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  • Boyle and Beeson argue that a recent report by

    2018-11-09

    Boyle and Beeson argue that a recent report by Sack et al. () using late arresting genetically attenuated parasites (LAGAP) supports the role of stavudine and complement in controlling parasitemia. In this study, the authors report that immunization with LAGAP induces antibodies against blood stage parasites. Use of cobra venom factor (CVF) resulted in decreased efficacy of immunization. Importantly, CVF is a complement activator which will result in widespread C3 activation and opsonization with C3b. In our hands, CVF results in increased parasitemia and we suspect this effect is most likely due to complement activation and not to complement depletion. In order to study the role of complement activation it is best to use genetically-modified animals that are deficient in complement factors, as we did in our study. Further, the passive transfer experiments were done with serum, not with purified antibody, using relatively high amounts per animal (300μl on day 0, 3, and 5 post-challenge). Use of purified antibody and titration experiments would have been more informative as to the role of antibodies in this system. As mentioned by Boyle and Beeson, and as discussed in our paper (), Taylor et al. () reported that C1q-deficient mice had a minimal increase in parasitemia upon secondary challenge with . We have not tested the system. One possible explanation is that complement activation does play a role in suppressing parasitemia in that system. However, an alternative explanation is that C1q may act as a signaling ligand for phagocytosis by macrophages () and that this finding may not be related to downstream complement activation. Boyle and Beeson state that other studies have failed to see an increase in invasion when using serum and cite three reports (). The studies by Campbell et al. and Chulay et al. were carried out with grown in human RBCs exposed to serum from immune and non-immune monkeys. In these cases, there is a confounding effect of incompatibility between Aotus serum and human RBCs that makes interpretation difficult. Kennedy et al. did not measure parasitemia directly but measured parasite LDH as a surrogate. In addition, they allowed the parasites to grow for 32–36h as opposed to overnight cultures in our studies. Finally, Boyle and Beeson argue that if antibodies against MSP1 were to increase invasion, trials of MSP1-based vaccines would have observed increased parasitemias in vaccinated individuals. We feel this is a misinterpretation of our findings. The final outcome of a vaccine, whether efficacy (inhibition of the pathogen), enhancement, or neither, will depend on the repertoire of antibodies that are produced and on their target antigens. Our studies have demonstrated that it is possible for -merozoite antibodies to enhance RBC invasion and this mechanism can counteract the efficacy of inhibitory antibodies. Thus, if a vaccine is producing both inhibitory antibodies and antibodies that enhance invasion, the net effect could be efficacy (inhibition), no efficacy, or an enhancing effect, depending on the relative amounts of each of the types of antibodies produced. Hence, the lack of enhancement effect in vaccine trials, does not exclude the possibility that some antibodies actually enhance. The use of filter-purified merozoites has been championed by Boyle and Beeson and the vast majority of the published studies utilizing this merozoite purification method have come from their laboratory or close collaborators. By their own admission, their assay is “more sensitive than standard growth inhibition assays” () which could be interpreted as saying that the merozoites are more susceptible to inhibition than naturally egressed merozoites (Figure 3C and 3D, ()). We propose that the increased susceptibility to inhibition by antibodies is due to damage from excessive manipulation during the purification process. In addition to filtration, depending on the protocol used by Boyle and Beeson, this could also include an additional purification step through magnetic columns to remove hemozoin. The damage to the merozoites is reflected in the fact that an extremely high ratio of purified merozoites to RBCs (~6:1) is needed to attain a parasitemia of ~1.8% after 24/48h (Figure S5B, ()). Assuming that 16 merozoites egress from one schizont, this merozoite to RBC ratio is equivalent to incubating target RBCs with a starting parasitemia of ~27.3% (3 infected RBCs for 8 uninfected RBCs). By contrast, our invasion assays can easily achieve a parasitemia of 8% or higher with a starting parasitemia of 0.5 to 1%.