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Abstract
The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field.
Highlights
The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest
The antibody-dependent respiratory burst (ADRB) assay assesses the ability of anti-merozoite antibodies to activate neutrophils/polymorphonuclear cells (PMNs) via Fc receptors (FcR) to release reactive oxygen species (ROS), a mechanism distinct from assays which assess antibody-induced neutrophil phagocytosis of either merozoites or iRBCs13,21–23
We have recently reported that the ADRB assay does not associate with protection against P. yoelii rodent malaria[24], ADRB activity has been strongly associated with a reduction in P. falciparum clinical disease in naturally-exposed individuals in Senegal[19] lending support to the utility of a reproducible, standardized protocol for use by the malaria research community
Summary
The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. We optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. In the case of Plasmodium falciparum malaria, over 200 million people are infected each year leading to approximately 0.6 million deaths[2,3] Despite this burden of disease, the immunological mechanisms which confer protection in vivo in humans remain highly debated and poorly understood[4], and vaccine development strategies often suffer from a lack of informed immunological guidance. We have recently reported that the ADRB assay does not associate with protection against P. yoelii rodent malaria[24], ADRB activity has been strongly associated with a reduction in P. falciparum clinical disease in naturally-exposed individuals in Senegal[19] lending support to the utility of a reproducible, standardized protocol for use by the malaria research community. ADRB activity is a plausible mediator of protection against P. falciparum malaria, supporting the reported association with clinical protection[19]
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