Development Of Antibody Dependent Respiratory Burst Assay As A Tool For Evaluating Cellular Immunity To Malaria

ABSTRACT

Malaria vaccine clinical trials in exposed populations have not always been consistent in

finding robust associations between the predicted immune responses and protection

against disease. The selection of most promising vaccine candidates have been based on

direct antibody inhibition assays that have performed dismally. This suggests that such

immune correlates employed in assessing their efficacies may not be surrogate markers of

protection. Immunity to asexual blood stage malaria is complex and likely involves

multiple mechanisms. IgG antibodies are thought to play a critical role and a

corresponding reliable in vitro correlate of antibody-mediated cellular immunity has long

been sought to facilitate malaria vaccine development. The aim of present work was to

establish an effector cell antibody-dependent respiratory burst assay as a correlate of

naturally acquired immunity that may be a useful tool to evaluate vaccine-induced

immunity in malaria studies. The specific objectives of the study were: to determine if

polymorphonuclear neutrophils (PMNs) and monocytes could be triggered by malaria

merozoites to produce reactive oxygen species (ROS); to determine the minimum number

of merozoites required for the trigger; to determine the reproducibility of the assay, to

determine the applicability of the assay by testing if the blood stage malaria vaccine

candidates MSP–1, MSP–2 and AMA–1 elicited antibodies that trigger effector cell

respiratory burst, and to associate total antibody titres to respiratory burst activity after

vaccination. This study adopted an experimental design where the characteristics of the

antibody mediated effector-cell-dependent respiratory burst induction were established

and the key variables determining the performance of the assay evaluated. Merozoites

opsonised with either malaria hyper immune IgG from Africa or IgGs raised from

vaccinations with either MSP-1, MSP-2 or AMA-1 were incubated with

PMNs/monocytes and production of ROS determined by isoluminol-amplified

chemiluminesence in triplicate assays. ELISA assays were performed alongside to

determine if total antibody titres elicited by vaccination had any association with the

magnitudes of ROS measured. Results show that monocytes (fresh or cryopreserved) and

PMNs are effective at respiratory burst induction of ROS. At equivalent cell numbers,

PMNs exhibited higher ROS production than autologous monocytes (p