Antigen selection and expression in yeast
Earlier TBV antigen discovery efforts using the rodent malaria system identified several candidate antigens, including PbPH, which contains a pleckstrin homology domain, and PbSOP26, a secreted protein from the ookinetes [13, 14]. Based on their excellent transmission-reducing (TR) activities, their orthologs in P. vivax PVX_098655 (PvPH) and PVX_101120 (PvSOP26) were selected for characterization. The sequences corresponding to amino acids (aa) 22–304 of PvPH and aa 30–272 of PvSOP26 of the Sal-I strain were synthesized and codon-optimized for expression in the yeast Pichia pastoris (GenScript Biotech Corp, China). They were cloned into the pPIC9K (Invitrogen, Carlsbad, CA, USA) vector, which was used to transform the P. pastoris GS115 strain . The yeast strains expressing the two recombinant proteins (rPvPH and rPvSOP26) were cultured in 1 L of buffered minimal medium and induced by methanol. After lysis of the yeast cells with an ATS high-pressure homogenizer (ATS Engineering Inc, Germany), the recombinant proteins were purified using Ni-NTA columns, and the purity of the recombinant proteins was estimated by SDS–PAGE . For negative control, we expressed a glutathione S-transferase (GST) protein as described previously .
Generation of anti-rPvPH and -rPvSOP26 sera
To generate antisera against the two recombinant proteins, BALB/c mice (n = 10 in each group) were injected subcutaneously with the purified rPvPH, rPvSOP26, or GST control protein (50 μg each) emulsified in the complete Freund's adjuvant (Sigma, St Louis, MI, USA). The mice were then given two booster immunizations with the same recombinant proteins (25 μg/mouse) emulsified in incomplete Freund's adjuvant (Sigma) at a 2-week interval. Two weeks after the final immunization, the antisera in each group of mice were collected via cardiac puncture and pooled.
The recombinant proteins rPvPH and rPvSOP26 were separated in a 12% SDS–PAGE gel under reduced conditions and electro-transferred onto a 0.22 μm polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 for 2 h, the blots were probed with the pooled mouse antisera against rPvPH or rPvSOP26 as the primary antibodies and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:10,000) as the secondary antibodies. A western blot kit (Thermo, Waltham, MA, USA) was used to visualize the protein bands .
Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed to determine the antibody titers of mouse immune sera. Microtiter plates were coated with the purified rPvPH or rPvSOP26 (5 μg/mL) at 4°C for more than 8 h. The plates were first blocked with 1% bovine serum albumin (BSA) for 2 h at 37°C, then incubated with the antisera from mice immunized with rPvPH and rPvSOP26, respectively, at 37°C for 2 h. These antisera were diluted in 1% BSA in phosphate-buffered saline (PBS) from 1:200 to 1:512,000. After two washes with PBS, 100 mL HRP-conjugated goat anti-mouse IgG antibodies (Invitrogen; 1:5,000) were added to each well and incubated for 2 h. After five washes with PBS, tetramethylbenzidine was added to wells, and the plate was kept it in the dark for 10 min. The reaction was stopped by adding 2 mM H2SO4. An ELISA plate reader was used to measure the absorbance at 490 nm . The endpoint titers were determined as the highest antiserum dilution with an optical reading greater than the average reading from a control serum (anti-GST) plus three standard deviations as the cut-off value .
P. vivax samples
The human-subject protocol for this study was approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (MUTM 2018-016).Patients with P. vivax malaria who were symptomatic for clinical malaria, slide-positive for P. vivax infection, 18 years or older, and not pregnant were considered eligible for inclusion in this study. Three P. vivax patients were enrolled after signing the informed consent. Before antimalarial treatment, 5–10 mL venous blood was collected into heparinized tubes and used to make blood smears and for SMFA .
Indirect immunofluorescent assay (IFA)
The expression and the location of PvPH were studied by IFA. The erythrocytes from the P. vivax patients were mixed with 47% Nycodenz/RPMI 1640 and centrifuged at 500 ×g for 25 min to obtain the parasite-infected erythrocytes at the grey interface. They were used to make thin smears, which were fixed with 4% paraformaldehyde for 30 min at 37℃. Skimmed milk (5%) in PBS was used to block the slides for 30 min. After three washes with PBS, the slides were incubated with mouse antisera against rPvPH, rPvSOP26 or the GST control for 1 h at room temperature. After three washes with cold PBS, the slides were incubated with FITC-conjugated goat anti-mouse antibodies (1:500, Invitrogen) for 1 h, and with 1 μg/mL 4’6-diamidino-2-phenylindole (DAPI; Invitrogen) for 30 min. After an additional wash with cold PBS, the slides were mounted with the ProLong®Gold Antifade Reagent kit (Invitrogen). Fluorescence images were obtained with an Olympus BX53 microscope [18, 21].
Quantification of TB activity
Antisera from mice immunized with rPvPH, rPvSOP26 or the GST control protein were diluted with heat-inactivated AB+ serum obtained from healthy donors in Thailand (1:1, v/v ratio). Erythrocytes of P. vivax patients were mixed with the diluted sera (1:1, v/v ratio) and incubated at 37oC for 15 min. Each reconstituted infected blood was then introduced to a glass feeder and kept at 37 oC. One hundred starved mosquitoes were allowed to feed on the blood mixture for 30 min at 37oC through the membrane feeder. After several hours, only fully engorged mosquitoes were kept on 10% sucrose solution in cotton balls at 20°C at 80% relative humidity for a week. Twenty mosquitoes from each group were randomly selected dissected on day 7 after blood feeding. Mosquito midguts were stained with 5% mercurochrome, and oocysts were counted [22, 23]. The infection prevalence, which is the proportion of oocyst-positive infected mosquitoes, was used to determine the TB activity. The intensity of infection, i.e. the number of oocysts per mosquito midgut, was used to determine the TR activity.
Analysis of genetic polymorphisms
For the parasite isolates used for SMFA, genetic polymorphisms of PvPH and PvSOP26 genes were determined. DNA was extracted from dried filter-paper blood spots using a QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). DNA fragments encoding rPvPH (22–304 aa) and rPvSOP26 (30–272 aa) were amplified by PCR . The primers were designed based on the P. vivax Sal-1 (PVX_083235) sequence: PvPH-F (GTCCCAATTAGAATCTGTTT) and PvPH-R (GTTCCTTCTGTTGGGTGTTT); PvSOP26-F (ACCTTGTAGCCTCTACACTT) and PvPH-R (AAATTTGTTGAAAAAATTAT). All amplified DNA products were purified with a QIAquick Gel Extraction Kit (Qiagen) and sequenced using the ABI Prism® BigDye™ cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) as previously described . Alignment of nucleotide sequences was done using the BioEdit software.
Statistical analyses were performed using SPSS version 22.0. The intensity of infection was calculated using a Mann–Whitney U test. The infection prevalence was compared using Fisher’s exact test. P values less than 0.05 were considered statistically significant.