Plasmid construction
The construct into which the 16E7 and 16E7SH genes were cloned for expression in plants (pTRAc-ZERA-eGFP) was made as follows: the eGFP gene was amplified from pEGFP (BD Biosciences) using a forward primer (5’-gatcccatggacgacgatgataaggtgagcaagggcgaggagctg-3’) which allowed for inclusion of an enterokinase cleavage site (DDDDK) at the 5’ terminus of eGFP (italicized sequence), and the following reverse primer: 5’ cggatccattacttgtacagctcgtccatgccgag 3’. The amplified product was subcloned into pUC18ZERA (provided by Era Biotech, Barcelona, Spain [26] using Nco I and BamH I restriction enzyme sites such that the Zera® sequence was in frame with the eGFP fusion to generate pZERA-GFP. This construct was amplified using primers (5’ actcatgagggtgttgctcgttgc 3’ and 5’ cggaccattacttgtacagct 3’) to enable cloning of the ZERA-eGFP fusion into the plant expression vector pTRAc (provided by Rainer Fischer, Fraunhofer Institute, Molecular Biology and Applied Ecology, Aachen, Germany) [19], using BspH I and BamH I restriction enzyme sites. The oncogenic HPV-16 E7 (16E7) gene (Figure 1A) was provided by J. Schiller (Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD, USA) and amplified using primers 5'-gatctcatgagtgacgacgatgataagatgcatggagatacacctacattg-3'and 5'-agggatccttatggtttctgagaacagatgg-3'. The product was cloned into the Nco I and BamH I sites of pTRAc-ZERA-eGFP (replacing eGFP) forming pTRAc-ZERA-16E7. The shuffled HPV 16E7 (16E7SH) gene (Figure 1A) [11] was amplified from pTH-16E7SH using the following primers: 5'-gatcccatggacgacgatgataagatgcacggcgacaccccc-3' and 5'- aaggatccttatggtttctgagaacagatggggcac-3'. The product was cloned into the Nco I and BamH I sites of pTRAc-ZERA-eGFP (replacing eGFP) forming pTRAc-ZERA-16E7SH. In addition, the modified 16E7SH fragment was cloned into the Afl III and BamH I sites of the vector pTRAc, yielding pTRAc-16E7SH.
To construct the recombinant DNA vaccine, ZERA-16E7SH was amplified from pTRAc-ZERA-16E7SH with primers 5'-aaaagcttcatgagggtgttgctcgttg-3' and 5'-atgaattctggatccttatggtttctgag-3' and cloned into the pTH vector [27] using Hind III and EcoR I to yield pTH-ZERA-16E7SH. The constructs utilised in this study are summarised in Figure 1B. For expression in E. coli, 16E7SH was cloned into pPROEX™ HT (Life Technologies) and for expression in insect cells it was cloned into flashBAC expression vector (Oxford Expression Technologies Ltd.).
Agrobacteriumtransformation
Three hundred ng of the pTRA constructs were individually electroporated into A. tumefaciens GV3101 strain with the pMP90RK helper plasmid as previously described [19]. Electroporated cells were incubated in 1 ml Luria–Bertani (LB) broth for 2 h then plated on LB medium containing, 50 μg rifampicin ml-1 and 30 μg kanamycin ml-1.
Agroinfiltration and transient expression
Agrobacterium LBA4404 with pBIN-NSs, containing the TSWV NSs silencing suppressor gene [28], and Agrobacterium tumefaciens GV3101::pMP90RK with the pTRAc constructs were grown in induction medium, prepared for infiltration and the Agrobacterium suspension was either injection- (small scale - a few leaves) or vacuum-infiltrated (large scale - whole plants) into the abaxial air spaces of 6-8 week old N. benthamiana leaves and left to grow under 16 h light, 8 h dark at 22°C growth conditions all previously described until the desired extraction time ranging from day 1 to day 10 post infiltration [19]. The constructs were either infiltrated alone or co-infiltrated with the LBA4404 pBIN-NSs.
Protein extraction
To screen leaf tissue for protein expression, five leaf discs (5 mm diameter ~ 0.05 g wet plant mass) were ground in liquid nitrogen, incubated in 200 μl of extraction buffer (100 mM Tris pH 8, 5% SDS, 5% β-ME, 200 mM NaCl) with 1× Complete Protease Inhibitor (EDTA-free; Roche) at 95°C for 20 min. Samples were then incubated at RT for 1 h followed by agitated incubation at 37°C overnight. The supernatant was clarified by centrifugation for 20 min (13000 rpm, desktop centrifuge, 4°C) and then detected by means of western blots.
Protein detection in leaf extracts
Samples were incubated at 85°C for 5 min in loading buffer, separated on 15% SDS-PAGE, then either stained with Coomassie blue or transferred onto a nitrocellulose membrane using the Trans-Blot® SD Semi-Dry Transfer Cell (Bio-rad) for western blot analysis. E7 proteins were detected with anti-E7 sera (1:4000) followed by goat anti-mouse-alkaline-phosphatase conjugate (1:10000; Sigma). Zera®-containing proteins were detected with polyclonal anti-Zera® sera (1:5000; ERA biotech) followed by goat anti-rabbit-alkaline-phosphatase conjugate (1:5000; Sigma). NBT/NCIP tablets (Roche) were used for final detection.
Proteins were quantified by measuring the density of the band on a western blot or Coomassie stained bands in comparison to a known protein concentration standard, using GeneTools software (SYNGENE) on scanned images. TSP was determined using the BioRAD assay according to the manufacturer's instructions.
Large-scale expression and sucrose gradient purification of protein bodies (PBs)
Large-scale expression and purification was required to produce vaccine dosages for animal trials. Seven days post vacuum infiltration, the leaves were cut, weighed, ground in liquid nitrogen using a pestle and mortar and resuspended in a ratio of 1:5 (w/v) of buffer PBP3 (100 mM Tris pH 8, 50 mM KCl, 6 mM MgCl2, 10 mM EDTA, 0.4 M NaCl) made up in 10% sucrose. Samples were centrifuged at 24 000 rpm for 10 min on ice and then filtered through a Miracloth™ (Calbiochem). The filtrate was loaded onto a sucrose step gradient (19%, 27%, 42%, 56% w/w) and ultracentrifuged at 80 000 g, 4°C for 2 h (Beckman SW32Ti rotor). Protein fractions (IF) of 2 ml were retrieved at the step interface and the pellet was resuspended in 2 ml PBP3 and analyzed by western blotting.
Expression of 16E7SH in E. coli
16E7SH protein for animal trials was expressed in E. coli as detectable levels of its expression in plants were never achieved. Competent DH5α E. coli cells were transformed and protein expression was induced as per manufacturer’s recommendations. The induced cells were pelleted and lysed (Tris-HCl 50 mM pH 8, NaCl 300 mM,5% glycerol,1 mM DTT). Inclusion bodies containing 16E7SH were solubilized with solubilization buffer (Tris-HCl 50 mM pH 7.6, NaCl 300 mM, 8 M Urea, 2 mM DTT). After solubilization, 16E7SH was IMAC-purified twice on a Ni column, washing bound samples with Triton X-114, both to purify the protein and to remove endotoxins. Eluted proteins were further purified twice by size exclusion chromatography (Superdex 200 column, GE Healthcare Life Sciences), the first in the presence of arginine, and the second with phosphate-buffered saline (PBS). The resulting protein was analyzed to verify the absence of LPS contamination with an Endosafe®-PTS™ test system (Charles River Ltd.).
Insect cell culture and baculovirus production of PBs
Spodoptera frugiperda (Sf9) insect cells (Invitrogen) were grown in suspension or as monolayers at 28°C in serum-free SF900 SFM Medium (Gibco). Recombinant baculoviruses were produced by co-transfection of Sf9 cells with flashBAC DNA and transfer vectors pBacPak8 containing the Zera®-encoding sequence, according to the manufacturer’s recommendations. Recombinant viruses were titrated and monolayer Sf9 cultures were infected with the recombinant baculovirus at a multiplicity of infection (MOI) of 5.
Zera® protein bodies were isolated from frozen Sf9 cell biomass previously infected with the selected baculovirus. Zera® PBs were recovered as described by Torrent et al. [26]. PBs washed with LPS-free water were characterized by SDS-PAGE and confocal and scanning electron microscopy.
Mammalian cell culture
Wildtype HPV-16 E7-expressing 2 F11 cells (C57BL/6 origin, H2b haplotype; [29] were cultured in RPMI 1640 supplemented with heat-inactivated 5% (v/v) foetal calf serum (FCS, Gibco, Eggenstein, Germany), 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml), G418 (0.8 mg/ml). RMA cells [30] were cultured with the same medium with the exception of G418.
C3 tumour cells derived from embryonic mouse cells transfected with the complete HPV-16 genome [31] were cultured in the same medium as 2 F11 cells, supplemented with kanamycin (0.1 mg/ml). Splenocytes were cultured in αMEM (Sigma, Deisenhofen, Germany) supplemented with 10% FCS, 0.1 mM β-mercaptoethanol, 4 mM glutamine and antibiotics as above for the first 4-5 days after splenectomy. Subsequently, the splenocytes were cultured in αMEM + supplemented with 2.5% supernatant of a concanavalin-A-induced rat spleen cell culture as a source of murine IL-2 and 25 mM methyl-α-mannopyranosid (Sigma, Deisenhofen, Germany).
Immunization of mice
Six-to-eight week old female C57BL/6 mice (owner bred) were kept under SPF isolation conditions and standard diet at the animal facilities of the University of Konstanz, Konstanz, Germany. In the case of DNA injections, agarose-gel verified plasmids (>95% supercoiled) of preparations containing less than 0.1 endotoxin units/μg plasmid DNA as tested earlier by Limulus endotoxin assay (QIAGEN EndoFree Plasmid Kit). PBs were thoroughly sonicated on ice. For co-inoculation with PBs and E7 proteins, PBs were mixed with the recombinant homogenized E7 protein by pipetting on ice directly prior to immunization (2-4 minutes). For CTL analysis animals were immunized once (100 μg DNA/per animal [50 μg DNA in 50 μl PBS per musculus tibialis anterior i.m.] or 5 μg ZERA-16E7SH +/- 100 μl Incomplete Freund’s adjuvant (IFA) or 2.5 μg 16E7SH +/- 2.5 μg Zera® PBs +/- 100 μl IFA per animal s.c. into the left flank). Ten to 12 days after vaccination animals were sacrificed and spleens were isolated.
Tumour regression experiments
C57BL/6 mice received 0.5 × 106 HPV-16 E7 expressing C3 cells [31] in 100 μl of PBS, subcutaneously in the right shaved flank (needles: 20G 1½” BD Microlance 3). When small tumours were palpable in all animals (12-15 days after tumour cell injection), the vaccine was injected i.m. in both musculus tibialis anterior for the DNA vaccines, or s.c. into the left flank for protein vaccines, as described above. Tumour sizes were measured with a caliper. Mice were sacrificed when the tumour size reached 400 mm2 or when tumours were bleeding. Tumour sizes of the mice within a group were calculated as arithmetic means with standard deviation (SD). All operations on live animals were performed under Isoflurane anaesthesia.
All animal experiments were performed with approval by and in accordance with regulatory guidelines and standards set by the institutional review board at Regierungspraesidium, Freiburg, Germany.
CTL and humoral responses in vaccinated mice
Data provided were obtained without in vitro restimulation ex vivo, all ELISPOT assays, or after one in vitro restimulation (51Cr-release assay). In the case of in vitro restimulation, 2 × 107 splenocytes (pretreated with ACT lysis buffer [17 mM Tris/HCl, 160 mM NH4Cl, pH 7.2] to deplete erythrocytes) were co-cultured with 2 × 106 irradiated (100 Gy) HPV-16 E7 wildtype-expressing 2 F11 cells [29] cells in 25 cm2 culture flasks for 5-6 days. Cultures were grown at 37°C and 7.5% CO2 in a humidified incubator.
IFN-γ/Granzyme B ELISPOT assays
Murine IFN-γ ELISPOT assays were performed ex vivo as previously described [11]. The Granzyme B ELISPOT assay was performed similarly to the IFN-γ ELISPOT assay. For this assay, the anti-mouse Granzyme capture antibody (100 ng/well, AF1865; R&D Systems, Minneapolis, USA) and the biotinylated anti-mouse Granzyme detection antibody (50 ng/well, BAF1865; R&D Systems, Minneapolis, USA) were used. Splenocytes were seeded in triplicate in 2-fold serial dilutions from 200 000 to 25 000 cells per well. One of the triplicates was left untreated (negative control), the second received 200 ng of pokeweed mitogen/well (Sigma, Deisenhofen, Germany) in 2 μl of PBS (positive control), whereas the third received 0.2 μmol of H2Db-restricted HPV-16 E749-57 peptide in 2 μl of PBS/well (test sample). Spots of the negative control (untreated) were subtracted from the spot number in the corresponding test sample.
51Cr-release assays
The 51Cr-release assays were performed after one in vitro restimulation of murine spleen cells. One × 104 Na2CrO4-labelled (0.05 mCi) target cells/well (RMA or E7 wildtype expressing 2 F11 cells) were incubated together with decreasing numbers of effector cells in 200 μl per well of a 96-well round bottom plate (Costar, Corning, USA) for 4 h. Subsequently, 50 μl of supernatant was harvested from each well and the released radioactivity was measured in a Microbeta counter (Wallac, Turku, Finland). Specific lysis was calculated according to the formula: percent specific lysis = [(cpm of the sample - spontaneous release)/(total release - spontaneous release)] × 100, where total release and spontaneous release are measured in counts per minute (cpm). Spontaneous chromium release was determined by using 51Cr-labeled target cells without effector cells, and total chromium release was determined by adding 2% Triton X-100 to lyse the labelled target cells.
Humoral antibody titre determination by ELISA
One μg/ml of recombinant HPV-16 E7-wildtype protein (ProteinX Lab, San Diego, CA, USA, Cat. No. 2003207) or Zera® protein diluted in PBS was used to coat round-bottom enzyme-linked immunosorbent assay (ELISA) plates (Becton Dickinson) by incubating at 4°C overnight. Wells containing PBS were used as a negative control. Plates were washed three times with PBS containing 0.05% Tween 20 and incubated for 1 h at 37°C with 100 μl of milk buffer (5% milk powder and 0.05% Tween 20 in PBS) per well. After the wells were washed three times with PBS, serum specimens diluted 1:10 in milk buffer, post-immunization [day of splenectomy] were added to two wells in a total volume of 50 μl per well, and incubated for 1 h at 37°C. Samples were removed and washed three times with PBS. In order to detect IgGs, HRP-conjugated rabbit anti-mouse IgG (heavy and light chains) (Zymed, San Francisco, Calif.) diluted 1:3000 were used. After incubation for 1 h at 37°C and three washes with PBS, substrate (200 μg of tetramethylbenzidine per ml in a solution of 0.1 M Na acetate [pH 6.0] and 0.03% H2O2) was added, the reaction was stopped with 1 M H2SO4, and the plates were assayed in an ELISA reader at 450 nm.
Statistical analysis
Differences of means between experimental and control group were considered statistically significant when p was less than 0.05 by unpaired Students t-test.