In vitro invasion inhibition assay using antibodies against Plasmodium knowlesi Duffy binding protein alpha and apical membrane antigen protein 1 in human erythrocyte-adapted P. knowlesi A1-H.1 strain

Fauzi Muh,Jin-Hee Han,Eun-Taek Han,Seong-Kyun Lee,Robert W Moon,Ji-Hoon Park,Yee Ling Lau,Mohammad Rafiul Hoque,Egy Rahman Firdaus

doi:10.1186/s12936-018-2420-4

Abstract

BackgroundThe rapid process of malaria erythrocyte invasion involves ligand–receptor interactions. Inducing antibodies against specific ligands or receptors that abrogate the invasion process is a key challenge for blood stage vaccine development. However, few candidates were reported and remain to be validated for the discovery of new vaccine candidates in Plasmodium knowlesi.MethodsIn order to investigate the efficacy of pre-clinical vaccine candidates in P. knowlesi-infected human cases, this study describes an in vitro invasion inhibition assay, using a P. knowlesi strain adapted to in vitro growth in human erythrocytes, PkA1-H.1. Recombinant proteins of P. knowlesi Duffy binding protein alpha (PkDBPα) and apical membrane antigen 1 (PkAMA1) were produced in Escherichia coli system and rabbit antibodies were generated from immune animals.ResultsPkDBPα and PkAMA1 recombinant proteins were expressed as insoluble and produced as a functional refolded form for this study. Antibodies against PkDBPα and PkAMA1 specifically recognized recombinant proteins and native parasite proteins in schizont-stage parasites on the merozoite organelles. Single and combination of anti-PkDBPα and anti-PkAMA1 antibodies elicited strong growth inhibitory effects on the parasite in concentration-dependent manner. Meanwhile, IgG prevalence of PkDBPα and PkAMA1 were observed in 13.0 and 46.7% in human clinical patients, respectively.ConclusionThese data provide support for the validation of in vitro growth inhibition assay using antibodies of DBPα and AMA1 in human-adapted P. knowlesi parasite PkA1-H.1 strain.

Highlights

The rapid process of malaria erythrocyte invasion involves ligand–receptor interactions
The bands migrated in different mobility between reduced (DTT +) and non-reduced condition (DTT −) on the SDS-PAGE (Fig. 1b)
Anti-PkDBPα, anti-PkAMA1 and anti-His antibodies recognized the recombinant proteins and parasite lysate by western blot analysis (Fig. 1c). These results suggest that the native condition of refolded proteins had been formed and its antibody can recognize the recombinant protein and native parasite protein

Summary

Introduction

The rapid process of malaria erythrocyte invasion involves ligand–receptor interactions. The Plasmodium life cycle is complex and involves ligand–receptor interactions for successful invasion and multiplication in host erythrocytes [7]. Plasmodium vivax and P. knowlesi depend on the presence of the Duffy antigen receptor for chemokine (DARC) on erythrocytes for invasion. P. knowlesi Duffy binding protein alpha (PkDBPα) is a ligand for DARC and pivotal in human infection [8, 9]. Antibodies against domain II of PkDBPα or against surface-exposed DARC epitope inhibited invasion of monkey and human erythrocytes [10]. The complex formed with the ectodomain of PkAMA1 (domain 1 and 2) and rhoptry neck protein 2 (RON2) is essential for invasion and as compared to Plasmodium falciparum and P. vivax, the RON2-binding site of PkAMA1 is much less polymorphic [13]

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Conclusion