Routine in vitro culture of P. falciparum gametocytes to evaluate novel transmission-blocking interventions

Abstract

The prevention of parasite transmission from the human host to the mosquito has been recognized as a vital tool for malaria eradication campaigns. However, transmission-blocking antimalarial drug and/or vaccine discovery and development is currently hampered by the expense and difficulty of producing mature Plasmodium falciparum gametocytes in vitro-the parasite stage responsible for mosquito infection. Current protocols for P. falciparum gametocyte culture usually require complex parasite synchronization and addition of stimulating and/or inhibitory factors, and they may not have demonstrated the essential property of mosquito infectivity. This protocol details all the steps required for reliable P. falciparum gametocyte production and highlights common factors that influence culture success. The protocol can be completed in 15 d, and particular emphasis is placed upon operating a gametocyte culture facility on a continuous cycle. In addition, we show how functionally viable gametocytes can be used to evaluate transmission-blocking drugs both in a field setting and at high throughput (HTP) for drug discovery.

New developments in anti-malarial target candidate and product profiles

“…Over the last 4 years, the ability to generate enough gametocytes to allow primary screening of large compound collections has been realized [108, 146–148]. Full characterization of compounds across a range of in vitro activities, such as exflagellation (male gamete formation), female gamete formation or oocyst inhibition provides additional insight.…”

Culture adaptation of malaria parasites selects for convergent loss-of-function mutants

“…Increased asexual growth can also result from a reduction in resources committed towards sexual stage gametocyte development, so a potential role in gametocytogenesis should also be considered for Epac and the ApiAP2 genes identified here (apart from AP2-G for which this function is already known). To address this experimentally, assays need to be developed to reliably phenotype gametocytogenesis responses to relevant induction treatments in culture, as processes previously used have been crude and experimentally variable31.…”

Novel lead structures with both Plasmodium falciparum gametocytocidal and asexual blood stage activity identified from high throughput compound screening

“…This deficit is partly due to difficulty in producing P. falciparum gametocytes in culture, a process that takes at least 12–14 days with very limited yield [8]. This hurdle limits the capacity of malaria gametocytes for compound screening even with the recent development of several high throughput assays [9–13]. Consequently, only limited compound collections have been screened, including two screens of FDA approved drugs collections [14, 15], several screens of MMV Malaria Box library [16], and additionally, three relatively large scale screens of ~10,000 molecules [9, 16, 17].…”