Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing

Jun-Hong Ch’Ng,Maria Del Pilar Quintana,Ulrika Qundos,Madle Sirel,Mats Wahlgren,Ingmarie Nilsson,Sherwin Chun Leung Chan,Kirsten Moll,Arash Zandian,Asa Tellgren-Roth,Peter Nilsson

doi:10.1038/srep43190

Abstract

Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.

Highlights

Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications
The function of repetitive interspersed families of polypeptides (RIFINs) in blood group A-specific rosetting has generated much interest concerning the importance of malaria in shaping geographical blood group profiles[1]
RIFINs have been classified into two subgroups, with 70% belonging to subgroup A (A-RIFINs), possessing a 25 amino acid insertion-deletion region, and 30% to subgroup B (B-RIFINs) which lack an indel[3]

Summary

Introduction

Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. We make use of ultra-dense peptide arrays to examine the specificities of anti-RIFIN IgG preparations, test the functionality of these antibodies in different antibody-based assays, and follow up with RNA sequencing (RNAseq) to determine RIFIN expression in laboratory-adapted parasite lines.

Results

Conclusion