(585c) Novel Protein Engineered for the Treatment of Malaria

Introduction: Malaria is the single most deadly infectious disease. Malaria is responsible for almost half of all historical human deaths. While the traditional ravages of malaria have been curbed by the rise of modern medicine, there are still 214 million annual cases, killing 438 thousand of those infected. As recently as the year 2000, malaria claimed almost 1 million lives a year. The stark reduction in annual malarial mortality seen over the last 17 years can be in part attributed to increased access to classical antimalarial drugs. While the spread of traditional medicine has saved numerous people, the overuse of these antimalarial agents has led to the rise of drug resistant malaria. There are now strains of malaria which are resistant to all pharmaceutical treatments. The spread of drug resistant malaria has breathed a new sense of urgency into the search for novel antimalarial agents.

As the malaria parasite destroys its host, an infected erythrocyte undergoes some of the classic hallmarks of apoptosis. A key hallmark of apoptosis is the presence of the anionic phospholipid phosphatidylserine (PS) on the surface of the erythrocyte. In healthy cells, PS is actively transported to the inner leaflet of the plasma membrane, preventing its accumulation on the outside of the cell. Damaged cells, such as those infected by Plasmodium parasites are unable to maintain the cellular processes necessary to prevent the exposure of PS to the outer leaflet of their plasma membrane. The protein annexin V (AV) has a remarkably high affinity to PS, and will selectively bind to cells expressing this anionic phospholipid on their surface. Endogenously administered AV will actively localize to PS expressing cells in vivo. A therapeutic enzyme cysthathionine-γ-lyase (mCGL) was incorporated with AV into a fusion protein (mCGL-AV) and assayed for antimalarial properties.

Materials and Methods: In vitro experiments were performed using blood harvested from 5 week old BALB/c mice infected with P. berghei, during the ascending phase of infection. Isolated erythrocytes harvested from BALB/c mice were then incubated with mCGL-AV under an oxygen reduced atmosphere to determine in vitro cytotoxicity. Localization of mCGL-AV to parasites was determined via fluorescence microscopy. Parasite cytotoxicity in reponse to mCGL-AV was determined through flow cytometry. Methemoglobinemia was determined through absorbance spectroscopy. In addition, Plasmodium-infected outbread albino CF-1 mice were treated with a single dose of the mCGL-AV fusion protein (10 mg/kg).

Results and Discussion: The fusion protein mCGL-AV selectively localizes to parasitized erythrocytes when observed with fluorescent microscopy. Healthy cells failed to localize mCGL-AV. The fusion protein mCGL-AV rapidly destroys parasites in vitro. Parasites incubated with mCGL-AV for 3 hours and then washed were then cultured for 12 hours and assayed for parasite death using propidium iodide. Malaria parasites were killed by low doses of mCGL-AV (MCC = 20 nM; CC50 = 35 nM). No significant oxidation (EC=100 nM) of the hemoglobin of healthy erythrocytes was observed at therapeutic doses as measured by absorbance spectroscopy. Additionally, healthy erythrocytes fixed and stained with Giemsa stain were undamaged by therapeutic levels of mCGL-AV as determined by DIC light microscopy. In the study in treating mice with a single dose of mCGL-AV, half of the mice treated were found to be cured. No mice in the untreated group survived beyond 10 days since the beginning of the infection.

Conclusions: A fusion protein consisting of annexin (AV) and the mutated enzyme cysthathionine-γ-lyase (mCGL) actively accumulates with great specificity on parasitized erythrocytes expressing phosphatidylserine. mCGL-AV localized to the malaria parasite then catalyzes the destruction of the parasite essential amino acid methionine, depriving the rapidly replicating parasite of a key metabolite. The combination of a therapeutic enzyme and a parasite specific delivery mechanism represents a novel treatment modality in the field of infectious disease.