In Vitro Culture of Plasmodium falciparum for the Production of Mature Gametocytes for Performing Standard Membrane Feeding Assay and Infection of Anopheles spp.

Plasmodium falciparum is the parasite responsible for the disease malaria. In vitro cultivation of mature gametocytes of P. falciparum plays a central role in evaluating and developing the transmission-blocking drugs and sexual stage vaccines. These types of preventive molecules are crucial for controlling malaria in the future. Among different Plasmodium species that are involved in human malaria, only P. falciparum is cultivable. Therefore, an efficient method is required for in vitro culture of P. falciparum producing mature and infective gametocytes. This chapter describes a reliable and efficient protocol for the production of adult and infective gametocytes that is suitable for small- and large-scale culture.

Characterization of Naturally Acquired Immunity to a Panel of Antigens Expressed in Mature P. falciparum Gametocytes.

Introduction: Naturally acquired immune responses against antigens expressed on the surface of mature gametocytes develop in individuals living in malaria-endemic areas. Evidence suggests that such anti-gametocyte immunity can block the development of the parasite in the mosquito, thus playing a role in interrupting transmission. A better comprehension of naturally acquired immunity to these gametocyte antigens can aid the development of transmission-blocking vaccines and improve our understanding of the human infectious reservoir. Methods: Antigens expressed on the surface of mature gametocytes that had not previously been widely studied for evidence of naturally acquired immunity were identified for protein expression alongside Pfs230-C using either the mammalian HEK293E or the wheat germ cell-free expression systems. Where there was sequence variation in the candidate antigens (3D7 vs a clinical isolate PfKE04), both variants were expressed. ELISA was used to assess antibody responses against these antigens, as well as against crude stage V gametocyte extract (GE) and AMA1 using archived plasma samples from individuals recruited to participate in malaria cohort studies. We analyzed antibody levels (estimated from optical density units using a standardized ELISA) and seroprevalence (defined as antibody levels greater than three standard deviations above the mean levels of a pool of malaria naïve sera). We described the dynamics of antibody responses to these antigens by identifying factors predictive of antibody levels using linear regression models. Results: Of the 25 antigens selected, seven antigens were produced successfully as recombinant proteins, with one variant antigen, giving a total of eight proteins for evaluation. Antibodies to the candidate antigens were detectable in the study population (N = 216), with seroprevalence ranging from 37.0% (95% CI: 30.6%, 43.9%) for PSOP1 to 77.8% (95% CI: 71.6%, 83.1%) for G377 (3D7 variant). Responses to AMA1 and GE were more prevalent than those to the gametocyte proteins at 87.9% (95% CI: 82.8%, 91.9%) and 88.3% (95% CI: 83.1%, 92.4%), respectively. Additionally, both antibody levels and breadth of antibody responses were associated with age and concurrent parasitaemia. Conclusion: Age and concurrent parasitaemia remain important determinants of naturally acquired immunity to gametocyte antigens. Furthermore, we identify novel candidates for transmission-blocking activity evaluation.

Design of antimalarial transmission blocking agents: Pharmacophore mapping of ligands active against stage-V mature gametocytes of Plasmodium falciparum.

The discovery of transmission-blocking (T-B) agents is crucial for preventing and complete removal of malaria infection. However, most of the existing antimalarials are only active against the asexual stages of Plasmodium parasite, but ineffective against the sexual stage (gametocytes). In this background, we have developed pharmacophore models against the stage-V mature gametocytes of P. falciparum parasites. The pharmacophore model (Hypo-1) showed five pharmacophoric features namely, one hydrogen bond donor (HBD), one hydrophobic aliphatic (HYAl), one ring aromatic (RA), and two hydrophobic aromatic (HYAr) essential for the anti-gametocytic activity. The amino, methyl, fused phenyl ring of the quinazoline heterocycle, two phenyl rings of biphenyl moiety (HBD, HYAl, HYAr1, HYAr2 and RA) are the crucial features responsible for the non-specific anti-gametocytic activity (PfG). Subsequently, the model (Hypo-2) developed against the stage-V female gametocytes (PffG) showed the contribution of three pharmacophoric features namely, two hydrogen bond acceptor (HYA) and one RA required for the anti-gametocytic activity. The sulfhydryl, imine and pyridyl groups are observed to be essential for anti-gametocytic activity against female gametocytes. Both the models (PfG and PfGG) showed the classification accuracies of 78.26 and 71.64% for training set compounds and 60.80 and 60.18% for the test set compounds, respectively, for classification of compounds into higher and lower active classes. Also, both the models were found to retain the higher active compounds (IC50 <100 nM) in top 1% of total compounds (actives and decoys) as observed after screening the decoy set compounds. Communicated by Ramaswamy H Sarma.

Insight into phagocytosis of mature sexual (gametocyte) stages of Plasmodium falciparum using a human monocyte cell line.

During natural infection malaria parasites are injected into the bloodstream of a human host by the bite of an infected female Anopheles mosquito. Both asexual and mature sexual stages of Plasmodium circulate in the blood. Asexual forms are responsible for clinical malaria while sexual stages are responsible for continued transmission via the mosquitoes. Immune responses generated against various life cycle stages of the parasite have important roles in resistance to malaria and in reducing malaria transmission. Phagocytosis of free merozoites and erythrocytic asexual stages has been well studied, but very little is known about similar phagocytic clearance of mature sexual stages, which are critical for transmission. We evaluated phagocytic uptake of mature sexual (gametocyte) stage parasites by a human monocyte cell line in the absence of immune sera. We found that intact mature stages do not undergo phagocytosis, unless they are either killed or freed from erythrocytes. In view of this observation, we propose that the inability of mature gametocytes to be phagocytized may actually result in malaria transmission advantage. On the other hand, mature gametocytes that are not transmitted to mosquitoes during infection will eventually die and undergo phagocytosis, initiating immune responses that may have transmission blocking potential. A better understanding of early phagocytic clearance and immune responses to gametocytes may identify additional targets for transmission blocking strategies.