Gametocyte Surface Antigens Table 2. Associations between relative recognition of Pfs48/45 sand Pfs230 antigens, and the presence of antibodies against either GSA or asexual parasite surface antigens, carriage of gametocytes at presentation and transmission-blocking immunity in 65 plasma from Gambian children. Mean difference in relative recognition (95% CI)* Antibodies to Pfs48/45 Antibodies to Mature gametocytes (Stage V) Antibodies to Asexual stages Gametocyte carriage at presentation Transmission blocking activity ** 0.533 (20.780–1.85) P = 0.420 0.875 (20.059–1.81) P = 0.066 0.740 (20.171–1.65) P = 0.110 0.56 (20.757–1.88) P = 0.393 Antibodies to Pfs230 1.47 (0.429–2.51) P = 0.006 1.18 (0.433–1.93) P = 0.003 0.754 (20.006–1.51) P = 0.052 1.47 (0.364–2.58) P = 0.011 *Relative recognition is a ratio, and has no units (see Materials & Methods); mean difference can be positive or negative. If the 95% CI is wholly positive, then the patient plasma indicated had a significantly higher mean fluorescence in the ELISA, whereas a wholly negative 95% CI would indicate significantly lower mean fluorescence in the ELISA. P value from 2-sided t-test of Student. **Data from samples collected in 2000 and infecting at least one mosquito are included (N = 35; see Table 1). doi:10.1371/journal.pone.0002280.t002 the presence of anti-GSA plasma IgG was associated with significantly lower gametocyte densities at day 28 (ratio of means 0.139, 95% C.I. 0.037–0.526; P = 0.004). This was not seen among the 67 children contributing to this analysis who received CQ/AS or AL (ratio of means 0.643, 95% C.I. 0.008–50.5; P = 0.84). Thus, in our studies at least, artemisinin treatment is a more important determinant of gametocyte persistence than the presence of anti-GSA antibodies. Gametocyte densities at day 28 were significantly higher among children receiving CQ, SP or CQ/SP with anti-asexual parasite antibodies present in the blood sample donated for DMFA (ratio of means 29.7 , 95% C.I. 5.08– 174; P ,0.001; N = 101), whereas this was not the case in children treated with ACT. Discussion In this study of malaria patients identified as gametocyte carriers, we have demonstrated that erythrocytes infected with mature gametocytes of P. falciparum carry surface antigens that are recognised by naturally-elicited antibody responses. Our results provide four lines of evidence that these GSA are distinct from surface antigens expressed by the asexual stages of cultured 3D7 parasites. Firstly, serum from a Dutch adult with a previously demonstrated high titre of transmission-blocking antibodies (T. Bousema, P. Schneider and R. Sauerwein, personal communication) strongly recognised the surface of erythrocytes infected with stage V gametocytes, but did not recognise the surface of erythrocytes infected with 3D7 asexual parasites (Fig. 2). Secondly, among the 200 plasma from Gambian donor children, carriage of IgG that recognised GSA was not associated with recognition of the surface of erythrocytes harbouring 3D7 asexual parasites. Thirdly, anti-GSA antibodies were signifi- cantly more common in plasma samples taken on days 10 or 14 after treatment, compared to day 7 plasma, whereas anti-asexual parasite antibodies were found only in day 7 samples. Finally, among children treated with non-artemisinin therapy, the presence of anti- GSA antibodies in day 7, 10 or 14 plasma was significantly associated with lower gametocyte densities at day 28, whereas anti- asexual parasite antibodies were associated with significantly higher gametocyte densities at day 28. These observations need to be interpreted in the light of the unique dynamics of gametocyte emergence in P. falciparum infections, where in successfully treated individuals there is a delayed peak of peripheral gametocytaemia approximately 7 days after clearance of asexual parasites[40], whereas the infectiousness of circulating gametocytes to mosquitoes is highest at 10–14 days [25]. Where patent or sub-patent persistence of asexual parasites occurs, gametocyte carriage is significantly extended [41], and therefore the length of time an individual is exposed to viable circulating gametocytes, and thus likely to acquire antibodies recognising GSA, will differ according to treatment outcome, and to the intrinsic anti-gametocyte effects of the treatment received. Plasma antibodies that block transmission of P. falciparum and P. vivax to mosquitoes were first described several decades ago, and are currently of interest for the development of transmission- blocking vaccines that target gamete antigens [42]. We measured transmission-blocking activity in our series of Gambian blood donors by DMFA [23], and found no association with recognition of GSA. However, the DMFA has several drawbacks for identification of transmission-blocking activity compared to the standardised membrane feeding assay, SMFA, [27], most notably that blood-fed mosquito cages in which no transmission occur are not informative. Therefore it would be useful to have our panel of plasma also tested in the SMFA. One of the unexpected findings of our study is that none of the sera or plasma tested recognised the surface of erythrocytes harbouring immature stages of 3D7 gametocytes. Serum antibod- ies from a Dutch adult with known transmission-blocking immunity were found to recognise the surface of erythrocytes infected with Stage V gametocytes that were demonstrably competent for exflagellation , but not of those infected with immature developing gametocytes. Similarly, none of the Gambian plasma recognised immature gametocytes. Therefore it appears that the GSA recognised by the antibodies we have described are distinct from the putative ligands responsible for sequestration of immature gametocytes during their development [20,34]. Possible explanations for this apparent contradiction include, firstly, that the adhesins of immature gametocytes are highly polymorphic and strain-specific, and those of clone 3D7 were not recognised by immune sera from The Gambia. Secondly, it may be that gametocyte adhesins are cryptic or non- immunogenic molecules, or there is a deficiency in the 3D7 clone that prevents the normal expression of gametocyte adhesins in vitro. Finally it remains possible that sequestration of immature gametocytes in vivo does not require the expression of adhesins on the erythrocyte surface. Experiments with gametocytes derived from parasites from a variety of genetic backgrounds are needed to test these possibilities, and to determine whether our observations made with the 3D7 clone are generally applicable. The identity of the GSA we have described remains unknown, and we cannot dismiss the possibility that altered host membrane proteins are targets of the recognition we have observed. There is an association between parasite-encoded STEVOR proteins and the erythrocyte membrane in late stage cultured gametocytes of the 3D7 line [34], and a subset of these, or of the related rifin PLoS ONE | www.plosone.org 6 May 2008 | Volume 3 | Issue 5 | e2280