Liposome-mediated vaccine delivery

Liposomes are spherical entities composed of a phospholipid bilayer shell with an aqueous core and the potency of liposomes depends on the number of lipid layers, electric charge, composition and method of preparation. For vaccine

delivery, an antigen (or adjuvant) maybe either encapsulated in the core of the liposome, buried within the lipid bilayer or adsorbed on the surface for presentation to antigen presenting cells. Thus, for vaccine purposes, these particles are considered most useful for delivering antigens and adjuvants [15].

Cationic liposomes have been used extensively in both drug delivery and vaccine research. This is based on the assumption that cationic liposomes are able to deliver the antigen to the antigen-presenting cells (APCs) and thus enhance antigen-specific immune responses. Moreover, Nakanishi et al. has demonstrated that cationic liposomes were much more potent than anionic or neutral liposomes for generating a cell-mediated immune response [16]. Hence, the adsorption of antigen onto the cationic liposome increase the efficiency of antigen presentation, it has been suggested by the primary adjuvant mechanism of cationic liposomes, which targeting the cell membrane of antigen-presenting cells, then subsequently leads to enhanced uptake and presentation of antigen [17]. An interesting preclinical study in mice conducted by Guan et al. evaluated the effect of the liposome formulation on the type of immune response generated for a MUC1 therapeutic cancer vaccine. This study revealed that liposome-associated (either encapsulated or surface-exposed MUC1 peptide (BP25) produced a strong specific CTL response [18]. In addition to cationic lipids such as DC-Chol and DDA have been effectively used as adjuvants. It has been claimed that DC-Chol is able to overcome the observed non-responsivness to hepatitis B vaccine. Brunel et al. have outlined its use in a liposomal adjuvant

where, if used in conjunction with genetically engineered Hepatitis B, the levels of specific antibodies (IgG1 and IgG2a) and cell factors can be increase [19].

Although liposomes constituted one of most studied antigen delivery systems, they are still the subject of novel results on enhancing strategies. The synergistic effect of liposomally co-entrapped DNA and protein has been shown to exceed the well-known adjuvant effects of plasmid DNA and liposomes. This new approach to vaccination has been termed ‘codelivery’ and it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell mode of presentation that would commonly occur with live viral pathogens, opening new uses for this technology.

Here, we evaluated the novel liposome-polymer transfection complex as a delivery system and applied in the development of DNA and subunit protein antigen vaccine. The delivery system had the advantages of ease manufacture and low cost in comparison to general cationic liposome of antigen delivery system. Thus, it showed the potential to apply in the development of animal vaccine for farm animals that need large quantity of vaccine product with low cost to prevent the infectious disease, particularly for species where a large number of animals with a relatively low commercial value are utilized such as chickens.

2. Materials & Methods

2.1 Chemicals

PEI (branched form, average molecular weight of 25 kDa) was purchased from Aldrich Chemical Co. (St. Louis, MO, USA), PEG (average molecular weight of 8,000) and Propium ioide (PI) were purchased from Sigma Co. (St.

Louis, MO, USA). Soybean oil was purchased from Uni-President^® Co. (Taiwan, ROC). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, antibiotics (penicillin 100U/ml; streptomycin 100 µg/ml), SYBER green and Trypan blue stain were purchased from GIBCO (Grand Island, NY, USA)

2.2 Cells and Culture, Plasmids

The mouse embryo fibroblast cell line Balb/3T3 was cultured in DMEM with 10% FBS and 1% PSA. The mouse lymphoblast cell line P338/D1 was cultured in RPMI with 10% FBS and 1% PSA. Cells were cultured in a 37°c incubator with 5% CO2. The plasmid of pAAV-MCS-hrGFP was from Dr.

Liao’s Lab (the department of biological science and technology, NCTU).

2.3 Mice and Immunogens

Female BALB/cByJNarI mice 3-5 week of age were purchased from National Laboratory Animal Center (NLAC). All mice were maintained under standard pathogen-free conditions. Female mice were used at 9-10 weeks of age.

For DNA vaccine, both of the plasmids of pCJ3- HpHsp60 and pCJ3-Urease B were gifts from Dr. Wu’s Lab (the department of food science, NTOU).

Plasmids DNA was purified by anion exchange chromatography (Qiagen-tip 2500 Mega Prep Kit, Qiagen. Germany). For subunit protein vaccine, recombinant H pylori heat-shock protein 60 (rHpHsp60) and recombinant Urease B protein (rUreB) were encoded by pET HpHsp60 and pET 30a-Urease B DNA which from Dr. Liao’s Lab (the department of biological science and technology, NCTU).

2.4 Preparation of Liposome-Polymer Transfection Complex

Liposome-polymer transfection complex (LPTC) was formed by two hydrophilic polymers (PEI, PEG) and soybean oil, two phases were mixed by sonication. Briefly, the first step was to make separately oil and aqueous phases.

In aqueous phase, polyethyleneimine (PEI) and polyethylene glycol (PEG) at 1:1 molar ratio dissolved in ddH₂O until the mixture dissolved well. After aqueous phase was prepared, soybean oil added to the aqueous phase with 10%

of total volume of mixture. Then, the mixture was vortexed for 10min and then, sonicated at 25W for 30min at room temperature until it formed milky white appearance and stored at 4°C.

2.5 Particle Size and Zeta-Potential Measurement

The particle size and zeta-potential of LPTC was measured by the laser light scattering measurement using a Malvern Zetasizer 3000HS (Malvern Instruments, UK). Here, we used N/P ratios to describe the PEI/DNA complexes (PEI contained in LPTC) that were the ratios of moles of the amine groups of PEI to those of the phosphate groups of DNA. The calculation of N/P ratios was described in the following section. The LPTC/DNA binary complexes were prepared at N/P ratios ranging from 1 to 30 containing 50µg DNA. Then the prepared complexes were incubated for 30 minutes and diluted by distilled water to 1ml volume for the size and zeta potential measure respectively. The distribution of particle size was measured by dynamic light scattering (90 plus, Brookhaven Instruments Corp., USA)

The calculation of the N/P ratio is based on the assumption that one repeating unit of PEI featuring one nitrogen corresponds to 43.1 g/mol, and one repeating unit of DNA featuring one phosphate corresponds to 330 g/mol. To calculate the N/P ratio for a 1 mg/mL solution of 25kDa PEI, the equation is as follows:

N/P= (µL PEI stock solution) × 23.2 mM nitrogen residues (µg plasmid DNA) × 3 nmol phosphate

In this example, for 100 µg plasmid DNA and 200 µg PEI (= 200 µL), the N/P ratio is 15.5.

2.6 Transmission Electron Microscopy (TEM)

The mixture was placed onto a 400 mesh copper grid coated with carbon.

About 2 min after deposition, the grid was tapped with a filter paper to remove surface water and placed into dry box for two days.

2.7 Gel Electrophoresis Assay

The LPTC/DNA binary complexes at varying N/P ratios ranging from 1 to 30 were prepared by adding appropriate volumes of LPTC to 300 ng plasmid DNA pre-stained by 1µl SYBR Green in ddH2O. The binary complexes were incubated at 37°C for 30min, and then heparin (Sigma, St. Louis, MO) was added in different dosage from 150 µg to 1 µg. After 10min, the complexes were in a total volume of 10 µl and then were loaded on the 0.8% (w/v) agarose gel with Tris-acetate (TAE) running buffer at 100V for 30min. DNA was visualized with a UV lamp using Uni-photo gel image system (EZ lab, Taiwan, ROC).

2.8 DNase I Protection Assay

LPTC was complexed with 300 ng DNA at N/P = 10, then the complexes were treated either (1) DNase I alone (1 units), (2) DNase I and heparin at the same time, (3) DNase I then heparin or (4) heparin alone. For the treatment of DNAase I, 300ng DNA was incubated with 1 units of DNase I in a 20 µl reaction mixture at 37°C for 30 min, and then 0.5 M EDTA was used to stop the

reaction followed by heparin or buffer. The DNA complexes were analyzed with 0.8% agarose gel eletrophoresis.

2.9 Plasmid DNA Extraction- Midi Preparation Method

The midipreparation was performed by NucleoBond PC 100 kit (Macherey-Nagel, Duran, Germany) following the manufacturer’s protocol. At first, a single colony of E. coli was inoculated in 100 ml of LB broth contained antibiotics and grew overnight at 37°C with agitation. The cells were recovered by centrifugation at 8,000 rpm for 15 minutes at 4°C. The pellet was collected, and 4 ml buffer S1 (with RNase) (Macherey-Nagel, Duran, Germany) was added to dispense the pellet. Then 4 ml buffer S2 (Macherey-Nagel, Duran, Germany) was added to the suspension. The lysate was mixed gently and incubated at room temperature for 3 minutes (no more than 5 minutes). The pre-cooled 4 ml buffer S3 (Macherey-Nagel, Duran, Germany) was then added to the solution and mixed gently until a homogeneous suspension containing an off-white flocculate was formed. The mixture was incubated on ice for 5 minutes and then spun at 13,000 rpm for 25 minutes at 4°C. The supernatant was loaded onto the NucleoBond AX 100 Midi column which was equilibrated with 2.5 ml buffer N2 (Macherey-Nagel, Duran, Germany). The flow-through was emptied by gravity flow and discarded. 10 ml buffer N3 (Macherey-Nagel, Duran, Germany) was added to wash the column twice. The DNA was eluted by 5 ml buffer N5 (Macherey-Nagel, Duran, Germany) Then 3.5 ml isopropanol

was added to precipitate the DNA. The mixture was incubated on ice for 10 minutes and recovered by centrifugation at 13,000 rpm for 30 minutes at 4°C. 6 ml 70% ethanol was added to the pellet and the solution was spun at 13,000 rpm for 5 minutes. Finally, the pellet was dissolved in appropriate amount of ddH2O and stored at -20°C.

2.10 Plasmid DNA Extraction- Mega Preparation Method

A single colony was picked from a freshly streaked selective plate and inoculated a starter culture of 5ml LB medium containing the appropriate selective antibiotic. After incubation for 8 hr at 37°C with vigorous shaking (225rpm), 2.5ml of starter culture was inoculated to 2.5 liters LB broth contained selective antibiotic for 12–16 h with vigorous shaking (225rpm). . The cells were recovered by centrifugation at 8,000 rpm for 15 minutes at 4°C. The cell pellet was resuspended in 50 ml buffer S1. Then 50 ml buffer S2 was added and mixed thoroughly by vigorously inverting 4–6 times, and incubated at room temperature for 5 min. The 50ml pre-cooled buffer S3 was added immediately and thoroughly by vigorously inverting 4–6 times, and incubated on ice for 30 min. After centrifugation at 12,000rpm for 30 min at 4°C, the supernatant was loaded on to a QIAGEN-tip 2500 column which was equilibrated by applying 35 ml buffer N2, and allowed the column to empty by gravity flow. 100 ml buffer N3 was added to QIAGEN-tip 2500 column and wash twice. DNA was eluted with 35 ml buffer N5, then adding 24.5ml (0.7 volumes)

room-temperature isopropanol to precipitate the eluted DNA. Mix and centrifuge immediately at 12,000 rpm for 30 min at 4°C. Carefully decant the supernatant.

Wash DNA pellet with 7 ml in room-temperature 70% ethanol, and centrifuge at 12,000 rpm for 10 min. Carefully decant the supernatant without disturbing the pellet. Air-dry the pellet for 10–20 min, and re-dissolve the DNA in a suitable volume of DDW

2.11 In Vitro Cytotoxicity Assay

The cytotoxicity assay was performed with Balb/3T3 cells by MTT assay.

Briefly, the cells were seeded in a 96-well plate at a density of 2 × 10⁴ cells/well, and then cells were incubated in 100 µl DMEM containing 10% FBS for 3hr prior to adding the LPTC. After LPTC were added for 3 days, the medium was replaced with 100µl of fresh medium and 10 µl MTT (5mg/ml) solutions was added to each well. After 4 hr of incubation, the medium was removed and 100µl DMSO was added. The absorbance was measured at 595 nm using a microplate reader (Tecan). The relative cell viability was calculated as:

OD595(control) was obtained in the absence of polymers and OD595(sample) was obtained in the presence of LPTC.

2.12 In Vitro Transfection Assay

Cell Viability (%) = ( OD595(sample) / OD595(control) ) ×100

Cells were seeded in the 6-well plate at a density of 2.5×10⁵ cells/well and cultured with 2ml growth medium for 24 hours respectively. Cells were transfected plasmid DNA encoding GFP gene by LPTC with various N/P ratios.

Briefly, 3 µg plasmid DNA and 5 µl of LPTC in different concentrations were each diluted into 100 µl of 150mM NaCl and vortexed. The LPTC solution was added into DNA solution for 5 minutes (Notice: not the reverse order), and then vortexed. After 20 minutes, the cells were rinsed and supplemented with 200 µl Opti-MEM I medium (Gibco, Grand Island, NY). The LPTC/DNA mixture was gently and equally added to each well. Finally, Opti-MEM I medium (600 µl) (Gibco, Grand Island, NY) were added to each well. After 12 hours incubation, 2ml fresh growth medium were added into each well. After 48 hours, the gene expressions were measured by FACScan flow cytometry (Becton Dickinson, Moutain View, CA).

2.13 Measurement of Transfection Efficiency by Flow Cytometry

After 48 hours of transfection, cells were harvested to measure the gene expression. Briefly, the medium was discarded and each well was rinsed with 1ml PBS. 1ml trypsin was then added, and the cells were incubated at 37°C for 5 minutes. 1ml PBS was added into each well and the cells were recovered by centrifugation at 1,500rpm for 5 minutes at 4°C. The supernatant was discarded and the pellet was re-suspended by 1ml PBS in FACS tube. The reporter gene

expression was measured by FACScan flow cytometry (Becton Dickinson, Moutain View, CA). Fluorescence intensities were analyzed with CELLQUEST software (Becton Dickinson).

2.14 In Vitro Cellular Uptake Assay

BSA-FITC (14 µg/ml) mixed with LPTC (4.8 µg) for 30min in room temperature and kept in dark. P338/D1 cells (2 × 10⁶ cells/ml) were pulsed with BSA-FITC/LTPC complexes and BSA-FITC alone in a humidified atmosphere at 5% CO₂ and 37°C or at 4°C. After incubation for 2hr, cell were centrifuged in 4,000 rpm for 5 min and washed with 1ml cold PBS for two times. After the final steps of washing procedure were finished, the supernatant was discarded and the pellet was re-suspended in 1ml cold PBS in FACS tube. To remove the surface-associated BSA-FITC on the cell membrane, 20 µl trypan blue were added to quench and kept in dark on ice for the following measurement of FACScan flow cytometry (Becton Dickinson, Moutain View, CA).

2.15 Recombinant HpHsp60 Protein Expression

pET-HpHsp60 were transformed into E. Coli BL21, then grown on LB plates containing kanamycin (30 mg/ml) at 37°C. After 16 hr incubation, five colonies were inoculated into 100ml LB medium containing kanamycin (30 mg/ml) at 37°C for 16 hr. The culture broth were refreshed in 900 ml LB

medium with shaking at 37°C, 225 rpm until the value of OD 600 reaches 0.6 (about 40 min). IPTG diluting from stock concentration of 800 mM to a final concentration of 1 mM were added to culture broth for 4 hr incubation. Cells were harvested by centrifugation at 8,000 rpm for 15 min at 4°C. Supernatant were discarded and the pellet were resuspended into 30 ml binding buffer. Then, total cell lysates were sonicated for 15 min and centrifuged at 12,000 rpm for 30 min at 4°C. The purification of protein was performed on HisTrap^TM HP column.

All of the buffer used in the experiment should be filtered with 0.45 µm syringe filter. Column were firstly washed by 5 column volumes of DDW and then equilibrated by 5 column volume of binding buffer at the flow rate about 1ml/min. Pretreated sample were loaded into column and were washed by wash buffer about 60 column volume. Then, column eluted with elution buffer and each fraction was collected for protein detection by coomasie reagent. The positive fractions were collected and loaded into G25 column to remove unnecessary salt from the solution with PBS. Each fraction was collected and was detected by coomasie reagent for protein content. Then the recombinant protein was checked by SDS-PAGE (Fig.14).

• Elution buffer (20 mM Na2HPO₄, 0.5 M NaCl, 200 mM imidazole, pH 7.4)

• Binding buffer (20 mM Na2HPO₄, 0.5 M NaCl, 40 mM imidazole, pH 7.4)

• Wash buffer (20 mM Na2HPO4, 0.5 M NaCl, 60 mM imidazole, pH 7.4)

2.16 Recombinant Urease B Protein Expression

pET-30-Urease B plasmid were transformed into E. Coli BL21, then grown on LB plates containing kanamycin (30 mg/ml) at 37°C. After 16 hr incubation, five colonies were inoculated into 100ml LB medium containing kanamycin (30 mg/ml) at 37°C for 16 hr. The culture broth were refreshed in 900 ml LB medium with shaking at 37°C, 225 rpm until the value of OD 600 reaches 0.6 (about 40 min). IPTG diluting from stock concentration of 800 mM to a final concentration of 1 mM were added to culture broth for 4 hr incubation. Cells were harvested by centrifugation at 8,000 rpm for 15 min at 4°C. Supernatant were discarded and the pellet were resuspended into 30 ml lysis buffer. Then, total cell lysates were sonicated for 15 min and centrifuged at 12,000 rpm for 30 min at 4°C. The pellet was resuspended by 10 ml wash buffer I and centrifugated at 12,000 rpm for 20 min. Repeat the same step of above mentioned process. Then, the pellet was resuspended by 10 ml wash buffer II and the mixture was shake in 4 °C for 20 min (55 rpm). After the mixture was well mixed, the mixture was centrifugated at 12,000 rpm for 20 min. Repeat the same step of above mentioned process, again. The collected pellet was resuspened by 10 ml lysis buffer and centrifugated at 12,000 rpm for 20 min.

The pellet was resuspened by 10 ml binding buffer (6N urea) and the mixture was shake in 4 °C for 12~16 hr (55 rpm). The mixture was collected and centrifugated at 12,000 rpm for 30 min. Then the supernant should be filtered with 0.45 µm syringe filter. The purification of protein was performed on

HisTrap^TM HP column. All of the buffer used in the experiment should be elution buffer and each fraction was collected for protein detection by coomasie reagent. The positive fractions were collected and loaded into dialysis membrane (Spectrum Laboratories. Inc., USA) to exchange the urea contented solution Each fraction was collected and was detected by coomasie reagent for protein content. Then the recombinant protein was checked by SDS-PAGE (Fig.

14).

2.17 Ex Vivo Splenocyte Stimulation of LPTC

Spleens were isolated aseptically in a laminar flow hood. Organs were cut in several pieces and clumps were further dispersed by drawing and expelling the suspension for several times through a sterile syringe with a 19-G needle.

Suspensions were filtered through sterile gauzes. After 5min centrifugation with 1500rpm, pellets were resuspended with 10ml ACK lysis buffer (1X) in DDW and incubated for 10min. Suspensions were centrifugated with 1500rpm for 10min, then the pellets were washed by 5ml PBS and centrifugated (1500rpm, 5min) to remove the supernatant. Cells were resuspended with RPMI-1640 medium and immediately used. Splenocytes were seeded in 24 well at a density of 4 × 10⁶ cells/ well, then 80µg LPTC were added into cells and further cultured with 1ml RPMI-1640 medium for 48 hr.

2.18 Cytokine Release From Splenocytes

TNF-alpha in cell-culture supernatants was collected by centrifugation and assayed by ELISA using a mouse TNF-alpha ELISA development kit (R&D).

The concentration of TNF-alpha was determined using a standard curve. ELISA was performed as according to the manufacturer's instruction.

2.19 In Vivo immunization regimen

For heterologous immunization protocol, naïve mice were prime immunized with DNA (ie, pCJ3-Hphsp60 and pCJ3-Urease B) and were boosted

with protein (ie, rHpHsp60 and rUreB) (Fig.11). Control group received sterile normal saline solution. DNA was administered as 12.5 µg/dose with or without formulated with 10µl LPTC (3.28 mg/mL) in 5% glucose PBS and then protein was administered as 100 µg/dose with or without formulated with 10µl LPTC (100 mg/mL) in 5% glucose PBS by subcutaneous inoculation. One additional group received 25 µg DNA with formulated with 20µl LPTC (3.28 mg/mL) in 5% glucose PBS and boosted by 100 µg protein formulated with 10µl LPTC (100 mg/mL) in 5% glucose PBS. Each mouse was inoculated with 100µl formulation.

For co-delivery of DNA and protein antigen immunization protocol, we injected 100 µg protein antigen (ie, rHpHsp60 and rUreB) and 50 µg DNA (ie, pCJ3-Hphsp60 and pCJ3-Urease B) in 5% glucose PBS, or formulated with 10µl LPTC (25 mg/mL) in 5% glucose PBS buffer via subcutaneous route (Fig.15). Control group received sterile normal saline solution. Each mouse was inoculated with 100µl formulation. Mice were immunized at 0 and 2 weeks.

Blood were collected before each immunization and 2 weeks after the last dose.

2.20 Determination of Serum Antibody Levels

Blood sample were collected from the retro-orbital plexus of mice. Serum anti-HpHsp60-specific antibodies and anti-Urease B-specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Briefly,

micro-ELISA plates (Nunc-Maxisorp, Nunc, Wiesbaden, Germany) were coated with 100 ng rHpHsp60 or rUreB protein per well in 100 µl PBS. Serial dilutions of

micro-ELISA plates (Nunc-Maxisorp, Nunc, Wiesbaden, Germany) were coated with 100 ng rHpHsp60 or rUreB protein per well in 100 µl PBS. Serial dilutions of