Malaria Immunity and Transmission
Malaria continues to be a critical world health problem resulting in 100-200 million clinical cases a year with over 0.5 million deaths, mainly children under the age of five. In the past decade the introduction of insecticide-treated bed nets and Artemisinin combination therapy have reversed the increase in mortality caused by the spread of chloroquine-resistant parasites and led to renewed hope for malaria eradication. Key to eradication efforts is the development of new strategies to control parasite transmission, which is the focus the Williamson lab.The Plasmodium parasite that causes malaria is transmitted from person to person by a mosquito (Fig. 1). Parasites are introduced into the blood stream by the bite of an infected mosquito and then travel to the liver. After development in liver cells, parasites are re-released to the blood stream where they now invade red blood cells and replicate, producing 16-32 new parasites every two days. Instead of continuing to replicate, some parasites differentiate into either male or female gametocytes over the course of 12 days. Gametocytes, after being taken up in a blood meal by a mosquito, are stimulated to fertilize and begin a developmental cascade in the mosquito that leads to the production of sporozoites, which are infectious to humans. Sporozoites are stored in the salivary gland of a mosquito until release during another blood meal, initiating a new infection.
The complex life cycle allows the parasite to avoid many standard defenses used by the human immune system, but also provides multiple points for interventions. Ongoing projects in the Williamson lab are directed toward: 1) Malaria immunity/systems biology, 2) In vitro and in vivo molecular signaling of sexual differentiation, and 3) Identification of gametocytocidal drugs.
1) Malaria immunity/systems biology: The host specificity of P. falciparum makes direct investigation of the infection in humans critical to the development of effective interventions. Over 50 years ago human passive transfer experiments (Cohen et al. 61) clearly demonstrated a key role for naturally acquired antibodies that protect against malaria. However, reproducing this immunity by vaccination has proven difficult due to antigenic variation, genetic polymorphism, limited immunogenicity of recombinant proteins, and difficulties producing recombinant proteins with native conformation. Utilizing advances in high throughput sequencing and multi-dimensional data analysis we are taking a systems biology approach to understand the human response to repetitive challenges with P. falciparum infected-mosquitoes as happens in the field. Insights into the cellular and antibody responses and their association with clinical symptoms will be used to develop alternative vaccinology approaches to induce protective immunity. This strategy bypasses the use of recombinant antigens produced in heterologous organisms for screening and seeks to identify the protective responses that have evolved in the human host, including the initial innate response and the maturation of the Ig variable region following infection. Ultimately, Ig variable region sequences associated with protection will be generated recombinantly to confirm anti-P.falciparum activity. Target antigens will also be identified and used in conjunction with the insights gained into the early innate and cellular immune responses to develop vaccine strategies that mimic the protective human response.
2) In vitro and in vivo molecular signaling of sexual differentiation: For malaria to be transmitted from human to human via a mosquito, an intraerythrocytic parasite must undergo sexual differentiation into either a male or female gametocyte. We identified the first Plasmodium falciparum gene found to be required for gametocyte development, Pfgdv1 and the set of genes differentially expressed in early gametocytes (Pfge genes) (Eski et al 2012). Localization studies demonstrate that in parasites undergoing gametocytogenesis PfGDV1 is located at the nuclear periphery adjacent to the replication origin protein MCM2 and histone modifying enzyme PfSir2 (Fig.2). This location could suggest that Pfgdv1 plays a role regulating gene expression during early gametocytogenesis. Currently, proteins and nucleic acids associated with this Pfgdv1 are being identified by chromatin and protein immunoprecipitation, as well as fluorescence in situ hybridization (FISH), and will be tested for a role in the regulation of Pfge genes. Pfge gene function is also being evaluated by targeted gene disruption and other molecular techniques adapted for P. falciparum by our laboratory (Ikadai et al 2013, Eksi et al. 2011, Morahan et al 2011, López‑Barragán et al. 2011). Combining these approaches will begin to define mechanisms the parasite uses to initiate sexual differentiation and develop into mature gametocytes. Moreover, field studies are being initiated to evaluate sexual differentiation in vivo by expression profiling and to determine if genetic polymorphisms in the Pfgdv1-related transcriptome correlate with transmission rates (Joice, R et al.2013, 2014). Such molecular relationships will be used to develop genetic or expression signatures of high transmission capacity to direct future control malaria control efforts.
Fig. 2) Immunofluorescence assay of P. falciparum parasites expressing PfGDV1 tagged with GFP that have been probed with anti-PfMCM2 antibodies and DAPI DNA stain. The image includes two parasites: one in the focal plane (1) and one out of the focal plane (2). The hemozoin crystal in the food vacuole (H) is the dense structure in the bright field (BF) image.
3) Identification of gametocytocidal drugs: A sensitive, fluorescence-based, gametocytocidal assay has been developed in my laboratory to facilitate screening molecules for activity against sexual stage parasites (Tanaka and Williamson 2011). The miniaturization of this viability assay (Tanaka et al 2013) allowed the screening of more than 6000 bioactive compounds for gametocytocidal activity (Wei et al. 2014, Tanaka et al. 2014). Seven compounds had IC50s of <50 nM against late stage (III-V) P. falciparum gametocytes, including Torin 2 (IC50 = 8 nM). This compound is >1,000 times less effective against mammalian HepG2 cells and completely blocks P. berghei oocyst production in a mouse model of transmission. Interestingly, a closely related derivative, Torin 1, which has similar activity as Torin 2 against the mammalian target, mTOR, was 200 times less effective against gametocytes. This finding coupled with the lack of mTOR homologs in Plasmodium led us to hypothesize that the human and P. falciparum Torin 2 targets are distinct and that this difference will allow the development of an effective P. falciparum specific drug. Additional Torin 2 analogs are being developed by collaborators at the National Center for Advancing Translational Science (NCATS) at NIH and this project has been selected by the Therapeutics for Rare and Neglected Diseases Program at NCATS for further drug development efforts.