DNA constructions and manipulations
Plasmids and baculovirus stocks bearing HPV 16L1 cDNA have previously been described. Deletions of 16L1 cDNA were created by first ligating the entire cDNA of 16L1 into the BglII-SmaI site of pSP72 (Promega) to generate pSS1. Using PCR SuperMix High Fidelity (Invitrogen) and pSS1, inverse PCR was used to generate 16L1 derivatives deleted for aa 404–436 (del1) and aa 413–430 (del2). To enable modular oligonucleotide-based constructions, a unique NheI site encoding the residues ...AS... was engineered in place of each of the deletions. The following oligonucleotide 5' and 3' pairs (with the NheI site underlined) were used in PCR amplifications: del1: 5'GGCCGCTAGCAAAGAAGATCCCCTTAAAAAATATACTT; and 5'GGCCGCTAGCATTCCAGTCCTCCAAAATAGT; del2: 5'GGCCGCTAGCCATACACCTCCAGCACCTAAAGAAGATC; and 5'GGCCGCTAGCGCCTCCTGGAGGAGGTTGTAAAC. The following PCR conditions were used: 94°C × 5 minutes × 1, then 35 cycles of 94°C × 1 minute, 55°C × 30 seconds, and 68°C × 5 minutes, followed by 68°C × 7 minutes and overnight storage at 4°C. The PCR amplicons were column purified (Qiagen), serially digested with NheI and DpnI (to remove the pSS1 template) and then self-ligated. The resulting plasmids were sequenced to confirm the existence of the respective deletions and NheI sites within the L1 cDNA. Thereafter, the modified L1 cDNAs were excised and ligated into the BglII-SmaI sites of pVL1392 (Orbigen), during which the NheI site remained unique within the resulting plasmids, termed pC-1 (bearing 16L1 del1) and pB-1 (bearing 16L1 del2).
To embed RSV F protein epitopes within the h4 domain of L1 derivatives, codon-optimized complementary oligonucleotides that encode aa 255–278 (SELLSLINDMPITNDQKKLMSNNV) and 423–438 (TASNKNRGIIKTFS) of RSV A2 strain F protein (GI: 333933) were used. These oligos bore NheI-compatible termini and were phosphorylated with T4 kinase (NEB), annealed, and ligated into the NheI site of pC-1 and pB-1. Sequencing of the resulting plasmids confirmed the existence of the appropriate oligonucleotide sequences and predicted to encode the aa sequence: AS...RSV F epitope...AS, in which the alanine and serine flanking the RSV-derived residues are derived from the NheI site. Note that the oligos were designed such that the first residue (S) of RSV F 255–278 starts from the serine incorporated from the NheI site, i.e. the amino terminus of the epitope-bearing sequence is: ...ASELL...
To construct the baculovirus stocks for expression of L1 proteins used in this study, pC-1 and pB-1 derivatives were co-transfected into Spodoptera frugiperda (Sf9; Invitrogen) cells with linearized baculovirus DNA (Baculo-Gold; BD Biosciences) and cellfectin (Invitrogen). After 72 hrs, the Sf9 serum-free media from each co-transfection was removed and the baculovirus stocks were serially propagated and plaque purified × 3 prior to use in T. ni cells.
Protein expression and purification
RSV F protein was purified as previously described. Infection of T. ni cells with baculovirus bearing the 16L1 cDNA and subsequent purification of L1 VLPs were performed as described. The purification of capsomere derivatives was based on previous protocols and is briefly described as follows. T.ni cells growing at log phase in 250 mL cultures (1 – 2 × 106 cells/mL) in serum free media (Express Five, Invitrogen) were infected with each of the appropriate baculovirus stocks at a multiplicity of infection (MOI) of ≥ 3. After 72 hrs, the cells were collected by centrifugation, resuspended in ice-cold PBS + Complete Protease Inhibitor cocktail (Roche), and lysed using a Dounce homogenizer × 20 strokes and and a sonicator (3 × 20–30 second bursts, continuous cycle, output 3–4). The resulting mixture was brought to 40% CsCl (Roche) in 1× PBS and subjected to isopycnic ultracentrifugation at 28,000 × rpm × 40 hrs at 4°C using a Beckman SW28.1Ti rotor. The visible L1 band within the CsCl gradient was removed, dialyzed against PBS/0.5 M NaCl for >1 hr, and then overlayed onto a 30%/63% sucrose cushion using PBS/0.5 M NaCl as the solvent. After centrifugation at 28,000 × rpm × 5 hr at 4°C in a SW28.1Ti rotor, the capsomere-enriched fraction at the 0%/30% sucrose interface was removed and dialyzed exhaustively against PBS/1 M NaCl prior to -80°C storage and subsequent analysis.
Immunological and structural characterizations of capsomere derivatives
For protein gel electrophoresis, capsomeres and L1 VLPs were mixed 1:1 with 2× SDS-sample buffer containing β-mercaptoethanol, heated at 95°C for 2–5 minutes, and then resolved on 10%/5% discontinuous SDS-PAGE using BioRad Protean Tetra-cell apparatus. Where appropriate, molecular weight markers (Novex and MagicMark; Invitrogen) were resolved in parallel and proteins were visualized by staining with Coomassie Brilliant Blue R-250. For immunoblots, the proteins were resolved on SDS-PAGE as above and then transferred onto nitrocellulose using a BioRad Trans-blot device (typically at 100V at room temperature for 1 hr). PBS + 0.1% Tween-20 (PBST) + 2% dried non-fat milk was used to block non-specific protein binding onto nitrocellulose. For detection of L1 protein, CAMVIR-1 (Santa Cruz Biotechnology) was used at 1:60,000 dilution followed by goat anti-mouse IgG heavy/light chain antibody-horseradish peroxidase (HRP) conjugate (Southern Biotech) at 1:20,000 in PBST. Antibody-antigen complexes were visualized by chemiluminescence (ECL; Pierce) and radiography (Kodak).
ELISAs were performed essentially as previously described. Typically, each protein for analysis was diluted with PBS and plated at 100 ng/well onto 96 well ELISA plates (Nunc) and incubated overnight at 4°C. Following incubation with primary antibodies as described in figure legends, alkaline phosphatase-conjugated goat anti-mouse secondary antibodies (Southern Biotech) and phosphatase substrate tablets (Sigma-Aldrich) were then used to visualize antigen-antibody complexes. The resulting colorimetric reactions were read at OD405 nm using a 96-well ELISA plate reader (Molecular Devices).
For electron microscopy analysis, capsomere samples (typically at 0.5–1 mg/ml in PBS/1 M NaCl) were diluted 1:10 in ice-cold PBS/1 M NaCl and then adsorbed onto carbon-coated grid for approximately 1 – 3 minutes. Excess fluid was then blotted with filter paper and the grids were negatively stained with 2% uranyl acetate. Images from grids were obtained using a Hitachi 7100 transmission electron microscope at 80 kV and 60,000 × – 100,000 × magnification. As control images for capsomeres, intact 16L1 VLPs were dissociated into capsomeres using previously described incubation conditions with dithiothreitol.
Sucrose gradients with standards (bovine catalase and E. coli β-galactosidase, Sigma-Aldrich; bovine serum albumin, VWR) were performed as described except that PBS/1 M NaCl was used as the sucrose solvent. Approximately 50 – 100 μg of each of the three standards were mixed with 100 μg of each of the capsomeres or intact L1 VLPs and subjected to ultracentrifugation at 41,000 × rpm × 16 – 20 hours at 4°C using a SW41.1Ti rotor (Beckman). The resulting gradient fractions (0.5 ml aliquots) were serially collected from the top of the ultracentrifuge tube, resolved on 10/5% SDS-PAGE, and then stained with Coomassie Brilliant Blue R-250 (for localization of standard peaks) or transferred to nitrocellulose and probed with CAMVIR-1 anti-L1 mAb prior to chemiluminescence detection.
Immunogenicity of capsomere derivatives in mice
The animals were fed standard diet and water ad libitum and housed a pathogen-free environment within the University of Rochester School of Medicine and Dentistry Vivarium. Prior to any immunogenicity studies, all animal care and use protocols used in this study were approved by the Institutional Animal Care and Use Committee at the University of Rochester Medical Center. Female 6 – 8 week old BALB/c mice (Jackson Laboratories) in groups of 4 – 5 mice per capsomere were injected intramuscularly with 50 ug of each of the capsomeres. For priming injection, the capsomeres were diluted 1:1 and emulsified with Freund's complete adjuvant, while for boosts at weeks 3 and 6, Freund's incomplete adjuvant was used at 1:1 dilution. At week 6, submandibular bleeds were performed on the mice and the resulting sera were analyzed for reactogenicity against purified RSV F protein and purifed 16L1 VLPs. For antisera against 16L1 VLPs, 50 μg of purified VLPs were injected intramuscularly into mice as above except that no adjuvants were used.