A Multi-DNA Preventive Vaccine for p53=Neu-DrivenCancer Syndrome

C. De Giovanni,1 G. Nicoletti,2 A. Palladini,1 S. Croci,1 L. Landuzzi,2 A. Antognoli,1

A. Murgo,1 A. Astolfi,1 S. Ferrini,3 M. Fabbi,3 A.M. Orengo,3 A. Amici,4 M.L. Penichet,5

L. Aurisicchio,6 M. Iezzi,7 P. Musiani,7 P. Nanni,1 and P.-L. Lollini8

Abstract

The highly aggressive cancer syndrome of female mice carrying a p53 knockout allele and a rat HER-2=neu(Neu) transgene (BALB-p53Neu) can be prevented by a cell vaccine presenting three components: Neu, inter-leukin (IL)-12 production, and allogeneic major histocompatibility complex (MHC) alleles (Triplex cell vaccine).Here we tested a second-generation Triplex DNA-based vaccine (Tri-DNA), consisting of the combination ofthree gene components (a transmembrane–extracellular domain fragment of the Neu gene, IL-12 genes, and theH-2Dq allogeneic MHC gene), carried by separate plasmids. The Tri-DNA vaccine was at least as effective as theTriplex cell vaccine for cancer immunoprevention, giving a similar delay in the onset of mammary cancer andcomplete protection from salivary cancer. Both vaccines induced anti-Neu antibodies of the murine IgG2aisotype at similar levels. The Tri-DNA vaccine gave more restricted immunostimulation, consisting of a fullyhelper T cell type 1 (Th1)-polarized response, with effective production of interferon (IFN)-g in response to thevaccine but no spontaneous production, and no induction of anti-Neu IgG3 antibodies. On the other hand, theTriplex cell vaccine induced both Th1 and Th2 cytokines, a strong increase in spontaneous IFN-g production, andhigh levels of IgG3 antibodies recognizing Neu-positive syngeneic cells. In conclusion, the Tri-DNA vaccine is aseffective as Triplex cell vaccine, exploiting a more restricted immune stimulation.

Introduction

Cancer immunoprevention aims at inducing prophy-lactic immune responses in healthy individuals at risk of

cancer (Lollini et al., 2006; Ryan et al., 2007). The most obviousapplication concerns cancer caused by infectious agents, buteffective immunoprevention obtained against gene-drivencarcinogenesis in transgenic mice suggested the possibility toapply immunopreventive strategies to hereditary tumors. Thepotential applications of immunoprevention, however, couldfurther expand to the prevention of common types of cancer,provided that suitable target antigens (also named oncoanti-gens) are identified and side effects avoided (Lollini and Forni,2003). Among transgenic models so far investigated, pre-vention of tumors by immunological means has been proven

to halt or significantly delay mammary carcinogenesis drivenby rat HER-2 (Neu). Various immunological stimuli wereadministered to healthy mice to prevent the onset of tu-mors (Lollini et al., 2006). One of the most effective im-munopreventive approaches consisted of a Neu-positiveallogeneic cell vaccine combined with systemic or transducedinterleukin (IL)-12, known as the Triplex vaccine (Nanni et al.,2001; De Giovanni et al., 2004). We demonstrated that lifelongvaccination with both the Neu antigen and the adjuvants(allogenicity and IL-12) determined an almost complete im-munoprevention. In a translational view, however, an allo-geneic cell vaccine to be administered lifelong could haveseveral problems, starting with logistics (goodmanufacturingpractice-certified cell culture facility, storage, and distri-bution) and including eligibility (depending on patient’s

1Cancer Research Section, Department of Experimental Pathology, Alma Mater Studiorum, University of Bologna, I-40126 Bologna, Italy.2Laboratory of Oncologic Research, Rizzoli Orthopedic Institute, I-40136 Bologna, Italy.3Istituto Nazionale per la Ricerca sul Cancro, I-16132 Genoa, Italy.4Department of Molecular, Cellular, and Animal Biology, University of Camerino, I-62032 Camerino, Italy.5Division of Surgical Oncology, Department of Surgery, and Department of Microbiology, Immunology, and Molecular Genetics, Jonsson

Comprehensive Cancer Center, David Geffen School of Medicine, University of California at Los Angeles (UCLA), Los Angeles, CA 90095.6Istituto di Ricerche di Biologia Molecolare (IRBM), I-00040 Pomezia, Italy.7CESI Aging Research Center, G. D’Annunzio University, I-66013 Chieti, Italy.8Department of Hematology and Oncologic Sciences, L., and A. Seragnoli, Alma Mater Studiorum, University of Bologna, I-40138 Bologna,

Italy.

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histocompatibility profile) and safety. Moreover, we demon-strated that all the components previously listed are neces-sary, because lack of any caused a fall in efficacy (Nanni et al.,2001; De Giovanni et al., 2004), but we never investigatedwhether they are also sufficient. In fact, it is conceivable that acell vaccine carries a portfolio of other immune stimuli, someof which could cause side effects. An optimized vaccineshould avoid unnecessary immune stimulation and givemaximal effects with the simplest schedule.

DNA vaccines could offer advantages in terms of feasibilityand safety: In fact, several anticancer therapeutic DNA vac-cines have already entered clinical testing, and are well tol-erated (Rice et al., 2008). Electroporation greatly increases theefficacy of DNA vaccines administered by the intramuscularroute (Fattori et al., 2002; Rice et al., 2008). The central role inDNA vaccination played by antigen-presenting cells throughdirect antigen presentation and cross-priming has beenclearly demonstrated, but electroporation seems also to elicitdirect presentation by transfected muscle cells (Shirota et al.,2007). It has been proposed that plasmidDNA-based vaccineshave intrinsic helper T cell type 1 (Th1)-inducing adjuvantproperties due to the presence of unmethylated CpG se-quences (Krieg et al., 1998), which mediate production of Th1-inducing cytokines via Toll-like receptor (TLR)-9 (Hemmiet al., 2000). In addition, it was reported that double-strandedDNA, delivered by electroporation, has an adjuvant effect initself through TANK-binding kinase-1 and interferon (IFN)regulatory factor-3-dependent innate activation of both im-mune and nonimmune cells to produce type I interferon andtheir inducible genes (Ishii et al., 2008). A cytosolic DNAsensor was found to activate these pathways (Wang et al.,2008).

Electroporated Neu DNA vaccines gave interesting resultsin transgenic murine models (Quaglino et al., 2004; Pupa et al.,2005; Jacob et al., 2006; Cho et al., 2008) and are being studiedin clinical trials in therapeutic approaches (Wei et al., 2008).We therefore designed a second-generation Triplex DNAvaccine (Tri-DNA) based on the combination of three plas-mids, each carrying one of the required components (Neu,IL-12, and an allogeneic histocompatibility gene), to be ad-ministered by intramuscular injection and electroporation. Totest both the immune response elicited by Tri-DNA vaccineand its cancer-preventive efficacy in comparison with Triplexcell vaccine, we choose a model system able to outline eitherbetter or worse effects. Mice knocked out for a p53 allele andtransgenic for Neu (BALB-p53Neu mice) quickly develop acancer syndrome, consisting of salivary and mammary tu-mors (in females) and of rhabdomyosarcoma and salivarytumors (in males). Early administration of the Triplex cellvaccine gave high prevention of both salivary and mammarytumors (Croci et al., 2004). We therefore compared Tri-DNAand Triplex cell vaccination in BALB-p53Neu females, usingshort vaccination schedules (5 to 14 weeks of age), and foundthat the Tri-DNA vaccine is as effective as Triplex cell vaccine,and can avoid for unnecessary immune stimulation.

Materials and Methods

Mice and cell lines

Mice knocked out for the p53 oncosuppressor gene andtransgenic for a mutant rat HER-2=neu oncogene driven by amouse mammary tumor virus (MMTV) long terminal repeat

(LTR) (hereafter referred to as BALB-p53Neu) were obtainedas reported (Croci et al., 2004). Parental BALB=c p53!=– mice(BALB=cJ-Trp53tm1Tyj) were purchased from Jackson Labora-tory (Bar Harbor, ME). BALB=c Neu-transgenic mice (BALB-neuT)were bred in our animal facilities (Nanni et al., 2001). Allof the experiments with animals fulfilled institutional guide-lines and were approved by the institutional committee foranimal use and care. Neu-positive TT12.E2 cells (here referredto as Neu=H-2q cells) and Neu-negative N202.1E cells (herereferred to as Neuneg=H-2q cells) derived from mammarycarcinomas arisen in FVB-NeuN#202 mice, transgenic for therat neu proto-oncogene (Nanni et al., 2000). Neu-positiveTUBO cells (here referred to asNeu=H-2d cells) derived from amammary carcinoma arisen in BALBneuT mice (Rovero et al.,2000). IL-12-engineered TT12.E2 cells (here referred to asNeu=H-2q=IL12) were obtained and described previously (DeGiovanni et al., 2004).

Plasmids

Plasmid pcDNA3 encoding a transmembrane–extracellulardomain fragment of rat HER-2=neu (here referred to as pNeu)has been described previously (Quaglino et al., 2004). Plasmidencoding both murine IL-12 genes (pIL12) was bicistronicpIL12-IRES1neo (De Giovanni et al., 2004; Faggioli et al., 2008);it induced a high IL-12 expression level when used to obtainstable IL-12-engineered cells (De Giovanni et al., 2004). Tocheck its ability to express IL-12 in vivo, we performed intra-muscular injection of 50mg of pIL12 followed by electro-poration (see below for conditions) and collected sera atvarious time points thereafter. Empty vector pIRES1neo wasused under the same experimental conditions. The presence ofIL-12 and IFN-g in sera was assessed through enzyme-linkedimmunosorbent assays (ELISAs) (R&D Systems,Minneapolis,MN).

To obtain a plasmid encoding an allogeneic MHC gene, wechose a gene of the same haplotype carried by the Triplex cellvaccine (H-2q, FVB=N strain) and cloned H-2Dq cDNA fromvarious sources (lymphocytes and tumor cells). Briefly, cellswere lysed in TRIzol (Invitrogen, Milan, Italy) and total RNAwas isolated with the RNeasy system (Qiagen, Chatsworth,CA). The open reading frame of H-2Dq was amplified by RT-PCR from total RNA, using the following primers: forward,50-CGATGGCTCCGCGCACGCTGCTC-30 and reverse, 50-CAGTCCAGGCAGCTGTCTTCACGC-30. H-2Dq cDNA wascloned in the PCR2.1 vector, using the TOPO-TA cloningsystem (Invitrogen), sequenced, and then subcloned into thepcDNA3 expression plasmid (Invitrogen): this plasmid ishereafter referred to as pDq. Sequencing of the various H-2Dq

cDNA clones obtained showed almost complete homologywith a reported Dq sequence (Pullen et al., 1992), with onlytwo mismatches leading to amino acid changes (CAG=AGGat codon 69 leading to a Q-to-R amino acid change, andCGC=GCC at codon 104 leading to an A-to-R change). Be-cause of the consistency of our sequences in different clones,mismatches can be interpreted either as mistakes in the pre-viously published sequence (Pullen et al., 1992) or as substrainvariations.

Transfection

To verify the ability of pDq to transfer H-2Dq expression, avariant of B16 melanoma (B78H1) was chosen, because it is a

454 DE GIOVANNI ET AL.

good transfection recipient and does not express MHC mol-ecules (Graf et al., 1984; De Giovanni et al., 1991). B78H1 cellswere seeded at the concentration of 0.25"106 cells per well in6-well plates (Falcon; BDBiosciences, Bedford,MA) and left toattach for 24 hr. pDq (2mg=well) was then transfected withLipofectamine 2000 (Invitrogen) at a 1:2.5 ratio (DNA [mg]:Lipofectamine [ml]) according to the manufacturer’s protocol.Two days after transfection H-2Dq expression was evaluatedon harvested cells, through indirect immunofluorescence andflow cytometry, using the monoclonal antibody 28-14-8S(Ozato et al., 1982) (Cedarlane Laboratories, Hornby, ON,Canada).

Complement-dependent cytotoxicity

BALB=c mice (H-2d) at 5 and 7 weeks of age were vacci-nated by intramuscular injection of pDq plasmid into the tibialmuscles (25 mg in 20ml, each muscle) followed by electro-poration (according to the same protocol reported below forDNA vaccination). One week after the second injection, pDq-vaccinated mice were bled and serum was stored at #808C.Aliquots of 0.5"106 lymphocytes, collected from BALB=c (H-2d) and FVB-NeuN#202 (H-2q) mice, were resuspended inRPMI! 5% fetal bovine serum (FBS) containing serum frompDq-vaccinated and untreated BALB=c mice (serum diluted1:10 in a final volume of 0.5ml) and incubated for 30min inice. A baseline control in RPMI! 5% FBS medium and apositive control, consisting of 28-14-8s monoclonal antibody(recognizing both H-2q and H-2d epitopes) at 1:10 dilution,were run in parallel. Lymphocytes were then washed oncewith phosphate-buffered saline (PBS) and incubated with a1:10 dilution of rabbit complement (Cedarlane Laboratories)for 30min in ice. Lymphocytes were then washed again andresuspended in RPMI! 5%FBS, and cell number and viabilitywere determined by erythrosin dye exclusion test. The per-centage of cytotoxicity was calculated as follows: 100# (cellyield in complement-containing RPMI! 5% FBS=cell yieldwith serum)"100.

DNA and cell vaccinations

Plasmid large-scale preparations were set up and purifiedwith Endofree Qiagen Plasmid-Giga kits (Qiagen). DNA wasprecipitated, dissolved in water at a concentration of 3–5mg=ml, and stored in monodose aliquots at#208C for use inimmunization protocols. For the Tri-DNA vaccine, 50-mgamounts of each plasmidweremixed together and diluted to afinal volume of 40ml per mouse in 0.9% NaCl and poly-glutamate (6mg=ml) (final concentrations). Groups treatedwith a combination of two plasmids or with pNeu alone re-ceived 50mg of each plasmid. Anesthetized mice received theinjection of DNA vaccine into the tibial muscles (20ml in eachmuscle) through a 28-gauge needle syringe and immediatelythereafter were subjected to electroporation, consisting of twosquare-wave 25-msec, 375-V=cm pulses generated with a T830electroporator (BTX, San Diego, CA). Mice received four vac-cinations (at 5, 7, 12, and 14 weeks of age). The control groupreceived mock vaccinations as described previously, butlacking plasmids. Mice were monitored weekly for mammaryor salivary tumor onset.

Triplex cell vaccine consisted of Neu-positive IL-12-engineered allogeneic cells (Neu=H-2q=IL12). Cells and

vaccine schedule were previously reported (Croci et al., 2004;De Giovanni et al., 2004), with the exception that only thefirst three vaccination cycles were performed (from 5 to 14weeks of age). Mice were then monitored as describedpreviously.

Cytokine production and cell-mediated cytotoxicity

Mice were killed 1 week after the first cycle of vaccinationand spleens were collected. Total spleen mononuclear cells, orsubpopulations (CD4! or CD8!) purified bymagnetic sorting(Miltenyi Biotec, Bergisch Gladbach, Germany), were cul-tured for 6 days alone or in the presence of proliferation-blocked restimulator cells (at a 10:1 lymphocyte:tumor cellratio) in RPMI 1640 supplemented with 10% fetal bovine se-rum and with recombinant IL-2 (20 units=ml), as described(Nanni et al., 2001). Culture supernatants were collected andconcentrations of IL-4, IL-5, IL-10, IL-12(p70), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-g, andtumor necrosis factor (TNF)-a were simultaneously deter-mined with the Bio-Plex 200 suspension array system(Bio-Rad, Milan, Italy). A commercially available premixedmultiplex cytokine set was used (mouse cytokine Th1=Th2panel) in accordancewith themanufacturer’s instructions andat a high-sensitivity setting. Briefly, 50-ml volumes of super-natants and serial dilutions of the cytokine standards wereincubated at room temperature for 30min with a mix of dif-ferent fluorescently dyed beads conjugated with monoclonalantibodies specific for the various cytokines. Afterwardsamples were incubated for 30min with 25ml of detectionantibody and then with 50 ml of streptavidin–phycoerythrin(PE) for 10min. Each step was followed by three washings.Finally, samples were resuspended in 125ml of assay bufferand analyzed with the Bio-Plex reader. A 50-ml volume wassampled from each well and the PE-fluorescent signal of aminimum of 100 beads per cytokine type was evaluated. Beaddoublets were excluded from analysis. Data were elaboratedwith Bio-Plex Manager Software 5.0. Extrapolating PE-fluo-rescent signals to a standard curve allowed quantitation ofeach cytokine in the samples. The experimental ranges ofdetection of the various cytokines were as follows: IL-4, 2100–0.2 pg=ml; IL–5, 500–0.1 pg=ml; IL-10, 1100–1 pg=ml; IL-12,1100–1pg=ml; GM-CSF, 500–2pg=ml; IFN-g, 1400–0.3pg=ml;TNF-a, 3700–3pg=ml. Production of murine IL-4 and IFN-gwas also determined in parallel by ELISAs (Endogen, Wo-burn, MA) with similar results. For the cytotoxicity assayspleen mononuclear cells of mice that had completed thevaccination cycles (15 weeks old) were restimulated by co-culture at a 50:1 ratio with proliferation-blocked Neu=H-2d

cells for 6 days in RPMI 1640 supplemented with 10% fetalbovine serum and recombinant IL-2 (20 units=ml). The abilityof restimulated lymphoblasts to lyse Neu=H-2d or Neu=H-2q

tumor cells was evaluated in a standard 51Cr release assay asdescribed (De Giovanni et al., 2004).

Antibody response

Mice were routinely bled via the tail vein and serum sam-ples were stored frozen at #808C. Production of antibodieswas then studied both by ELISA and by indirect immuno-fluorescence of syngeneic and allogeneic Neu-positive cells.An ELISA to detect antibodies recognizing rat HER-2 was

DNA VACCINES FOR p53/Neu CANCER PREVENTION 455

performed in Maxisorp NUNC 96-well microplates as re-ported (Cipriani et al., 2008). Plates coated with rat Neu wereincubated overnight with serum at 1:300 dilution, washed,incubated with alkaline phosphatase-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO) for30min at room temperature, washed again, and developedwith p-nitrophenyl phosphate (Sigma-Aldrich). A standardcurve with anti-c-ErbB2=Neu mouse monoclonal antibody(clone 7.16.4; EMD Biosciences, San Diego, CA) was run inparallel (0.10–250 ng=ml). For immunofluorescence studies,Neu=H-2d and Neu=H-2q cells were incubated with sera at a1:65 dilution for 30min in ice, and thenwashed and incubatedwith an Alexa Fluor 488 F(ab0)2 fragment of goat anti-mouseIgG (H-L) chains antibody (Invitrogen Molecular Probes,Eugene, OR) to evaluate total immunoglobulin binding. Forisotype subclass analysis, the following secondary fluoresceinisothiocyanate (FITC)-conjugated monoclonal antibodieswere used (BD Biosciences): anti-mouse IgG1 clone A85-1,anti-mouse IgG2a clone R19-15, anti-mouse IgG2b clone R12-3, and anti-mouse IgG3 clone R40-82. Cytofluorometricanalysis was performed with a FACScan flow cytometer (BDBiosciences). Rat HER2=neu expression of target cells wasevaluated in each set of experiments with anti-c-ErbB2=Neumouse monoclonal antibody. Data from each sample werenormalized over target cell expression.

Histology and immunohistochemistry

For histologic evaluation, tissue samples were fixed in 10%neutral-buffered formalin, embedded in paraffin, sectioned at4 mm, and stained with hematoxylin and eosin (H&E). Forimmunohistochemistry, paraffin-embedded sections wereimmunostained with anti-c-erbB-2 (Dako, Milan, Italy). Afterwashing, sections were overlaid with biotinylated anti-rabbitimmunoglobulin ( Jackson ImmunoResearch Europe, Suffolk,UK ) for 30min. Unbound immunoglobulin was removed bywashing, and slides were incubated with ready-to-use per-oxidase streptavidin (Lab Vision, Bio-Optica Milan, Milan,Italy) and diaminobenzidine (Dako).

Statistical analysis

The Mantel–Haenzel test was used to compare tumor la-tency Kaplan–Meier curves; the Student t test or Wilcoxonnonparametric test was used for other comparisons.

Results

Plasmid validation

We checked the ability of the plasmid encoding murine IL-12 (pIL12) to give rise to cytokine production in vivo. Micereceived an intramuscular injection of 50 mg of pIL12 andelectroporation under the same conditions chosen for vacci-nation, and then were bled at several time points. A rise inserum IL-12 (and secondary induction of serum IFN-g) fol-lowed pIL12 injection and remained at detectable levels forsome days (Fig. 1A). Similar data have been reported in theliterature (Faggioli et al., 2008). Serum IL-12 or IFN-g levelswere undetectable in untreated mice as well as in mice treatedwith empty pIRESneo, pNeu, and pDq vectors at up to150 mg=mouse administered via intramuscular injection pluselectroporation (data not shown). Transfection of a MHC-negative cell line with plasmid encoding H-2Dq (pDq) gave

rise to cells with surface expression of H-2Dq (Fig. 1B). In-tramuscular injection of pDq followed by electroporation inH-2dmice determined complement-dependent cytotoxic activitytoward H-2q, but not H-2d, lymphocytes (Fig. 1C). ThereforepDq plasmid is able to transfer H-2Dq expression and to elicit aspecific antibody response. Plasmid encoding a transmembrane–extracellular domain fragment of rat HER-2=neu (pNeu), al-ready used as a single DNA vaccine in our work and in otherstudies reported in the literature (Rovero et al., 2000; Quaglinoet al., 2004; Pannellini et al., 2006; Cho et al., 2008), inducedeffective anti-Neu antibody responses in Neu-transgenicmice.

Immunoprevention of cancer syndromethrough DNA vaccines

Tri-DNA vaccine consisted of the combination of the threeplasmid vectors pNeu, pIL12, and pDq (also referred to aspNeu!pIL12!pDq). BALB-p53Neu female mice receivedvaccinations with Tri-DNA at 5, 7, 12, and 14 weeks of age, incomparison with Triplex cell vaccine administered in thesame time window (Fig. 2). Groups of mice were also vacci-nated with DNA vaccines lacking allogeneic MHC gene orboth adjuvants (referred to as pNeu!pIL12 and pNeu, re-spectively). Vaccinated mice were observed weekly, with re-cording of the occurrence of the two types of cancer typical ofsuch a model, that is, salivary and mammary cancer. BALB-p53Neu females, either untreated (Croci et al., 2004) or sub-jected to mock vaccination and electroporation (Fig. 2, dashedline), showed a fast and almost simultaneous occurrence ofsalivary andmammary cancers (with latencies of 13–15weeksfor the former and slightly longer for the latter). All the vac-cines studied, both DNA based and cellular, induced highlysignificant delays in the onset of both types of tumors. Vac-cinated mice reached a median tumor-free survival time of45–55 weeks for mammary cancer. A 90–100% prevention ofsalivary tumors was obtained with Triplex cell and Tri-DNAvaccines. Overall survival for the different groups and therelative frequency of the two types of tumors at sacrifice areshown in Fig. 3. Almost all untreatedmice developed salivarytumors, rapidly arising and leading to death (see also Fig. 4A–D), and in about half of the cases combined with mammarytumors (Fig. 3, bottom). In vaccinated mice the situation wasalmost reversed: Salivary tumors rarely occurred alone evenin mice treated with the simplest DNA vaccines. In particular,with the Tri-DNA vaccine salivary cancer was fully preventedand mouse survival was determined by mammary carcinomaonset. Similar results were obtained with the Triplex cellvaccine. Tri-DNA-vaccinated mice in fact showed morpho-logically normal salivary glands with well-defined ducts andacini that were enclosed in delicate stromal tissue (Fig. 4E).On-going inflammatory aspects or inflammation effects (e.g.,scars) were not detected. Neu expression was not found insalivary glands from Tri-DNA-vaccinated mice (Fig. 4F), al-though it was observed in hyperplastic ducts before vacci-nation (Fig. 4B). On the other hand, Tri-DNA-vaccinated miceshowed several Neu-positive mammary cancer nodules withinflammatory cells in their stroma (Fig. 4G and H).

Because key mechanisms of the immunopreventive effectin Neu transgenic mice rely on IFN-g production and anti-bodies (Nanni et al., 2004), we focused on thesemechanisms tocompare multi-DNA and cell vaccines.

456 DE GIOVANNI ET AL.

Cytokine production and cell-mediated cytotoxicity

To study how the various vaccines drive the cellular im-mune responses, we studied the cytokine production profile ofvaccinated mouse spleen cells cultured alone (spontaneousproduction) or on in vitro restimulation (Fig. 5A). The Triplexcell vaccine induced high production of IFN-g, already evidentwithout in vitro restimulation, but at the same time inductionof IL-4 together with other Th2 cytokines such as IL-5 and IL-10was observed. Spleen cells from the Tri-DNAvaccine groupshowed significant IFN-g production only on restimulationwith cells presenting allogeneic MHC alone or combined withNeu expression. Spleen cells from the other DNA vaccinegroups (pNeu!pIL12 and pNeu alone) as well as those frommock-electroporated control mice (data not shown) showedbarely detectable spontaneous cytokine production levels;in vitro stimulation did not increase IFN-g or IL-4.

The production of IFN-g and IL-4 by CD4! and CD8!

populations was also studied (Fig. 5B). Triplex cell vaccineelicited both IFN-g- and IL-4-producing CD4! cells; in vitrorestimulation with Neu-negative allogeneic cells, but not withNeu-positive allogeneic cells, led to a significant decrease inIL-4 production by CD4! cells. CD8! splenocytes from theTriplex cell group showed low spontaneous IFN-g produc-tion, which increased on restimulation, reaching high levelsonly in the presence of allogeneic Neu-positive stimulatorcells. In the DNA vaccine groups CD4! cells showed low IL-4production even after in vitro restimulation; IFN-g productionwas found to be significant only in cultures restimulated withallogeneic cells, with the highest values in CD8! cultures.

Neither the Triplex cell nor Tri-DNA vaccine gave rise tocytotoxic effectors against Neu=H-2d cell targets (data notshown).

On the whole, the Triplex cell vaccine gave rise to powerfulTh1 plus Th2 immune stimulation (also comprising cytokineswith immunosuppressive activity, such as IL-10), both ofwhich were increased by culture with Neu-positive syngeneiccells. On the other hand, the Tri-DNAvaccine induced a highlyrestricted Th1 response, stimulated mainly by allorecognition.

Humoral response

Antibodies recognizing the Neu antigen were specificallydetected by ELISA (Fig. 6A). All the mice in the Tri-DNAvaccine group (solid squares) already showed detectablelevels of anti-Neu antibodies after the first vaccination cycle,whereas in the groups receiving DNA vaccines lacking one orboth adjuvants as well as in the Triplex cell vaccine groupsome late-responder mice were also found. After the secondvaccination cycle all the DNA vaccines studied producedsimilar ranges of anti-Neu antibody levels. At the end of thevaccination cycles the highest antibody levels were obtainedwith the Triplex cell vaccine (Fig. 6A, open squares).

The use of a cell vaccine such as the allogeneic Triplexvaccine could induce antibodies recognizing other cell surfacemoieties, some of which could be downstream to Neu.Transduction of muscle cells could elicit antibodies recogniz-ing molecules induced by the Neu pathway: in fact, skeletalmuscle cells naturally show some level of HER-2 expressionand can respond to its pathway (LeBrasseur et al., 2005). Tostudy the antibody response directed against Neu-expressingcells, we then tested the ability of sera from vaccinated miceto bind syngeneic or allogeneic neu-positive cells (Neu=H-2d

FIG. 1. Validation of pIL12 and pDq vectors. (A) Inductionof serum IL-12 (solid circles) and IFN-g (open triangles) afterintramuscular injection and electroporation of 50mg of pIL12(treatments indicated by arrows). Shown are means andstandard error from three mice. Horizontal line indicates thesensitivity threshold of the test. (B) Cytofluorometric evalu-ation of H-2Dq expression in B78H1 cells 48 hr after trans-fection with pDq (boldface profile). Dotted profile: untreatedB78H1 cells. The x axis reports the fluorescence intensity(arbitrary units) and the y axis reports the number ofevents. (C) Complement-dependent cytotoxic (CDC) activityagainst H-2q or H-2d lymphocytes by sera of H-2d micesubjected to intramuscular injection and electroporation of50mg of pDq plasmid (solid columns, two mice) or untreatedcontrols (open columns, two mice). Hatched columns: 28-14-8S positive control serum. Mean and standard error areshown.

DNA VACCINES FOR p53/Neu CANCER PREVENTION 457

and Neu=H-2q cells, respectively; Fig. 6B and C). Kinetics ofinduction of humoral reactivity were similar to those foundwith ELISAs. Mock-electroporated control mice did notdevelop any humoral response, both when tested in ELISAand as binding to Neu-positive cells (data not shown). Thereactivity against Neu=H-2q cells observed with sera fromgroups including allogeneic MHC in vaccine formulationobviously comprises anti-histocompatibility antibodies; how-ever, a minor contribution to binding is attributable to histo-compatibility because such sera tested against a Neu-negativeH-2Dq-expressing target cell had low binding activity (datanot shown). In fact, DNA vaccines lacking an allogeneic MHCgene (pNeu!pIL12 and pNeu alone) could induce antibodiesalmost as effectively as the DNA vaccine including the H-2Dq

gene.On the whole, the various DNA vaccines did not reach

remarkably different antibody levels, but the presence ofadjuvants in the Tri-DNA vaccine seems to confer a faster andmore homogeneous response. Sera from the Triplex cell vac-cine group reached significantly higher levels of binding bothto syngeneic and to allogeneic Neu-positive cells.

To further investigate whether the various vaccines couldalso lead to qualitatively different humoral responses, westudied the isotype profile of the antibodies binding syngeneicNeu-positive cells (Fig. 7). All the vaccines studied inducedcomparable levels of IgG2a antibodies, but only the Triplex

cell vaccine caused a high level of IgG3 antibodies, togetherwith a slight increase in IgG1 antibodies.

Discussion

The Tri-DNA vaccine was designed as a second-generationTriplex vaccine, because it combines three plasmids each car-rying one of the three components conferring the high im-munopreventive effectiveness of the Triplex cell vaccine: theNeu target antigen and, as adjuvants, IL-12 and allogeneicMHCmolecules. The Tri-DNA vaccine was as effective as the Triplexcell vaccine, but inducedmore restricted immunostimulation. Inparticular, the Tri-DNA vaccine gave a fully Th1-polarizedresponse, with effective production of IFN-g in response to thevaccine but no spontaneous production, and no induction ofIgG3 antibodies binding syngeneic Neu-positive cells. On theother hand, the Triplex cell vaccine induced both Th1 and Th2cytokines, high spontaneous IFN-g production, and highlevels of IgG3 antibodies against syngeneic cells.

The Tri-DNA vaccine has controlled antigenicity, in com-parison with the Triplex cell vaccine. In fact, a cellular vaccinein principle could carry a portfolio of immune stimuli, otherthan desired, whereas our Tri-DNA vaccine carries only thethree genes chosen. In particular, an allogeneic cell vaccinecarries multiple different MHC genes. In the Tri-DNA vaccinea single MHC gene was used as adjuvant instead of a com-plete haplotype. TheMHCgene chosen,H-2Dq, has about 88%homology with the corresponding MHC gene of the hostd haplotype; nevertheless, it gave effective stimulation aswell,as determined on the basis of the high IFN-g response by allo-

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FIG. 2. Cancer-immunopreventive activity of DNA vac-cines and Triplex cell vaccine in BALB-p53Neu female mice(6–11 mice per group). Vaccination cycles are shown witharrows (DNA vaccines) or boxes (Triplex cell vaccine). Da-shed line: Mock-electroporated control mice. Significance ofdifferences with Mantel–Haenzel test: any vaccine versusmock, p< 0.05 at least (both salivary and mammary carci-noma); pNeu!pIL12!pDq versus pNeu!pIL12 or pNeu,p$ 0.074 or p$ 0.096, respectively (salivary carcinoma only).

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458 DE GIOVANNI ET AL.

FIG. 4. Histology (A, C, E, and G) and immunohistochemistry (B, D, F, and H) of salivary glands (A–F) and mammaryglands (G and H) of BALB-p53Neu female mice. The salivary gland obtained from a 5-week-old nonvaccinated mouse (A)shows several foci of small duct hyperplasia (arrows) that are intensively positive for Neu (B). The salivary gland of an 18-week-old nonvaccinated mouse is almost completely occupied by a scarcely differentiated Neu-positive carcinoma (C andD).The salivary gland obtained from a 38-week-old Tri-DNA-vaccinated mouse is morphologically normal with well-definedducts and acini (E) constituted by cells without expression of Neu (F), whereas the corresponding mammary tissue showsinvasive carcinoma (G) with cells clearly expressing Neu on membranes (H). A well-vascularized stroma (G and H, arrows)with inflammatory cells is present at the periphery of the mammary tumor. In (F) and (H) within the stroma some false-positive cells with morphological features of macrophages can be observed.

DNA VACCINES FOR p53/Neu CANCER PREVENTION 459

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restimulated spleen cells. Such IFN-g induction could takeplace even in vivo after each vaccination cycle, contributing tothe effectiveness of the vaccine.

DNAvaccines are generallywell tolerated (Rice et al., 2008).However, potential side effects must be taken into consider-ation, such as the risk for a stable integration potentiallyleading to insertional mutagenesis on the one hand (Rice et al.,2008) and prolonged antigen expression leading to immuneanergy on the other (Radcliffe et al., 2007). Our choice tocombine a HER-2 DNA vaccine with an allogeneic MHC genevaccine could overcome both concerns, because allorecogni-tion, besides its Th1-driving effect (Wallgren et al., 2005),should lead to rapid elimination of transfected cells (Groneviket al., 2005). Plasmids carrying the IL-12 genes are currentlybeing tested to correct Th2-mediated pathologies such as al-lergic asthma (Malheiro et al., 2008).

Monoantigenic anti-Neu-electroporatedDNAvaccineshavebeen reported to confer protection against carcinogenesis dri-ven by Neu alone (Quaglino et al., 2004; Cipriani et al., 2008).Carcinogenesis induced by the combination of Neu and p53knockout is much more aggressive and difficult to prevent(Croci et al., 2004; Pannellini et al., 2006). In thismodel Tri-DNAvaccine was more effective than the monoantigenic Neu vac-cine in keeping allmice free of salivary tumors. The presence of

the two adjuvants (IL-12 and allogeneic MHC gene) did notmodify the long-term level of antibodies induced, but gave afaster and homogeneous rise of antibodies after only onevaccination cycle. In fact, the response to DNAvaccines showsheterogeneity among individuals ( Jacob et al., 2006; Ciprianiet al., 2008) that is generally circumvented by repeated boost-ing. Because immunoprevention in Neu-transgenic modelswas reported to be related both to the individual antibodylevel (Cipriani et al., 2008) and to the precociousness of theresponse induced (Croci et al., 2004; Pannellini et al., 2006), it islikely that the more homogeneous response induced by theTri-DNA vaccine combined with the high IFN-g boost deter-mined by allorecognition could make the difference.

In contrast to salivary carcinogenesis, however, no superioreffect of the Tri-DNA vaccine was observed for mammarycarcinogenesis. We hypothesize that a more defined temporalwindow for salivary carcinogenesis could take place, so ourtemporally defined schedule of vaccination (ending at 14weeks of age) could be sufficient to completely prevent sali-vary carcinogenesis, whereas a longer or even life-longschedule may be required to halt carcinogenesis in the con-tinuously evolving mammary gland.

The Tri-DNA vaccine induced a Th1-biased response andled to clarification of the antibody isotypes involved in

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FIG. 5. Cytokine production by spleen cells collected after the first vaccination cycle and cultured for 6 days alone or in thepresence of the indicated restimulator cells. (A) Total unseparated spleen cells; (B) CD4! and CD8! cells. No IL-4 productionby CD8! cells was observed (data not shown). Mean and standard error from two or three mice per group is shown.‰

DNA VACCINES FOR p53/Neu CANCER PREVENTION 461

immunoprevention.We previously demonstrated that murineIgG2a was required and that IgG1 is dispensable (Nanni et al.,2001; De Giovanni et al., 2004), but no data on the role ofmurine IgG3 have been reported so far in the literature. Herewe found that IgG3 is not necessary for immunoprevention,because DNA vaccines, which did not induce IgG3 at all, wereat least as effective as the Triplex cell vaccine, which inducedhigh levels of IgG3. Murine IgG3 is the predominant murineIgG subclass elicited by bacterial polysaccharide antigens, andis likely to correspond to the human IgG2 isotype (Yoo et al.,2003). Murine IgG3 has mechanisms of action different fromthose of the other isotypes: it does not bind the Fc receptor, butcan activate the complement system through Fc–Fc interac-tions (Getahun and Heyman, 2006; Nimmerjahn and Ravetch,2008). Some data on the involvement of murine IgG3 in au-toimmunity have also been reported (Baudino et al., 2006).Autoimmunity could be a serious side effect of cancer im-

munoprevention ( Jacob et al., 2006; Wei et al., 2008), becausecancer antigens, HER-2 included, can be expressed in normaltissues. We did not observe any sign of autoimmunity in miceof our vaccinated series (both with cell and DNA vaccines),suggesting that the high level of IgG3 antibodies elicited by theTriplex cell vaccinewas not required for tumor prevention, butat the same time was not detrimental to mouse health.

In conclusion, DNA vaccines were as effective as the Triplexcell vaccine, exploiting a more restricted mode of immunestimulation.The combinationofmultipleplasmidsand thehighefficacy obtained through electroporation makes the systemhandy and flexible. Our results suggest that this novel strategymay be effective in the prevention of HER-2-expressing tumorsin humans at high risk, but also suggest its potential efficacy inthe therapeutic setting. In fact, DNA-based combined ap-proaches are being studied for therapeutic approaches (Martel-Renoir et al., 2003; Feng et al., 2005). Multi-DNA vaccines could

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FIG. 6. Humoral response of BALB-p53Neu female mice vaccinated with the indicated DNA vaccines compared with Triplexcell vaccine. Vaccination cycles are shown with arrows (DNA vaccines) or boxes (Triplex cell vaccine). Data from five to nineindividual mice are shown in each panel. (A) Anti-Neu antibodies, measured by ELISA on sera at 1:300 dilution. Dashed line:Sensitivity of the test. Significance of differences between groups (nonparametric Wilcoxon test): pNeu!pIL12!pDq versuspNeu!pIL12 or pNeu at 7 weeks (one vaccination), p$ 0.06 and p$ 0.04, respectively; any DNA vaccine versus Triplex cellvaccine at 16 weeks, p< 0.05. (B) Cytofluorometric analysis of serum binding activity toward Neu=H-2d cells. Significance ofdifferences between groups (nonparametric Wilcoxon test): pNeu!pIL12 versus pNeu at 7 weeks, p< 0.05; any DNA vaccineversus Triplex cell vaccine at 16weeks, p< 0.01. (C) Cytofluorometric analysis of serumbinding activity towardNeu=H-2q cells.Significance of differences between groups (nonparametric Wilcoxon test): pNeu!pIL12!pDq versus pNeu at 7 weeks,p< 0.05; any DNA vaccine versus Triplex cell vaccine at 16 weeks, p< 0.01.

462 DE GIOVANNI ET AL.

in principle be more suitable to tailor-cut treatment, allowingcombinations of the right panel of tumor antigen, adjuvantcytokine, and adjuvant allogeneic MHC gene for each tumortype or even each individual.

Acknowledgments

This work was supported by grants from the following: theItalian Association for Cancer Research (Milan, Italy); theItalianMinistry for Education, University, and Research PRINprojects; the University of Bologna (Bologna, Italy); the Al-maMedicina Foundation (Bologna, Italy); and the PallottiFunds, Department of Experimental Pathology, University ofBologna. A. Palladini is the recipient of a fellowship from theItalian Foundation for Cancer Research (Milan, Italy). S. Crociis the recipient of a research fellowship (Assegno di Ricerca)from the University of Bologna. A. Antognoli is the recipientof a Ph.D. fellowship from the University of Bologna.A. Murgo is the recipient of a research fellowship from theW. Vanini–S. Cavagnino Foundation, InterdepartmentalCentre for Cancer Research, University of Bologna.

Author Disclosure Statement

For all authors, no competing financial interests exist.

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pNeu+pIL12pNeu

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Address reprint requests to:Dr. Carla De GiovanniCancer Research Section

Department of Experimental PathologyViale Filopanti 22

I-40126 Bologna, Italy

E-mail: carla.degiovanni@unibo.it

Received for publication October 22, 2008;accepted after revision February 12, 2009.

Published online: April 1, 2009.

464 DE GIOVANNI ET AL.