Elsevier

Vaccine

Volume 30, Issue 11, 2 March 2012, Pages 1972-1980
Vaccine

Antibodies against a Plasmodium falciparum antigen PfMSPDBL1 inhibit merozoite invasion into human erythrocytes

https://doi.org/10.1016/j.vaccine.2012.01.010Get rights and content

Abstract

One approach to develop a malaria blood-stage vaccine is to target proteins that play critical roles in the erythrocyte invasion of merozoites. The merozoite surface proteins (MSPs) and the erythrocyte-binding antigens (EBAs) are considered promising vaccine candidates, for they are known to play important roles in erythrocyte invasion and are exposed to host immune system. Here we focused on a Plasmodium falciparum antigen, PfMSPDBL1 (encoded by PF10_0348 gene) that is a member of the MSP3 family and has both Duffy binding-like (DBL) domain and secreted polymorphic antigen associated with merozoites (SPAM) domain. Therefore, we aimed to characterize PfMSPDBL1 as a vaccine candidate. Recombinant full-length protein (rFL) of PfMSPDBL1 was synthesized by a wheat germ cell-free system, and rabbit antiserum was raised against rFL. We show that rabbit anti-PfMSPDBL1 antibodies inhibited erythrocyte invasion of wild type parasites in vitro in a dose dependent manner, and the specificity of inhibitory activity was confirmed using PfMSPDBL1 knockout parasites. Pre-incubation of the anti-PfMSPDBL1 antibodies with the recombinant SPAM domain had no effect on the inhibitory activity suggesting that antibodies to this region were not involved. In addition, antibodies to rFL were elicited by P. falciparum infection in malaria endemic area, suggesting the PfMSLDBL1 is immunogenic to humans. Our results suggest that PfMSPDBL1 is a novel blood-stage malaria vaccine candidate.

Highlights

► PfMSPDBL1, expressed on the surface of Plasmodium falciparum merozoite, has both DBL and SPAM domains. ► Anti-PfMSPDBL1 antibody reduced the merozoite invasion into RBC. ► PfMSPDBL1 is a novel blood-stage malaria vaccine candidate of P. falciparum.

Introduction

Malaria is a serious infectious disease caused by a protozoan parasite of the genus Plasmodium. It causes approximately 300 million illnesses and 1 million deaths annually [1]. The appearance of malaria parasites with resistance to antimalarial drugs and of mosquito vector with resistance to insecticides has highlighted the importance of developing a malaria vaccine. Protective immunity against Plasmodium falciparum develops after repeated exposure and prevents severe disease and symptomatic episodes by controlling blood-stage parasitemia [2]. Antibodies are important effectors of protective immunity against malaria as evidenced by experimental animal models and, most importantly, passive transfer studies in which antibodies from malaria-immune adults were successfully used to treat patients with severe malaria [3], [4]. These findings provide a strong rationale that an effective vaccine is achievable against the asexual blood-stage parasites by inducing an immune response. Although a number of blood-stage vaccines have been developed and tested in preclinical and clinical trials, only limited clinical success has been achieved with blood-stage vaccines to date [5], [6]. Therefore, discovery of novel blood-stage vaccine candidates is an important step towards control of malaria.

One approach to discover a novel malaria vaccine candidate is to target proteins that play critical roles in the invasion process into erythrocytes. The invasion involves multiple steps, including initial attachment, apical reorientation, and tight junction formation, followed by the entry of the merozoite into the erythrocyte. The initial attachment between a free merozoite and an erythrocyte is a reversible interaction and thought to be mediated by merozoite surface proteins (MSPs) [7]. On the other hand, the tight junction formation requires the function of erythrocyte-binding antigens (EBAs). The EBAs are members of the Duffy binding-like (DBL) superfamily, EBA175 (encoded by MAL7P1.176 gene), EBA140/BAEBL (by MAL13P1.160 gene) and EBA181/JESEBL (by PFA0125c gene) are expressed within P. falciparum [8]. The EBAs are stored in the micronemes and are secreted onto the merozoite surface just before invasion, and serve as ligands that bind to receptors on the surface of erythrocytes [9], [10], [11]. A number of studies have shown that antibodies to these merozoite antigens are thought to function in vivo by inhibiting merozoite invasion of erythrocyte (ligand-blocking), opsonizing merozoites for phagocytosis, and inducing antibody-dependent cellular inhibition (ADCI) [12], [13], [14], [15]. Since the MSPs and EBAs are exposed to the host immune system, these proteins are considered as promising vaccine candidates and some of these are at various stages of development for clinical trials [16].

Here, we have focused on a novel antigen PfMSPDBL1 (encoded by PF10_0348 gene). This protein has a DBL domain and secreted polymorphic antigen associated with merozoites (SPAM) (Fig. 1A). A previous report [17] showed that the PfMSPDBL1 protein is localized on the merozoite surface and it can bind to the erythrocytes and hence suggested that PfMSPDBL1 may play a role in initial attachment of the erythrocyte by merozoites and that the DBL domain may be a potential target of ligand-blocking antibodies as well as DBL domains of other erythrocyte binding proteins. However, there were not any assays performed in the previous study to prove the invasion inhibitory effect of the anti-PfMSPDBL1 antibodies [17]. On the other hand, recently, a new MSP3 multi-gene family was predicted, and PfMSPDBL1 is one of the members of this family [18]. Because of the high conservation of the MSP3-family in the SPAM domain, antibody against one member cross-reacted to the other MSP3 family members. Importantly, these “cross-reactive” human antibodies inhibited parasite growth in antibody dependent cellular inhibition (ADCI) assays [18]. However, the inhibition was attributed to the “cross-reactive” antibodies, so it is still not known if antibodies that specifically bind to PfMSPDBL1 contributed to this inhibition. In this study, we have investigated the effects of specific antibodies against PfMSPDBL1 protein on erythrocyte invasion by merozoites using growth inhibition assays.

Section snippets

Recombinant plasmid construction for protein expression

All recombinant plasmids were constructed using pEU plasmids that are designed specifically for the wheat germ cell-free protein expression system (CellFree Sciences, Matsuyama, Japan) [19]. The nucleotide sequence of the cloned inserts were confirmed using ABI PRISM® 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA). A gene encoding full length [FL, amino acid positions (aa) 26–697] without N-terminal signal peptide of the PfMSPDBL1 (Fig. 1A) and a gene fragment encoding PfMSP1

Synthesis of recombinant PfMSPDBL1 proteins using a wheat germ cell-free system

We designed two recombinant proteins, i.e., rFL and rSPAM of PfMSPDBL1 (Fig. 1A) and expressed them using the wheat germ cell-free system. Fig. 1C shows the recombinant proteins resolved in a 12.5% SDS-polyacrylamide gel. Almost all of the recombinant PfMSPDBL1 proteins were recovered in the soluble fraction and easily purified as a single dominant band (Fig. 1C, arrows) by affinity chromatography. The yields of purified rFL and rSPAM proteins were 73 and 62 μg/6.0 ml of the reaction mixture,

Discussion

The striking feature of the PfMSPDBL1 protein is the presence of both DBL and SPAM domains. The well-characterized merozoite proteins with DBL domains, such as EBA175, are known to play crucial roles in erythrocyte invasion [7]. On the other hand, the proteins with SPAM domains, such as MSP3, are targets of protective immune response [31], [32]. The aim of this study was to investigate the effects of antibodies against PfMSPDBL1 protein, which has both DBL and SPAM, on merozoite invasion of

Acknowledgments

We are grateful to Guy A. Schiehser, David Jacobus, and Walter Reed Army Institute of Research for the drug WR99210. We thank Thangavelu U. Arumugam for the critical reading of the manuscript and Daisuke Ito for the technical assistance. We also thank the Japanese Red Cross Society for providing us the human erythrocytes and human plasma. AGM is an ARC Australian Research Fellow. The work performed at WEHI was made possible through Victorian State Government Operational Infrastructure Support

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  • Cited by (0)

    1

    Present address: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.

    2

    Present address: Mahidol Vivax Research Center, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

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