Clinical Protection from Falciparum Malaria Correlates with Neutrophil Respiratory Bursts Induced by Merozoites Opsonized with Human Serum Antibodies

Background: Effective vaccines to combat malaria are urgently needed, but have proved elusive in the absence of validated correlates of natural immunity. Repeated blood stage infections induce antibodies considered to be the main arbiters of protection from pathology, but their essential functions have remained speculative. Methodology/Principal Findings: This study evaluated antibody dependent respiratory burst (ADRB) activity in polymorphonuclear neutrophils (PMN) induced by Plasmodium falciparum merozoites and antibodies in the sera of two different African endemic populations, and investigated its association with naturally acquired clinical protection. Respiratory bursts by freshly isolated PMN were quantified by chemiluminescence readout in the presence of isoluminol, which preferentially detects extra-cellular reactive oxygen species (ROS). Using a standardized, high throughput protocol, 230 sera were analyzed from individuals of all age groups living in meso-(Ndiop) or holo-endemic (Dielmo) Senegalese villages, and enrolled in a cross-sectional prospective study with intensive follow-up. Statistical significance was determined using non-parametric tests and Poisson regression models. The most important finding was that PMN ADRB activity was correlated with acquired clinical protection from malaria in both high and low transmission areas (P = 0.006 and 0.036 respectively). Strikingly, individuals in Dielmo with dichotomized high ADRB indexes were seventeen fold less susceptible to malaria attacks (P = 0.006). Complementary results showed that ADRB activity was (i) dependent on intact merozoites and IgG opsonins, but not parasitized erythrocytes, or complement, (ii) correlated with merozoite specific cytophilic IgG1 and IgG3 antibody titers (P < 0.001 for both), and (iii) stronger in antisera from a holo-endemic compared to a meso-endemic site (P = 0.002), and reduced in asymptomatic carriers (P < 0.001). Conclusions/Significance: This work presents the first clearly demonstrated functional antibody immune correlate of clinical protection from Plasmodium falciparum malaria, and begs the question regarding the importance of ADRB by PMN for immune protection against malaria in vivo

Relationship between Antibody Levels, IgG Binding to Plasmodium falciparum-Infected Erythrocytes, and Disease Outcome in Hospitalized Urban Malaria Patients from Dakar, Sénégal

Background. Management of clinical malaria requires the development of reliable diagnostic methods and efficient biomarkers for follow-up of patients. Protection is partly based on IgG responses to parasite antigens exposed at the surface of infected erythrocytes (iRBCs). These IgG responses appeared low during clinical infection, particularly in severe disease. Methods. We analyzed the IgG binding capacity to the surface of live erythrocytes infected by knob positive FCR3 strain. Sera from 69 cerebral malaria (CM) and 72 mild malaria (MM) cases were analyzed by ELISA for IgG responses to five antigens from iRBC and by flow cytometry for IgG binding as expressed in labeling index ratio (LIR). The relationship between IgG levels, LIR, parasitemia, age, and the clinical outcomes was evaluated. Results. We found a significant decrease of LIR in adult CM fatal cases compared to surviving patients (p=0.019). In MM, LIRs were correlated to IgG anti-iRBC and anti-PfEMP3/5 levels. In CM, no correlation was found between LIR, IgG levels, and parasitemia. Conclusion. The IgG binding assay was able to discriminate outcome of cerebral malaria cases and it deserves further development as a potential functional-associated assay for symptomatic malaria analysis

ADRB chemiluminescence profiles can be standardized.

<p>(A-C) Chemiluminescence profiles from 3 different ADRB experiments (3 PMN pools) tested without serum (blank, green trace), or in the presence of NIS (grey trace), HIS (red trace) and the same individual sample serum (S1, blue trace). The ADRB index for S1, calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009871#s2" target="_blank">Materials and Methods</a> using HIS as a positive internal standard, is shown for each experiment.</p

Comparison of ADRB index profiles for Dielmo and Ndiop.

<p>ADRB index values for Dielmo and Ndiop were analyzed as a function of parasite carriage at the time of sampling. Statistical significance was determined using the Mann-Whitney test and significant <i>P</i> values are noted.</p

Dichotomized ADRB indexes have an age-dependent impact on clinical episodes at both low and high transmission sites.

<p>Analyses of associations between ADRB index and the number of clinical episodes experienced during a 5.5-month follow-up period, gave the best Poisson regression model with a cut-off of 250. The cumulative mean no. of clinical episodes/person/day at monthly intervals across the follow-up period were plotted for different age groups as a function of ADRB index <250 (blue trace) or ≥250 (red trace). (A) Ndiop: age groups: <15 years, 15–29 years, and ≥30 years (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009871#pone-0009871-t001" target="_blank">Table 1</a>); (B) Dielmo: age groups: <7 years, 7–14 years, ≥15 years (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009871#pone-0009871-t003" target="_blank">Table 3</a>).</p

Poisson regression analysis of anti-merozoite IgG levels and clinical outcome for non-parasitized Ndiop inhabitants.

<p>Bivariate and multivariate Poisson regression models were built to analyze the relation between anti-merozoite IgG levels and acute malaria morbidity. The bivariate model includes morbidity and analysis of one variable. The multivariate model includes morbidity and other variables such as age and IgG levels, where the impact of the different variables is analyzed together. The <i>P</i> value in parenthesis refers to the overall impact of age on the multivariate model. (NS: non significant.)</p

PMN respiratory burst activity depends on merozoites and immune antibodies.

<p>ADRB indexes determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009871#s2" target="_blank">Materials and Methods</a> using a single pool of PMN: with immune (HIS) or non-immune (NIS) sera only (blue); with merozoites and immune (HIS, S2, S3, or S4) or non-immune (NIS, S5, S6) sera (green) or the same with fMLP activated PMN (yellow); with normal (light red), parasitized (dark red) RBC, or lysed parasitized RBC (3x freeze-thawed) (grey) in the presence of immune or non-immune sera.</p

Serum IgG, but not complement, mediates ADRB activity.

<p>ADRB indexes were determined individually using 7 different immune sera either untreated (lane 1), after inactivation of complement (lane 2), after IgG depletion (lane 3), or after purification of IgG (lane 4). Distribution and median values are indicated by vertical and horizontal lines respectively.</p