title: A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals.
abstract: BACKGROUND
  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, 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.
  
  
  METHODS
  Fixed 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.
  
  
  RESULTS
  Fixation, 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).
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Institute of Tropical Medicine, University of Tübingen, Wilhelmstraße 27, Tübingen D-72074, Germany.
authors.forename: Anthony
authors.initials: A
authors.lastname: Ajua
authors.forename: Thomas
authors.initials: T
authors.lastname: Engleitner
authors.forename: Meral
authors.initials: M
authors.lastname: Esen
authors.forename: Michael
authors.initials: M
authors.lastname: Theisen
authors.forename: Saadou
authors.initials: S
authors.lastname: Issifou
authors.forename: Benjamin
authors.initials: B
authors.lastname: Mordmüller
mesh_terms: D000328:Adult; D000465:Algorithms; D000913:Antibodies, Protozoan / Q000097:blood*; D002675:Child, Preschool; D004311:Double-Blind Method; D004912:Erythrocytes / Q000276:immunology / Q000469:parasitology; D005434:Flow Cytometry / Q000379:methods* / Q000706:statistics & numerical data; D005455:Fluorescent Antibody Technique / Q000379:methods / Q000706:statistics & numerical data; D005681:Gabon; D006801:Humans; D007223:Infant; D017780:Malaria Vaccines / Q000008:administration & dosage; D016778:Malaria, Falciparum / Q000276:immunology* / Q000517:prevention & control; D010963:Plasmodium falciparum / Q000276:immunology*
chemical_list: D000913:Antibodies, Protozoan; D017780:Malaria Vaccines