Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based ‘launch’ vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20–30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the ‘launch’ vector technology for the production of VLP-based recombinant vaccines against infectious diseases.

Malaria transmission blocking vaccines (TBV) directed against proteins expressed on sexual stages of Plasmodium falciparum in the mosquito midgut are considered an effective means to reduce malaria transmission. Antibodies induced by TBV block sporogonic development in the mosquito, and thus transmission to the next human host. The Pfs25 protein, expressed on the surface of gametes, zygotes and ookinetes, is one of the primary targets for TBV development. Using a plant virus-based transient expression system, we have successfully produced Pfs25 fused to a modified lichenase (LicKM) carrier in Nicotiana benthamiana, purified and characterized the protein (Pfs25-FhCMB), and evaluated this vaccine candidate in animal models for the induction of transmission blocking antibodies. Soluble Pfs25-FhCMB was expressed in plants at a high level, and induced transmission blocking antibodies that persisted for up to 6 months post immunization in mice and rabbits. These data demonstrate the potential of the new malaria vaccine candidate and also support feasibility of expressing Plasmodium antigens in a plant-based system

Platelet accretion into arterial thrombus in stenotic arterial vessels involves shear-induced platelet activation and adhesion. The Cone and Plate(let) Analyzer (CPA) is designed to simulate such conditions in vitro under a rotating high shear rate in whole blood. In the present study, we evaluated various experimental conditions (including aspirin, temperature, and calcium concentration) and investigated the effects of small molecules along with peptide glycoprotein IIb/IIIa antagonists on platelet adhesion using the CPA system. Concentration-dependent effect of glycoprotein IIb/IIIa antagonists on shear-induced platelet adhesion showed marked differences in potencies: IC 50 ϭ 34, 35, 91, 438, and 606 nM for DPC802 (a specific glycoprotein IIb/IIIa antagonist), roxifiban, sibrafiban, lotrafiban, and orbofiban (free acid forms), respectively, and IC 50 values of 43, 430, and 5781 nM for abciximab, tirofiban, and eptifibatide, respectively. Parallel study was also conducted to evaluate the effect of glycoprotein IIb/IIIa inhibitors using optical aggregometry. The potency of fibans in blocking shear-induced platelet adhesion correlated well with their binding affinity to the resting and activated glycoprotein IIb/IIIa receptors, as well as their "off-rates". Nevertheless, none of these fibans was able to effectively block shear-induced platelet adhesion at targeted clinical dosing regimens except for abciximab. These data suggest that glycoprotein IIb/IIIa antagonists that show similar efficacy in the inhibition of platelet aggregation in a static in vitro assay may differ substantially in a shear-based system of platelet adhesion. The clinical significance of this phenomenon awaits further investigation.