A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax

Abstract

Currently, there are no reliable red blood cells invasion assays to guide the discovery of vaccines against Plasmodium vivax, the most prevalent malaria parasite in Asia and South America. Here we describe a protocol for an ex vivo P. vivax invasion assay that can be easily deployed in laboratories located in endemic countries. The assay is based on mixing enriched cord blood reticulocytes with matured, trypsin treated P. vivax schizonts concentrated from clinical isolates. The reliability of this assay was demonstrated using a large panel of P. vivax isolates freshly collected from patients in Thailand.

Host Erythrocyte Environment Influences the Localization of Exported Protein 2, an Essential Component of the Plasmodium Translocon

“…This may be partly explained by the limited conservation of known proteins associated with the P. falciparum trafficking machinery in other Plasmodium species (58). In addition, there has been only limited success in setting up short-term culture systems to support such basic studies of P. vivax (57,(59)(60)(61). However, it has not been shown if the transport machinery utilized by P. vivax is unique or if the reticulocyte cytoplasm influences export.…”

Invasion-Inhibitory Antibodies Elicited by Immunization with Plasmodium vivax Apical Membrane Antigen-1 Expressed in Pichia pastoris Yeast

“…The first portion was smeared onto glass slides, air dried, and fixed with cold acetone for 15 min and stored at Ϫ20°C until needed (These were used in the "Indirect fluorescence assay" section above). The remainder of the schizont concentrate was then utilized in a P. vivax invasion assay that utilized reticulocytes enriched from one isolate of human cord blood (37). In addition to the treatment (AMA-1 plus Quil A [1:100]) and untreated control, it is vital to use the positive control (25 g/ml of antibody 2C3, a monoclonal antibody against the Duffy antigen receptor [DARC]), which almost always blocks P. vivax invasion in this isolate (a kind gift from Yves Colin and Olivier Bertrand, INSERM UMR-S665 and Institut National de la Transfusion Sanguine, Paris, France).…”

Plasmodium vivax: restricted tropism and rapid remodeling of CD71-positive reticulocytes

“…We excluded CD71 as a receptor for P vivax invasion because in assays conducted in the presence of anti-CD71 monoclonal antibodies or with trypsinized reticulocytes (CD71 is trypsin-sensitive), the invasion efficacy was not altered. 18 The experiments described rely on cell sorting, are expensive, and provide limited quantities of reticulocytes. We remedied this obstacle by immunomagnetic sorting of Percoll-enriched reticulocytes labeled with anti-CD71 antibodies, yielding relatively large volumes of CD71 2 and CD71 1 fractions.…”

“…These 3 erythrocytic subsets were then used in a standardized P vivax reinvasion assay modified to use cryopreserved isolates and magnetically enriched schizonts. 18 The schizont preparation was added to equal numbers of the RBC fractions sorted as described previously. After an incubation period of 24 hours, the newly infected RBCs were quantified by fluorescent and light microscopy ( Figure 1B).…”

“…16 Indeed, the use of fresh and frozen fetal reticulocytes isolated from cord blood has allowed the development of reliable P vivax invasion assays. [17][18][19] To define the fine scale tropism of P vivax, we conducted ex vivo invasion assays on highly purified reticulocyte subsets (defined by TO staining and CD71 expression) isolated from cord blood. We further documented the remodeling of the reticulocyte following the invasion by the parasite.…”