Fig. 4. Immunogenic gametocyte antigens identified by three complementary approaches.

(A) Surface-depleted versus surface-intact uninfected and infected RBC membranes were probed with Malawian plasma samples and naïve U.S. sera by Western blot. Each lane represents protein extract from 2.5 × 10⁶ uninfected RBCs (uRBCs) or iRBCs. Differential band patterns between the +trypsin and −trypsin samples are marked with red arrows. (B) Volcano plot showing human and Plasmodium proteins identified by comparing surface-intact versus surface-depleted giRBC membranes. The x axis represents log₂ fold change of −trypsin/+trypsin, and the y axis shows the t test P value (P < 0.05 corresponds to P = 0.0004 after Benjamin-Hochberg correction) of −trypsin/+trypsin biological replicates (n = 3). Plasmodium proteins with a log₂ fold change of >1.25 are marked in red, and significant Plasmodium proteins across three replicates are marked in blue. (C) Surface-depleted (+trypsin/chymotrypsin) versus surface intact (−trypsin/chymotrypsin) uRBC and giRBC membranes were probed with sera from mice (six per group) immunized with surface-intact or surface-depleted giRBCs by Western blot. Each lane represents protein extract from 2.5 × 10⁶ uRBCs or iRBCs. Differential band patterns between the trypsin(+) and trypsin(−) giRBC samples are shown in red. (D) The array described in Fig. 1 was probed with sera from mice immunized with either surface-depleted or surface-intact giRBC membranes. Responses were normalized to controls and then quantile normalized. (E) PTP6 and GEXP21 differential responses between sera from mice immunized with surface-intact (−trypsin) giRBC membranes and surface-depleted (+trypsin) giRBC membranes. (F) GEXP07 and GEXP10 differential responses from sera from mice immunized with intact and surface-depleted membranes. See table S7 for complete dataset.