Multiplexed Microsphere-Based Flow Cytometric Assay to Assess Strain Transcending Antibodies to Plasmodium vivax Duffy Binding Protein II Reveals an Efficient Tool to Identify Binding-Inhibitory Antibody Responders.

Jéssica R S Alves,Francis B Ntumngia,Andréa Teixeira-Carvalho,Fernanda F De Araújo,Camilla V Pires,John H Adams,Flora S Kano,Letícia M Torres,Barbara A S Lima,Luzia H Carvalho

doi:10.3389/fimmu.2021.704653

Abstract

Malaria remains a major public health problem worldwide, and Plasmodium vivax is the most widely distributed malaria parasite. Naturally acquired binding inhibitory antibodies (BIAbs) to region II of the Duffy binding protein (DBPII), a P. vivax ligand that is critical for reticulocyte invasion, are associated with a reduced risk of clinical malaria. Owing to methodological issues in evaluating antibodies that inhibit the DBPII–DARC interaction, a limited number of studies have investigated DBPII BIAbs in P. vivax-exposed populations. Based on the assumption that individuals with a consistent BIAb response are characterized by strain-transcending immune responses, we hypothesized that detecting broadly reactive DBPII antibodies would indicate the presence of BIAb response. By taking advantage of an engineered DBPII immunogen targeting conserved DBPII neutralizing epitopes (DEKnull-2), we standardized a multiplex flow cytometry-based serological assay to detect broadly neutralizing IgG antibodies. For this study, a standard in vitro cytoadherence assay with COS-7 cells expressing DBPII was used to test for DBPII BIAb response in long-term P. vivax-exposed Amazonian individuals. Taken together, the results demonstrate that this DBPII-based multiplex assay facilitates identifying DBPII BIAb carriers. Of relevance, the ability of the multiplex assay to identify BIAb responders was highly accurate when the positivity for all antigens was considered. In conclusion, the standardized DBPII-based flow cytometric assay confirmed that DBPII-BIAb activity was associated with the breadth rather than the magnitude of anti-DBPII antibodies. Altogether, our results suggest that multiplex detection of broadly DBPII-reactive antibodies facilitates preliminary screening of BIAb responders.

Highlights

In recent years, we have witnessed remarkably increased levels of global funding to combat malaria, which resulted in a significant reduction in the burden of the disease [1]
BIAb activity (>90% of inhibition in the COS-7 cells) and ELISAdetected antibodies; each positive pool included four individual samples; (ii) DBPII-negative pools from malaria endemic area, which were selected from individuals living in an endemic area (Amazon Basin) but with no detectable DBPII antibody response; and (iii) DBPII-negative pools from individuals living in a nonendemic area of malaria (Belo Horizonte, Minas Gerais, Brazil) and who have never been exposed to malaria transmission
As recombinant DBPII proteins were covalently bound to beads, we further assessed the availability of surface-exposed epitopes using two DBPII-specific monoclonal antibodies (mAbs), including one that recognizes DBPII conformational inhibitory epitopes

Summary

Introduction

We have witnessed remarkably increased levels of global funding to combat malaria, which resulted in a significant reduction in the burden of the disease [1]. Among the same population, it is possible to find a small portion of a stable strain-transcending DBPII inhibitory response [15, 17,18,19], which is associated with the presence of BIAbs [20] and reduced risk of P. vivax clinical malaria [19, 21, 22]. These findings suggest that a broadly reactive DBPII inhibitory response should be pursued in all DBPII-based vaccine strategies. We recently demonstrated that a second-generation engineered DBPII immunogen lacking most variant strain-specific epitopes (termed DEKnull-2) retained good immunogenicity and induced a broadly reactive BIAb response [20]

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Conclusion