A novel bispecific antibody platform to direct complement activity for efficient lysis of target cells.

Harnessing complement-mediated cytotoxicity by therapeutic antibodies has been limited because of dependency on size and density of antigen, structural constraints resulting from orientation of antibody binding, and blockade of complement activation by inhibitors expressed on target cells. We developed a modular bispecific antibody platform that directs the complement-initiating protein C1q to target cells, increases local complement deposition and induces cytotoxicity against target antigens with a wide-range of expression. The broad utility of this approach to eliminate both prokaryotic and eukaryotic cells was demonstrated by pairing a unique C1q-recruiting arm with multiple targeting arms specific for Staphylococcus aureus, Pseudomonas aeruginosa, B-cells and T-cells, indicating applicability for diverse indications ranging from infectious diseases to cancer. Generation of C1q humanized mice allowed for demonstration of the efficacy of this approach to clear disease-inducing cells in vivo. In summary, we present a novel, broadly applicable, and versatile therapeutic modality for targeted cell depletion.

Quantification of sporozoite invasion, migration, and development by microscopy and flow cytometry.

There is an important role for in vitro assays to better understand the initial steps of malaria infection. In this section, we describe both microscopy-based and flow cytometry-based sporozoite invasion, migration and development assays with the rodent malaria parasites, Plasmodium berghei and Plasmodium yoelii, and the human malaria parasite, Plasmodium falciparum.

Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade host cells.

Malaria infection is initiated when Anopheles mosquitoes inject Plasmodium sporozoites into the skin. Sporozoites subsequently reach the liver, invading and developing within hepatocytes. Sporozoites contact and traverse many cell types as they migrate from skin to liver; however, the mechanism by which they switch from a migratory mode to an invasive mode is unclear. Here, we show that sporozoites of the rodent malaria parasite Plasmodium berghei use the sulfation level of host heparan sulfate proteoglycans (HSPGs) to navigate within the mammalian host. Sporozoites migrate through cells expressing low-sulfated HSPGs, such as those in skin and endothelium, while highly sulfated HSPGs of hepatocytes activate sporozoites for invasion. A calcium-dependent protein kinase is critical for the switch to an invasive phenotype, a process accompanied by proteolytic cleavage of the sporozoite's major surface protein. These findings explain how sporozoites retain their infectivity for an organ that is far from their site of entry.

Antimalarial activity of allicin, a biologically active compound from garlic cloves.

The incidence of malaria is increasing, and there is an urgent need to identify new drug targets for both prophylaxis and chemotherapy. Potential new drug targets include Plasmodium proteases that play critical roles in the parasite life cycle. We have previously shown that the major surface protein of Plasmodium sporozoites, the circumsporozoite protein (CSP), is proteolytically processed by a parasite-derived cysteine protease, and this processing event is temporally associated with sporozoite invasion of host cells. E-64, a cysteine protease inhibitor, inhibits CSP processing and prevents invasion of host cells in vitro and in vivo. Here we tested allicin, a cysteine protease inhibitor found in garlic extracts, for its ability to inhibit malaria infection. At low concentrations, allicin was not toxic to either sporozoites or mammalian cells. At these concentrations, allicin inhibited CSP processing and prevented sporozoite invasion of host cells in vitro. In vivo, mice injected with allicin had decreased Plasmodium infections compared to controls. When sporozoites were treated with allicin before injection into mice, malaria infection was completely prevented. We also tested allicin on erythrocytic stages and found that a 4-day regimen of allicin administered either orally or intravenously significantly decreased parasitemias and increased the survival of infected mice by 10 days. Together, these experiments demonstrate that the same cysteine protease inhibitor can target two different life cycle stages in the vertebrate host.

The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion.

The circumsporozoite protein (CSP) is the major surface protein of Plasmodium sporozoites, the infective stage of malaria. Although CSP has been extensively studied as a malaria vaccine candidate, little is known about its structure. Here, we show that CSP is proteolytically cleaved by a papain family cysteine protease of parasite origin. Our data suggest that the highly conserved region I, found just before the repeat region, contains the cleavage site. Cleavage occurs on the sporozoite surface when parasites contact target cells. Inhibitors of CSP processing inhibit cell invasion in vitro, and treatment of mice with E-64, a highly specific cysteine protease inhibitor, completely inhibits sporozoite infectivity in vivo.

The enteric parasite Entamoeba uses an autocrine catecholamine system during differentiation into the infectious cyst stage.

Enteric amoebae of the genus Entamoeba travel from host to host in an encysted form. We previously showed that in vitro cyst development of Entamoeba invadens requires the addition of defined amounts of multivalent galactose-terminated molecules, such as mucin, to the cultures. The amoeba surface lectin that binds mucin is presumed to convey transmembrane signals when clustered by the ligand, but the signaling molecules that function downstream of the lectin are not known. We report here that Entamoeba encystation was induced in the absence of galactose ligand when catecholamines were added to the encystation medium. Micromolar amounts of both epinephrine and norepinephrine induced encystation. Of a variety of synthetic catecholamine agonists tested, only beta(1)-adrenergic receptor agonists supported encystation, whereas alpha- and beta(2)-adrenergic receptor agonists did not. Only beta(1)-adrenergic receptor antagonists inhibited encystation, and did so even when exogenous catecholamines were not added, indicating that catecholamine binding is required for encystation and suggesting an endogenous source of the ligand. High performance liquid chromatography analysis of Entamoeba extracts showed that the amoebae themselves contain catecholamines and at least one of these is released when the cells are stimulated to encyst with galactose-terminated ligands. The presence of catecholamine binding sites on the surface of amoeba trophozoites was confirmed using radiolabeled catecholamine antagonist. Amoeba encystment was inhibited by addition of beta(1)-adrenergic receptor antagonist to cells that were stimulated to differentiate with either galactose ligand or catecholamines, but not with dibutyryl cAMP. This suggests that the amoeba catecholamine receptor functions downstream of the galactose lectin and upstream of adenylyl cyclase. This enteric protozoan parasite, therefore, contains the components of an autocrine catecholamine ligand-receptor system that may act in conjunction with a galactose lectin to regulate differentiation into the infectious cyst stage.