The animal use protocol was reviewed and approved by the Faculty of Medicine, Institutional Animal Care and Use Committee, University of Malaya (2019-201010/PARA/R/TJH).
In silico analysis on CSP and selection of target peptides
Amino acid sequences of CSP of P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi were obtained from GenBank with the accession number: AAN87622 (PfCSP), AGN05254 (PvCSP), ARF20132 (PmCSP), SBT00176 (PoCSP), and XP_002259002 (PkCSP), respectively. Two peptide sequences that were specific to PkCSP were selected based on sequence alignment using Clustal Omega, namely PkCSP peptide I and PkCSP peptide II. Percentage of identity of both selected peptides with other human Plasmodium species were determined using NCBI global alignment. Relative surface accessibility and solubility of the peptides were assessed using NetSurfP-2.0 and PepCalc.com respectively. PkCSP peptide I sequence was synthesized and used as immunogen in monoclonal antibody production by ABclonal, Wuhan, China. PkCSP peptide II was synthesized by Bio Basic Canada Inc, Canada and used as immunogen for generation of polyclonal antibody in rabbit. Both peptide epitopes were synthesized with an additional cysteine in N-terminal for conjugation to keyhole limpet hemocyanin (KLH) as the carrier protein.
Construction of recombinant PkCSP plasmid
Plasmodium knowlesi genomic DNA was extracted from P. knowlesi strain A1H1 culture using blood extraction kit (QIAGEN, Hilden, Germany). Primers for PkCSP were designed based on the PkCSP gene sequence from P. knowlesi strain H genome assembly (NCBI reference sequence: NC_011909). DNA encoding for the signal peptide of PkCSP was excluded during the design of primers. The CSP gene was amplified using polymerase chain reaction (PCR) with the primer pair PkCSP FP: 5’-GGATCCACACACTTCGAACATAATG-3’ and PkCSP RP: 5’-GGATCCTTAATTGAATAATGCTAGGAC-3’. The PCR conditions were as follows: initial denaturing step at 95°C for 4 minutes; 35 cycles at 95°C for 30 seconds, 51°C for 45 seconds, and 72°C for 80 seconds; final elongation step at 72°C for 10 minutes. PCR product was cloned into pGEM®-T plasmid vector (Promega Corporation, USA). Then, the recombinant plasmid was digested using BamHI restriction enzyme (New England Biolabs, Inc., USA) and subsequently cloned into Novagen® pET-30a(+) (Merck KGaA, Germany), USA). Recombinant plasmid was transformed into Escherichia coli expression host T7 Express lysY/Iq (New England Biolabs, Inc., USA). All the recombinant plasmids constructed were sent for sequencing to verify the gene identity.
Expression of PkCSP
Single colony T7 host cell containing recombinant PkCSP plasmid was propagated overnight in Luria-Bertani broth containing kanamycin (30 µg/ml) and chloramphenicol (35 µg/ml) at 37°C with constant shaking at 250 rpm. Next day, the overnight culture was sub-cultured until the optical density at 600 nm (OD600) reached 0.4-0.6. The culture was induced with 1 mM isopropyl β-D-1-thiogalactopyanoside (IPTG) and allowed to propagate for another 4 hours. Cell pellets were harvested by centrifugation at 6500 rpm for 10 minutes and stored at -80°C.
Purification, dialysis and quantification of recombinant PkCSP
Recombinant PkCSP (rPkCSP) was affinity purified under hybrid condition of the ProBondTM purification system (Invitrogen, USA). Cell pellet was resuspended in 6 M guanidinium lysis buffer (pH 7.8) and sonicated on ice to break the cells. Purification column containing nickel-NTA agarose resin was prepared under denaturing condition. Protein lysate was added to the column and incubated on ice for 2 hours. Then, the column was washed with denaturing binding buffer (pH 7.8), denaturing wash buffer (pH 6.0) and native wash buffer (pH8.0) according to the manufacturer’s instruction. The purified rPkCSP was eluted with native elution buffer (pH 8.0). Purified protein was dialysed and the concentration of purified rPkCSP was determined using Quick StartTM Bradford Protein Assay (Bio-Rad Laboratories, USA).
SDS-PAGE, Coomassie brilliant blue staining and Western blot
Crude protein lysate and purified rPkCSP were resolved in 12% SDS-PAGE under reducing conditions. The gels were stained with coomassie brilliant blue to reveal the protein bands. Separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and blocked overnight in 5% blocking buffer (Tris-buffered saline (TBS) containing 5% skimmed milk) at 4°C. The membranes were probed with His-Tag® monoclonal antibody (EMD Millipore Corp., USA) diluted with 2.5% blocking buffer (1:2500 dilution) for one hour at room temperature. The membranes were washed three times with 0.2% TBS-T (TBS containing 0.2% Tween-20) and incubated with biotin-labelled goat anti-mouse IgG (1:2500 dilution) for one hour, followed by alkaline phosphatase (AP)-conjugated streptavidin (1:2500 dilution) for one hour. Finally, nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) was added in the dark to develop colour. Purified rPkCSP was excised from SDS-PAGE gel and sent for protein identification using MALDI/TOF.
The synthesized PkCSP peptide II was conjugated to mariculture KLH via carboxyl-reactive carbodiimide crosslinker (EDC) using Thermo ScientificTM ImjectTM EDC mcKLH spin kit. A 12-week old female New Zealand white rabbit was used for immunization. Pre-immune serum was collected on day 0. On the same day, KLH-conjugated PkCSP peptide II, 200 µg was mixed with complete Freund’s adjuvant (CFA) (Sigma-Aldrich, USA) at 1:1 ratio and injected into the rabbit subcutaneously at four different sites (50 µg each site) to minimize adverse reactions post-immunization. Four boosters prepared in incomplete Freund’s adjuvant (IFA) (200 µg each booster) were administered using the same administration route as primary immunization at week 2, 4, 6 and 8. Endpoint titre assay was carried out using pre-immune serum as negative control. Maximum amount of blood were collected from the rabbit when the endpoint antibody titre is higher than 1:51200. Antiserum was collected by centrifuging the blood at 2,000 rpm for 10 mins and was stored in aliquots at -80°C until use.
Evaluation of reactivity of mice and rabbit antiserums against rPkCSP and detection of PkCSP in P. knowlesi total protein extract
A total of five mice were immunized with KLH-conjugated PkCSP peptide I using the company’s protocol for monoclonal antibody production. The reactivity of the rabbit antiserum and most reactive mouse antiserum against rPkCSP was evaluated using western blot assay. rPkCSP protein (100 ng) was resolved in 12% SDS-PAGE and transferred onto PVDF membrane. The membrane was blocked overnight with 5% blocking buffer at 4°C. On the next day, the membrane was cut into strips and treated individually with pre-immune serum and antiserum (1:250 dilution) of both rabbit and mouse for 2 hours at room temperature. The strips probed with rabbit serum were incubated in horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (1:2500 dilution) for one hour. TMB 1-component membrane peroxidase substrate was added in dark to develop colour.
For strips that were probed with mouse serum, biotin-labelled goat anti-mouse IgG + IgM (1:1000 dilution) was added and incubated for one hour. Following this, the strips were incubated in AP-conjugated streptavidin (1:2500 dilution) for one hour. Lastly, colour was developed using NBT/BCIP in dark.
Plasmodium knowlesi total protein extract was extracted from P. knowlesi strain A1H1 culture (5% haematocrit, 5% parasitaemia) according to Cooper (2002) . Western blot assay was carried out as mentioned previously by probing anti-PkCSP peptide I antiserum (1:250 dilution) against P. knowlesi total protein extract. Recombinant PkCSP was included as a control of this assay.
Mice immunization and evaluation of PkCSP mice antisera reactivity
Six to eight-week old female BALB/c mice were used for immunization (n=3). Purified PkCSP, 30 µg, was mixed with CFA at 1:1 ratio and the mixture was injected into mice as primary immunization (day 0). For booster injections, 30 µg were mixed with IFA and administered on day 14 and 21. All injections were given subcutaneously. Serum of each mouse was collected on day 0, 14, 21 and 31. Endpoint titre assay was carried out using day 31 mice antiserum. Antiserum from the most reactive mouse was used in western blot assay as described in the previous method. For this assay, purified rPkCSP and P. knowlesi total protein extract were resolved on 12% SDS-PAGE. pET-30a(+) lysate and crude rPkCSP lysate were included as controls. Dilution of biotin-labelled goat anti-mouse IgG + IgM used was increased to 1:2500 to reduce nonspecific background.