The VSA expressed on the surface of unselected 3D7-and HB3-IEs were significantly better recognized by plasma IgG from P. falciparum -exposed donors of both sexes than by IgG from nonexposed donors, whereas levels in exposed men and multigravidae were not significantly different from each other. Furthermore, IE surface-reactive IgG levels did not depend on parity (Table 1 and Figure 1a ). Thus, unselected 3D7 and HB3 both showed typical non-PAM-type plasma antibody recognition patterns of IE surface-expressed VSA. This was confirmed by the nonreactivity of 3D7-and HB3-IEs with all eight VSA PAM -specific monoclonal antibodies (Figure 1d ). When VSA expression was re-assessed after three rounds of selection (about 6 weeks after the first round of selection), the recognition of 3D7-and HB3-IEs selected on either PAM1·4 or PAM8·1 all showed an indeterminate VSA phenotype, where one but not both criteria for sex-specific antibody recognition were met (Table 1 and Figure 1b ). PAM8·1-selected 3D7 remained nonreactive with the four monoclonal antibodies used for testing at this time (PAM1·4, PAM3·10, PAM4·7, and PAM8·1), whereas the other three parasite lines showed reactivity with at least one of them (Figure 1e ). These results suggested that further rounds of selection might lead to definite VSA PAM expression, at least for PAM1·4-selected 3D7, PAM1·4-and PAM8·1-selected HB3. Indeed, PAM1·4-selected 3D7, as well as PAM1·4-and PAM8·1-selected HB3 had all acquired a typical sex-specific and parity dependent VSA PAM expression pattern after four additional rounds of selection (Table 1 and Figure 1c ), and reacted with all the monoclonal antibodies except the VAR2CSA DBL5-ε -specific PAM4·7 (Figure 1f ). In contrast, 3D7 selected seven times for reactivity with PAM8·1 retained an indeterminate sex-specificity pattern also seen after three rounds of selection, did not acquire the parity-dependent pattern typical of VSA PAMexpressing lines (Table 1) , and did not react with any of the eight VSA PAM -specific monoclonal antibodies (Figure 1f ). Thus, PAM8·1 could not be used to select 3D7 for VSA PAM reactivity, consistent with the absence of the predicted epitope for this antibody in the VAR2CSA DBL3-X domain of this parasite (3).