DNA constructions and yeast clone selection
A yeast codon optimized sequence encoding the tandem core construct including lysine linkers in both MIRs was designed and synthesized at GeneArt and cloned in the pPICZC vector (Invitrogen) using BstBI and AgeI restriction sites. Silent mutations were introduced up- and downstream of the two MIRs to create unique restriction sites (XbaI/NotI in Core 1 and EcoRI/NheI in Core 2). The resulting plasmid pPICZC PHe7 K1,K1 (K1-K1, for short) was used as a template for other MIR insertions. The lysine-encluding linker in Core 1 was replaced by yeast-optimized DNA sequence encoding the 55 amino acid long influenza H3N2 virus (A/Hong Kong/1/1968, Accession № AAK51718) HA stalk domain (corresponding to HA amino acids 420–474), using XbaI and NotI restriction sites. Resulting pPICZC coHe(LAH3,K1) construct (LAH3-HBc, for short) was linearized with PmeI to transform P. pastoris KM71H electrocompetent cells via electroporation. High copy number clones were selected in 96 well plates with liquid YPD medium containing 0.2 mg/mL (first 48 h) and 2 mg/mL (following 48 h) of zeocin. Cultures showing highest optical density (OD) at A = 600 nm were selected and spread on YPD agar plates in order to isolate single cell colonies. Selected clones were analysed for insert copy number by quantitative real time PCR based on the zeocin gene. The highest copy clone was selected and used to generate a Research Cell Bank.
Fermentation conditions for LAH3-HBc
For seed culture, 2 × 250 mL of buffered glycerol-complex medium (BMGY; 1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM potassium phosphate buffer pH 6.0, 1.34% (w/v) YNB, 0.0004% (w/v) biotin, and 1% (v/v) glycerol) was inoculated with 1.8 mL of cell bank suspension (BMGY culture in 30% (v/v) glycerol, OD = 25.0) in 2 L baffled Nalgene® shakeflasks. After 16–18 h the OD of the flask was between 15 and 20 and a 5% transfer volume was used to innoculate the bioreactor.
Invitrogen’s fermentation protocol for Pichia pastoris MutS strains was used to generate experimental material in a 30 L BIOSTAT Cplus bioreactor (Sartorius). The reactor was filled with Basal Salts medium (26.7 mL/L phosphoric acid (85%), 0.93 g/L CaSO4, 18.2 g/L K2SO4, 14.9 g/L MgSO4·7H2O, 4.13 g/L KOH, 40 g/L glycerol), plus 4.35 mL PTM1 trace salts per litre of Basal Salts media to achieve a total starting working volume of 10 L post-inoculation. The PTM1 trace salts contained: CuSO4·5H2O, 6.0 g/L; KI, 0.08 g/L; MnSO4·H2O, 3.0 g/L; Na2MoO4·2H2O, 0.2 g/L; H3BO3, 0.02 g/L; ZnCl2, 20.0 g/L; FeCl3, 13.7 g/L; CoCl2 · 6H2O, 0.9 g/L; H2SO4, 5.0 mL/L; and biotin, 0.2 g/L.
The bioreactor was run in batch-mode after inoculation. The dissolved oxygen tension (DOT) was maintained at 30% and was controlled in a sequence cascade by agitating the impeller between 400 to 1000 rpm followed by oxygen gas blending in ratio mode at a constant volumetric gas flowrate of 0.51 vvm. The pH range was maintained between 4.75–5.0 and pre-induction temperature at 30 ± 0.1 °C. A 20% drop in carbon evolution rate (CER) and spike in DOT, indicating depletion of carbon source, triggered the fed-batch induction phase. This was observed 28.5 h after bioreactor inoculation.
This fed-batch induction phase was maintained for 48 h at a fixed flowrate of 50 mL/h. The induction media itself is compromised of a 60:40 ratio of 50% (v/v) glycerol and pure methanol repectively, plus 12 mL PTM1 salts per liter of induction media. PPG2000 was used to prevent extensive foam formation throughout the fermentation. After 48 h of induction the culture was cooled to 12 °C to minimize proteolytic activity. Fermentation broth was harvested at 3000 g, 20 min and 4 °C. The wet pellets were weighed and stored at −20 °C.
Purification and characterization of chimeric VLPs
Yeast cells were resuspended in lysis buffer (20 mM Tris HCl, 100 mM NaCl, 0.1% Triton X-100, pH = 8.0) at a proportion of 15% (w/v) and disrupted by French Press (4 cycles, 10,000 psi). The soluble fraction was separated by centrifugation (30 min, 18,000 g, +4 °C).
For purification of HBc K1-K1 VLPs, solid ammonium sulfate was added until 35% of saturation by continious stirring for 5 min following centrifugation (20 min, 18,000 g, +4 °C). The precipitate was dissolved in a minimal volume of 20 mM tris HCl pH = 8.0, subjected to thermal treatment (30 min at 55 °C) and centrifuged again. The supernatant was passed through an anion-exchange HiPrep 16/10 DEAE Fast Flow column and the flow-through fraction was collected. All chromatography runs were monitored and controlled by an ÄKTA FPLC chromatography device (GE Healthcare).
For purification of chimeric LAH3-HBc VLPs, PEG6000 50% solution in 20 mM Tris HCl, pH = 8.0 (w/v) was added dropwise to the cell supernatant under continious stirring until the final concentration reached 5% (w/v) and incubated 1 h at +4 °C. After centrifugation (20 min, 18,000 g, +4 °C) the precipitate was dissolved in a minimal volume of 20 mM tris HCl, 2 M urea and loaded onto a size-exclusion Sepharose 4 FF matrix (XK26/40 column, bed height 25 cm) in column buffer A (20 mM Tris HCl, 100 mM NaCl, 1 M urea, pH = 8.0) at V = 1 mL/min. 10 mL fractions were collected and analyzed by native agarose gel, denaturing PAGE, and electron microscopy. Selected fractions were pooled and loaded onto anion-exchange Fractogel EMD TMAE (M) column (Tricorn 10/50 column, 3.5 mL bed volume) equilibrated with column buffer A. Column-bound proteins were eluted with a linear gradient of 10 column volumes using column buffer A containing 1 M NaCl. Fractions containing VLPs were dialized (10 kDa MWCO membrane) against 100× excess of 20 mM Tris HCl, 100 mM NaCl, pH = 8.0, with two buffer exchanges, for 48 h at +4 °C. Dialized material was pooled and concentrated with Amicon 100 kDa MWCO filter until it reached concentration of about 1 mg/mL. The VLP preparation was aliquoted, slowly frozen at −70 °C in a Mr. Frosty™ container and used for immunization of mice.
Protein concentrations were estimated by the Bradford assay. The purity of protein samples was analyzed by SDS-PAGE according to standard protocols with a 4% stacking and 15% separating polyacrylamide gel (PAAG). To visualize protein bands, the gels were stained with Coomassie Brilliant Blue G-250. Alternatively, separated proteins were transferred onto nitrocellulose membrane and detected by immunoblotting with the monoclonal anti-HBc antibody and the anti-mouse IgG peroxidase conjugate (DAKO). To assess nucleic acid content, samples were subjected to native 1% agarose gel electrophoresis in TAE buffer (pH = 8.4) for approximately 0.5 h at 5 V/cm. Nucleic acids in the agarose gels were visualized by ethidium bromide staining. A 1 kb DNA ladder (Thermo Fischer Scientific) was used as a marker. For transmission electron microscopy, the protein samples (at c = 0.1–0.5 mg/mL) were adsorbed on carbon-Formvar-coated copper grids and negatively stained with 1% uranyl acetate aqueous solution. The grids were examined with a JEM-1230 electron microscope (JEOL Ltd., Tokyo, Japan) at 100 kV. Electron micrographs were recorded digitally using a side-mounted Morada camera (Olympus - Soft Imaging System GmbH, Munster, Germany) with iTEM software (version 3.2, Soft Imaging System GmbH).
Mouse immunizations
All mouse experiments were performed in accordance with protocols approved by the Animal Welfare Structure of the Minister of Agriculture, Viticulture and the Consumer Protection of the Grand Duchy of Luxembourg (Ref. LNSI-2014-02). Female BALB/c mice were purchased from Harlan Laboratories, Inc. 8-week-old BALB/c mice were immunized using a standard protocol including 3 intraperitoneal injections at 2 weeks interval with 30 μg of chimeric LAH3-HBc VLPs for primary immunisation, and with twice 15 μg for the second and third injection. The antigen was dissolved in 100 μL phosphate buffered saline adjuvanted with 100 μL MF59 (Addavax, Invivogen) containing 20 μg CpG (OD2395 Vaccigrad, Invivogen) totaling 200 μL per injected dose.
Enzyme-linked immunosorbent assay (ELISA)
Influenza-specific IgG in mouse serum was measured by indirect ELISA using the following purified recombinant group 1 and group 2 HA antigens at the given concentrations: A/Texas/36/1991 (Tex91) (H1, 1.5 μg/ml), A/California/4/2009 (Cal09) (H1, 3.5 μg/ml), A/Japan/305/57 (JP57) (H2, 1.25 μg/ml), A/Perth/16/2009 (Perth09) (H3, 0.625 μg/ml), A/Swine/Ontario/01911–1/99 (Onta99) (H4, 1.25 μg/ml), A/Vietnam/1203/04 (VN04) (H5, 1.25 μg/ml), A/Netherlands/219/2003 (Neth03) (H7, 2.5 μg/ml), A/Hong Kong/1073/99 (HK99) (H9, 1.25 μg/ml), A/Jiangxi-Donghu/346/2013 (JX13) (H10, 2.5 μg/ml), A/mallard /Alberta/294/1977 (Alb77) (H11, 1.25 μg/ml), A/mallard/Astrakhan/263/1982 (Astrak82) (H14, 1 μg/ml), and A/duck/AUS/341/1983 (AUS83) (H15, 2 μg/ml) all purchased from Sino Biological Inc. Subsaturating coating concentrations were determined with a serum of a mouse sublethally challenged either with pH1N1 (group 1 HA) or H3N2 (group 2 HA) or with specific monoclonal antibodies against HA H1N1, HA H3N2, HA H5N1, HA H7N9 and HA H9N2 from the same supplier, after coating ELISA plates with threefold dilution series of each antigen.
Wells of 384-well microtiter plates (Greiner) were coated overnight at 4 °C with 20 μL/well of 3.5 μg/mL purified HA proteins in carbonate buffer (100 mM, pH = 9.6) or with carbonate buffer alone as a background control. Irrelevant antigens (Cytomegalovirus grade 2 antigen, 2 μg/ml; purified EBV capsid protein, 30 ng/ml; Toxoplasma gondii antigen, 2 μg/ml; Microbix, Mississauga, Canada) served as negative antigen controls. Reactivity against “empty” HBc VLPs (20 μl per well of 2 μg/ml) was used as positive control. All subsequent steps were performed at room temperature. Wells were washed sequentially in washing buffer (Tris HCl containing 1% Tween 20) and blocked for 2 h with 1% BSA in Tris buffer. After washing, sera (starting with a 100-fold dilution) were added, incubated for 90 min, and washed. Alkaline phosphatase conjugated goat anti-mouse IgG (1/750 dilution, ImTec Diagnostics) was added for 90 min, washed and developed using 2-amino-2-methyl-1-propanol. Absorbance was measured at 405 nm (Spectromax Plus, Sopachem) after 60 min incubation.