Bocavirus sequence and modification.
The open reading frame of the bocavirus capsid proteins (or virus proteins), encoded by the cap
gene, is based on the previously published HBoV-st2 sequence (GenBank accession number DQ000496) (2
). By introducing several sequence modifications into the capsid protein genes, a bicistronic mRNA was utilized to produce VP1 and VP2 in the baculovirus system (Fig. ). A single open reading frame encodes both the large and small capsid proteins, with the larger capsid protein (VP1) and internal translational initiation producing the smaller, major capsid protein. To prevent translation initiation at a noninitiating AUG, the three out-of-frame ATG triplets in the VP1 unique sequence were altered without changing the amino acid. Thus, the first methionine codon that scanning ribosomes encounter is the initiation codon for the major coat protein. Achieving the typical parvovirus-like stoichiometry of the major and minor capsid proteins in the heterologous insect cell system was accomplished by changing the predicted VP1 initiation codon from AUG to ACG. Members of the Dependovirinae
(AAV) utilize non-AUG initiation codons to regulate the level of VP2 produced from a VP2/VP3 bicistronic mRNA, and this strategy has proven successful for recombinant AAV produced in BEV-insect cell cultures (50
). The threonine (ACG) codon requires the proper context for translational initiation; therefore, the nonanucleotide immediately upstream of the wild-type AAV2 VP2 ACG initiation codon (CCTGTTAAG, corresponding to nucleotides 2606 to 2613 of AAV2) was inserted upstream of the VP1 initiation site in the bocavirus VP expression cassette (Fig. ). In addition, a glycine codon (GGT) was inserted in the second codon, creating a Kozak-compatible motif. A silent transversion in the first position of the second codon was introduced to improve the translational initiation of the major coat protein (Fig. ). To facilitate the cloning in vector pVDF, two restriction enzyme sites (EcoRI and NotI) were included at either end of the VP gene. The new HBoV capsid gene was synthesized (BioBasic, Inc., Markham, Ontario, Canada) and cloned into plasmid pUC 59.
FIG. 1. Optimization of the HBoV genome for production of virus proteins in a baculovirus system. The VP1 unique region and a VP1 and VP2 amino terminus common region were encoded in a single open reading frame. To prevent translation initiation at a noninitiating (more ...) Cloning and baculovirus production.
The synthetic capsid gene was amplified by PCR using 5′-CGCACCACAAAACACCTCAGG and 5′-GGTGACCATTCTGAATTGTG as the upstream and downstream primers, respectively, yielding a 2,200-bp fragment. An aliquot of the PCR mixture was digested with EcoRI and NotI and then purified (PCR purification kit; Qiagen, Inc., Valencia, CA). The digested PCR product was ligated (Fast Ligation; New England BioLabs, Ipswich, MA) with EcoRI- and NotI-digested pFastBac (Invitrogen Corp., Carlsbad, CA), yielding pFB-Boca. Recombinant baculovirus was generated using the Bac-to-Bac system, which produces infectious, recombinant baculovirus DNA in Escherichia coli strain DH10Bac (Invitrogen). The “bacmids” from several DH10Bac colonies were isolated and used to transfect Sf9 cells according to the manufacturer's protocol (Invitrogen). Briefly, 30 μg of bacmid DNA was mixed with 600 μl of Grace's medium (Invitrogen) and combined with 36 μl of Cellfectin (Invitrogen) premixed in 600 μl of Grace's medium. The final mixture (1.2 ml) was added to each well of six-well plates containing approximately 1 × 106 Sf9 cells per well. After 3 days, the supernatant of each well was collected, presumably containing BEV generation P1. The P1 stock was amplified (1:100, by volume) in Sf9 cell suspension cultures (2 × 106 cells/ml) for 3 days, generating P2 BEV. Infectious BEV titers were determined by plaque assays. The P2 stock, titers of >1 × 108 PFU/ml, were filtered (0.22 μm) and stored at 4°C in opaque, dark-sided, 50-ml conical tubes (Greiner Bio-One, Monroe, NC).
VLP production and purification.
The capsid or virus proteins of HBoV were produced by infecting 100 ml of Sf9 cells (2 × 106
cells/ml) with clonally isolated baculovirus at a multiplicity of infection of 3 PFU/cell. At 72 h postinfection, cells and supernatant were separated by centrifugation (900 × g
for 15 min). The VLP in the supernatant was recovered by precipitation in 2.5% (final concentration) polyethylene glycol (PEG) (5 ml of 50% PEG 8000 [Sigma-Aldrich, St. Louis, MO] added to 95 ml of supernatant) and incubated for 3 h at 4°C with gentle agitation. Cell pellets were resuspended in 7 ml of phosphate-buffered saline with 2 mM MgCl2
and disrupted using a 7-ml Dounce homogenizer. The cell lysates were clarified by centrifugation (2,000 × g
for 15 min) and concentrated by precipitation in 2.5% PEG as described above. The PEG-precipitated material was recovered by centrifugation (45 min at 2,600 × g
), and the pellets were resuspended in 11 ml of CsCl solution (refractive index [RI] = 1.372 or ρ = 1.41 g/cm3
). The CsCl solutions were centrifuged to equilibrium (72 h at 222,000 × g
) in a swinging-bucket rotor (SW41 rotor; Beckman Coulter, Inc., Fullerton, CA). Each centrifuge tube was fractionated dropwise via bottom puncture using a 26-gauge butterfly needle set, 0.5-ml fractions were collected, and the RI of each fraction was measured with a digital refractometer (AR 200; Leica Microsystems, Inc., Buffalo, NY). Typical parvovirus empty-particle densities ranged from 1.30 to 1.32 g/cm3
, corresponding to an RI of 1.362 to 1.364 (21
). Size exclusion column chromatography (Sephadex 200, 10/300; GE Healthcare Bioscience Division, Piscataway, NJ) provided the final purification step. The homogeneity of the HBoV VLP was assessed by silver-staining sodium dodecyl sulfate (SDS)-polyacrylamide gels.
Larger amounts of VLP were produced using 5-liter bioreactors (Wave mixer; GE Healthcare Bioscience Division, Piscataway, NJ). The purification protocol described above was modified for scale but otherwise remained the same.
The theoretical, 280-nm molar extinction coefficient (117,855 M−1
) was determined using ProtParam software (18
) and used to calculate the protein concentration by UV absorption.
Antibody production and purification.
Rabbit anti-bocavirus VLP immune serum was produced using a standard 70-day prime-boost regimen. In brief, 200 μg of purified HBoV VLP was administrated intramuscularly to the rabbit followed by three intramuscular boosts at 21, 35, and 49 days. Initially, a preimmunization sample was obtained, and subsequently, serum samples were collected at 44, 59, and 63 days postimmunization. The mean specific antibody concentration was estimated to be 0.15 to 0.5 mg/ml.
Purification of HBoV-specific antibody from human serum and rabbit serum was performed with either an affinity column prepared by covalently attaching HBoV-VLP to a 1-ml HiTrap N-hydroxysuccinimide-activated HP Sepharose column (GE Healthcare Biosciences) or using a 1-ml protein G-Sepharose column (GE Healthcare Biosciences) as indicated.
PAGE and Western blotting.
Polyacrylamide gel electrophoresis (PAGE) was used for determining the apparent molecular mass and estimating VLP homogeneity. Ten microliters of loading buffer and 4 μl of reducing agent (NuPage system; Invitrogen Corp.) were added to 26 μl of sample and heated at 70°C for 10 min prior to electrophoresis. Samples were applied (10 μl per lane) to a precast 4 to 20% polyacrylamide gel and electrophoretically fractionated in a morpholineethanesulfonic acid-SDS buffer system (NuPage; Invitrogen) at 150 V (constant voltage) for 60 min. Protein bands were visualized with either Coomassie brilliant blue (SimplyBlue SafeStain; Invitrogen) or silver staining (SilverQuest; Invitrogen) according to the recommendations of the manufacturer. For Western blot analysis, proteins were electroblotted from the gel onto a nitrocellulose membrane according to the manufacturer's protocols (iBlot system; Invitrogen).
The Electron Microscopy Core Facility of the National Heart, Lung, and Blood Institute of the National Institutes of Health (NIH) (Mathew P. Daniels, Director) supported the ultrastructure analysis of the HBoV VLP specimens using a transmission electron microscope (JEM1200EX; Jeol, Ltd., Tokyo, Japan) equipped with an AMT XR-60 digital camera (Advanced Microscopy Techniques, Danvers, MA). The VLP samples were diluted, and a small aliquot, e.g., 10 to 20 μl, was pipetted onto paraffin film. An aliquot of each sample (5 μl) was placed onto a carbon-coated 200-mesh copper grid for 1 min and then washed with 10 drops of distilled water. Staining was achieved by adding 5 drops of 2% (wt/vol) uranyl acetate. Excess staining solution was immediately wicked away with blotting paper, and the grids were then air dried. The grids were examined with a transmission electron microscope, and micrographs of randomly selected fields were taken at various magnifications.
The optimum conditions for ELISA, including coating concentration of the VLP protein, serum dilution, conjugate dilution, incubation times, temperature, and blocking reagent, were determined in preliminary checkerboard titration experiments. To optimize ELISA, bocavirus-VLP proteins were diluted to 2 mg/ml in 0.2 M carbonate-bicarbonate buffer, pH 9.6 (Sigma-Aldrich Corp., St. Louis, MO), and 100 μl was added to each well of 96-well polystyrene flat-bottom plates (MaxiSorp; Nunc) and incubated overnight at 4°C. After the antigen solution was removed, 200 μl of blocking solution (2% bovine serum albumin in carbonate-bicarbonate buffer [pH 9.6]) was added and incubated overnight at 4°C. Sufficient plates were prepared for human serum tests in advance and stored at 4°C. Human serum and preimmune rabbit serum samples were serially fourfold diluted from 1/100 to 1/409,600, whereas bocavirus rabbit antiserum was serially fourfold diluted from 1/1,600 to 1/6,553,600 in PBS-T (phosphate-buffered saline, 1% bovine serum albumin, 0.05% Tween 20). The plates were incubated at 37°C for 1 h on a platform shaker with the diluted sera and then washed four times with PBS-T. The secondary goat anti-human IgG antibody for the human serum samples (Chemicon Division, Millipore, Corp., Billerica, MA) and goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO) were obtained as horseradish peroxidase (HRP) conjugates and used after 1:30,000 dilution in PBS-T. One hundred microliters of the diluted appropriate secondary IgG-HRP was added to each well. The nonadsorbed IgG-HRP was removed by washing with PBS-T. After five washes, 100 μl of 3,3′,5,5′-tetramethylbenzidine liquid substrate (Sigma-Aldrich) was added to each well and incubated at room temperature in the dark for 15 min. Peroxidase cleaves 3,3′,5,5′-tetramethylbenzidine, and by adding 1 N sulfuric acid the mixture (100 μl to each well), the peroxidase reaction was terminated, producing a chromogenic end product that absorbs at 450 nm. An automated plate reader (Spectramax M2 and Softmax Pro software; Molecular Devices) measured the absorption of each well at 450 nm with a reference wavelength set at 570 nm.
Human sera were obtained from healthy blood bank donors at the Warren G. Magnuson Clinical Center of the NIH under a protocol approved by the Internal Review Board of the National Cancer Institute. Patient identifiers were removed, and only age and sex data were accessible.
The AAV VLPs used in this study were produced in insect cells using the baculovirus expression system. The AAV cap
gene expression constructs were described previously (50
). The production and purification of AAV-VLPs using a 5-liter-bag Wave bioreactor were performed for HBoV-VLP production.
Chi-squared or Fisher's exact test was performed to determine differences between females and males and as a function of age of the donors (1