Measuring naturally acquired immune responses to candidate malaria vaccine antigens in Ghanaian adults

Daniel Dodoo,Harini Geneshan,Ben Gyan,Susan Adu-Amankwah,Sanjai Kumar,Esteban Abot,Dorothy Anum,Glenna Banania,Martha Sedegah,Jennylyn Legano,Donald Brambilla,Renato Sayo,Denise L Doolan ,Thomas L Richie ,Kwadwo A Koram ,Michael R Hollingdale ,William O Rogers ,Joseph Ocran ,Bartholomew D Akanmori ,Judith E Epstein

doi:10.1186/1475-2875-10-168

Abstract

BackgroundTo prepare field sites for malaria vaccine trials, it is important to determine baseline antibody and T cell responses to candidate malaria vaccine antigens. Assessing T cell responses is especially challenging, given genetic restriction, low responses observed in endemic areas, their variability over time, potential suppression by parasitaemia and the intrinsic variability of the assays.MethodsIn Part A of this study, antibody titres were measured in adults from urban and rural communities in Ghana to recombinant Plasmodium falciparum CSP, SSP2/TRAP, LSA1, EXP1, MSP1, MSP3 and EBA175 by ELISA, and to sporozoites and infected erythrocytes by IFA. Positive ELISA responses were determined using two methods. T cell responses to defined CD8 or CD4 T cell epitopes from CSP, SSP2/TRAP, LSA1 and EXP1 were measured by ex vivo IFN-γ ELISpot assays using HLA-matched Class I- and DR-restricted synthetic peptides. In Part B, the reproducibility of the ELISpot assay to CSP and AMA1 was measured by repeating assays of individual samples using peptide pools and low, medium or high stringency criteria for defining positive responses, and by comparing samples collected two weeks apart.ResultsIn Part A, positive antibody responses varied widely from 17%-100%, according to the antigen and statistical method, with blood stage antigens showing more frequent and higher magnitude responses. ELISA titres were higher in rural subjects, while IFA titres and the frequencies and magnitudes of ex vivo ELISpot activities were similar in both communities. DR-restricted peptides showed stronger responses than Class I-restricted peptides. In Part B, the most stringent statistical criteria gave the fewest, and the least stringent the most positive responses, with reproducibility slightly higher using the least stringent method when assays were repeated. Results varied significantly between the two-week time-points for many participants.ConclusionsAll participants were positive for at least one malaria protein by ELISA, with results dependent on the criteria for positivity. Likewise, ELISpot responses varied among participants, but were relatively reproducible by the three methods tested, especially the least stringent, when assays were repeated. However, results often differed between samples taken two weeks apart, indicating significant biological variability over short intervals.

Highlights

To prepare field sites for malaria vaccine trials, it is important to determine baseline antibody and T cell responses to candidate malaria vaccine antigens
immunofluorescence antibody assays (IFA) positivity has generally been defined by titres equal to or higher than the dilution of control sera not giving a positive immunofluorescence with the test antigen[4,5], while enzyme linked immunosorbent assays (ELISA) positivity has generally been defined as titres greater than the mean + 3 standard deviations (SD) of the negative control samples ("classical approach”)
Sera were tested in IFA and ELISA

Summary

Introduction

To prepare field sites for malaria vaccine trials, it is important to determine baseline antibody and T cell responses to candidate malaria vaccine antigens. Defining background responses is useful for planning vaccine trials in endemic areas, due to the need to distinguish vaccine-induced responses from the baseline of naturally acquired responses once the vaccine is administered. Knowing this baseline and comparing immune responses post immunization with the responses obtained in malaria-naïves helps to assess whether naturally-acquired responses can be boosted by the candidate malaria vaccine. It has been pointed out that there are problems associated with the classical approach when negative and positive samples are not well separated and the background levels of controls are variable; in this case, a latent class model may better estimate the proportion of positive samples [6]. Because the “the classical method” yielded many positive samples due to the small SD of negative controls, a second, more stringent method was applied, in which a sample was deemed positive if it met criteria for the first method and simultaneously showed an optical density (OD) ≥ 0.5 at a dilution of >1/100

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