For immunization with CSP27, CSP9 or CSP9NVDP, or CSP27-NP conjugates 30 μg protein or as described in the relevant figure legend was emulsified in Imject Alum according to the manufacturer’s instructions (ThermoFisher Scientific) and delivered intra-peritoneally.

Spleens were collected from mice after cervical dislocation, and single cell suspension was prepared after passing through a 70 μM cell strainer into FACS buffer. Cells were briefly blocked for 30 minutes using 1μg/mL Streptavidin and 10 μg/mL TruStain fcX antibody diluted in FACS buffer. An antibody master mix was prepared in FACS buffer to stain the surface antigens of cells as per standard protocol using antibodies and B cell tetramers (prepared in house) for 30 minutes in dark. Cells were then lyzed using ACK lysis buffer and washed twice before staining with 1% 7AAD as Live/dead dye. Samples were acquired with a BD Fortessa or X20 flow cytometer, and data was analyzed using FlowJo software. The universal gating strategy is shown Figure S2, after which the further gating to study the B cells of interest is provided in the results.

For adoptive transfer experiments with Ighg2A10 and B1-8 cells, single cell suspensions of splenocytes were prepared and an aliquot used for flow cytometry using probes as described below to determine the proportion of cells that were antigen-specific B cells. The concentration of splenocytes was then normalized to allow for the transfer of the desired number of antigen specific cells.

Blood was collected from mice via either tail vein or retro-orbital bleeds and left to clot overnight at 4 °C, and the following day sera was collected by spinning the blood at 2000 g for 15 minutes at 4 °C. Next, Maxisorp Nunc-Nucleon 96 flat bottom plates were coated with 1 μg/ml streptavidin overnight at 4 °C in dark. The following day, plates were washed in wash buffer consisting 0.05% tween20 in PBS, and were incubated for 1 hour with different biotinylated peptides listed in pertaining to diverse sections of CSP protein. The peptide sequences were as follows, CSPNterm: Biotin-Ahx-QEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLKKNSRSLGENDDGNNEDNEKLRKPKHKKLKQPADG; CSPRepeat: Biotin-Ahx-NANPNANPNANPNANPNANPNANP NANPNANPNANP, and CSPCterm: Biotin-Ahx-NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSSTEWSPCSVTCGNGIQVRI KPGSANKPKDELDYANDIEKKICKMEKCS. Additional peptides also carrying a Biotin-Ahx linker are described in Figure S6. Wells were blocked for 1 hour before incubating with initial sera (dilution of 1/100 times, followed by 1 in 4 serial dilutions down the plate) for one hour. After washing, anti IgG detection antibody conjugated to HRP was diluted 1/2000 times and was incubated for one hour. The plates were washed, then developed with Peroxidase Substrate Kit for 15 minutes, and the reaction was stopped using 50 μL/ well stop solution, consisting of 10% SDS in PBS. Absorbance at 405 nm (A405) was measured using an Infinite PRO Tecan plate reader, and the data was expressed as area under the curve (AUC) calculated in Prism 7 from the log(dilution) on the x axis and the A405 on the y axis, fitting a sigmoidal curve.

Splenocytes was harvested from naive C57BL/6 or MD4 mice. Cells were then incubated for 30 minutes with a mixture of tetramers consisting full length recombinant CSP conjugated to PE and domain specific region of CSP, conjugated to APC. The cells were washed to remove unbound tetramers, and were enriched for PE after incubating with anti-PE microbeads and passing over the LS magnetic column (Miltenyi) according to the manufactures recommendation. Subsequently, antibody staining was performed on the enriched cells as per established protocol. Prior to acquisition on a BD X-20 flow cytometer, 1 × 104 CountBright absolute counting beads (Invitrogen) were added, to normalize the number of tetramer specific cell events obtained in each sample.

CSP27 or CSP9 was initially concentrated using an Amicon ultra-15 centrifugal filter unit (10 kD molecular weight cut-off, MWCO). Briefly, the filter was washed by spinning 10 mL of MQ at 4000 g for 20 minutes at 4 °C then discarding the flow through and any retained water. 2 mg of CSP27 or 1 mg CSP9 was added to the membrane and made up to 10 mL in 3% NaHCO3, then spun at 4000 g for 60 minutes at 4 °C. The flow-through was discarded and the retained protein made up to 10 mL in 3% NaHCO3, then respun at 4000 g for 60 minutes at 4 °C. The flow-through was discarded and the retained protein was made up to final volume of 1 mL per mg of original starting material in 3% NaHCO+. This was then transferred into a pre-soaked 3.5 kD MWCO dialysis tubing and dialysed overnight, then for 4 hours in 1 L 3% NaHCO3 at 4 °C.

Next, NP-ε-Aminocaproyl-Osu (NP-CAP-Osu) was dissolved in DMF to a final concentration of 10 mg/mL. We determined empirically that a 2:1 ratio of NP:CSP in the final conjugated product required a 20:1 ratio of NP:CSP at the conjugation step. For other NP:CSP ratios (6:1 and 10:1) this 10-fold increase during conjugation was also used; i.e., 60:1 and 100:1, respectively. The dialysed CSP and required NP-CAP-Osu were combined in sterile 2 mL tubes, covered with foil to protect from light, then rotated for 4 hours at RT to conjugate. The conjugated products were then dialysed in 1 L 3% NaHCO3 at 4 °C for 5 hours, overnight then 4 hours before dialysis in 1 L PBS for 4 hours and overnight. Conjugated products were stored at 4 °C with 0.1% sodium azide to prevent bacterial growth.

Since NP-CAP-Osu has an absorption maximum of 430nm with extinction coefficient of 4230, the concentration of NP was determined via NanoDrop. Briefly, 1.5 μL aliquots of CSP27-NP/ CSP9-NP were analyzed on a NanoDrop ND-1000 Spectrophotometer to measure protein at 280 nm and NP dye at 430 nm. An average of two reads was used to calculate the concentration of NP.

The absorbance of NP influenced the absorbance of proteins at 280 nm, therefore NanoDrop was not an accurate method for quantification of protein concentration (CSP27/CSP9) in the conjugated products. Instead, the concentration of CSP27 or CSP9 was determined by sandwich ELISA, taking advantage of histidine-tags (His-tag) attached to both CSP27 and CSP9 (Figure S1). Briefly, 96-well Maxisorp Nunc-Nucleon plates were coated overnight with 1 μg/mL of anti His-tag antibody. The following day, plates were washed and blocked with 1% BSA for 1 hour. The NP-CSP conjugated products were initially diluted 1/100 in 1% BSA along with a 1/100 dilution of a CSP27 or CSP9 standard with known concentration, and serially diluted 1 in 5 times down the plate. Next, a 1/2000 dilution of 2A10 primary antibody (Zavala et al., 1983) was incubated for one hour, washed and incubated with secondary antibody (Anti IgG detection antibody conjugated to HRP) for one hour. After washing, the plates were developed with Peroxidase Substrate Kit for 15 minutes and read at 405 nm using a Tecan Infinite 200Pro plate reader. The reaction was stopped using 50 μL/ well stop solution, consisting of 10% SDS in PBS. The concentration of CSP27 or CSP9 in the conjugated products was interpolated from a sigmoidal standard curve of CSP27 or CSP9.

To detect B cells to specific epitopes in CSP we used tetramers based on peptide probes described above for the CSPRepeat, CSPNterm, or CSPCterm region conjugated to phycoerythrin (PE) or allophycocyanin (APC). The tetramers were generated by mixing biotin-conjugated peptides with streptavidin-conjugated PE or streptavidin-conjugated APC in a 4:1 molar ratio. Briefly, 2.17 nM of peptide was made up to 50 μL in PBS. Then 8.68 nM of PE or APC were added in 4 equal aliquots every 15 minutes, incubating at room temperature (RT) in darkness between aliquots. Tetramers were stored at 4°C in dark until use.

NP-specific B cells were detected using 4-hydroxy-5-iodo-3-nitrophenol (NIP) conjugated to PE or APC. Briefly, 1 mg of Native R-Phycoerythrin protein or Natural Allophycocyanin protein were transferred into pre-soaked 3.5 kD MWCO dialysis tubing and dialysed for 5 hours, overnight, then for 4 hours in 1 L 3% NaHCO3 at 4°C. NIP- ε -Aminocaproyl-Osu (NIP-CAP-Osu) was dissolved in DMF to a concentration of 10mg/mL. The NIP-CAP-Osu was added to the dialysed PE or APC at a ratio of 20 μg:1 mg and rotated at RT for 4 hours protected from light with aluminum foil. The conjugated NP-PE and NP-APC were then dialysed in 1 L 3% NaHCO3 at 4°C for 5 hours, overnight then 4 hours before dialysis in 1 L PBS for 4 hours and overnight. NP probes were stored at 4°C in dark till use.

Immunisations were conducted with the following amounts of antigen: NP-CSP27 = 15 μg/mouse, NP-CSP9 = 11.93 μg/mouse. Negative control groups were immunized with vehicle (PBS in alum). Antigens were emulsified in alum (2:1 volumetric ratio antigen: alum) to a total volume of 150 μL per mouse. The resultant solution was vortexed slowly at RT for 30 minutes to ensure the immunisations were fully emulsified. Immunisations were delivered intraperitoneally (IP), 150 μL total delivered in 75 μL aliquots on each side of abdomen into C57BL/6 recipient mice.

Three different constructs of CSP were used for immunization, they include CSP9NVDP, CSP9, and CSP27 (Figure S1). C57BL/6 recipient mice were randomly separated in four groups, three of them had 15 mice per group, and were immunized with one of the CSP constructs. The fourth group had 11 mice and was the negative control. Each mouse received 30 μg of a CSP construct. These were emulsified in alum to a 2:1 volumetric ratio of antigen: alum and a resultant solution of 200 μL, before vortexing at RT for 30 minutes to ensure complete emulsification. Mouse were then IP immunized with 200 μL, 100 μL on left and right side, respectively. The immunisation regimen consisted of one priming and two boaster doses, each separated by an interval of 5 weeks. One day before immunization, blood was collected from mice via tail vein or retro-orbital bleeds for assessing antibody response. 2 weeks after the mice received their final booster, they were challenged via mosquito bite.

Controlled malaria infection challenge was performed via bite of Anopheles stephensi mosquitoes infected with Pb-PfSPZ a P. berghei parasite strain that expresses P. falciparum CSP (Espinosa et al., 2017). Also, the parasites express GFP or mCherry, allowing infected mosquitoes to be visually identified via microsopy, and 5 positive mosquitoes were sorted onto separate containers. These mosquitoes were fed with sucrose for the first 6 hours and then with water solution a day before the challenge. The following day, mice were anaesthetised and placed on top of the containers to allow the mosquitoes to blood feed for 30 minutes. In some experiments, 42 hours post mosquito bite, mice were euthanised via cervical dislocation and liver was collected, washed twice in PBS, and homogenized in 4 mL denaturing working stock. In other experiments mice were monitored daily for the presence of parasites in the blood which was detected via flow cytometry of blood collected via tail nick. In this case a mouse was considered patent in the day the proportion of RBCs that were GFP+ exceeded 0.1%.

To extract parasite rRNA, 60 μL of 2 M Sodium Acetate was added to a 600 μL aliquot of homogenized liver and vortexed to mix. Then 750 μL Acid Phenol:Chloroform was added and vortexed before incubating on ice for 15 minutes. The samples were spun at 15000 g for 20 minutes at 4°C and the upper aqueous phase transferred into a clean 1.5 mL tube. The RNA was precipitated via addition of 400 μL isopropanol, vortexing then incubating at −20°C for 1 hour. The RNA was pelleted at 15000 g for 20 minutes at 4°C then the pellet washed twice with 1 mL cold 70% ethanol (EtOH) then dried for 10 minutes before resuspension in ultra-pure water. RNA concentration was measured via Nanodrop, and was diluted to make 100 μL aliquots at 50 ng/μL and stored at 4°C.

cDNA was synthesized from the RNA using iScript cDNA Synthesis Kit according to the manufacturer’s protocol. Briefly, each sample was run in a 20 μL reaction in individual dome-capped PCR tubes containing 10 μL ultra-pure water, 4 μL 5 x iScript Reaction Mix, 1 μL iScript Reverse Transcriptase and 5 μL RNA (50 ng/μL). The samples were run on an Eppendorf ProS Mastercycler at 25°C for 5 minutes, 42°C for 30 minutes, 85°C for 5 minutes then hold at 4°C.

RT qPCR was run using Power SYBR Green PCR Master Mix. Briefly, a PCR mastermix was made for all samples plus 2 no template controls (NTC) and 5 standards (STD) with the following volume per one reaction: 4.4 μL ultra-pure water, 5 μL Power SYBR Green PCR Master Mix, 0.05 μL P. berghei forward primer and 0.05 μL P. berghei reverse primer. 38 μL aliquots of mastermix were transferred into 1.5 mL tubes for each condition; 2 NTC, 5 STD and x cDNA samples. For DNA templates for P. berghei 18S qPCR 2 μL of the following was added for each condition; NTC – ultra-pure water, STD – plasmid standards (107, 106, 105, 104 and 103), samples – cDNA. The PCR was plated in triplicates of 10 μL/well in a MicroAmp 384 well reaction plate. The plate was sealed with MicroAmp optical adhesive film and spun at 500 g for 15 s to ensure all samples were at the bottom of each well. The qPCR was run on a 7900HT Fast Real-Time PCR System using the following conditions; 50°C for 2 minutes, 95°C for 10 minutes then 40 cycles of 95°C for 15 s and 60°C for 1 minute followed by 95°C for 15 s and 60°C for 15 s. The above qPCR reaction was repeated for glyceraldehyde 3-phosphate dehydrogenase (GapDH) using GapDH primers. However, the standards consisted instead of a pool of 2 μL cDNA from each sample that was serially diluted for the following concentrations; 1.0, 0.5, 0.25, 0.125, 0.0625. qPCR data was read using sDS2.4 software, P. berghei 18S was normalized to the GapDH reference gene before further calculating means in Prism 7.

CSPRepeat-specific B cells were FACS purified from Ighg2A10 knock-in mouse splenocytes (McNamara et al., unpublished data), and were cultured in complete RPMI for 16 hours. Then the cells were labeled in RPMI media containing 2 μM/ml Indo-1 and 7AAD for 20 min at 37°C. Following2 washes, the signal at BUV395 channel (Indo-1 bound) and BUV496 channel (Indo-1 free) was collected for 60 s to define baseline Ca2+ levels as the ratio of Indo-1 (bound/free). B cells were then stimulated for additional 360 s with either 0.5 μM/ml CSP or 10 μg/ml OVA-HEL. 1 μg/ml ionomycin was used as positive control. Data were collected in a FACS Fortessa instrument (BD) and analyzed using the Kinetics tool in FlowJo software (Tree Star). To analyzed the result, the baseline Ca2+ level was defined by the mean value of Indo-1 (bound/free) within 0-60 s. The calcium influx was then calculated by the mean values of every 30 s divided by the baseline value, and the plot was made by the calcium influx versus the mean value of this time frame.

Surface plasmon resonance saturation experiments were performed on a Biacore 8K instrument (GE Healthcare) at 25°C using a Series S Sensor Chip NTA (GE Healthcare) and SPR running buffer (10 mM HEPES, 150 mM NaCl, 50 μM EDTA, 0.05% v/v Tween 20, pH 7.4). Solutions of His6-tagged CSP27 and CSP9 were prepared in SPR running buffer at concentrations of 0.1 μg/ml and 0.4 μg/ml, respectively. His6-tagged CSP27 and CSP9 were immobilized on separate channels on the sensor chip surface as per the manufacturer’s recommendations: a pre-conditioned chip was first activated with 500 μM NiCl2, and a solution of the His6-tagged ligand was subsequently passed over the chip using a flow rate of 5 μl/min for 120 s. This yielded approximately 150 RU and 50 RU of immobilized CSP27 and CSP9, respectively. A saturating solution of mAb 2A10 (2 μM in SPR running buffer) was then passed over the chip for 400 s using a flow rate of 30 μl/min, followed by a 400 s dissociation period. The increase in response units (RU) corresponding to ligand immobilization (RUlig) and analyte binding (RUanalyte) in the reference-subtracted (reference = blank surface) sensorgrams was measured, and the binding stoichiometry (n, molar ratio of antibody to antigen in the complex under saturating concentrations of mAb 2A10) was estimated using Equation (1) as previously described (Oda and Azuma, 2000), using molecular weights (MW) calculated using ProtParam (Wilkins et al., 1999):CSP27 (35.4 kDa), CSP9 (28.2 kDa) and mAb 2A10 (145.9 kDa).

All buffers were filtered and degassed prior to use. Following each cycle, the chip was completely regenerated using sequential washes of 500 mM imidazole, 350 mM EDTA (pH 8.5) and 100 mM NaOH. Each experiment was performed in duplicate (n = 2), on separate channels on the SPR chip.

Mice were intraperitoneally immunized with 30 μg of a CSP construct in alum, and on Day10 the spleens were harvested to prepare single cell splenocytes suspension. Individual mouse splenocytes were then stained with either of the CSP epitope specific tetramer, and were enriched using magnetic LS column (Miltenyi), according to the manufacturer’s directions. Subsequently they were stained with antibody master mix as describer previously, and the Germinal Center B cells were sorted using BD FACs Aria I or II (Becton Dickinson) machine. The RNA was then extracted using the PicoPure RNA Extraction Kit (Applied Biosystems), and on the same day cDNA was prepared using the iScript cDNA synthesis kit (BioRad), and were stored in −20°C until further use. BCR sequenced was amplified, and indexed (Nextera indexing kit, Illumina) as previously described approach (Fisher et al., 2017). Sequencing was performed on the Illumina MiSeq sequencing platform. The obtained paired end sequence reads were aligned to Mus musculus reference genome at IMGT (Lefranc, 2001) through MiXCR (Bolotin et al., 2015) to obtain the assembled sequences. Top 99% of these sequences were then filtered to remove non coding, or frameshift mutations in the receptor sequences using VDJ tools (Shugay et al., 2015). In the heavy and the light chain aligned and filtered sequences, we then measured the V gene uses and their repertoire overlap using the immunarch package in R ( The mutation frequencies in the light chain were plotted by listing the number of substitution, insertion and deletion observed in the top 99% of the sequences while aligning the raw sequencing reads to the germline variant in IMGT through MiXCR (Bolotin et al., 2015; Lefranc, 2001).

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