Coadministration of a Plasmid Encoding HIV-1 Gag Enhances the Efficacy of Cancer DNA Vaccines

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1 originl rtile The Amerin Soiety of Gene & Cell Therpy Codministrtion of Plsmid Enoding HIV-1 Gg Enhnes the Effiy of Cner DNA Vines Lure Lmriht 1, Kevin Vnvrenerg 1, Ans De Beukeler 2, Lien Vn Hoeke 2,3, John Grooten 2, Bernrd Ukr 1, Psle Lipnik 4, Niek N Snders 5,6, Stefn Lienenklus 7,8, Véronique Prét 1 nd Gëlle Vndermeulen 1 1 Advned Drug Delivery nd Biomterils, Louvin Drug Reserh Institute, Université tholique de Louvin, Brussels, Belgium; 2 Deprtment of Biomedil Moleulr Biology, Ghent University, Ghent, Belgium; 3 VIB Medil Biotehnology Center, Ghent University, Ghent, Belgium; 4 Bio nd Soft Mtter, Université tholique de Louvin, Louvin-L-Neuve, Belgium; 5 Lortory of Gene Therpy, Ghent University, Ghent, Belgium; 6 Cner Reserh Institute Ghent, Ghent, Belgium; 7 Institute for Lortory Animl Siene, Hnnover Medil Shool, Hnnover, Germny; 8 Institute for Experimentl Infetion Reserh, Centre for Experimentl nd Clinil Infetion Reserh, TWINCORE, Hnnover, Germny DNA vintion holds gret promise for the prevention nd tretment of ner nd infetious diseses. However, the linil ility of DNA vines is still ontroversil due to the limited immune response initilly oserved in humns. We hypothesized tht eletroportion of plsmid enoding the HIV-1 Gg virl psid protein would enhne ner DNA vine poteny. DNA eletroportion used to deliver plsmids in vivo, indued type I interferons, therey supporting the tivtion of innte immunity. The odministrtion of ovlumin (OVA) nd HIV-1 Gg enoding plsmids modulted the dptive immune response. This strtegy fvored ntigen-speifi Th1 immunity, delyed B16F1-OVA tumor growth nd improved mouse survivl in oth prophylti nd therpeuti vintion pprohes. Similrly, prophylti DNA immuniztion ginst the melnom-ssoited ntigen gp1 ws enhned y the odelivery of the HIV-1 Gg plsmid. The djuvnt effet ws not driven y the formtion of HIV-1 Gg virus-like prtiles. This work highlights the ility of oth eletroportion nd the HIV-1 Gg plsmid to stimulte innte immunity for enhning ner DNA vine immunogeniity nd demonstrtes interesting trks for the design of new trnsltionl geneti djuvnts to overome the urrent limittions of DNA vines in humns. Reeived 19 Novemer 215; epted 9 June 216; dvne online pulition 19 July 216. doi:1.138/mt INTRODUCTION Hrnessing the power of the immune system to tret or prevent diseses vi vine dministrtion is one of the most suessful puli helth interventions. DNA vines onsist of reominnt ntigen enoded y engineered DNA plsmids nd re expressed in vivo to soliit immunity ginst pthogens or ner ells. 1 DNA vines re promising for ner immunotherpy s they indue rod immune response, 2 inluding oth T helper type 1 (Th1) response nd ytotoxi T lymphoytes (CTLs), the most potent effetors ginst ner ells. 3,4 They lso prime CD4+ T ells, therey supporting the tivity of CTLs nd memory T ells. 5 Currently, two DNA vines re liensed for veterinry use. Onept (Meril, Lyon, Frne) is ommerilized for the tretment of melnom in dogs nd Apex-IHN (Novrtis, Bsel, Switzerlnd) for prophylti vintion of slmon ginst infetious hemtopoieti nerosis virus. Despite the ommeril use of DNA vine in veterinry mediine, DNA vintion is still under investigtion for use in humns euse the immunogeniity is often lower thn other onventionl vines. In reent yers, severl dvnements hve een mde to improve DNA vine immunogeniity in humns, leding to n inresing numer of linil trils. 6 Prtiulrly, plsmid delivery y in vivo eletroportion (EP) nd the use of genetilly enoded immune djuvnts hve enhned DNA vine effiy. 7 First, EP is the most ommonly used nd the most powerful nonvirl delivery method for DNA vines. 6,8 This method ws used to improve DNA vine effiy in lrge nimls. 7,9 EP effiieny is not only ttriuted to n enhned gene trnsfer 7 ut lso to the indution of trnsient tissue inflmmtion nd the susequent lol reruitment of immune ells. 1,1 Seond, geneti djuvnts, i.e., plsmid vetors enoding immunomodultory moleules, im t enhning the immunogeniity of ntigens in vivo. They stimulte the innte immune system to trigger pproprite dendriti ell (DC) mturtion nd therey roust, speifi, nd long-lsting dptive immune response. Indeed, the type of insult tht drives DC mturtion ffets whether nd how T ell will respond to n ntigen, s T-ell priming, expnsion, funtion, nd loliztion re ontrolled y sequentil signls provided y DCs. 11 These geneti djuvnts inlude ytokines, hemokines, or immune stimultory moleules, suh s toll-like reeptor (TLR) gonists or interferon (IFN) regultory ftors. 7,12 14 Most of these djuvnts re used in prelinil studies, nd even though they re promising, only few of them hve een evluted in linil trils until now. Therefore, there is still ritil need of linilly pplile geneti djuvnts. Correspondene: Véronique Prét, Advned Drug Delivery nd Biomterils, Louvin Drug Reserh Institute, Université tholique de Louvin, Avenue Mounier 73, te B , 12 Brussels, Belgium. E-mil: veronique.pret@ulouvin.e vol. 24 no. 9, sep. 216

2 The Amerin Soiety of Gene & Cell Therpy HIV-1 Gg Plsmid to Potentite Cner DNA Vine Plsmids enoding HIV-1 Gg (pgg) hve een widely studied during the pst dedes in the ontext of HIV vine development. In linil trils, their sfety nd immunogeniity hve een demonstrted The HIV-1 Gg proteins re le to selfssemle leding to virus-like prtile (VLP) formtion, 18 nd these psid proteins my onstitute generl pthogen-ssoited moleulr pttern (PAMP) for innte sensing. 19 Moreover, omining gp16-enoding DNA vine with pgg enhned the immune response to the gp16 envelop ntigen of HIV-1, 2 therey suggesting the pgg s djuvnt potentil. In this study, we hypothesize tht the in vivo EP of pgg ould enhne ner DNA vine immunogeniity. First, we tested whether ell trnsfetion with pgg led to susequent VLP formtion. Then, we nlyzed whether EP indued innte immunity in vivo. The effets of pgg on the immune response ginst model ntigen (ovlumin (OVA)) nd tumor-ssoited ntigen (TAA) (gp1) were evluted in B16F1-OVA melnom. Finlly, we evluted whether the in vivo pgg immunomodultory effets were medited y the formtion of VLPs. RESULTS Cell trnsfetion with plsmid oding for HIV-1 Gg leds to VLP formtion in vitro To ssess whether ell trnsfetion with the pgg vetor enoding the full length nd unmodified HIV-1 Gg protein would led to the expression of HIV-1 Gg proteins nd susequent VLP formtion, this plsmid ws trnsfeted y lipofetion in HEK 293T ells in vitro. Prtiles isolted from the ell superntnt were hrterized. After negtive stining of the smples, spheril prtiles were oserved under trnsmission eletron mirosope (Figure 1). Dynmi light sttering nlyses showed uniform popultion of prtiles with size of 18 ± 1 nm nd polydispersity index of.6 ±.3 (men ± SD, n = 3) (Figure 1). Finlly, prtile omposition ws ssessed y Western lotting. The HIV-1 Gg protein ws deteted in the purified smples (Figure 1). Plsmid DNA EP indues type I IFN responses in vivo Type I IFNs re ntivirl ytokines endowed with mny iologil effets, inluding ntitumor tivity. 21 The effet of EP on the indution of type I IFNs ws ssessed using IFN-β reporter mie in whih luiferse gene is pled under the ontrol of the IFNβ promoter. Vi in vivo ioluminesene imging, the indution of IFN-β n e lolized nd quntified. EP pplied fter n injetion of phosphte uffered sline (PBS) did not signifintly stimulte IFN-β indution. The injetion of n empty vetor without EP indued low type I IFN indution 24 hours fter the injetion, ut only the omintion of EP nd n empty plsmid DNA (pdna) vetor strongly indued the IFN-β promoter (Figure 2,). The luiferse expression levels inresed drstilly during the first 6 hours then remined stle nd strted deresing fter 48 hours. IFN-β indution remined lolized t the injetion site (Figure 2). Codelivery of the HIV-1 Gg plsmid promotes the Th1 polriztion of the immune response To investigte the ility of the HIV-1 Gg plsmid to modulte the immune response, the OVA model ntigen ws initilly Intensity (%) MW, kd nm 1 1, Size (d.nm) P1 1, Figure 1 The hrteriztion of HIV-1 Gg virus-like prtiles (VLPs). VLPs purified from the superntnts of HEK 293T ells trnsfeted with HIV-1 Gg plsmid. () Prtiles oserved y trnsmission eletron mirosopy fter purifition nd negtive stining of smples. This imge is representtive of dt from three replite experiments. Bre sle: 2 nm. () Individul mesurement of prtile size y DLS (n = 3). Prtile size is lulted from the trnsltionl diffusion oeffiient using the Stokes Einstein eqution. () Western lot nlysis of three replite produtions of VLPs purified y ultrentrifugtion (P1, P2, P3) with rit polylonl ntiodies to HIV-1 Gg s primry ntiodies nd polylonl got nti-rit immunogloulins leled with iotin s seondry ntiodies. The HIV-1 Gg protein ws deteted t ~56 kd. hosen euse it llows for oth immune response hrteriztion nd tumor growth studies. Tking into ount the high immunogeniity of OVA, mie were immunized with low dose (1 µg) of OVA enoding plsmid (). The sme mount of pgg ws odelivered with or not to determine if it would impt the immune response in vivo. After priming, lood smples nlysis indited tht the humorl response remined very low while it slightly inresed fter the first nd seond oost (Figure 3). The delivery of lone led to higher totl nti-ova ntiody titers ompred to mie where pgg ws odelivered (Figure 3). Anlysis of immunogloulins G1 nd G2 showed tht these lower ntiody titers orrelted with shift of nti-ova ntiody isoforms towrd IgG2 (Figure 3). In mie immunized with oth nd pgg, P2 P3 HIV-Gg Moleulr Therpy vol. 24 no. 9 sep

3 HIV-1 Gg Plsmid to Potentite Cner DNA Vine The Amerin Soiety of Gene & Cell Therpy Rdine (p/se/m 2 /sr) PBS + EP PBS EP pempty + EP pempty EP 2 4 Time (hours) 6 PBS EP PBS + EP pempty EP pempty + EP Luminesene 1, hour 9, 3 hours 8, 6 hours 7, 24 hours 6, 48 hours 5, Rdine (p/se/m 2 /sr) Figure 2 The effet of plsmid eletroportion on type 1 interferon (IFN) expression. Groups of nive heterozygous luiferse reporter mie (IFN-β+/Δβ-lu) were treted with phosphte uffered sline (PBS) or n empty plsmid (pempty) efore pplying eight 2 ms nd 2 V/m eletri pulses with plte eletrodes (+EP) or not ( EP). Type 1 IFN expression ws quntified y in vivo ioluminesene imging following i.p. injetion of luiferin. () Rdine ws oserved t, 3, 6, 24, nd 48 hours following tretment. The results re presented s the men ± SEM (n = 4). The sterisks indite signifint differenes ompred to the ontrol group PBS without EP (P <.5) (Friedmn test nd Dunn s post-ho test). () Representtive imging of the mie s funtion of tretment nd time. Color presenttion indites the intensity of ioluminesene, s shown in the r. the men IgG2/IgG1 rtio ws 54 times higher thn in mie immunized with lone. In ddition, 2 weeks fter the lst oost, IFN-γ levels were nlyzed t different time points following restimultion of splenoytes with OVA. Signifintly higher IFN-γ onentrtions were oserved in the groups where ws omined with pgg (Figure 3). Finlly, the odelivery of pgg tended to improve the ell-medited quired immune defense ginst ells expressing the vine ntigen. Indeed, higher killing rte (P vlue:.9) of OVA-expressing trget ells ws oserved in mie immunized with pgg nd s ompred to mie tht reeived the DNA vine lone (Figure 3d). Codelivery of the HIV-1 Gg plsmid during prophylti nti-ova DNA immuniztion delyed B16F1-OVA melnom tumor growth To explore if odelivery of pgg would effetively impt ner DNA vine effiieny, mie were prophyltilly immunized with 1 µg omined or not with 1 µg pgg. After hllenge, tumor growth nd mouse survivl were followed (Figure 4). The medin survivl in nive mie ws 27 dys, nd no nive mie were live fter 56 dys. Survivl ws signifintly etter in mie immunized with. The medin survivl time rehed 54 dys with finl survivl rtio of 1/6 (Figure 4). Importntly, the survivl ws fr etter in mie immunized with the omintion of vol. 24 no. 9 sep. 216

4 The Amerin Soiety of Gene & Cell Therpy HIV-1 Gg Plsmid to Potentite Cner DNA Vine Anti-OVA IgG tot tire 1, 1, 1, 1 1 IgG2 / IgG1 1, 1, IFN-γ onentrtion (pg/ml) hours hours 72 hours 15, 1, 5, d Perent killing OVA ells (%) P =,9 Figure 3 The effet of pgg odelivery during nti-ovlumin (OVA) immuniztion on the immune response. C57BL/6 mie were immunized in regimen of one prime nd two oosts t 2-week intervl with the ntigeni OVA plsmid omined or not with the HIV-1 Gg plsmid. () OVA-speifi totl IgG titers were mesured y enzyme-linked immunosorent ssys (ELISAs) in the ser of mie olleted t experimentl dys 1, 24, nd 38 (1 dys fter eh vine delivery, onsidering dy s the priming dy). The error rs indite men ± SEM (n = 6). The sterisks indite signifint differenes etween groups (P <.1) (Mnn Whitney U-test nd Bonferroni tests). () Antiody isotypes were nlyzed in ser of mie rndomly olleted 1 dys fter the lst oost. Individul IgG1 nd IgG2 titers were mesured y ELISAs nd eh mouse ws hrterized y n IgG2/IgG1 rtio (P <.5) (Mnn Whitney U-test). () To nlyze OVA-speifi IFN-γ levels, mie were immunized nd srified 1 week fter the lst vine dministrtion. IFN-γ onentrtions mesured in the superntnt of mie splenoytes tht hd een restimulted with OVA protein 24, 48, nd 72 hours efore. The errors rs indite men ± SEM (n = 6). The sterisks indite signifint differenes etween groups (P <.1, P <.1) (Kruskl Wllis nd Dunn s post-ho test). (d) The perentge of ntigen-speifi killing ws nlyzed y in vivo ytotoxi ssy. Immunized mie were doptively trnsferred with two popultions of leled splenoytes: MHC-I OVA peptide-pulsed-trget ells nd MHC-I irrelevnt-peptide-pulsed ells. Two dys fter trnsfer, the speifi killing of trget ells ws otined y ompring the reltive derese of the two popultions. Perentges of OVA trget ell killing were ompred using the Mnn Whitney U-test. nd pgg. In tht se, the medin survivl ws 83 dys nd hlf of the mie were still live t the end of the experiment. The tumor volume s funtion of time profile shows tht vintion strongly delyed tumor growth (Figure 4). Tumor volumes were signifintly lower in vinted mie ompred to nives, nd the size ws smller in mie immunized with the two plsmids, with signifint differenes oserved from dy 26 to 7. No progression of the implnted tumor ws oserved in mie immunized with omined with pgg for more thn 2 weeks longer thn mie immunized with the DNA vine lone. The men tumor size rehed 5 mm 3 23 dys lter in the group immunized with the two plsmids, ompred to mie immunized with lone. Codelivery of the HIV-1 Gg plsmid during prophylti nti-gp1 DNA immuniztion delyed B16F1-OVA melnom tumor growth Next, the ility of pgg to improve DNA vine effetiveness in the se of less immunogeni TAA ws ssessed. Prophylti immuniztion with 5 µg of plsmid oding for the gp1 melnom ntigen (pgp1) omined or not with 1 µg pgg ws performed efore hllenging the mie (Figure 5). Tumor growth ws slower when pgg ws odelivered with pgp1 (Figure 5). At dy 14 fter hllenge, the implnted tumor ws still not visile in four out of the six mie when the pgp1 vine ws odelivered with pgg, while only two out of six mie did not disply progression of the tumor implnted when Moleulr Therpy vol. 24 no. 9 sep

5 HIV-1 Gg Plsmid to Potentite Cner DNA Vine The Amerin Soiety of Gene & Cell Therpy Vintion Chllenge Srifie Follow-up tumor growth nd survivl Survivl (%) 15 2, 1, , 5 Figure 4 The effet of pgg odelivery during prophylti nti-ovlumin (OVA) immuniztion on the ntitumor tivity. () Experimentl pln. C57BL/6 mie were immunized in regimen of one prime nd two oosts t 2-week intervl with the ntigeni OVA plsmid omined or not with pgg. Two weeks fter the lst vintion, they were hllenged with B16F1-OVA ells. Tumor growth nd mouse survivl were ssessed for 3 months. () Survivl rtes monitoring fter hllenge. The sterisks indite signifint differenes ompred with nive mie (P <.5, P <.1) (omprison of survivl urves, Mntel Cox test). () Tumor growth follow-up fter hllenge in mie immunized with nd omined with pgg. The results re expressed s men SEM (n = 6). The sterisks show signifint differenes etween groups (P <.1, P <.1) (nlysis of vrine, Dunnett s post-ho test) Vintion Chllenge Srifie Follow-up tumor growth nd survivl Survivl (%) GP1 15 2, pgp1 + pgg 1 5 1,5 1, 5 pgp1 pgp1 + pgg Figure 5 The effet of pgg odelivery during prophylti nti-gp1 immuniztion on the ntitumor tivity. () Experimentl pln. C57BL/6 mie were immunized in regimen of one prime nd two oosts t 2-week intervl with the ntigeni GP1 plsmid omined or not with pgg. Two weeks fter the lst vintion, they were hllenged with B16F1-OVA ells nd tumor growth nd mouse survivl were monitored. () Survivl rtes monitoring fter hllenge. The sterisks indite signifint differenes ompred with nive mie (P <.5) (omprison of survivl urves, Mntel Cox test). () Tumor growth follow-up fter hllenge in mie immunized with pgp1 nd pgp1 omined with pgg. The results re expressed s men SEM (n = 6). The sterisks show signifint differenes etween groups (P <.5) (nlysis of vrine, Dunnett s post-ho test) vol. 24 no. 9 sep. 216

6 The Amerin Soiety of Gene & Cell Therpy HIV-1 Gg Plsmid to Potentite Cner DNA Vine mie were immunized with pgp1 lone. All tumors were growing in nive mie t tht time. Additionlly, 3 weeks fter hllenge the men tumor volume in the pgp1 nd pgg immunized mie ws lower (555 mm 3 ± 31) (men ± SEM, n = 6) thn in the pgp1 immunized (1,83 mm 3 ± 27; P =.2) nd nive (1,339 mm 3 ± 161; P =.4) mie. Survivl urves onfirmed the positive effet of pgg (Figure 5), s signifint improvement in survivl ws visile following pgg odelivery, wheres no signifint differene ws oserved in the se of the gp1-enoding plsmid delivered lone. Codelivery of the HIV-1 Gg plsmid during therpeuti nti-ova DNA immuniztion delyed B16F1-OVA melnom tumor growth Finlly, the therpeuti potentil of DNA vine omined or not with pgg ws evluted. To evlute suh therpeuti effet, the DNA vine ws seleted. The low immunogeniity of the pgp1 plsmid nd the rpid growth of B16F1-OVA tumors predited poor effiieny of gp1 therpeuti DNA vine. As therpeuti immuniztion onsists of vine dministrtion fter the disese onset, the tretment strted 2 dys fter the implnttion of B16F1-OVA tumors (Figure 6) nd onsisted of 1 µg of with or without 1 µg pgg. The mix of nd pgg signifintly delyed tumor growth (Figure 6). Indeed, it took 17 dys for tumors to reh 5 mm 3 in mie immunized with nd 24 dys when pgg ws dded. At dy 21, the men tumor volume (±SEM) ws 217 ± 68 mm 3, 73 ± 228 mm 3, nd 989 ± 28 mm 3 with 9%, 56%, nd 4% of tumors tht were smller thn 5 mm 3 for mie treted with the omintion of nd pgg, with lone nd for nonimmunized mie, respetively. Mouse survivl ws lso signifintly inresed when pgg ws odelivered (Figure 6). These results demonstrte tht therpeuti DNA immuniztion with odelivered with pgg, ut not with lone, promoted tumor growth dely. The formtion of HIV-1 Gg VLPs is not required for djuvnt immunomodultory effet To ssess whether the djuvnt effet of pgg ould e medited y the formtion of VLPs, plsmid enoding mutted version of HIV-1 Gg (pgg) ws onstruted. pgg enoded protein very lose in sequene to the ntive Gg protein, ut three speifi points muttions mke it neither le to ind to the plsm memrne, nor to self-ssemle properly in VLP. 22 First, prophylti immuniztion experiment followed y tumor hllenge (Figure 5) demonstrted tht pgg nd pgg, when omined to, hd the sme effet on tumor growth (Figure 8 ). The sme onlusion ws drwn from the therpeuti immuniztion experiments s the omintion of with pgg or pgg ws eqully effiient (Figure 7 ). This suggests tht the djuvnt role of pgg on the vine-ntigen speifi immune response is proly not medited y the formtion of HIV-1 Gg VLPs in vivo. Interestingly, EP of pgg or pgg lone hd delying impt on tumor growth during therpeuti (Figure 7,) ut not prophylti (Figure 8,) immuniztions. This result further supports the role of Gg enoding plsmid s stimultor of the innte immune system. DISCUSSION Despite promising results in niml models, the linil effiy of DNA vines remins to e proven due to the wekness of the immune response oserved in humns. There is therefore ritil need to find new strtegies to enhne DNA vine Vintion Chllenge Srifie Follow-up tumor growth nd survivl Survivl (%) ,5 1, Figure 6 The effet of pgg odelivery during therpeuti nti-ovlumin (OVA) immuniztion on the ntitumor tivity. () Experimentl pln. C57BL/6 mie were hllenged with B16F1-OVA ells. Two dys lter, they were immunized in regimen of one prime nd two oosts t 1-week intervl with the ntigeni OVA plsmid omined or not with HIV-1 Gg plsmid. Tumor growth nd mouse survivl were ssessed fter hllenge. () Survivl rtes monitoring fter hllenge. The sterisks indite signifint differenes ompred with nive mie (P <.5) (omprison of survivl urves, Mntel Cox test). () Tumor growth follow-up fter hllenge in mie immunized with nd omined with pgg. The results re expressed s men SEM (n = 1). The sterisks show signifint differenes etween groups (P <.5) (nlysis of vrine, Dunnett s post-ho test). Moleulr Therpy vol. 24 no. 9 sep

7 HIV-1 Gg Plsmid to Potentite Cner DNA Vine The Amerin Soiety of Gene & Cell Therpy Survivl (%) , 1,5 1, 5 2, 1,5 1, pgg pgg Figure 7 Comprison of the effet of pgg nd pgg during therpeuti nti-ovlumin (OVA) immuniztion on the ntitumor tivity. () Survivl rte monitoring fter hllenge. The sterisks indite signifint differenes ompred with nive mie (P <.1) (omprison of survivl urves, Mntel Cox test). (,) Tumor growth follow-up fter hllenge in mie immunized respetively with omined with pgg or pgg nd mie immunized with pgg or pgg lone. The results re expressed s men SEM (n = 1). The sterisks show signifint differenes etween groups (P <.1, P <.1) (nlysis of vrine, Dunnett s post-ho test). immunogeniity nd to hve them trin the immune system to generte potent immune responses. As EP nd the use of geneti djuvnts were shown previously to enhne DNA vine effiy, we investigted whether the EP of plsmid enoding the HIV-1 Gg protein ould inrese ner DNA vine immunogeniity. We showed tht odelivery of pgg y EP enhned the poteny of the immune response ginst the vine ntigens, suggesting strong djuvnt effet of this strtegy. Firstly, we showed tht ell trnsfetion with pgg led to the prodution of VLPs y the ellulr mhinery. This result ws expeted s HIV-1 Gg proteins re responsile for virl psid pgg pgg ssemly. 18 These prtiles re similr in size, omposition, nd struture to intt infetious virus (Figure 1 ). Nevertheless, they do not present ny infetivity risk s they re devoid of the virl genome. 23 Severl studies hve een highlighting the immunomodultory properties of HIV-1 Gg proteins. On the one hnd, HIV-1 Gg VLPs were shown to improve DC mturtion nd T-ell priming. These prtiles effiiently promoted mturtion of DCs, inresing the expression of MHC-I nd II nd ostimultory moleules suh s CD8 nd CD86. They lso indued the nturl killer ells in the mie, whih re known to ply ruil role in the ntitumor response. 24 Another study demonstrted tht HIV-1 VLPs n propgte innte immune signling y pkging GAMP, messenger tht tivtes ntivirl signling pthwys in response to ytosoli sensing of DNA, nd delivering it to uninfeted trget ells. 25 On the other hnd, the host my sense the invding virl protein vi pttern reognition reeptors (PRRs) tht reognize PAMPs. The HIV-1 psid n e deteted vi ylophilin A nd TRIM5 reeptors, tivting innte immune signling pthwys nd susequent DC mturtion. 26,27 It ws lso found tht the p17 nd p24 domins of the HIV-1 Gg protein serve s PAMPs for TLR2 heterodimers, 28 signifintly inresing the innte immune tivtion. Agonists of TLR2 nd TLR3 re promising djuvnts for vine immunotherpy of ner, to sfely enhne ntitumor immunity. 29 Seond, we demonstrted tht EP s delivery method used for DNA vintion indued innte immunity. Indeed, EP of pdna led to the indution of type I IFNs in vivo, wheres EP without pdna or injetion of pdna without EP did not strongly indue the IFN-β promoter (Figure 2). An explntion of this phenomenon my e n inrese in DNA plsmid trnsfetion 7 nd thus higher sensing of errnt ytosoli DNA. In most ses, the reognition of ytosoli DNA results in the indution of the innte immune response through the STING TBK1 signling sde, nd thus the susequent expression of type I IFNs. 13 These ytokines ply ruil role in the indution of protetive ntitumor immune response. 21 Adjuvnt properties of EP hd lredy een highlighted previously in series of studies, showing its pity to trigger 1-fold inrese of ntigen expression 7 ut lso to reruit DCs to the vinted re nd indue lol tissue inflmmtion. 1,1 Here, we reported diret indution of type I IFNs y EP in vivo, whih is shown for the first time to our knowledge. This result supports the ruil role of using EP in DNA vintion protools to further enhne the immune response ginst DNA vine-enoded ntigens. Then, the potentil of pgg to t s n djuvnt in the ontext of DNA vine EP ws investigted. Initil evidene of pgg s djuvnt potentil me from the nlysis of the dptive immune response triggered y the nti-ova DNA vintion. The odelivery of pgg led to ler shift of the nti-ova immune response towrd Th1 polriztion. This finding ws hrterized oth y derese in the totl nti-ova IgG titers nd n inrese in the IgG2/IgG1 rtio, s well s n inrese in ntigenspeifi IFN-γ levels in the presene of pgg nd n improved indution of ntigen-speifi CTLs (Figure 3). This regultion of the Th1/Th2 homeostsis driven y pgg fvored the ellulr immunity nd is therefore more likely to indue effiient elimintion of nerous ells. 3 pgg s ility to inrese DNA vine vol. 24 no. 9 sep. 216

8 The Amerin Soiety of Gene & Cell Therpy HIV-1 Gg Plsmid to Potentite Cner DNA Vine poteny ws further proven s it inresed protetion ginst tumor hllenge. The prophylti immuniztion of mie with n nti-ova DNA vine slowed down the growth of B16F1- OVA tumors nd improved mouse survivl when the ntigeni plsmid ws omined with pgg (Figure 4). Moreover, the effiy of this strtegy ws onfirmed in less immunogeni TAA model y omining pgg with vine oding for the gp1 melnom ntigen. The results indited tht mie developed more potent immune response to the B16F1-OVA tumor only when pgg ws odelivered with pgp1 (Figure 5). In this se, the delivery of the pgp1 enoding plsmid lone did not offer tumor protetion, highlighting gin the importne of the djuvnt plsmid in induing n effetive immune response. Finlly, therpeuti immuniztion of the mie ws tested with the nti- OVA DNA vine model. One gin, tumor growth dely ws oserved only with the omintion of ntigeni nd djuvnt plsmids (Figure 6). These results demonstrted the ility of pgg to regulte dptive immunity, s the ddition of this plsmid promotes Th1 immune response nd, in onsistent wy, improves the protetive immunity ginst tumors. Finlly, the mehnisms lying ehind the immune modultion indued y pgg were further studied. First of ll, the improved vine effiy ws speifi to pgg odelivery nd not due to n inrese of DNA mount used for immuniztions. Indeed, using 1 µg or 1 µg of DNA vine led to similr protetion effiy following B16F1-OVA hllenge (see Supplementry Figure S1). As pgg djuvnt effet ould e driven y the formtion of VLPs in vivo, the requirement of prtiles formtion ws nlyzed. Immuniztion of mie with plsmid enoding mutted form of the HIV-1 Gg protein tht prevents VLP formtion 22 led to similr results thn immuniztion with the plsmid oding for the ntive protein (Figures 7 nd 8). This suggests tht the djuvnt effet is proly medited y immune reognition of the DNA or protein sequene of HIV-1 Gg rther thn y VLPs. For instne the HIV-1 Gg plsmid my ontin CpG motifs tht n e reognized y the TLR9 nd tivte innte immunity. 31 Aout HIV-1 Gg protein, some of its domins were shown to t s PAMPs nd indue innte immunity. In ddition, the protein my ontin MHC-II restrited epitopes tht n indue strong T-helper response. 32 The ntitumor effet is medited y the ntigen-speifi response, s delivery of pgg lone did not offer protetion ginst tumor hllenge fter prophylti immuniztion (Figure 8). Interestingly, pgg seemed to hve n ntitumor effet y itself in therpeuti vintion setting (Figure 7). This supports the importnt role plyed y pgg on immune system meditors. Future work my im t etter hrterizing the mehnisms involved. Tken together, these results support n djuvnt role for pgg when odelivered y EP with tumor ntigen enoding plsmid. This strtegy seemed to effiiently stimulte innte immunity, tking dvntge of oth the ility of DNA EP to stimulte innte immunity nd of the immunomodultory effets indued y pgg. This pproh led to n effiient priming of n dptive immune response nd llowed for the ugmenttion of ner DNA vine effiy. The use of pgg s geneti djuvnt is of prtiulr interest from trnsltionl point of view. Indeed, geneti djuvnts Survivl (%) , 1,5 1, 5 2, 1,5 1, pgg pgg pgg pgg Figure 8 Comprison of the effet of pgg nd pgg during prophylti nti-ovlumin (OVA) immuniztion on the ntitumor tivity. () Survivl rte monitoring fter hllenge. The sterisks indite signifint differenes ompred with nive mie (P <.1) (omprison of survivl urves, Mntel Cox test). (,) Tumor growth follow-up fter hllenge in mie immunized respetively with omined with pgg or pgg nd mie immunized with pgg or pgg lone. The results re expressed s men SEM (n = 6). The sterisks show signifint differenes etween groups (P <.1) (nlysis of vrine, Dunnett s post-ho test). hold higher potentil for enhning DNA vine poteny thn trditionl ones. First, they possess intrinsi djuvnt properties due to their reognition y ytosoli DNA sensors. In ddition, oth ntigens nd djuvnts often need to e delivered t the sme time to the ptient to otin oordinted priming of the immune response. As the ntigeni plsmid of DNA vines is not the physil ntigen ut its oding sequene, it first needs to e expressed in suffiient mounts y the host ells. Beuse mny onventionl djuvnts work immeditely fter injetion, their effet might e lower t the time the ntigen is present. 33 In ontrst, the use of plsmid enoding immunomodultory proteins Moleulr Therpy vol. 24 no. 9 sep

9 HIV-1 Gg Plsmid to Potentite Cner DNA Vine The Amerin Soiety of Gene & Cell Therpy permits the oordinted delivery of ntigens nd djuvnts, tiloring the immune response to the demnds of eh prtiulr disese. 7 Until now, most of the geneti djuvnts used in trils enode proteins tht tivte innte immune responses diretly, suh s ytokines, hemokines, signling or ostimultory moleules. Here, the strtegy is different nd innovtive s pgg most likely indues innte immune responses indiretly, through PAMP reognition y pttern reognition reeptors. Lst ut not lest, HIV-1 Gg hs lredy een involved in linil trils in the ontext of HIV vine development, where it ws shown to e sfe. 17 Therefore, the trnsition from prelinil to linil evlution of this strtegy would e esier. In onlusion, this study supports the djuvnt effet of plsmid EP in vivo. It demonstrtes tht immuniztion with pgg fvors Th1 immunity ginst the DNA vine-enoded ntigen nd onsistently delys tumor urden. This work highlights the powerful djuvnt potentil of pgg for enhning DNA vine poteny nd opens interesting trks for the design of new geneti djuvnts tht ould e used in linil setting in n ttempt to overome the urrent limittions of DNA vines in humns. MATERIALS AND METHODS Plsmids. Full length nd odon-optimized gene sequenes (see Supplementry Mterils nd Methods) of HIV-1 Gg, OVA, nd humn gp1 were designed using GeneOptimizer nd otined y stndrd gene synthesis from GeneArt (Thermo Fisher Sientifi, Wlthm, MA). These sequenes were suloned in the pvax2 vetor s previously desried. 34 A mutnt version of the HIV-1 Gg enoding plsmid ws otined y introduing three point muttions (lnine muttions t the residues G 2, W 316, nd M 317 ) in the gene sequene using the QuikChnge Multi Site- Direted Mutgenesis Kit (Agilent Tehnologies, Snt Clr, CA) nd following the mnufturer s protool. The primers used were (Primer Nme Primer Sequene) g35 5ʹ-gtgggtggt-3ʹ for G2A muttion nd t976g_g977_979g_t98 5ʹ-ggtgtttgtg ggtttttttggtg-3ʹ for W316A nd M317A muttions. The mutted sequene ws heked y Snger DNA sequening (Bekmn Coulter Genomis, Dnvers, MA). The plsmids were prepred using the EndoFree Plsmid Gig Kit (Qigen, Venlo, Netherlnds) ording to the mnufturer s protool. Plsmid dilutions were performed in Duleo s PBS (1 ) (Life Tehnologies, Crlsd, CA). The qulity of the purified plsmid ws ssessed y the rtio of optil densities (26 nm/28 nm) nd y.5% grose gel eletrophoresis. DNA onentrtion ws determined y optil density t 26 nm. The plsmids were stored t 2 C. Cell ulture. B16F1-OVA, melnom ell line from C57BL/6 mie tht stly expresses OVA, ws ultured in MEM medium supplemented with GlutMAX with 1% fetl ovine serum, 1 μg/ml streptomyin, nd 1 U/ml peniillin (Life Tehnologies). HEK 293 T ells were grown in omplete DMEM medium supplemented with GlutMAX (with 1% fetl ovine serum, 2 mmol/l l-glutmine, 1 μg/ml streptomyin, nd 1 U/ml peniillin) (Life Tehnologies). VLP prodution. In totl, HEK 293 T ells were ultured for 2 dys in T75 flsks (Sigm-Aldrih, St. Louis, MO) nd then trnsfeted with Lipofetmine 2 following the mnufturer s instrutions (Invitrogen, Crlsd, CA). Seventeen mirogrms of pgg nd 58 µl of lipofetmine were used for lipofetion. The medium ws repled 24 hours fter trnsfetion, nd the superntnts were olleted t dys 2, 3, nd 4 posttrnsfetion. Cellulr deris ws removed y low-speed entrifugtion nd y filtrtion through.45 μm filter. Filtered superntnts were then ultrentrifuged (5,g, 2.5 hours, 4 C) through 2% surose. Purified VLPs were suspended in 5 μl PBS, stored t 4 C overnight, nd pled t 8 C for long-term storge. The totl protein ontent of eh preprtion ws determined y Miro BCA ssy ording to the mnufturer s instrution (Thermo Fisher Sientifi). VLP hrteriztion. Prtile size distriution ws determined y dynmi light sttering using NnoSizer ZS (Mlvern Instruments, Mlvern, UK). Purified smples were diluted 8 times in PBS, nd the mesurement ws performed in triplite. The dt were nlyzed using the Dispersion Tehnology Softwre 5.. For prtile imging y trnsmission eletron mirosopy, the smples were dsored onto 4 mesh formvr-oted EM grids (Ted Pell, Redding, CA) for 1 minutes nd then wshed three times with distilled wter. Exess liquid ws removed using filter pper, nd the negtive ontrst ws otined y pplying 2% phosphotungsti id (Sigm-Aldrih) t ph 7 for 2 minutes. The grids were ir dried for 1 minutes nd then exmined in LEO 922 eletron mirosope with CCD mer. Western lot nlyses were performed on 1 µg of protein from the purified VLP smples. The proteins were dentured in 2 Lemmli Smple Buffer for 7 minutes t 95 C. After loding the smples on Mini-PROTEAN TGX Prest Gels 4 2% (Bio-Rd, Herules, CA), eletrophoresis ws performed t 3 V for 15 minutes using 1% Tris-glyin.1% sodium dodeyl sulfte running uffer (Sigm-Aldrih). The proteins were then trnsferred to nitroellulose memrne using the trns-blot Turo Trnsfer System following the mnufturer s instrution (Bio-Rd). After loking, primry rit polylonl ntiodies to HIV-1 Gg nd OVA (Fitzgerld, Aton, MA) were dded, ll diluted 1:2,, for n hour. Biotinylted nti-rit seondry ntiodies (Vetor Lortories, Cmridgeshire, UK) t 1:1, were dded for 1 hour nd followed y streptvidin horserdish peroxidse for 2 minutes. Visuliztion ws performed with 5:5 ECL Western Blotting Sustrte nd SuperSignl West Femto Mximum Sensitivity Sustrte solution (Thermo Sientifi). Animls. Six-week-old C57BL/6 femle mie were otined from Jnvier Ls (Le Genest Sint Isle, Frne) nd housed in miniml disese fility with d liitum ess to food nd wter. For the immuniztion studies, the mie were 7 weeks old t the eginning of the experiments. For tumor implnttion nd EP, the mie were nesthetized y intrperitonel (i.p.) injetion of 15 µl of solution of 1 mg/ml ketmine nd 1 mg/ml xylzine. Heterozygous luiferse reporter mie (IFN-β+/Δβ-lu) with BALB/ kground were red with d liitum ess to food nd wter. Mle nd femle mie were ged 12 weeks t the eginning of the experiments. For imging, the mie were nesthetized y isoflurne (Aott Animl Helth; Medini NV). The ethil ommittee for Animl Cre nd Use of the Medil Setor of the Université Ctholique de Louvin pproved our experimentl protools (UCL/MD/211/7). Type 1 IFN indution: in vivo ioluminesene imging. Heterozygous luiferse reporter mie were nesthetized y i.p. injetion of 15 µl of solution of 1 mg/ml ketmine nd 1 mg/ml xylzine. After removing the hir using rodent shver (AgnTho s, Lidingö, Sweden), the left tiil rnil musle ws injeted with 3 µl of PBS or 3 µl of empty plsmid DNA solution diluted in PBS. This injetion ws followed or not y EP. For EP, ondutive gel ws pplied on the leg to ensure eletril ontt with the skin (EKO-GEL, ultrsound trnsmission gel, Egn, Itly) nd eight 2 ms nd 2 V/m eletril pulses were delivered through 4 mm sped plte eletrodes y Cliniportor system with frequeny of 2 Hz (Cliniportor, IGEA, Crpi, Itly). For in vivo ioluminesene imging, the mie were injeted i.p. with 15 mg/kg of VivoGlo luiferin (Promeg, Fithurg, WI) in PBS nd monitored using n IVIS Lumin II imging system. Photon flux ws quntified using the Living Imge 4.4 softwre (ll from Perkin Elmer, Wlthm, MA). Mouse immuniztion. C57BL/6 mie were shved, nd their left tiil rnil musle ws injeted with plsmid DNA solution. The solutions vol. 24 no. 9 sep. 216

10 The Amerin Soiety of Gene & Cell Therpy HIV-1 Gg Plsmid to Potentite Cner DNA Vine were omposed of 1 µg or 5 µg of ntigeni OVA or GP1 plsmid lone or with 1 µg HIV-1 Gg enoding plsmid. One mirogrm of plsmid oding for mutted form of HIV-1 Gg ws lso used for immuniztion, omined or not with 1 µg of. A ondutive gel ws pplied, nd EP ws performed s previously desried. The plsmids were dministered three times, every week or every 2 weeks for therpeuti nd prophylti DNA immuniztion, respetively. Tumor implnttion nd tumor growth mesurement. In totl, B16F1-OVA ells diluted in 1 μl PBS were injeted suutneously into the right flnk of eh C57BL/6 mouse 2 dys efore the first plsmid dministrtion or 2 weeks fter the lst dministrtion for therpeuti nd prophylti DNA immuniztion studies, respetively. The tumor size ws mesured three times week with n eletroni digitl lliper. Tumor volume ws lulted s the length width height (in mm 3 ). The mie were srified when the volume of the tumor rehed 1,5 mm 3 or when they were in poor ondition nd expeted to die shortly. Immunogloulin titers. Ten dys fter the lst plsmid dministrtion, lood smples were olleted nd ser were isolted y entrifugtion t 3,7g for 15 minutes t 4 C nd stored t 2 C until use. OVA-speifi IgG, IgG1, nd IGg2 levels were determined y enzyme-linked immunosorent ssy s previously desried. 35 Briefly, OVA ws oted on Nun Mxisorp pltes. Severl ser dilutions were mde, nd the detetion of nti-ova ntiodies ws performed using peroxidse-leled rt ntimouse immunogloulin G, G1, nd G2 (LO-IMEX, Brussels, Belgium). IgG titers were defined s the logrithm of the inverse of the ser dilution orresponding to n sorne equl to the lnk vlue plus 1 times the stndrd devition. Antigen-speifi IFN-γ levels. Mie were immunized nd killed 2 weeks fter the third vine dministrtion. The spleens were removed septilly, nd splenoytes were isolted s previously desried. 36 Then, 96-well pltes were seeded with ells in 1 μl of medi per well. The ells were stimulted y dding 1 µl of 1 µg/ml of OVA solution (Sigm- Aldrih). The pltes were inuted t 37 C in humidified inutor t 5% CO 2. The superntnts were olleted fter 24, 48, nd 72 hours of restimultion, nd ytokine prodution ws ssessed using DuoSet ELISA Development kits for IFN-γ (R&D Systems, Aingdon, UK) ording to the mnufturer s instrutions. The onentrtions, expressed in pg/ml, were determined y referene to ytokine stndrd urves. In vivo ytotoxiity ssy. Splenoytes from femle C57BL/6 mie were pulsed with 1 µg/ml of MHC-I OVA peptide or MHC-I gp1 peptide (Anspe) efore leling with 5 µmol/l (hi) or.5 µmol/l (low) CFSE (Invitrogen, Mereleke, Belgium), respetively. Lelled ells were mixed t 1:1 rtio, nd totl of ells were doptively trnsferred into the immunized mie. Two dys fter trnsfer, the spleens of the host mie were isolted nd nlyzed y flow ytometry fter stining with α-f4/8 (BD Biosienes, Sn Diego, CA) to exlude uto-fluoresent mrophges. The perentge ntigen-speifi killing ws determined using the following formul: 1 1 ((% CFSE hi ells/% CFSE low ells) immunized mie /(% CFSE hi ells/% CFSE low ells) nonimmunized mie ). 37 Sttistil nlysis. The results re presented s the mens ± SEM. The differenes in mens etween groups were nlyzed for signifine using pproprite tests (GrphPd Prism Softwre). Nonprmetri tests were used to study type I IFN indution, ntiody titers, INF-γ onentrtion, nd OVA-peptide-pulsed-trget ells speifi killing s four to six mie per group were used in those experiments. Survivl urves were ompred vi Mntel Cox (log-rnk) test. Finlly, repeted mesures nlysis of vrine ws pplied for B16F1-OVA tumor growth nlysis, ssuming Gussin distriution of the dt for tht tumor model. SUPPLEMENTARY MATERIAL Figure S1. The effet of dose during prophylti nti-ova immuniztion on the ntitumour tivity. Supplementry Mterils nd Methods ACKNOWLEDGMENTS L.L. is reserh fellow nd G.V. is postdotorl reserher of the Fonds de l Reherhe Sientifique-FNRS, Belgium. The uthors would like to thnk Srin El Bhiri for her help with the sttistil nlysis. N.N.S. knowledges funding from Reserh Fund Flnders (FWO, grnt no. G.621.1) nd Bijzonder Onderzoeksfonds (BOF) from Ghent University. The uthors delre no onflit of interest. Referenes 1. Clvet, CY, Andre, FM nd Mir, LM (214). Dul therpeuti enefit of eletroportion-medited DNA vintion in vivo: enhned gene trnsfer nd djuvnt tivity. Onoimmunology 3: e Donnelly, JJ, Whren, B nd Liu, MA (25). DNA vines: progress nd hllenges. J Immunol 175: Brumüller, H, Wieder, T, Brenner, E, Aßmnn, S, Hhn, M, Alkhled, M et l. (213). T-helper-1-ell ytokines drive ner into senesene. Nture 494: Rosenerg, SA, Ynnelli, JR, Yng, JC, Toplin, SL, Shwrtzentruer, DJ, Weer, JS et l. (1994). 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