Effect of vesicle size on tissue localization and immunogenicity of liposomal DNA vaccines

Vaccine 29 (2011) 4761–4770

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Effect of vesicle size on tissue localization and immunogenicity of liposomal DNA vaccines Myrra G. Carstens a , Marcel G.M. Camps b , Malou Henriksen-Lacey c , Kees Franken d , Tom H.M. Ottenhoff d , Yvonne Perrie c , Joke A. Bouwstra a , Ferry Ossendorp b , Wim Jiskoot a,∗ a

Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research (LACDR), Einsteinweg 55, 2333 CC Leiden, The Netherlands Department of Immunohematology and Blood Transfusion, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands c Aston Pharmacy School, School of Life and Health Sciences, Aston University, Birmingham B4 7ET, United Kingdom d Department of Infectious Diseases, Leiden University Medical Center (LUMC), Leiden, The Netherlands b

a r t i c l e

i n f o

Article history: Received 18 January 2011 Received in revised form 4 April 2011 Accepted 21 April 2011 Available online 10 May 2011 Keywords: DNA vaccines Cationic liposomes Biodistribution Vaccine carrier

a b s t r a c t The formulation of plasmid DNA (pDNA) in cationic liposomes is a promising strategy to improve the potency of DNA vaccines. In this respect, physicochemical parameters such as liposome size may be important for their efficacy. The aim of the current study was to investigate the effect of vesicle size on the in vivo performance of liposomal pDNA vaccines after subcutaneous vaccination in mice. The tissue distribution of cationic liposomes of two sizes, 500 nm (PDI 0.6) and 140 nm (PDI 0.15), composed of egg PC, DOPE and DOTAP, with encapsulated OVA-encoding pDNA, was studied by using dual radiolabeled pDNA-liposomes. Their potency to elicit cellular and humoral immune responses was investigated upon application in a homologous and heterologous vaccination schedule with 3 week intervals. It was shown that encapsulation of pDNA into cationic lipsomes resulted in deposition at the site of injection, and strongest retention was observed at large vesicle size. The vaccination studies demonstrated a more robust induction of OVA-specific, functional CD8+ T-cells and higher antibody levels upon vaccination with small monodisperse pDNA-liposomes, as compared to large heterodisperse liposomes or naked pDNA. The introduction of a PEG-coating on the small cationic liposomes resulted in enhanced lymphatic drainage, but immune responses were not improved when compared to non-PEGylated liposomes. In conclusion, it was shown that the physicochemical properties of the liposomes are of crucial importance for their performance as pDNA vaccine carrier, and cationic charge and small size are favorable properties for subcutaneous DNA vaccination. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction The capability of DNA vaccines to induce both humoral and cellular immune responses makes them an attractive alternative to subunit vaccines. This is particularly important for the application of vaccines as anti-cancer therapy, or in the protection against intracellular diseases like TBC and malaria, for which effective vaccines are lacking [1–4]. However, promising results in the preclinical setting have not translated into successful application in humans, and clinical products have not been registered so far [2,5]. This lack of success has been related to the poor immunogenicity of DNA vaccines, and/or their limited ability to express sufficient levels of antigen. Therefore, several strategies are being developed to overcome these limitations, such as optimization of the expres-

∗ Corresponding author. Tel.: +31 71 5274314; fax: +31 71 5274565. E-mail address: [email protected] (W. Jiskoot). 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.04.081

sion vector, and the application of advanced delivery methods and improved formulations [6–10]. The use of a formulation based on cationic liposomes was shown to be an effective approach to improve the performance of DNA vaccines [9]. By encapsulating antigen-encoding plasmid DNA (pDNA) into cationic liposomes composed of PC, DOPE and DOTAP, improved antibody responses and antigen specific cytotoxic Tlymphocyte (CTL) responses were obtained after intramuscular administration in mice, as compared to naked pDNA [11–13]. Besides, this formulation improved the immune response after administration of antigen-encoding pDNA via the subcutaneous or oral route [14,15]. The increased potency of liposome-encapsulated pDNA as a vaccine was ascribed to the protective effect of cationic liposomes against enzymatic degradation of pDNA, and improved interaction with negatively charged cellular membranes. This leads to improved transfection and consequently higher antigen levels [13]. Moreover, it has been reported that cationic lipids possess immunostimulatory properties and are able to activate immune

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cells, which also accounts for their successful application in the formulation of subunit vaccines [16–18]. Besides an improved antigen expression and immunostimulatory effects, liposomes may play a role in persistence of its payload at the injection site. This was shown to correlate with enhanced efficacy of a liposomal subunit vaccine [19]. It has also been suggested as one of the mechanisms responsible for the increased potency of pDNA complexed with polymeric microparticles [20], but has never been investigated for liposomal pDNA vaccines so far. The efficacy of liposomal pDNA vaccines may be further improved by optimizing their physicochemical characteristics. From the gene delivery field it is well known that the particle size and charge are of crucial importance for the cellular uptake route and trafficking in somatic cells, thereby affecting the transfection efficiency [21–23]. Also the interaction of particles with the immune system is largely influenced by their size and charge [24–27]. For example, it has been established that particle diameters below 0.5 ␮m were optimal for uptake by antigen presenting cells (APCs), and particle uptake was enhanced by a positive surface charge in an in vitro model [26]. Various in vivo vaccination studies, mainly with subunit vaccines, indicated that the size of particulate vaccine carriers does not only affect the strength, but also the type of immune response that is elicited [24,27]. On the other hand, one should be cautious with generalizations, as recently the optimal particle characteristics have been shown to strongly depend on the route of administration [28–32]. Recently we described the preparation of pDNA-encapsulating liposomes with a size below 200 nm, which showed enhanced transfection efficiency in vitro when compared to larger liposomes [33]. The aim of the current study was to evaluate the effect of vesicle size on the tissue distribution of pDNA using a dual radiolabeled tracking method, and to correlate vesicle size and tissue distribution to the potency of pDNA-liposomes to elicit cellular and humoral immune responses, upon subcutaneous vaccination in mice. Our results demonstrate that the liposome size largely influences both cellular and humoral immunogenicity, as well as the tissue localization of the pDNA-liposomes. Furthermore, the results from the tissue distribution studies suggested that a strong depoteffect of pDNA at the injection site may hamper the induction of an immune response, rather than improving it.

FITC-coupled anti-CD4, APC-coupled anti-IFN␥, anti-CD28, and anti-CD3␧. Horseradish peroxidase (HRP) conjugated goat antimouse IgG (␥ chain specific), IgG1 (␥1 chain specific) and IgG2a (␥2a chain specific) were purchased from Southern Biotech (Birmingham, USA). Chromogen 3,3 ,5,5 -tetramethylbenzidine (TMB) and the substrate buffer were purchased from Invitrogen (Breda, The Netherlands). SOLVABLE® , Ultima Gold scintillation fluid, and [␣-32 P]-dATP (111 TBq/mmol), were purchased from Perkin Elmer (Milan, Italy). All chemicals were used as received, buffers were filtered through a 0.2 ␮m Millex® GP PES-filter (Millipore, Cork, Ireland) prior to use. 2.2. Liposome preparation and characterization 2.2.1. Preparation of pDNA-liposomes Liposomes encapsulating pDNA were prepared by the dehydration–rehydration method, according to Gregoriadis et al. [35], and sized by the optimized extrusion method developed in our lab as described elsewhere [33]. Briefly, small unilamellar vesicles were prepared by the film-hydration method followed by sonication, using chloroform as organic solvent and demineralized water in the hydration step. Typically, 28 ␮mol lipid was used to prepare 1 mL of liposome dispersion, at a molar ratio of egg PC/DOPE/DOTAP of 8/4/2. Subsequently, pDNA (3 mg/mL solution in PBS) was added, the mixture was frozen in liquid nitrogen, and freeze-dried overnight. To ensure equal amounts of pDNA and lipid in the final preparations used for in vivo studies, the starting concentration of pDNA used in the preparation of extruded liposomes was typically 200 ␮g/mL, and for non-extruded liposomes 100 ␮g/mL. Dehydration–rehydration liposomes were formed by controlled hydration of the lipid-DNA cake in HEPES buffered glucose solution (5% (g/v), pH 7.4, HBG). Sizing of the liposomes was performed by high-pressure extrusion at 30 ◦ C through NuclePore polycarbonate Track Etch membranes (Whatman, ‘s-Hertogenbosch, The Netherlands), with pore sizes of 400 nm (2×) and 200 nm (4×), using a LipexTM extruder (Northern Lipids, Burnaby, Canada). Non-encapsulated pDNA was removed by washing, using ultracentrifugation or a VivaSpin 2 centrifugational concentrator [33]. PEGylated liposomes were prepared by the same preparation method. During the formation of the lipid film, 10 mole % of PEG2000-DSPE was added, and typically the starting concentration of pDNA was 130 ␮g/mL.

2. Materials and methods 2.1. Reagents and antibodies The phospholipids dioleoyl-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and poly(ethylene glycol)2000-distearoyl phosphoethanolamine (PEG2000-DSPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Egg l-␣-phosphatidylcholine (egg PC), glucose, 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Pontamin Blue, and hydrogen peroxide were obtained from Sigma–Aldrich. The plasmid DNA (pDNA) was a V1Jns vector of 6.5 kBp, encoding ovalbumin (OVA), and was produced as described previously [34]. Synthetic unmethylated CpGoligonucleotide (CpG-ODN 1826) was obtained from Invivogen (Toulouse, France), OVA grade VII from Calbiochem (Merck KGaA, Darmstadt, Germany), sterile phosphate buffered saline (PBS) from Braun (‘s Hertogenbosch, The Netherlands) and chloroform (HPLC grade) was from Fisher Scientific (Leicestershire, UK). 1,2 dipalmitoyl I-3-phosphatidyl[N-methyl-3 H]choline ([3 H]DPPC, 1.5–3.33 TBq/mmol), Nick Translation Kit (N5500) and illustra ProbeQuant G-50 Micro Columns were obtained from GE Healthcare (UK). The following antibodies were purchased from BD Pharmingen (San Diego, USA): PE-coupled anti-CD8b.2,

2.2.2. Physicochemical characterization of pDNA-liposomes Particle size (diameter, Zave , and polydispersity index, PDI) and zeta potential (ZP) were determined by dynamic light scattering (DLS) and laser Doppler velocimetry, respectively, using a NanoSizer ZS (Malvern Instruments, Malvern, UK). Samples were diluted in 20 mM HEPES buffer pH 7.4, to obtain a slightly opalescent dispersion. The lipid content in the formulations was confirmed by phosphate determination according to Rouser [36], and in line with our previous results, no changes in lipid concentration during the preparation method were observed [33]. The content of double stranded DNA (dsDNA) in the liposomes was determined by a PicoGreenTM assay (Invitrogen® , Breda, The Netherlands), according to the instructions of the supplier, after destruction of the liposomes by 100–500 fold dilution in TE-buffer (10 mM Tris, 1 mM EDTA, pH 8.0) containing 0.1% (g/v) Triton X-100 [33]. The loading efficiency is expressed as percentage of the initial pDNA content. 2.2.3. Radiolabeling of pDNA-liposomes To enable the investigation of the in vivo biodistribution of the different pDNA-liposomes, dual radiolabeled formulations were prepared. Liposomes were labeled by incorporation of [3 H]-DPPC into the lipid bilayer by adding 0.75 kBq of [3 H]-DPPC dissolved in toluene/ethanol 1/1 to 28 ␮mol of lipids dissolved in chlo-

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roform before formation of the lipid film. Plasmid DNA was labeled by the incorporation of [␣-32 P] labeled dATP by a nick translation procedure. Thereto a Nick Translation Kit (GE Healthcare) was used, according to the instructions of the supplier, using 2 ␮g of pDNA and 66 pmole of [␣-32 P]-dATP. After the reaction, the free label was removed using an Illustra ProbeQuant G-50 Micro Column. Subsequently an appropriate amount of the resulting [32 P]-labeled pDNA was mixed with unlabeled pDNA, and this solution was added to the liposomes, followed by the dehydration–rehydration procedure and sizing as described above. 2.3. In vivo studies 2.3.1. Investigation of the biodistribution The biodistribution of the different DNA vaccine formulations was studied in female 6–8 week old BALB/c mice (Charles River, Margrate, UK), using radiolabeled formulations. Experimentation strictly adhered to the 1986 Scientific Procedures Act (UK). All protocols have been subject to stringent ethical review and were carried out in a designated establishment. Three days before the injection with pDNA vaccine (day −3), mice were injected subcutaneously (s.c.) into the neck scruff with 200 ␮L Pontamin Blue dissolved in PBS (0.5%, w/v). This is an in vivo marker for monocytes [19,37]. At day 0 mice were injected s.c. into the right flank with 20 ␮g [32 P]-labeled pDNA (either in PBS or in [3 H]-labeled liposomes with a total lipid dose of 5.6 ␮mol), corresponding to a dose of 150 kBq of [32 P] and 150 kBq of [3 H]. At time-points 1 day, 4 days and 8 days post-injection (p.i.), mice were killed by cervical dislocation and tissues were collected. Tissue from the site of injection (SOI), inguinal lymph nodes (draining DLN and nondraining or blank BLN), spleen, liver and kidney were removed and weighed into glass scintillation vials to which 1.5 mL of SOLVABLE® (Perkin Elmer) was added. Fully digested tissue samples were bleached using hydrogen peroxide, and assessed for [32 P] content by Cerenkov counting. Next, 10 mL of Ultima Gold was added, and samples were stored for 3 months at 4 ◦ C to allow decay of [32 P] (t1/2 = 14 days) before the amount of [3 H]-DPPC was quantified. In addition, a known sample of the injected dose was measured. The data are expressed as percentage of the injected dose (%ID). 2.3.2. Vaccination studies 2.3.2.1. Immunization and sampling schedules. Female C57BL/6Jico mice, 8 weeks old at the start of the immunization study, were obtained from Charles River (St. Germain sur l’Arbresle, France), and kept at the animal facility of the Leiden University Medical Center. Studies were conducted in accordance with national legislation and under supervision of the animal ethic committee of Leiden University. In the homologous DNA prime-boost regimen, mice were injected three times s.c. into the right flank with 20 ␮g of OVA-encoding pDNA, either in PBS (Naked pDNA) or entrapped in large, non-extruded or small, extruded liposomes, both at a total lipid dose of 5.6 ␮mol. Each group of 5 mice received 3 immunizations of the same formulation at 3 week intervals. The heterologous DNA prime-protein boost regimen was composed of 2 immunizations with 20 ␮g of OVA-encoding pDNA (Naked pDNA or liposomal pDNA), and a 3rd injection with 30 ␮g of OVA-protein dissolved in PBS, at 3 week intervals [38]. As control, mice received 3 injections with PBS (negative control), a mixture of 50 ␮g of OVA-protein and 30 ␮g CpG-ODN 1826 in PBS (positive control), or a single injection of 30 ␮g of OVA-protein (boost only control). One day before each immunization, blood samples were collected from the tail vein. Three weeks after the last vaccination mice were euthanized, and blood samples and lymphatic organs were collected to analyze the immune response.

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2.3.2.2. Determination of serum IgG. The presence of OVA-specific antibodies (IgG, IgG1 and IgG2a ) in the mouse sera was assessed by sandwich ELISA as described in detail elsewhere [39]. Briefly, 96 well plates (Microlon® , Greiner Bio-one, Alphen a/d Rijn, The Netherlands) were coated overnight at 4 ◦ C with 100 ng OVA per well, followed by blocking with 1% (w/v) BSA in PBS with 0.05% Tween 20 for 1 h at 37 ◦ C. Subsequently, two-fold serial dilutions of sera from individual mice were added to the plates and incubated for 2 h at 37 ◦ C. Antibodies were detected using HRP-conjugated goat anti-mouse IgG, IgG1 or IgG2a , using TMB as a substrate. Antibody titers are expressed as the reciprocal of the sample dilution that corresponds to half of the maximum absorbance at 450 nm of a complete s-shaped absorbance – log dilution curve. 2.3.2.3. Determination of the endogenous CTL response. To determine the endogenous cytotoxic T-lymphocyte (CTL) response upon vaccination, splenocytes (10 × 106 cells) were stimulated in vitro with 1 × 106 OVA-expressing EG7-cells that were treated with mitomycin C (50 ␮g/mL, 37 ◦ C, 1 h) and irradiated at 3000 rad [40]. After 7 days incubation, splenocytes were isolated by Ficoll density centrifugation, and stained for H-Kb tetramer (TM) OVA257–264 , CD8b2 (clone 53–5.8) and propidium iodide to exclude dead cells, as described before [41]. In addition, the production of IFN-␥ was determined by intracellular cytokine staining (ICS) as described previously [40]. Briefly, restimulated and purified splenocytes were incubated in vitro with or without 1 ␮g/mL OVA257–264 peptide overnight in the presence of Brefeldin A. The cells were washed in FACS buffer and stained with PE-conjugated CD8b2 antibody. ICS was conducted using the Cytofix/Cytoperm kit according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA, USA) and APC-coupled anti-IFN␥ antibody. Cells were analyzed by flow cytometry, using FACSCalibur with FACSDiva 6.2.1 software. 2.4. Statistical analysis Statistical analysis was performed using Prism 4 for Windows (Graphpad, San Diego, USA). Data are presented as mean ± SD, and statistical significance was determined either by a one or two way analysis of variance (ANOVA) with a Bonferroni post-test. 3. Results 3.1. Physicochemical characterization of the pDNA-liposomes The particle size, zeta potential and loading efficiency of the different formulations that were investigated in this study are shown in Table 1. In line with previous results [33], DNA-encapsulating liposomes formed by the dehydration–rehydration method were heterodisperse liposomes with an average diameter (Zave ) of about 500 nm and a PDI of 0.6. After sizing by the extrusion procedure as described previously [33], small monodisperse liposomes were obtained with a Zave and PDI of about 140 nm and 0.15, respectively. Both non-extruded and extruded pDNA-liposomes were positively charged, with a zeta-potential of 73 and 52 mV, respectively. The loading efficiency of extruded liposomes was 47%, whereas in nonextruded liposomes 100% loading efficiency was obtained. 3.2. Effect of liposome size on biodistribution To gain insight into the effect of the particle size on the pharmacokinetic profile of the liposomes and of the encapsulated pDNA, the tissue localization of the liposomal pDNA-formulations was studied after s.c. injection. Mice were injected with dual radiolabeled pDNA-liposomes (i.e. [32 P]-pDNA and [3 H]-DPPC-containing liposomes), and on days 1, 4 and 8 the organs were harvested and analyzed.

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Table 1 Physicochemical characteristics of the pDNA-liposomes. Formulationa

Zave (nm)b

PDIb

ZP (mV)b

Loading efficiency (%)c

Non-extruded LS Extruded LS PEG-LS

560 ± 200 142 ± 7 174 ± 14

0.55 ± 0.27 0.14 ± 0.02 0.15 ± 0.04

73 ± 5 52 ± 2 28 ± 10

100 ± 6 47 ± 8 64 ± 14

a b c

Liposomes (LS) were composed of egg PC, DOPE and DOTAP in a molar ratio of 8:4:2. PEG-LS contain 10 mole% of PEG2000-DSPE. Particle size (diameter, Zave ), polydispersity index (PDI) and zeta-potential (ZP), expressed as mean ± SD, n = 5–7 individual preparations. Loading efficiency of pDNA, expressed as % of the initial pDNA-content (mean ± SD, n = 5–7 individual preparations).

After s.c. injection with naked [32 P]-pDNA, less than 0.5% of the injected dose was detected 1 day p.i. at the site of injection and negligible levels in the other tissues as well (data not shown), indicating a rapid clearance. In contrast, s.c. administration of pDNA-liposomes resulted in a strong retention of the pDNA at the site of injection, i.e. 40 ± 4% (mean ± SD, n = 4) and 22 ± 5% of the injected pDNA-dose present at day 1, when encapsulated in non-extruded and extruded liposomes, respectively (Fig. 1a, Graph A). After 8 days, still 9.6 ± 3.8% and 1.0 ± 0.4% of the injected amount of pDNA was remaining at the injection site for the non-extruded and extruded liposomes, respectively. It was observed that encapsulation of pDNA into large, non-extruded liposomes resulted in significantly stronger retention at the site of injection than when small, extruded liposomes were used (P < 0.001, ANOVA). From

graph B it can be seen that the faster clearance coincided with a higher accumulation of pDNA in the draining lymph nodes, but differences were only significant at 1 day p.i. Analysis of [3 H]-DPPC in the different tissues (Fig. 1b) revealed similar trends for the liposome levels as for the pDNA levels. At 1 day after s.c. injection with large, non-extruded liposomes, 57 ± 5% (mean ± SD, n = 4) of the injected dose of lipid was detected at the site of injection. After 8 days, still 30 ± 8% of the injected amount of lipid was still present (Fig. 1a, Graph A), indicating a strong depot-formation. Subcutaneous injection of small, extruded liposomes resulted in significantly lower levels of [3 H]-DPPC at the site of injection (31 ± 9% ID and 2.0 ± 1.0% ID at day 1 and 8 respectively, P < 0.001, ANOVA). The results suggest that the pDNA-retention correlates with the formation of a lipid depot at the site of injection, and

a. pDNA (B) Draining lymph node 0.012

45

Non-extruded LS

40

Extruded LS

*

0.010 0.008 0.006 0.004 0.002 0.000

30

1

***

25

4

8

Time p.i. (days) (C) Spleen 0.15

20 15

%ID pDNA

%ID pDNA

35

%ID pDNA

(A) Site of injection

***

10 5

***

0 1

4

0.10

0.05

0.00 1

8

4

8

Time p.i. (days)

Time p.i. (days) (B) Draining lymph node

b. Lipid

0.05

(A) Site of injection 60

Extruded LS

%ID Lipid

0.04

Non-extruded LS 50

0.03 0.02 0.01 1

4

8

Time p.i. (days) (C) Spleen 0.35

30

***

0.30

20

%ID Lipid

%ID Lipid

0.00

***

40

***

10

***

0 1

4

8

0.25 0.20 0.15 0.10 0.05 0.00 1

4

8

Time p.i. (days)

Time p.i. (days) Fig. 1. Effect of liposome size on the pDNA-levels (a) and liposome deposition (b) at the site of injection (graphs A), in the draining lymph node (graphs B) and spleen (graphs C), after s.c. injection of mice with [32 P]-pDNA encapsulated in non-extruded (light bars) and extruded [3 H]-labeled liposomes (LS, dark bars). Results are expressed as percentage of the injected dose of pDNA (a) and lipid (b) (% ID pDNA and % ID Lipid respectively, mean ± SD, n = 4 mice). Significance between both formulations is indicated by ***p < 0.001 and *p < 0.05 (ANOVA with Bonferroni post-test).

M.G. Carstens et al. / Vaccine 29 (2011) 4761–4770

Total IgG

5/5

*** Homologous vaccination Heterologous vaccination

3

2/5

1/5 1

1/5 1/5

0/5

N

N

C on N a on ked trol * -e xt pD ru NA Ex d e tr d L ud S ed LS

0/5

titer that was 250-fold higher than in the other groups (P < 0.001, ANOVA, Fig. 2). These data indicate that the prime and first boost with pDNA encapsulated in extruded liposomes resulted not only in specific B-cell activation, but also in the induction of OVA-specific Thelper -cells since IgG antibodies were efficiently raised. 3.4. Cellular immune response

1/5

2

C on N a on ked trol * -e xt pD ru NA Ex d e tr d L ud S ed LS

Log 10 IgG titration

4

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Formulation Fig. 2. Antibody titers after homologous (dark bars) and heterologous (light bars) vaccination schedule, composed of 3 weekly s.c. injections with pDNA-formulation (naked pDNA, or pDNA encapsulated in non-extruded liposomes (LS) and extruded LS), and/or OVA-protein. *Controls represent the negative control (dark bar), and boost only control (light bar). Total anti-OVA-IgG titers are shown after the after the last injection (2nd boost), and presented as mean ± SD of 5 mice. Numbers represent the number of positive mice. Significance is shown by ***p < 0.001 (ANOVA with Bonferroni post-test).

that mean vesicle size and size distribution are important factors in the induction of a depot-effect by cationic liposomes. 3.3. Humoral immune response The immunogenicity of the different pDNA-formulations was studied in a 3-weekly vaccination schedule. Thereto, naïve mice were vaccinated 3× with the same pDNA-formulation (primeboost-boost) with three week intervals (homologous vaccination schedule). In addition, the pDNA-liposomes were applied in a heterologous vaccination schedule, composed of 2 vaccinations with the same pDNA-formulation (prime-1st boost) followed by a 3rd vaccination with OVA-protein (2nd boost). Various vaccination studies demonstrated that a heterologous DNA-prime proteinboost regimen may be more effective than DNA or protein alone [38,42–45]. Besides the detection of higher antibody titers in for example HIV, influenza and TBC vaccination studies [38,44,45], it has been reported that also the avidity of the antigen-specific antibody response may be improved by a heterologous vaccination regimen [43]. The induction of a humoral immune response was analyzed by measuring OVA-specific IgG-antibodies in serum. The levels of total IgG after the first 2 injections (prime and 1st boost) of pDNA, either non-formulated or encapsulated in non-extruded or extruded cationic liposomes were below the detection limit of the assay (results not shown). After a 2nd boost with pDNA (homologous schedule), OVA-specific IgG antibodies were detected in 1 out of 5 mice injected with naked pDNA and 2 out of 5 mice injected with small, extruded pDNA-liposomes (Fig. 2). Mice that received 3 injections with large, non-extruded pDNA-liposomes did not show detectable IgG-levels. In the heterologous vaccination schedule, injection of 30 ␮g OVA-protein into mice that received two prior injections of naked pDNA or non-extruded pDNA-liposomes resulted in a low level of IgG in 2 out 5 mice, whereas injection of naive mice with 30 ␮g OVA-protein (boost only control) did not induce detectable IgG levels (Fig. 2). Importantly, all mice that received 2 vaccinations with extruded pDNA-liposomes and 1 vaccination with OVA-protein showed significant levels of OVA-specific IgG, with an average

The induction of an endogenous specific CTL response in the different vaccination schedules was studied by quantification of the percentage of OVA257–264 -specific T-cells in in vitro stimulated splenocytes obtained 3 weeks after the last injection. The induction of cellular immunity is crucial for application as antitumor vaccine, or to protect against diseases like TBC and malaria, and is considered one of the main advantages of DNA vaccines over subunit vaccines. Fig. 3a shows the percentage of OVA257–264 -specific CD8+ T-cells in the individual mice upon homologous vaccination with the different formulations followed by specific in vitro restimulation. It can be observed that triple vaccination with 20 ␮g pDNA (Naked pDNA) resulted in a highly variable percentage of OVA257–264 -specific CD8+ T-cells, ranging from 0 to 55% of the total number of CD8+ T-cells. The large, non-extruded pDNA-liposomes induced no or moderate amounts of specific CD8+ T-cells. Importantly, three s.c. injections of small, extruded pDNA-liposomes resulted in significantly higher percentages of OVA257–264 -specific CD8+ T-cells, and the variations between the individual mice were small (45 ± 7%, mean ± SD, n = 5; P < 0.001, ANOVA), indicating a robust induction of OVA-specific CD8+ T-cells. The functionality of the induced T-cells was studied by analyzing their ability to produce IFN␥, using intracellular cytokine staining. Fig. 3b shows a significantly higher IFN␥ production in the T-cells from mice that received three s.c. injections of small, extruded pDNA-liposomes (24 ± 6%) than those from the group injected with large, non-extruded pDNA-liposomes (P < 0.01, ANOVA). Naked pDNA induced highly variable percentages of IFN␥-producing T-cells (24 ± 20%). The results corroborate that homologous vaccination with extruded pDNA-liposomes results in the robust induction of an endogenous specific CTL response. Importantly, in the splenocytes obtained from the mice that were vaccinated with extruded liposomes, a significant percentage of functional OVA-specific CD8+ T-cells was already detected by ICS ex vivo (0.15 ± 0.08%, P < 0.05, ANOVA), i.e. directly after harvesting the spleens (Fig. 4, lower right panel). Taken together, these data suggest that not only the induction of humoral immunity, but also the induction of cellular immunity is promoted by formulating pDNA into cationic liposomes with a size below 200 nm. In addition to a homologous vaccination schedule, we analyzed the induction of a cellular immune response after a heterologous DNA prime-protein boost vaccination schedule. In line with the results described above, we observed stronger induction of endogenous OVA-specific CD8+ T-cells in the mice that received pDNA encapsulated into extruded liposomes compared to the other formulations (results not shown). The observed responses were lower than those observed after the homologous vaccination schedule, but comparison of the different formulations revealed similar trends in the CD8+ T-cell response. 3.5. Effect of PEGylation of cationic liposomes on biodistribution and immunogenicity The vaccination studies demonstrated that extruded pDNAliposomes induced stronger and more robust immune responses than non-extruded pDNA-liposomes. Considering the results of the tissue localization studies, this may be related to a higher mobility of the small, extruded liposomes when compared to the large ones. As the liposomes’ drainage to the lymph nodes

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Fig. 3. Priming of endogenous CD8+ T-cells upon 3 s.c. injections with PBS (䊉), naked pDNA (), or pDNA encapsulated in non-extruded liposomes (LS, ), and extruded LS (). (a) Presence of CD8+ T-cells in stimulated splenocytes, capable of interacting with H-Kb -OVA257–264 tetrameric complexes. The y-axis displays the percentage of tetramer-positive cells out of total CD8+ cells for the individual mice. The right hand panel shows a representative FACS-plot of splenocytes obtained from mice vaccinated with non-extruded LS and extruded LS. Cells were gated on CD8+ and the numbers in the upper right quarter are the percentages of tetramer positive cells of total CD8+ cells. (b) IFN␥-producing cells in stimulated splenocytes. Data are presented as in (a), y-axis displays the percentage of IFN␥-producing cells out of total CD8+ cells. Significance is shown by ***p < 0.001, **p < 0.01, *p < 0.05 (ANOVA with Bonferroni post-test).

in vivo can be enhanced by shielding of the surface charge by a PEG-layer, this may be a good strategy to improve their immunogenicity further. Indeed, in a dermal vaccination study, van den Berg et al. demonstrated that PEGylated pDNA-loaded cationic nanoparticles induced stronger antigen-specific CTL responses than non-PEGylated nanoparticles [46]. In view of this, we also explored the effect of the incorporation of 10 mole % PEG2000DSPE into the liposome bilayer on the tissue distribution and immunogenicity of extruded pDNA-liposomes after s.c. vaccination. These PEGylated pDNA-liposomes had a diameter of 170 ± 14 nm (mean ± SD, n = 3, Table 1). The zeta-potential was reduced to 28 ± 10 mV (mean ± SD, n = 3, Table 1), indicating partial

shielding of the cationic charge. Analysis of the tissue localization after subcutaneous administration of dual radiolabeled PEGylated pDNA-lipsomes indicated significantly faster clearance from the site of injection than non-PEGylated pDNA-liposomes of the same size. Moreover, the levels of pDNA and liposomes in the draining lymph node and spleen were significantly enhanced (Fig. 5). These small, extruded PEGylated pDNA-liposomes were applied in the vaccination studies as described above. As shown in Fig. 6a, significant levels of OVA-specific IgG antibodies were detected in 3 and 4 out of 5 mice, in the homologous and heterologous vaccination schedule, respectively. This is comparable to the results obtained with non-PEGylated liposomes of the same size. Fig. 6b

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Fig. 4. IFN␥-producing CD8+ T-cells in splenocytes from vaccinated mice, measured directly ex vivo by ICS. Representative FACSplots are shown of splenocytes obtained from mice after 3 s.c. injections with PBS, naked pDNA, or pDNA encapsulated in non-extruded liposomes (LS) or extruded LS. Cells were gated on CD8+ and the numbers in the upper right quarter are the percentages of IFN␥-producing cells of total CD8+ cells.

demonstrates that the percentage of OVA-specific CD8+ T-cells and IFN␥-producing T-cells is slightly lower upon homologous vaccination with PEGylated pDNA-liposomes than with non-PEGylated liposomes. These results indicate that in this setting, PEGylation of the small sized pDNA-liposomes does improve their mobility in vivo, but does not result in improved humoral or cellular immune responses. 4. Discussion DNA vaccines have several advantages over other types of vaccines, including a good safety profile and the ability to induce both humoral and cellular immunity. This makes them a promising class of vaccines for immunization against diseases such as AIDS, TBC and malaria, and to apply as therapeutic vaccines in the fight against cancer and other chronic infectious diseases. However, their potency is limited, due to poor transfection efficiency and/or limited immunogenicity, which can be overcome by the development of advanced formulation strategies [6–10]. Cationic liposomes are well-known vaccine carriers, and have been applied in the formulation of both subunit vaccines and DNA vaccines [9,16,47]. Their cationic charge does not only result in condensation and protection of pDNA against degradation, but it also increases the interaction with cells, and it has been suggested to have an immunostimulatory effect. Next to charge, liposome size may be important for their efficiency as DNA vaccine carrier, but this aspect has received little attention so far. In this manuscript we describe the effect of liposome size on the immunogenicity of liposomal DNA vaccines after subcutaneous administration. Previously we described the preparation of cationic liposomes with a size smaller than 200 nm, and entrapped pDNA, by high-

pressure extrusion. These small sized, extruded pDNA-liposomes showed higher transfection efficiency in vitro, and stronger activation of the important pathogen recognition receptor TLR9 than larger, non-extruded pDNA-liposomes or naked pDNA [33]. The present study focused on the effect of liposome size on the performance in vivo. Our vaccination studies clearly demonstrate that small sized, extruded cationic pDNA-liposomes are able to induce good cellular and humoral immune responses, both in a homologous and heterologous (protein boost) vaccination schedule. We did not only observe higher antibody titers, but also an enhanced induction of functional, OVA-specific CD8+ T-cells, when compared to larger pDNA-liposomes or naked pDNA. The positive effect of a smaller size on the induction of an immune response has been observed previously with a polymeric carrier system: Singh et al. showed that i.m. vaccination with DNA-loaded CTAB-coated PLGA-particles of 300 nm induced higher antibody titers than particles of 1 ␮m [48]. In the vaccination studies with the non-extruded pDNA-liposomes, we did not detect significant antibody titers, and even a weaker, rather than a stronger induction of a cellular immune response was observed when compared to naked pDNA. This is in contradiction with previous findings, which indicated a favorable effect of pDNA encapsulation into large cationic liposomes when compared to naked pDNA [12,15]. Although the same type of liposomes were used as in our studies, there were differences in the lipid-to-pDNA ratio, in the type of plasmid and antigen that was encoded. Besides, differences in the vaccination protocol with regard to pDNA dose, administration frequency, number of injections, site of injection and read-out may explain the discrepancy. As tissue localization may play a role in the efficacy of DNA vaccines [20], we studied the biodistribution at different time points

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a. pDNA

(B) Draining lymph node 0.020

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Time p.i. (days) Fig. 5. Effect of liposome PEGylation on the pDNA-levels (a) and liposome deposition (b) at the site of injection (graphs A), in the draining lymph node (graphs B) and spleen (graphs C), after s.c. injection of mice with [32 P]-pDNA encapsulated in extruded, non-PEGylated (filled bars) and extruded PEGylated [3 H]-labeled liposomes (PEG-LS, empty bars). Results are expressed as percentage of the injected dose of pDNA (a) and lipid (b) (% ID pDNA and % ID lipid respectively, mean ± SD, n = 4 mice). Significance between both formulations is indicated by ***p < 0.001, **p < 0.01 and *p < 0.05 (ANOVA with Bonferroni post-test).

after subcutaneous administration of dual radiolabeled pDNAliposomes. Our results indicate that encapsulation of pDNA into cationic liposomes results in its retention at the site of injection after subcutaneous administration, related to the formation of a lipid depot. The depot-effect of cationic liposomes has been described previously for subunit vaccines, and is ascribed to the liposomes’ interaction with proteins and their aggregation in vivo. In this respect, their positive charge is considered the predominant factor [16,19,47]. It was observed previously that a small particle size favors the lymphatic uptake of neutral or negatively charged

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liposomes in rats [49,50]. Our data point to the vesicle size being of significant importance for the depot-effect of cationic liposomes as well. In liposome-adjuvanted subunit vaccines, the formation of a depot contributes to their enhanced efficacy by prolonging the exposure to the antigen and improving the recruitment of monocytes [16,19,51], which has also been suggested to play a role in the improved efficacy of DNA vaccines formulated with CTAB–PLG microparticles [20]. In line with this, the enhanced potency of pDNA encapsulated in small liposomes compared to naked pDNA, can be,

60

Extruded LS Extruded PEG-LS

50 40 30 20 10 0

TM+ cells

IFNγ+ cells

Fig. 6. Effect of PEGylation on the induction of humoral (a) and cellular (b) immune response. (a) Antibody titers after homologous (left) and heterologous (right) vaccination schedule, using extruded pDNA-liposomes (LS, filled bars) or PEGylated extruded pDNA-LS (Extruded PEG-LS, empty bars). Total anti-OVA-IgG titers are shown after the last injection (2nd boost), and presented as mean ± SD of 5 mice. Numbers represent the number of positive mice. (b) Priming of endogenous CD8+ T-cells upon 3 s.c. injections with extruded LS () or extruded PEG-LS (). TM+ cells represent the percentage of tetramer-positive cells out of total CD8+ cells for the individual mice (left), and IFN␥+ cells represent the percentage of IFN␥-producing cells out of total CD8+ cells after in vitro stimulation.

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at least partly, related to retention at the injection site. However, vaccination with non-extruded pDNA-liposomes failed to induce an immune response, despite a strong depot-effect. Likely, the immobilization of the pDNA-liposomes at the site of injection prevents the pDNA from sufficiently reaching the secondary lymphatic organs and hampers the antigen to be produced. Besides, the difference in size may have influenced uptake by APCs. It has been established in vitro that the optimal particle diameter for uptake by APCs is below 0.5 ␮m [26], and the enhanced immunogenicity of DNA-loaded CTAB/PLGA-particles of 300 nm over 1 ␮m particles has been ascribed to improved APC uptake as well [48]. The introduction of a PEG-coating can further enhance the mobility of cationic pDNA-carriers, and, as demonstrated in a dermal vaccination study, thereby improve their immunogenicity [46]. In our studies, we indeed demonstrated that PEGylation of the small sized pDNA-liposomes improved their lymphatic drainage in vivo. However, this did not translate into improved humoral or cellular immune responses. This may be related to an insufficiently long residence time due to fast clearance from the lymph nodes as well. Moreover, despite the positive influence of PEG on the stability and mobility of the pDNA-liposomes in vivo, PEG is also known to negatively affect the interaction with cells. This may result in lower transfection efficiency and lower immunogenicity, thereby explaining the lack of an improved response. These limitations might be overcome by the use of sheddable coatings [52], or by the coupling of APC-specific targeting ligands to these liposomes [53]. These are promising strategies to further improve the potency of liposomal pDNA vaccines. 5. Conclusion The results presented in this paper clearly indicate that the physicochemical characteristics, such as vesicle size and size distribution of liposomal DNA vaccines play a key role in their performance in vivo. A robust cellular and humoral immune response was induced upon s.c. vaccination with pDNA encapsulated in liposomes with a small size and a cationic surface charge, indicating that these are favorable properties for s.c. administered liposomal DNA vaccine carriers. Our localization studies suggest that liposomal DNA vaccines do not benefit from a strong depot-effect, or from rapid accumulation in the draining lymph nodes, pointing to a delicate balance between retention and lymphatic uptake. Besides, other events such as antigen expression and activation of immune cells in vivo may be of equal or higher importance for the efficacy of particulate DNA vaccines, underlining the complexity of their mechanism of action. It is important to understand not only the effect of the physicochemical properties on each of these events, but also to know their contribution to the final efficacy as a DNA vaccine. Joint efforts of pharmaceutical scientists and immunologists are needed to increase our insight. This will allow further optimization of pDNA vaccine carriers, in order to develop clinically effective pDNA vaccines that provide protective immunity at low pDNA doses. References [1] Rice J, Ottensmeier CH, Stevenson FK. DNA vaccines: precision tools for activating effective immunity against cancer. Nat Rev Cancer 2008;8(February (2)):108–20. [2] Kutzler MA, Weiner DB. DNA vaccines: ready for prime time? Nat Rev Genet 2008;9(October (10)):776–88. [3] Ottenhoff TH. Overcoming the global crisis: “yes, we can”, but also for TB? Eur J Immunol 2009;39(August (8)):2014–20. [4] Laddy DJ, Weiner DB. From plasmids to protection: a review of DNA vaccines against infectious diseases. Int Rev Immunol 2006;25(May–August (3–4)):99–123. [5] Liu MA, Ulmer JB. Human clinical trials of plasmid DNA vaccines. Adv Genet 2005;55:25–40.

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