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Direct Immunization of Malaria DNA Vaccine into theLo

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Biochemical and Biophysical Research Communications 271, 107–115 (2000)

doi:10.1006/bbrc.2000.2558, available online at http://www.idealibrary.com on

iver by Gene Gun Protects against Lethal Challengef Plasmodium berghei Sporozoite

higeto Yoshida,*,1 Shin-Ichiro Kashiwamura,† Yoshinori Hosoya,‡ Enjie Luo,*iroyuki Matsuoka,* Akira Ishii,* Akio Fujimura,§ and Eiji Kobayashi§,¶

Department of Medical Zoology, ‡Department of Surgery, §Department of Clinical Pharmacology, and ¶Division of Organeplacement Research, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachimachi,ochigi 329-0498, Japan; and †Institute of Advanced Medical Science, Laboratory of Host Defences,yogo College of Medicine, 1-1 Mukogawacho, Nishimomiya 663-8501, Japan

eceived March 15, 2000

effective vaccines. Immunization with irradiated Plas-mmaiplswwzRDnst

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The liver is the first target organ for malaria para-ites immediately after the bite of an infected mos-uito. We studied local immunization of malaria DNAaccines at the site of the liver using a gene gun as aseful tool for in vivo transfection of foreign genes. Aalaria DNA vaccine consisting of the Plasmodium

erghei circumsporozoite protein (PbCSP) gene plushe mouse IL-12 gene was bombarded directly by aene gun into mouse liver once or into the skin twice.marked protective effect was induced by gene bom-

ardment into the liver (more than 71%) comparedith that into the skin (less than 33%). A Th1-type

mmune response and high production of iNOS werebserved in the hepatic lymphocytes from mice bom-arded into the liver, resulting in more effective pro-ection compared with those bombarded into the skin.hese results provide an important implication on theevelopment of efficient malaria vaccine strategies.2000 Academic Press

Key Words: malaria; Plasmodium berghei; DNA vac-ine; liver; CSP; cytokine; IL-12; nitric oxide.

Malaria is one of the worst health problems in theorld. Approximately 300 to 500 million people are

nfected with malaria annually, and 1.5 to 2.7 millionives are lost in each year. The main hope to controlhis disease may reside in the development of safe and

Abbreviations used: PbCSP, P. berghei circumsporozoite protein;O, nitric oxide; iNOS, inducible nitric oxide synthase; GPI, glyco-

ylphosphatidylinositol; CD, dendritic cells; HVJ, hemagglutinatingirus of Japan.

1 To whom correspondence should be addressed at Department ofedical Zoology, Jichi Medical School, 3311-1 Yakushiji, Minami-

awachimachi, Tochigi 329-0498, Japan. Fax: 81-285-44-6489.-mail: [email protected].

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odium spp. sporozoites induce sterile protective im-unity against malaria in humans, monkeys, mice,

nd chickens (reviewed in Ref. 1). Protective immunitys directed against the pre-erythrocytic stage of thearasite (i.e., sporozoites and liver stages). In particu-ar, IFN-g, provided by malaria-specific CD81 T cells,timulates infected cells to produce nitric oxide (NO)hich destroys infected hepatocytes or parasitesithin these cells (2–4). However, the irradiated sporo-

oite vaccine is not practical for large scale human use.ecent studies support the importance of pursuing aNA vaccine development for malaria control (5–8). Aew DNA vaccine should aim to protect immunizedubjects against malaria comparable to that induced byhe irradiated sporozoite vaccine.

Our primary strategy is to deliver DNA vaccinesirectly into the liver, where malaria parasites developnto exoerythrocytic forms and multiply by schizogony.ntroduction of a target DNA into the liver may resultn direct transfection of professional antigen present-ng cells like Kupffer cells and/or the presentation of-cell epitopes encoded by the DNA vaccines in associ-tion with MHC-I molecules on the surface of hepato-ytes. Among several methods of such liver-directedene transfer reported for gene therapy (reviewed inef. 9), we used a gene-gun mediated gene transfer into

he live on the basis of our and Williams et al.’s resultshowing that gene-gun delivery of DNA into the liver isn efficient method of foreign gene introduction (10,1). In addition to transfection efficacy, multiple geneonstructs can be simultaneously delivered in desir-ble molar ratios, and accurately in situ into targetrgans within only 10 s.We chose the Plasmodium berghei circumsporozoite

rotein (PbCSP) and the mouse IL-12 genes as a DNA

0006-291X/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 271, No. 1, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

evelopmental stages namely sporozoites and infectedepatocytes (1, 12) and is one of the most extensivelytudied malaria vaccine candidate antigens (13). Im-unization with a DNA vaccine encoding CSP by in-

ramuscular injection conferred 68% protection againstporozoite challenge in the P. yoelii model, and therotection was dependent on CD81 T cells (8). Tochieve protective immunity against malaria parasitest the site of the liver, the induction of IFN-g by CD81

cells and subsequent NO production have beenhown to be important for the destruction of infectedepatocytes or the parasite within these cells (2, 3, 6,4–16). IL-12 has been reported to be an importantnducer and enhancer of Th-1 type responses includingFN-g production and cytotoxic activities (reviewed inef. 17). Coadministration of DNA vaccine with an

L-12 expression plasmid by intramuscular immuniza-ion significantly enhanced cell-mediated immunity byh-1 cell activation (18–20). Moreover, a single subcu-

aneous injection of recombinant human IL-12 twoays before challenge with P. cynomolgi sporozoitesrotected 100% of rhesus monkeys, suggesting thatecombinant human IL-12 protects monkeys throughhe IFN-g-dependent elimination of P. cynomolgi-nfected hepatocytes (21).

In the present study, we demonstrate for the firstime that direct immunization with malaria DNA vac-ine into the liver effectively protected mice against thehallenge of P. berghei-infected mosquito bites. Theseata clearly indicate that optimal vaccines designed tonduce protective immune responses in infected hepa-ocytes should be considered to improve current ma-aria vaccine strategies.

ATERIALS AND METHODS

Mice and parasite. Female 6- to 8-week-old BALB/c miceSEASCO, Saitama, Japan) were used in all experiments. Plasmo-ium berghei ANKA strain was maintained by cyclical passagehrough BALB/c mice and Anopheles stephensi (SDA 500 stain).

DNA vaccine. The PbCSP gene was amplified from genomic DNAf P. berghei ANKA strain by PCR using a sense primer59-GGAGGGCTAGCATGGAGACAGACACACTCCTGCTATGGG-ACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGCGGATCCA-TGCAGGACTACAAGGACGACGTAGACAAGGGATATGGACAAA-TAAAAGCATCCAAGCCC-39: a newly created NheI site is broken-nderlined, the mouse Igk secretion signal sequence (22) is underlinednd the FLAG encoding sequence (23) is double-underlined) and anntisense primer (59-GGAGGGCGGCCGCATCCCGGGTTTTCT-ATTTGAACCTTTTCGTTTTCTAACTCTTATACCAGAACC-39: aewly created NotI site is broken-underlined). The PCR was performedith Pfu DNA polymerase (Stratagene GmbH, Heidelberg, Germany)nder the following conditions for 30 cycles (denaturation at 94°C for0 s, annealing at 55°C for 1 min, elongation at 72°C for 2 min). TheCR product encodes PbCSP fused to the mouse Igk secretion signalequence in the place of its native signal sequence and without its-terminal glycosylphosphatidylinositol (GPI) anchor. After purifica-

ion the PCR product was digested with NheI and NotI, and ligated intoheI–NotI site in pcDNA3.1 (Invitrogen, San Diego, CA). The resultinglasmid was designated pcDNA-CS87 (Fig. 1, A1).

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IL12p35p40, was constructed as follows. Both mouse IL-12 p35nd p40 cDNAs (kindly provided by Dr. S. Wolf, Genetic Institute,ambridge, MA) were modified to clone into pcDNA3.1. The prim-rs for IL-12 p35 were 59-GAGGAGGATCCATGTGTCAA-CACGCTACCTCCTCTTTTTGG-39 (a newly created BamHI site

s broken-underlined) and 59-GGGGAGGGCCCTCAGGCG-AGCTCAGATAGCCCATCAC-39 (a newly created ApaI site isroken-underlined); the primers for IL-12 p40 were 59-AGGAGGATCCATGTGTCCTCAGAAGCTAACCATCTCC-39 (aewly created BamHI site is broken-underlined) and 59-GGG-AGGGCCCCTAGGATCGGACCCTGCAGGGAACACATGC-39

a newly created ApaI site is broken-underlined). After purifica-ion the PCR product encoding IL-12 p35 was digested withamHI and ApaI, and ligated into BamHI–ApaI site in pcDNA3.1.he resulting plasmid was designated pcDNA-mIL12p35. Simi-

arly, the PCR product encoding IL-12 p40 was digested withamHI and ApaI, and ligated into BamHI–ApaI site in pcDNA3.1.he resulting plasmid was designated pcDNA-mIL12p40. A 1.8-kbunI–NaeI fragment from pcDNA-mIL12p35 was ligated intounI–NruI site in pcDNA-mIL12p40 to generate pcDNA-IL12p35p40. The luciferase gene was removed from pT3/T7 luc

CLONTECH, Palo Alto, CA) by digestion with BamHI and NotInd inserted into BamHI–NotI site in pcDNA3.1 to generatecDNA-Luc. The sequences of the PbCSP and the IL-12 genesere verified by DNA sequencing. The sequence of the PbCSPene obtained from PCR was identical with that reported forNKA strain PbCSP gene (24), except for a GAA insertion into themino acid position between 81 and 82. Plasmid DNAs wereroduced in bacteria and purified using Qiagen Maxi Prep kitsQiagen, Santa Clara, CA).

Transient transfection in vitro. Proteins expression from pcDNA-S87 and pcDNA-mIL12p35p40 were analyzed by immunofluores-ent staining following transfection into HepG2 cells (American Typeell Culture, Rockville, MD) using Qiagen lipofectin (Qiagen) accord-

ng to the manufacturer’s protocol. Briefly, 2 days after transfection,ells growing in cover slips were fixed with acetone/methanol (3:2/v) and blocked with 3% BSA in PBS. The pcDNA-CS87- transfectedells were incubated with mAb 3D11 (kindly provided by Dr. R.ussenzweig, New York University School of Medicine) followedith a goat anti-mouse IgG-FITC conjugate. The pcDNA-IL12p35p40-transfected cells were incubated with rat anti-mouse

L-12 p40 (kindly provided by Dr. S. Wolf, Genetic Institute, Cam-ridge, MA) followed with a goat anti-rat IgG-FITC conjugate. Flu-rescence was observed using a Zeiss fluorescence microscope.

Gene immunization. Gold particles coated with plasmid DNAsnd their cartridges were prepared as described previously (10).ight-week-old BALB/c female mice were anesthetized by injectionith a mixture of Rompun-Vetalar (1:2 ratio; Bayer and Park-Daviseterinary, respectively). For liver bombardment, the abdomen washaved and the liver surface was exposed by a transverse skinncision. DNA-coated gold particles (1 mg plasmid DNA per shot)ere delivered into the left lateral lobe of the liver using a Heliosene Gun (Nippon Bio-Rad Laboratory, Tokyo, Japan) at a heliumischarge pressure of 200 psi. After bombardment the lobe wasepositioned, and the incision sutured. For skin bombardment, thebdomen of the skin was shaved and DNA-coated gold particles (1 mglasmid DNA per shot) were delivered twice (one at time 0 and the 2eeks later) into the abdomen with the gene gun at a helium dis-

harge pressure of 300 psi.

Protection against challenge. For Experiment 1, two weeks afterhe second immunization by skin bombardment with pcDNA-Luclone or pcDNA-CS87 plus pcDNA-mIL12p35p40, or 4 weeks afterhe immunization by liver bombardment with pcDNA-CS87 pluscDNA-mIL12p35p40, 3 groups of 10 mice were challenged by theites of P. berghei-infected Anopheles stephensi. Sedated mice werelaced on a nylon-mesh screened container containing infected mos-

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osquito feedings over 5 min. We chose the bites of P. berghei-nfected mosquitoes for the challenge to emulate the natural route of

alaria infection. Mice were considered protected if malaria Giemsa-tained thin blood films taken from day 2 until 14 after challengeere negative by microscopy. One mouse from the group bombardedith pcDNA-CS87 plus pcDNA-mIL12p35p40 into the liver died 2ays after challenge. No malaria infection was found in this mouse.o address the immune responses which conferred protectiongainst sporozoite challenge, protected mice were rechallenged byore than 20 bites of P. berghei-infected mosquitoes and examined T

ell subsets in the liver.For Experiment 2, the same procedure as in Experiment 1 was

epeated except that 5 groups including non-treated control mice andice bombarded with pcDNA-Luc into the liver were examined. Each

roup consisted of 10 mice except for 8 nontreated control mice.orty-two hours after sporozoite challenge, 3 mice from each groupere sacrificed to examine cytokine production in the liver and

pleen. Protected mice were rechallenged by more than 20 bites of P.erghei-infected mosquitoes to examine the induction of inducibleitric oxide synthase (iNOS) mRNA.Serum samples from both experiments were collected by tail vein

leeding immediately prior to each DNA bombardment and imme-iately prior to challenge, and assayed for the presence of anti-bCSP Abs.

Flow cytometric analysis of hepatic lymphocytes. Hepatic lym-hocytes from the protected mice in Experiment 1 were isolated fromerfused livers 42 h after sporozoite rechallenge according to theethod described by Goossens et al. (25). The surface phenotype of

ells was analyzed using mAbs specific for FITC-conjugated CD4 andE-conjugated CD8 (PharMingene, San Diego, CA) in conjunc-ion with a two-color immunofluorescence test and analyzed with aACScalibur flow cytometer using CellQuest software (Becton–ickinson, San Jose, CA).

Cytokine production assay. For the in vitro cytokine productionssay, single-cell suspensions of splenic and hepatic lymphocytesrom mice bombarded in Experiment 2 at 42 h after sporozoitehallenge were prepared by routine methods (25, 26). Cell popula-ions were plated at 3 3 106 cells per well in 24-well plates (Nunc,oskilde, Denmark) and cultured in the presence of medium alone or

mmobilized anti-CD3 mAb (PharMingen) at a final concentration ofmg/ml. All cell cultures were incubated at 37°C in an atmosphere of% CO2 in air. Cell-free supernatant fluids were harvested fromhese cell cultures at 6 h and assayed for IFN-g or IL-4 activities.FN-g levels were measured by standard sandwich ELISA as de-cribed previously (27). IL-4 levels were determined by proliferationf IL-4 dependent CT4S cell bioassay as described previously (28). Inhe case of IL-4, hepatic lymphocytes were stimulated by anti-CD3Ab because no IL-4 production was detected without the stimula-

ion in any groups. IFN-g or IL-4 levels were calculated from stan-ard curves constructed by using recombinant mouse IFN-g or IL-4.

Ab reagents for in vivo depletion and neutralization. Six of 10rotected mice with no parasitemia in Experiment 2 were rechal-enged to detect iNOS mRNA in the livers. In experiments assessinghe importance of CD41, CD81 T-cells and IFN-g on expression ofNOS mRNA, another 4 protected mice were given the followingreatment individually before rechallenge. For depletion of CD41

-cells, one of the 4 mice was treated ip on days 26 and 23 and theay of sporozoite rechallenge with 200 mg of the rat anti-mouse CD4Ab, GK1.5 (29). For depletion of CD81 T-cells, one of the 4 miceere treated iv on days 26 and 23 and the day of sporozoite rechal-

enge with 200 mg of the rat anti-mouse CD8 mAb, lyt2.2 (the kindift of Dr. T. Aji, Okayama University Medical School). For neutral-zation of IFN-g, one of the 4 mice was treated iv on days 26 and 23nd the day of sporozoite rechallenge with 200 mg of the rat anti-ouse IFN-g mAb. For an appropriate control mAb, one of the 4 miceas treated iv on days 26 and 23 and the day of sporozoite rechal-

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can Type Culture Collection, Rockville, MD). Mice were sacrificed4 h after sporozoite rechallenge and the livers were removed forNOS mRNA analysis. Meanwhile, spleen cells were prepared fromach mice and the efficacy of CD41 and CD81 T cell depletion wasssessed by flow cytometric analysis (.95%).

Detection of inducible nitric oxide synthase mRNA by RT-PCR.ivers removed from mice were frozen in liquid nitrogen and theotal liver RNA was extracted as described previously (2). Primerequences used for mouse iNOS detection were 59-CCCTTCCG-AGTTTCTGGCAGCAGC-39 forward and 59-GGCTGTCAGAG-CTCGTGGCTTTGG-39 reverse. For mouse b-actin detection, therimer pair was 59-TGGAATCCTGTGGCATCCATGAAAC-39 for-ard and 59-AACGCAGCTCAGTAACAGTCCGCCTA-39 reverse. Cy-

le parameters were 30 cycles of 95°C for 1 min, 55°C for 1 min, and2°C for 1 min. The identity of the specific PCR products was verifiedy Southern blot hybridization. The cDNAs encoding iNOS and-actin were labeled with enzyme horseradish peroxidase using anCL direct nucleic acid labeling kit (Amersham Pharmacia Biotech,uckinghamshire, UK). A series of the PCR products was run on a.5% agarose gel. After transfer to a nylon membrane, the PCRroducts were hybridized to the probes. High stringency conditionsere used in the hybridization and washing. Positive signals wereetected by an ECL detection system (Amersham Pharmacia Bio-ech) followed by exposure using IR LAS-1000 Lite V1.1 (FUJIFILM,okyo, Japan). The amounts of the bands on autoradiographs wereuantified by densitometric scanning using Image Gauge V3.3 (FU-IFILM).

Statistical analysis. The statistical significances of differences inurvival between groups of mice were analyzed by the x2 test andaplan–Meier test.

ESULTS

Construction and expression of PbCSP and IL-12 initro. Plasmid maps for pcDNA-CS87 and pcDNA-IL12p35p40 are shown in Fig. 1. pcDNA-CS87 (Fig.

, A1) contains the PbCSP gene lacking 59-signal se-uence and 39-GPI anchor sequence, which corre-ponds to the amino acid coding region 21 to 299. Thisene fragment is fused to the mouse Igk signal se-uence. Transcription initiates from the CMV pro-oter, continues through the mouse Igk signal se-

uence, the FLAG encoding sequence, the maturebCSP protein coding sequence and terminating at aovine growth hormone polyadenylation sequence.cDNA-mIL12p35p40 contains the sequences for the35 and p40 subunits of mouse IL-12, linked tandemlyn the same direction and each driven by CMV pro-

oter, and a bovine growth hormone polyadenylationequence (Fig. 1, B1).To verify the construction of pcDNA-CS87 and

cDNA-mIL12p35p40, HepG2 cells were transfectedith one or other. Positive immunofluorescence wasbserved in cells transfected with pcDNA-CS87 andcDNA-mIL12p35p40 by the indirect fluorescent Abest (Fig. 1, A2 and B2). A BamHII–NotI 900-kb frag-ent form pcDNA-CS87 was inserted into BamHII–otI site in a baculovirus expression vector pBACgus-3.1scFv (30). Proteins produced in the recombinantaculovirus-infected cells were secreted into mediumnd recognized with 3D7 mAb in Western blots (data

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ot shown). Purified recombinant PbCSP was thereforesed as an ELISA antigen to detect antibodies ob-ained from mice immunized with plasmid DNA.

Protection after immunization. We specifically chosehe IL-12 gene as a “genetic adjuvant” for malaria DNAaccine because the coadministration of DNA vaccinesith IL-12 expression plasmids preferentially activates

FIG. 1. Transient Expression of the PbCSP gene and the mousecDNA-mIL12p35p40, respectively. A schematic representation of tecretion signal, a portion of the PbCSP gene (corresponding aminoA1). The nucleotide sequence present at the junction between the CMhe mouse Igk secretion signal and FLAG sequences are underlinontaining the mouse IL12p35 gene under the control of a CMV proms shown in (B1). Indirect immunofluorescence assay of acetone-

IL12p35p40. HepG2 cells were transfected either with pcDNA-CS8nd anti-mouse IL-12p40 mAb, respectively.

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h1-type cells (18–20), and Th1 type immune responsesan play a critical role in protection against intrahepaticarasites (31, 32). We therefore focused on the coadmin-stration of DNA vaccine with the IL-12 expression plas-

id into the liver and did not address the contribution ofbCSP alone or IL-12 alone to protection. The liver sur-

ace of BALB/c mice was exposed surgically and bom-

-12 gene in HepG2 cells using expression vectors pcDNA-CS87 andpcDNA-CS87 plasmid containing a CMV promoter, the mouse Igkd position 21–299) and an SV40 polyadenylation signal is shown inpromoter and the PbCSP gene is illustrated above the plasmid map.A schematic representation of the pcDNA-mIL12p35p40 plasmid

r and the mouse IL12p40 gene under the control of a CMV promoterd HepG2 cells transfected either with pcDNA-CS87 or pcDNA-

2) or pcDNA-mIL12p35p40 (B2), and stained with anti-PbCSP mAb

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arded with the DNA vaccine by gene gun. Anotherroup was similarly bombarded twice with the sameNA vaccine into the skin. Mice were challenged by theites of P. berghei-infected mosquitoes 4 weeks after liverombardment or 2 weeks after the second skin bombard-ent (Fig. 2A). The same experiment was repeated with

FIG. 2. Survival of mice bombarded with different regimes usinggene gun after lethal sporozoite challenge. Two experiments were

erformed. (A) Experiment 1: Mice were bombarded twice withcDNA-Luc into the skin (N 5 9), twice with pcDNA-CS87 pluscDNA-mIL12p35p40 into the skin (N 5 9) or once with pcDNA-S87 plus pcDNA-mIL12p35p40 into the liver (N 5 8). (B) Exper-

ment 2: Mice were bombarded twice with pcDNA-Luc into the skinN 5 7), twice with pcDNA-CS87 plus pcDNA-IL12p35p40 into thekin (N 5 7), once with pcDNA-Luc into the liver (N 5 7), once withcDNA-CS87 plus pcDNA-mIL12p35p40 into the liver (N 5 7) oron-immunized mice as a control (N 5 5). Mice were challenged byhe bites of P. berghei-infected mosquitoes 2 weeks (mice bombardednto the skin) or 4 weeks (mice bombarded into the liver) after theast immunization. Malaria-infected mice were monitored daily forurvival up to 35 or 40 days after challenge. Asterisks indicate thatroups of mice immunized with pcDNA-CS87 plus pcDNA-L12p35p40 into the liver (CS 1 IL12/Liver) have significantlyigher protection rates than groups of mice bombarded with theontrol plasmid pcDNA-Luc into the skin (Luc/Skin) at P , 0.05,sing the x2 test.

111

nto the liver (Fig. 2B). We have previously shown that 2ays after bombardment with the lacZ gene into the rativer, b-galactosidase activity was detected to a depth of 2

m below the liver surface (10). In the two experiments,ice bombarded with the DNA vaccine into the liver had

tatistically significant protection (73%) compared withhose into the skin (31%). All nonimmunized mice andice bombarded with pcDNA-Luc into the liver died.lthough we checked parasitemia daily from 3 to 14ays after challenge in Experiment 1, those animalshich became infected showed no significant delay

n parasitemia in any group (Fig. 3). It is of interest toote that although all mice once showing parasitemiaied of infection, the period of life varied from 13 to 34ays after challenge in Experiment 1 (Fig. 2A) or 16 to 25ays in Experiment 2 (Fig. 2B), even in the controlroups.We assessed the relationship between humoral re-

ponses to PbCSP and protection in mice. Serum sam-les were collected immediately prior to challenge andweeks after challenge. No detectable ELISA antibody

esponses to recombinant PbCSP were induced in anymmunized mice, whereas immunization with thebCSP plasmid alone by intramuscular injection in-uced relatively high antibody to PbCSP (data nothown). This is consistent with the low-level Ab re-ponse induced by coadministration of HIV gag geneith IL-12 gene (20).Taken together, these data show that direct immu-

ization into the liver resulted in more effective pro-ection compared to immunization into the skin. Inddition, the lack of association of protection with Abesponse suggested that the immunity was dependentn cell-mediated responses to PbCSP.

Increase in the number of CD81 T cells in the liver ofrotected mice after rechallenge. To test whetherporozoite rechallenge affected the relative proportionf infiltrating T cell subsets, splenic and hepatic lym-hocytes from the protected mice of Experiment 1ere isolated 42 h after rechallenge and analyzed byow cytometry. Two-color staining for CD8 and CD4howed that CD8 single-positive cells in the liver wereubstantially increased both in mice bombarded intohe liver and skin following rechallenge compared withhose in the control mice (Fig. 4). Neither significanthange in CD3/IL-2R b/TCR-gd subsets nor change inD8/CD4 subsets was observed in the spleens (dataot shown).

Induction of IFN-g in the hepatic lymphocytes frommmunized mice after challenge. Splenic and hepaticymphocytes from mice bombarded in Experiment 2 at2 h after challenge were cultured in the presence ofedium alone or cross-linked anti-CD3 antibody. Su-

ernatant fluids were assayed for IFN-g or IL-4 pro-

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uction. Hepatic lymphocytes from mice bombardednto the liver produced without stimulation almost-fold greater IFN-g production than did cells obtainedrom mice bombarded into the skin (Fig. 5). Controlice, in contrast, produced no detectable IFN-g. In

ddition, no IFN-g was detectable in any culture su-ernatant fluid from spleen cells of any group (data nothown).All culture supernatant fluids collected were also

ssayed for IL-4. There was no detectable IL-4 in anyroup without stimulation. Curiously, hepatic lympho-ytes from mice bombarded into the liver produceduch less IL-4 than did cells obtained from another

roup in response to anti-CD3 antibody stimulation.hus, the cytokine assays suggested that coadminis-ration of the PbCSP gene with the IL-12 gene induceTh1-type immune response in the liver.

FIG. 3. Parasitemia profiles in groups of mice bombarded with dxperiment 1. Mice were bombarded with pcDNA-Luc into the skin

N 5 9) or pcDNA-CS87 plus pcDNA-mIL12p35p40 into the liverarasitemia percentage was monitored on Giemsa-stained blood film

FIG. 4. Flow cytometry analysis of hepatic lymphocytes isolateosquitoes. Mice bombarded into the liver or skin protected challeng

B) and skin-bombarded mice (C) were rechallenged by the bites ofrom three mice per group 42 h after rechallenge. Naive mice (A) werells were stained with PE-coupled anti-CD4 mAb and FITC-coupledepatic lymphocytes were generated after exclusion of dead cells by

112

Detection of iNOS mRNA by RT-PCR. Previousorks had demonstrated that in addition to CD81 T

ells and IFN-g, nitric oxide (NO) is required for therotection induced by immunization with irradiated P.erghei sporozoites in mice (2). To determine whetherhe protected liver-bombarded mice induce iNOS in theiver after sporozoite invasion, RNA was isolated fromhe livers of protected mice bombarded into the liver inxperiment 2 at 24 h after sporozoite rechallenge andnalyzed for the presence of iNOS mRNA by RT-PCRFig. 6). iNOS was not detectable in the livers of eitherormal mouse or mouse bombarded into the liverlanes 1 and 3), whereas iNOS was induced in theivers of mice bombarded into the liver 24 h after re-hallenge (lane 4). A faint level of iNOS mRNA wasetected in the liver of normal mouse 24 h after chal-enge (lane 2).

erent regimes using a gene gun after lethal sporozoite challenge in(N 5 9), pcDNA-CS87 plus pcDNA-mIL12p35p40 into the skin (B)) (N 5 9), and challenged as described in the legend to Fig. 2A.or individual animals from 3 to 14 days after challenge.

rom mice 42 h after challenge by the bites of P. berghei-infecteds described in the legend to Fig. 2B. The protected liver-bombardederghei-infected mosquitoes, and hepatic lymphocytes were isolatedallenged and the hepatic lymphocytes were prepared as (B) and (C).i-CD8 mAb. Two-color immunofluorescence analysis dot blots for the

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Since increases in IFN-g and CD81 T cells in theivers of mice bombarded into the liver were observed,e assessed the relationship between CD81 T cells,

FN-g and liver iNOS. The protected liver-bombardedice were rechallenged following treatment with ei-

her anti-IFN-g, anti-CD8, or anti-CD4 mAb. iNOSRNA was detected in the livers of mice administrated

oth with control mAb and anti-CD4 mAb (lanes 7 and). In contrast, we found a significant decrease in thebility of the protected mice to express iNOS in re-

FIG. 5. Comparison of IFN-g and IL-4 production levels from hhallenge of P. berghei-infected mosquito bites. Groups of three miceorty-two hours after challenge, hepatic lymphocytes from three micend IL-4 (left) productions were determined in the supernatant at 12ice (Control), mice skin-immunized with control plasmid (Luc/Ski

ombarded with the DNA vaccine (CS 1 IL12/Skin) and mice liver-biddle of the panel. Data represent means 6 standard deviations a

FIG. 6. Detection of iNOS mRNA expression. Mice bombardedith pcDNA-CS87 plus pcDNA-mIL12p35p40 into the liver wererotected against challenge as described in the legend to Fig. 2B. Therotected mice were inoculated with different mAbs and then rechal-enged by the bites of P. berghei-infected mosquitoes. RNA wasxtracted from the livers of a control mouse (lane 1), a protectedouse (lane 3), a control mouse 24 h after challenge (lane 2) and a

rotected mouse 24 h after rechallenge (lane 4). Other protected miceere rechallenged following the treatment with anti- IFN-g mAb

lane 5), anti-CD8 mAb (lane 6), anti-CD4 mAb (lane 7) and controlAb (lane 8). RNA was extracted from livers 24 h after rechallenge.outhern hybridization was performed to detect the expression of

NOS and b-actin mRNA from livers following amplification by RT-CR as described under Materials and Methods. Each lane repre-ents data from an individual mouse.

113

ponse to sporozoite challenge by administration ofnti-CD8 antibody and anti-IFN-g antibody (lanes 5nd 6). Therefore, induction of iNOS mRNA was de-endent on CD81 T cells and IFN-g.

ISCUSSION

Protective immunity against intrahepatic malariaarasites is induced by immunization with irradiatedporozoites, thereby preventing blood stage malaria. Inn attempt to pursue development of vaccines thatnduce immune responses comparable to those elicitedy immunization with irradiated sporozoites, geneticmmunization with naked DNA has been shown tonduce humoral as well as cellular immune responsesith high efficiency, emphasizing the enormous poten-

ial of this strategy for vaccination purposes (5–8). Weypothesized that induction of local immunity at theite of the liver could be important in preventing theevelopment of exoerythrocytic stages and subsequentlood infection. In this study, we introduced malariaNA vaccine into the liver using a gene gun-mediatedene delivery system and examined the protective ef-ects. Protection obtained by gene gun delivery into theiver once (73%) was significantly higher than that byhe material into the skin twice (31%), suggesting thatirect gene transfer of DNA vaccine to the liver surfaces potentially a more effective approach of generatingrotective immunity against malaria parasites withhe CSP immunogen.

One obvious advantage of immunization with a DNAaccine into the liver, over immunization with the same

tic lymphocytes of mice bombarded with DNA vaccine followed byre bombarded and challenged as described in the legend to Fig. 2B.re collected and cultured with or without anti-CD3 Ab. IFN-g (right)y ELISA and bioassay, respectively. Five groups consisting of naivemice liver-bombarded with control plasmid (Luc/Liver), mice skin-arded with the DNA vaccine (CS 1 IL12/Liver) are indicated at the

are representative of two replicative experiments.

epaweweh b

n),ombnd

DNA vaccine into the skin is that malaria antigens ex-pcsg(ebtftmm5ihlpitcia

fsapeACllrsttrlh

lviommeal(iksgwd

portance of efficient uptake of DNA vaccine by Kupfferc

dtdtgsse

A

mmmSoic

R


ressed on the cell surface from DNA constructs can beorrectly presented to the immune system in a nativetate at the appropriate site. Thus, immunization withenes encoding malaria liver stage antigens, such as CSP8), TRAP (33), LSA-1 (34), and HEP17 (6), can in theorylicit a whole range of immune responses, closely resem-ling the process of natural sporozoite infection. In addi-ion, the success of DNA vaccination depends upon trans-ection and activation of dendritic cells (DC) followed byhe initiation of T cell response (35). Gene gun bombard-ent into the skin results in direct transfection of bonearrow-derived Langerhans cells as DC, which are only

% of the cells in the epidermis (36). These DC are deeplynvolved in priming native T cells. In contrast to the skin,igher frequencies of Kupffer cells as resident DC in the

iver to support the recruitment and activation of antigenresenting cells may account for the liver playing a crit-cal role in DNA immunization. Although it is unlikelyhat this approach will ultimately be used for DNA vac-ination in humans, this information may potentiallynfluence the choice of clinical trials of DNA vaccinationgainst not only malaria, but hepatic diseases.The local cytokine environment of the liver may af-

ect the type of immune responses generated at thisite. Manipulation of the cytokine environment by co-dministration with cytokine-encoding plasmids hasotential to allow the tailoring of a DNA vaccine tonsure that protective immune response is initiated.nalysis of cell-mediated immunity revealed thatD81 T cells, IFN-g and iNOS were induced in the

ivers of the protected mice immediately after rechal-enge. In addition, the induction of iNOS in the liverequires CD81 T cells and IFN-g. Thus, our resultstrongly support the notion that coadministration ofhe PbCS gene with the IL-12 gene may induce Th1-ype immune responses into the hepatic lymphocytes,esulting in local secretion of IFN-g and stimulation ofiver cells to produce NO for the destruction of infectedepatocytes or the parasite within these cells.We demonstrate here for the first time the efficacy of

ocal immunization into the liver with malaria DNAaccines by gene gun directly in eliciting protectivemmune responses. Our data indicate that introductionf DNA to target organs such as the liver in the case ofalaria may improve the prospects for the develop-ent of an efficient vaccination strategy for the pre-

rythrocytic antigens. To extend the present study asn application for clinical use, we used an anioniciposome containing hemagglutinating virus of JapanHVJ-liposome) to deliver DNA vaccine into the liverntravenously. This gene transfer method has beennown to be efficient for recovery from rat liver cirrho-is by inoculation of HVJ-liposome with the hepatocyterowth factor gene (37). This method, however, did notork well for a malaria DNA vaccine compared withirect immunization into the liver, suggesting the im-

114

ells in the liver (data not shown).Further work is required to establish a novel DNA

elivery system acceptable for clinical use. Moreover,he liver is an important and attractive target for theevelopment of not only malaria vaccine, but geneherapy strategy. Widespread application of hepaticene transfer in humans awaits the development of aafe and simple delivery system and inducible tissue-pecific promoters that can direct efficient transgenexpression in vivo.

CKNOWLEDGMENTS

We thank Dr. S. Wolf for the mouse IL-12 gene and anti-IL-12Ab, Dr. T. Aji for anti-CD8 mAb, and Dr. R. Nussenzweig for 3D11Ab. We also thank Dr. R. E. Sinden for critical review of theanuscript. This work was supported in part by a Grant-in-Aid forcientific Research on Priority Areas (08281104) from the Ministryf Education, Science, Culture, and Sports of Japan and by a Grant-n-Aid for Scientific Research (10671129) from the Ministry of Edu-ation, Science, Culture, and Sports of Japan.

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