Antibody generation has therefore remained an important goal for blood-stage malaria vaccination efforts. Simple anti-repeat region antibodies correlate fairly well with the protection afforded by the most advanced malaria vaccine candidate to date, GlaxoSmithKline’s RTS,S, but the paradoxically consistent presence of individuals who are protected from challenge with barely detectable antibody responses makes this a less than ideal predictive biomarker [
5] for this sporozoite-stage vaccine. For blood-stage antigens, although high antibody titers can protect against challenge with the cognate antigens [
6], overall titers against the dominant merozoite surface and invasion antigens have correlated poorly with protection from re-infection or disease when measured by simple ELISA [
7], making this assay an unreliable surrogate for protection. To date, the only
in vitro methods to evaluate the functionality of antibodies to the blood-stage parasite are the growth inhibition assay (GIA), which measures the ability of antibodies to slow the expansion of
P. falciparum parasites cultured in human erythrocytes in the incubator [
8], and the antibody-dependent cellular cytotoxicity (ADCC) assay, which has been difficult to generalize to the field [
9]. In their current paper, Osier and colleagues [
10] have described a new assay, the opsonic phagocytosis assay (OPA), taking advantage of the logical necessity that
in vivo, responses include not just the antibodies themselves but the other immune cells with which they interact, thus making this assay more biologically relevant. This new functional assay measures the ability of antibodies to opsonize freshly isolated live merozoites for phagocytosis by macrophages in short-term culture. The authors have carefully characterized this assay, and found that it works with both freshly isolated macrophages within lymphocyte mixtures and with an isolated macrophage cell line. It appears readily reproducible to any laboratory with cell and malaria culture capabilities and a flow cytometer.
The most compelling evidence presented by Osier and colleagues are the correlations within the context of two independent longitudinal cohort studies conducted on the coast in Kenya. The naturally acquired responses measured by this OPA correlated with the ability to resist clinical illness in both groups of children residing in this malaria endemic area. Activity in OPA significantly correlated with IgG ELISA but had a weak correlation with GIA (Spearman’s rho −0.358, P = 0.041). The addition of this novel functional assay as a biomarker seems a logical step towards a better understanding of the various specificities involved in the development of protective immunity to malaria.
Although the correlation between naturally acquired partial protection and the results of OPA is very suggestive, the link predicting protection against malaria induced by a vaccine presenting a limited number of antigens remains untested. It is possible that a vaccine candidate might induce antibodies that perform well in the OPA, yet fail to predict the outcome of a challenge infection, as has been the case with GIA and anti-malarial antibodies in general. Caution in generalizing to the in vivo situation for vaccine development is warranted because of the relatively short transit time (less than 10 minutes) of merozoites between schizont rupture and re-invasion, although the short duration of the assay may functionally mimic this. Because it is an assay that utilizes a whole, living merozoite, its applicability to screen for efficacy of a single-allele, single-antigen vaccine is difficult to determine. Thus, this tool should be championed as a functional assay that, perhaps through future antigen add-back or blocking experiments, may help dissect the diverse antibody responses developing in sequentially exposed individuals to determine which of them are functionally important in resisting disease. This work also sets the stage to develop similar opsonization assays against the infected red blood cell, which is presented to the host immune responses for a considerably longer duration.