family may be recognised by anti-GSA antibodies. Other possible protein targets of anti-GSA antibodies include members of the PfEMP1 family, as a subset of var genes are known to be transcribed in gametocytes [37], and the LCCL-domain protein pSLAP, which is associated with the plasma membrane of erythrocytes infected with late stage P. falciparum gametocytes [43]. Future work will employ recombinant forms of candidate GSA, and specific antibodies raised against these reagents, to identify the key molecules involved in this natural immune response to the transmissible form of P. falciparum. The identity of the antigens recognised on the surface of erythrocytes infected with asexual parasites in our experiments is also unclear. In the absence of phenotypic selection using anti- VSA antibodies or adhesion panning, 3D7 cultures express a surface phenotype characterised by relatively low level CD36- dependent endothelial adhesion, and these parasites are poorly recognised by plasma antibodies from semi-immune children [44]. This explains the lack of association between the age of our plasma donors and recognition of the surface of erythrocytes infected with asexual parasites. Therefore it is possible that asexual parasites with a surface antigen phenotype dominated by the highly recognised Type A var genes, such as the antibody-selcted 3D7- Dodowa1 line [11], may have been more commonly recognised by plasma antibodies from children who recognised GSA. What we can say with confidence is that mature 3D7 gametocytes in our synchronised cultures expressed molecules on the host erythrocyte surface that were not found on erythrocytes harbouring 3D7 asexual parasites from the same cultures. A subset of our test plasma (those collected in 2000) were analysed for recognition of recombinant forms of the gamete antigens Pfs48/45 and Pfs230. Recognition of the latter, but not the former, was significantly associated with antibodies recognising both GSA and asexual antigens in the flow cytometry assay, and with transmission-blocking activity in the DFMA. This is partially consistent with previous work, which has shown an association between the presence of antibodies recognising these two gamete antigens with transmission-blocking activity in the SMFA [14,15]. In most studies, prevalence of antibodies to Pfs230 increases with age and exposure [38], but it is unknown whether acquisition of antibodies to GSA may follow a similar pattern. An exploratory analysis suggests that the presence of anti-GSA antibodies 1 to 2 weeks after treatment of clinical malaria with non- ACT regimens are associated with lower gametocyte densities References 1. Epstein JE, Giersing B, Mullen G, Moorthy V, Richie TL (2007) Malaria vaccines: are we getting closer? Curr Opin Mol Ther. 9: 12–24. 2. Roberts DJ, Craig AG, Berendt AR, Pinches R, Nash G, et al. (1992) Rapid switching to multiple antigenic and adhesive phenotypes in malaria. Nature 357: 647–648. 3. Brannan LR, Turner CM, Phillips RS (1994) Malaria parasites undergo antigenic variation at high rates in vivo. Proc Biol Sci 256: 71–75. 4. Baruch DI, Pasloske BL, Singh HB, Bi X, Ma XC, et al. (1995) Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 82: 77–87. 5. Smith JD, Chitnis CE, Craig AG, Roberts DJ, Hudson-Taylor DE, et al. (1995) Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82: 101–110. 6. Marsh K, Otoo L, Hayes R, Carson D, Greenwood B (1989) Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection. Trans R Soc Trop Med Hyg 83: 293–303. 7. Bull PC, Lowe BS, Kortok M, Molyneux CS, Newbold CI, et al. (1998) Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med 4: 358–360. 8. Giha HA, Staalsoe T, Dodoo D (2000) Antibodies to variable Plasmodium falciparum infected erythrocyte surface antigens are associated with protection from novel malaria infections. Immunol Lett 71: 117–126. Gametocyte Surface Antigens 4 weeks after treatment. Interestingly, anti-asexual parasite antibod- ies were associated with higher day 28 gametocyte densities in this same group of treated children, suggesting that in these individuals sub-patent asexual parasitaemia may be persisting, giving rise to both antibody responses and subsequent gametocytaemia [41]. The role of anti-GSA antibodies in controlling gametocytaemia in vivo needs to be further investigated in longitudinal studies of gametocyte carriage, but it does appear from results presented here that such an effect may only be significant in the absence of ACT treatment. Further longitudinal studies and cross-sectional serological surveys of anti- GSA antibodies in children with and without gametocytaemia are needed to further test the association between these antibodies and gametocyte carriage. Our results present the first, albeit partial, picture of the immune response to GSA. The antibodies that recognise GSA are more common in older children, and are distinct from immune responses directed against asexual parasite surface antigens in our unselected cultures. Further, parasitological follow-up data suggest anti-GSA antibodies are associated with ability to control gametocyte densities in vivo. We propose that the generation of vaccine-elicited immunity targeting GSA may represent a feasible new strategy aimed at blocking the transmission of P. falciparum in endemic settings, and thus further epidemiological and biological studies of anti-GSA responses are needed. Acknowledgments We are indebted to P. Schneider, J.T. Bousema and R. Sauerwein at the Unversity of Nijmegen, the Netherlands, for helpful discussions and for providing previously characterised transmission-blocking serum. We particularly note the crucial contribution of the late J.T. Lensen to this area of investigation. We thank M. Jawara, G. Walraven and M. Pinder at the MRC Laboratories, The Gambia for their substantial contributions to our mosquito infectivity studies at Farafenni. We also extend our thanks to Trine Staalsoe at the Center for Medical Parasitology (Institute for Medical Microbiology and Immunology), University of Copenhagen, for advice on the preparation of samples for analysis by flow cytometry, and for supplying Ghanaian immune sera. Author Contributions Conceived and designed the experiments: CS GT CD MS. Performed the experiments: CD MS WR. Analyzed the data: CS CD MS WR. Contributed reagents/materials/analysis tools: CS GT WR. Wrote the paper: CS GT CD MS WR. 9. Dodoo D, Staalsoe T, Giha H (2001) Antibodies to variant antigens on the surfaces of infected erythrocytes are associated with protection from malaria in Ghanaian children. Infect Immun 69: 3713–3718. 10. Ofori MF, Dodoo D, Staalsoe T (2002) Malaria-induced acquisition of antibodies to Plasmodium falciparum variant surface antigens. Infect Immun 70: 2982–2988. 11. Jensen AT, Magistrado P, Sharp S, Joergensen L, Lavstsen T, et al. (2004) Plasmodium falciparum Associated with Severe Childhood Malaria Preferentially Expresses PfEMP1 Encoded by Group A var Genes. J Exp Med. 199: 1179–90. 12. Mendis KN, Munesinghe YD, de Silva YN, Keragalla I, Carter R (1987) Malaria transmission-blocking immunity induced by natural infections of Plasmodium vivax in humans. Infect Immun 55: 369–372. 13. Graves PM, Wirtz RA, Carter R, Burkot TR, Looker M, et al. (1988) Naturally occurring antibodies to an epitope on Plasmodium falciparum gametes detected by monoclonal antibody-based competitive enzyme-linked immunosorbent assay. Infect Immun 56: 2818–2821. 14. Roeffen W, Geeraedts F, Eling W, Beckers P, Wizel B, et al. (1995) Transmission blockade of Plasmodium falciparum malaria by anti-Pfs230-specific antibodies is isotype dependent. Infect Immun 63: 467–471. 15. Roeffen W, Mulder B, Teelen K, Bolmer M, Eling W, et al. (1996) Association between anti-Pfs48/45 reactivity and P. falciparum transmission-blocking activity in sera from Cameroon. Parasite Immunol 182: 103–109. 16. Boudin C, Olivier M, Molez JF, Chiron JP, Ambroise-Thomas P (1993) High human malarial infectivity to laboratory-bred Anopheles gambiae in a village in Burkina Faso. Am J Trop Med Hyg 48: 700–706. PLoS ONE | www.plosone.org 7 May 2008 | Volume 3 | Issue 5 | e2280