Inhibition of parasite invasion by monoclonal antibody against epidermal growth factor-like domain of Plasmodium vivax merozoite surface protein 1 paralog

Abstract

The Plasmodium vivax merozoite surface protein 1 paralog (PvMSP1P), which has epidermal growth factor (EGF)-like domains, was identified as a novel erythrocyte adhesive molecule. This EGF-like domain (PvMSP1P-19) elicited high level of acquired immune response in patients. Antibodies against PvMSP1P significantly reduced erythrocyte adhesion activity to its unknown receptor. To determine PvMSP1P-19-specific antibody function and B-cell epitopes in vivax patients, five monoclonal antibodies (mAbs) and 18-mer peptides were generated. The mAb functions were determined by erythrocyte-binding inhibition assay and invasion inhibition assay with P. knowlesi. B-cell epitopes of PvMSP1P-19 domains were evaluated by peptide microarray. The pvmsp1p-19 sequences showed limited polymorphism in P. vivax worldwide isolates. The 1BH9-A10 showed erythrocyte binding inhibitory by interaction with the N-terminus of PvMSP1P-19, while this mAb failed to recognize PkMSP1P-19 suggesting the species-specific for P. vivax. Other mAbs showed cross-reactivity with PkMSP1P-19. Among them, the 2AF4-A2 and 2AF4-A6 mAb significantly reduced parasite invasion through C-terminal recognition. The linear B-cell epitope in naturally exposed P. vivax patient was identified at three linear epitopes. In this study, PvMSP1P-19 N-terminal-specific 1BH9-A10 and C-terminal-specific 2AF4 mAbs showed functional activity for epitope recognition suggesting that PvMSP1P may be useful for vaccine development strategy for specific single epitope to prevent P. vivax invasion.

Figure 1

Schematic structure of PvMSP1P-19 and sequence diversity in worldwide isolate. (a) The schematic diagram shown PvMSP1P primary structure. The signal peptide (SP, black box), tandem repeat (TR, orange box), polymorphic Glu/Gln-rich region (PR, yellow box), epidermal growth factor like (EGF, Gray box), and the glycosylphosphatidylinositol (GPI, blue box) indicates. The 18-mer peptides indicated with amino acid position and sequence for peptide microarray. (b) Sliding window plot showing nucleotide diversity (π) values of PvMSP1P using 66 worldwide isolates. The grey box represents two EGF-like domains at amino acid positions 1751 to 1834.

Figure 2

PvMSP1P-19 monoclonal antibody production and validation. (a) Three clones were successfully hybridized to produce monoclonal antibodies, and hybridoma culture supernatants were obtained. The OD values were measured by indirect ELISA at 405 nm. Antigen was used at concentrations of 1 µg/ml. (b) A western blot showing five monoclonal antibodies reacting with PvMSP1P-19. The approximately 14 kDa specific band indicates rPvMSP1P-19 (arrow head). His, penta-anti-His antibody; lanes 1–5, anti-PvMSP1P-19 monoclonal antibodies as follows: lane 1, 1BH9-A10; lane 2, 2AF4-A2; lane 3, 2AF4-A6; lane 4, 3BC6-A5; and lane 5, 3BC6-B12. (c) Reactivity detection for monoclonal antibodies with native PvMSP1P by immunofluorescence assay. The mature schizont of P. vivax was dual labelled with PvMSP1P-19 monoclonal antibodies (green) and rabbit immune sera against PvMSP1-19 (red, merozoite surface marker). Nuclei are visualized with DAPI (blue). Bar indicate 5 μm.

Figure 3

PvMSP1P-19 monoclonal antibody epitope mapping. The normalized mean fluorescence intensity (MFI) was calculated by comparing the no peptide printed slide well MFI value with each peptide printed array well MFI value. Data are shown as the mean ± S.D. of four independent experiments.

Figure 4

Erythrocyte binding inhibitory effect of PvMSP1P-19 monoclonal antibodies. The percentage of relative binding was calculated by comparing the number of rosettes between no antibody-treated and monoclonal antibody-treated samples. The PvMSP1P-19 specific monoclonal antibody, 1BH9-A10, showed red blood cell binding inhibitory activity. Data are shown as the mean ± S.D. of three independent experiments.

Figure 5

Cross-reactivity of PvMSP1P-19 monoclonal antibodies with P. knowlesi. (a) MSP1P EGF-like domain (MSP1P-19) amino-acid sequence comparison between PvMSP1 and PkMSP1. The red bar indicates identical sequence and blue bar indicates diverse sequence. (b) A western blot showing five monoclonal antibodies reacting with recombinant PkMSP1P-19. The approximately 37 kDa specific band indicates rPkMSP1P-19 (arrow head). GST, anti-GST antibody; lanes 1–5, anti-PvMSP1P-19 monoclonal antibodies as follows: lane 1, 1BH9-A10; lane 2, 2AF4-A2; lane 3, 2AF4-A6; lane 4, 3BC6-A5; and lane 5, 3BC6-B12. (c) A western blot showing five monoclonal antibodies reacting with P. knowlesi parasite lysate. The clear multiple band indicates processed naïve PkMSP1P-19. Lanes 1–5, anti-PvMSP1P-19 monoclonal antibodies as follows: lane 1, 1BH9-A10; lane 2, 2AF4-A2; lane 3, 2AF4-A6; lane 4, 3BC6-A5; and lane 5, 3BC6-B12. P, P. knowlesi lysate; R, normal RBC extract. (d) Reactivity observed by PvMSP1P-19 monoclonal antibodies with P. knowlesi merozoite at schizont stage by immunofluorescence assay. The mature schizont of P. knowlesi was dual labelled with PvMSP1P-19 monoclonal antibodies (green) and rabbit immune sera against PvMSP1-19 (red, merozoite surface marker). Nuclei are visualized with DAPI (blue). Bar indicate 5 μm.

Figure 6

P. knowlesi invasion inhibition activities of PvMSP1P-19 monoclonal antibodies. (a) P. knowlesi stages were confirmed by morphology under light microscopy. The black arrow head indicates the mature schizont stage of P. knowlesi before invasion. The black arrow points to an unhealthy parasite, and the red arrow indicates the healthy ring stage of the parasite after re-invasion. (b) FACS gating strategy of ring stage parasitaemia evaluation. The schizont stage parasites are considered to have SYBR green signals of more than log 10⁶. (c) The P. knowlesi invasion inhibition efficacy was confirmed by an invasion inhibition assay. Data are shown as the invasion inhibition rate mean ± standard deviation (S.D.) with pre-immune sera (PI) and anti-2C3 (murine anti-Fy6) and PvMSP1P-19 monoclonal antibodies. Significant differences between PI and anti-2C3 or monoclonal antibodies calculated with a one-way ANOVA with the Tukey post-test. ***p < 0.001.