Abstract
BackgroundAn understanding of the mechanisms mediating protective immunity against malaria in humans is currently lacking, but critically important to advance the development of highly efficacious vaccines. Antibodies play a key role in acquired immunity, but the functional basis for their protective effect remains unclear. Furthermore, there is a strong need for immune correlates of protection against malaria to guide vaccine development.MethodsUsing a validated assay to measure opsonic phagocytosis of Plasmodium falciparum merozoites, we investigated the potential role of this functional activity in human immunity against clinical episodes of malaria in two independent cohorts (n = 109 and n = 287) experiencing differing levels of malaria transmission and evaluated its potential as a correlate of protection.ResultsAntibodies promoting opsonic phagocytosis of merozoites were cytophilic immunoglobulins (IgG1 and IgG3), induced monocyte activation and production of pro-inflammatory cytokines, and were directed against major merozoite surface proteins (MSPs). Consistent with protective immunity in humans, opsonizing antibodies were acquired with increasing age and malaria exposure, were boosted on re-infection, and levels were related to malaria transmission intensity. Opsonic phagocytosis was strongly associated with a reduced risk of clinical malaria in longitudinal studies in children with current or recent infections. In contrast, antibodies to the merozoite surface in standard immunoassays, or growth-inhibitory antibodies, were not significantly associated with protection. In multivariate analyses including several antibody responses, opsonic phagocytosis remained significantly associated with protection against malaria, highlighting its potential as a correlate of immunity. Furthermore, we demonstrate that human antibodies against MSP2 and MSP3 that are strongly associated with protection in this population are effective in opsonic phagocytosis of merozoites, providing a functional link between these antigen-specific responses and protection for the first time.ConclusionsOpsonic phagocytosis of merozoites appears to be an important mechanism contributing to protective immunity in humans. The opsonic phagocytosis assay appears to be a strong correlate of protection against malaria, a valuable biomarker of immunity, and provides a much-needed new tool for assessing responses to blood-stage malaria vaccines and measuring immunity in populations.
Highlights
An understanding of the mechanisms mediating protective immunity against malaria in humans is currently lacking, but critically important to advance the development of highly efficacious vaccines
We demonstrated that antibody-mediated opsonic phagocytosis: (1) was specific to the immunoglobulin G (IgG) fraction of malaria-exposed sera in a dose-dependent manner; (2) was inhibited by pre-treatment of THP-1 cells with cytochalasin D, which is a known inhibitor of macrophage and monocyte phagocytosis; (3) was comparably quantified using either THP-1 cells or freshly isolated human peripheral blood mononuclear cells (PBMC); and (4) robustly measured the internalization of merozoites into phago-lysosomes, as demonstrated by staining merozoites with the pHdependent dye pHrodoTM, which only becomes fluorescent in the acid environment of phago-lysosomes (Figure 1A-D)
Opsonic phagocytosis was optimally detected at 10 minutes [see Additional file 1: Figure S1], and our opsonic phagocytosis assays (OPA) gave results that were reproducible over a wide range of phagocytic activity [see Additional file 1: Figure S2]; we demonstrated activity using merozoites purified from several different P. falciparum isolates
Summary
An understanding of the mechanisms mediating protective immunity against malaria in humans is currently lacking, but critically important to advance the development of highly efficacious vaccines. Antibodies play a key role in acquired immunity, but the functional basis for their protective effect remains unclear. There is a strong need for immune correlates of protection against malaria to guide vaccine development. Knowledge of the mechanisms that mediate protective immunity against Plasmodium falciparum malaria in humans is currently very limited, and this has been a major barrier to vaccine development. The mechanisms mediating protective humoral immunity and the key targets of functional antibodies remain poorly understood. There is a lack of strong immune correlates of protective immunity for use in vaccine development and population monitoring in malaria control programs [9]. A neutrophil-based antibody-dependent respiratory burst (ADRB) assay was shown to be a correlate of acquired immunity in two endemic populations in Senegal, but these findings have not as yet been reproduced in other cohort studies in Africa [14]
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