Abstract:
Malaria is an infectious disease of global health importance with approximately half a million global annual deaths. The sexual forms (gametocytes) are responsible for transmission of the malaria parasite Plasmodium from the human host to the mosquito vector. Despite their importance for transmission and therefore maintenance of the cycle, no safe and highly effective drug nor a transmission blocking vaccine against Plasmodium gametocytes including P. falciparum, the most important plasmodial species, are available. In addition, only little is known about the biology, susceptibility, longevity and recognition by the immune system of these essential stages. In this PhD work, the lifespan of P. falciparum gametocytes and natural antibody response to late-stage gametocytes were systematically investigated. Additionally, susceptibility of mature gametocytes to new compounds was evaluated.
Previously, P. falciparum gametocyte lifespan was estimated in vivo; mostly by mathematical modelling. Here, longevity of gametocytes was measured for the first time in vitro by measuring parasite viability in gametocyte culture for an extended period. It was observed that mature gametocytes can survive a maximum of 16-32 days from the day of maturation (representing the in vivo circulation time) with an average half-life of 2.6 to 6.5 days as evaluated by the different viability assays. In addition, mature male gametocytes were found to be able to exflagellate for two weeks, which shows their viability and sustained potential capability to infect mosquitoes.
In line with this, the naturally acquired antibody (Ab) response towards gametocytes was also explored in clinical settings. As expected, Ab recognition increased after fixation and permeabilization of gametocyte-infected erythrocytes. Ab recognition between the laboratory strain (NF54) and a clinical isolate (JH013) was similar. Adults showed a higher Ab response and sero-prevalence compared to children. A significant lower level of Ab response was observed in adults vaccinated with the malaria vaccine candidate GMZ2 (but not in children) after 84 days compared to baseline. When testing the association between the anti-gametocyte Ab response to the Ab response to whole asexual antigens, a positive correlation was observed in adults but not in children. The evaluation of the effect of intestinal helminths infection showed that children infected with Ascaris had a significantly higher anti-gametocyte Ab response compared to uninfected children.
Additionally, to evaluate the sensitivity of mature gametocytes to new compounds, activity of synthetic dyes was tested against mature gametocytes. Five fluorescent dyes showed activity at submicromolar concentration against P. falciparum mature gametocytes of the culture-adapted 3D7 clone and two clinical isolates. 3D7 was observed to be more sensitive than the clinical isolates. Three mitochondrial dyes (MitoRed, DiOC6, and rhodamine B) were even more active, with 50% inhibitory concentrations (IC50s) below 200nM, than the control dye methylene blue (770nM) that is known to have gametocidal activity. Among the three, MitoRed was most active against 3D7 and clinical isolates, with a 3-fold higher inhibition when incubated for 48h than for 24h. The effect of incubation time on the activity of drugs was also pronounced with the control drugs, epoxomicin and artesunate.
In a different project, the gametocidal effect of a new myxobacterial compound, chlorotonil A, was investigated. Mature gametocytes were sensitive to chlorotonil A at a low nanomolar concentration, which is similar to its activity against asexual blood stage parasites and superior to most other antimalarials in development. This underlines the potential of chlorotonil A as a drug development candidate.
Altogether in this PhD work, it was shown that P. falciparum gametocytes survive in vitro 2-4 weeks after maturation. This provides an estimate of the in vivo circulation time that can be used to model transmission and the effect of transmission-blocking interventions. These interventions could be immunological or drug-mediated. The second set of investigations showed that naturally acquired antibodies against gametocyte-infected erythrocyte surface antigens were affected by vaccination and helminth co-infections. In the third and fourth subproject, screening of compounds showed that mitochondrial fluorescent dyes and chlorotonil A had high inhibitory activity against P. falciparum mature gametocytes and may be developed further.
gametocytes
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