Biological materials
Unique biological materials described in this study that are not available from standard commercial resources are available from the authors upon request.
Bacterial strains and growth conditions
Details of all cells and strains used in this study can be found in Supplementary Table.
E. coli Top 10, a molecular cloning host, was grown in Luria Bertani (LB) media
(Difco, USA) containing ampicillin (100 µg/ml) at 37 °C. ClearColi BL21 (DE3) (Lucigen, USA), an endotoxin-free host, was engineered for BP production. For BP production 200 ml pre-culture in synthetic media was used to inoculate the 1 L main culture (synthetic media) in the Bioflo®320 (Eppendorf, Germany). This was followed by fed-batch cultivation where DO and pH were maintained for 48 h until harvesting of biomass.
Plasmid construction
Details of all plasmids used in this study can be found in Supplementary Table 4. Cloning techniques such as plasmid preparation, restriction digests, gel purification, ligations, competent cell preparations and transformations were performed as per user manuals and as described elsewhere61. The DNA fragments encoding two repeats of the B-cell epitope (NANPNANPNANP) or the B/T-cell epitope with amino acid sequence (DPNANPNVDPNANPNVNANPNANPNANPEYLNKIQNSLSTEWS PCSVT) derived from circumsporozoite protein (CSP) were synthesised (Biomatik, Canada) and subcloned into plasmid pPolyN generating the final plasmids pET-14b B12N PhaC and pET-14b BT2N PhaC. The DNA sequence was confirmed by the Griffith University DNA Sequencing Facility (Griffith University, Australia). The sequence confirmed plasmids were transformed into the endotoxin-free production host, ClearColi BL21 (DE3) (Lucigen, USA) that was previously engineered for BP production60.
BP purification and sterilisation
BP harbouring biomass was mechanically lysed using a microfluidizer M-110P (Microfluidics, USA). Cell lysate was centrifuged at 8000 x g for 30 min at 4oC to sediment the BPs, which were then sequentially washed 5 mM EDTA and detergent 2% (v/v) Triton X-100 containing wash buffers (10 mM Tris.HCl, pH 7.5)38. Purified BPs were stored in 10 mM Tris buffer (pH 7.5) containing 20% (v/v) ethanol at 4 °C for further analysis. BPs were then washed three times with (10 mM Tris.HCl, pH 7.5) before being resuspended in 10 mM Tris buffer pH 7.5, with 20% ethanol. One-hundred microlitres of the 20% (w/v) BP suspension was plated onto Thermo Scientific™ Blood Agar (TSA with Sheep Blood) (Thermo Fisher, USA) Medium to assess sterility.
BP protein analysis
BP-associated fusion proteins were analysed by SDS-PAGE as described elsewhere62. Briefly, protein samples were separated in 10% (vol/vol) polyacrylamide separating gels with 4% (vol/vol) polyacrylamide stacking gels at 170 V for 45 min. The molecular mass of the samples was estimated using GangNam-STAIN prestained protein ladder (iNtRON Biotechnology) or Novex™ Sharp Pre-stained Protein Standard (Invitrogen). SDS-PAGE were stained by incubating overnight in Staining solution (0.05% (wt/vol) coomassie brilliant blue R-250 dye, 50% (vol/vol) ethanol, and 10% (vol/vol) acetic acid), then rinsed and destained in 50% (vol/vol) ethanol and 10% (vol/vol) acetic acid for 6 h. Images were taken using a BIO-RAD Gel Doc XR + with the Image LabTM Software (Bio-Rad).
Protein quantification
Protein concentration and purity was determined via densitometry on SDS-PAGE using Image J version 1.52a (National Institute of Health). Bands were compared to a standard curve prepared from known concentrations of bovine serum albumin (BSA) standard as described elsewhere47. Additionally, protein concentration was also determined using the Quick StartTM Bradford 1x Dye Reagent (Bio-Rad) as per manufacturer’s instructions. Absorbance at 595 nm was measured using the Biotek Synergy 2 microplate reader (Biotek). Binding and purification capacity were calculated as μg of protein per mg of BP. Cleavage efficiency was calculated by dividing the amount of purified protein by the amount protein bound and multiplying by 100.
Mass spectroscopy quad time of flight (MALDI-TOF) analysis
Purified protein bands from the SDS-PAGE gel were excised and subjected to tryptic hydrolysis as described63. The resulting extracted tryptic peptide samples were then analysed by matrix-assisted laser desorption ionisation time-of-flight/mass spectrometry (MALDI-TOF/MS).
Transmission electron microscope (TEM)
BPs were processed and sectioned for TEM. Briefly, the samples were fixed in 2.5% glutaraldehyde, and embedded in 2% agarose. All subsequent processing was done in a Biowave processing microwave (PELCO). The samples were post fixed with 1% osmium tetroxide with 4 times 2 min on/off cycles at 80 W. They were washed twice in water at 80 W 40 s with vacuum. Dehydration through a graded series of ethanols, 30, 50, 70, 90, 2 × 100% all at 150 W for 40 s with vacuum. The samples were then infiltrated with Epon resin using graded mixes with ethanol, 3:1, 1:1, 1:3 then 2 times 100% resin all at 250 W for 3 min with vacuum. Samples were polymerised at 60 °C for 2 days. Ultrathin sections of 60 nm were cut using a diamond knife on an Ultracut UC6 ultramicrotome (Leica) and mounted on Formvar coated copper grids. The sections were stained with 5% uranyl acetate in 50% ethanol 5 min then rinsed in water and stained again with Reynolds Lead Citrate for 3 min and rinsed 21 in water again before being air dried. TEM micrographs of the sectioned samples were imaged using a Hitachi HT7700 (Hitachi) at 80 kV.
Size and zeta potential measurements
BP particle size was determined by dynamic light scattering (DLS) analysis using the Litesizer 500(Anton Paar) at room temperature (25 °C). Zeta potential of the BP was determined by electrophoretic light scattering coupled with phase analysis light scattering using Litesizer (Anton Paar). All BP samples were measured in 0.1% (wt/vol) of the wet particles in 50 mM Tris-HCl, pH 7.5, and the soluble protein samples were measured in 50 mM Tris-HCl, pH 7.5. Three technical replicates were performed.
PHB content analysis
Analysis of cellular PHB content and purified BP samples was based on the method of Karr et al.64. Twenty-five micolitre samples of culture were pelleted by immediate centrifugation (5000 × g for 10 min at 4 °C), rinsed with 5 ml of milli-Q water, and centrifuged again. Pellets were stored at −80 °C until further analysis. Pellets were then freeze-dried and weighed. Next, 2 ml of concentrated sulphuric acid was added to the pellet and PHB was acid hydrolysed to crotonic acid for 1 h at 90 °C. The samples were filtered using a 0.22 μm filter (Merck Millipore) and diluted 1 in 500. Adipic acid was added in 1:1 to the sample (400 mg/L final concentration) and used as an internal standard in HPLC analysis. For this, 30 μL of sample was injected into an Agilent Hiplex H column (300 × 7.7 mm, PL1170–6830) with a guard cartridge (Security Guard Carbo-H, 4 × 3 mm, Phenomenex PN: AJO-4490) using an Agilent 1200 HPLC system equipped with high-performance autosampler (Agilent HiP-ALSSL, G1367C), binary pump (Agilent Bin Pump, G1312A), degasser (Agilent Degasser, G1379B), thermostatted column compartment (Agilent TCC, G1316B), multiwavelength (Agilent MWD, G1365B) and refractive index (Agilent RID, G1362A) detectors. Analytes were eluted with 4 mM sulphuric acid at 0.6 ml/min flow rate, column temperature of 65 °C, and monitored at 210 nm UV and positive polarity at 40 °C on the RID, over an isocratic run of 30 min. Crotonic acid was used as the standard for quantification. Peak areas were integrated using ChemStation (Rev B.03.02 [341]). The PHB % w/w was calculated using the determined crotonic acid concentration in the sample (based on Karr et al.)64 and the analysed samples dry weight.
Vaccine formulation and immunogenicity study
Vaccine formulations contained either empty BPs, B-cell epitope peptide, B/T-cell epitope peptide, B-cell epitope-coated BPs or B/T-cell epitope-coated BPs. B- and B/T-cell epitope peptides were ordered from GenScript. All vaccines were formulated to contain 55.6% v/v of the adjuvant Xstend III and B- and B/T-cell epitope doses of 25 µg and 50 µg per injection, respectively. The empty BP group was formulated so that the amount of PhaC injected would equal the maximum amount of PhaC for any other group. The groups were double blinded, and injections were given to eight female Merino sheep per group on three occasions (days 0, 14 and 28), serum samples were taken on day 0, 14, 21, 28, 42, and 56 by jugular vein venipuncture. Approximately 18–20 ml serum was collected from each sheep, which was processed within 4 h of collection by accredited or trained personnel. Samples were aliquoted into cryovials labelled with a unique specimen number and stored in dry ice or at −80 oC until needed. This study protocol received ethical approval (approval numbers ELA1800507 and ELA1900168) from the Animal Research Authority of the Yarrandoo Elanco Animal Ethics Committee. This study complied with all relevant ethical regulations for animal testing and research.
Enzyme-linked immunosorbent assay (ELISA)
Serum antibody responses were analysed by ELISA. High-binding plates (Greiner Bio-One, Germany) were coated overnight at 4 °C with 100 µl of 5 µg/ml B-cell epitope- or B/T-cell epitope-coated BPs in phosphate-buffered saline containing 0.05% (v/v) Tween 20, pH 7.5 (PBST). As controls, plates were also coated overnight at 4 °C with corresponding amounts of empty BPs in PBST or only PBST. Plates were washed three times with PBST using the ELx 405 Select cw plate washer (BioTek, USA) and blocked with 3% (w/v) BSA for 1 h at 25 °C. Plates were washed with PBST three times again and incubated with primary polyclonal antibodies, sera taken from individual sheep, diluted with PBST at concentrations ranging from 1/400 to 1/409,600 at 25 °C for 1 h. When analysing antibody responses against empty BPs, primary antibody dilutions ranged from 1/400 to 1/1600. Additional control ELISAs to assess BP surface exposure of epitopes were performed using monoclonal anti-NANP3 antibodies (clone 2A10, MRA-183A, BEI resources) as the primary antibody instead of sheep sera at the same dilutions and using Rabbit Anti-Mouse IgG H&L (HRP) (Abcam, UK, Catalogue No.ab6728) diluted 1:1000 as secondary antibody. After three more washes with PBST, plates were incubated with secondary HRP-conjugated antibodies, Rabbit Anti-Sheep IgG H&L (HRP) (Abcam, UK, Catalogue No. ab6747), diluted 1/20,000 with PBST and incubated for 1 h at 25 °C to detect bound sheep antibodies. After washing, o-phenylenediamine substrate (Abbott Diagnostics, IL, USA) was added at a concentration of 0.7 mg/ml in Stable Peroxide buffer (Thermo Fisher, USA) on plates and incubated for 15 min at 25 °C. The reaction was stopped by adding 50 µL of 0.5 N H2SO4, and the results were measured at 490 nm on the Synergy™ 2 Multi-Mode Microplate Reader (BioTek, USA). The blank values were subtracted, and the values converted to EC50, which gave half the maximum response and absorbance (mean values ± SEM, n = 6) and estimated by a sigmoid curve fitted with a straight line (y = mx + c) using linear interpolation.
Minitab 19 (Minitab, State, College, PA, USA) was used for statistical analysis of data. Statistical differences between two groups were determined with a nonparametric Mann–Whitney test. Each data point represented the mean of eight sheep ± the standard error. Statistical significance was determined when p < 0.05.
Immunoblotting
To investigate the specificity of the IgG response, pooled sera from sheep immunised with soluble B-cell epitope, soluble B/T-cell epitope, empty BPs, B-cell epitope-coated BPs or B/T-cell epitope-coated BPs were diluted 10,000-fold and used for immunoblotting against E. coli whole-cell lysate containing various BPs and enriched BPs after they were transferred from Bis-Tris gel to nitrocellulose membranes (Life Technology, USA). E. coli whole-cell lysate not producing any fusion protein, i.e., no BPs, served as negative control. Rabbit Anti-Sheep IgG H&L (HRP) (Abcam, UK, Catalogue No. ab6747) was diluted 20,000-fold and used for detection of bound IgG antibodies.
Signal was developed by incubating the membrane with SuperSignal West Pico StablePeroxide Solution and SuperSignal West Pico Luminol/Enhancer Solution (Thermo Scientific, USA) for 5 min. The image was then captured with the ODYSSEY Fc imaging system (LI-COR, USA).
IgG purification
IgG was purified from pooled sheep serum using our previously developed BPs coated with the protein-G IgG-binding domain (GB1)352. BPs were centrifuged at 6000 x g for 4 m before being washed twice by resuspending in PBS buffer. BPs were then resuspended to 5% w/v using day 56 pooled sheep serum and incubated at 25 oC for 30 min at 200 rpm. BPs were then centrifuged and washed three times with PBS buffer. To release pure IgG, BPs were resuspended to 5% w/v in 50 mM glycine (pH2.7) and incubated at 25 oC for 5 min at 200 rpm. BPs were then centrifuged at 15,000 x g for 4 min to separate soluble IgG. In all, 20 mM K2HPO4 was added to neutralise the solution. Samples were analysed by SDS-PAGE to confirm IgG presence and stored at −20 oC.
Parasite maintenance
P. falciparum NF54 asexual stages were maintained in human type O-positive erythrocytes (Melbourne Red Cross) in RPMI-HEPES supplemented with 10% heat-inactivated human serum (Melbourne Red Cross) at 37 °C. Gametocytes for transmission to mosquitoes on day 17 were generated using the “crash” method with daily media changes65.
Mosquito infection and parasite development
Five to 7-day-old female Anopheles stephensi mosquitoes were fed on asynchronous gametocytes, diluted to 0.3%–0.6% stage V gametocytemia. Mosquitoes were sugar starved for 2 days after blood meal to select for blood-fed mosquitoes. Surviving mosquitoes were provided 5% glucose ad libitum via filter paper wicks or sugar cubes. Oocyst numbers were enumerated from midguts dissected from cold-anaesthetised and ethanol-killed mosquitoes 7 days post-infection and stained with 0.1% mercurochrome. Salivary glands were dissected from mosquitoes (days 16–20 after blood meal), crushed using a pestle, and glass wool filtered to obtain sporozoites.
Sporozoite staining
Salivary gland sporozoites were fixed in 4% paraformaldehyde solution for 20 min at room temperature (RT), washed and dried on glass slides. Samples were blocked in 2% BSA, 0.1% Triton-X-100 in PBS for 1 h at room temp (RT). Primary antibodies were incubated at 10 µg/ml in blocking buffer for 1 h at RT. Samples were washed three times with PBS and incubated in secondary anti-sheep (AF488 donkey anti-sheep IgG: (CiteAb, Catalogue No. A11015)) or anti-mouse (AF488 donkey anti-mouse IgG: (CiteAb, Catalogue No. A11029)) Alexa 488 antibodies (1:500 dilution) at RT before washing three times in PBS, staining for 5 min in 2 µg/ml DAPI, washing three times and air-drying. Slides were mounted beneath cover glasses with Prolong gold antifade reagent. All micrographs were acquired on a Zeiss LSM880 confocal microscope with Airyscan, processed with ImageJ and are presented with identical scaling.
Hepatocyte cell traversal assay with sporozoites
Human hepatoblastoma cells (HC-04 hepatocytes)66 were maintained on Iscove’s Modified Dulbecco’s Media (IMDM) and supplemented with 5% heat-inactivated foetal bovine serum (FBS) at 37 oC in 5% CO2. Cells were split 1:6 every 2–3 days once they reached 90% confluency. The traversal activity of sporozoites was measured using a standard cell-wounding assay53. In brief, 1 × 105 HC-04 cells were seeded into wells of a 96-well plate (Corning, Sigma-Aldrich). The following day, wells were seeded with 3 × 104 sporozoites (MOI 0.3) for 3 h in the presence of 1 mg/ml FITC-labelled dextran (10,000 molecular weight [MW]; Sigma-Aldrich). Cells were washed to remove sporozoites, trypsinized to obtain a single-cell suspension and FITC-dextran positive cells enumerated by flow cytometry. Antibodies were incubated at 10 µg/ml. For each condition, triplicate samples of 10,000 cells were counted by FACS in each of the three independent experiments and data statistically analysed using Graphpad Prism 7. An exemplification of the gating strategy is provided in the Supplementary Information (Supplementary Fig. 1).
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
