-cell responses leding to improved overll therpeutic outcomes. Disppointingly, survivl ws not extended when mice were treted with 5 dys fter tumor engrftment s compred to or mock tretments (Figure 3). Our immunologicl nlyses indicted tht DCT-specific CD8 + or CD4 + T-cell responses induced y were rely detectle either in tumor-ering or in tumor-free mice Dys B16-F c d Log1 pfu/g Spleen No tumor Lung tumor Hours fter injection Log1 pfu/g Lymph nodes No tumor Lung tumor Hours fter injection Virus titering Figure 2 Mr MG1 selectively replictes t the B16-F1 melnom metstses site in vivo. () C57Bl/6 mice received syngeneic B16-F1 cells y intrvenous injection in order to estlish syngeneic lung melnom metstses. Fourteen dys fter tumor chllenge, ws dministered y intrvenous injection t dose of 1 9 pfu. To ddress Mr tumor selectivity, virus repliction in tumor-free mice ws compred to repliction in lung metstses-ering nimls. Virl titers were quntified t vrious timepoints fter infection in the lungs (d), where metstses re locted in tumor-ering nimls, s well s in secondry lymphoid orgns: () spleen nd (c) inguinl lymph nodes. Titers were quntified in three smples per tissue nd illustrted s men ± SD. P <.1, P <.1. GFP, green fluorescent protein; NS, nonsignificnt; pfu, plque-forming units. Log1 pfu/g Lungs 8 No tumor 7 Lung tumor Hours fter injection c Percent survivl B16-F1 lung metstses 4 2 PBS Dys fter tumor chllenge % IFNg+ CD8 + T cells DCT(SVY) TF TF TB TB PBS PBS MG1-hDTC % IFNg+ CD8 + T cells DCT(KFF) TF TF TB TB PBS PBS MG1-hDTC Figure 3 Mr lone is insufficient to improve the therpeutic outcome nd to induce ntitumor immunity. () C57Bl/6 mice received syngeneic B16-F1 cells y intrvenous injection (i.v.) in order to estlish syngeneic lung melnom metstses. Five dys fter tumor chllenge, Mr MG1 expressing either GFP or the melnom ntigen hdct ws dministered i.v. t dose of 1 9 pfu; untreted mice received phosphte-uffered sline (PBS). Despite virl repliction t the tumor site, filed to increse mouse survivl s illustrted y Kpln Meier curves (n = 5 per group). (,c) C57Bl/6 mice received or not syngeneic B16-F1 cells y i.v. injection in order to estlish syngeneic lung melnom metstses. Five dys lter, tumor-free (TF) nd tumor-ering (TB) mice were dministered i.v. with 1 9 pfu or PBS. Nine dys fter Mr injection, immune responses ginst the tumor ntigen DCT were mesured in the lood. () Percentge of CD8 + T cells secreting IFN-γ fter ex vivo exposure to the MHC-I restricted DCT immunodominnt epitope SVYDFFVWL (SVY). (c) Percentge of CD4 + T cells secreting IFN-γ fter ex vivo exposure to the MHC-II restricted DCT immunodominnt epitope KFFHRTCKCTGNFA (KFF). Box plots representing percentile including medin nd whiskers illustrting the rnge etween miniml nd mximl vlues (n = 5 mice per group). P vlue considered nonsignificnt (NS) when >.5. DCT, dopchrome tutomerse; GFP, green fluorescent protein; IFN-γ, interferon-gmm; pfu, plque-forming units vol. 22 no. 2 fe. 214

4 The Americn Society of Gene & Cell Therpy Mr Virus s Potent Oncolytic Vccine Vector (Figure 3,c) suggesting tht Mr MG1 is wek vccine vector for priming significnt T-cell responses ginst the trnsgene ntigen. Mr MG1 is potent vector for oosting DCT-specific responses We hve previously reported tht nother oncolytic rhdovirus, vesiculr stomtitis virus (VSV), is highly effective s ooster of pre-existing immunity. 13 We speculted tht Mr virus might shre the sme iologicl property. To test this possiility, we chose recominnt denovirl vector expressing hdct () s priming vector due to its ility to induce strong primry CD8 + T-cell responses. In this cse, ws dministered y intrmusculr injection (i.m.) t dose of pfu, s optimized previously. 13,16 DCT-specific T-cell responses were mesured t dy 1 fter Ad injection in the lood, which corresponds to the pek time of the primry response elicited y Ad vectors. As expected, no DCT-specific T-cell response ws detected in control mice injected with the empty Ad vector (Figure 4) while dministrtion generted strong DCT-specific CD8 + T-cell response (% IFN-γ-secreting CD8 + T cells: men ± SEM = 6.5% ±.6, Figure 4,). As demonstrted previously, 13 i.v. dministrtion of 1 9 pfu of VSV-hDCT 12 dys fter priming generted strong systemic secondry CD8 + T-cell response ( + VSVhDCT: 19.32% ± 2.72; Figure 4). Interestingly, the sme dose of elicited n even stronger recll response confirming tht rhdoviruses shre common chrcteristic s oosting vccine vectors ( + : 3.59% ± 3.49; Figure 4,). We susequently tested other routes of Mr dministrtion including i.v., i.m., nd intrnsl (i.n.). Results indicted tht the i.v. route ws fr superior to other routes in oosting CD8 + T-cell response demonstrting systemic increses in DCT-specific immune responses in oth lood nd spleen (Figure 5,c): 3.59% ± 3.49 y i.v. versus 4.73% ± 1.52 i.n. versus 13.84% ± 1.88 i.m. in the lood. In ddition to CD8 + T cells, the ws lso le to oost systemic CD4 + T-cell responses following i.v. dministrtion (Figure 5,d): % IFN-γ secreting CD4 + T-cells in the lood =.31% ±.12; Figure 5. Mr MG1 vccine ccelertes secondry CD8 + T-cell responses The ility of Mr MG1 vector to rpidly mplify CD8 + T-cell responses prompted us to evlute the outcome of shortening the prime-oost intervl on the DCT oost response. MG1- hdct ws injected i.v. t dose of 1 9 pfu, 12, 9 or 4 dys fter prime. Interestingly, n intervl s short s 9 dys ws not only sufficient to oost the secondry responses ut ctully elicited much higher mgnitude of DCT-specific CD8 + T cells thn the 12-dy intervl (9-dy intervl: 37.53% ± 3.66 versus 12-dy intervl: 3.59% ± 3.49; Figure 6). More surprisingly, significnt oost could e chieved within 4 dys fter Ad priming (Figure 6, 11.99% ± 2.26) suggesting tht Mr MG1 my represent unique oost vector for certin diseses where rpid mplifiction of CD8 + T cells is required. Additionlly, while rely detectle y shortening the primeoost intervl to 4 dys, the DCT-specific CD4 + T-cell oost response ws mintined y reducing the intervl from 12 to 9 dys (12-dy intervl:.31% ±.12 versus 9-dy intervl:.32% ±.8 versus 4-dy intervl:.9% ±.3; Figure 6). ing with Mr in lung metsttic model genertes potent ntitumor immunity nd extended survivl We susequently evluted the therpeutic efficcy of MG1- hdct dministered s oosting vector in lung metsttic % IFN-γ+ CD8 + T-cells DCT(SVY) VSV-hDCT + IFN-γ CD8 + Figure 4 Mr MG1 is potent vector for oosting DCT-specific response. () C57Bl/6 mice received pfu of y intrmusculr injection (DCT prime) efore receiving, or not, 1 9 pfu of VSV-hDCT or Mr y intrvenous injection (DCT oost). Negtive controls received pfu of. DCT-specific T-cell responses were mesured 1 dys fter Ad (pek of the prime response) nd 5 dys fter VSV/MG1 (pek of the oost response). Immune responses represented s the percentge of CD8 + T cells secreting IFN-γ fter ex vivo exposure to SVY peptide corresponding to the MHC-I restricted immunodominnt epitope of DCT (n = 5 mice for nd + VSV-hDCT groups, n = 1 mice for nd + groups). () Representtive dot plots of DCT-specific CD8 + T-cell responses mesured in nd + immunized mice. Box plots representing percentile including medin nd whiskers illustrting the rnge etween miniml nd mximl vlues. P <.5 nd P <.1. Ad, denovirl vector; DCT, dopchrome tutomerse; IFN-γ, interferon-gmm; pfu, plque-forming units. Moleculr Therpy vol. 22 no. 2 fe

5 Mr Virus s Potent Oncolytic Vccine Vector The Americn Society of Gene & Cell Therpy % IFN-γ+ CD8 + T cells No oost Blood - DCT(SVY) i.v. i.n. i.m. % IFN-γ+ CD4 + T cells No oost Blood - DCT(KFF) i.v. i.n. i.m. c % IFN-γ+ CD8 + T cells No oost Spleen - DCT(SVY) i.v. i.n. i.m. d % IFN-γ+ CD4 + T cells No oost Spleen - DCT(KFF) i.v. i.n. i.m. Figure 5 Systemic dministrtion of Mr MG1 vccine is required for generting optiml tumor-specific oost responses. C57Bl/6 mice received pfu y intrmusculr injection (DCT prime). Twelve dys lter, 1 9 pfu of Mr were dministered through vrious routes (DCT oost): intrvenous (i.v.), intrnsl (i.n.), or intrmusculr (i.m.). DCT-specific T-cell responses were mesured 5 dys fter Mr injection in the lood (,) nd in the spleen (c,d). Immune response mesured t the sme timepoint in prime only nimls is indicted ( No oost ). (,c) Percentge of CD8 + T cells secreting IFN-γ fter ex vivo exposure to the MHC-I restricted DCT immunodominnt epitope SVY. (,d) Percentge of CD4 + T cells secreting IFN-γ fter ex vivo exposure to the MHC-II restricted DCT immunodominnt epitope KFF. Box plots representing percentile including medin nd whiskers illustrting the rnge etween miniml nd mximl vlues (n = 1 mice for the i.v. group, n = 5 for the i.n. nd i.m. groups). P vlue considered nonsignificnt (NS) when >.5, P <.5, P <.1, nd P <.1. Ad, denovirl vector; DCT, dopchrome tutomerse; IFN-γ, interferon-gmm; pfu, plque-forming units. % IFN-γ+ CD8 + T cells No oost 12 dys DCT(SVY) DCT(KFF) dys 4 dys % IFN-γ+ CD4 + T cells No oost 12 dys 9 dys 4 dys Figure 6 Mr MG1 vccine ccelertes secondry CD8 + T-cell responses. C57Bl/6 mice received pfu y intrmusculr injection (DCT prime). At 12, 9 or 4 dys fter prime, ws dministered y intrvenous injection t dose of 1 9 pfu. DCT-specific T-cell responses were mesured 5 dys fter Mr injection in the lood. Immune response mesured t the sme timepoint in prime only nimls is indicted ( No oost ). () Percentge of CD8 + T cells secreting IFN-γ fter ex vivo exposure to the MHC-I restricted DCT immunodominnt epitope SVY. () Percentge of CD4 + T cells secreting IFN-γ fter ex vivo exposure to the MHC-II restricted DCT immunodominnt epitope KFF. Box plots representing percentile including medin nd whiskers illustrting the rnge etween miniml nd mximl vlues (n = 1 mice per group). P vlue considered nonsignificnt (NS) when >.5, P <.5 nd P <.1. Ad, denovirl vector; DCT, dopchrome tutomerse; IFN-γ, interferon-gmm; pfu, plque-forming units vol. 22 no. 2 fe. 214

6 The Americn Society of Gene & Cell Therpy Mr Virus s Potent Oncolytic Vccine Vector model. Five dys following i.v. injection of B16-F1 cells, nimls received sequentil dministrtion of nd t 9-dy intervl (Figure 7). We confirmed tht prime- oost vccintion generted very strong DCT-specific CD8 + T-cell response (men % IFN-γ + CD8 + T cells = ± 2.17, Figure 7) tht ws 14 times higher thn in Dys Survivl B16-F1 Immune nlysis - - DCT(SVY) d % IFN-γ+ CD8 + T cells c % IFN-γ+ CD4 + T cells f DCT(KFF) e Percent survivl g B16-F1 lung metstses + + B16-F1 lung metstses Dys fter tumor chllenge + + (-CD8) + (-CD4) Dorsl Ventrl + + Percent survivl Dys fter tumor chllenge Figure 7 Mr dministered in heterologous prime-oost setting llowed to generte potent ntitumor immunity nd to extend survivl of melnom lung tumor-ering nimls. () C57Bl/6 mice were chllenged intrvenously with B16-F1 cells in order to estlish syngeneic lung melnom metstses. Five dys lter, mice received pfu y intrmusculr injection. Control mice received empty Ad vector. Nine dys fter Ad injection, nimls were dministered intrvenously with 1 9 pfu or its GFP control. Immune responses ginst the tumor ntigen DCT were mesured 5 dys fter Mr injection in the lood nd mouse survivl ws monitored dily. Percentge of CD8 + or CD4 + T cells recting to SVY () or KFF (c) peptide exposure, respectively. Box plots representing percentile including medin nd whiskers illustrting the rnge etween miniml nd mximl vlues. Pooled dt from severl experiments: n = 9 for group, n = 23 for group, n = 29 for + group, n = 11 for + group. (d) Kpln Meier curves illustrting survivl of treted melnom lung-tumor ering mice. Pooled dt from severl experiments: n = 16 for group, n = 23 for group, n = 3 for + group, n = 11 for + group. (e) T-cell popultions were selectively depleted to evlute their respective therpeutic contriution. Kpln Meier curves illustrting survivl of treted melnom lung-tumor ering mice (n = 5 per group). (-CD8) indictes CD8 + T-cells depletion while (-CD4) indictes CD4 + T-cells depletion. (f) Lungs extrcted t dy 19 from untreted nd + treted mice. (g) Autoimmunity (vitiligo) induced in long-term survivors following + tretment. Pictures tken 28 dys (mice on the left) or 22 dys (mice on the right) fter tumor chllenge. P vlue considered nonsignificnt (NS) when >.5, P <.5, P <.1, nd P <.1. Ad, denovirl vector; GFP, green fluorescent protein; DCT, dopchrome tutomerse; IFN-γ, interferon-gmm; pfu, plque-forming units. Moleculr Therpy vol. 22 no. 2 fe

7 Mr Virus s Potent Oncolytic Vccine Vector The Americn Society of Gene & Cell Therpy nonoosted mice (1.95% ±.29 in group nd 1.91% ±.59 in + group, Figure 7). Similrly, DCT-specific CD4 + T-cell responses were mesured in MG1- hdct oosted nimls while rrely detected in primed only mice (men % IFN-γ + CD4 + T cells =.25% ±.6 in + group versus <.5% in nd + groups, Figure 7c). Compred to control, or + tretment slightly prolonged survivl time (Figure 7d). However, oosting with the MG1-DCT vccine drmticlly improved the medin survivl, nd more thn 2% of mice were cured of their tumors (Figure 7d). Figure 7f is n exmple where decrese of the size nd numer of melnom lung metstses ws evident t 5 dys fter injection, coincident with the pek of secondry CD8 + T-cell responses. To chrcterize the respective contriution of CD4 + nd CD8 + T-cell responses to the therpeutic efficcy, ech T-cell comprtment ws selectively depleted using corresponding ntiodies. As shown on Figure 7e, depletion of the CD8 + T-cell popultion t the time of the oost rogted the therpeutic enefit of MG1- hdct dministrtion. On the contrry, CD4 + T-cells depletion ppered not to ffect significntly the therpeutic efficcy indicting tht CD8 + T cells ply criticl role in controlling tumor growth following the comintion therpy with nd. We noted tht ll long-term survivors developed vitiligo of vrile severity (Figure 7g), mnifesttion of melnocyte destruction, reinforcing the potency of nti-dct immune responses oosted y. Some long-term survivors were rechllenged with B16-F1 melnom cells. As shown in the Supplementry Figure S3, none of them developed tumors illustrting the existence of nti-dct immune memory tht cn protect from tumor recurrence. Additionlly, we evluted the efficcy of (i) the two homologous prime-oost involving either or vccines nd (ii) the + VSV-hDCT heterologous primeoost in the melnom lung-metstsis model. The ltter hs previously shown efficcy ginst melnom rin tumors 13 ut hs never een pplied yet to tret melnom lung tumors. As shown in the Supplementry Figure S4, oth nd filed to oost their homologous prime nd did not improve the therpeutic efficcy: medin survivl = 2 dys upon tretment versus 23 dys with + nd 27 dys with versus 28 dys with +. When compring the two oncolytic rhdovirl vccines, MG1- hdct ppered more potent thn VSV-hDCT to oost pre-existing DCT-specific CD8 + T-cell responses. This stronger ntitumor immunity trnslted into n improved tumor outcome: 73.5 dys upon Ad-DCT + tretment versus 44.5 dys with + VSV-hDCT. As consequence, these results fvor Mr MG1 over VSV s cndidte for further clinicl evlution of our oncolytic prime-oost pproch. ing with Mr significntly improves therpeutic outcomes in n intrcrnil tumor model Finlly, we lso evluted the efficcy of our prime-oost strtegy involving Mr vccine in very chllenging 5-dy-old melnom rin metsttic model. Immune responses resulting from the prime- ooster tretment were gin remrkly strong (29.11% ± 2.17 IFN-γ + CD8 + T cells, Figure 8) nd of similr mplitude to tht seen in lung-metstsis ering mice (P =.61 versus + group in Figure 7). medited immunotherpy significntly improved survivl of mice ering melnom rin metstsis, with % IFN-γ+ CD8 + T cells DCT(SVY) + Percent survivl B16-F1 rin tumor Dys fter tumor chllenge + Figure 8 Mr efficiently oosted DCT-specific response in melnom rin tumor-ering mice nd significntly extended their survivl. C57Bl/6 mice were chllenged with 1 3 B16-F1 cells y intrcrnil injection in order to estlish syngeneic rin melnom tumor. Five dys lter, mice received pfu y intrmusculr injection while control mice received n empty Ad vector. Nine dys fter Ad injection, nimls were dministered intrvenously with 1 9 pfu. Mouse survivl ws monitored dily. () Percentge of CD8 + T cells secreting IFN-γ in response to DCT-specific SVY peptide exposure mesured 5 dys fter Mr injection in the lood. Box plots representing percentile including medin nd whiskers illustrting the rnge etween miniml nd mximl vlues. Pooled dt from surviving mice t dy 19 fter tumor chllenge from severl experiments: n = 2 for group (only 2 control mice out of 17 still live t the time of immune nlysis), n = 8 for group, n = 15 for + group. () Kpln Meier curves illustrting survivl of treted melnom rin-tumor ering mice. Pooled dt from severl experiments: n = 17 for group, n = 1 for group, n = 15 for + group. P <.1, nd P <.1. Ad, denovirl vector; DCT, dopchrome tutomerse; IFN-γ, interferon-gmm; pfu, plque-forming units vol. 22 no. 2 fe. 214

8 The Americn Society of Gene & Cell Therpy Mr Virus s Potent Oncolytic Vccine Vector medin extended from 15 dys for controls to 25.5 dys for the group (Figure 8). The dditionl dministrtion of Mr oncolytic ooster further improved tumor outcome with medin survivl reching 42 dys together with cures oserved in 21.4% of treted nimls ( + group, Figure 8). DISCUSSION Cncer vccines offer gret promise for systemic cncer therpy nd long-term tumor protection. While pssive immunotherpy relies on repeted dministrtion of short-lived molecules (ntiody, cytokine) or immune cells, cncer vccines im t developing ctive ntitumor immunity y stimulting ptient s immune system to rect ginst self ntigens ssocited with the mlignnt phenotype. Due to immune tolernce, generting n dptive ntitumor immunity tht results in therpeutic enefit is chllenging. To rech this gol, efforts re eing mde to identify immunogenic tumor ntigens nd chrcterize potent vccine pltforms. Severl virl vccine vectors re currently under clinicl evlution for cncer therpy. Cndidtes express immunogenic tumor-ssocited ntigens (e.g., CEA, NY-ESO-1, 5T4, or PSA). These recominnt viruses re mostly repliction-defective (e.g., TRICOM nd TroVx poxvirl vectors). 17 In this context, therpeutic ctivity strictly relies on their ility to mount n efficient dptive ntitumor immunity. While the efficcy of OV therpy relies on oncolytic virl repliction in tumors, it very likely enefits from the induction of n ntitumor immunity. Vrious strtegies hve een proposed to improve the therpeutic impct of these viruses y influencing these two spects. 12 We hve recently identified Mr virus s new oncolytic vesiculovirus with more potent oncolytic ctivity thn its prototypicl cousin VSV. 9,1 In order to stimulte tumorspecific dptive immunity, we hve developed the pproch of inserting trnsgene encoding tumor ntigen into the genome of n OV. However, similr to VSV-sed oncolytic vccines, 13 Mr MG1 expressing DCT (MG1-DCT) filed to induce meningful DCT-specific CD8 + T-cell responses in tumor-free or tumor-ering nimls. We speculte tht replicting OVs will prove to e wek priming vccine vectors s the immune response ginst the virl vector is dominted y responses ginst highly immunogenic virl ntigens tht must e expressed for repliction. 8,13 To overcome this issue, we introduced our Mr vccine s oosting vector in heterologous prime-oost regimen. In this setting, the response ginst the tumor ntigen trnsgene is secondry response nd is therefore more roust nd cn compete ginst the primry response ginst the vector itself. 8,13 Our tem nd others hve demonstrted tht denovirl vccines re potent priming vectors cple of reking immune tolernce. 13,18 2 Coupled to, Mr remrkly oosted Ad primed DCT-specific responses. When the comintion vccine ws pplied for tretment of melnom tumor-ering mice, tumor-specific T-cell responses generted continued to e potent despite tumor-induced immune suppression. In fct, we hve previously demonstrted tht using oncolytic VSV s tumor vccine ooster leds to lrger ntitumor immune responses in tumor-ering nimls, 13 nd now show tht this is even further enhnced when using Mr MG1 virus s n oncolytic vccine (Supplementry Figure S4). Systemic delivery of the OV ppered criticl for its vccine potentil implying tht this route llowed for enggement of ntigen-presenting cells with strong ility to oost immune responses. Regrding the strong uptke of in the spleen, this lymphoid tissue could e involved in the phenomenon. Investigtions re ongoing in the lortory. As well, Mr virus repliction in the tumor ed my chnge the locl tumor microenvironment nd ffect its relted immunosuppression. Tumor vsculture shutdown nd/ or locl induction of n immunostimultory cytokine storm hve repetedly een ssocited with OV tumor infection nd my contriute to the phenomen In the B16-F1 murine melnom model, Mr oncolysis hd no impct on overll survivl. This oservtion my not e surprising given the reltively wek nd ephemerl repliction of Mr t the tumor site. While B16- F1 cells were permissive for Mr infection in vitro, it would pper tht B16-F1 cells were poorly permissive to Mr in vivo. However, Mr MG1 oncolysis previously showed potent efficcy for treting other mouse models of cncer 9 indicting tht this reltively poor oncolytic effect is model specific. Importntly, the oncolytic vccine pproch introduced in heterologous prime-oost setting compensted for the trnsient virl oncolysis. The strong tumor-specific cytotoxic T-cell immunity induced drmticlly extended survivl. Despite the ggressiveness of the melnom tumor models used, Ad prime-mr MG1 oost vccine comintion llowed long-term complete remission in more thn 2% of the nimls treted. Tking into ccount our previous work involving VSV, 13 we confirmed here with Mr MG1 tht vesiculoviruses cn e potent vccine vectors for cncer immunotherpy, displying oth oncolytic ctivity nd n impressive ility to oost dptive cell immunity. Their ssocition with Ad vccine in heterologous prime-oost regimen ppered impressively efficient regrding the therpeutic enefit. However, this setting cn still e optimized. Numerous vritions need to e evluted. Among them, it might e possile to extend the prime-oost intervl. Due to constrints linked to the ggressiveness of the tumor, such n option remined inccessile with the B16-F1 melnom model. As cells infected y the oncolytic vccine overexpressed the trgeted ntigen, they represent prticulrly immunogenic trget for the ntigen-specific cytotoxic T cells. Extending the intervl should llow one to hve reduced popultions of these effector T cells t the time of the oost. This should llow for n extended window of virl spred with potentilly incresed oost efficcy. Another strtegy for improvement could consist of replcing the repliction-deficient Ad vector with nother heterologous oncolytic vccine vector. Also, we could modulte the immune system with drugs prior to, or long with, the oncolytic prime-oost vccintion. As proof-of-concept, we recently succeeded in drmticlly enhncing the therpeutic efficcy of the Ad prime-vsv oost protocol y dministering n HDAC inhiitor long with the ooster vccintion. 27 Thus, our oncolytic Mr vccine ppered to hve unprecedented oosting ility. Its comintion with repliction-deficient denovirl vccine vectors showed impressive therpeutic efficcy illustrting its therpeutic potentil. We hve now shown tht MG1 Mr virus displys greter oncolytic potency 9 s Moleculr Therpy vol. 22 no. 2 fe

9 Mr Virus s Potent Oncolytic Vccine Vector The Americn Society of Gene & Cell Therpy well s n enhnced ility to oost ntitumorl immunity leding to enhnced therpeutic impct reltive to oncolytic VSV (Supplementry Figure S4). Our group is currently conducting preclinicl sfety study of the Ad + Mr MG1 oncolytic primeoost pproch in nonhumn primtes. Clinicl evlutions in ptients with dvnced tumor expressing chrcterized tumorssocited ntigen will follow. MATERIALS AND METHODS Mice. Femle C57BL/6 mice (8 1 weeks old t study initition) were purchsed from Chrles River Lortory (Wilmington, MA) nd housed in specific pthogen-free fcility. All niml studies complied with Cndin Council on Animl Cre guidelines nd were pproved y McMster University s Animl Reserch Ethics Bord. Recominnt viruses. Recominnt viruses involved in the study hve een descried previously. nd re repliction-deficient denoviruses (E1/E3-deletion) sed on the humn serotype 5. 28,29 hdct trnsgene encodes the full-length humn melnom ntigen DCT. hs no trnsgene. Recominnt Mr nd VSV were generted y trnsgene insertion etween the G nd L virl genes. VSV-hDCT derives from the wild-type Indin strin of the VSV. 16,3 nd derive from the ttenuted strin MG1 of Mr virus. 9 Mr hs een developed for the purpose of the study. Cell culture. Murine melnom B16-F1 cells (expressing the murine DCT ntigen) were grown in F11-MEM contining 1% fetl ovine serum, 2 mmol/l l-glutmine, 1 mmol/l sodium pyruvte nd vitmin solution,.1 mmol/l nonessentil mino-cids, 5 mmol/l β-mercptoethnol, 1 U/ ml penicillin, nd 1 mg/ml streptomycin (ll from Invitrogen, Grnd Islnd, NY). Vero cells were cultured in lph-mem contining 1% fetl ovine serum, 2 mmol/l l-glutmine, 1 U/ml penicillin, nd 1 mg/ml streptomycin (ll from Invitrogen, Grnd Islnd, NY). Cell trnsduction B16-F1 cells per well were seeded in six-well pltes. The monolyer cell culture ws trnsduced with Mr t multiplicity of infection (MOI) of.1. Incution ws performed in 1 μl for 1 hour efore dding 2.9 ml of culture medium. A 2 μl of culture superntnt were collected for virus titrtion t 8, 12, 24, nd 36 hours nd followed y cell imging under fluorescent microscope. Virus titer in culture superntnt were determined y plque ssy on Vero cells using stndrd techniques. Viility of infected cells ws evluted t ech timepoint y MTT ssy. MTT ssy. A cell suspension of B16-F1 cells per ml ws incuted with t MOI.1 for n hour under shking cells were then seeded per well of 96-well plte in 2 μl culture medium. At 1.5 hour prior to timepoint, 1 μl solule MTT t 5 mg/ml (Sigm-Aldrich, Okville, ON) ws dded nd incuted for 3 hours. Formzn slts, resulting from the conversion of MTT y live cells, were resuspended in dimethylsulfoxide nd their concentrtion determined y opticl density t 57 nm. Mice infection/immuniztion. Rhdoviruses Mr, MG1- hdct nd VSV-hDCT were injected intrvenously (i.v.) in 2 μl phosphte-uffered sline t dose of 1 9 pfu. For denovirus injection, mice were nesthetized in seled chmer contining 5% inhltion isoflurne. Adenovirl vectors nd were dministered intrmusculrly (i.m.) t totl dose of pfu (1 1 8 pfu in 5 μl phosphte-uffered sline per qudriceps). Tumor chllenge. For lung metstses engrftment, B16-F1 cells were injected i.v. in 2 μl sline wter. For rin tumors, mice were nesthetized y inhltion of 5% isoflurne through nose cone nd engrfted y intrcrnil (i.c.) injection of 1 3 B16-F1 cells in 2 μl phosphte-uffered sline using stereotctic frme s previously descried. 13 Mice were monitored dily nd euthnized for tissue hrvest or upon signs of moridity. Tissue homogentes. To mesure virus repliction in vivo, tissues (lungs, spleen, nd inguinl lymph nodes) were hrvested 1 3 dys fter i.v. inocultion of Mr, weighted nd immeditely frozen t 8 C. After thwing, ech tissue ws homogenized in 1 ml phosphte-uffered sline using n Ultr Turrx T25 homogenizer (14, rpm for 1 seconds). Virl titers in homogentes were quntified y plque ssy on Vero cells nd expressed s log pfu/g of tissue. Peptides. Peptides corresponding to the immunodominnt epitopes of DCT: (i) DCT SVYDFFVWL/ SVY tht inds to H-2K ; 1% conserved etween humn nd murine DCT nd (ii) DCT KFFHRTCKCTGNFA/ KFF from humn DCT tht inds to I-A ; differing from the murine epitope in position 92: His (humn) Asn (murine). Peptides were synthesized y Biomer Technologies (Sn Frncisco, CA). Antiodies. Monoclonl ntiodies used in flow cytometry ssys: nti-cd16/cd32 (clone 2.4G2) to lock Fc receptors, nti-cd3 (clone 145-2C11), nti-cd8 (clone ), nd nti-cd4 (clone RM4-5) for detecting cell surfce mrkers, nti-ifn-γ (clone XMG1.2) for intrcellulr cytokine stining. All regents were purchsed from BD Phrmingen (Mississug, ON). For T-cell depletion, 2 μg of nti-cd8 (clone 2.43) nd nti-cd4 (clone GK1.5) ntiodies were injected in the peritonel cvity three times t 2 dys intervl strting 2 dys efore dministrtion. Selective T-cell depletion ws confirmed y flow cytometry on lood smples (dt not shown). Detection of ntigen-specific T-cell responses. DCT-specific T-cell responses were mesured 1 dys post-prime nd 5 dys post-oost s followed. Peripherl lood mononucleted cells or splenocytes were incuted in complete RPMI (RPMI medium supplemented with fetl clf serum 1%, Penicillin-Streptomycin 1% nd l-glutmine 1%) with SVY peptide (2 μg/ml) or KFF peptide (15 μg/ml) for DCT-specific CD8 + or CD4 + T-cell (re-)stimultion, respectively. Incution ws performed in incutor (37 C, 5% CO 2, 95% humidity) for 5 hours, with refeldin A (1 μg/ml, GolgiPlug BD Phrmingen) during the lst 4 hours. Cells were treted with ntiodies trgeting CD16/CD32 efore stining with fluorescent-leled ntiodies trgeting T-cell surfce mrkers. Then, cells were permeilized nd fixed with Cytofix/Cytoperm (BD Phrmingen) nd stined for intrcellulr cytokines. Dt were cquired using FACSCnto flow cytometer with FACSDiv softwre (BD Phrmingen) nd nlyzed with FlowJo Mc softwre (Treestr, Ashlnd, OR). Sttistic nlysis. GrphPd Prism version 6 for Windows (GrphPd Softwre, Sn Diego, CA) ws used for grphing nd sttisticl nlyses. Immune responses dt were grphed s ox plots (25 75 percentile with medin) with whiskers illustrting the rnge etween miniml nd mximl vlues. Student s two-tiled t-test were used to nlyze immune dt nd compre virus titers. Survivl curves were represented ccording to the Kpln Meier method, nd compred using the log-rnk test. Differences etween groups were considered significnt when P.5 (P <.5, P <.1 nd P <.1). SUPPLEMENTARY MATERIAL Figure S1. Induction of the interferon response in B16-F1 cells protects from Mr MG1 infection Figure S2. Mr vector construction nd expression of DCT in trnsduced Vero cells. Figure S3. Tumor-specific immune memory resulting from the + tretment protects cured mice from tumor recurrence. Figure S4. + heterologous prime-oost induces stronger ntitumor immunity nd shows etter therpeutic efficcy thn homologous prime-oost strtegies or thn the heterologous prime-oost involving VSV-hDCT vol. 22 no. 2 fe. 214

10 The Americn Society of Gene & Cell Therpy Mr Virus s Potent Oncolytic Vccine Vector ACKNOWLEDGMENTS Supported y grnts to B.D.L. from the Cndin Cncer Society nd the Ontrio Institute for Cncer Reserch nd to Y.W. from the Cndin Institute for Helth Reserch. The uthors declred no conflict of interest. REFERENCES 1. Russell, SJ, Peng, KW nd Bell, JC (212). Oncolytic virotherpy. Nt Biotechnol 3: Heo, J, Reid, T, Ruo, L, Breitch, CJ, Rose, S, Bloomston, M et l. (213). Rndomized dose-finding clinicl tril of oncolytic immunotherpeutic vccini JX-594 in liver cncer. Nt Med 19: Krpngiotou, EM, Roulstone, V, Twigger, K, Bll, M, Tny, M, Nutting, C et l. (212). Phse I/II tril of cropltin nd pclitxel chemotherpy in comintion with intrvenous oncolytic reovirus in ptients with dvnced mlignncies. Clin Cncer Res 18: Hrrington, KJ, Hingorni, M, Tny, MA, Hickey, J, Bhide, SA, Clrke, PM et l. (21). Phse I/II study of oncolytic HSV GM-CSF in comintion with rdiotherpy nd cispltin in untreted stge III/IV squmous cell cncer of the hed nd neck. Clin Cncer Res 16: Miest, TS, Yiw, KC, Frenzke, M, Lmpe, J, Hudcek, AW, Springfeld, C et l. (211). Envelope-chimeric entry-trgeted mesles virus escpes neutrliztion nd chieves oncolysis. Mol Ther 19: White, CL, Twigger, KR, Vidl, L, De Bono, JS, Coffey, M, Heinemnn, L et l. (28). Chrcteriztion of the dptive nd innte immune response to intrvenous oncolytic reovirus (Dering type 3) during phse I clinicl tril. Gene Ther 15: Lw, M, Hollinshed, R nd Smith, GL (22). Antiody-sensitive nd ntiodyresistnt cell-to-cell spred y vccini virus: role of the A33R protein in ntiodyresistnt spred. J Gen Virol 83(Pt 1): Chen, Y, Yu, DC, Chrlton, D nd Henderson, DR (2). Pre-existent denovirus ntiody inhiits systemic toxicity nd ntitumor ctivity of CN76 in the nude mouse LNCP xenogrft model: implictions nd proposls for humn therpy. Hum Gene Ther 11: Brun, J, McMnus, D, Lefevre, C, Hu, K, Flls, T, Atkins, H et l. (21). Identifiction of geneticlly modified mr virus s n oncolytic rhdovirus. Mol Ther 18: Mhoney, DJ, Lefevre, C, Alln, K, Brun, J, Snei, CA, Bird, S et l. (211). Virustumor interctome screen revels ER stress response cn reprogrm resistnt cncers for oncolytic virus-triggered cspse-2 cell deth. Cncer Cell 2: Melcher, A, Prto, K, Rooney, CM nd Bell, JC (211). Thunder nd lightning: immunotherpy nd oncolytic viruses collide. Mol Ther 19: Pol J, Rességuier J nd Lichty B (211). Oncolytic viruses: step into cncer immunotherpy. Vir Adpt Tret 212: Bridle, BW, Li, J, Jing, S, Chng, R, Lichty, BD, Brmson, JL et l. (21). Immunotherpy cn reject intrcrnil tumor cells without dmging the rin despite shring the trget ntigen. J Immunol 184: Pulido, J, Kottke, T, Thompson, J, Glivo, F, Wongthid, P, Diz, RM et l. (212). Using virlly expressed melnom cdna lirries to identify tumor-ssocited ntigens tht cure melnom. Nt Biotechnol 3: Stojdl, DF, Lichty, B, Knowles, S, Mrius, R, Atkins, H, Sonenerg, N et l. (2). Exploiting tumor-specific defects in the interferon pthwy with previously unknown oncolytic virus. Nt Med 6: Bridle, BW, Boudreu, JE, Lichty, BD, Brunellière, J, Stephenson, K, Koshy, S et l. (29). Vesiculr stomtitis virus s novel cncer vccine vector to prime ntitumor immunity menle to rpid oosting with denovirus. Mol Ther 17: Drper, SJ nd Heeney, JL (21). Viruses s vccine vectors for infectious diseses nd cncer. Nt Rev Microiol 8: Aurisicchio, L, Mennuni, C, Ginnetti, P, Clvruso, F, Nuzzo, M, Ciprini, B et l. (27). Immunogenicity nd sfety of DNA prime/denovirus oost vccine ginst rhesus CEA in nonhumn primtes. Int J Cncer 12: Fcciene, A, Aurisicchio, L, Eli, L, Plomo, F, Mennuni, C, Cilierto, G et l. (26). DNA nd denovirl vectors encoding crcinoemryonic ntigen fused to immunoenhncing sequences ugment ntigen-specific immune response nd confer tumor protection. Hum Gene Ther 17: Mennuni, C, Clvruso, F, Fcciene, A, Aurisicchio, L, Storto, M, Scrselli, E et l. (25). Efficient induction of T-cell responses to crcinoemryonic ntigen y heterologous prime-oost regimen using DNA nd denovirus vectors crrying codon usge optimized cdna. Int J Cncer 117: Breitch, CJ, De Silv, NS, Flls, TJ, Aldl, U, Evgin, L, Pterson, J et l. (211). Trgeting tumor vsculture with n oncolytic virus. Mol Ther 19: Wng, LC, Lynn, RC, Cheng, G, Alexnder, E, Kpoor, V, Moon, EK et l. (212). Treting tumors with vccini virus expressing IFNß illustrtes the complex reltionships etween oncolytic ility nd immunogenicity. Mol Ther 2: Endo, Y, Ski, R, Ouchi, M, Onimtsu, H, Hioki, M, Kgw, S et l. (28). Virusmedited oncolysis induces dnger signl nd stimultes cytotoxic T-lymphocyte ctivity vi protesome ctivtor upregultion. Oncogene 27: Errington, F, Steele, L, Prestwich, R, Hrrington, KJ, Pndh, HS, Vidl, L et l. (28). Reovirus ctivtes humn dendritic cells to promote innte ntitumor immunity. J Immunol 18: Benenci, F, Courrèges, MC, Frser, NW nd Coukos, G (28). Herpes virus oncolytic therpy reverses tumor immune dysfunction nd fcilittes tumor ntigen presenttion. Cncer Biol Ther 7: Benenci, F, Courrèges, MC, Conejo-Grcí, JR, Mohmed-Hdley, A, Zhng, L, Bucknovich, RJ et l. (25). HSV oncolytic therpy upregultes interferon-inducile chemokines nd recruits immune effector cells in ovrin cncer. Mol Ther 12: Bridle, BW, Chen, L, Lemy, CG, Dillo, JS, Pol, J, Nguyen, A et l. (213). HDAC inhiition suppresses primry immune responses, enhnces secondry immune responses, nd rogtes utoimmunity during tumor immunotherpy. Mol Ther 21: Lne, C, Leitch, J, Tn, X, Hdjti, J, Brmson, JL nd Wn, Y (24). Vccintioninduced utoimmune vitiligo is consequence of secondry trum to the skin. Cncer Res 64: Ng, P, Beuchmp, C, Evelegh, C, Prks, R nd Grhm, FL (21). Development of FLP/frt system for generting helper-dependent denovirl vectors. Mol Ther 3(5 Pt 1): Lwson, ND, Stillmn, EA, Whitt, MA nd Rose, JK (1995). Recominnt vesiculr stomtitis viruses from DNA. Proc Ntl Acd Sci USA 92: Moleculr Therpy vol. 22 no. 2 fe