Abstract
BackgroundAntibodies play a central role in naturally acquired immunity against Plasmodium falciparum. Current assays to detect anti-plasmodial antibodies against native antigens within their cellular context are prone to bias and cannot be automated, although they provide important information about natural exposure and vaccine immunogenicity. A novel, cytometry-based workflow for quantitative detection of anti-plasmodial antibodies in human serum is presented.MethodsFixed red blood cells (RBCs), infected with late stages of P. falciparum were utilized to detect malaria-specific antibodies by flow cytometry with subsequent automated data analysis. Available methods for data-driven analysis of cytometry data were assessed and a new overlap subtraction algorithm (OSA) based on open source software was developed. The complete workflow was evaluated using sera from two GMZ2 malaria vaccine trials in semi-immune adults and pre-school children residing in a malaria endemic area.ResultsFixation, permeabilization, and staining of infected RBCs were adapted for best operation in flow cytometry. As asexual blood-stage vaccine candidates are designed to induce antibody patterns similar to those in semi-immune adults, serial dilutions of sera from heavily exposed individuals were compared to naïve controls to determine optimal antibody dilutions. To eliminate investigator effects introduced by manual gating, a non-biased algorithm (OSA) for data-driven gating was developed. OSA-derived results correlated well with those obtained by manual gating (r between 0.79 and 0.99) and outperformed other model-driven gating methods. Bland-Altman plots confirmed the agreement of manual gating and OSA-derived results. A 1.33-fold increase (p=0.003) in the number of positive cells after vaccination in a subgroup of pre-school children vaccinated with 100 μg GMZ2 was present and in vaccinated adults from the same region we measured a baseline-corrected 1.23-fold, vaccine-induced increase in mean fluorescence intensity of positive cells (p=0.03).ConclusionsThe current workflow advances detection and quantification of anti-plasmodial antibodies through improvement of a bias-prone, low-throughput to an unbiased, semi-automated, scalable method. In conclusion, this work presents a novel method for immunofluorescence assays in malaria research.
Highlights
Antibodies play a central role in naturally acquired immunity against Plasmodium falciparum
Malaria is a major cause of morbidity and mortality in endemic countries with African children carrying the major burden of the disease
It is hypothesized that antiplasmodial Ab concentrations similar to those acquired upon natural exposure are required to attain semiimmunity, a type of non-sterile but robust immunity that protects from clinical complications and excessive parasite replication [1,3]
Summary
Antibodies play a central role in naturally acquired immunity against Plasmodium falciparum. Current assays to detect anti-plasmodial antibodies against native antigens within their cellular context are prone to bias and cannot be automated, they provide important information about natural exposure and vaccine immunogenicity. It is hypothesized that antiplasmodial Ab concentrations similar to those acquired upon natural exposure are required to attain semiimmunity, a type of non-sterile but robust immunity that protects from clinical complications and excessive parasite replication [1,3]. The main evidence for the role of Abs in semi-immunity comes from studies where purified Abs from African malaria-immune adults were successfully used to treat non-immune malaria patients [4,5] within Africa or, as an extension of this, in SouthEast Asia [5]. The mechanisms, properties, and specificities of Abs that mediate protection in malaria, remain unknown [3]
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