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Fulltext 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

Muh F, Lee SK, Hoque MR, Han JH, Park JH, Firdaus ER, et al.

Malar J, 2018 Jul 27;17(1):272.
PMID: 30049277 DOI: 10.1186/s12936-018-2420-4

BACKGROUND: The 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.
METHODS: In 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.
RESULTS: PkDBPα 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.
CONCLUSION: These 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.
Fulltext De Novo Assembly of Plasmodium knowlesi Genomes From Clinical Samples Explains the Counterintuitive Intrachromosomal Organization of Variant SICAvar and kir Multiple Gene Family Members

Oresegun DR, Thorpe P, Benavente ED, Campino S, Muh F, Moon RW, et al.

Front Genet, 2022;13:855052.
PMID: 35677565 DOI: 10.3389/fgene.2022.855052

Plasmodium knowlesi, a malaria parasite of Old World macaque monkeys, is used extensively to model Plasmodium biology. Recently, P. knowlesi was found in the human population of Southeast Asia, particularly Malaysia. P. knowlesi causes uncomplicated to severe and fatal malaria in the human host with features in common with the more prevalent and virulent malaria caused by Plasmodium falciparum. As such, P. knowlesi presents a unique opportunity to develop experimental translational model systems for malaria pathophysiology informed by clinical data from same-species human infections. Experimental lines of P. knowlesi represent well-characterized genetically stable parasites, and to maximize their utility as a backdrop for understanding malaria pathophysiology, genetically diverse contemporary clinical isolates, essentially wild-type, require comparable characterization. The Oxford Nanopore PCR-free long-read sequencing platform was used to sequence and de novo assemble P. knowlesi genomes from frozen clinical samples. The sequencing platform and assembly pipelines were designed to facilitate capturing data and describing, for the first time, P. knowlesi schizont-infected cell agglutination (SICA) var and Knowlesi-Interspersed Repeats (kir) multiple gene families in parasites acquired from nature. The SICAvar gene family members code for antigenically variant proteins analogous to the virulence-associated P. falciparum erythrocyte membrane protein (PfEMP1) multiple var gene family. Evidence presented here suggests that the SICAvar family members have arisen through a process of gene duplication, selection pressure, and variation. Highly evolving genes including PfEMP1family members tend to be restricted to relatively unstable sub-telomeric regions that drive change with core genes protected in genetically stable intrachromosomal locations. The comparable SICAvar and kir gene family members are counter-intuitively located across chromosomes. Here, we demonstrate that, in contrast to conserved core genes, SICAvar and kir genes occupy otherwise gene-sparse chromosomal locations that accommodate rapid evolution and change. The novel methods presented here offer the malaria research community not only new tools to generate comprehensive genome sequence data from small clinical samples but also new insight into the complexity of clinically important real-world parasites.
Fulltext Cross-species analysis of apical asparagine-rich protein of Plasmodium vivax and Plasmodium knowlesi

Muh F, Ahmed MA, Han JH, Nyunt MH, Lee SK, Lau YL, et al.

Sci Rep, 2018 04 10;8(1):5781.
PMID: 29636493 DOI: 10.1038/s41598-018-23728-1

The Plasmodium falciparum apical asparagine (Asn)-rich protein (AARP) is one of malarial proteins, and it has been studied as a candidate of malaria subunit vaccine. Basic characterization of PvAARP has been performed with a focus on its immunogenicity and localization. In this study, we further analyzed the immunogenicity of PvAARP, focusing on the longevity of the antibody response, cross-species immunity and invasion inhibitory activity by using the primate malaria parasite Plasmodium knowlesi. We found that vivax malaria patient sera retained anti-PvAARP antibodies for at least one year without re-infection. Recombinant PvAARP protein was strongly recognized by knowlesi malaria patients. Antibody raised against the P. vivax and P. knowlesi AARP N-termini reacted with the apical side of the P. knowlesi merozoites and inhibited erythrocyte invasion by P. knowlesi in a concentration-dependent manner, thereby suggesting a cross-species nature of anti-PvAARP antibody against PkAARP. These results can be explained by B cell epitopes predicted in conserved surface-exposed regions of the AARP N-terminus in both species. The long-lived anti-PvAARP antibody response, cross-reactivity, and invasion inhibitory activity of anti-PvAARP support a critical role of AARP during the erythrocyte invasion and suggest that PvAARP induces long-lived cross-species protective immunity against P. vivax and P. knowlesi.
Fulltext Cross-species reactivity of antibodies against Plasmodium vivax blood-stage antigens to Plasmodium knowlesi

Muh F, Kim N, Nyunt MH, Firdaus ER, Han JH, Hoque MR, et al.

PLoS Negl Trop Dis, 2020 06;14(6):e0008323.
PMID: 32559186 DOI: 10.1371/journal.pntd.0008323

Malaria is caused by multiple different species of protozoan parasites, and interventions in the pre-elimination phase can lead to drastic changes in the proportion of each species causing malaria. In endemic areas, cross-reactivity may play an important role in the protection and blocking transmission. Thus, successful control of one species could lead to an increase in other parasite species. A few studies have reported cross-reactivity producing cross-immunity, but the extent of cross-reactive, particularly between closely related species, is poorly understood. P. vivax and P. knowlesi are particularly closely related species causing malaria infections in SE Asia, and whilst P. vivax cases are in decline, zoonotic P. knowlesi infections are rising in some areas. In this study, the cross-species reactivity and growth inhibition activity of P. vivax blood-stage antigen-specific antibodies against P. knowlesi parasites were investigated. Bioinformatics analysis, immunofluorescence assay, western blotting, protein microarray, and growth inhibition assay were performed to investigate the cross-reactivity. P. vivax blood-stage antigen-specific antibodies recognized the molecules located on the surface or released from apical organelles of P. knowlesi merozoites. Recombinant P. vivax and P. knowlesi proteins were also recognized by P. knowlesi- and P. vivax-infected patient antibodies, respectively. Immunoglobulin G against P. vivax antigens from both immune animals and human malaria patients inhibited the erythrocyte invasion by P. knowlesi. This study demonstrates that there is extensive cross-reactivity between antibodies against P. vivax to P. knowlesi in the blood stage, and these antibodies can potently inhibit in vitro invasion, highlighting the potential cross-protective immunity in endemic areas.