Antibody recognition of Plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the Brazilian Amazon.

Alessandra Sampaio Bassi Fratus,Stefanie Costa Pinto Lopes,Fabio Trindade Maranhão Costa,Fernanda Janku Cabral,Márcia Melo Medeiros,Wesley Luzetti Fotoran,Rosimeire Dalla Martha,Luiz Hildebrando Pereira Da Silva,Bianca Cechetto Carlos,Gerhard Wunderlich

doi:10.1590/0074-0276140027

Abstract

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Highlights

Human plasma samples from symptomatic and asymptomatic individuals - Human plasma samples were obtained during an epidemiological survey from persons living in the surroundings of Porto Velho, the capital of the state of Rondônia (RO) (Brazil)
Cytoadherent-selected isolates show in part identical var DBLα sequences, but no selection of determined var type subclasses - In order to identify an association between var gene transcription and cytoadherent phenotypes in the Amazonian field isolates tested in this study, we used Reverse transcription (RT)-polymerase chain reaction (PCR) followed by cloning/sequencing of the amplified products
Panning of the parasites on CHO-ICAM, which should ideally result in DBLα distinct sequence identifiers (DSID) type 3 (PF11_0521) expressing parasites, failed to result in such parasites and all parasites panned over this receptor showed only low frequencies of DBLα DSID 1-3 expressing parasites that were previously associated with severe malaria (Bull et al 2005) (Fig. 1)

Summary

Introduction

Human plasma samples from symptomatic and asymptomatic individuals - Human plasma samples were obtained during an epidemiological survey from persons living in the surroundings of Porto Velho, the capital of the state of Rondônia (RO) (Brazil). Parasites and static cytoadherence assay - P. falciparum field isolates S20, 99, 106 and 134 were collected in RO (Albrecht et al 2006). Cytoadherence was measured by the incubation of 5 x 106 trophozoite stage parasites over the cells for 1 h. Reverse transcription (RT)-PCR and sequencing analysis - For determining the dominant var transcript in field isolates and 3D7 parasites, the RNAs of 108 ring stage parasites were recovered from each parasite line using the Trizol® method (Invitrogen). Immunofluorescence - Live parasite immunofluorescence assays were conducted with enriched P. falciparum trophozoites and human plasmas from asymptomatic and symptomatic carriers. Trophozoite/schizont stage parasites were incubated with 40 μg/mL of 4’-6diamidino-2-phenylindole and HCS CellMask in 100 μL RPMI/1% bovine serum albumin for 60 min at 37oC, followed by incubation with plasma (dilution 1:25) and an Alexa Fluor labelled antihuman-IgG antibody (Molecular Probes, 1:200 dilution). We employed the antiglycophorin antibody (1:1,000 dilution) (Molecular Probes) and a secondary antibody, antimouse-IgG 1:500 (Alexa Fluor 594, Molecular Probes)

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