Immunoglobulin G responses to variant forms of Plasmodium vivax merozoite surface protein 9 upon natural infection in Thailand

Merozoite surface protein 9 (MSP9) constitutes a ligand complex involved in erythrocyte invasion by malarial merozoites and is a promising vaccine target. Plasmodium vivax MSP9 (PvMSP9) is immunogenic upon natural malaria exposure. To address whether sequence diversity in PvMSP9 among field isolates could affect natural antibody responses, the recombinant proteins representing two variants each for the N- and the C-terminal domains of PvMSP-9 were used as antigens to assess antibody reactivity among 246 P. vivax-infected patients’ sera from Tak and Ubon Ratchathani Provinces in Thailand. Results revealed that the seropositivity rates of IgG antibodies to the N-terminal antigens were higher than those to the C-terminal antigens (87.80% vs. 67.48%). Most seropositive sera were reactive to both variants, suggesting the presence of common epitopes. Variant-specific antibodies to the N- and the C-terminal antigens were detected in 15.85% and 16.70% of serum samples, respectively. These seropositivity rates were not significant difference between provinces. The seropositivity rates, levels and avidity of anti-PvMSP9 antibodies exhibited positive trends towards increasing malaria episodes. The IgG isotype responses to the N- and the C-terminal antigens were mainly IgG1 and IgG3. The profile of IgG responses may have implications for development of PvMSP9-based vaccine.


Expression of the N-and the C-terminal PvMSP9 constructs
The recombinant proteins were expressed as described previously [47]. In brief, protein expression was induced with 1 mM of isopropyl-β-D thiogalactopyranoside (IPTG) at 30°C for 6 h under constant agitation. For recombinant proteins produced in inclusion bodies, the cell pellets were re-suspended in 20 μL of 1M Tris-HCl pH 7.5, 250 mM EDTA, 1% Triton X-100 and 250 mM phenylmethylsulfonyl fluoride (PMSF), vortexed and sonicated on ice. After adding lysozyme, the lysates were incubated for 60 min at 30°C under gentle shaking, sonicated on ice and washed for 5 times with buffer containing 1M Tris-HCl pH 7.5, 250 mM EDTA and 1% Triton X-100. The cell pellets were dissolved in solubilization buffer containing 500 mM 3-(cyclohexyamino)-1 propanesulfonic acid (CAPS) pH 11, 1, 1M dithiothreitol (DTT) and 5% Nlauroylsarcosine.

Purification of recombinant proteins
Purification of recombinant PvMSP9 proteins essentially followed the procedures reported by Kosuwin and colleagues [47]. After cell debris was removed from the protein suspension by centrifugation, refolding of recombinant PvMSP-9 proteins was carried out by multistep dialysis of solubilized inclusion bodies. An initial step was done in 2 liters of dialysis buffer (1M Tris-HCl pH 7.4) with 0.1mM DTT for 3 h at 4°C, followed by an overnight dialysis at 4°C with fresh buffer. The solubilized protein was then dialyzed in buffer without DTT for 3 h and repeated twice. Final dialysis was performed in redox refolding buffer containing 0.2 mM oxidized glutathione and 1 mM reduced glutathione at 4°C overnight. The purified recombinant PvMSP-9 proteins were aliquoted and kept at -80°C until use. Meanwhile, purification of soluble proteins was done by affinity chromatography using His-Bind Resin (Novagen, Germany) according to the manufacturer's recommendation. In brief, the cell pellets were harvested by centrifugation at 6,000 g for 30 min at 4°C, allowed to drain dry completely and resuspended in binding buffer containing 20 mM Tris-HCl pH 7.5, 5 mM imidazole, 500 mM NaCl and 250 mM PMSF. After vortexing and sonicating on ice, lysozyme solution (20 mg/ml) was added, incubated at 30°C 60 min and sonicated again. After centrifugation at 6000 g for 30 min at 4°C, the supernatant was passed through a filter with pore size of 0.45 μm and kept at room temperature for 30 min before further purification with His-Bind Resin column following the manufacturer's recommendation and kept at -80 °C until use. Protein concentration was estimated using DC Protein Assay (Bio-Rad, Japan).

Analysis of recombinant PvMSP9 proteins
Recombinant proteins were analyzed by using 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing (with β-mercaptoethanol) and non-reducing (without β-mercaptoethanol) conditions with a standard molecular weight maker.
Electrophoresis was performed at a constant voltage (100V). The gel was stained with Coomassie Brilliant Blue solution for 30 minutes at room temperature and detained with solution containing 10% glacial acetic and 30% methanol until the protein bands could be clearly visible.
The procedure for ELISA was performed as described previously by Kosuwin and colleages [47]. In brief, PolySorp immunoplates (Nunc, Wiesbaden, Germany) were coated in duplicate wells with 0.5 µg per well of the affinity purified PvMSP-9 protein in a 0.06 M carbonate-bicarbonate buffer containing 15mM Na 2 CO 3 , 35mM NaHCO 3 , 0.2 M NaCl pH 9.6, and incubated at 4°C overnight. After washing with phosphate buffer saline (PBS) buffer containing 0.05% Tween 20 (PBS/T), the microplates were blocked with of 3% skim milk in PBS at room temperature for 1 h, washed with PBS buffer and incubated with 100 μl of individual plasma samples in duplicate wells at 1:100 dilutions at room temperature for 1 h.
After washing with PBS/T, the plates were incubated with horseradish peroxidase-conjugated goat anti-human IgG diluted to 1:1000 with 3% skimmed milk for 1 h at room temperature and washed again with PBS/T. The presence of bound IgG antibodies was detected by using ophenylenediamine dihydrochloride-hydrogen peroxide (Sigma-Aldrich, Germany) and the reaction was terminated with 2 N sulphuric acid. The reaction was analyzed by measurement of absorbance at 490 nm using a microplate reader (Biotech, Winooski, VT, USA). Categories I -IX represent positive antibodies to one or both variants of the N-and the Cterminal antigens (n = 154).
Categories X -XV display positive antibodies to either the N-or the C-terminal antigens (n = 74).
Category XVI denotes no antibody response to both N-and C-terminal antigens (n = 18).      No data  Figure S1. Conserved domain I of PvMSP9 (Belem strain) and its corresponding sequences of P. cynomolgi (PcyMSP9), P. knowlesi (PkMSP9) and P. falciparum (PfMSP9)(GenBank accession nos AF435853, , KC935411, AF435855 and LN999947, respectively). Erythrocytebinding regions in PfMSP9 [8] are shown in red. Amino acid substitutions among Thai P. vivax isolates are shown underneath the Belem sequence whose identical residues are indicated by dots [15]. The region for the N-terminal antigen is marked with asterisks above the alignment. Amino acid substitutions in antigens TMS102 and CT1186 are shaded.