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1 Development of repliction-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8 + T cell immunity Luks Fltz 1 3, Ahmed N Hegzy,4,5,16, Andres Bergthler 1,,6,16, Admr Verschoor,7,16, Christin Clus 8, Mrylise Fernndez 1,9, Luc Gttinoni 1, Susn Johnson 1,9, Florin Kreppel 11, Stefn Kochnek 11, Mries vn den Broek,1, Andres Rdruch 4,5, Frédéric Lévy 13,15, Pul-Henri Lmert 9, Clire-Anne Siegrist 1,9,14, Nichols P Restifo 1, Mx Löhning,4,5, Adrin F Ochsenein 8, Gry J Nel 3 & Dniel D Pinschewer 1,,9 Lymphocytic choriomeningitis virus (LCMV) exhiits nturl tropism for dendritic cells nd represents the prototypic infection tht elicits protective CD8 + T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we hve hrnessed the immunoiology of this renvirus for vccine delivery. By using producer cells constitutively synthesizing the virl glycoprotein (GP), it ws possile to replce the gene encoding LCMV GP with vccine ntigens to crete repliction-defective vccine vectors. These rlcmv vccines elicited CTL responses tht were equivlent to or greter thn those elicited y recominnt denovirus 5 or recominnt vccini virus in their mgnitude nd cytokine profiles, nd they exhiited more effective protection in severl models. In contrst to recominnt denovirus 5, rlcmv filed to elicit vector-specific ntiody immunity, which fcilitted re-dministrtion of the sme vector for ooster vccintion. In ddition, rlcmv elicited T helper type 1 CD4 + T cell responses nd protective neutrlizing ntiodies to vccine ntigens. These fetures, together with low seroprevlence in humns, suggest tht rlcmv my show utility s vccine pltform ginst infectious diseses nd cncer. Vccines represent one of the most successful interventions in modern medicine, hving led to the control of mny devstting infectious diseses nd recently lso to the prevention of cervicl cncer. Recominnt DNA technology nd ioengineering hve mde it possile to exploit virl vectors for optimized vccine delivery. Attenuted or repliction-incompetent viruses such s modified vccini virus Ankr nd recominnt denovirus 5 re geneticlly modified to express vccine ntigen of interest. Mny vectors hve limittions, nd their ility to counter infection y HIV, heptitis C virus nd tuerculosis or tret tumors remins uncertin. The induction of protective nd long-lived cytotoxic T lymphocytes (CTLs) is n unmet yet importnt gol of vccintion ginst mny of these diseses, which hve considerle effect on humn helth. Poxvirus-sed vectors, for exmple, effectively induce multifunctionl CTL responses 1, ut they exhiit limited ility to elicit CTL responses of high mgnitude. Conversely, recominnt denovirus (rad)-sed vccines stimulte high-frequency responses tht tend to e less multifunctionl 3. Moreover, significnt proportion of the glol popultion crries denovirus 5 neutrlizing ntiodies from nturl infection, which cn ffect vector immunogenicity 4,5. Even in the sence of preexisting immunity, vccintion with rad- or poxvirus-sed vccines elicits potent vector-specific ntiody immunity, which interferes with homologous oosting 5. Thus, there remins need to develop virl vectors tht elicit effective T cell immunity without concomitnt induction of ntiodies tht lock vector re-dministrtion. The prototypic renvirus lymphocytic choriomeningitis virus (LCMV) contins isegmented negtive-strnd RNA genome encoding the virl proteins glycoprotein (LCMV-GP), nucleoprotein (NP), Z nd L in n misense strtegy 6 (Fig. 1). LCMV-GP medites receptor inding nd cell entry nd represents the only trget for LCMV-neutrlizing ntiodies 6. LCMV-induced T cell responses re rod nd long-lived 7, nd LCMV infection hs een widely studied s model of CTL-medited protection. The neutrlizing ntiody response to LCMV is extrordinrily wek, nd convlescent serum fils to prevent re-infection 6,8. These chrcteristics suggested tht modified LCMV might serve s vector for the induction of CTL immunity. 1 Deprtment of Pthology nd Immunology, University of Genev, Genev, Switzerlnd. Institute of Experimentl Immunology, University Hospitl of Zurich, Zurich, Switzerlnd. 3 Vccine Reserch Center, Ntionl Institute of Allergy nd Infectious Diseses, Ntionl Institutes of Helth, Bethesd, Mrylnd, USA. 4 Experimentl Immunology, Deprtment of Rheumtology nd Clinicl Immunology, Chrité University Medicine, Berlin, Germny. 5 Deutsches Rheum-Forschungszentrum, Berlin, Germny. 6 Institute for Systems Biology, Settle, Wshington, USA. 7 Institute for Medicl Microiology, Immunology nd Hygiene, Technicl University Munich, Munich, Germny. 8 Tumor Immunology, Deprtment of Clinicl Reserch, University of Berne, Berne, Switzerlnd. 9 World Helth Orgniztion Collorting Center for Neontl Vccinology, University of Genev, Genev, Switzerlnd. 1 Center for Cncer Reserch, Ntionl Cncer Institute, US Ntionl Institutes of Helth, Bethesd, Mrylnd, USA. 11 Division of Gene Therpy, University of Ulm, Ulm, Germny. 1 Oncology, University Hospitl of Zurich, Zurich, Switzerlnd. 13 Ludwig Institute for Cncer Reserch, Eplinges, Switzerlnd. 14 Deprtment of Peditrics, University of Genev, Genev, Switzerlnd. 15 Present ddress: Deiophrm, Lusnne, Switzerlnd. 16 These uthors contriuted eqully to this work. Correspondence should e ddressed to D.D.P. (dniel.pinschewer@gmx.ch) or L.F. (luks.fltz@gmil.com). Received 5 June 9; ccepted 1 Octoer 9; pulished online 7 Ferury 1; doi:1.138/nm.14 nture medicine VOLUME 16 NUMBER 3 MARCH 1 339

2 Z 5 Vccine ntigen or reporter gene GP S (3.4 k) 5 3 L (7. k) L NP 3 Vccine ntigen or reporter gene pol-i-sv pol-i-prom. S segment pol-i-termin. pol-i-l pol-i-prom. L segment pol-i-termin. pol-ii-l Actin-prom. intron L-ORF pol-ii-np Actin-prom. intron NP-ORF poly/a poly/a c Plsmids Producer cell Producer cell 7 h GP GP Vector prticles d 93T-GP e Virus (PFU per ml) <5 93T-GP 93T Time fter infection (h) Recently, it hs ecome possile to mnipulte the LCMV genome y recominnt cdna technology 9,1. Here we report on moleculr strtegy for rendering the virus repliction incompetent to confer n cceptle sfety profile while llowing the incorportion of diverse ntigens tht cn confer protection ginst infections nd cncers. RESULTS Genertion of repliction-defective rlcmv vectors To generte repliction-deficient LCMV (rlcmv) vectors, we hypothesized tht the virl GP open reding frme could e replced y gene encoding vccine ntigen (Fig. 1). We generted rlcmv vectors in which GP ws deleted using four-plsmid co-trnsfection system previously used to generte wild-type LCMV 1. The short (S) nd long (L) vector RNA genome segments were expressed intrcellulrly from plsmid expression vectors under control of n RNA polymerse I promoter nd termintor (Fig. 1). The miniml virl trns-cting fctors (NP nd L) were co-expressed from n RNA polymerse II promoter in dditionl plsmids (Fig. 1). We used these four plsmids for trnsient trnsfection of BHK-1 cells stly trnsfected with n LCMV-GP expression vector ( producer cells ; Fig. 1c). In these cells, the GP-deficient virl genomes were complemented in trns nd gve rise to vectors whose RNA could e mplified nd expressed in trget cells, ut they could not give rise to spreding virus nd propgte infection. To confirm expression of the heterologous gene, we generted n rlcmv with the green fluorescent protein (GFP) reporter gene in plce of LCMV-GP (rlcmv-gfp). Infection of norml BHK-1 or 93T cells with rlcmv-gfp yielded individul fluorescent cells tht did not increse in numer over time, wheres pssge of GP-expressing BHK-1 or 93T producer cells yielded expnding fluorescent foci indictive of virl propgtion (Fig. 1d nd dt not shown). This fvirl copies in spleen (log 1 ) < Time fter infection (d) g Helthy nimls / nimls tested 3/3 /3 1/3 Intrcererl infection wtlcmv / Time fter virus infection (d) Figure 1 Genertion of rlcmv vectors, repliction in cultured cells nd rpid elimintion nd lck of pthogenicity in vivo. () Wild-type LCMV with its two genome segments (S nd L) nd the four open reding frmes (ORFs). Sustitution of GP for vccine ntigen or reporter gene cretes the rlcmv vectors. () Expression cssettes of plsmids used for the recovery of rlcmv vectors. In pol-i-sv nd pol-i-l 1, the mouse polymerse I promoter (pol-i-prom.) nd termintor (pol-i-termin.) drive intrcellulr expression of the LCMV vector S segment nd L segment, respectively; pol-ii-l 1 nd pol-ii-np 1 express the respective virl proteins under control of n ctin promoter (Actin-prom.)-driven expression cssette with intron nd polydenyltion (poly(a)) signl. ORF, open reding frme. (c) Process to recover rlcmv vectors in producer cells. LCMV-GP protein (GP) on the surfce of producer cells is incorported into vector prticles. (d) Fluorescence microscopy of GFP expression in 93T cells nd GP-expressing 93T cells (93T-GP) infected for 48 h with rlcmv-gfp t multiplicity of infection of.5. Scle rs, 1 µm. (e) Infectious progeny prticles in superntnts of the 93T cells nd GP-expressing 93T cells in d t 7 h fter infection. Dt re representtive of three individul culture wells (men ± s.e.m.). (f) Rel-time RT-PCR nlysis of vector S segment copies, mesured in totl RNA from spleen of mice vccinted with rlcmv-gfp. Ech symol represents n individul mouse; smll horizontl lines indicte the men. (f) Disese development in mice infected intrcererlly with wild-type LCMV or (three mice per group). Animls displying clinicl signs of lymphocytic choriomeningitis were killed in ccordnce with the Swiss lw for niml protection. 93T finding suggested tht rlcmv prticles formed in norml cells were GP deficient nd repliction incompetent, s expected. The growth curves of rlcmv-gfp confirmed the presence of vector in the superntnts of producer cells ut not those of norml cells (Fig. 1e). In C57BL/6 mice inoculted intrvenously with rlcmv- GFP, virl RNA copies in spleen declined rpidly nd erly, nd within 1 d, they pproched the limit of detection (Fig. 1f). To determine whether this vector induced disese in the centrl nervous system, we injected n rlcmv vector expressing ovlumin () intrcererlly. This vector filed to elicit clinicl signs of choriomeningitis, wheres wild-type LCMV uniformly cused ftl disese (Fig. 1g). Mice with comined genetic deficiency in type I nd type II interferon receptors (Ifnr1 / Ifngr / ; clled Ifngr / here) re uniquely permissive for LCMV, nd Ifngr / mice hve therefore een used to detect very low-level LCMV repliction 11. We inoculted Ifngr / mice with rlcmv-gfp nd killed them, 7 or 1 d lter. We smpled spleen, liver, lung, kidney nd rin tissues without detecting infectivity t ny time point (Supplementry Fig. 1), which further documented tht the rlcmv vectors were repliction incompetent in vivo. The rlcmv vector lso did not inhiit CTL or ntiody responses to vesiculr stomtitis virus (VSV; Supplementry Fig. 1), which indicted tht rlcmv vectors did not dversely ffect generl immunocompetence. rlcmv vectors elicit potent CD8 + T cell responses To chrcterize the immunogenicity of proteins encoded y rlcmv, we injected incresing doses of or rad vector expressing OVA () into C57BL/6 mice nd ssessed CD8 + T cell frequencies specific for OVA (SIINFEKL epitope) in the peripherl lood fter 8 d. At lower vector doses of 3 1 or plqueforming units (PFU), elicited higher frequencies of 34 VOLUME 16 NUMBER 3 MARCH 1 nture medicine

3 H-K SIINFEKL of CD8 + (%) 14 n.s n.s or dose (PFU) H-K SIINFEKL of CD8 + (%) or dose (PFU) c Immune serum trnsfered on dy 1 d Anti- Anti- Immuniztion dy D 14: H-K SIINFEKL + of CD8 + (%) Cytokine + of CD8 + (%) Figure The rlcmv vectors elicit CD8 + T cell responses of high frequency nd functionlity tht re efficiently mplified in homologous prime-oost vccintion. (,) SIINFEKL-specific CD8 + T cell frequencies in the lood of mice vccinted (primed) with or (dose, horizontl xis) on dy nd oosted with the sme vector-dose comintion on dy 38, ssessed on rvacc-ova dy 8 fter vccintion () nd 7 d fter oost (). (c) SIINFEKL-specific CD8 + IFNγ + TNFα + T cell frequencies in peripherl lood: serum (5 µl) from mice vccinted with IFNγ + only or 4 weeks previously ws trnsferred into nive mice; 1 d fter trnsfer, oth groups nd controls without serum trnsfer were vccinted with the sme two vectors, nd T cell frequencies were determined 14 d lter. Dt re representtive of three mice per group (men ± s.d.). (d) Intrcellulr cytokine ssy of SIINFEKL-specific CD8 + T cells producing IFN-γ lone (IFN-γ + only) or oth IFN-γ nd TNF-α (IFN-γ + TNF-α + ) in spleen, ssessed on dy 14 fter vccintion with, or VACC-OVA. Dt re representtive of three to six mice per group (men ± s.e.m.). P vlues of less thn.5 () were considered sttisticlly significnt, nd P vlues of less thn.1 () were considered highly significnt. specific CD8 + T cells thn did, wheres t higher doses of or PFU, the responses were similr (Fig. ). A further increse in dose ws techniclly possile only for nd resulted in exhustion of T cell responses (Fig., PFU), similr to previous reports 1. An rlcmv vector expressing the HLA-A.1-restricted CD8 + T cell epitope MelnA6 35(7L) (rlcmv MelnA6 35(7L)) stimulted vigorous responses in HLA-A.1-trnsgenic mice (Supplementry Fig. ), which demonstrted tht rlcmv cn trigger responses of cliniclly relevnt specificity. The route of vector dministrtion my ffect immune responses. We used n intrvenous route of immuniztion here, ut responses of similr mgnitude hve een oserved fter intrderml, sucutneous, intrmusculr or intrperitonel dministrtion of rlcmv vectors (Supplementry Fig. 3 nd dt not shown). CTL induction y ws olished y ultrviolet irrdition of vector (dt not shown), which indicted tht its immunogenicity depended on gene expression in vivo. When rlcmv- nd rad-induced CTL responses were oosted with homologous vector on dy 38, elicited significntly higher specific CD8 + T cell frequencies thn did t ll doses (Fig. ). The reltive increse in specific CD8 + T cell frequency fter oosting ws prticulrly evident t lower doses ut ws lso oserved t stndrd doses nd ws higher for thn for (,.46-fold ±.54-fold;, 1.58-fold ±.33- fold (men ± s.d. of three experiments); P <.1; dt not shown). Furthermore, we found tht three dministrtions of t monthly intervls mintined OVA-specific CD8 + T cell frequencies t ~3 4% of totl CD8 T + cells 76 d fter the finl immuniztion (Supplementry Fig. 4). We estlished the functionlity of such memory cells in n in vivo cytotoxicity ssy on dy 168 fter the third immuniztion. Trnsfusion of syngenic splenocytes incuted with the OVA-derived CTL peptide SIINFEKL resulted in lmost complete rejection of these cells within 4 h, wheres control cells without peptide persisted in the circultion (Supplementry Fig. 4). n.s. More thn 8% of -induced memory CTLs hd high expression of the ctivtion mrker CD44. Aout hlf were KLRG-1 +, 4% were positive for the interleukin 7 (IL-7) receptor (CD17 + ) nd 15% were CD6L + centrl memory cells 45 d fter single immuniztion (Supplementry Fig. c). By dy 196 fter oosting, over 9% hd ecome positive for the IL-7 receptor, nd the frction of CD6L + centrl memory cells hd incresed to slightly over 3%. Unlike ntiodies to denovirus 5 (ref. 5), ntiodies to LCMV hve limited cpcity to prevent reinfection 6,8, property tht could hve explined the superior performnce of in homologous prime-oost immuniztions (Fig. ). To test this hypothesis, we pssively trnsferred serum from -immunized mice into nive mice. After susequent immuniztion with, the CD8 + T cell response ws similr to or only modertely lower thn tht of control mice tht received nive serum (Fig. c). In contrst, pssive trnsfer of -immune serum mrkedly decresed the specific CD8 + T cell response fter immuniztion, consistent with previous studies of preexisting immunity to denovirus 5 (ref. 5). We otined nlogous results with immune serum collected 1 d fter the lst of three monthly immuniztions with rlcmv- OVA or (Supplementry Fig. 5), which confirmed tht ntiodies with the cpcity to lock rlcmv vector immunogenicity were inefficiently induced. To evlute the functionlity of rlcmv-induced CTLs, we mesured OVA-specific interferon-γ (IFN-γ)-secreting CD8 + T cells nd CD8 + T cells producing oth IFN-γ nd tumor necrosis fctor-α (TNF-α) in the spleen. We found tht elicited responses t higher frequencies thn did immuniztion with or recominnt vccini virus expressing OVA (VACC-OVA, Fig. d), which ttested to the qulity of rlcmv-induced CTLs. Induction of CD4 + T cells nd protective ntiodies To exmine CD4 + T cells nd ntiody responses, we generted n rlcmv vector expressing defective form of the VSV G envelope nture medicine VOLUME 16 NUMBER 3 MARCH 1 341

4 Thy No vccine rlcmv-indg61 Dy 3 Dy 6 CFSE Thy of CD4 + (%) Detection limit Time fter immuniztion (d) c IFN-γ TNF-α Figure 3 The rlcmv vectors trigger rpid prolifertion, T helper type 1 differentition nd memory formtion of CD4 + T cells nd elicit durle neutrlizing ntiody responses to vccine ntigen. () Prolifertion (ssessed s CFSE dilution) in lood of nive syngeneic recipients given CFSE-leled SMARTA TCR-trnsgenic splenocytes crrying the Thy-1.1 mrker, then vccinted with rlcmv-indg61 or left unvccinted (No vccine), ssessed on dys 3 nd 6. Green indictes trnsferred epitope-specific (Thy ) cells. (,c) Frequency of SMARTA1 CD4 + T cells in peripherl lood over time () nd cytokine profile of SMARTA1 CD4 + T cells fter restimultion with phorol 1-myristte 13-cette nd ionomycin on dy 9 (c), ssessed for cells from the mice in. (d) VSV-neutrlizing titers of totl immunogloulin (Totl Ig) nd immunogloulin G (IgG) in serum of IL IL Helthy nimls / nimls tested 4/4 3/4 /4 1/4 d VSV na titer 1/496 1/14 1/56 1/64 1/16 >1/8 / IgG Totl Ig Time fter immuniztion (d) rlcmv-ind61 Time fter VSV chllenge (d) C57BL/6 mice vccinted with rlcmv-indg61. na, neutrlizing ntiody. Dt re representtive of three to six mice per group (,d; men ± s.e.m.). (e) Disese development in type I interferon receptor deficient mice vccinted with rlcmv-indg61 or left untreted, then chllenged intrvenously with 1 6 PFU VSV 4 weeks lter (four mice per group). Animls with terminl myeloencephlitis were killed. e Vccine 13 glycoprotein (rlcmv-indg61), in which the immunodominnt I-A -restricted epitope of LCMV-GP (GP61) ws inserted in the ectodomin of the VSV G envelope glycoprotein. We leled trnsgenic CD4 + T cells specific for GP61 (SMARTA1 T cells 13 ) with the fluorescent dye CFSE nd trnsferred the cells into nive C57BL/6 recipients. Administrtion of rlcmv-indg61 stimulted rpid cell division evident in the dilution of CFSE on 98% of the SMARTA1 CD4 + T cells within 3 d of vccintion (Fig. 3). Similrly, doptively trnsferred GP33-specific CD8 + T cells from T cell receptor (TCR)- trnsgenic mice underwent rpid cell division fter immuniztion with rlcmv (Supplementry Fig. 6). After initil popultion expnsion, rlcmv-indg61-ctivted SMARTA1 T cells slowly decresed in frequency during the oservtion period of months, similr to CD4 + T cells fter infection with wild-type LCMV 7,13 (Fig. 3). When SMARTA1 CD4 + T cells were restimulted 9 d fter rlcmv-indg61 vccintion, >95% of the cells synthesized TNF-α nd mny lso coexpressed IFN-γ (Fig. 3c, left), wheres secretion of IL-4 nd IL-1 ws minimlly detectle (Fig. 3c, right), indictive of T helper type 1 polriztion in rlcmv-triggered CD4 + T cell responses 13. Immuniztion with rlcmv-indg61 lso stimulted potent neutrlizing ntiody response to VSV tht reched titers of ~1:1, within 4 d (Fig. 3d). Isotype clss switching to immunogloulin G ws evident 7 d fter vccintion, which reflected efficient CD4 + T cell help. Titers in the rnge of 1:5, were mintined throughout the oservtion period of 54 d, which demonstrted the durility of this ntiody response fter rlcmv vccintion. We confirmed the protective cpcity of rlcmv-induced ntiodies in mice deficient in type I interferon receptors. Immuniztion with rlcmv-indg61 protected these mice ginst chllenge with 1 6 PFU VSV (>1,-fold the 5% lethl dose 14 ), wheres non-immune controls rpidly developed terminl myeloencephlitis (Fig. 3e). Efficcy nd longevity of rlcmv-induced CTL responses We next exmined the protective cpcity of rlcmv-induced CTL immunity. We vccinted C57BL/6 mice with, or VACC-OVA, chllenged them with recominnt Listeri monocytogenes expressing OVA (rlm-ova 15 ) on dy 16 fter immuniztion nd mesured cteril titers in the liver 3 d lter 16. Immuniztion with or resulted in cteril titers tht were nerly undetectle. In contrst, immuniztion with VACC-OVA filed to confer protection (Fig. 4). When we chllenged the mice 58 d fter single vccintion or d fter prime-oost immuniztion, we otined similr results, which confirmed the durility of protection of nd. This protection ws produced in dose-response mnner. When we chllenged the mice either 18 d fter single immuniztion or d fter prime-oost vccintion, rlcmv- OVA fforded nticteril protection t doses 3- to 1,-fold lower thn those of (Supplementry Fig. 7 ). Next we ssessed the cytolytic efficcy of rlcmv-, rad- nd VACCinduced CTLs in solid tissues. RIP-OVA mice express OVA ntigen in pncretic islet cells 17 nd exhiit centrl tolernce to OVA, ut doptively trnsferred OVA-specific TCR-trnsgenic CD8 + T cells (OT-I cells 18 ) cn destroy pncretic et cells nd cuse dietes if ppropritely stimulted y OVA-specific vccintion. We gve RIP- OVA mice OT-I splenocytes (~1 1 OVA-specific CD8 + T cells) nd then immunized the mice with, or VACC-OVA (Fig. 4). In two independent experiments, immunized mice uniformly exhiited loss of glucose control (nine of nine mice), wheres VACC-OVA-immunized mice (one of six mice) nd -immunized mice (none of six mice) remined mostly unffected. This finding demonstrted tht rlcmv ws more effective thn rad nd VACC in stimulting specific CTLs tht destroyed ntigen-expressing cells in solid tissue. To test the cpcity of rlcmv vectors to promote CTL-medited rejection of peripherl solid tumors, we injected B16.F1 melnom cells expressing the CTL epitope GP33 sucutneously into the flnk of C57BL/6 mice 19. Eight dys lter, tumor msses were plple, nd we treted mice with rlcmv-, rad- or VACC-sed vectors expressing the GP33 epitope (rlcmv-gp33, rad-gp33 or VACC- GP33, respectively) nd used s negtive control. Within 8 d of injection, the tumor msses were significntly smller in rlcmv-gp33-treted mice thn in ll other groups (Fig. 4c, top). Further, ll 11 rlcmv-gp33-treted mice survived until dy (14 d fter immunotherpy; termintion of the experiment), with tumors shrinking or stilizing. In contrst, most mice vccinted with rad- GP33 or VACC-GP33 or with the control hd died or reched the endpoint criteri of tumor volume (Fig. 4c, ottom). We used vccini virus chllenge to ssess the ility of rlcmv to confer ntivirl protection. We immunized C57BL/6 mice with incresing doses of rlcmv-gp33 or rad-gp33. Then, 18 d lter, we chllenged the mice intrperitonelly with the recominnt vccini virus VACC-G expressing full-length LCMV-GP (contining GP33) 34 VOLUME 16 NUMBER 3 MARCH 1 nture medicine

5 Figure 4 Efficcy nd protective cpcity of rlcmv-induced CTL responses. () Bcteril titers in the livers of C57BL/6 mice vccinted with, or VACC-OVA, then chllenged with rlm-ova 16 or 58 d fter single immuniztion or d fter homologous primeoost vccintion (dys nd 38); titers were mesured 3 d fter chllenge. Immuniztion with rlcmv-cre (chllenge on dy 16 or 58) nd no immuniztion (chllenge on dy ) serve s controls. Ech symol represents n individul mouse; smll verticl lines indicte the men. () Blood glucose concentrtions of RIP-OVAtrnsgenic mice trnsfused with OT-I TCR-trnsgenic splenocytes on dy 1 nd immunized with, VACC-OVA or on dy. Dt re from one representtive of two experiments (men ± s.e.m. of three to six mice). (c) Tumor volume nd survivl of C57BL/6 mice injected sucutneously in oth flnks with B16.F1 melnom cells expressing the CTL epitope GP33 (ref. 19); 8 d lter, tumor msses were plple, nd rlcmv-gp33, rad-gp33 or VACC-GP33 ws given for immunotherpy. Immuniztion with serves s control. Tumor volume ws clculted y the formul V = π c / 6, where, nd c re the orthogonl dimeters. For survivl nlysis, deth nd humne endpoints (tumor volume, tumor exulcertion, cchexi) were counted s events. Broken lines (top) indicte loss of mice to follow-up (ottom), which cused is towrd smller tumors t susequent time nd mesured VACC-G titers in the ovries 6 d fter chllenge. Even t the lowest vccine dose, VACC-G titers in rlcmv-gp33- immunized mice were three orders of mgnitude lower thn those of non-immune control mice (Fig. 4d). Vccintion with rad-gp33 lso conferred protection ut required doses of PFU, 1-fold higher thn the miniml protective dose of rlcmv-gp33. rlcmv vectors trget nd ctivte dendritic cells in vivo The trgeting nd ctivtion of dendritic cells (DCs) is key for CTL induction,1, nd wild-type LCMV exhiits tropism for DCs. To understnd the mechnism y which rlcmv vectors might elicit dptive immune responses, we studied the cellulr tropism of n rlcmv expressing Cre recominse (rlcmv-cre). After injection of rlcmv-cre into trnsgenic reporter mice 3, Cre-medited removl of loxp-flnked trnscription stop signl llowed expression of n ctin promoter-driven GFP. Flow cytometry of GFP + cells identified out rlcmv-cre-infected DCs per spleen, in contrst to round fluorescent mcrophges, T cells nd B cells (Fig. 5). This indicted tht rlcmv vectors showed tropism for DCs, similr to wild-type LCMV. DCs infected with rlcmv-cre (GFP + ) exhiited higher surfce levels of CD86 thn did uninfected (GFP ) DCs in the sme mice (Fig. 5; men fluorescence indices, 15.5 ±.7 (GFP + ) versus 5.3 ±.4 (GFP ); P <.1), nd the ltter were similr to DCs in mice tht received control (men fluorescence index, 4.6 ±.; P =.14), which suggested tht rlcmv vectors ctivted Blood glucose (mm) Listeri chllenge: Vccintion rvacc-ova rlcmv-cre or none d rvacc-g ovry titer (PFU log 1 ) D 16 CFU in liver (log 1 ) < Immuniztion Dietes threshold 1 5 rvacc-ova Time fter vccintion (d) <1.7 3x x No vccine rlcmv-gp33 or rad-gp33 vccine dose (PFU) D 58 CFU in liver (log 1 ) < c Tumor volume (mm 3 ) Survivl (%) ND, 1,5 1, minly infected DCs. To nlyze DC ctivtion y rlcmv further, we used the trnsgenic mouse model ST33 (ref. 1), which expresses GP33 fter intrcellulr expression of Cre. Expression of GP33 in resting ST33 DCs triggers specific CD8 + T cell tolernce, wheres GP33 expression in ctivted DCs results in CD8 + T cell priming 1. Administrtion of rlcmv-cre in ST33 mice resulted in.8% GP33- specific CD8 + T cells on dy 1 nd dy 7, nd this ws sent in ST33 mice inoculted with (Fig. 5c). Susequent chllenge with VACC-G triggered rpid popultion expnsion of the GP33-specific CTLs tht produced IFN-γ upon in vitro restimultion. These dt confirmed tht rlcmv not only trgeted DCs ut lso functionlly ctivted them in vivo. When rlcmv vectors were incuted with humn peripherl lood mononucler cells (PBMCs) in vitro, they infected over 6% of myeloid DCs, plsmcytoid DCs nd monocytes, compred with less thn % of B cells, CD4 + nd CD8 + T cells (Supplementry Fig. 8), which resemled the vector tropism in mice (Fig. 5). Infection of humn myeloid DCs lso incresed expression of the ctivtion mrker CD86 (Supplementry Fig. 8). Finlly, incution of PBMCs from humn melnom ptients with rlcmv MelnA6 35(7L) in vitro stimulted sustntil HLA-A1-restricted MelnA6 35(7L)-specific CD8 + T cell response (Supplementry Fig. 8c), which exceeded the response triggered y MelnA6 35(7L) peptide. These experimentl oservtions indicte tht rlcmv will e immunogenic in humns. 6 Therpeutic immuniztion D CFU in liver (log 1 ) < ND Time fter tumor trnsplnttion (d) Immuniztion rvcc-gp33 rad-gp33 rlcmv-gp33 rlcmv-gp33 rad-gp33 rvcc-gp points. Dt re representtive of initilly 18 tumors from 9 11 mice per group (men ± s.e.m.). (d) Virl titers in the ovries of C57BL/6 mice vccinted with rlcmv-gp33 or rad-gp33 (dose, horizontl xis) or left untreted, then given 1 6 PFU of VACC-G intrperitonelly 18 d lter nd killed 6 d lter. Sttisticl nlysis identifies groups tht differ significntly from unvccinted controls. Dt re representtive of five mice (men ± s.d.). P vlues of less thn.1 () were considered highly significnt. nture medicine VOLUME 16 NUMBER 3 MARCH 1 343

6 Z/EG reporter mice rlcmv-cre Forwrd sctter CD11c + F4/8 + CD3 + CD ±135 7 ±1 GFP DISCUSSION The properties of rlcmv suggest tht it holds promise s vector for vccintion nd immunotherpy in infectious diseses nd cncer. Its desirle fetures stem minly from three properties: (i) its ility to trget nd functionlly ctivte DCs, (ii) its stimultion of potent CD8 + T cell responses nd (iii) its reltive insensitivity to ntiody neutrliztion nd thus its efficcy in homologous prime-oost vccintion. A crdinl spect of rlcmv immunogenicity is its trgeting to DCs, followed y ctivtion of these cells, which is proly responsile for the potent CTL induction. The ility of rlcmv-immunized mice to respond to homologous oosting reflects the poor induction of neutrlizing ntiodies, n intrinsic feture of the LCMV envelope glycoprotein 8. In ddition, the limited numer of rlcmv vector prticles dministered for vccintion here provided reltively low mounts of LCMV-GP, nd exposure of the immune system to LCMV-GP ws of short durtion. The gene encoding LCMV-GP is sent from rlcmv vectors, nd the vccine cnnot produce further LCMV-GP in vivo to trigger GP-specific neutrlizing ntiody responses. Moreover, even ntiodies tht potently neutrlize LCMV in vitro exhiit only limited cpcity to interfere with T cell induction in vivo 4. Together these considertions proly explin why even multiple dministrtions of rlcmv over prolonged periods of time hve filed to elicit ntiodies tht interfere with vector immunogenicity. In ddition to the limited interference y preexisting humorl immunity, ll the existing evidence suggests tht humn seroprevlence to LCMV is generlly elow 5% (refs. 5 8). In contrst, high-titer protective neutrlizing ntiody responses re generted to the trnsgene encoded y the vector, which indictes its likely utility s vccine vector in humns. Wild-type LCMV cn replicte nd cuse disese in immunosuppressed trnsplnt recipients 9. The lck of rlcmv repliction suggests therefore tht these vectors will provide suitle sfety profile for further development in humns. Possile pplictions for rlcmv include protective vccines, for exmple, ginst HIV, heptitis C virus, tuerculosis nd mlri, s well s cncer immunotherpy. Preliminry dt in mice suggest tht rlcmv vectors elicit CD8 + T cell responses to HIV ntigens t high frequency nd functionlity (L.F. nd G.J.N., unpulished 191 ±6 3 ±65 Cell numers Immuniztion GFP Stin c D 1 D 7 rlcmv-cre pos. α-cd86.78 rlcmv-cre neg. α-cd86 ±.1 neg. α-cd86 rlcmv-cre pos. Isotype rlcmv-cre neg. Isotype CD86 or isotype control ST33 mice rlcmv-cre H-D GP33 tetrmer.5 ±.1 CD8.81 ±.15.3 ±.1 D 34 (dy 6 fter rvcc/g) dt). Future studies should determine whether rlcmv vectors re prticulrly dvntgeous in eliciting CD8 + T cell responses to self ntigens on tumors, in which immunotherpy cn e compromised y T cell tolernce. The choice of n optiml vccine vector depends on oth the properties of the vector nd the mechnisms of protection for specific diseses. Additionl prmeters, including ese of storge, lrge-scle mnufcturing, cost effectiveness nd the size of the gene pylod, cn e limiting nd remin to e determined for rlcmv. We hve generted rlcmv vectors crrying foreign genes up to.6 kiloses in size without technicl difficulties. If size limits for inserts existed, multiple vectors crrying individul ntigens could e comined in single vccine, s successfully performed with recominnt denovirus 5 sed vectors 3. Given ll of these oservtions, rlcmv represents n ttrctive vector for stimulting oth cellulr nd humorl immunity. Thus, it provides pltform tht my e used to prevent or tret mlignnt nd infectious diseses for which existing vccintion strtegies hve yet to confer protection. Methods Methods nd ny ssocited references re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Medicine wesite. Acknowledgments We thnk H. Hengrtner nd R. Zinkerngel for criticl comments, suggestions, discussions nd long-term support; E. Horvth for technicl ssistnce; S.A. Rosenerg nd J.R. Wunderlich for smples from ptients with melnom; M. Roederer nd K. Foulds for regents nd flow cytometry support; A. Oxenius, R. Spörri nd N. Joller for providing ccess to their flow cytometry fcility; A. Pegu, R. Roychoudhuri nd C. Cheng for discussions nd dvice on humn DC cultures; D. von Ler (Georg-Speyer-Hus) for plsmid M369 nd GP-expressing 93T cells; H. Shen (University of Pennsylvni School of Medicine) for rlm-ova; M. Groettrup (University of Constnce) for VACC-OVA, originlly generted y J. Yewdell (Ntionl Institute of Allergy nd Infectious Diseses); nd R. Schirmeck (University of Ulm) for StT-OVA-G cdna. L.F. ws supported y fellowship of the Schweizerische Stiftung für medizinisch-iologische Stipendien. A.N.H. is fellow of GRAKO111 of the Germn Reserch Foundtion. M.L. is Lichtenerg fellow funded y the Volkswgen Foundtion. A.B. ws supported y PhD scholrship of the Boehringer Ingelheim Fonds nd y post-doctorl fellowship of the Roche Reserch Foundtion. D.D.P. holds stipendiry professorship of the IFN-γ (GP33-stimulted) 1.4 ±4.4.9 ±.5 1. ±.7. ±.1 TNF-α (GP33-stimulted) Figure 5 The rlcmv vectors trget nd ctivte DCs. () Flow cytometry of GFP expression in DCs (CD11c + ), mcrophges (F4/8 + ), T cells (CD3 + ) nd B cells (CD19 + ) in Z/EG trnsgenic reporter mice 3 d fter vccintion with rlcmv-cre or (control vector). Numers in outlined res indicte totl numer of fluorescent cells per spleen (men ± s.e.m.); gtes were set such tht no positive cells were recorded for mice vccinted with. Dt re representtive of four Z/EG mice per group. () CD86 surfce expression on CD11c + DC popultions from the mice in, identified s GFP + (pos.) or GFP (neg.) nd stined with ntiody to CD86 (α-cd86) or isotype-mtched control ntiody (Isotype). (c) GP33-specific CD8 + T cell frequencies in lood of ST33 mice vccinted with rlcmv-cre or control vector, ssessed y mjor histocomptiility complex clss I (H-D ) tetrmer stining on dys 1 nd 7 fter vccintion (left), nd GP33-specific recll responses in spleen fter mice were chllenged with VACC-G on dy 8 of the experiment, ssessed 6 d lter y intrcellulr cytokine ssy. Numers in plots indicte percent tetrmer-positive CD8 + T cells (left; top right qudrnt) or percent IFN-γ + CD8 + T cells (right; top left qudrnt) or IFN-γ + TNF-α + CD8 + T cells (right; top right qudrnt). Dt re from one representtive of two similr experiments (men ± s.d. of three to four mice per group). 344 VOLUME 16 NUMBER 3 MARCH 1 nture medicine

7 Swiss Ntionl Science Foundtion (PPA ) nd ws supported y grnt 31A-1467/1 of the Swiss Ntionl Science Foundtion. AUTHOR CONTRIBUTIO L.F., A.N.H., A.B., A.V., C.C., M.F., L.G., S.J., F.K. nd D.D.P. performed experiments; L.F., A.N.H., A.B., A.V., C.C., L.G., P.-H.L., C.-A.S., N.P.R., M.L., A.F.O., G.J.N. nd D.D.P. designed experiments; S.K., M.v.d.B., A.R. nd F.L. contriuted regents; nd L.F., G.J.N. nd D.D.P. wrote the mnuscript. COMPETING INTERESTS STATEMENT The uthors declre competing finncil interests: detils ccompny the full-text HTML version of the pper t Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Hrri, A. et l. An HIV-1 clde C DNA prime, NYVAC oost vccine regimen induces relile, polyfunctionl, nd long-lsting T cell responses. J. Exp. Med. 5, (8).. Peters, B.S. et l. Studies of prophylctic HIV-1 vccine cndidte sed on modified vccini virus Ankr (MVA) with nd without DNA priming: effects of dosge nd route on sfety nd immunogenicity. Vccine 5, 1 17 (7). 3. Drrh, P.A. et l. Multifunctionl TH1 cells define correlte of vccine-medited protection ginst Leishmni mjor. Nt. Med. 13, (7). 4. Kostense, S. et l. Adenovirus types 5 nd 35 seroprevlence in AIDS risk groups supports type 35 s vccine vector. AIDS 18, (4). 5. Roerts, D.M. et l. Hexon-chimeric denovirus serotype 5 vectors circumvent pre-existing nti-vector immunity. Nture 441, (6). 6. Buchmeier, M.J., Bowen, M.D. & Peters, C.J. Arenviride: The viruses nd their repliction. in Fields Virology (eds. Knipe, D.M. nd Howley, P.M.) (Lippincott Willims & Wilkins, Phildelphi, Pennsylvni, 1). 7. Homnn, D., Teyton, L. & Oldstone, M.B. Differentil regultion of ntivirl T-cell immunity results in stle CD8 + ut declining CD4 + T-cell memory. Nt. Med. 7, (1). 8. Pinschewer, D.D. et l. Kinetics of protective ntiodies re determined y the virl surfce ntigen. J. Clin. Invest. 114, (4). 9. Pinschewer, D.D., Perez, M., Snchez, A.B. & de l Torre, J.C. Recominnt lymphocytic choriomeningitis virus expressing vesiculr stomtitis virus glycoprotein. Proc. Ntl. Acd. Sci. USA 1, (3). 1. Fltz, L., Bergthler, A., de l Torre, J.C. & Pinschewer, D.D. Recovery of n renvirus entirely from RNA polymerse I/II-driven cdna. Proc. Ntl. Acd. Sci. USA 13, (6). 11. Ciure, A. et l. Persistence of lymphocytic choriomeningitis virus t very low levels in immune mice. Proc. Ntl. Acd. Sci. USA 96, (1999). 1. Kres, P., Scndell, E., Odermtt, B. & Ludewig, B. Rpid functionl exhustion nd deletion of CTL following immuniztion with recominnt denovirus. J. Immunol. 174, (5). 13. Lohning, M. et l. Long-lived virus-rective memory T cells generted from purified cytokine-secreting T helper type 1 nd type effectors. J. Exp. Med. 5, (8). 14. Steinhoff, U. et l. Antivirl protection y vesiculr stomtitis virus-specific ntiodies in lph/et interferon receptor-deficient mice. J. Virol. 69, (1995). 15. Pope, C. et l. Orgn-specific regultion of the CD8 T cell response to Listeri monocytogenes infection. J. Immunol. 166, (1). 16. Jensen, E.R., Shen, H., Wettstein, F.O., Ahmed, R. & Miller, J.F. Recominnt Listeri monocytogenes s live vccine vehicle nd proe for studying cellmedited immunity. Immunol. Rev. 158, (1997). 17. Kurts, C. et l. CD8 T cell ignornce or tolernce to islet ntigens depends on ntigen dose. Proc. Ntl. Acd. Sci. USA 96, (1999). 18. Hogquist, K.A. et l. T cell receptor ntgonist peptides induce positive selection. Cell 76, 17 7 (1994). 19. Prevost-Blondel, A. et l. Tumor-infiltrting lymphocytes exhiiting high ex vivo cytolytic ctivity fil to prevent murine melnom tumor growth in vivo. J. Immunol. 161, (1998).. Steinmn, R.M. Dendritic cells: verstile controllers of the immune system. Nt. Med. 13, (7). 1. Prost, H.C., Lgnel, J., Kollis, G. & vn den Broek, M. Inducile trnsgenic mice revel resting dendritic cells s potent inducers of CD8 + T cell tolernce. Immunity 18, (3).. Sevill, N. et l. Immunosuppression nd resultnt virl persistence y specific virl trgeting of dendritic cells. J. Exp. Med. 19, (). 3. Novk, A., Guo, C., Yng, W., Ngy, A. & Loe, C.G. Z/EG, doule reporter mouse line tht expresses enhnced green fluorescent protein upon Cre-medited excision. Genesis 8, (). 4. Seiler, P. et l. Induction of protective cytotoxic T cell responses in the presence of high titers of virus-neutrlizing ntiodies: implictions for pssive nd ctive immuniztion. J. Exp. Med. 187, (1998). 5. de Lmllerie, X., Fulhorst, C.F. & Chrrel, R.N. Prevlence of ntiodies to lymphocytic choriomeningitis virus in lood donors in southestern Frnce. Trnsfusion 47, (7). 6. Lledo, L., Gegundez, M.I., Sz, J.V., Bhmontes, N. & Beltrn, M. Lymphocytic choriomeningitis virus infection in province of Spin: nlysis of ser from the generl popultion nd wild rodents. J. Med. Virol. 7, (3). 7. Elers, A.R. et l. Low prevlence of ntiodies ginst the zoonotic gents Brucell ortus, Leptospir spp., Streptococcus suis serotype II, hntvirus, nd lymphocytic choriomeningitis virus mong veterinrins nd pig frmers in the southern prt of The Netherlnds. Vet. Q. 1, 5 54 (1999). 8. Stephensen, C.B. et l. Prevlence of serum ntiodies ginst lymphocytic choriomeningitis virus in selected popultions from two U.S. cities. J. Med. Virol. 38, 7 31 (199). 9. Fischer, S.A. et l. Trnsmission of lymphocytic choriomeningitis virus y orgn trnsplnttion. N. Engl. J. Med. 354, (6). 3. Semn, M.S. et l. Multiclde humn immunodeficiency virus type 1 envelope immunogens elicit rod cellulr nd humorl immunity in rhesus monkeys. J. Virol. 79, (5). nture medicine VOLUME 16 NUMBER 3 MARCH 1 345

8 ONLINE METHODS Genertion nd titrtion of rlcmv vectors. The rlcmv vectors were recovered s descried 1, with modifictions s outlined in Results (use of GP-expressing BHK-1 cells). The GP-expressing 93T nd BHK-1 producer cell lines re stle trnsfectnts crrying plsmid M369 tht expresses codonoptimized LCMV-GP cdna 31. Plsmids pol-i-l, pc-l nd pc-np hve een reported 1. For genertion of the vrious psv plsmids directing intrcellulr expression of vector S segments, the respective open reding frmes were mplified y PCR nd were inserted into psbsm( ) 9 y cloning strtegy previously outlined in detil 9. The plsmid psvindg61 (for intrcellulr expression of the rlcmv-indg61 S segment) ws generted y circulr mutgenesis PCR on psr 9. By this procedure, cdna sequence encoding GP mino cids 61 8 (GLNGPDIYKGVYQFKSVEFD) flnked y upstrem nd downstrem glycine residues ws inserted into the INDG open reding frme upstrem of the sequence encoding mino cid 19. Two types of were generted expressing either full-length OVA (the sme s in VACC-OVA) or the StT-OVA-G fusion construct (the sme s in ). Both types of were tested in prllel in the mjority of experiments. The immune responses were indistinguishle nd hence for simplicity the results of the two groups were pooled. The vector rlcmv MelnA6 35(7L) ws generted y incorportion into the S segment of fusion gene tht hs een descried 3 (identicl to VACC-MelnA6 35(7L); descried elow).the vector rlcmv- GP33 ws generted y incorportion of chimeric protein contining the GP33 epitope s descried 33. The rlcmv vectors were grown nd titrted y immunofocus ssy 8 on 93T-GP cells. Bcteri, viruses, virl vectors nd quntifiction. Recominnt L. monocytogenes expressing OVA 15, wild-type LCMV (strin clone 13) nd VSV serotype Indin hve een descried 1. The rad vectors with deletion of E1 were grown nd purified s descried 34. The vector expresses StT-OVA-G, truncted OVA open reding frme fused N-terminlly to T77 domin nd C-terminlly to EGFP. Immuniztions with rad were performed on the sis of prticle titers nd re expressed in PFU, with the ssumption tht 1 PFU 1 prticles y pproximtion. The vector VACC/MelnA6 35(7L) expresses the MelnA6 35(7L) peptide ELAGIGILTV in fusion construct s reported 3. The recominnt vccini viruses VACC-G, expressing LCMV-GP, VACC-GP33 nd VACC-OVA, expressing full-length OVA (ll WR strin), hve een descried nd were propgted nd titrted on BSC4 cells. A TqMn RT-PCR protocol trgeting the gene encoding LCMV-NP (to e descried elsewhere) quntified rlcmv S segment copies in the spleen of vccinted mice. Mice, niml experiments, immuniztion nd infectious chllenge. SMARTA1 mice 13, P14 mice 38, HHD mice 39, OT-I mice 18, RIP-OVA mice 17, Z/EG mice 3, ST33 mice 1, mice deficient in type I interferon receptors 14 on C57BL/6 ckground, Ifngr / mice 11 nd C57BL/6 wild-type mice were red t the Institute for Lortory Animl Sciences of the University of Zurich, Switzerlnd. Animl experiments were performed t the University of Genev, t the University of Zurich nd t the University of Bern, with permission y the respective cntonl uthorities. Immuniztion with rlcmv vectors ws performed t dose of to PFU given intrvenously, nd to prticles of were given intrvenously unless specified otherwise (referred to s stndrd dose in the text). The vector VACC-OVA ws dministered intrperitonelly t dose of 1 6 PFU. For chllenge with rlm-ova, colony-forming units were given intrvenously. Assessment of T cell nd ntiody responses. Intrcellulr cytokine ssys nd mjor histocomptiility complex clss I tetrmer stining were performed s descried 1,13. Tetrmers were from Beckmn Coulter nd ntiodies were from BD Biosciences, ebioscience nd Biolegend. Smples were nlyzed using FlowJo (TreeStr). VSV-neutrlizing ntiodies were mesured s reported 1. Sttisticl nlysis. Two groups were compred with t-tests (unpired, twotiled), nd single vlues of multiple groups were compred y one-wy nlysis of vrince with Bonferroni s post-test for multiple comprisons or with Dunnett s post-test for comprison ginst reference. Time courses of multiple groups were compred y two-wy nlysis of vrince. Sttisticl nlysis ws performed with GrphPd Prism softwre version 4. nd SPSS version 13.. P vlues of less thn.5 () were considered sttisticlly significnt, nd P vlues of less thn.1 () were considered highly significnt. Induction of humn MelnA6 35(7L)-specific CD8 + T cells. PBMCs from HLA-A1 + ptients with melnom (Surgery Brnch, Ntionl Cncer Institute) were stimulted with rlcmv MelnA6 35(7L) in culture medium contining 3 IU ml 1 of IL- (Chiron). MelnA6 35(7L)-specific CD8 + T cell frequencies were evluted y flow cytometry efore stimultion nd weeks fter stimultion. Infection of DCs nd PBMCs from helthy donors. Procedures for mdc isoltion from lood hve een descried 4. Briefly, enriched popultions of lymphocytes nd monocytes were otined y counterflow centrifugl elutrition. The mdcs were isolted from elutrited monocytes with the CD1c mgnetic ed isoltion kit (Miltenyi Biotec). PBMCs were prepred from uffy cots of helthy norml lood donors y grdient centrifugtion (Lympholyte; Cedrlne Lortories Limited). DCs nd PBMCs were cultured in complete medium (RPMI-164; Gico) supplemented with mmol l 1 l-glutmine, 1% streptomycin nd penicillin, nd 1% FCS (Invitrogen Life Technologies). To mintin viility, the medium ws further supplemented with 1 ng ml 1 recominnt humn GM-CSF (for mdcs) or ng ml 1 IL- (for PBMCs; oth PeproTech). The vector rlcmv MelnA6 35(7L) ws dded immeditely fter sorting t multiplicity of infection of 1. Intrcellulr stining for LCMV-NP ws performed 48 h fter infection y using phycoerythrin-conjugted VL-4 ntiody to LCMV-NP. 31. Mtter, M. et l. Decresed tumor surveillnce fter doptive T-cell therpy. Cncer Res. 67, (7). 3. Vlmori, D. et l. Induction of potent ntitumor CTL responses y recominnt vccini encoding meln-a peptide nlogue. J. Immunol. 164, (). 33. Bellier, B. et l. DNA vccines encoding retrovirus-sed virus-like prticles induce efficient immune responses without djuvnt. Vccine 4, (6). 34. Wortmnn, A. et l. Fully detrgeted polyethylene glycol-coted denovirus vectors re potent genetic vccines nd escpe from pre-existing nti-denovirus ntiodies. Mol. Ther. 16, (8). 35. Anton, L.C. et l. Dissocition of protesoml degrdtion of iosynthesized virl proteins from genertion of MHC clss I-ssocited ntigenic peptides. J. Immunol. 16, (1998). 36. Hny, M. et l. Anti-virl protection nd prevention of lymphocytic choriomeningitis or of the locl footpd swelling rection in mice y immuniztion with vccinirecominnt virus expressing LCMV-WE nucleoprotein or glycoprotein. Eur. J. Immunol. 19, (1989). 37. Prost, H.C. et l. Immunodominnce of n ntivirl cytotoxic T cell response is shped y the kinetics of virl protein expression. J. Immunol. 171, (3). 38. Pircher, H., Burki, K., Lng, R., Hengrtner, H. & Zinkerngel, R.M. Tolernce induction in doule specific T-cell receptor trnsgenic mice vries with ntigen. Nture 34, (1989). 39. Pscolo, S. et l. HLA-A.1-restricted eduction nd cytolytic ctivity of CD8 + T lymphocytes from β microgloulin (β m) HLA-A.1 monochin trnsgenic H-D β m doule knockout mice. J. Exp. Med. 185, (1997). 4. Lore, K. et l. Toll-like receptor lignds modulte dendritic cells to ugment cytomeglovirus- nd HIV-1-specific T cell responses. J. Immunol. 171, (3). nture medicine doi:1.138/nm.14