Cationic microparticles are a potent delivery system for a HCV DNA vaccine

Vaccine 23 (2004) 672–680

Cationic microparticles are a potent delivery system for 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 USA b 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 2004 Available online 9 August 2004

Abstract We initially evaluated in mice the ability of naked DNA encoding intracellular forms of the E1E2 envelope proteins from HCV to induce antibody responses and compared the responses induced with the same plasmid adsorbed onto cationic poly (lactide co-glycolide) (PLG) microparticles. Although naked DNA was only able to induce detectable responses at the 100 ␮g dose level, making this approach impractical for evaluation in larger animals, PLG/DNA induced detectable responses at 10 ␮g. In addition, the PLG/DNA microparticles induced significantly enhanced responses to naked DNA when compared at the same dose level. Remarkably, PLG/DNA induced comparable responses to recombinant E1E2 protein adjuvanted with the emulsion MF59. Furthermore, PLG/DNA effectively primed for a booster response with protein immunization, while naked DNA did not. Therefore, PLG/DNA was selected for further evaluation in a non-human primate model. In a study in rhesus macaques, PLG/DNA induced seroconversion in 3/3 animals following three immunizations. Although the antibody responses appeared lower than those induced with recombinant protein adjuvanted with MF59, following a fourth dose, PLG/DNA and protein induced comparable responses. However, a single booster dose of recombinant protein administered to the animals previously immunized with PLG/DNA induced much higher responses. In addition, one of three animals immunized with PLG/DNA showed a cytotoxic T lymphocyte response in peripheral blood lymphocytes. In conclusion, cationic PLG microparticles with adsorbed HCV DNA generates potent immune responses. © 2004 Elsevier Ltd. All rights reserved. Keywords: PLG microparticles; DNA vaccine; HCV vaccine

1. Introduction The hepatitis C virus (HCV) was identified over a decade ago and is now recognized as the leading cause of parenterally-transmitted non-A and non-B viral hepatitis [1,2]. HCV is now known to infect approximately 3% of the worlds population, an estimated 200 million people [3]. Currently, about 30,000 newly acquired HCV infections occur in the US annually, most of which have IV drug use as a risk factor [4]. However, in the remaining cases, multiple ∗

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0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.06.037

sexual partners are a defined risk factor as too is any activity subjecting individuals to exposure to contaminated blood, e.g. health care workers, dialysis patients, organ transplant recipients, etc. [4,5]. In many developing countries, there is a huge incidence of HCV infection, often due to problems of needle re-use and contamination. Although the immune response is capable of clearing HCV infection, the majority of infections become chronic. However, most acute infections remain asymptomatic and liver disease usually occurs only after years of chronic infection [6,7]. Currently, there is no vaccine available to prevent HCV infection and the only available therapies, IFN-␣ and ribavirin, are effective in less than half the patients treated [8,9]. Therefore, there is an

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urgent need for the development of an efficacious vaccine to prevent HCV infection and also for the development of immunotherapies to be used as an alternative, or in conjunction with existing therapies. Although there is little firm data to correlate immunity with prevention of infection, there are growing indications for the role of HCV-specific immune responses in the resolution or amelioration of infection and disease. Several studies have suggested that T cell immunity to HCV can determine the outcome of HCV infection and disease [10–13]. One study concluded that individuals displaying predominant Th0/Th1 CD4+ T helper responses resolved their HCV infections, while those with Th2 type responses tended to progress to chronicity [14]. In addition, it has been shown that there is an inverse correlation between the frequency of HCV-specific cytotoxic T lymphocytes (CTLs) and viral load [15]. Recently, control of HCV in chimpanzees was associated with a Th1 T cellular immune response [16]. Therefore, accumulated evidence suggests an important role for HCV-specific T cell responses in controlling HCV infection. Nevertheless, chimpanzee’s immunized with the recombinant envelope glycoprotein (E1E2) were protected against experimental challenge with homologous virus [17]. In addition, protection was associated directly with the titer of anti-E1E2 antibodies, suggesting a likely role for antibodies in protection [17]. A role for antibodies in protection has also been suggested from rare cases of spontaneous resolution of chronic infection in patients [18]. Furthermore, administration of human immunoglobulin preparations containing anti-HCV antibodies has also been shown to ameliorate acute hepatitis C in experimentally infected chimpanzees [19] as well as reducing the incidence of transmission of HCV in liver transplant recipients [20], transfusion recipients [21,22] and sexual partners [23]. Hence, induction of potent antibody responses against HCV would appear to be an attractive strategy, although T cells, particularly of the Th1 type would also appear to be beneficial. In the relatively recent past, DNA vaccines have emerged as an attractive approach for inducing potent long term CTL and Th1 cellular responses in a range of animal models [24]. However, although DNA vaccines have been administered to human volunteers in a number of clinical trials and appear safe, their potency has been low relative to the responses achieved in smaller animal models [24]. In particular, although detectable CTL and Th1 responses have been induced in human volunteers, even high doses of DNA (2.5 mg) on several occasions, have failed to induce detectable antibody responses [25,26]. Antibody responses were not detected in human volunteers even when a needle-free jet injection device was used for DNA delivery in an attempt to improve potency [27]. Hence, although the reasons for lack of potency of DNA vaccines in human subjects and non-human primates remains poorly understood, there is a clear need for the potency of DNA vaccines to be improved, particularly in relation to the humoral response. We recently described a novel approach involving cationic microparticles

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as a delivery system for DNA vaccines, which dramatically improved vaccine potency [28]. We used the biodegradable polymer, PLG and a charged surfactant to prepare cationic microparticles to which DNA strongly adsorbed, allowing high loading efficiency with several plasmids, and protection against degradation for the adsorbed DNA [29]. In a number of studies, involving plasmids encoding antigens from HIV, we showed that microparticles enhanced the potency of DNA vaccines for both humoral and T cell responses in a range of animal models [28–30]. However, the HIV plasmids were designed for high level of antigen secretion from cells [31]. In the current studies, we evaluated cationic microparticles as a delivery system for a DNA vaccine encoding the HCV envelope proteins E1E2 as a non-secreted intracellular heterodimer, and compared the responses induced with naked DNA and with adjuvanted protein in mice. In addition, we also evaluated DNA prime and protein boost regimens. Following encouraging studies in mice, preliminary studies were undertaken to evaluate the microparticle formulation in rhesus macaques and the responses obtained were compared to those obtained using E1E2 protein adjuvanted with MF59 emulsion [32,33].

2. Experimental procedures 2.1. Plasmid design The plasmid pCMVtpaE1E2p7 (6275 bp) was constructed by cloning HCV-I encoding amino acids 192–809 with the upstream tissue plasminogen activator signal sequence into the pnewCMV-II expression vector, but also had the natural transmembrane anchor domains at the c-terminii of the E1 and E2. The vector contains the human cytomegalovirus enhancer/promoter, intron A, bovine growth hormone polyadenylation sequence and an ampicillin resistance gene. 2.2. Cloning, expression and purification of E1E2 protein E1E2192–809 was expressed from recombinant CHO cells as described previously [34]. E1E2 antigen was extracted from inside the CHO cells with Triton X-100 detergent. The E1E2 antigen was purified using Galanthus nivalis lectin agarose (Vector Laboratories, Burlingame, CA) chromatography and fast flow S-Sepharose cation-exchange chromatography (Pharmacia) as previously described [1]. The oil-in-water adjuvant MF59 was manufactured at Chiron Vaccines, Marburg and was previously described [33]. 2.3. Chemicals reagents PLG (RG 504, 50:50 lactide:glycolide monomer ratio) was obtained from Boehringer Ingelheim, USA. CTAB was obtained from Sigma Chemical Co., St. Louis, USA and

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was used as shipped. For the CTL assays, 54 peptides (each twenty amino acids in length overlapping by 10 amino acids with the preceding/subsequent peptide) spanning HCV-1a E1 and E2 amino acids 191–740 were synthesized with free amine N-termini and free acid C termini by Chiron Mimotopes Pty. Ltd. (Clayton, Australia). The lyophilized peptides were resuspended in 10% DMSO in water, and then each was diluted to 2 mg/ml. Using equal volumes of each peptide, 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 acids 461–740). The amount of peptide that was used in the assay is indicated in the description that follows. U96-Nunc Maxisorp plates (Nalgene Nunc International, Rochester, NY), Goat anti-Mouse IgG-HRP conjugate (Caltag Laboratories, Burlingame, CA), and TMB Microwell Peroxidase Substrate System (Kirkegaard & Perry Laboratories, Gaithersburg, MD) were used for the ELISA. 2.4. The preparation and characterization of PLG/CTAB microparticles The PLG/CTAB microparticles were prepared using a solvent evaporation technique essentially as described previously [28,29]. Briefly, the microparticles were prepared by emulsifying 10 ml of a 6% (w/v) polymer solution in methylene chloride with 1 ml of TE buffer at high speed using an IKA homogenizer. The primary emulsion was then added to 50 ml of distilled water containing CTAB (0.5%, w/v). This resulted in the formation of a water/oil/water emulsion which was stirred at 6000 rpm for 12 h at room temperature, allowing the methylene chloride to evaporate. The resulting microparticles were washed in distilled water by centrifugation at 10,000 × g and freeze dried. The size distribution of the microparticles was measured using a particle size analyzer (Malvern Instruments, Malvern, UK). The HCV E1E2 plasmid was adsorbed onto the microparticles by incubating 100 mg of microparticles with a 200 ug/ml solution of DNA in 1× TE buffer under gentle stirring at 4 ◦ C for 12 h. The microparticles were then separated by centrifugation, followed by lyophilization. Two different loading levels (1 and 4%, w/w) were prepared for the mouse and rhesus studies. The amount of adsorbed DNA was determined by hydrolysis of the PLG/DNA microparticles in 0.5N NaoH/1% SDS solution followed by measurement of absorbance at A260 nm. Blank PLG microparticles controls were run simultaneously to deduct background value. The size distribution of the microparticles was determined using a particle size analyzer (Malvern Instruments, Malvern, UK). The zeta potential was measured on a Zetasizer 2000 (Malvern Instruments, Malvern, UK). The total amount of CTAB in PLG/CTAB microparticles was estimated after hydrolysis by HPLC. The 24 h release was estimated by incubating 10 mg of freeze dried PLG/CTAB/DNA microparticles in PBS at 37 ◦ C and estimating the released DNA by measuring the supernatant at A260 nm.

2.5. Mouse studies The first study was designed to determine if the plasmid construct, which was designed to produce non-secreted E1E2, was actually able to induce antibody responses. Therefore, groups of 10 female CB6F1 mice aged 6–8 weeks and weighing about 20–25 g were immunized with naked DNA or PLG/DNA at high doses (10 and 100 ␮g), at days 0 and 28. The formulations were injected in saline into the anterior tibialis muscle of the two hind legs (50 ␮l per site) of each animal. Mice were bled on day 42 through the retro-orbital plexus and the sera were separated. HCV E1E2 specific serum IgG titers were quantified by ELISA. Since we were successful in inducing antibody responses with plasmid adsorbed to PLG at a reasonable dose (10 ␮g), in a second study we further explored the dose range for PLG/DNA and compared the responses to immunization with adjuvanted protein. In groups of 10 mice, we compared 1 and 10 ␮g of PLG/DNA to immunization with 2 ␮g of recombinant protein in MF59 at 0 and 28 days. An additional group of mice was immunized with 10 ␮g of naked DNA to compare with the previous data. Mice were bled and sera was separated for assay on day 42. The total dose of microparticles at a 1% loading level was 1 mg of PLG. In the third study in mice, we challenged our own observations that PLG/DNA was comparable to protein for antibody responses by increasing the protein dose level, and also looking at prime/boost studies combining DNA and protein immunization. The initial immunizations in five different groups of 10 mice were performed with naked DNA (10 ␮g), PLG/DNA (10 ␮g) or 5 ␮g of E1E2 protein adjuvanted in MF59. In the overall study design, three different groups of 10 mice were immunized three times with PLG/DNA, naked DNA, or E1E2 protein in MF59. In addition, two further groups of mice received two doses of either PLG/DNA or naked DNA (10 ␮g), and both groups were boosted with a third immunization, consisting of a single dose of E1E2 protein (5 ␮g) adjuvanted in MF59. All five groups of animals were immunized on three occasions, separated by 4 weeks and sera was collected on day 70. 2.6. Rhesus study In the mouse studies, we had established that PLG/DNA was significantly better than naked DNA and very surprisingly, appeared to be similar to adjuvanted protein for the induction of antibody responses. Therefore, in the study in non-human primates, we challenged this observation and compared PLG/DNA to immunization with adjuvanted protein. Hence, three rhesus macaques in two groups were immunized with PLG/DNA (1 mg), or 50 ␮g of E1E2 protein in MF59 at weeks 0, 4, 8 and 24. The total dose of microparticles at a 4% (w/w) loading level was 25 mg of PLG microparticles. In addition, to assess the prime/boost approach that was also evaluated in mice, all animals were boosted with 40 ␮g of E1E2 protein in MF59 at week 64.

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2.7. HCV E1E2 antibody assays The antibody responses against HCV E1E2 in mice were measured on the sera collected 2 weeks after each immunization by ELISA. Microtiter plates were coated with 200 ␮l of the purified HCV E1E2 at 0.625 ␮g/ml overnight at 4 ◦ C. The coated wells were blocked for 1 h at 37 ◦ C with 300 ␮l of 1% BSA in phosphate-buffered saline (PBS). The plates were washed five times with a washing buffer (PBS, 0.3% Tween20), tapped, and dried. Serum samples and a serum standard were initially diluted in the blocking buffer and then transferred into coated, blocked plates in which the samples were serially diluted three-fold with the same buffer. Plates were washed after 1-h incubation at 37 ◦ C. Horseradish peroxidase conjugated goat anti-mouse IgG gamma chain specific (Caltag Laboratories, Inc.) diluted 1:30,000 in phosphate buffer was used to determine the total IgG titer. After the 1-h incubation at 37 ◦ C, plates were washed to remove unbound antibodies. OPD substrate was used to develop the plates, and the color reaction was blocked after 30 min by the addition of 4N HCl. The titers of IgG antibodies were expressed as the reciprocal of the dilution in which the optical density of the diluted sample equaled 0.5 at 492 and 620 nm. The antibody responses against HCV E1E2 in rhesus macaques were measured following the protocol described above, except that goat anti-rhesus (Southern Biotech Association, Inc.) was used as secondary antibody.

peptides and 50 mCi 51 Cr for 1.5 h, washed three times, and plated into a 96-well plate at 5 × 103 cells/well. The CD8+ T cells were plated at three effector to target (E:T) cell ratios in duplicate. Effectors and targets were incubated together for 4 h in the presence of 3.75 × 105 unlabeled targets per well that were included to minimize lysis of B-LCLs by H. papio and/or endogenous foamy virus-specific CTLs. Supernatants (50 ml) were transferred to Lumaplates (Packard Bioscience, Meriden, CT), and radioactivity was measured with a Wallac Microbeta 1450 scintillation instrument (PerkinElmer, Boston, MA). Percent specific lysis was calculated as 100× [(mean experimental release − mean spontaneous release)/(mean maximal release − mean spontaneous release)]. CTL responses were scored as positive when percent specific lysis at the two highest E:T cell ratios was greater than or equal to the percent lysis of control targets plus 10%. 2.10. Statistical analysis Data values are expressed as mean ± standard error of the mean unless otherwise indicated. Serum antibody titers are reported as geometric mean titer. Significant differences among groups were ascertained using the ANOVA factorial test at the 95% confidence interval (StatView 4.4 software; Abacus Concepts, Inc.).

2.8. Cells and cell lines for rhesus studies

3. Results and discussion

Peripheral blood was drawn from the femoral vein while the animals were under anesthesia. PBMCs were obtained after centrifugation over a Ficoll-Hypaque gradient and were cultured in 24-well dishes at 5 × 106 cells/well. Of those cells, 1 × 106 were sensitized with 10 ␮M of a peptide pool (consisting of individual peptides) for 1 h at 37 ◦ C, washed and added to the remaining 4 × 106 untreated PBMCs in 2 ml of culture medium (RPMI 1640, 10% heat-inactivated FBS, and 1% antibiotics) supplemented with 10 ng/ml of IL7 (R&D Systems, Minneapolis, MN). After 48 h, 5% (final) IL2-containing supernatant (T-STIM without PHA, Becton Dickinson Biosciences, Discovery Labware, San Jose, CA) and 50 U/ml (final) of rIL-2 (Chiron) were added to the cultures. Cultures were fed every 3–4 days. After 10 days in culture, CD8+ T cells were isolated using anti-CD8 Abs bound to magnetic beads (Dynal, Oslo, Norway) according to the manufacturer’s instructions. Purified CD8+ cells (>93% pure as determined by flow cytometry) were cultured for another 2–3 days before being assayed for cytotoxic activity. B-LCLs were derived from each animal using supernatants from the Herpesvirus papio producer cell line S394.

3.1. Microparticle characterization

2.9. CTL assay Cytotoxic activity was assessed in a standard 51 Cr release assay. Autologous B-LCLs were incubated with 9.25 mg/ml

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PLG/DNA microparticles were prepared at 1% (w/w) loading levels for the mouse studies and at 4% (w/w) for the rhesus study. The size as measured by laser light scaterring was determined to be 1.5 ± 0.6 ␮m for blank PLG/CTAB microparticles and 8.6 ± 3.4 ␮m after DNA adsorption. The increase in size after DNA adsorption was attributed to DNA–DNA bridging after adsorption. The zeta potential of the PLG/DNA formulation re-constituted in waterfor-injection at pH 6.5 was measured to be +12 mV. The 24 h release was measured to be 12–20% for the 1% (w/w) formulationa and 42% for the 4% (w/w) load formulation. The actual amount of CTAB was estimated to be about 0.96% (w/w) to PLG polymer. 3.2. Mouse studies In the first study, we showed that despite encoding for a non-secreted antigen, the plasmid construct was able to induce antibody responses to E1E2. In addition, we showed that significantly enhanced antibody responses to E1E2 were induced by adsorbing the plasmid to PLG microparticles in comparison to immunization with naked DNA. It was clear that 10 ␮g of naked DNA was below the threshold dose needed to induce a detectable immune response. In contrast,

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Fig. 1. Serum IgG titers following immunization of mice at 0 and 4 weeks with 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/DNA was 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 to induce a significantly enhanced response over naked DNA at 10 ␮g, (P < 0.05) but also showed that PLG/DNA did not induce a potent response at 1 ␮g. Therefore, 10 ␮g of PLG/DNA was the appropriate dose to evaluate in subsequent studies. Remarkably, this study also showed that PLG/DNA (10 ␮g) induced a comparable antibody response to 2 ␮g of E1E2 protein adjuvanted with MF59 (Fig. 2). The third study served to confirm and extend the remarkable observations from the earlier studies. As previously reported, PLG/DNA was significantly more potent than naked DNA at 10 ␮g after two or three doses (P < 0.05), but more

Fig. 3. Serum IgG titers in mice 2 weeks post second (2wp2) and 2 weeks post third immunization (2wp3), following immunization at 0, 4 and 8 weeks with naked DNA or PLG/DNA (10 ␮g), or E1E2 recombinant protein (5 ␮g) in MF59 adjuvant. In addition, two groups of mice were immunized twice with naked DNA or PLG/DNA (10 ␮g) at 0 and 4 weeks, and boosted with E1E2 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 three immunizations). Also PLG/DXDXP was significantly better (P < 0.05) than DXDXP after three immunizations.

notably was comparable to immunization with 5 ␮g E1E2 protein in MF59. In addition, although three doses of 10 ␮g of naked DNA did not induce a detectable response, two doses of PLG/DNA (10 ␮g) induced a potent response (Fig. 3). Moreover, two doses of PLG/DNA (10 ␮g) primed for a potent response following boosting with E1E2 protein in MF59, while naked DNA (10 ␮g) was less effective as a priming regimen. Furthermore, three doses of PLG/DNA (10 ␮g) was equally potent to two doses of PLG/DNA (10 ␮g), followed by a boost with a single dose of 5 ␮g protein in MF59 (Fig. 3). Hence, the rather surprising conclusion from the mouse studies was that PLG/DNA was comparable to adjuvanted protein for inducing antibody responses to E1E2. 3.3. Rhesus study

Fig. 2. Serum IgG titers following immunization of mice at 0 and 4 weeks with naked DNA at 10 ␮g, PLG/DNA at 1 and 10 ␮g, or E1E2 recombinant protein in MF59 adjuvant at 2 ␮g (N = 10, ±S.E.M.) (P < 0.05) for PLG/DNA and DNA at 10 ␮g dose.

All three rhesus immunized with E1E2 protein in MF59 showed serum IgG responses 2 weeks after the second immunization, which were boosted with a third immunization. However, only 2/3 rhesus immunized with PLG/DNA responded 2 weeks after the second immunization, but all three animals responded following a third immunization (Table 1). Therefore, seroconversion was achieved in all three rhesus immunized with PLG/DNA following a third dose. However, the responses to PLG/DNA appeared to be lower than the responses to protein immunization. In addition, there was no evidence of boosting for the two responding animals from the DNA group for the third dose, although boosting was seen following the fourth dose of PLG/DNA in all animals (Table 2). This finding suggested that the third dose of DNA may have been spaced too close to the second to achieve

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

SFBR animal no.

Formulation

Dose (route)

Immunization schedule (weeks)

1

AY922 BB227 BB230

HCV E1E2 PLG/DNA

1 mg (IM)

0, 4, 8, 24 and 64 (40 ␮g protein boost)

2

15862 15863 15864

HCV E1E2 protein/MF59

50 ␮g (IM)

0, 4, 8, 24 and 64 (40 ␮g protein boost)

Table 2 Serum 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

Pre 2w post1st 2w post2nd 2w post 3rd 14w post 3rd 2w post 4th 40w post 4th 2w post 5th

Animal immunized with E1E2 PLG/DNA

15862

15863

15864

AY922

BB227

BB230

<5 NT 550 988 113 813 25 475

<5 NT 638 763 50 625 13 575

<5 NT 538 2488 250 6525 188 1388

<5 <5 150 125 <5 375 <5 925

<5 <5 <5 25 <5 63 <5 363

<5 <5 75 75 <5 375 <5 3075

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

effective boosting. There was a much greater delay between the third and fourth doses, and boosting was achieved following the fourth dose with both protein and DNA. After the fourth doses, the response to PLG/DNA was quite encouraging in comparison to protein. Although there was one low responder in the PLG/DNA group (BB227) and one high responder in the protein group (15864), the remaining four animals, two from the protein group and two from the PLG/DNA group all had similar titers in the 300–800 range. A single dose of E1E2 protein induced excellent boosting in rhesus previously immunized with PLG/DNA, while the same dose of protein given to the animals previously immunized four times with protein did not induce a similar level of boosting. Hence, following five immunizations, we achieved comparable serum antibody responses in both groups of animals which were immunized with protein alone in MF59, or immunized with PLG/DNA followed by a single booster dose of protein in MF59. Two weeks after the fourth immunization with PLG/DNA, we evaluated CTL responses from PBMCs in all animals. One animal (BB227) out of the three immunized with PLG/DNA showed a peptide-specific CTL response (Table 3). Perhaps surprisingly, this animal (BB227) was the weakest responder for antibodies. The ability of cationic PLG microparticles with adsorbed DNA to induce significantly enhanced antibody titers to HCV E1E2 plasmid in comparison to immunization with naked DNA in mice is consistent with earlier observations with HIV plasmids [28,29] However, the current plasmid evaluated for E1E2 was not designed to produce secreted antigen, unlike the HIV plasmids previously evaluated. The current

findings contrasted sharply with a previous study, in which a plasmid encoding a c-terminal truncation of HCV E2 that facilitated secretion did not induce detectable antibody responses in mice even at a 100 ␮g dose level and a protein booster dose was required to induce seroconversion [35]. Although the E1E2 plasmid evaluated in the current studies was able to induce detectable titers at a dose of 100 ␮g as naked DNA in mice, the cationic PLG microparticles with adsorbed DNA were much more potent than naked DNA. Remarkably, PLG/DNA was comparable to immunization with recombinant E1E2 protein adjuvanted with MF59, an approach that has previously been shown to be protective against viral challenge [17]. Although this observation is consistent with previous data on HIV plasmids adsorbed to PLG microparticles [30], the E1E2 antigen expressed from the plasmid is very different from antigens previously evaluated in conjunction with PLG. The env plasmid previously evaluated [29,30] was codon optimized for high level expression in mammalian cells, with optimal secretion of antigen [36], while the gag plasmid [28,30] was also codon optimized and is efficiently Table 3 Cytotoxic T lymphocyte response in rhesus macaque (BB227) immunized with PLG/DNA two weeks after the fourth immunization Effector/Target cell ratio

Un-sensitized controls

Lysis with pool 1 sensitized targets (%)

40/1 13/1 4/1

5 <1 <1

24 14 12

Percent specific lysis at different effector/target cell ratios.

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secreted from cells [31]. In contrast, the E1E2 plasmid used in the current studies results in production of the antigen intracellularly [34]. The presence of the natural transmembrane anchor domains at the c-terminii of E1 and E2 results in the newly-synthesized E1E2 heterodimer glycoprotein being tightly anchored in the lumen of the endoplasmic reticulum [37,38]. Previous studies with naked DNA had shown that the immunogenicity of the E1E2 plasmid was not enhanced by codon optimization (unpublished observations). Hence, a novel and surprising observation in the current studies is the ability of the PLG microparticles to induce enhanced antibody responses to an antigen which is not designed to be secreted from the cells. In the third mouse study, we evaluated the ability of naked DNA versus PLG/DNA to prime for a potent antibody response following a boost with recombinant protein in MF59 adjuvant. Although naked DNA was able to prime for a boost response by protein, even three doses of naked DNA (10 ␮g) could not initiate a primary response. In contrast, two doses of PLG/DNA (10 ␮g) induced a potent serum antibody response. In addition, PLG/DNA was also more effective at priming for a boost response to protein than naked DNA. Furthermore, a very surprising observation was that three doses of PLG/DNA was comparable to two doses, followed by a protein boost. On several previous occasions, DNA has been shown to be ineffective at inducing potent antibody responses, but the responses have been significantly enhanced by a protein boost [35,39]. The ability of PLG/DNA alone to induce significantly enhanced antibody responses in comparison to naked DNA alone, and it’s ability to provide a more potent prime for an emulsion adjuvanted protein boost in mice were notable observations and encouraged us to move forward with the PLG/DNA approach into rhesus macaques. Because naked DNA was ineffective for inducing responses in mice at lower doses (10 ␮g), we did not move this approach forward into the rhesus study. Instead, we decided to challenge the observations from the mouse studies, showing that PLG/DNA was comparable to protein, and compared PLG/DNA with immunization with E1E2 recombinant protein adjuvanted with MF59 in rhesus. In the rhesus macaque study, we were very encouraged to observe that the PLG/DNA microparticles induced seroconversion in 3/3 animals, following three immunizations, and that the responses were boosted after a fourth dose. Although there was little boosting of the response to DNA following the third immunization, the third dose did induce seroconversion in the one remaining animal which had not yet responded. Nevertheless, the absence of significant boosting raises questions about the optimal regimen for immunization with DNA vaccines in non-human primates and humans. Certainly with PLG/DNA, there is not yet enough experience to determine which is the optimal regimen, but the regimen employed in the current studies may well be sub-optimal. Although, the serum IgG responses induced with PLG/DNA were significantly less than the responses induced by the recombinant E1E2 protein in MF59 after two or three doses, the ability

of PLG/DNA to induce seroconversion in rhesus macaques is both striking and encouraging. Since these observations need to be viewed in light of the previous poor efficacy of DNA vaccines for the induction of antibody responses in primates, even following large doses on multiple occasions [24]. In addition, following four doses of PLG/DNA, the responses induced were generally comparable to those induced by adjuvanted protein. While the relative lack of potency of DNA vaccines generally seen for antibody induction in primates has not been fully explained, the dose relative to body mass appears to be an important contributing factor. In mice, 10 ␮g of E1E2 as naked DNA was unable to induce a significant antibody response, but 100 ␮g did induce seroconversion. If the 100 ␮g dose was scaled to a rhesus from a mouse based on body mass alone (rhesus ∼5 kg), we would predict that a dose of about 20 mg of DNA would be needed to induce seroconversion, which is clearly impractical. Particularly if one considers the relative weight of a rhesus in comparison to a human and assuming additional dose scaling would prove necessary for human immunization. In contrast, the PLG/DNA induced detectable antibody responses in mice in the 1–10 ␮g dose range. If the 10 ␮g dose level is scaled to a rhesus based on body mass alone, then a dose of 2 mg of PLG/DNA might be expected to induce detectable responses. This was indeed the case and the 1mg dose level evaluated for PLG/DNA induced seroconversion in 3/3 rhesus. However, it is reasonable to expect that the dose level, or the potency of the PLG/DNA formulation might need to be further enhanced to induce detectable responses in human subjects. Nevertheless, the dose scaling of DNA based on body mass alone is a simplistic approach to determine why DNA is less potent in primates and other issues need to be considered, including volume of administration relative to the muscle mass into which the vaccine is injected [40] and relative proximity to ancillary lymph nodes, etc. Overall, these studies suggest that PLG microparticles enhanced the potency of the HCV plasmid sufficiently that DNA may be considered as an option for immunization of primates. In contrast, naked DNA does not appear to be an option if an important objective is to induce antibody responses to the encoded antigen. Furthermore, following a single booster dose with recombinant protein in MF59, the PLG/DNA group had comparable serum IgG titers to the rhesus which had been immunized exclusively with E1E2 protein in MF59. Since E1E2 is produced as an intracellular antigenic complex [41], there may be challenges in manufacturing it at the levels required for a universal HCV vaccine. Therefore, the ability of PLG/DNA to induce potent antibody responses, and to prime a response that can be boosted with a single dose of E1E2 protein provides a protein dose-sparing option for vaccine development. In addition, DNA vaccines have become an established option to prime CTL responses which may also be important in the protective immune response against HCV [12]. Apart from ISCOMadjuvanted vaccines [42], protein based vaccines have been largely ineffective for the induction of CTL responses in nonhuman primates and humans [43]. In one of the three rhesus

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macaques immunized with PLG/DNA we were able to detect a CTL response following the fourth immunization. Although CTL was not evaluated in the E1E2/MF59 immunized animals, we have sufficient experience with this adjuvant to be confident that a CTL response would not have been induced (unpublished data). A number of studies have been undertaken to determine the mechanism of action for cationic PLG microparticles to induce enhanced responses to adsorbed DNA vaccines. We have shown that PLG/DNA, but not naked DNA are able to mediate transfection of DC in vitro [44]. In addition, PLG/DNA protects DNA against degradation and enhances gene expression in muscle and local lymph nodes [28,29,44]. Furthermore, PLG/DNA but not naked DNA is able to recruit significant numbers of activated APC to the injection site following immunization [44]. Which of these mechanisms is most important for enhancing responses to the non-secreted E1E2 plasmid is currently unknown. The data obtained in the current studies were sufficiently encouraging to move the PLG/DNA approach forward into a non-human primate challenge model with HCV. These studies are ongoing, but initial data have confirmed that PLG/DNA can induce seroconversion and T cell responses in non-human primates (Houghton et al., unpublished data). The results from these studies will be reported in detail when the challenge studies are completed.

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

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