MALARVX'S UNIQUE IN VITRO CULTURE SYSTEM FOR PRODUCTION OF PLASMODIUM MOSQUITO-STAGES
Pf life cycle in the mosquito
In the natural life cycle of Plasmodium falciparum, gametocytes are ingested during a blood meal and transform in the mosquito midgut into gametes that fuse to form a zygote.
The zygote undergoes DNA replication and maturation into an ookinete that traverses the mosquito midgut epithelium and finally embeds into the basal lamina on the midgut periphery. The embedding is thought to trigger transformation of the zygote into the early oocyst (Arrighi and Hurd 2002; Adini and Warburg 1999; Vlachou et al. 2001).
The mosquito midgut supports development of oocysts, leading to the formation of up to several thousand sporozoites.
Once released, sporozoites travel to the salivary glands through the hemocoel and are inoculated into the second host during a blood meal (Sinden 1984).
In vitro Pf ookinetes
We described detailed methodology for in vitro culturing of Pf ookinetes to oocyst mosquito stages, identification of a unique Pf oocyst protein marker (Cap380), and development of an anti-PfCap380 antibody to study both culture-derived (in vitro) and mosquito-derived (in vivo) oocysts.
In vitro Pf zygotes
We described a methodology to obtain large quantities of highly purified zygotes by using in vitro culture together with purification methods including MACS magnetic column purification followed by Percoll density gradient centrifugation.
These straightforward and effective methods provide ample material for studies to further understanding of zygote biology and identify novel zygote surface markers for evaluation in transmission-blocking vaccines.
In vitro Pf oocyst
MalarVx has developed a culture platform that includes a 3-dimensional (3D) matrix to provide an attachment substrate for ookinetes, allowing growth of oocysts to mature sporozoites. In brief, our in vitro culture system proceeds from standard red blood cell culture to induction of gametocytes. Gamete maturation is determined by examining male gamete emergence.
Once this occurs, fertilization is promoted. Zygotes appear within 6 hours and ookinetes after 24 hours. We also optimized the ookinete growth medium and conditions and support their in vitro differentiation into oocysts.
Manuscript: submitted
In vitro Pf sporozoites
To produce sporozoites, ookinetes were purified by MACS column and seeded into a 3D scaffold well. Following oocyst maturation, the first in vitro sporozoites (IVS) are released following the timeframe of in vivo sporozoites development.
MalarVx see this newly IVS system to be a great value to the field of Malaria Research as a substitute to the intensive cost, time, men power and efficiency of running an insectarium facility to produce infected sporozoites.