In order to test the

In order to test the effect of anti-merozoite SCR7 in complement-mediated RBC invasion we used two types of antibodies, an anti-merozoite mouse monoclonal (mAb5.2) and polyclonal IgG from MSP142 vaccines (Otsyula et al., 2013). Use of mAb5.2 resulted in enhancement of RBC invasion above that of FS alone. 3min HIS eliminated this enhancement and addition of both C2 and fB was required for rescue. Because C2 is an integral part of the CP and fB of the AP amplification loop, these data provide strong evidence that both of these pathways of complement activation play a role in antibody-mediated enhancement of RBC invasion. The involvement of the CP is not surprising given its reliance on antibodies. However, antibodies are also known to play an important role in stabilizing the nascent AP convertase (Lutz and Jelezarova, 2006). In addition, the AP serves as the main amplification loop for the majority of complement deposition even when the CP is the initial source of complement activation (Harboe et al., 2004; Harboe and Mollnes, 2008). The important role of C3 was further demonstrated by the effect of the C3 specific inhibitor compstatin. Use of this inhibitor eliminated antibody-mediated enhancement of invasion (Fig. 2b). Although compstatin had no effect on enhancement by FS alone, we cannot rule out that other complement factors such as C1q, MBL, or C4 also play a role here. Our experiments using single factor depleted and reconstituted heterologous serum (Fig. S8) suggest that heterologous serum is not as effective as autologous FS in producing enhancement of invasion. Possible reasons include the presence of minor incompatibilities between serum and RBCs or residual effects from the method of depletion. Thus, further studies should be done with single factor-depleted autologous serum. Enhancement of invasion has been described with the use of antibodies that block other antibodies that inhibit the processing of MSP1 (Guevara Patino et al., 1997). However, the mechanism that we describe here is clearly different. In an effort to validate the relevance of antibody and complement-mediated enhancement of RBC invasion in humans we purified total IgG from serum from MSP142 vaccine recipients collected two weeks after three doses of vaccine (Otsyula et al., 2013). Due to the limited amounts of sample obtained we were unable to carry out extensive assays. Nonetheless, we measured the RBC invasion inhibitory activity of total IgG in C3/C4-inactivated or reconstituted serum. Consistent with previous results (Otsyula et al., 2013), we found very little inhibitory activity in total IgG. However, unlike IgG from non-immunized controls, IgG from vaccine recipients showed reversal of inhibitory activity and enhancement of RBC invasion upon reconstitution of serum with C3/C4. Thus, these results demonstrate that enhancement of RBC invasion can occur with human anti-merozoite antibodies. Contrary to our data, Boyle et al. (Boyle et al., 2015) recently reported that complement activation enhances the inhibitory activity of human anti-merozoite antibodies. They determined that the critical inhibitory step was fixation of C1q on the merozoite surface. One major difference between the approach of these investigators and ours is their use of filter-purified merozoites as opposed to allowing merozoites to naturally egress from schizonts. Consequently, we carried out invasion assays with filter-purified merozoites as described by Boyle et al. (Boyle et al., 2015). We showed that filtered-purified merozoites appear to be highly defective and more sensitive to complement activation than naturally egressed merozoites (Fig. S15). To demonstrate the role of CR1 in the process of complement-mediated invasion we used sCR1 as a competitor. We observed that in the presence of sCR1 there was not only complete reversal of antibody-mediated enhancement in FS but significant amount of inhibition was observed in comparison to the isotype (Fig. 3). The inhibitory activity of sCR1 may be attributed to its diverse functions. Firstly, sCR1 competes for binding sites with erythrocyte surface CR1, which may potentially form large C3b-sCR1 complexes on the merozoite surface and induce steric hindrance of ligand receptor interactions in the presence of these complexes. Secondly, sCR1 is able to bind to PfRh4 on the merozoite surface thereby inhibiting the direct interaction of erythrocyte surface CR1 with PfRh4. Thirdly, sCR1 possesses complement regulatory activity by destabilizing membrane assembled C3 convertases through its intrinsic decay accelerating activity and by acting as a cofactor for Factor I, which breaks down C3b into iC3b. The role of CR1 in complement-dependent invasion was further supported by our co-localization studies that clearly show aggregation of CR1 at the site of merozoite attachment but only in the presence of C3.