Improvement of the antibody-dependent respiratory burst assay for assessing protective immune responses to malaria

Abstract

The antibody-dependent respiratory burst (ADRB) assay is a sensitive isoluminol-based chemiluminescence (CL) functional assay designed to assess the capacity of opsonizing antibodies against merozoites to induce neutrophil respiratory burst. ADRB was shown to measure protective immunity against malaria in endemic areas, but the assay needed further improvement to ensure better sensitivity and reproducibility. Here, we adjusted parameters such as the freezing-thawing procedure of merozoites, merozoites's concentration and the buffer solution's pH, and we used the improved assay to measure ADRB activity of 207 sera from 97 and 110 individuals living, respectively, in Dielmo and Ndiop villages with differing malaria endemicity. The improvement led to increased CL intensity and assay sensitivity, and a higher reproducibility. In both areas, ADRB activity correlated with malaria endemicity and individual's age discriminated groups with and without clinical malaria episodes, and significantly correlated with in vivo clinical protection from Plasmodium falciparum malaria. Our results demonstrate that the improved ADRB assay can be valuably used to assess acquired immunity during monitoring by control programmes and/or clinical trials.

Keywords: Plasmodium falciparum; functional assay; malaria; merozoites; neutrophils; respiratory burst.

Figure 1.

Effect of freezing method of merozoites on chemiluminescence intensity of ADRB Chemiluminescence intensity profile of neutrophils antibody-dependent respiratory burst activity performed with merozoites frozen at −20°C (a) and merozoites frozen in glycerolyte (b). 40 µl merozoites were opsonized with 10 µl standard positive control sera (HIS) and unopsonized with negative controls (blank and NIS) before activate 100 µl neutrophils (1.5 × 10⁶ cells ml⁻¹).

Figure 2.

Effect of various concentrations of merozoites on chemiluminescence intensity of antibody-dependent respiratory burst activity. Chemiluminescence profiles response of various concentrations of merozoites opsonized with 10 µl of hyper-immune serum (HIS, red circle line) and unopsonized with 10 µl of non-immune serum (NIS, blue solid line) or without addition of serum (blank, green dash line) before activate 100 µl human neutrophils (1.5 × 10⁶ cells ml⁻¹).

Figure 3.

Determination of the optimal concentration of merozoite required to assess neutrophils ADRB activity. The effects of different concentrations of merozoites decreasing (70 to 30 × 10⁶ mz ml⁻¹) on chemiluminescence intensity and on the signal-to-noise ratio (HIS/Blank, yellow line) were assessed at pH 7.0 and at constant temperature (37°C). Each point represents the average of RLU max and the average of signal-to-noise ratio obtained in three different ADRB experiments on three consecutive days. Best signal-to-noise ratio was obtained with the concentration of 50 × 10⁶ mz ml⁻¹.

Figure 4.

Effect of the increase of pH on chemiluminescence intensity of neutrophil antibody-dependent respiratory burst activity and on the signal strength of assay. (a) Chemiluminescence profiles response of neutrophil antibody-dependent respiratory burst activity (using a same PMNs pool of 1.5 × 10⁶ cells ml⁻¹ and 50.10⁶ mz ml⁻¹) performed at pH 7.0 and pH 8.0, in the presence of hyper-immune serum (HIS, red solid line) and non-immune serum (NIS, blue trace) and without addition of serum (blank, green trace). (b) Enhancement of the signal strength of ADRB assay to pH 8.0.

Figure 5.

Schematic representation of the antibody-dependent respiratory burst assay procedure optimized.

Figure 6.

ADRB activity as a function of the endemic level of malaria. ADRB indexes of sera from villagers living in a holoendemic area (Dielmo, n = 97) and in a mesoendemic area (Ndiop, n = 110) were calculated as described in the Material and methods section using HIS as a positive internal standard in presence of negative controls (NIS). The statistics for difference of mean ADRB responses between sera from high (Dielmo) and low (Ndiop) malaria transmission are given: p < 0.0001, Mann–Whitney test. The significance was determined at p < 0.05.

Figure 7.

ADRB activity as a function of age group. Mean ADRB indexes of sera from Dielmo and Ndiop by age groups. Age of villagers was categorized according to parasitological and clinical data gathered throughout the longitudinal study of the Dielmo–Ndiop project into three age groups: 3–6, 7–15, and greater than or equal to 15 years for Dielmo, and 4–14, 15–30, and greater than or equal to 30 years for Ndiop. The mean of ADRB increased with age (p = 0.01 and 0.04 respectively for Dielmo and Ndiop). The non-parametric Kruskal–Wallis test was used for statistical analysis with a significance determined at p < 0.05.

Figure 8.

ADRB activity as function of occurrence of malaria episodes during the follow-up period (a) Mean ADRB indexes of sera from Dielmo (n = 97) and Ndiop (n = 110) in individuals group with no malaria episode and in those who experienced at least one malaria episode. In both study areas, the mean ADRB indexes were significantly higher in the ‘no malaria episode’ group compared with the ‘malaria episodes’ group. The non-parametric Wilcoxon test was used for statistical analysis with a significance determined at p < 0.05. (b) Mean ADRB indexes of sera from Dielmo and Ndiop for each group of malaria attacks episodes subdivided according to the number of malaria attacks into: ‘no malaria episode’, ‘one malaria episode’ and ‘multiple malaria episodes’. There was no significant difference in the protective association between the three groups in Ndiop. Statistically significant differences were determined by the non-parametric Kruskal–Wallis test. The significance was determined at p < 0.05.