Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient.

BACKGROUND: Plasmodium falciparum zygotes develop in the mosquito midgut after an infectious blood meal containing mature male and female gametocytes. Studies of mosquito-produced P. falciparum zygotes to elucidate their biology and development have been hampered by high levels of contaminating mosquito proteins and macromolecules present in zygote preparations. Thus, no zygote-specific surface markers have been identified to date. Here, a methodology is developed to obtain large quantities of highly purified zygotes using in vitro culture, including purification methods that include magnetic column cell separation (MACS) followed by Percoll density gradient centrifugation. This straightforward and effective approach provides ample material for studies to enhance understanding of zygote biology and identify novel zygote surface marker candidates that can be tested as transmission blocking vaccine (TBV) candidates. METHODS: Plasmodium falciparum gametocyte cultures were established and maintained from asexual cultures. Gametocytes were matured for 14 days, then transferred into zygote media for 6 h at 27 ± 2 °C to promote gamete formation and fertilization. Zygotes were then purified using a combination of MACS column separation and Percoll density gradient centrifugation. Purity of the zygotes was determined through morphological studies: the parasite body and nuclear diameter were measured, and zygotes were further transformed into ookinetes. Immunofluorescence assays (IFA) were also performed using the ookinete surface marker, Pfs28. RESULTS: After stimulation, the culture consisted of transformed zygotes and a large number of uninfected red blood cells (RBCs), as well as infected RBCs with parasites at earlier developmental stages, including gametes, gametocytes, and asexual stages. The use of two MACS columns removed the vast majority of the RBCs and gametocytes. Subsequent use of two Percoll density gradients enabled isolation of a pure population of zygotes. These zygotes transformed into viable ookinetes that expressed Pfs28. CONCLUSION: The combined approach of using two MACS columns and two Percoll density gradients yielded zygotes with very high purity (45-fold enrichment and a pure population of zygotes [approximately 100%]) that was devoid of contamination by other parasite stages and uninfected RBCs. These enriched zygotes, free from earlier parasites stages and mosquito-derived macromolecules, can be used to further elucidate the biology and developmental processes of Plasmodium.

The Development of Whole Sporozoite Vaccines for Plasmodium falciparum Malaria.

Each year malaria kills hundreds of thousands of people and infects hundreds of millions of people despite current control measures. An effective malaria vaccine will likely be necessary to aid in malaria eradication. Vaccination using whole sporozoites provides an increased repertoire of immunogens compared to subunit vaccines across at least two life cycle stages of the parasite, the extracellular sporozoite, and intracellular liver stage. Three potential whole sporozoite vaccine approaches are under development and include genetically attenuated parasites, radiation attenuated sporozoites, and wild-type sporozoites administered in combination with chemoprophylaxis. Pre-clinical and clinical studies have demonstrated whole sporozoite vaccine immunogenicity, including humoral and cellular immunity and a range of vaccine efficacy that depends on the pre-exposure of vaccinated individuals. While whole sporozoite vaccines can provide protection against malaria in some cases, more recent studies in malaria-endemic regions demonstrate the need for improvements. Moreover, challenges remain in manufacturing large quantities of sporozoites for vaccine commercialization. A promising solution to the whole sporozoite manufacturing challenge is in vitro culturing methodology, which has been described for several Plasmodium species, including the major disease-causing human malaria parasite, Plasmodium falciparum. Here, we review whole sporozoite vaccine immunogenicity and in vitro culturing platforms for sporozoite production.

Host Cell Copper Transporters CTR1 and ATP7A are important for Influenza A virus replication.

BACKGROUND: The essential role of copper in eukaryotic cellular physiology is known, but has not been recognized as important in the context of influenza A virus infection. In this study, we investigated the effect of cellular copper on influenza A virus replication. METHODS: Influenza A/WSN/33 (H1N1) virus growth and macromolecule syntheses were assessed in cultured human lung cells (A549) where the copper concentration of the growth medium was modified, or expression of host genes involved in copper homeostasis was targeted by RNA interference. RESULTS: Exogenously increasing copper concentration, or chelating copper, resulted in moderate defects in viral growth. Nucleoprotein (NP) localization, neuraminidase activity assays and transmission electron microscopy did not reveal significant defects in virion assembly, morphology or release under these conditions. However, RNAi knockdown of the high-affinity copper importer CTR1 resulted in significant viral growth defects (7.3-fold reduced titer at 24 hours post-infection, p = 0.04). Knockdown of CTR1 or the trans-Golgi copper transporter ATP7A significantly reduced polymerase activity in a minigenome assay. Both copper transporters were required for authentic viral RNA synthesis and NP and matrix (M1) protein accumulation in the infected cell. CONCLUSIONS: These results demonstrate that intracellular copper regulates the influenza virus life cycle, with potentially distinct mechanisms in specific cellular compartments. These observations provide a new avenue for drug development and studies of influenza virus pathogenesis.