Acquisition of Growth-Inhibitory Antibodies against Blood-Stage Plasmodium falciparum Fiona J. McCallum 1,2 , Kristina E. M. Persson 1 , Cleopatra K. Mugyenyi 3 , Freya J. I. Fowkes 1 , Julie A. Simpson 4 , Jack S. Richards 1,2 , Thomas N. Williams 3 , Kevin Marsh 3 , James G. Beeson 1 * 1 The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, 2 Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia, 3 Kenya Medical Research Institute, CGMRC/Wellcome Trust Collaborative Program, Kilifi, Kenya, 4 Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, University of Melbourne, Carlton, Victoria, Australia Abstract Background: Antibodies that inhibit the growth of blood-stage Plasmodium falciparum may play an important role in acquired and vaccine-induced immunity in humans. However, the acquisition and activity of these antibodies is not well understood. Methods: We tested dialysed serum and purified immunoglobulins from Kenyan children and adults for inhibition of P. falciparum blood-stage growth in vitro using different parasite lines. Serum antibodies were measured by ELISA to blood- stage parasite antigens, extracted from P. falciparum schizonts, and to recombinant merozoite surface protein 1 (42 kDa C- terminal fragment, MSP1-42). Results: Antibodies to blood-stage antigens present in schizont protein extract and to recombinant MSP1-42 significantly increased with age and were highly correlated. In contrast, growth-inhibitory activity was not strongly associated with age and tended to decline marginally with increasing age and exposure, with young children demonstrating the highest inhibitory activity. Comparison of growth-inhibitory activity among samples collected from the same population at different time points suggested that malaria transmission intensity influenced the level of growth-inhibitory antibodies. Antibodies to recombinant MSP1-42 were not associated with growth inhibition and high immunoglobulin G levels were poorly predictive of inhibitory activity. The level of inhibitory activity against different isolates varied. Conclusions: Children can acquire growth-inhibitory antibodies at a young age, but once they are acquired they do not appear to be boosted by on-going exposure. Inhibitory antibodies may play a role in protection from early childhood malaria. Citation: McCallum FJ, Persson KEM, Mugyenyi CK, Fowkes FJI, Simpson JA, et al. (2008) Acquisition of Growth-Inhibitory Antibodies against Blood-Stage Plasmodium falciparum. PLoS ONE 3(10): e3571. doi:10.1371/journal.pone.0003571 Editor: Colin J. Sutherland, London School of Hygiene & Tropical Medicine, United Kingdom Received July 16, 2008; Accepted October 7, 2008; Published October 29, 2008 Copyright: ß 2008 McCallum et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: National Health and Medical Research Council of Australia (Project grant and Career Development Award to J. Beeson; Postgraduate Research Fellowships to F. McCallum and J. Richards), the Welcome Trust (Project grant to K. Marsh and J. Beeson; Program grant to K. Marsh; Fellowships to K. Marsh and T. Williams), the Miller Fellowship of the Walter and Eliza Hall Institute (to J. Beeson), the Teggerstiftelsen, Maud and Birger Gustavssons Stiftelse (K. E. M. Persson) and Wenner-Gren Fellowship (K. E. M Persson). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: beeson@wehi.edu.au Introduction Plasmodium falciparum malaria is a leading cause of childhood mortality, with around 1 million deaths annually [1]. In malaria- endemic areas effective immunity against malaria develops after repeated exposure that limits blood-stage parasitemia and prevents symptomatic illness and severe complications [2]. Antibodies that inhibit blood stage replication of P. falciparum are believed to be important in mediating both acquired immunity and immunity generated by candidate blood-stage vaccines [3,4,5]. Serum antibodies that inhibit parasite growth in vitro have been isolated from clinically immune individuals, but are rarely detected in malaria-naı ¨ve individuals [3,5]. Early studies suggest that these ‘‘growth-inhibitory’’ antibodies are acquired in an isolate-specific manner following convalescence from acute infection [6]. However, the role of growth-inhibitory antibodies in protection from clinical disease in humans, or their function in vivo, has not been established. Furthermore, there is limited knowledge of the acquisition of these antibodies and it is unclear whether inhibitory antibodies can be acquired quickly after limited exposure or instead require repeated exposure over an extended period. Inhibitory antibodies are thought to mainly act by inhibiting erythrocyte invasion through targeting merozoite surface antigens and invasion ligands [7,8]. Data from animal studies support an important role for antibodies against merozoite antigens in immunity [9,10]. Merozoite surface protein 1 (MSP1), apical membrane antigen 1 (AMA1), erythrocyte binding antigens (EBAs) and P. falciparum reticulocyte-binding homologues (PfRh proteins) are implicated as important targets of acquired human inhibitory antibodies [11,12,13,14], and polyclonal and monoclonal anti- bodies produced in experimental animals against these antigens can inhibit erythrocyte invasion in vitro [7,10,12,15,16,17]. Studies PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3571