Vaccine 23 (2004) 672–680

Cationic microparticles are a potent delivery systemfor a HCV DNA vaccine

Derek T. O’Hagana,∗, Manmohan Singha, Christine Donga, Mildred Ugozzolia, Kim Bergera,Edward Glazera, Mark Selbyb, Mark Winingera, Philip Nga, Kevin Crawforda,

Xavier Paliardc, Steven Coatesa, Michael Houghtona

a Vaccines Research, Chiron Corporation, 4560 Horton St., M/S 4.3, Emeryville, CA 94608 USAb Medarex, Milpitas, CA, USA

c Gryphon Therapeutics, South San Francisco, CA, USA

Received 14 November 2003; received in revised form 10 June 2004; accepted 15 June 2004Available online 9 August 2004

Abstract

inducea lide) (PLG)m icalf nif-i responses tor ith proteini a study inr s appearedl mparabler A inducedm peripheralb©

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We initially evaluated in mice the ability of naked DNA encoding intracellular forms of the E1E2 envelope proteins from HCV tontibody responses and compared the responses induced with the same plasmid adsorbed onto cationic poly (lactide co-glycoicroparticles. Although naked DNA was only able to induce detectable responses at the 100�g dose level, making this approach impract

or evaluation in larger animals, PLG/DNA induced detectable responses at 10�g. In addition, the PLG/DNA microparticles induced sigcantly enhanced responses to naked DNA when compared at the same dose level. Remarkably, PLG/DNA induced comparableecombinant E1E2 protein adjuvanted with the emulsion MF59. Furthermore, PLG/DNA effectively primed for a booster response wmmunization, while naked DNA did not. Therefore, PLG/DNA was selected for further evaluation in a non-human primate model. Inhesus macaques, PLG/DNA induced seroconversion in 3/3 animals following three immunizations. Although the antibody responseower than those induced with recombinant protein adjuvanted with MF59, following a fourth dose, PLG/DNA and protein induced coesponses. However, a single booster dose of recombinant protein administered to the animals previously immunized with PLG/DNuch higher responses. In addition, one of three animals immunized with PLG/DNA showed a cytotoxic T lymphocyte response inlood lymphocytes. In conclusion, cationic PLG microparticles with adsorbed HCV DNA generates potent immune responses.2004 Elsevier Ltd. All rights reserved.

eywords:PLG microparticles; DNA vaccine; HCV vaccine

. Introduction

The hepatitis C virus (HCV) was identified over aecade ago and is now recognized as the leading causef parenterally-transmitted non-A and non-B viral hepati-

is [1,2]. HCV is now known to infect approximately 3% ofhe worlds population, an estimated 200 million people[3].urrently, about 30,000 newly acquired HCV infections oc-ur in the US annually, most of which have IV drug use asrisk factor[4]. However, in the remaining cases, multiple

∗ Corresponding author. Tel.: +1 510 923 7662; fax: +1 510 923 2586.E-mail address:derekohagan@chiron.com (D.T. O’Hagan).

sexual partners are a defined risk factor as too is any acsubjecting individuals to exposure to contaminated ble.g. health care workers, dialysis patients, organ transrecipients, etc.[4,5]. In many developing countries, therea huge incidence of HCV infection, often due to probleof needle re-use and contamination. Although the immresponse is capable of clearing HCV infection, the majoof infections become chronic. However, most acute intions remain asymptomatic and liver disease usually oconly after years of chronic infection[6,7]. Currently, thereis no vaccine available to prevent HCV infection andonly available therapies, IFN-� and ribavirin, are effective iless than half the patients treated[8,9]. Therefore, there is a

264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2004.06.037

D.T. O’Hagan et al. / Vaccine 23 (2004) 672–680 673

urgent need for the development of an efficacious vaccine toprevent HCV infection and also for the development of im-munotherapies to be used as an alternative, or in conjunctionwith existing therapies.

Although there is little firm data to correlate immunitywith prevention of infection, there are growing indications forthe role of HCV-specific immune responses in the resolutionor amelioration of infection and disease. Several studies havesuggested that T cell immunity to HCV can determine theoutcome of HCV infection and disease[10–13]. One studyconcluded that individuals displaying predominant Th0/Th1CD4+ T helper responses resolved their HCV infections,while those with Th2 type responses tended to progress tochronicity[14]. In addition, it has been shown that there is aninverse correlation between the frequency of HCV-specificcytotoxic T lymphocytes (CTLs) and viral load[15]. Re-cently, control of HCV in chimpanzees was associated witha Th1 T cellular immune response[16]. Therefore, accumu-lated evidence suggests an important role for HCV-specificT cell responses in controlling HCV infection. Nevertheless,chimpanzee’s immunized with the recombinant envelope gly-coprotein (E1E2) were protected against experimental chal-lenge with homologous virus[17]. In addition, protectionwas associated directly with the titer of anti-E1E2 antibod-ies, suggesting a likely role for antibodies in protection[17].A stedf ctioni m-m iesh xper-i ei ipi-e rs[ ainstH h Tc beb

rgeda CTLaH ed toh pears nsesa -t ducedi ) ons ibodyr d inh n de-v vep po-t manp needf icu-l de-s cles

as a delivery system for DNA vaccines, which dramaticallyimproved vaccine potency[28]. We used the biodegradablepolymer, PLG and a charged surfactant to prepare cationicmicroparticles to which DNA strongly adsorbed, allowinghigh loading efficiency with several plasmids, and protectionagainst degradation for the adsorbed DNA[29]. In a numberof studies, involving plasmids encoding antigens from HIV,we showed that microparticles enhanced the potency of DNAvaccines for both humoral and T cell responses in a range ofanimal models[28–30]. However, the HIV plasmids weredesigned for high level of antigen secretion from cells[31].

In the current studies, we evaluated cationic microparti-cles as a delivery system for a DNA vaccine encoding theHCV envelope proteins E1E2 as a non-secreted intracellularheterodimer, and compared the responses induced with nakedDNA and with adjuvanted protein in mice. In addition, wealso evaluated DNA prime and protein boost regimens. Fol-lowing encouraging studies in mice, preliminary studies wereundertaken to evaluate the microparticle formulation in rhe-sus macaques and the responses obtained were compared tothose obtained using E1E2 protein adjuvanted with MF59emulsion[32,33].

2. Experimental procedures

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role for antibodies in protection has also been suggerom rare cases of spontaneous resolution of chronic infen patients[18]. Furthermore, administration of human i

unoglobulin preparations containing anti-HCV antibodas also been shown to ameliorate acute hepatitis C in e

mentally infected chimpanzees[19] as well as reducing thncidence of transmission of HCV in liver transplant recnts[20], transfusion recipients[21,22] and sexual partne

23]. Hence, induction of potent antibody responses agCV would appear to be an attractive strategy, althougells, particularly of the Th1 type would also appear toeneficial.

In the relatively recent past, DNA vaccines have emes an attractive approach for inducing potent long termnd Th1 cellular responses in a range of animal models[24].owever, although DNA vaccines have been administeruman volunteers in a number of clinical trials and apafe, their potency has been low relative to the respochieved in smaller animal models[24]. In particular, al

hough detectable CTL and Th1 responses have been inn human volunteers, even high doses of DNA (2.5 mgeveral occasions, have failed to induce detectable antesponses[25,26]. Antibody responses were not detecteuman volunteers even when a needle-free jet injectioice was used for DNA delivery in an attempt to improotency[27]. Hence, although the reasons for lack of

ency of DNA vaccines in human subjects and non-hurimates remains poorly understood, there is a clear

or the potency of DNA vaccines to be improved, partarly in relation to the humoral response. We recentlycribed a novel approach involving cationic microparti

.1. Plasmid design

The plasmid pCMVtpaE1E2p7 (6275 bp) was construy cloning HCV-I encoding amino acids 192–809 w

he upstream tissue plasminogen activator signal sequnto the pnewCMV-II expression vector, but also hadatural transmembrane anchor domains at the c-terf the E1 and E2. The vector contains the human

omegalovirus enhancer/promoter, intron A, bovine groormone polyadenylation sequence and an ampicillin r

ance gene.

.2. Cloning, expression and purification of E1E2rotein

E1E2192–809 was expressed from recombinant CHO cs described previously[34]. E1E2 antigen was extract

rom inside the CHO cells with Triton X-100 detergehe E1E2 antigen was purified usingGalanthus nivalisectin agarose (Vector Laboratories, Burlingame, Chromatography and fast flow S-Sepharose cation-exchhromatography (Pharmacia) as previously described[1].he oil-in-water adjuvant MF59 was manufactured at Chaccines, Marburg and was previously described[33].

.3. Chemicals reagents

PLG (RG 504, 50:50 lactide:glycolide monomer raas obtained from Boehringer Ingelheim, USA. CTAB wbtained from Sigma Chemical Co., St. Louis, USA

674 D.T. O’Hagan et al. / Vaccine 23 (2004) 672–680

was used as shipped. For the CTL assays, 54 peptides (eachtwenty amino acids in length overlapping by 10 amino acidswith the preceding/subsequent peptide) spanning HCV-1aE1 and E2 amino acids 191–740 were synthesized withfree amine N-termini and free acid C termini by ChironMimotopes Pty. Ltd. (Clayton, Australia). The lyophilizedpeptides were resuspended in 10% DMSO in water, and theneach was diluted to 2 mg/ml. Using equal volumes of eachpeptide, two pools of twenty-seven peptides each were made:pool 1 (peptides #1–27 comprised of amino acids 191–470)and pool 2 (peptides #28–54 comprised of amino acids461–740). The amount of peptide that was used in the assayis indicated in the description that follows. U96-Nunc Max-isorp plates (Nalgene Nunc International, Rochester, NY),Goat anti-Mouse IgG-HRP conjugate (Caltag Laboratories,Burlingame, CA), and TMB Microwell Peroxidase SubstrateSystem (Kirkegaard & Perry Laboratories, Gaithersburg,MD) were used for the ELISA.

2.4. The preparation and characterization ofPLG/CTAB microparticles

The PLG/CTAB microparticles were prepared using a sol-vent evaporation technique essentially as described previ-ously [28,29]. Briefly, the microparticles were prepared bye y-l anI d to5 isr ichw low-i mi-c tiona them lyzer(

mi-c h a2 r-r atedb ntl usea s de-t in0 t ofa trolsw Thes singa K).

2000( t ofC hy-d ubat-i inP ringt

2.5. Mouse studies

The first study was designed to determine if the plas-mid construct, which was designed to produce non-secretedE1E2, was actually able to induce antibody responses. There-fore, groups of 10 female CB6F1 mice aged 6–8 weeks andweighing about 20–25 g were immunized with naked DNAor PLG/DNA at high doses (10 and 100�g), at days 0 and28. The formulations were injected in saline into the anteriortibialis muscle of the two hind legs (50�l per site) of eachanimal. Mice were bled on day 42 through the retro-orbitalplexus and the sera were separated. HCV E1E2 specific serumIgG titers were quantified by ELISA.

Since we were successful in inducing antibody responseswith plasmid adsorbed to PLG at a reasonable dose (10�g),in a second study we further explored the dose range forPLG/DNA and compared the responses to immunization withadjuvanted protein. In groups of 10 mice, we compared 1 and10�g of PLG/DNA to immunization with 2�g of recombi-nant protein in MF59 at 0 and 28 days. An additional group ofmice was immunized with 10�g of naked DNA to comparewith the previous data. Mice were bled and sera was sepa-rated for assay on day 42. The total dose of microparticles ata 1% loading level was 1 mg of PLG.

In the third study in mice, we challenged our own obser-v an-t , anda pro-t ntgP inM s of1 kedD erg orn h at pro-t lsw eeksa

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mulsifying 10 ml of a 6% (w/v) polymer solution in methene chloride with 1 ml of TE buffer at high speed usingKA homogenizer. The primary emulsion was then adde0 ml of distilled water containing CTAB (0.5%, w/v). Thesulted in the formation of a water/oil/water emulsion whas stirred at 6000 rpm for 12 h at room temperature, al

ng the methylene chloride to evaporate. The resultingroparticles were washed in distilled water by centrifugat 10,000× g and freeze dried. The size distribution oficroparticles was measured using a particle size ana

Malvern Instruments, Malvern, UK).The HCV E1E2 plasmid was adsorbed onto the

roparticles by incubating 100 mg of microparticles wit00 ug/ml solution of DNA in 1× TE buffer under gentle stiing at 4◦C for 12 h. The microparticles were then separy centrifugation, followed by lyophilization. Two differe

oading levels (1 and 4%, w/w) were prepared for the mond rhesus studies. The amount of adsorbed DNA wa

ermined by hydrolysis of the PLG/DNA microparticles.5N NaoH/1% SDS solution followed by measuremenbsorbance at A260 nm. Blank PLG microparticles conere run simultaneously to deduct background value.ize distribution of the microparticles was determined uparticle size analyzer (Malvern Instruments, Malvern, UThe zeta potential was measured on a Zetasizer

Malvern Instruments, Malvern, UK). The total amounTAB in PLG/CTAB microparticles was estimated afterrolysis by HPLC. The 24 h release was estimated by inc

ng 10 mg of freeze dried PLG/CTAB/DNA microparticlesBS at 37◦C and estimating the released DNA by measu

he supernatant at A260 nm.

ations that PLG/DNA was comparable to protein foribody responses by increasing the protein dose levellso looking at prime/boost studies combining DNA and

ein immunization. The initial immunizations in five differeroups of 10 mice were performed with naked DNA (10�g),LG/DNA (10�g) or 5�g of E1E2 protein adjuvantedF59. In the overall study design, three different group0 mice were immunized three times with PLG/DNA, naNA, or E1E2 protein in MF59. In addition, two furthroups of mice received two doses of either PLG/DNAaked DNA (10�g), and both groups were boosted wit

hird immunization, consisting of a single dose of E1E2ein (5�g) adjuvanted in MF59. All five groups of animaere immunized on three occasions, separated by 4 wnd sera was collected on day 70.

.6. Rhesus study

In the mouse studies, we had established that PLG/as significantly better than naked DNA and very surp

ngly, appeared to be similar to adjuvanted protein fornduction of antibody responses. Therefore, in the sn non-human primates, we challenged this observand compared PLG/DNA to immunization with adjuvanrotein. Hence, three rhesus macaques in two groups

mmunized with PLG/DNA (1 mg), or 50�g of E1E2rotein in MF59 at weeks 0, 4, 8 and 24. The total dosicroparticles at a 4% (w/w) loading level was 25 mgLG microparticles. In addition, to assess the prime/bpproach that was also evaluated in mice, all animalsoosted with 40�g of E1E2 protein in MF59 at week 64.

D.T. O’Hagan et al. / Vaccine 23 (2004) 672–680 675

2.7. HCV E1E2 antibody assays

The antibody responses against HCV E1E2 in mice weremeasured on the sera collected 2 weeks after each immuniza-tion by ELISA. Microtiter plates were coated with 200�l ofthe purified HCV E1E2 at 0.625�g/ml overnight at 4◦C. Thecoated wells were blocked for 1 h at 37◦C with 300�l of 1%BSA in phosphate-buffered saline (PBS). The plates werewashed five times with a washing buffer (PBS, 0.3% Tween-20), tapped, and dried. Serum samples and a serum standardwere initially diluted in the blocking buffer and then trans-ferred into coated, blocked plates in which the samples wereserially diluted three-fold with the same buffer. Plates werewashed after 1-h incubation at 37◦C. Horseradish peroxidaseconjugated goat anti-mouse IgG gamma chain specific (Cal-tag Laboratories, Inc.) diluted 1:30,000 in phosphate bufferwas used to determine the total IgG titer. After the 1-h in-cubation at 37◦C, plates were washed to remove unboundantibodies. OPD substrate was used to develop the plates,and the color reaction was blocked after 30 min by the addi-tion of 4N HCl. The titers of IgG antibodies were expressedas the reciprocal of the dilution in which the optical densityof the diluted sample equaled 0.5 at 492 and 620 nm.

The antibody responses against HCV E1E2 in rhesusmacaques were measured following the protocol describeda sso-c

2

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peptides and 50 mCi51Cr for 1.5 h, washed three times, andplated into a 96-well plate at 5× 103 cells/well. The CD8+T cells were plated at three effector to target (E:T) cell ratiosin duplicate. Effectors and targets were incubated togetherfor 4 h in the presence of 3.75× 105 unlabeled targets perwell that were included to minimize lysis of B-LCLs byH.papioand/or endogenous foamy virus-specific CTLs. Super-natants (50 ml) were transferred to Lumaplates (Packard Bio-science, Meriden, CT), and radioactivity was measured witha Wallac Microbeta 1450 scintillation instrument (Perkin-Elmer, Boston, MA). Percent specific lysis was calculated as100× [(mean experimental release− mean spontaneous re-lease)/(mean maximal release− mean spontaneous release)].CTL responses were scored as positive when percent specificlysis at the two highest E:T cell ratios was greater than orequal to the percent lysis of control targets plus 10%.

2.10. Statistical analysis

Data values are expressed as mean± standard error ofthe mean unless otherwise indicated. Serum antibody titersare reported as geometric mean titer. Significant differencesamong groups were ascertained using the ANOVA factorialtest at the 95% confidence interval (StatView 4.4 software;Abacus Concepts, Inc.).

3

3

/w)l r ther erringwm .T utedt en-t er-f 24 hr ) for-m hea 96%(

3

or an to in-d wedt werei s inc eart sen ntrast,

bove, except that goat anti-rhesus (Southern Biotech Aiation, Inc.) was used as secondary antibody.

.8. Cells and cell lines for rhesus studies

Peripheral blood was drawn from the femoral vein whe animals were under anesthesia. PBMCs were obtfter centrifugation over a Ficoll-Hypaque gradient and wultured in 24-well dishes at 5× 106 cells/well. Of thoseells, 1× 106 were sensitized with 10�M of a peptide pooconsisting of individual peptides) for 1 h at 37◦C, washednd added to the remaining 4× 106 untreated PBMCs iml of culture medium (RPMI 1640, 10% heat-inactivaBS, and 1% antibiotics) supplemented with 10 ng/ml of(R&D Systems, Minneapolis, MN). After 48 h, 5% (fin

L2-containing supernatant (T-STIM without PHA, Becickinson Biosciences, Discovery Labware, San Jose,nd 50 U/ml (final) of rIL-2 (Chiron) were added to the c

ures. Cultures were fed every 3–4 days. After 10 days inure, CD8+ T cells were isolated using anti-CD8 Abs bouo magnetic beads (Dynal, Oslo, Norway) according toanufacturer’s instructions. Purified CD8+ cells (>93% pures determined by flow cytometry) were cultured for ano–3 days before being assayed for cytotoxic activity. B-Lere derived from each animal using supernatants fromerpesvirus papio producer cell line S394.

.9. CTL assay

Cytotoxic activity was assessed in a standard51Cr releasessay. Autologous B-LCLs were incubated with 9.25 mg

. Results and discussion

.1. Microparticle characterization

PLG/DNA microparticles were prepared at 1% (woading levels for the mouse studies and at 4% (w/w) fohesus study. The size as measured by laser light scatas determined to be 1.5± 0.6�m for blank PLG/CTABicroparticles and 8.6± 3.4�m after DNA adsorptionhe increase in size after DNA adsorption was attrib

o DNA–DNA bridging after adsorption. The zeta potial of the PLG/DNA formulation re-constituted in wator-injection at pH 6.5 was measured to be +12 mV. Theelease was measured to be 12–20% for the 1% (w/wulationa and 42% for the 4% (w/w) load formulation. Tctual amount of CTAB was estimated to be about 0.w/w) to PLG polymer.

.2. Mouse studies

In the first study, we showed that despite encoding fon-secreted antigen, the plasmid construct was ableuce antibody responses to E1E2. In addition, we sho

hat significantly enhanced antibody responses to E1E2nduced by adsorbing the plasmid to PLG microparticleomparison to immunization with naked DNA. It was clhat 10�g of naked DNA was below the threshold doeeded to induce a detectable immune response. In co

676 D.T. O’Hagan et al. / Vaccine 23 (2004) 672–680

Fig. 1. Serum IgG titers following immunization of mice at 0 and 4 weekswith naked DNA or PLG/DNA at 10 and 100�g (N = 10,±S.E.M.) (P <0.05) for PLG/DNA and DNA at 10�g dose and (P > 0.05) at 100�g dose.

PLG/DNA induced a potent response at 10�g (Fig. 1) (P< 0.05). At the higher dose of 100�g, though, PLG/DNAwas not significantly different from naked DNA (P > 0.05).Therefore, PLG/DNA was appropriate for further evaluation,since the dose level needed to induce an antibody response(10�g) was able to be scaled into larger animals.

The second study confirmed the ability of PLG/DNA toinduce a significantly enhanced response over naked DNAat 10�g, (P < 0.05) but also showed that PLG/DNA didnot induce a potent response at 1�g. Therefore, 10�g ofPLG/DNA was the appropriate dose to evaluate in subsequentstudies. Remarkably, this study also showed that PLG/DNA(10�g) induced a comparable antibody response to 2�g ofE1E2 protein adjuvanted with MF59 (Fig. 2).

The third study served to confirm and extend the remark-able observations from the earlier studies. As previously re-ported, PLG/DNA was significantly more potent than nakedDNA at 10�g after two or three doses (P < 0.05), but more

F eksw ntpa

Fig. 3. Serum IgG titers in mice 2 weeks post second (2wp2) and 2 weekspost third immunization (2wp3), following immunization at 0, 4 and 8 weekswith naked DNA or PLG/DNA (10�g), or E1E2 recombinant protein (5�g)in MF59 adjuvant. In addition, two groups of mice were immunized twicewith naked DNA or PLG/DNA (10�g) at 0 and 4 weeks, and boosted withE1E2 recombinant protein (5�g) in MF59 at 8 weeks (N = 10,±S.E.M.).In the legend, D: DNA (10�g) and P: protein (5�g) in MF59 (P < 0.05)for PLG/DNA and DNA at 10�g dose at both time points (two and threeimmunizations). Also PLG/DXDXP was significantly better (P< 0.05) thanDXDXP after three immunizations.

notably was comparable to immunization with 5�g E1E2protein in MF59. In addition, although three doses of 10�g ofnaked DNA did not induce a detectable response, two dosesof PLG/DNA (10�g) induced a potent response (Fig. 3).Moreover, two doses of PLG/DNA (10�g) primed for a po-tent response following boosting with E1E2 protein in MF59,while naked DNA (10�g) was less effective as a priming reg-imen. Furthermore, three doses of PLG/DNA (10�g) wasequally potent to two doses of PLG/DNA (10�g), followedby a boost with a single dose of 5�g protein in MF59 (Fig. 3).Hence, the rather surprising conclusion from the mouse stud-ies was that PLG/DNA was comparable to adjuvanted proteinfor inducing antibody responses to E1E2.

3.3. Rhesus study

All three rhesus immunized with E1E2 protein in MF59showed serum IgG responses 2 weeks after the second im-munization, which were boosted with a third immunization.However, only 2/3 rhesus immunized with PLG/DNA re-sponded 2 weeks after the second immunization, but all threeanimals responded following a third immunization (Table 1).Therefore, seroconversion was achieved in all three rhesusimmunized with PLG/DNA following a third dose. However,the responses to PLG/DNA appeared to be lower than the re-s s noe romt wass als( NAm hieve

ig. 2. Serum IgG titers following immunization of mice at 0 and 4 weith naked DNA at 10�g, PLG/DNA at 1 and 10�g, or E1E2 recombinarotein in MF59 adjuvant at 2�g (N= 10,±S.E.M.) (P< 0.05) for PLG/DNAnd DNA at 10�g dose.

ponses to protein immunization. In addition, there wavidence of boosting for the two responding animals fhe DNA group for the third dose, although boostingeen following the fourth dose of PLG/DNA in all animTable 2). This finding suggested that the third dose of Day have been spaced too close to the second to ac

D.T. O’Hagan et al. / Vaccine 23 (2004) 672–680 677

Table 1Immunization regimen for two groups of three rhesus macaques immunized with E1E2 PLG/DNA, or E1E2 recombinant protein in MF59, including animalnumbers

Group SFBR animal no. Formulation Dose (route) Immunization schedule (weeks)

1 AY922 HCV E1E2 1 mg (IM) 0, 4, 8, 24 and 64 (40�g protein boost)BB227 PLG/DNABB230

2 15862 HCV E1E2 protein/MF59 50�g (IM) 0, 4, 8, 24 and 64 (40�g protein boost)1586315864

Table 2Serum IgG antibody responses in rhesus macaques immunized with E1E2 PLG/DNA or E1E2 protein in MF59, including animal numbers

E2 antibody titers

Animal immunized with E1E2 + MF59 Animal immunized with E1E2 PLG/DNA

15862 15863 15864 AY922 BB227 BB230

Pre <5 <5 <5 <5 <5 <52w post1st NT NT NT <5 <5 <52w post2nd 550 638 538 150 <5 752w post 3rd 988 763 2488 125 25 7514w post 3rd 113 50 250 <5 <5 <52w post 4th 813 625 6525 375 63 37540w post 4th 25 13 188 <5 <5 <52w post 5th 475 575 1388 925 363 3075

All animals received a dose of protein in MF59 at week 64.

effective boosting. There was a much greater delay betweenthe third and fourth doses, and boosting was achieved fol-lowing the fourth dose with both protein and DNA. After thefourth doses, the response to PLG/DNA was quite encour-aging in comparison to protein. Although there was one lowresponder in the PLG/DNA group (BB227) and one high re-sponder in the protein group (15864), the remaining four ani-mals, two from the protein group and two from the PLG/DNAgroup all had similar titers in the 300–800 range.

A single dose of E1E2 protein induced excellent boostingin rhesus previously immunized with PLG/DNA, while thesame dose of protein given to the animals previously immu-nized four times with protein did not induce a similar level ofboosting. Hence, following five immunizations, we achievedcomparable serum antibody responses in both groups of an-imals which were immunized with protein alone in MF59,or immunized with PLG/DNA followed by a single boosterdose of protein in MF59. Two weeks after the fourth immu-nization with PLG/DNA, we evaluated CTL responses fromPBMCs in all animals. One animal (BB227) out of the threeimmunized with PLG/DNA showed a peptide-specific CTLresponse (Table 3). Perhaps surprisingly, this animal (BB227)was the weakest responder for antibodies.

The ability of cationic PLG microparticles with adsorbedDNA to induce significantly enhanced antibody titers to HCVE kedD ithH lu-a tigeu rent

findings contrasted sharply with a previous study, in whicha plasmid encoding a c-terminal truncation of HCV E2 thatfacilitated secretion did not induce detectable antibody re-sponses in mice even at a 100�g dose level and a proteinbooster dose was required to induce seroconversion[35]. Al-though the E1E2 plasmid evaluated in the current studies wasable to induce detectable titers at a dose of 100�g as nakedDNA in mice, the cationic PLG microparticles with adsorbedDNA were much more potent than naked DNA. Remark-ably, PLG/DNA was comparable to immunization with re-combinant E1E2 protein adjuvanted with MF59, an approachthat has previously been shown to be protective against viralchallenge[17]. Although this observation is consistent withprevious data on HIV plasmids adsorbed to PLG micropar-ticles [30], the E1E2 antigen expressed from the plasmid isvery different from antigens previously evaluated in conjunc-tion with PLG. The env plasmid previously evaluated[29,30]was codon optimized for high level expression in mammaliancells, with optimal secretion of antigen[36], while the gagplasmid[28,30]was also codon optimized and is efficiently

Table 3Cytotoxic T lymphocyte response in rhesus macaque (BB227) immunizedwith PLG/DNA two weeks after the fourth immunization

Effector/Target Un-sensitized Lysis with pool 1

1E2 plasmid in comparison to immunization with naNA in mice is consistent with earlier observations wIV plasmids[28,29] However, the current plasmid evated for E1E2 was not designed to produce secreted annlike the HIV plasmids previously evaluated. The cur

n,

cell ratio controls sensitized targets (%)

40/1 5 2413/1 <1 144/1 <1 12

Percent specific lysis at different effector/target cell ratios.

678 D.T. O’Hagan et al. / Vaccine 23 (2004) 672–680

secreted from cells[31]. In contrast, the E1E2 plasmid usedin the current studies results in production of the antigenintracellularly [34]. The presence of the natural transmem-brane anchor domains at the c-terminii of E1 and E2 results inthe newly-synthesized E1E2 heterodimer glycoprotein beingtightly anchored in the lumen of the endoplasmic reticulum[37,38]. Previous studies with naked DNA had shown thatthe immunogenicity of the E1E2 plasmid was not enhancedby codon optimization (unpublished observations). Hence,a novel and surprising observation in the current studies isthe ability of the PLG microparticles to induce enhanced an-tibody responses to an antigen which is not designed to besecreted from the cells.

In the third mouse study, we evaluated the ability of nakedDNA versus PLG/DNA to prime for a potent antibody re-sponse following a boost with recombinant protein in MF59adjuvant. Although naked DNA was able to prime for a boostresponse by protein, even three doses of naked DNA (10�g)could not initiate a primary response. In contrast, two dosesof PLG/DNA (10�g) induced a potent serum antibody re-sponse. In addition, PLG/DNA was also more effective atpriming for a boost response to protein than naked DNA.Furthermore, a very surprising observation was that threedoses of PLG/DNA was comparable to two doses, followedby a protein boost. On several previous occasions, DNA hasb y re-s ancebt com-p e am oosti movef ues.B nsesi -p ed toc wingt aredP o-t

ed too con-v andt ought thet sioni ded.N quest NAv withP inew d int thes nif-i inantE ility

of PLG/DNA to induce seroconversion in rhesus macaquesis both striking and encouraging. Since these observationsneed to be viewed in light of the previous poor efficacy ofDNA vaccines for the induction of antibody responses in pri-mates, even following large doses on multiple occasions[24].In addition, following four doses of PLG/DNA, the responsesinduced were generally comparable to those induced by ad-juvanted protein. While the relative lack of potency of DNAvaccines generally seen for antibody induction in primateshas not been fully explained, the dose relative to body massappears to be an important contributing factor. In mice, 10�gof E1E2 as naked DNA was unable to induce a significantantibody response, but 100�g did induce seroconversion. Ifthe 100�g dose was scaled to a rhesus from a mouse basedon body mass alone (rhesus∼5 kg), we would predict thata dose of about 20 mg of DNA would be needed to induceseroconversion, which is clearly impractical. Particularly ifone considers the relative weight of a rhesus in compari-son to a human and assuming additional dose scaling wouldprove necessary for human immunization. In contrast, thePLG/DNA induced detectable antibody responses in mice inthe 1–10�g dose range. If the 10�g dose level is scaled toa rhesus based on body mass alone, then a dose of 2 mg ofPLG/DNA might be expected to induce detectable responses.This was indeed the case and the 1mg dose level evaluated forP ver,i cy oft cedt erthe-l ne isa tenti clud-i intowa t thatP las-m fori nota ucea

om-b bles d ex-c ceda l-l rsalH cep can beb oteind tion,D rimeC ctiveia eenl on-h us

een shown to be ineffective at inducing potent antibodponses, but the responses have been significantly enhy a protein boost[35,39]. The ability of PLG/DNA alone

o induce significantly enhanced antibody responses inarison to naked DNA alone, and it’s ability to providore potent prime for an emulsion adjuvanted protein b

n mice were notable observations and encouraged us toorward with the PLG/DNA approach into rhesus macaqecause naked DNA was ineffective for inducing respo

n mice at lower doses (10�g), we did not move this aproach forward into the rhesus study. Instead, we decidhallenge the observations from the mouse studies, shohat PLG/DNA was comparable to protein, and compLG/DNA with immunization with E1E2 recombinant pr

ein adjuvanted with MF59 in rhesus.In the rhesus macaque study, we were very encourag

bserve that the PLG/DNA microparticles induced seroersion in 3/3 animals, following three immunizations,hat the responses were boosted after a fourth dose. Althhere was little boosting of the response to DNA followinghird immunization, the third dose did induce seroconvern the one remaining animal which had not yet responevertheless, the absence of significant boosting raises

ions about the optimal regimen for immunization with Daccines in non-human primates and humans. CertainlyLG/DNA, there is not yet enough experience to determhich is the optimal regimen, but the regimen employe

he current studies may well be sub-optimal. Although,erum IgG responses induced with PLG/DNA were sigcantly less than the responses induced by the recomb1E2 protein in MF59 after two or three doses, the ab

d

-

LG/DNA induced seroconversion in 3/3 rhesus. Howet is reasonable to expect that the dose level, or the potenhe PLG/DNA formulation might need to be further enhano induce detectable responses in human subjects. Nevess, the dose scaling of DNA based on body mass alo

simplistic approach to determine why DNA is less pon primates and other issues need to be considered, inng volume of administration relative to the muscle masshich the vaccine is injected[40] and relative proximity toncillary lymph nodes, etc. Overall, these studies suggesLG microparticles enhanced the potency of the HCV pid sufficiently that DNA may be considered as an option

mmunization of primates. In contrast, naked DNA doesppear to be an option if an important objective is to indntibody responses to the encoded antigen.

Furthermore, following a single booster dose with recinant protein in MF59, the PLG/DNA group had comparaerum IgG titers to the rhesus which had been immunizelusively with E1E2 protein in MF59. Since E1E2 is produs an intracellular antigenic complex[41], there may be cha

enges in manufacturing it at the levels required for a univeCV vaccine. Therefore, the ability of PLG/DNA to induotent antibody responses, and to prime a response thatoosted with a single dose of E1E2 protein provides a prose-sparing option for vaccine development. In addiNA vaccines have become an established option to pTL responses which may also be important in the prote

mmune response against HCV[12]. Apart from ISCOM-djuvanted vaccines[42], protein based vaccines have b

argely ineffective for the induction of CTL responses in numan primates and humans[43]. In one of the three rhes

D.T. O’Hagan et al. / Vaccine 23 (2004) 672–680 679

macaques immunized with PLG/DNA we were able to detecta CTL response following the fourth immunization. AlthoughCTL was not evaluated in the E1E2/MF59 immunized ani-mals, we have sufficient experience with this adjuvant to beconfident that a CTL response would not have been induced(unpublished data).

A number of studies have been undertaken to determinethe mechanism of action for cationic PLG microparticlesto induce enhanced responses to adsorbed DNA vaccines.We have shown that PLG/DNA, but not naked DNA areable to mediate transfection of DC in vitro[44]. In addition,PLG/DNA protects DNA against degradation and enhancesgene expression in muscle and local lymph nodes[28,29,44].Furthermore, PLG/DNA but not naked DNA is able to recruitsignificant numbers of activated APC to the injection site fol-lowing immunization[44]. Which of these mechanisms ismost important for enhancing responses to the non-secretedE1E2 plasmid is currently unknown.

The data obtained in the current studies were sufficientlyencouraging to move the PLG/DNA approach forward intoa non-human primate challenge model with HCV. Thesestudies are ongoing, but initial data have confirmed thatPLG/DNA can induce seroconversion and T cell responsesin non-human primates (Houghton et al., unpublished data).The results from these studies will be reported in detail whent

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[9] McHutchison JG, Gordon SC, Schiff ER, et al. Interferon alfa-2balone or in combination with ribavirin as initial treatment for chronichepatitis C. Hepatitis Interventional Therapy Group. N Engl J Med1998;339(21):1485–92.

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[16] Major ME, Mihalik K, Puig M, et al. Previously infected and re-covered chimpanzees exhibit rapid responses that control hepatitis Cvirus replication upon rechallenge. J Virol 2002;76(13):6586–95.

[17] Choo QL, Kuo G, Ralston R, et al. Vaccination of chimpanzeesagainst infection by the hepatitis C virus. Proc Natl Acad Sci USA1994;91(4):1294–8.

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he challenge studies are completed.

cknowledgements

We would like to thank Nelle Cronen for her help in prepng the manuscript.

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