Preparation of constructs
All the chimeric constructs are based on the L. lactis pSS1 plasmid vector32. Codon-optimized Pro-6C-His containing different linkers (Fig. 1a and Supplementary Fig. S1) between Pro and 6C was synthesized by (GeneArt® Life Technologies, Germany) and cloned into pSS1 with the help of BamHI/SalI cloning sites. For the development of Ni++ independent purification of the selected constructs, His tag was replaced with C tag (EPEA) by PCR. All the constructs were verified by sequencing and subsequently transformed into L. lactis MG1363 (Bioneer A/S, Denmark) by electroporation as described42. Briefly, competent L. lactis cells were thawed on ice and 40-µl cells were mixed with 2 µl of DNA in a microcentrifuge tube. The mixture was then transferred to an ice-cold electroporation cuvette and pulsed using the following settings: voltage, 2.0 kV; capacitance, 25 µF; resistance, 200 ohms; and time constant of 4–5. Following discharge, 0.96 mL of ice-cold SGM17 media is added to the cuvette, transferred to a microcentrifuge tube, and incubated at 30 °C for 2 h. The suspension was plated and incubated at 30 °C for up to three days for colony screening.
Screening, fermentation and protein purification
Screening for expression of chimeric constructs was performed as previously described24 with slight modifications. Briefly, L. lactis MG1363, containing chimeric Pro-6C constructs with linker, was grown overnight at 30 °C in 5 ml of LAB medium broth (3.5% yeast extract, 0.05 mM FeSO4 7 H2O, 2.5 μM CaCl2, 2.65 μM MgCl2, 0.5 mM citric acid 2H2O, 1.38 mM ammonium sulfate, 14.7 mM sodium acetate, and 20 mM KH2PO4 buffer pH 7.4) containing 1 μg/ml erythromycin, 5% glucose, and 4% disodium glycerophosphate. Culture supernatants were clarified by centrifugation at 9000 g for 20 min. Analysis of all the constructs was performed by Coomassie-stained SDS-PAGE gel and Western blotting with mAb45.1. Fermentation of L. lactis MG1363/chimeric constructs was performed in a 0.5-L-scale fermenter at 30 °C with gentle stirring (150 rpm). Cell-free culture filtrates were concentrated fivefold and buffer-exchanged into HEPES buffer (20 mM HEPES, 50 mM NaCl, pH 8.0, supplemented with 15 mM imidazole) using a Quix-Stand Benchtop system (Hollow fiber cartridge with cutoff at 10,000 Da, surface area 1400 cm2, GE Healthcare, Sweden) followed by filtration through a Nalgene Rapid-Flow Sterile Disposable filter units with PES membrane 0.22-μm pore size. Proteins were purified on a 5-ml His-Trap HP column (GE Healthcare, Sweden) followed by an anionic exchange column as described previously24. Purified proteins containing monomers with the highest amount of mAb45.1-reactive protein were concentrated by a VIVA spin column 10-kDa cutoff (GE Healthcare, Sweden), and kept in 20 mM HEPES, 300 mM NaCl, and 1 mM EDTA, pH 8.0, at −80 °C until use.
SDS-PAGE and Western blot
Samples were diluted with 6X SDS (sodium dodecyl sulfate, Sigma-Aldrich) sample buffer, heated for 10 min at 98 °C, and loaded onto NuPAGE Novex 4–12% Bis-Tris precast gels (Invitrogen, USA) according to the manufacturer’s instructions. Gels were run at 150–200 V for 35–50 min in 1X MOPS SDS running buffer and stained with Coomassie-staining procedures.
Following SDS-PAGE, proteins were transferred to nitrocellulose membrane (Bio-Rad, USA) and blocked in 1% skim milk in TBS containing 0.05% Tween 20 (TBST) at room temperature for 1 h. Primary antibody, conformational, and reduction-sensitive Rat mAb 45.1 was used at 0.5 µg/ml in TBST and incubated for 1 h at room temperature. The membranes were washed with TBST (three times for 5 min) and secondary antibody (1:4000 dilution) of goat anti-rat IgG–HRP conjugated (Cat # 31470, Invitrogen, USA) in TBST was incubated at room temperature for 1 h. The membranes were again washed with TBST (three times for 5 min), developed using HRP kit (SERA CARE, USA).
All blots and gel are derived from the same experiment and processed in parallel. Raw image files for gels and Western blot images are provided in Supplementary materials (Supplementary Figure S4-S7).
Production of ProC6C at the bench scale
Fermentation of L. lactis MG1363/ProC6C construct was performed as previously described32 with minor modifications. Briefly, a 15-ml culture tube containing 5 mL of basic lab medium with 4% disodium glycerophosphate was inoculated with 100 µl of working cell bank and incubated for 4–6 h at 30 °C, until culture OD600 reached ≥1.0. The fermenter (BIOFLO 310, New Brunswick Scientific) containing 1 L of fermentation medium was inoculated with 0.4 ml of preculture. After 4 h of inoculation, the fermenter was supplied with 50% glucose continuously at the rate of 8 ml/h to maintain 5% glucose in the medium, until the end of the fermentation. The pH was maintained at 6.5 ± 0.2 using 2 M NaOH. Cultivation was carried out at 30 °C with gentle stirring (150 rpm) for 15–18 h, until an OD600 of 12–15 was reached. After 15–18 h of growth, the bulk cell mass was removed by centrifugation (9000 × g, 4 °C, 30 min). The culture supernatant was concentrated 5-fold and buffer exchanged in buffer (20 mM HEPES, 5% glucose, 50 mM Sod. borate, 10 mM l-arginine, and 1 mM EDTA, pH 6.5) using a Quix-Stand Benchtop system (hollow-fiber cartridge with cutoff at 10,000 Da, surface area 1400 cm2, GE Healthcare, Sweden) followed by filtration through a Nalgene Rapid-Flow Sterile Disposable filter units with PES membrane 0.22-μm pore size.
For the isolation of ProC6C from cell culture media a 3-step purification procedure: (1) capturing by IEC on a HiPrep Q HP column (GE Healthcare, Sweden), (2) removal of HCP on a CaptureSelect™ C-tagXL column (ThermoFisher, USA), and (3) separation of the monomer fraction by IEC on a HiPrep Q HP column was used. First step. The first step was optimized to capture 80–90% of the target antigen, ProC6C, with minimal binding of unwanted HCP. To ensure maximum recovery from the capturing step, the loading density was maintained at 20 mg/mL of resin or less. The clarified concentrated and buffer-exchanged fermentation supernatant was applied to a HiPrep Q-HP (16/10) column, washed with five column volumes (CVs) of 50 mM NaCl in Buffer A followed by 8 CVs of 100 mM NaCl in the same buffer (Supplementary Table S1). Bound material was eluted with 12 CVs of 200 mM NaCl and fractions containing the desired protein were pooled and diluted 8-fold with Buffer B (Supplementary Table S1) to bring the conductivity below 8 mS/cm for application to the subsequent column. Second step. The diluted eluate from the capturing step was then applied onto a CaptureSelect™ C-tagXL prepacked 3 × 5 ml column-flow rate 2.5 mL/min. The column was washed with 10 CVs of 140 mM MgCl2 in Buffer B and bound protein was eluted with 10 CVs of 700 mM MgCl2 in the same buffer (Supplementary Table S1). Fractions containing high concentration of ProC6C were pooled, resulting in a mixture of monomeric and multimeric forms of ProC6C. Third step. The eluted material from the second column was diluted 8-fold with Buffer C and applied again to a HiPrep Q-HP (16/10). The column was washed with 5 CVs of 150 mM NaCl in Buffer C followed by 8 CVs of 270 mM NaCl in the same buffer, thereby removing residual HCPs and smaller MW protein fragments resulting from proteolytic degradation of the target protein. Bound protein was eluted with 10 CVs of 310 mM NaCl (Supplementary Table S1). Fractions containing a single band of monomeric ProC6C were pooled and diluted in formulation buffer (20 mM HEPES, 5% glucose, 300 mM NaCl, and 1 mM EDTA, pH 8.0) and were stored at −80 °C until further use. Fractions were analyzed by SDS-PAGE and immune blotting with mAb45.1 against Pfs48/45 conformational epitope I. Protein concentration was measured by NanoDrop (A280). Column chromatography was performed using an NGC 10 Medium Pressure Chromatography system (Biorad, USA).
Process scale-up
Fermentation and sample preparation were done as described above. Protein-purification process and column size of each step of purification from 5L scale was performed as mentioned in Supplementary Table S1.
Protein characterizations and analytical methods
Reversed-phase high-performance liquid chromatography (RP-HPLC)
RP-HPLC was performed with minor modifications as described previously27. Briefly, RP-HPLC was performed using an Agilent 1100 Series HPLC System (Agilent Technologies, USA) equipped with an Agilent Poroshell 120EC-C18 column, 4 µm 4.6 × 100 mm (Agilent Technologies, USA). About 210 pmol of protein (nonreduced) was injected and eluted (flow rate 1 ml/min) with a linear gradient of 3–95% over 20 min of 0.1% trifluoroacetic acid (TFA), 20% isopropanol, and 70% acetonitrile. The absorbance was measured at 214 nm and chromatographic peaks were integrated by HPLC ChemStation (Agilent Technologies, USA).
Analytical size-exclusion chromatography (SE-HPLC)
Analytical SE-HPLC was performed as described previously27 with slight modification. Briefly, SE-HPLC of purified protein was performed with an Agilent 1100 Series HPLC System (Agilent Technologies, USA) equipped with a TSKgel G3000SWXL (TOSOH Biosciences, USA). An injection volume of ten microliters was used with a mobile phase of 20 mM HEPES, 310 mM NaCl, 5% glucose, and 1 mM EDTA, pH 8.0, at a flow rate of 1 mL/min. The absorbance was measured at 280 nm and chromatographic peaks were integrated by HPLC ChemStation (Agilent Technologies, USA). Protein standards (Sigma Aldrich) were also run using the same conditions mentioned above for sizing of the purified recombinant proteins.
Kinetic endotoxin assay
Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific, USA) was used to quantify endotoxin content of purified proteins.
Host-cell protein
L. lactis HCP kit (Cat # F490, Cygnus Technologies, USA) was used to quantify L. lactis HCPs as impurities in the purified proteins as described by the manufacturer.
Amino acid analysis (AAA)
AAA was performed in triplicate using 30 μL of sample (30 µg) for each hydrolysis (6 N HCl, 110 °C; 20 h in sealed, evacuated glass tubes). Cysteine and tryptophan were not determined.
Mass spectrometry
Accurate molecular mass of ProC6C was measured by LC–ESI–MS under both non-reducing and reducing conditions as previously described24. Briefly, 30 pmol of protein were loaded on a C4 precolumn (Acquity UPLC Protein BEH C4 Vanguard, 1.7, 2.1 × 5 mm, Waters, UK) and eluted onto a Q-TOF mass spectrometer (Synapt G2 HDMS, Waters, UK) with a chromatographic gradient. LC–MS data were recorded and analyzed by MassLynx software (Waters, UK). Mass spectra were deconvoluted using the MaxEnt 1 algorithm in the MassLynx software.
Analytical size-exclusion high-performance liquid chromatography coupled with multiangle static laser light scattering (SEC-MALS)
A Dionex (Thermo Scientific, USA) HPLC system connected in-line with a UV detector (Thermo Scientific DionexTM Ultimate 3000, MWD3000), a DAWN HELEOS 8+ multiangle laser light-scattering detector, and a Optilab T-rEX (Wyatt Technology Corporation) refractive index detector. Using a Superdex 200 Increase 10/300 GL column (GE Healthcare, Sweden) at room temperature in 20 mM HEPES, 310 mM NaCl, 5% glucose, and 1 mM EDTA, pH 8.0, 100 µl of proteins were injected with a flow rate of 0.5 ml/min. The ASTRA (version 6.1.17) software (Wyatt Technology Corporation, USA) was used to collect UV, refractive index, and light-scattering data. The weight average molecular mass was determined across the elution profile from static light-scattering measurements using ASTRA software and a Zimm model that relates the amount of scattered light to the weight average molecular mass of the solute, the concentration of the sample, and the square of the refractive-index increment (dn/dc) of the sample.
Dynamic light scattering (DLS)
DLS experiments were performed on a DynaPro NanoStar cuvette-based instrument (Wyatt Technology Corporation, USA) operated with Dynamics software (version 7.8.1.3). The data were collected at 20, 25, and 37 °C and a total of 10 measurements were performed at each temperature with an acquisition time of 5 s and read interval of 1 s.
Animals and immunogenicity studies
In the first immunogenicity study, groups of six female CD-1 mice (Janvier Labs, Denmark) were immunized s.c. 3 times at three-week interval with 10 µg of chimeric antigens on 70% Montanide ISA720 (Seppic, France). Mice were sacrificed two weeks after the last immunization and serum was collected for ELISA and SMFA analysis. In the second study, groups of five female CD-1 mice (Charles River, Germany) were immunized i.m. in the right thigh with 50 µl of vaccine (ProC6C), two times with a four-week interval containing 0.4 or 2.0 µg of ProC6C. Alhydrogel (InvivoGen, USA) formulations contained 75 micrograms of Alhydrogel and were mixed by pipetting for 5 min. Matrix-M™ adjuvant (Novavax AB, Uppsala, Sweden) formulations contained 5 micrograms of Matrix-M adjuvant per injection and were mixed by pipetting shortly. Formulations that contained both Alhydrogel and Matrix-M adjuvant were prepared by first adsorbing ProC6C to Alhydrogel as described above and then adding Matrix-M adjuvant. Fourteen days after the last immunization, mice were sacrificed, and serum was collected for ELISA and SMFA analysis.
Animal study ethics statement
All animal procedures complied with national regulations and ethical regulations for animal testing and research. The experiments included in this study received ethical approval, including the ethics committee of the University of Copenhagen (approval number P 19-308) and the Radboud University Medical Center (approval number 2016-0020).
Enzyme-linked immunosorbent assay (ELISA)
Antibody responses in mice were quantified by ELISA with plates coated by the vaccine antigen13 or full-length CSP27. Briefly, microtiter plates (Nunc MaxiSorp, Denmark) were coated with 0.33 µg/ml of recombinant protein in carbonate buffer, pH 9.2, and incubated overnight at 4 °C. Plates were blocked for 1 hr at room temperature (RT) using 3% skimmed milk in 1xPBS with 0.05% Tween 20 (1xPBST), washed twice, and incubated for 1 hr with 100 μl of test serum or a pool of negative control from prebleed mice serum, diluted 1:500 in 1% skimmed milk in 1xPBST. Subsequently, antigen-specific bound antibodies were probed with HRP-conjugated rabbit anti-mouse IgG–HRP (Cat # P0260, DAKO, Denmark) at 1:4000 dilution in 1% skimmed milk in 1xPBST for 1 hr. Plates were washed three times in 1x PBST between each step. ELSA was developed with 100 μl of 3, 3, 5, 5-tetramethylbenzidin (TMB) X-tra substrate (Kem-En-Tec, 4800 A). The color reaction was stopped with 100 μl of 0.2N H2SO4 and absorbance was measured at 450 nm. Data were collected on a BioSan HiPo MPP-96 microplate reader (BioSan, Latvia). Antibody midpoint titer (EC50) was calculated using sigmoidal curve fitting on GraphPad Prism version 8.4.3 (San Diego, USA).
Standard membrane feeding assays (SMFA)
Transmission-reducing activity of antibodies was assessed in SMFA. Cultured Plasmodium falciparum NF54 gametocytes were fed to Anopheles stephensi mosquitoes. Each blood meal contained 150 µL of packed red blood cells with gametocytes, 30 µL of naive human serum containing active complement, and test serum that was diluted in fetal calf serum. After 6–8 days, oocysts in 20 mosquitoes were counted by microscopy. TRA estimates from two independent experiments were generated using generalized linear mixed models (GLMMs) with zero-inflated negative binomial error structure43,44. Statistical analyses were performed using R studio (version 3.2.4, The R Foundation, Boston, USA).
Hepatocyte invasion-inhibition assay
In vitro invasion-inhibition experiments were conducted as previously described45. Plasmodium falciparum NF54 sporozoites were incubated with heat-inactivated (56 °C, 30 min) mouse sera on ice for 30 min and subsequently incubated with human hepatoma cells (HC-04, MR4-BEI Resources) for 3 h at 37 °C. Supernatant containing noninvading sporozoites was removed and hepatocytes were stained with eBioscience™ Fixable Viability Dye eFluor™ 780 (1:200 dilution, ThermoFisher cat no. 65-0865-14). Hepatocytes were permeabilized with Fixation/Permeabilization buffer (eBioscience) for 30 min. at 4 °C and then washed with Permeabilization buffer (eBioscience). Intracellular sporozoites were stained with 3SP2 antibody that was labeled using Alexa Fluor488 antibody labeling kit (ThermoFisher)45, 30 min. at 4 °C. Cells were washed with Permeabilization (eBioscience), fixed with 1% PFA, and analyzed by flow cytometry (Gallios, Beckman Coulter). For each experiment, three independent replicates were analyzed with at least 2000 single live hepatocytes. Invasion inhibition was normalized against a sporozoite only control (0% inhibition).
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
