P.o. curtisi and P. o. wallikeri infected blood samples were obtained from local hospitals in Jiangsu Province (China) between 2012 and 2016 from febrile patients who had returned from work in malaria endemic areas of sub-Saharan Africa  . Identification of the isolates was confirmed by polymerase chain reaction (PCR) analysis, and parasite species were distinguished using real-time TaqMan PCR .
PCR amplification and sequencing of pomsp4 genes
Genomic DNA extracted from P. ovale-infected individual blood samples was previously preserved in our laboratory. A total of 46 P. ovale spp. genomes (P. o. curtisi, n=23 and P. o. wallikeri, n=23) were randomly selected for amplification. Information on the imported P. ovale spp. specimens is given in Additional file 1: Table S1. Full nucleotide sequences of pocmsp4 and powmsp4 were amplified via PCR using primers designed as follows: pocmsp4 forward (5′-ATG AGG GTA CTC CAA TTT TTA TTA C-3′), pocmsp4 reverse (5′-TTA ATT TAT TGA CGC TAA AAT G-3′), powmsp4 forward (5′-ATG AGG GTA CTC CAA TTT TTA TTA C-3′), and powmsp4 reverse (5′- TTA ATT TAT TGA CGC TAA AAT G-3′). Pocmsp4 (Plasmodium Genomics Resource database, PocGH01_04023000) and powmsp4 (National Centre for Biotechnology Information GenBank database, accession number: LT594508.1) were used as reference gene sequences. Reactions were carried out in a volume of 20 μL, including 1 μL of genomic DNA, 0.8 μL of each primer (10 µM), 7.4 μL of double-distilled water, 0.5 units of DNA polymerase, and 2 mM deoxynucleoside triphosphate within 10 μL of premix (2× Phanta® Max Master Mix, Vazyme). PCR amplification was performed in a Mastercycler (Eppendorf) as follows: denaturation at 95 °C for 3 min; 35 cycles of 95 °C for 15 s, 51 °C for 30 s, and 72 °C for 30 s; and final extension at 72 °C for 5 min. PCR products were analysed in 1% agarose gel electrophoresis, visualized under an ultraviolet transilluminator (Bio-Rad ChemiDoc MP), and sequenced by Genewiz.
The N-terminal of PoMSP1 [pocmsp1 (GenBank: KC137343) and powmsp1 (GeneBank: KC137341)] and full length of PoAMA1 [pocama1 (PlasmoDB: PocGH01_09039800) and powama1 (GenBank: SBT36045.1)] merozoite surface proteins, which were previously expressed and preserved in our laboratory, were used for specificity tests of PoMSP4 protein-raised antibodies.
Construction of recombinant pomsp4 clones
PoMSP4-predicted open reading frames (ORFs) without the EGF-like domain consisting of 1–119 (PocMSP4) and 1–97 (PowMSP4) amino acids, for which a P. falciparum ortholog has been reported as highly immunogenic [30, 31, 37], were selected. Genomic DNA from P. ovale isolates was used as template for PCR amplification of pomsp4 ORFs. Primers were as follows: pocmsp4 forward (5′-ATG AGG GTA CTC CAA TTT TTA TTA C-3′), pocmsp4 reverse (5′-AGG CGA TGC TAT CGG TTT TG-3′), powmsp4 forward (5′-ATG AGG GTA CTC CAA TTT TTA TTA C-3′), and powmsp4 reverse (5′-TGC TAT ACC TAG GAC ATT TTT ACC C-3′). The reaction was performed in a 20 µL volume as described above on a Mastercycler (Eppendorf) with the following temperature profile: initial denaturation at 95 °C for 3 min; 35 cycles of 95 °C for 15 s, 56 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min. PCR products were also analysed as described above.
The amplified fragments were cloned into the pUC57 vector, sequenced by Genewiz on an ABI 3730xl DNA analyzer (Thermo Fisher Scientific) using universal primers (M13F: 5′-TGT AAA ACG ACG GCC AGT-3′, M13R: 5′-CAG GAA ACA GCT ATG AC-3′), and subcloned into pET32a expression plasmid vector (YouLong Biotech). Recombinant plasmids were transformed into expression host Escherichia coli strain BL21 (DE3) pLysS and sequenced using universal primers T7 through Genewiz.
Protein expression and purification
Escherichia coli BL21 (DE3) pLysS cells containing recombinant plasmid pET32apomsp4 were cultured in Luria–Bertani (supplemented with 50 mg/mL ampicillin) at 37 °C with shaking until optical density (OD) of 600 nm reached 0.6–0.8. The culture was induced with 0.5 mM isopropyl β-D-1 thiogalactopyranoside and allowed to grow for another 3 h at 37 °C. Cells were harvested through centrifugation at 4000 × g for 30 min. Protein purification was performed by YouLong Biotech using the following technique. Thawed cells were suspended in purification buffer (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, and 10 mM imidazole) and lysed by sonication. The insoluble fraction was separated by centrifugation at 15,000 × g for 15 min at 4 °C. The soluble fraction was applied to a column containing 1.0 mL of Ni-nitrilotriacetic acid-agarose (Qiagen) and then washed with 10 mL of purification buffer containing 20 mM imidazole. Recombinant proteins were eluted from the column with purification buffer containing 250 mM imidazole and then exchanged into Tris-HCl storage buffer (50 mM Tris-HCl [pH 8.5], 100 mM NaCl, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, and 10% [v/v] glycerol) using a 30 kDa ultrafiltration tube (Millipore). Proteins were stored at −80 °C until use.
Analyses of protein
The concentration of recombinant proteins (rPoMSP4) was determined through the Bradford method using bovine serum albumin (BSA) as standard (Bradford protein assay kit, Solarbio). Purified proteins were analysed by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining (Beyotime Biotech) to assess the expression level and immunoreactivity. The separated proteins from SDS-PAGE were electrophorectically transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon) and blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% skimmed milk at 4 °C. The membranes were probed with anti-His antibody (ABclonal) at 1:5000 dilution along with primary antibody dilution buffer (Meilunbio) overnight at 4 °C. Membranes were washed three times with 0.1% TBST and treated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution for 90 min. Finally, the membranes were analysed with a ChemiDoc MP imaging system (Bio-Rad).
Antibody raising and immunodetection
Six- to eight-week-old female BALB/c mice were used for immunizations as follows. Mice were grouped into the rPoMSP4-immunized (n=5 per group) and negative control groups (n=3 per group). Each mouse was intraperitoneally injected with 50 µg of rPocMSP4, rPowMSP4, or PBS, all of which were diluted in PBS with complete Freund’s adjuvant (Sigma). An equal volume of antigen with incomplete Freund’s adjuvant (Sigma) was used for subsequent boosters, which were administered on days 21 and 42 post-immunization intraperitoneally. The control group was administered an equal amount of PBS and adjuvant. Mouse blood samples were collected from the tip of the tail on days 0, 7, 14, 28, 35, and 49. Sera were obtained via centrifugation for 20 min at 2000 × rpm and stored at −80 °C.
Purified rPoMSP4 was tested against sera from rPocMSP4- or rPowMSP4-infected mice to assess anti-PoMSP4 IgG antibodies through Western blot analysis. During the assays, PVDF membranes were incubated with antisera (1:2000 dilutions) from the rPoMSP4-immunized group or negative control group, followed by HRP-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution.
Specificity of anti-PoMSP4 antibodies
Levels of IgG antibodies targeting PoMSP4 in mouse sera were detected via enzyme-linked immunosorbent assays (ELISA). In brief, 96-well ELISA plates were coated with 50 ng of rPoMSP4 antigen dissolved in coating buffer solution (15 mM sodium carbonate and 35 mM sodium bicarbonate in distilled water) overnight at 4 °C. After three times wash with PBS containing 0.1% of Tween-20 (PBST), the plates were blocked with 1% BSA in PBS and incubated at room temperature for 2 h. Thereafter, individual mouse sera (100 µL) diluted at different dilutions were added on the plate and incubated at room temperature for 2 h. The plates were washed again three times with PBST and HRP-conjugated goat anti-mouse IgG antibodies (Southern Biotech) at 1:5000 dilution and incubated for 1 h 30 min at room temperature. The plates were finally washed three times with PBST and incubated with 3, 3’, 5, 5’-tetramethylbenzidine (Invitrogen) substrate for a few minutes in the dark, and 2 M H2SO4 was added to stop the reaction. The absorbance at OD of 450 nm was measured using a microplate reader (Synergy, BioTeK). Furthermore, anti-PoMSP4 IgG antibodies were tested against rPoMSP1 and rPoAMA1 antigens via ELISA to test for the specificity of antibodies.
Lymphocyte proliferation assays
Lymphocyte proliferation was measured using a cell counting kit-8 (CCK-8, Beyotime Biotech). A certain amount of 5 × 105 cells/well of PoMSP4- and PBS-immunized cells was treated with 10 µL of PocMSP4 (5 µg/mL), 10 µL of PowMSP4 (5 µg/mL), or 10 µL of concanavalin A (Con A, 2 μg/mL), as positive control, in 96-well flat-bottom microtitre plates and then incubated for 72 h at 37 °C with 5% CO2. Thereafter, 10 µL of CCK-8 was added to each well, and the plates were incubated for 2 h at 37 °C and measured at 450 nm using a microplate reader.
Sequence alignment and analysis of data
Full nucleotide sequences of pomsp4 genes from all clinical isolates were translated to the deduced amino acid sequences using the MegAlign module of Lasergene 7 software package (DNAstar) and then aligned with reference sequences to assess conservation within PoMSP4. Amino acid sequences of the segments of PocMSP4 and PowMSP4 isolates were aligned with those of the PocGH01_04023000 and LT594508.1 reference strains, respectively. Sequence alignment for all P. ovale isolates was performed using MEGA v.7.0 software.
Statistical analysis and graphing were conducted using GraphPad Prism software version 5.0 (Graph Pad software, Inc.). SPSS v.16.0 was performed to analyse cross-reaction and antibody responses. Student’s t-test with probability (P) value of <0.05 indicated a significant difference.